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Targeting ornithine decarboxylase in Myc-induced lymphomagenesis prevents tumor formation
Abstract
Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.
... Indeed, forced overexpression of ODC in NIH 3T3 fibroblast cells is sufficient to induce tumors in immune compromised mice [24] and increased expression of ODC accelerates tumor development in premalignant epidermal cells [25]. In contrast, depletion of polyamines by knockout of Odc or treatment with an ODC suicide inhibitor coined α-difluoromethylornithine (DFMO) [26], markedly impairs tumor development in Eµ-Myc mice, a validated model of MYC-driven B cell lymphoma [27] and in MYCN-driven neuroblastoma [28,29]. Moreover, DFMO maintenance therapy can prevent relapse following completion of standard therapy and improves overall survival in pediatric patients with high-risk neuroblastoma (HRNB) [30]. ...
... ODC is the rate limiting enzyme of polyamine biosynthesis and ODC levels are tightly controlled and are promptly adjusted to cellular needs by several mechanisms, including transcription, mRNA stability, translation and degradation [54]. ODC is a direct transcription target of MYC oncoproteins and is overexpressed in MYCand MYCN-driven cancers [27][28][29]55]. The MYC-ODC-polyamine axis has been well studied and reviewed by others [56,57]. ...
... AMD1, which directs production of the aminopropyl donor necessary for the generation of SPD and SPM from PUT and SPD, respectively (Figure 1) [75], is also a transcription target induced by MYC oncoproteins [27]. Furthermore, AMD1 translation is also highly regulated. ...
Polycationic polyamines are present in nearly all living organisms and are essential for mammalian cell growth and survival, and for development. These positively charged molecules are involved in a variety of essential biological processes, yet their underlying mechanisms of action are not fully understood. Several studies have shown both beneficial and detrimental effects of polyamines on human health. In cancer, polyamine metabolism is frequently dysregulated, and elevated polyamines have been shown to promote tumor growth and progression, suggesting that targeting polyamines is an attractive strategy for therapeutic intervention. In contrast, polyamines have also been shown to play critical roles in lifespan, cardiac health and in the development and function of the brain. Accordingly, a detailed understanding of mechanisms that control polyamine homeostasis in human health and disease is needed to develop safe and effective strategies for polyamine-targeted therapy.
... 23 These tumors are generally clonal, implying that additional oncogenic lesions are required for lymphoma onset. Indeed, deregulated MYC not only promotes proliferation (eg, [145][146][147] ), but also generates oncogenic stress signals that elicit apoptosis, 148,149 linked to activation of the tumor suppressor p53. 150 Most importantly in this context, multi- Besides apoptosis, p53 engages alternative cellular responses in the presence of oncogenic stress: one of these is oncogene-induced senescence (OIS), linked to tumor suppression, to the response to therapy, as well as to the acquisition of aggressive cancer cell phenotypes. ...
... 183 While typically observed following activation of different oncogenes, such as RAS, 184 OIS contributed to limit the progression of MYC-driven lymphomas in response to TGF-beta signaling (either autocrine, or stroma-derived) 185,186 and could be elicited under specific circumstances, such as chemo-or anti-CD20 therapy, [187][188][189] or MYC activation in the absence of the cyclin-dependent kinase CDK2. 190 It is noteworthy here that, besides CDK2, other cell cycle regulators were shown to impact MYC-induced lymphomagenesis: for instance, decreased expression of the CDK-inhibitory factor p27 Kip1 was reported in BL 146 and p27 Kip1 deletion accelerated lymphomagenesis in Eμ-myc mice, while deletion of Cks1-a factor involved in p27 Kip1 degradation-delayed it. 191,192 In line with these observations deletion of Ornithine decarboxylase (Odc), a well-established MYC target gene overexpressed in BL, delayed lymphomagenesis by reducing the inhibition of p27 Kip1 by MYC. ...
... 191,192 In line with these observations deletion of Ornithine decarboxylase (Odc), a well-established MYC target gene overexpressed in BL, delayed lymphomagenesis by reducing the inhibition of p27 Kip1 by MYC. 146 Nonetheless, the clinical relevance of senescence or other cytostatic effects in lymphoma development or therapy remains to be addressed. ...
The rearrangement of immunoglobulin loci during the germinal center reaction is associated with an increased risk of chromosomal translocations that activate oncogenes such as MYC, BCL2 or BCL6, thus contributing to the development of B‐cell lymphomas. MYC and BCL2 activation are initiating events in Burkitt's (BL) and Follicular Lymphoma (FL), respectively, but can occur at later stages in other subtypes such as Diffuse Large‐B Cell Lymphoma (DLBCL). MYC can also be activated during the progression of FL to the transformed stage. Thus, either DLBCL or FL can give rise to aggressive double‐hit lymphomas (DHL) with concurrent activation of MYC and BCL2. Research over the last three decades has improved our understanding of the functions of these oncogenes and the basis for their cooperative action in lymphomagenesis. MYC, in particular, is a transcription factor that contributes to cell activation, growth and proliferation, while concomitantly sensitizing cells to apoptosis, the latter being blocked by BCL2. Here, we review our current knowledge about the role of MYC in germinal center B‐cells and lymphomas, discuss MYC‐induced dependencies that can sensitize cancer cells to select pharmacological inhibitors, and illustrate their therapeutic potential in aggressive lymphomas—and in particular in DHL, in combination with BCL2 inhibitors.
... Powder-formulated DFMO (eflornithine hydrochloride) was reconstituted with sterile water to a 100 mg/ml solution and given orally three times daily. For dose levels 1 and 2 (21day courses), chemotherapy was days 1-5 and DFMO was given for 14 days out of 21 (held for days [8][9][10][11][12][13][14]. Due to delayed platelet recovery, subsequent dose levels 2a-4a used 28-day courses, with DFMO given days 1-14 and 22-28, chemotherapy days 8-12 (the same regimen as dose levels 1 and 2 but with 7 additional days of DFMO to begin each cycle). ...
... Still, post-relapse PFS, recommended as an optimal primary endpoint for early phase neuroblastoma trials [48], is comparable to both realworld data with the COG chemoimmunotherapy regimem in multiply relapsed patients [2-year PFS 28%; [49]] and GD2directed chimeric antigen receptor therapy (GD2-CART01 2-year PFS 27%; [50]). Polyamines can also be imported from the tumor microenvironment to rescue polyamine homeostasis in DFMO-treated tumors [8,9]. AMXT1501 is a polyamine transport inhibitor that synergizes with DFMO in preclinical neural tumor models [12,51] and is currently in human Phase 1/2 testing combined with DFMO (NCT03536728). ...
Background
MYC genes regulate ornithine decarboxylase (Odc) to increase intratumoral polyamines. We conducted a Phase I trial [NCT02030964] to determine the maximum tolerated dose (MTD) of DFMO, an Odc inhibitor, with celecoxib, cyclophosphamide and topotecan.
Methods
Patients 2–30 years of age with relapsed/refractory high-risk neuroblastoma received oral DFMO at doses up to 9000 mg/m ² /day, with celecoxib (500 mg/m ² daily), cyclophosphamide (250 mg/m ² /day) and topotecan (0.75 mg/m ² /day) IV for 5 days, for up to one year with G-CSF support.
Results
Twenty-four patients (median age, 6.8 years) received 136 courses. Slow platelet recovery with 21-day courses (dose-levels 1 and 2) led to subsequent dose-levels using 28-day courses (dose-levels 2a-4a). There were three course-1 dose-limiting toxicities (DLTs; hematologic; anorexia; transaminases), and 23 serious adverse events (78% fever-related). Five patients (21%) completed 1-year of therapy. Nine stopped for PD, 2 for DLT, 8 by choice. Best overall response included two PR and four MR. Median time-to-progression was 19.8 months, and 3 patients remained progression-free at >4 years without receiving additional therapy. The MTD of DFMO with this regimen was 6750 mg/m ² /day.
Conclusion
High-dose DFMO is tolerable when added to chemotherapy in heavily pre-treated patients. A randomized Phase 2 trial of DFMO added to chemoimmunotherapy is ongoing [NCT03794349].
... One MYC regulated metabolic pathway that appears nearly universally up-regulated in cancer is polyamine biosynthesis, which directs the production of putrescine, spermidine and spermine. Notably, polyamine levels are generally elevated in tumors (3,4), and targeting the first enzyme of the pathway, ornithine decarboxylase (ODC), impairs the development of MYC-driven lymphoma and MYCN-driven neuroblastoma (5)(6)(7). Furthermore, the ODC suicide inhibitor αdifluoromethylornithine (DFMO) has shown activity as a chemoprevention agent in colorectal and prostate cancer trials (8,9). However, DFMO has little activity as a single agent in cancer therapeutic trials (10), and this likely reflects the fact that polyamines are abundant in the diet and their intracellular levels can be restored by polyamine transport (11). ...
... The myriad of mutations that are manifest in aggressive hematological malignancies, including in MYC-driven lymphoma (2), make these tumors particularly challenging to treat, and they are highly prone to developing resistance. Underscoring this fact, while several downstream targets of MYC have been shown to contribute to lymphoma development in the Eμ-Myc model (5,(61)(62)(63)(64)(65)(66), to date none of them are absolutely required for disease development as is the case for DHPS. Our findings suggest this profound phenotype is due to the pleiotropic effects of blocking eIF5A hypusination, which is required for the efficient translation of cooperating oncogenes and critical regulators of the cell cycle (Fig. 7K). ...
The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given this, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine–hypusine circuit, which posttranslationally modifies the eukaryotic translation factor eIF5A. The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where the loss of eIF5A hypusination abolishes malignant transformation of MYC-overexpressing B cells. Mechanistically, integrating RNA sequencing, ribosome sequencing, and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1–S phase cell-cycle progression and DNA replication. This circuit thus controls MYC's proliferative response, and it is also activated across multiple malignancies. These findings suggest the hypusine circuit as a therapeutic target for several human tumor types.
Significance
Elevated EIF5A and the polyamine–hypusine circuit are manifest in many malignancies, including MYC-driven tumors, and eIF5A hypusination is necessary for MYC proliferative signaling. Notably, this circuit controls an oncogenic translational program essential for the development and maintenance of MYC-driven lymphoma, supporting this axis as a target for cancer prevention and treatment.
See related commentary by Wilson and Klein.
... In addition to ODC1, other genes in the polyamine metabolism pathway regulated by c-MYC include SRM, SMS, AMD1, and OAZ2, an ornithine decarboxylase antizyme [178][179][180]. c-MYC also indirectly modulates AdoMetDC/AMD1 expression by promoting mTOR activation [181][182][183], which stabilizes AdoMetDC [184]. ...
... c-MYC also indirectly modulates AdoMetDC/AMD1 expression by promoting mTOR activation [181][182][183], which stabilizes AdoMetDC [184]. Regarding polyamine catabolism, c-MYC has been demonstrated to repress the expression of SSAT and SMOX [178,179], thus maintaining high cellular polyamine levels. ...
Simple Summary
This review informs on how the MYC signaling is dysregulated and participates in ovarian cancer progression, and the unmet challenge to directly target MYC for ovarian cancer treatment. Therefore, we proposed to alternatively target essential downstream polyamine metabolism pathway of MYC. In this review we include the metabolism of polyamine, the regulation of polyamine metabolism by MYC signaling, the utility of polyamine as therapeutic targets and cancer biomarkers.
Abstract
c-MYC and its paralogues MYCN and MYCL are among the most frequently amplified and/or overexpressed oncoproteins in ovarian cancer. c-MYC plays a key role in promoting ovarian cancer initiation and progression. The polyamine pathway is a bona fide target of c-MYC signaling, and polyamine metabolism is strongly intertwined with ovarian malignancy. Targeting of the polyamine pathway via small molecule inhibitors has garnered considerable attention as a therapeutic strategy for ovarian cancer. Herein, we discuss the involvement of c-MYC signaling and that of its paralogues in promoting ovarian cancer tumorigenesis. We highlight the potential of targeting c-MYC-driven polyamine metabolism for the treatment of ovarian cancers and the utility of polyamine signatures in biofluids for early detection applications.
... It was also shown that while the drug efficacy was the greatest in subjects with a low Spd/Spm ratio in rectal mucosa at the beginning of the trial, no relationship was found between DFMO/sulindac-induced changes in polyamine levels and drug efficacy [7]. In mouse studies, DFMO-treatment and ODC heterozygosity were both able to impair lymphomagenesis in Eμ-Myc transgenic mice, while only DFMO was effective in suppressing MYCN-driven neuroblastoma, suggesting that DFMO might target also additional factors than polyamines [8]. Previously it was shown that in addition to depleting polyamine pools, DFMO treatment led to a reduction of cellular nucleoside and nucleotide pools, especially that of thymidine-5′-monophosphate (dTMP) [9]. ...
... It was also shown that while the drug efficacy was the greatest in subjects with a low Spd/Spm ratio in rectal mucosa at the beginning of the trial, no relationship was found between DFMO/sulindac-induced changes in polyamine levels and drug efficacy [7]. In mouse studies, DFMO-treatment and ODC heterozygosity were both able to impair lymphomagenesis in Eµ-Myc transgenic mice, while only DFMO was effective in suppressing MYCN-driven neuroblastoma, suggesting that DFMO might target also additional factors than polyamines [8]. ...
Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072–81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.
... However, although direct inhibition of MYC is difficult because of its flat structure, indirect targeting of its degradation or biosynthesis has been challenging due to the multiple compensatory mechanisms that restore its intracellular content. An alternative pursued strategy is the targeting of MYC-regulated pathways that are required for tumor growth 10 . In this regard, inhibition of Ornithine decarboxylase (ODC), the first and ratelimiting enzyme in the polyamine biosynthesis pathway, and a direct MYC transcriptional target 11 has been proposed as a potential therapeutic option in malignancies driven by the MYC oncogenes, such as lymphoma and neuroblastoma 10,12 . ...
... An alternative pursued strategy is the targeting of MYC-regulated pathways that are required for tumor growth 10 . In this regard, inhibition of Ornithine decarboxylase (ODC), the first and ratelimiting enzyme in the polyamine biosynthesis pathway, and a direct MYC transcriptional target 11 has been proposed as a potential therapeutic option in malignancies driven by the MYC oncogenes, such as lymphoma and neuroblastoma 10,12 . ...
Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APC Min/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.
... Also, additional polyamines are stored nearby in astrocytes and glial cells if needed. However, altered polyamine levels in the brain of AD patients have been reported, including increased levels of ODC, the initial enzyme in polyamine synthesis [49], with partial localization of ODC from the nucleus to the cytoplasm [50], and increased accumulation of the ODC antizyme inhibitor 2 (AZIN2) in AD brains [51]. In murine models of tauopathies, over expression of AZIN2 can cause an abnormal polyamine response with increase in acetylated polyamines and abnormal tau deposition [41]. ...
A hypothesis of Alzheimer’s disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli. Polyamines are essential in nucleolar functions, such as RNA folding and ribonucleoprotein assembly. Changes in the nucleolar pool of anionic RNA and cationic polyamines acting as counterions can cause significant nucleolar dynamics. Polyamine synthesis reduces S-adenosylmethionine which, at low levels, triggers tau phosphorylation. Also, polyamine recycling reduces acetyl-CoA needed for acetylcholine, which is low in Alzheimer’s disease. Extraordinary nucleolar expansion and/or contraction can disrupt epigenetic control in peri-nucleolar chromatin, such as chromosome 14 with the presenilin-1 gene; chromosome 21 with the amyloid precursor protein gene; chromosome 17 with the tau gene; chromosome 19 with the APOE4 gene; and the inactive X chromosome (Xi; aka “nucleolar satellite”) with normally silent spermine synthase (polyamine synthesis) and spermidine/spermine-N1-acetyltransferase (polyamine recycling) alleles. Chromosomes 17, 19 and the Xi have high concentrations of Alu elements which can be transcribed by RNA polymerase III if positioned nucleosomes are displaced from the Alu elements. A sudden flood of Alu RNA transcripts can competitively bind nucleolin which is usually bound to Alu sequences in structural RNAs that stabilize the nucleolar heterochromatic shell. This Alu competition leads to loss of nucleolar integrity with leaking of nucleolar polyamines that cause aggregation of phosphorylated tau. The hypothesis was developed with key word searches (e.g., PubMed) using relevant terms (e.g., Alzheimer’s, lupus, nucleolin) based on a systems biology approach and exploring autoimmune disease tautology, gaining synergistic insights from other diseases.
... To confirm that polyamines control CD69 expression, we tested the effects of inhibiting ODC using difluoromethylornithine (DFMO), an irreversible suicide inhibitor of ODC (28) ( Figure 3A). Similar to glutamine deprivation, DFMO treatment resulted in increased and sustained CD69 expression in activated CD8 + T cells, and the stimulatory effects of DFMO were abolished by co-treatment with polyamines ( Figure 3B). ...
Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine and spermine. These metabolites play critical roles in activation-induced T-cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eIF5A. Here, we demonstrated that the glutamine-polyamine-hypusine axis controls the expression of CD69, an important regulator of tissue resident memory T cells (TRM). Inhibition of this circuit augmented the development of TRM cells ex vivo and in vivo in the bone marrow, a well-established niche for TRM cells. Furthermore, blocking the polyamine-hypusine axis augmented CD69 expression and IFN-γ and TNF-α production in human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes, as well as in human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate TRM cells for therapeutic benefit.
... Young Eµ-myc mice show a characteristic expansion of pre-tumoral B-cells, counter-balanced by a concomitant increase in apoptosis (Nilsson et al, 2005). Monitoring of bone marrow B220 + CD19 + B-cells revealed that their fraction was significantly increased in the Pcgf6 f/f background (Fig 2A) correlating with an impairment in Mycinduced apoptosis ( Fig 2B). ...
Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4, and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6–but not Mga–accelerates Myc-induced lymphomagenesis in Eµ- myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.
... To initially assess if MYC controlled the autophagy-lysosomal pathway, three independent expression profiling datasets from the Eμ-Myc transgenic mouse (34,36,37), a validated model of human B cell lymphoma with MYC involvement (38), were queried for expression of target genes of TFEB, which coordinately controls expression of components of this recycling center (20,21). Wild type (WT) B220 + splenic B cells express high levels of Tfeb and many Tfeb target genes (Fig. 1A). ...
MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eμ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA transport. Reactivation of the autophagy-lysosomal pathway by TFEB disabled the malignant state by disrupting mitochondrial functions, proteasome activity, AA transport, and AA and nucleotide metabolism, leading to metabolic anergy, growth arrest, and apoptosis. This phenotype provides therapeutic opportunities to disable MYC-driven malignancies, including AA restriction and treatment with proteasome inhibitors.
Significance:
MYC suppresses TFEB and autophagy and controls amino acid homeostasis by upregulating amino acid transport and the proteasome, and reactivation of TFEB disables the metabolism of MYC-driven tumors.
... Other contexts relevant to cellular potency also manifest small magnitude transcript-level changes that may be common in cellular potency transitions. Trans-activation by MYC induces relevant target gene expression less than two-fold (Baluapuri et al., 2020;Nilsson et al., 2005) and mouse pluripotency factor; Esrrb apparently causes less than two-fold upregulation of embryonic-stem-cell-specific genes (Chronis et al., 2017). The average negative effect of the transcription regulator, Polycomb, is about two-fold (Berrozpe et al., 2017), and its associated histone modification, H3K27me3, modulates cellular potency and imprinting (Bernstein et al., 2006;Santini et al., 2021). ...
In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.
... Young Eµ-myc mice show a characteristic expansion of pre-tumoral B-cells, counterbalanced by a concomitant increase in apoptosis (Nilsson et al. 2005). Monitoring of bone marrow B220 + CD19 + B-cells revealed that their fraction was significantly increased in the Pcgf6 f/f background ( Fig. 2A) correlating with an impairment in Myc-induced apoptosis (Fig. 2B). ...
Max is an obligate dimerization partner for the Myc transcription factors and for several repressors, such as Mnt, Mxd1-4 and Mga, collectively thought to antagonize Myc function in transcription and oncogenesis. Mga, in particular, is part of the variant Polycomb group repressive complex PRC1.6. Here, we show that ablation of the distinct PRC1.6 subunit Pcgf6 - but not Mga - accelerates Myc-induced lymphomagenesis in Emu-myc transgenic mice. Unexpectedly, however, Pcgf6 loss shows no significant impact on transcriptional profiles, in neither pre-tumoral B-cells, nor lymphomas. Altogether, these data unravel an unforeseen, Mga- and PRC1.6-independent tumor suppressor activity of Pcgf6.
... One salient example is ornithine decarboxylase (ODC). In the original proof-of-principle paper, deleting the ODC gene or inhibiting this enzyme with difluoromethylornithine (DFMO) delayed lymphoma development in the Eμ-MYC mouse model [44]. However, in the realm of experimental oncology, this approach appears to be redirected towards MYCNdriven tumors, such as neuroblastoma [45]. ...
MYC oncoprotein promotes cell proliferation and serves as the key driver in many human cancers; therefore, considerable effort has been expended to develop reliable pharmacological methods to suppress its expression or function. Despite impressive progress, MYC-targeting drugs have not reached the clinic. Recent advances suggest that within a limited expression range unique to each tumor, MYC oncoprotein can have a paradoxical, proapoptotic function. Here we introduce a counterintuitive idea that modestly and transiently elevating MYC levels could aid chemotherapy-induced apoptosis and thus benefit the patients as much, if not more than MYC inhibition.
... As polyamine is crucial in cancer development, the mechanism of how the polyamine pathway interacts with oncogenes becomes an important focus in cancer study [22,46]. The MYC family (c-MYC, N-MYC, and L-MYC) was shown to be activated in about 70% of human cancers [47,48] and regulate ODC transcriptionally, leading to upregulation of polyamine levels and induction of hyper-proliferation [48]. ...
Polyamines are essential for the proliferation, differentiation, and development of eukaryotes. They include spermine, spermidine, and the diamine precursor putrescine, and are low-molecular-weight, organic polycations with more than two amino groups. Their intracellular concentrations are strictly maintained within a specific physiological range through several regulatory mechanisms in normal cells. In contrast, polyamine metabolism is dysregulated in many neoplastic states, including cancer. In various types of cancer, polyamine levels are elevated, and crosstalk occurs between polyamine metabolism and oncogenic pathways, such as mTOR and RAS pathways. Thus, polyamines might have potential as therapeutic targets in the prevention and treatment of cancer. The molecular mechanisms linking polyamine metabolism to carcinogenesis must be unraveled to develop novel inhibitors of polyamine metabolism. This overview describes the nature of polyamines, their association with carcinogenesis, the development of polyamine inhibitors and their potential, and the findings of clinical trials.
... Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines (putrescine, spermidine, spermine), which are positively charged metabolites that contribute to normal cell cycle regulation and when elevated can stimulate increased cell proliferation [9][10][11][12][13][14]. ODC is a validated drug target in NB [6] and other MYC-deregulated cancers [15][16][17]. The ODC gene is directly activated by MYC transcription factors through interactions with the E-box elements of ODC1 gene [18][19][20][21]. Given the high relevance of MYCN gene amplification in NB, we postulated in 2004 that ODC is an important drug target for this cancer type and that inhibition of polyamine biosynthesis via ODC inhibition might be a novel therapeutic strategy for NB [22,23]. ...
Background
Neuroblastoma (NB) is the most common extracranial solid tumor in children. Interference with the polyamine biosynthesis pathway by inhibition of MYCN-activated ornithine decarboxylase (ODC) is a validated approach. The ODC inhibitor α-difluoromethylornithine (DFMO, or Eflornithine) has been FDA-approved for the treatment of trypanosomiasis and hirsutism and has advanced to clinical cancer trials including NB as well as cancer-unrelated human diseases. One key challenge of DFMO is its rapid renal clearance and the need for high and frequent drug dosing during treatment.Methods
We performed in vivo pharmacokinetic (PK), antitumorigenic, and molecular studies with DFMO/probenecid using NB patient-derived xenografts (PDX) in mice. We used LC–MS/MS, HPLC, and immunoblotting to analyze blood, brain tissue, and PDX tumor tissue samples collected from mice.ResultsThe organic anion transport 1/3 (OAT 1/3) inhibitor probenecid reduces the renal clearance of DFMO and significantly increases the antitumor activity of DFMO in PDX of NB (P < 0.02). Excised tumors revealed that DFMO/probenecid treatment decreases polyamines putrescine and spermidine, reduces MYCN protein levels and dephosphorylates retinoblastoma (Rb) protein (p-RbSer795), suggesting DFMO/probenecid-induced cell cycle arrest.Conclusion
Addition of probenecid as an adjuvant to DFMO therapy may be suitable to decrease overall dose and improve drug efficacy in vivo.
... The transcription of many genes, including c-Jun and c-Myc, are regulated by PAs [39]. Likewise, selective PAs are responsible for the regulation of AdoMetDC, AZ, and SSAT for the translation of various mRNA sections [51][52][53]. Additionally, several studies showed the effect of PAs on the cell signaling pathways by affecting the status and levels of main regulatory proteins such as CDK-4, GSK-3β, p53, p27Kip, p21Cip1, Src, EGFR, Mdm2, Akt/protein kinase B, and importin-α1 [54][55][56][57]. ...
Putrescine, spermine, and spermidine are the important polyamines (PAs), found in all
living organisms. PAs are formed by the decarboxylation of amino acids, and they facilitate cell growth and development via different cellular responses. PAs are the integrated part of the cellular and genetic metabolism and help in transcription, translation, signaling, and post-translational modifications. At the cellular level, PA concentration may influence the condition of various diseases in the body. For instance, a high PA level is detrimental to patients suffering from aging, cognitive impairment, and cancer. The levels of PAs decline with age in humans, which is associated with different health disorders. On the other hand, PAs reduce the risk of many cardiovascular diseases and increase longevity, when taken in an optimum quantity. Therefore, a controlled diet is an easy way to maintain the level of PAs in the body. Based on the nutritional intake of PAs, healthy cell functioning can be maintained. Moreover, several diseases can also be controlled to a higher extend via maintaining the metabolism of PAs. The present review discusses the types, important functions, and metabolism of PAs in humans. It also highlights the nutritional role of PAs in the prevention of various diseases.
... Elevated levels of AURKA upregulates c-Myc transcription affecting cellular proliferation and ATP production, important factors on FLHCC tumorigenesis [32,33]. c-Myc overexpression leads to increased ornithine decarboxylase (ODC) activity [34]. This results in increased ornithine consumption to polyamines synthesis [35], reducing ornithine bioavailability that results in urea cycle disorder due to ornithine transcarboxylase (OTC) dysfunction and consequent hyperammonemia [26]. ...
Purpose
hyperammonemic encephalopathy is a potentially fatal condition associated with fibrolamellar hepatocellular carcinoma. The mechanism involved in hyperammonemia in patients with fibrolamellar carcinoma was unclear until a possible physiopathological pathway was recently proposed. An ornithine transcarboxylase dysfunction was suggested as a result of increased ornithine decarboxylase activity induced by c-Myc overexpression. This c-Myc overexpression resulted from Aurora Kinase A overexpression derived from the activity of a chimeric kinase that is the final transcript of a deletion in chromosome 19, common to all fibrolamellar carcinomas.
Methods
we performed the analysis of the expression of all enzymes involved and tested for the mutation in chromosome 19 in fresh frozen samples of fibrolamellar hepatocellular carcinoma, non-tumor liver and hepatic adenomatosis.
Results
specific DNAJB-PRKACA fusion protein that results from the recurrent mutation on chromosome 19 common to all fibrolamellar carcinoma was detected only in the fibrolamellar carcinoma sample. Fibrolamellar carcinoma and adenomiomatosis samples presented increased expression of Aurora Kinase A, c-MYC and ornithine decarboxylase when compared to normal liver, while ornithine transcarbamylase was decreased.
Conclusion
The proposed physiopathological pathway is correct and that overexpression of c-Myc may also be responsible of hyperammonemia in patients with other types of rapidly growing hepatomas. This gives further evidence to apply new and adequate treatment to this severe complication.
... Radiolabelled spermidine transport assays. Polyamine transport in tumor cells was evaluated as described in published protocols 55,56 . Briefly, cells were plated in triplicate and grown to approximately 70% confluence. ...
Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG. Diffuse intrinsic pontine glioma (DIPG) is an almost incurable malignant childhood brain tumor. Here, the authors show that the polyamine synthetic pathway is activated in DIPG and that the dual targeting of polyamine synthesis and uptake results in prolonged survival in animal models.
... ODC is a short-lived protein that is rapidly degraded by the 26S proteasome within 30 min. ODC activity and degradation are delicately controlled by antizyme (AZ), which is a natural ornithine decarboxylase inhibitor that binds, inhibits, and promotes the protein degradation of ODC by the 26S proteasome [18,19]. High polyamine concentrations trigger a +1 ribosomal frameshifting of AZ mRNA to generate mature full-length AZ proteins [20][21][22]. ...
Background:
Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention.
Methods:
We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies.
Results:
Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC50 values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC50, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells.
Conclusions:
Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.
... In addition, DFMO depletes tumours of polyamines and inhibits the growth of MYC-deregulated tumours in animals. 162,163 Pre-emptive blockade of polyamine synthesis effectively prevents neuroblastoma formation in the transgenic TH-MYCN model. 162 DFMO as a cancer chemopreventive agent that has attracted much interest in recent years since ODC is transactivated by the c-myc oncogene in malignant tumours. ...
Autophagy is a mechanism that enables cells to maintain cellular homeostasis by re-
moving damaged materials and mobilizing energy reserves in conditions of starvation.
Although nutrient availability strongly impacts the process of autophagy, the spe-
cific metabolites that regulate autophagic responses have not yet been determined.
Recent results indicate that S-adenosylmethionine (SAM) represents a critical inhibi-
tor of methionine starvation–induced autophagy. SAM is primarily involved in four
key metabolic pathways: transmethylation, transsulphuration, polyamine synthesis
and 5′-deoxyadenosyl 5′-radical–mediated biochemical transformations. SAM is the
sole methyl group donor involved in the methylation of DNA, RNA and histones,
modulating the autophagic process by mediating epigenetic effects. Moreover, the
metabolites of SAM, such as homocysteine, glutathione, decarboxylated SAM and
spermidine, also exert important influences on the regulation of autophagy. From our
perspective, nuclear-cytosolic SAM is a conserved metabolic inhibitor that connects
cellular metabolic status and the regulation of autophagy. In the future, SAM might
be a new target of autophagy regulators and be widely used in the treatment of vari-
ous diseases.
... 74 Moreover, MYC stimulates spermine synthase and spermidine synthase to promote spermidine and spermine biosynthesis (Fig. 4). 75 This MYC-mediated regulation of polyamine biosynthesis has been confirmed in multiple cancers, such as leukemias, lung carcinomas, neuroblastomas, and breast cancers. [76][77][78][79] Recent studies revealed that mTORC1 stabilizes pro-S-adenosyl methionine (AdoMet) decarboxylase (pro-AdoMetDC), leading to increased AdoMetDC to further promote polyamine biosynthesis in prostate cancer. ...
Cancer cells must rewire cellular metabolism to satisfy the demands of unbridled growth and proliferation. As such, most human cancers differ from normal counterpart tissues by a plethora of energetic and metabolic reprogramming. Transcription factors of the MYC family are deregulated in up to 70% of all human cancers through a variety of mechanisms. Oncogenic levels of MYC regulates almost every aspect of cellular metabolism, a recently revisited hallmark of cancer development. Meanwhile, unrestrained growth in response to oncogenic MYC expression creates dependency on MYC-driven metabolic pathways, which in principle provides novel targets for development of effective cancer therapeutics. In the current review, we summarize the significant progress made toward understanding how MYC deregulation fuels metabolic rewiring in malignant transformation.
... The two transcription factors MYC and HIF-1A have been shown to be critical for the induction of glycolytic transcriptional programs in mouse T cells and human cancer cells (29,36), and MYC has been reported to be a transcriptional activator of genes in the polyamine synthesis pathway (37,38). We observed that MYC and HIF-1A were transcriptionally induced in activated primary human CD4 + T cells and that this effect was significantly reduced by metformin + 2-DG treatment (Fig. 3C). ...
Metabolic reprogramming plays a central role in T cell activation and differentiation, and the inhibition of key metabolic pathways in activated T cells represents a logical approach for the development of new therapeutic agents for treating autoimmune diseases. The widely prescribed antidiabetic drug metformin and the glycolytic inhibitor 2-deoxyglucose (2-DG) have been used to study the inhibition of oxidative phosphorylation and glycolysis, respectively, in murine immune cells. Published studies have demonstrated that combination treatment with metformin and 2-DG was efficacious in dampening mouse T cell activation-induced effector processes, relative to treatments with either metformin or 2-DG alone. In this study, we report that metformin + 2-DG treatment more potently suppressed IFN-γ production and cell proliferation in activated primary human CD4+ T cells than either metformin or 2-DG treatment alone. The effects of metformin + 2-DG on human T cells were accompanied by significant remodeling of activation-induced metabolic transcriptional programs, in part because of suppression of key transcriptional regulators MYC and HIF-1A. Accordingly, metformin + 2-DG treatment significantly suppressed MYC-dependent metabolic genes and processes, but this effect was found to be independent of mTORC1 signaling. These findings reveal significant insights into the effects of metabolic inhibition by metformin + 2-DG treatment on primary human T cells and provide a basis for future work aimed at developing new combination therapy regimens that target multiple pathways within the metabolic networks of activated human T cells.
... As mentioned above, ODC is overexpressed as a key rate-limiting enzyme in polyamine synthesis pathway and regulated at the transcriptional level by tumor-promoting agents in a variety of cancers [56]. ODC is the target of the oncogene Myc as a potential oncogene because it can be overexpressed in transformed mammalian cell lines alone or in combination with other oncogenes [57,58]. Transgenic mouse models overexpressing ODC in skin have demonstrated that ODC can lead to the formation of spontaneous skin tumors, suggesting its possible association with cancerous cells [59]. ...
Spermidine, as a natural component from polyamine members, is originally isolated from semen and also existed in many natural plants, and can be responsible for cell growth and development in eukaryotes. The supplementation of spermidine can extend health and lifespan across species. Although the elevated levels of polyamines and the regulation of rate-limiting enzymes for polyamine metabolism have been identified as the biomarkers in many cancers, recent epidemiological data support that an increased uptake of spermidine as a caloric restriction mimic can reduce overall mortality associated with cancers. The possible mechanisms between spermidine and cancer development may be related to the precise regulation of polyamine metabolism, anti-cancer immunosurveillance, autophagy, and apoptosis. Increased intake of polyamine seems to suppress tumorigenesis, but appears to accelerate the growth of established tumors. Based on these observations and the absolute requirement for polyamines in tumor growth, spermidine could be a rational target for chemoprevention and clinical therapeutics of cancers.
... Myc is a master regulator of metabolic transcription programs 44 and induces polyamine biosynthesis 45 via transcriptional induction of Odc1, which is elevated in Crbn 2/2 T cells ( Figure 3B; supplemental Figure 3G). Further, in activated T cells, Myc induces the expression of transporters and enzymes that direct glutamine catabolism and flux into polyamines, 26,38,45 and provokes increases in cell size. Interestingly, Crbn 2/2 CD8 1 T cells were significantly larger, based on forward scatter area, compared with Crbn 1/1 CD8 1 T cells (supplemental Figure 4B). ...
Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only known molecular target for this drug class, where drug-induced ubiquitin-dependent degradation of "neo-substrates" such as IKAROS, AIOLOS, and CK1α accounts for their biological activity. Far less clear is whether these Cereblon E3 ligase modulating compounds disrupt the endogenous functions of CRBN. Strikingly, here we report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T-cells augments their central metabolism manifest as elevated bioenergetics, with supraphysiological levels of polyamines secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Importantly, treatment with Celeblon-modulatingcompounds similarly augments central metabolism of human CD8+T-cell. Notably, the metabolic control of CD8+ T-cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn deficient T-cells have augmented antigen-specific cytolytic activity versus melanoma tumor cells ex vivo and in vivo and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of activated CD8+ T-cells and this phenotype can be exploited by treatment with drugs.
... Polyamines also promote the Z-DNA conformation and stabilize the DNA quadruplex conformation of c-myc, resulting in the overexpression of c-myc [8,9]. This acts as a positive feedback loop due to the ODC, SRM, and SMS genes being transcriptional targets of c-myc [10,11]. Spermidine has been shown to be essential for cell proliferation in eukaryotes, specifically for the hypusination of eIF5A, an initiation factor that regulates translation of a variety of cellular proteins. ...
As obligate intracellular parasites, viruses rely on host cells for the building blocks of progeny viruses. Metabolites such as amino acids, nucleotides, and lipids are central to viral proteins, genomes, and envelopes, and the availability of these molecules can restrict or promote infection. Polyamines, comprised of putrescine, spermidine, and spermine in mammalian cells, are also critical for virus infection. Polyamines are small, positively charged molecules that function in transcription, translation, and cell cycling. Initial work on the function of polyamines in bacteriophage infection illuminated these molecules as critical to virus infection. In the decades since early virus-polyamine descriptions, work on diverse viruses continues to highlight a role for polyamines in viral processes, including genome packaging and viral enzymatic activity. On the host side, polyamines function in the response to virus infection. Thus, viruses and hosts compete for polyamines, which are a critical resource for both. Pharmacologically targeting polyamines, tipping the balance to favor the host and restrict virus replication, holds significant promise as a broad-spectrum antiviral strategy.
... The metabolic pathways under Myc control include glycolysis, glutaminolysis, and polyamine synthesis (Le et al., 2012). Ornithine decarboxylase, which promotes polyamine synthesis, is a Myc trigger (Nilsson et al., 2005). Glutamine metabolism is a complementary pathway to glycolysis and coordinates increased amino acids, lipids and nucleotide biosynthesis while also providing energy to support these processes. ...
Myc has emerged as a pivotal transcription factor for four metabolic pathways: aerobic glycolysis, glutaminolysis, polyamine synthesis, and HIF-1α/mTOR. Each of these pathways accelerates the utilization of sugar. The BCG vaccine, a derivative of Mycobacteria-bovis, has been shown to trigger a long-term correction of blood sugar levels to near normal in type 1 diabetics (T1D). Here we reveal the underlying mechanisms behind this beneficial microbe-host interaction. We show that baseline glucose transport is deficient in T1D monocytes but is improved by BCG in vitro and in vivo. We then show, using RNAseq in monocytes and CD4 T cells, that BCG treatment over 56 weeks in humans is associated with upregulation of Myc and activation of nearly two dozen Myc-target genes underlying the four metabolic pathways. This is the first documentation of BCG induction of Myc and its association with systemic blood sugar control in a chronic disease like diabetes. : Immune System; Diabetology; Transcriptomics Subject Areas: Immune System, Diabetology, Transcriptomics
... (5) Almost all of the effects of MYC on the expression of specific target genes are weak, often below twofold even when MYC levels are manipulated to increase over several orders of magnitude. Although interference with the expression of MYC target genes can abolish MYCdriven tumorigenesis, there are very few studies that show that a twofold change in target gene expression is relevant: examples are 60S ribosomal protein L24 and ornithine decarboxylase, both of which are haploinsufficient to support MYC-induced lymphomagenesis but not for normal growth 63,64 . ...
Oncoproteins of the MYC family are major drivers of human tumorigenesis. Since a large body of evidence indicates that MYC proteins are transcription factors, studying their function has focused on the biology of their target genes. Detailed studies of MYC-dependent changes in RNA levels have provided contrasting models of the oncogenic activity of MYC proteins through either enhancing or repressing the expression of specific target genes, or as global amplifiers of transcription. In this Review, we first summarize the biochemistry of MYC proteins and what is known (or is unclear) about the MYC target genes. We then discuss recent progress in defining the interactomes of MYC and MYCN and how this information affects central concepts of MYC biology, focusing on mechanisms by which MYC proteins modulate transcription. MYC proteins promote transcription termination upon stalling of RNA polymerase II, and we propose that this mechanism enhances the stress resilience of basal transcription. Furthermore, MYC proteins coordinate transcription elongation with DNA replication and cell cycle progression. Finally, we argue that the mechanism by which MYC proteins regulate the transcription machinery is likely to promote tumorigenesis independently of global or relative changes in the expression of their target genes. The MYC oncoproteins are transcription factors, but the molecular mechanism of their oncogenic activity is unclear. MYC proteins promote transcription termination in stress conditions, which is proposed to increase cellular resilience to stress and to promote tumorigenesis independently of changes in the expression of their target genes.
... To validate this notion, we analyzed publically available MYC-ChIPseq data of HepG2 liver cancer cells from ENCODE (ENCSR000DLR) and found that of the 3509 genes with MYC-binding sites, 512 genes were deregulated by HES5wt ( Supplementary Fig. S7 and Supplementary Table S6). Among these, HES5-regulated genes with the highest difference were the well-known MYC targets ODC1 and LDHA, which constitute metabolic genes essential for rapid cell proliferation (Fig. 3a) [25][26][27][28][29]. Consistently, we found that ODC1 and LDHA were significantly downregulated at the protein level by HES5 (Fig. 3c, d). ...
NOTCH receptor signaling plays a pivotal role in liver homeostasis and hepatocarcinogenesis. However, the role of NOTCH pathway mutations and the NOTCH target gene HES5 in liver tumorigenesis are poorly understood. Here we performed whole-exome sequencing of 54 human HCC specimens and compared the prevalence of NOTCH pathway component mutations with the TCGA-LIHC cohort (N = 364). In addition, we functionally characterized the NOTCH target HES5 and the patient-derived HES5-R31G mutation in vitro and in an orthotopic mouse model applying different oncogenic backgrounds, to dissect the role of HES5 in different tumor subgroups in vivo. We identified nonsynonymous mutations in 14 immediate NOTCH pathway genes affecting 24.1% and 16.8% of HCC patients in the two independent cohorts, respectively. Among these, the HES5-R31G mutation was predicted in silico to have high biological relevance. Functional analyses in cell culture showed that HES5 reduced cell migration and clonogenicity. Further analyses revealed that the patient-derived HES5-R31G mutant protein was non-functional due to loss of DNA binding and greatly reduced nuclear localization. Furthermore, HES5 exhibited a negative feedback loop by directly inhibiting the NOTCH target HES1 and downregulated the pro-proliferative MYC targets ODC1 and LDHA. Interestingly, HES5 inhibited MYC-dependent hepatocarcinogenesis, whereas it promoted AKT-dependent liver tumor formation and stem cell features in a murine model. Thus, NOTCH pathway component mutations are commonly observed in HCC. Furthermore, the NOTCH target gene HES5 has both pro- and anti-tumorigenic functions in liver cancer proposing a driver gene dependency and it promotes tumorigenesis with its interaction partner AKT.
... Polyamine metabolism is also associated with many aspects of immune function. In particular, polyamine metabolism is enhanced in activated T-cells as a function of c-Myc induction of ornithine decarboxylase (ODC), which converts the non-proteinogenic amino acid ornithine to putrescine (Nilsson et al., 2005). ODC-mediated stimulation of polyamine metabolism is required for T-cell proliferation and cytolytic activity (Bowlin et al., 1987;Ehrke et al., 1986;Wang et al., 2011). ...
Metabolic dysregulation is an appreciated hallmark of cancer and a target for therapeutic intervention. Cellular metabolism involves a series of oxidation/reduction (redox) reactions that yield the energy and biomass required for tumor growth. Cells require diverse molecular species with constituent sulfur atoms to facilitate these processes. For humans, this sulfur is derived from the dietary consumption of the proteinogenic amino acids cysteine and methionine, as only lower organisms (e.g., bacteria, fungi, and plants) can synthesize them de novo. In addition to providing the sulfur required to sustain redox chemistry, the metabolism of these sulfur-containing amino acids yield intermediate metabolites that constitute the cellular antioxidant system, mediate inter- and intracellular signaling, and facilitate the epigenetic regulation of gene expression, all of which contribute to tumorigenesis.
... 16 Nevertheless, in the cancerous state, the requirement for these polyamines rises manifold and the polyamine metabolism pathway can be targeted through the use of polyamines from the external source resulting in cell death. 17,18 During the recent few years, the development of polyamine analogues as chemotherapeutic agents against different cancers has fascinated many researchers. [18][19][20][21][22] Many polyamine analogues targeting epigenetic modifications of the chromatin were evaluated previously and have shown promising results. ...
... Moreover, it is closely correlated with carcinogenesis both experimentally and clinically. For example, the overexpression of ODC in NIH/3T3 cells can spur transformation in vitro [92], as is the case for lymphomas [93] and skin tumors [94]. More strong correlations have been found from two clinical investigations of breast cancer, in which ODC activity levels were found to be significantly higher than those of normal tissues and to be correlated with higher levels of polyamine content and with histological grade, peritumoral lymphatic or blood vessel invasion [95]. ...
While the metabolic changes in cancer tissues were first observed by Warburg Otto almost a century ago, altered metabolism has recently returned as a focus of cancer research. 5'-Methylthioadenosine (MTA) is a naturally occurring sulfur-containing nucleoside found in numerous species. While MTA was first isolated several decades ago, a lack of sensitive and specific analytical methodologies designed for its direct quantification has hampered the study of its physiological and pathophysiological features. Many studies indicate that MTA suppresses tumors by inhibiting tumor cell proliferation, invasion, and the induction of apoptosis while controlling the inflammatory micro-environments of tumor tissue. In this review, we assessed the effects of MTA and of related materials on the growth and functions of normal and malignant cells. © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
... Ornithine decarboxylase (ODC) is a key regulatory enzyme in the biosynthesis of polyamines. Like c-Jun, also ODC is essential for mammalian development and tumor formation [20]. In our earlier studies, we have shown that the overexpression of human ODC induces morphological Research Paper www.oncotarget.com ...
We have previously shown that proto-oncoprotein c-Jun is activated in ornithine decarboxylase (ODC)- and RAS-transformed mouse fibroblasts, and that the transformed morphology of these cells can be reversed by expressing the transactivation domain deletion mutant of c-Jun (TAM67). Here, we found that lysyl oxidase (Lox), encoding an extracellular matrix-modifying enzyme, is downregulated in a c-Jun-dependent manner in ODC-transformed fibroblasts (Odc cells). In addition to Lox, the Lox family members Lox-like 1 and 3 (Loxl1 and Loxl3) were found to be downregulated in Odc as well as in RAS-transformed fibroblasts (E4), whereas Lox-like 4 (Loxl4) was upregulated in Odc and downregulated in E4 cells compared to normal N1 fibroblasts. Tetracycline-regulatable LOX re-expression in Odc cells led to inhibition of cell growth and invasion in three-dimensional Matrigel in an activity-independent manner. On the contrary, LOX and especially LOXL2, LOXL3, and LOXL4 were found to be upregulated in several human melanoma cell lines, and LOX inhibitor B-aminopropionitrile inhibited the invasive growth of these cells particularly when co-cultured with fibroblasts in Matrigel. Knocking down the expression of LOX and especially LOXL2 in melanoma cells almost completely abrogated the invasive growth capability. Further, LOXL2 was significantly upregulated in clinical human primary melanomas compared to benign nevi, and high expression of LOXL2 in primary melanomas was associated with formation of metastases and shorter survival of patients. Thus, our studies reveal that inactive pro-LOX (together with Lox propeptide) functions as a tumor suppressor in ODC- and RAS-transformed murine fibroblasts by inhibiting cell growth and invasion, and active LOX and LOXL2 as tumor promoters in human melanoma cells by promoting their invasive growth. Copyright: Kielosto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... 16 Nevertheless, in the cancerous state, the requirement for these polyamines rises manifold and the polyamine metabolism pathway can be targeted through the use of polyamines from the external source resulting in cell death. 17,18 During the recent few years, the development of polyamine analogues as chemotherapeutic agents against different cancers has fascinated many researchers. [18][19][20][21][22] Many polyamine analogues targeting epigenetic modifications of the chromatin were evaluated previously and have shown promising results. ...
Natural polyamines such as putrescine, spermidine, and spermine are crucial in the cell proliferation and maintenance in all the eukaryotes. However, the requirement of polyamines in tumor cells is stepped up to maintain tumorigenicity. Many synthetic polyamine analogues have been designed recently to target the polyamine metabolism in tumors to induce apoptosis. N4-Erucoyl spermidine (designed as N4-Eru), a novel acylspermidine derivative, has been shown to exert selective inhibitory effects on both hematological and solid tumors, but its mechanisms of action are unknown. In this study, RNA sequencing was performed to investigate the anticancer mechanisms of N4-Eru-treated T-cell acute lymphoblastic leukemia (ALL) cell line (Jurkat cells), and gene expression was examined through different tools. We could show that many key oncogenes including NDRG1, CACNA1G, TGFBR2, NOTCH1,2,3, UHRF1, DNMT1,3, HDAC1,3, KDM3A, KDM4B, KDM4C, FOS, and SATB1 were downregulated, whereas several tumor suppressor genes such as CDKN2AIPNL, KISS1, DDIT3, TP53I13, PPARG, FOXP1 were upregulated. Data obtained through RNA-Seq further showed that N4-Eru inhibited the NOTCH/Wnt/JAK-STAT axis. This study also indicated that N4-Eru-induced apoptosis could involve several key signaling pathways in cancer. Altogether, our results suggest that N4-Eru is a promising drug to treat ALL.
... 16 Nevertheless, in the cancerous state, the requirement for these polyamines rises manifold and the polyamine metabolism pathway can be targeted through the use of polyamines from the external source resulting in cell death. 17,18 During the recent few years, the development of polyamine analogues as chemotherapeutic agents against different cancers has fascinated many researchers. [18][19][20][21][22] Many polyamine analogues targeting epigenetic modifications of the chromatin were evaluated previously and have shown promising results. ...
Natural polyamines such as putrescine, spermidine, and spermine are crucial in the cell proliferation and maintenance in all the eukaryotes. However, the requirement of polyamines in tumor cells is stepped up to maintain tumorigenicity. Many synthetic polyamine analogues have been designed recently to target the polyamine metabolism in tumors to induce apoptosis. N4-Erucoyl spermidine (designed as N4-Eru), a novel acylspermidine derivative, has been shown to exert selective inhibitory effects on both hematological and solid tumors, but its
mechanisms of action are unknown. In this study, RNA sequencing was performed to investigate the anticancer mechanisms of N4-Eru-treated T-cell acute lymphoblastic leukemia (ALL) cell line (Jurkat cells), and gene expression was examined through different tools. We could show that many key oncogenes including NDRG1, CACNA1G, TGFBR2, NOTCH1,2,3, UHRF1, DNMT1,3, HDAC1,3, KDM3A, KDM4B, KDM4C, FOS, and SATB1 were downregulated, whereas several tumor suppressor genes such as CDKN2AIPNL, KISS1, DDIT3, TP53I13, PPARG, FOXP1 were upregulated. Data obtained through RNA-Seq further showed that N4-Eru inhibited the NOTCH/Wnt/JAK-STAT axis. This study also indicated that N4-Eru-induced apoptosis could involve several key signaling pathways in cancer. Altogether, our results suggest that N4-Eru is a promising drug to treat ALL.
... The ODC1 gene harbors canonical MYC binding sites in its promoter that contain the conserved Ebox motif. The same group later demonstrated in vivo in Eμ-Myc transgenic mice that ODC1 is a critical MYC transcription target and that targeting ODC with DFMO prevents tumor formation in MYC-induced lymphogenesis (132). In this regard, it is noteworthy that Zell et al. described a single nucleotide polymorphism (SNP) in the region of the ODC1 gene Eboxes that affects MYC and MAD binding to ODC1 and this SNP is linked to colon cancer recurrence (133). ...
This paper is in recognition of the 100th birthday of Dr. Herbert Tabor, a true pioneer in the polyamine field for over 70 years, who served as the editor-in-chief of the Journal for Biological Chemistry from 1971 to 2010. We review current knowledge of MYC proteins (c-MYC, MYCN, and MYCL) and focus on ornithine decarboxylase 1 (ODC1), an important bona fide gene target of MYC and encoding the sentinel, rate-limiting enzyme in polyamine biosynthesis. Although notable advances have been made in designing inhibitors against the "undruggable" MYC, its downstream targets and pathways are currently the main avenue for therapeutic anticancer interventions. To this end, the MYC-ODC axis presents an attractive target for managing cancers such as neuroblastoma, a pediatric cancer in which MYCN gene amplification correlates with poor prognosis and high-risk disease. ODC and polyamine levels are often up-regulated and contribute to tumor hyperproliferation, especially of MYC-driven cancers. We therefore had proposed to repurpose α-difluoromethylornithine (DFMO), an FDA-approved, orally available ODC inhibitor, for management of neuroblastoma, and this intervention is now being pursued in several clinical trials. We discuss the regulation of ODC and polyamines, which besides their well-known interactions with DNA and tRNA/rRNA, are involved in regulating RNA transcription and translation, ribosome function, proteasomal degradation, the circadian clock, and immunity, events that are controlled by MYC proteins.
... Production of drugs to target the MYC family directly has not yet been successful perhaps due to the wide variety of critical genes influenced by them. Studies by Cleveland and colleagues using transgenic mouse models and DFMO have shown that ODC is a key downstream target of MYC in both lymphoma (78) and neuroblastoma (79). The ongoing attempts to improve therapy for pediatric neuroblastoma in which MYCN gene amplification is a common event by using DFMO are described in detail in this article (26). ...
Polyamines have a long history in biochemistry and physiology, dating back to 1678 when Leeuwenhoek first reported crystals that were composed of spermine phosphate in seminal fluid. Their quantification and biosynthetic pathway were first described by Herb and Celia Tabor in collaboration with Sanford Rosenthal in the late 1950s. This work led to immense interest in their physiological functions. The 11 minireviews in this collection illustrate many of the wide-ranging biochemical effects of polyamines. This series provide a fitting tribute to Herb Tabor on the occasion of his 100th birthday demonstrating clearly the importance and growth of the research field that he pioneered in the late 1950s and has contributed to for many years. His studies of the synthesis, function and toxicity of polyamines have yielded multiple insights into fundamental biochemical processes and formed the basis of successful and continuing drug development. This minireview series reviews the highly diverse properties of polyamines in bacteria, protozoa, and mammals, highlighting the importance of these molecules in growth, development, response to the environment and their involvement in diseases including cancer and those caused by parasitic protozoans.
This perspective delves into the investigation of synthetic and naturally occurring inhibitors, their patterns of inhibition, and the effectiveness of newly utilized natural compounds as inhibitors targeting the Ornithine decarboxylase enzyme. This enzyme is known to target the MYC oncogene, thereby establishing a connection between polyamine metabolism and oncogenesis in both normal and cancerous cells. ODC activation and heightened polyamine activity are associated with tumor development in numerous cancers and fluctuations in ODC protein levels exert a profound influence on cellular activity for inhibition or suppressing tumor cells. This perspective outlines efforts to develop novel drugs, evaluate natural compounds, and identify promising inhibitors to address gaps in cancer prevention, highlighting the potential of newly designed synthetic moieties and natural flavonoids as alternatives. It also discusses natural compounds with potential as enhanced inhibitors.
There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence¹ and poor prognosis². Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.
A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy. Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation. Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.
Background:
Hand, foot, and mouth disease (HFMD) is a common viral childhood illness caused most commonly by enterovirus 71 (EV71) and coxsackievirus A16. The pathogenesis of EV71 has been extensively studied, and the regulation of the host immune response is suspected to aggravate the serious complications induced by EV71. Our previous research showed that EV71 infection significantly increased the release of circulating interleukin (IL)-6, IL-10, IL-13, and IL-27. Notably, these cytokines are related to the EV71 infection risk and clinical stage. Polyamines are compounds that are ubiquitous in mammalian cells and play a key role in various cellular processes. Several studies have shown that targeting polyamine metabolic pathways can reduce infections caused by viruses. However, the significance of polyamine metabolism in EV71 infection remains largely unknown.
Methods:
Serum samples from 82 children with HFMD and 70 healthy volunteers (HVs) were collected to determine the polyamine metabolites spermidine (SPD) and spermine (SPM), and IL-6 levels. In addition, peripheral blood mononuclear cells (PBMCs) were treated with EV71 viral protein 1 (VP1) and EV71 VP4, and the cells and supernatant were then collected to analyze the expression of polyamine metabolism-related enzymes by western blot. The data were analyzed using GraphPad Prism 7.0 software (USA).
Results:
The serum polyamine metabolites SPD and SPM were elevated in the HFMD patients, especially in the EV71-infected children. Further, a positive correlation was found between serum SPD and IL-6 levels in the EV71-infected children. We also found that the upregulation of peripheral blood polyamine metabolites in the EV71-infected HFMD children was related to EV71 capsid protein VP1, but not VP4. VP1 may promote the expression of polyamine metabolism-related enzymes and promote the production of polyamine metabolites, thereby upregulating the SPD/nuclear factor kappa B/IL-6 signaling pathway. However, VP4 has the opposite effect in this process.
Conclusions:
Our results suggest that EV71 capsid protein may regulate the polyamine metabolic pathways of infected cells in a variety of ways. This study provides insights into the mechanism of EV71 infection and polyamine metabolism and has good reference value for the development of EV71 vaccine.
Oncogenic overexpression of MYC leads to the fatal deregulation of signaling pathways, cellular metabolism, and cell growth. MYC rearrangements are found frequently among non-Hodgkin B-cell lymphomas enforcing MYC overexpression. Genetically engineered mouse models (GEMMs) were developed to understand MYC-induced B-cell lymphomagenesis. Here, we highlight the advantages of using Eµ-Myc transgenic mice. We thoroughly compiled the available literature to discuss common challenges when using such mouse models. Furthermore, we give an overview of pathways affected by MYC based on knowledge gained from the use of GEMMs. We identified top regulators of MYC-induced lymphomagenesis, including some candidates that are not pharmacologically targeted yet.
Prostate cancer is the most frequent malignant tumour among males (19%), often clinically silent and of difficult prognosis. Although several studies have highlighted the diagnostic and prognostic role of circulating biomarkers, such as PSA, their measurement does not necessarily allow the detection of the disease. Within this context, many authors suggest that the evaluation of circulating polyamines could represent a valuable tool, although several analytical problems still counteract their clinical practice. In particular, agmatine seems particularly intriguing, being a potential inhibitor of polyamines commonly derived from arginine. The aim of the present work was to evaluate the potential role of agmatine as a suitable biomarker for the identification of different classes of patients with prostate cancer (PC). For this reason, three groups of human patients—benign prostatic hyperplasia (BPH), precancerous lesion (PL), and prostate cancer (PC)—were recruited from a cohort of patients with suspected prostate cancer (n = 170), and obtained plasma was tested using the LC-HRMS method. Statistics on the receiver operating characteristics curve (ROC), and multivariate analysis were used to examine the predictive value of markers for discrimination among the three patient groups. Statistical analysis models revealed good discrimination using polyamine levels to distinguish the three classes of patients. AUC above 0.8, sensitivity ranging from 67% to 89%, specificity ranging from 74% to 89% and accuracy from 73% to 86%, considering the validation set, were achieved. Agmatine plasma levels were measured in PC (39.9 ± 12.06 ng/mL), BPH (77.62 ± 15.05 ng/mL), and PL (53.31 ± 15.27 ng/mL) patients. ROC analysis of the agmatine panel showed an AUC of 0.959 and p ≤ 0.001. These results could represent a future tool able to discriminate patients belonging to the three different clinical groups.
Polyamines are ubiquitous molecules that are involved in a number of important cellular processes. Aberrations in their function or metabolism play a role in diseases such as cancer and parasitic infection. A number of validated drug targets have been identified, including enzymes in the polyamine biosynthetic and catabolic pathways and the S-adenosylmethionine synthetic and salvage pathways. Polyamine Drug Discovery is the first comprehensive volume to cover all aspects of the design and development of potential therapeutics targeting polyamine metabolism. The book details research progress from 1975 to the present date and discusses the design and use of polyamine metabolism inhibitors as therapeutic agents. Various polyamine-containing drugs are described that can be used in chemotherapy, and as treatments for infections including trypanosomiasis, leishmaniasis and malaria. Finally, the roles of polyamine analogues in chemoprevention, polyamine-containing vectors for gene delivery, and the design of polyamine-based epigenetic modulators are detailed. Each chapter addresses a different aspect of polyamine drug discovery and all are written by medicinal and biological chemists with particular expertise in developing agents that modulate polyamine metabolism or function. The book will increase the visibility of polyamine drug discovery among pharmaceutical researchers and provide a valuable reference for everyone working in the field.
MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression post-transcriptionally by binding to the 3’UTR of their target mRNAs. The evolutionarily conserved microRNA-125a (miR-125a) is highly expressed in both murine and human hematopoietic stem cells (HSCs) and previous studies have shown that miR-125 strongly enhances self-renewal of HSCs and progenitors. In this study we explored whether temporary overexpression of miR-125a would be sufficient to permanently increase HSC self-renewal, or rather whether persistent overexpression of miR-125a is required. We used three complementary in vivo approaches to reversibly enforce expression of miR-125a in murine HSCs. Additionally, we interrogated the underlying molecular mechanisms responsible for the functional changes that occur in HSCs upon overexpression of miR-125a. Our data show that continuous expression of miR-125a is required to enhance HSC activity. Our molecular analysis confirms changes in pathways that explain the characteristics of miR-125a overexpressing HSCs. Moreover, it provides several novel putative miR-125a targets, but also highlights the complex molecular changes that collectively lead to enhanced HSC function.
Tumor metabolism and its specific alterations have become an integral part of understanding functional alterations leading to malignant transformation and maintaining cancer progression. Here, we review the metabolic changes in B-cell neoplasia, focusing on the effects of tumor metabolism on the tumor microenvironment (TME). Particularly, innate and adaptive immune responses are regulated by metabolites in the TME such as lactate. With steadily increasing therapeutic options implicating or utilizing the TME, it has become essential to address the metabolic alterations in B-cell malignancy for therapeutic approaches. In this review, we discuss metabolic alterations of B-cell lymphoma, consequences for currently used therapy regimens, and novel approaches specifically targeting metabolism in the TME.
Pyridoxal 5’-phosphat (PLP)-abhängige Enzyme katalysieren vielfältige Umwandlungen von Aminosäuren, wie Transaminierung, α-Decarboxylierung und Razemisierung sowie β/γ- Eliminierungs- und Substitutionsreaktionen. Vitamin B6-abhängige Enzyme spielen eine zentrale Rolle im Aminosäure- und Aminmetabolismus und sind interessante Zielobjekte für Medikamente, um auf spezifische, zu pathologischen Zuständen führende Prozesse einzuwirken. Hemmstoffe, welche der Struktur des Übergangszustands entsprechen, binden mit hoher Affinität an ihre Zielenzyme. Unter Anwendung dieses Prinzips wurde in dieser Doktorarbeit eine Strategie erarbeitet, um spezifische, intrazellulär wirkende Inhibitoren für bestimmte PLP-abhängige Enzyme zu entwickeln. Die PLP-abhängige Ornithindecarboxylase (ODC) ist ein Schlüssenenzym der Polyaminsynthese und wurde als erstes Zielobjekt ausgewählt. ODC ist in vielen Tumorzellen überexprimiert und deren Hemmung ist daher von grossem therapeutischem Interesse. Im ersten Teil der Doktorarbeit werden Design und Synthese eines solchen ODC-Inhibitors vorgestellt. Eine genaue Analyse der 3-D-Struktur von hODC offenbarte eine zusätzliche hydrophobe Tasche, welche die ε-Aminogruppe des Substrats Ornithins umhüllt. Molekülmodellierung mit einem BOC-geschützten Pyridoxyl-Ornithin-Konjugat (POB) in der aktiven Stelle der hODC deuteten auf eine starke Wechselwirkung des Enzyms mit der BOC- Gruppe hin. Das synthetisierte und gereinigte POB hemmte die Aktivität neu induzierter ODC in verschiedenen Tumorzellen. Das Ausmass der Hemmung der ODC-Aktivität korrelierte mit der zeitabhängigen Hemmung des Zellwachstums. Menschliche nicht-cancerogene glatte Muskelzellen der Aorta wurden nicht gehemmt. Die Proliferationshemmung von Tumorzellen durch POB war beträchtlich wirksamer als die von α-DL-Difluoromethylornithine (DFMO), des medizinisch am häufigsten verwendeten irreversiblen ODC-Inhibitors. Um die hemmende Wirkung dieser Art von intrazellulärem ODC-Inhibitoren zu verbessern, wurden im zweiten Teil der Doktorarbeit die strukturellen Anforderungen für das Binden und Hemmen von hODC und für die Zellproliferation genauer untersucht. Strukturell unterschiedliche Enzym-Substrat-Analoge wurden entwickelt, synthetisiert und auf Hemmwirkung getestet. Von 23 untersuchten Konjugaten zeigten zwei, nämlich Phosphorpyridoxyl- und Pyridoxyl-Tryptophanmethylester (pPTME, PTME), eine signifikant grössere Zellproliferationshemmung als POB und insbesondere eine viel grössere als DFMO. Alle aktiven Verbindungen besitzen ein hydrophobes Seitenkettenfragment und ein polyaminähnliches Motiv. Wie sich aus Untersuchungen mittels molekularem Modellieren vermuten liess, haben pPTME und PTME eine grössere Bindungsaffinität zu ODC als POB und hemmen die intrazelluläre ODC-Aktivität auch stärker. Zusätzlich induzieren diese drei Verbindungen, wie bereits von Polyaminanalogen bekannt, die Aktivitäten der Polyaminoxidase und der Spermin/Spermidin-N1-Acetyltransferase bis zu 250 % bzw. 780 %. Beide Enzyme sind im katabolen Polyaminstoffwechsel wirksam. Die duale Wirkung dieser Verbindungen, d.h. die Hemmung der ODC und die Induktion der katabolischen Enzyme, beeinflusst die intrazellulären Polyaminpools und könnte der Grund sein für den im Vergleich zu DFMO viel besseren Hemmeffekt auf die Zellproliferation. Histamin, ein biogenes Amin mit wichtigen biologischen Funktionen, wird durch die Decarboxylierung von Histidin mittels Histidin-Decarboxylase (HDC), einem PLP- abhängigen Enzym, gebildet. HDC ist demzufolge ein wichtiges Zielenzym für Hemmstoffe, um die Histaminproduktion in bestimmten pathologischen Zuständen zu verringern. Die Entwicklung neuer Inhibitoren der hHDC und die Aufklärung von deren strukturellen Voraussetzungen ist eine grosse Herausforderung, da die 3D-Struktur der HDC von Säugetieren noch immer unbekannt ist. Im letzten Teil der Dissertation wurden potentiell membrandurchlässige Pyridoxylsubstratkonjugate entwickelt, synthetisiert und als Inhibitoren der hHDC getestet. Ferner wurde ein Modell der aktiven Stelle der hHDC entworfen, das mit den experimentellen Daten kompatibel ist. Von den 9 mit menschlichen Mastzellen getesteten strukturellen Varianten hemmte das Pyridoxyl-Histidin-Methylesterkonjugat (PHME) die HDC am stärksten, was darauf schliessen lässt, dass die Bindungsstelle von hHDC keine andere Gruppe als die Imidazol-Seitenkette von Histidin akzeptiert. PHME vermochte vor allem die induzierte neu synthetisierte HDC zu hemmen und zeigte auch Hemmwirkung in Zellextrakten. Das vorgeschlagene Modell des aktiven Zentrums der hHDC, welches Phosphopyridoxyl-Histidin enthält, erklärte die Bindungsspezifizität von HDC für ihr Substrat und erhellt die Struktur-Aktivitätsbeziehung der entwickelten und untersuchten Verbindungen. Zusammenfassend zeigen die Ergebnisse, dass die Anwendung strukturell verschiedener Verbindungen zur Abklärung der Bindungsanforderungen der Zielproteine ein effizienter Weg zur Entwicklung neuer Inhibitoren von PLP-abhängiger Enzyme ist. ODC-Inhibitoren, wie sie hier vorgestellt wurden, können von Zellen aufgenommen werden und das Zellwachstum klinisch wichtiger bösartiger Tumorzelllinien, wie Gliomazellen, effizient hemmen. Pyridoxal 5’-phosphate (PLP)-dependent enzymes catalyze manifold transformations of amino acids such as transamination, α-decarboxylation, and racemization, as well as β- or γ- elimination and replacement reactions. Vitamin B6-dependent enzymes play a central role in amine and amino acid metabolism and are interesting targets for drugs to intervene in specific processes that lead to pathological states. As transition-state-based inhibitors bind with high affinity to their target enzymes, a strategy was explored to develop inhibitors on the basis of mimics of a transition-state intermediate of PLP-dependent enzymic reactions. The PLP-dependent ornithine decarboxylase (ODC) is the key enzyme in polyamine synthesis and was chosen as the first target. ODC is overexpressed in many tumor cells and thus a potential drug target. The thesis reports the design and synthesis of an analog of covalent coenzyme-substrate adducts as a novel precursor inhibitor of ODC. Structural analysis of the crystal structure of hODC disclosed a hydrophobic pocket adjacent to the ε- amino group of its substrate ornithine. Molecular modeling showed favorable interactions of a BOC-protected pyridoxyl-ornithine conjugate, termed POB, in the active site of hODC. The synthesized and purified POB at the concentration of 100 µM completely inhibited the activity of newly induced ODC in glioma LN229 and COS7 cells. In correlation with the inhibition of intracellular ODC activity, a time-dependent inhibition of cell growth was also observed in various tumor cell lines, but not in non-tumorigenic human aortic smooth muscle cells. POB was much more efficient in inhibiting proliferation of several types of tumor cells than α-DL-difluoromethylornithine (DFMO), the most widely used irreversible inhibitor of ODC. In the second part of the thesis, the structural requirements for binding to and inhibiting hODC and for inhibition of cell proliferation were elucidated in more detail to improve the inhibitory effect of this kind of inhibitors of intracellular hODC. Structurally diverse enzyme- substrate mimics were designed, synthesized and tested for their inhibitory potential. Out of 23 conjugates, phosphopyridoxyl- and pyridoxyl-tryptophan methyl ester (pPTME, PTME) showed a significantly more potent cell proliferation inhibition of glioma cells than POB and particularly more than DFMO. All the active compounds possess a hydrophobic side chain and polyamine motif (-NH-(CHX)4-NH-). As predicted from the molecular modeling results, pPTME and PTME have an improved binding affinity for ODC in comparison to POB and strongly inhibited the intracellular ODC activity of glioma cells. Additionally, these three compounds induce, as polyamine analogs do, the activity of the enzymes of polyamine catabolism, polyamine oxidase and spermine/spermidine N1-acetyltransferase, up to 250% and 780%, respectively. The dual mode of these compounds in cells, i.e. inhibition of ODC and induction of polyamine catabolic enzymes, influences the intracellular polyamine pools and might be the cause for their inhibitory effect on cell proliferation that compares favorably with that of DFMO. Histamine, a biogenic amine with important biological functions, for example in gastric acid secretion and allergic reactions, is produced from histidine by histidine decarboxylase (HDC), a PLP-dependent enzyme. HDC is thus a potential target to attenuate histamine production in certain pathological states. Targeting mammalian HDC with novel inhibitors and elucidating the structural basis of their specificity for HDC are particularly challenging tasks, because the 3D structure of mammalian HDC is still unknown. In the last part of my dissertation, potentially membrane-permeable pyridoxyl-substrate conjugates were designed, synthesized and tested as inhibitors for human HDC and an active site of hHDC was modeled that is compatible with the experimental data. The most potent inhibitory compound among 9 tested structural variants was the pyridoxyl-histidine methyl ester conjugate (PHME), indicating that the binding site of hHDC does not tolerate other groups than the imidazole side chain of histidine. PHME inhibited induced HDC in human HMC-1 cells and proved also inhibitory in cell extracts. The proposed model of hHDC, containing phosphopyridoxyl- histidine in the active site, explains the binding specificity of HDC towards its substrate and the structure-activity relationship of the investigated compounds. In conclusion, analogs of covalent coenzyme-substrate adducts and their structural variations provide promising lead compounds for the development of inhibitors for PLP- dependent enzymes such as ODC and HDC. ODC inhibitors of the kind presented in this thesis seem to be efficient agents for preventing proliferation of clinically interesting malignant tumor cell lines, such as gliomas.
Hedgehog (Hh) signaling is a critical developmental regulator and its aberrant activation,due to somatic or germline mutations of genes encoding pathway components, causes Basal CellCarcinoma (BCC) and medulloblastoma (MB). A growing effort has been devoted at theidentification of druggable vulnerabilities of the Hedgehog signaling, leading to the identificationof various compounds with variable efficacy and/or safety. Emerging evidence shows that anaberrant polyamine metabolism is a hallmark of Hh-dependent tumors and that itspharmacological inhibition elicits relevant therapeutic effects in clinical or preclinical models ofBCC and MB. We discuss here the current knowledge of polyamine metabolism, its role in cancerand the available targeting strategies. We review the literature about the connection betweenpolyamines and the Hedgehog signaling, and the potential therapeutic benefit of targetingpolyamine metabolism in two malignancies where Hh pathways play a well-established role: BCCand MB.
Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27Kip1, which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27Kip1 gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc ‘super-repressor’, with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp −20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation.
The cyclin-dependent kinase inhibitor p21(WAF1/CIP1)
inhibits proliferation both in vitro and in
vivo, and overexpression of p21 in normal and tumor cell lines
results in cell cycle arrest. In contrast, ectopic expression of Myc
alleviates G1 cell cycle arrest. Recent studies showed that
Myc can repress p21 transcription, thereby overriding a p21-mediated
cell cycle checkpoint. We found that activation of a Myc-estrogen
receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted
in suppression of endogenous p21 transcription. This effect
was observed in the absence of de novo protein synthesis
and was independent of histone deacetylase activity. In transient
transfection studies, Myc effectively repressed p21 promoter constructs
containing only 119 bp of sequence upstream of the transcription start
site. This region contains multiple Sp1-binding sites and a potential
initiator element, but no canonical Myc DNA-binding sites. Deletion of
the potential initiator element does not affect repression of the p21
promoter by c-Myc. Coimmunoprecipitation and glutathione
S-transferase pull-down experiments demonstrate that
c-Myc may form complexes with Sp1/Sp3. We found that the central
region of c-Myc interacts with the zinc finger domain of Sp1. Because
Sp1 is required for p21 transcription, it is possible that Myc may
down-regulate p21 transcription, at least in part, by sequestering Sp1.
Repression of the p21 promoter may contribute to the ability of c-Myc
to promote cell proliferation.
Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27kip1 with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2–Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad–Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.
Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. We have shown that its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Here we report the identification of the protease responsible for ODC degradation. Using a cell-free degradation system, we demonstrate that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis.
Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the human c-myc gene is located on chromosome 8. The analysis of hybrids between rodent cells and human Burkitt lymphoma cells, which carry a reciprocal translocation between chromosomes 8 and 14, allowed the mapping of the human c-myc gene on region (q24 leads to qter) of chromosome 8. This chromosomal region is translocated to either human chromosome 2, 14, or 22 in Burkitt lymphoma cells.
We show here that c-Myc antagonizes the cyclin-dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c-Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin-mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK-inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1-cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1-3, E or A) or of the Cdc25A phosphatase did not overcome p27-induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc-Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions.
Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.
The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check.
Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Emu) develop B-cell lymphoma and exhibit a mean survival time of approximately 6 months. The protracted latent period before the onset of frank disease likely reflects the ability of c-Myc to induce a p53-dependent apoptotic program that initially protects animals against tumor formation but is disabled when overtly malignant cells emerge. In cultured primary mouse embryo fibroblasts, c-Myc activates the p19(ARF)-Mdm2-p53 tumor suppressor pathway, enhancing p53-dependent apoptosis but ultimately selecting for surviving immortalized cells that have sustained either p53 mutation or biallelic ARF deletion. Here we report that p53 and ARF also potentiate Myc-induced apoptosis in primary pre-B-cell cultures, and that spontaneous inactivation of the ARF-Mdm2-p53 pathway occurs frequently in tumors arising in Emu-myc transgenic mice. Many Emu-myc lymphomas sustained either p53 (28%) or ARF (24%) loss of function, whereas Mdm2 levels were elevated in others. Its overexpression in some tumors lacking p53 function raises the possibility that Mdm2 can contribute to lymphomagenesis by interacting with other targets. Emu-myc transgenic mice hemizygous for ARF displayed accelerated disease (11-week mean survival), and 80% of these tumors lost the wild-type ARF allele. All ARF-null Emu-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Emu-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, cancelling its protective checkpoint function and accelerating progression to malignancy.
The c-Myc oncoprotein is a transcription factor involved in cellular transformation as well as apoptotic cell death. We show here that over-expression of c-Myc delivered by an adenovirus vector up-regulates endogenous proapoptotic bax mRNA and protein expression in human cells. In contrast, the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand induces cell death without up-regulating bax expression. c-Myc/Max heterodimers bind to canonical E-box elements located in the bax promoter region as demonstrated by electrophoretic mobility shift analysis and DNaseI foot-printing assays. Analysis of bax regulatory region mutants suggests a model involving myc-dependent activation as well as relief of repression through distinct E-box elements. c-Myc-null cells are deficient in bax-promoter activation as compared with wild-type c-Myc-expressing cells. Overexpression of c-Myc in serum-starved human or mouse embryonic cells leads to apoptosis which is significantly reduced in the presence of growth factor-containing serum. c-Myc-induced apoptosis appears to be deficient in bax-null as compared with bax-wild-type mouse embryonic fibroblasts. The results suggest that the cell death-promoting gene bax is directly downstream of c-Myc in a pathway leading to apoptosis.
Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.
The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARF's nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.
Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the
synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively
suppresses the expression of the Bcl-XL or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-XL RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-XL and Bcl-2 expression is inhibited by Myc in precancerous B cells from Eμ-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression
of Bcl-2 or Bcl-XL by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-XLlevels are markedly elevated in over one-half of all lymphomas arising in Eμ-myc transgenic mice. Bcl-2 and/or Bcl-XL overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways
are inactivated independently. Therefore, the suppression of Bcl-XL or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.
The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in Eμ-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and
inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in Eμ-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type Eμ-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions inARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in Eμ-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective ofBax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas fromBax-null Eμ-myc transgenics lackedp53 alterations, whereas 27% of the tumors inBax
+/− Eμ-myctransgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection ofp53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating
that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily
the case for ARF and Mdm2.
The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.
Previous studies have demonstrated both oncogenic and tumor suppressive properties for the E2F1 transcription factor. In this study, E2f1-null mice were crossed with transgenic mice expressing Myc under the control of an epithelial-specific keratin 5 promoter to determine whether the absence of E2F1 would modulate the oncogenic activity of Myc. Inactivation of E2f1 was found to significantly accelerate tumor development in keratin 5 Myc transgenic mice. Acceleration of tumorigenesis occurred despite the fact that apoptosis levels were increased in transgenic tissue and tumors null for E2f1, whereas Myc-induced proliferation was unaffected by the status of E2f1. These findings provide new insight into the tumor suppressive activity of E2F1 and identify for the first time a specific oncogenic alteration that cooperates with the loss of E2F1 in tumorigenesis.
The cyclin-dependent kinase (CDK) inhibitors p21(Cip1) and p27(Kip1) are induced in response to anti-proliferative stimuli and block G(1)/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors the relative contribution of p21(Cip1) and p27(Kip1) to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G(1)/S transition and its expression is frequently altered in tumors. Previous reports suggested that p27(Kip1) is a crucial G(1) target of MYC. Our study shows that in mice, deficiency for p27(Kip1) but not p21(Cip1) results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27(Kip1) deficient lymphomas an increased frequency of Myc activation is observed. p27(Kip1) deficiency was also shown to collaborate with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27(Kip1) loss. We show that in lymphocytes, MYC overexpression and p27(Kip1) deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis.
The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. We now show that transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc(-/-) cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, we show that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. Our data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation.
Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are approximately 0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (>or=10 microM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.
The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycERTM in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis—changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycERTM.
Myc proteins are key regulators of mammalian cell proliferation. They are transcription factors that activate genes as part of a heterodimeric complex with the protein Max. This review summarizes recent progress in understanding how Myc stimulates cell proliferation and how this might contribute to cellular transformation and tumorigenesis.
The enzyme ornithine decarboxylase is the key regulator of the synthesis of polyamines which are essential for cell proliferation. Expression of this enzyme is transiently increased upon stimulation by growth factors, but becomes constitutively activated during cell transformation induced by carcinogens, viruses or oncogenes. To test whether ornithine decarboxylase could be a common mediator of transformation and oncogenic itself, we transfected NIH3T3 cells with expression vectors carrying the complementary DNA encoding human ornithine decarboxylase in sense and antisense orientations. The increased expression of the enzyme (50-100-times endogenous levels) induced not only cell transformation, but also anchorage-independent growth in soft agar and increased tyrosine phosphorylation of a protein of M(r) 130K. Expression of ornithine decarboxylase antisense RNA was associated with an epithelioid morphology and reduced cell proliferation. Moreover, blocking the endogenous enzyme using specific inhibitor or synthesizing antisense RNA prevented transformation of rat fibroblasts by temperature-sensitive v-src oncogene. Our results imply that the gene encoding ornithine decarboxylase is a proto-oncogene central for regulation of cell growth and transformation.
Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.
In the murine interleukin 3 (IL-3)-dependent myeloid cell line 32D, down-regulation of c-myc and ornithine decarboxylase (ODC) expression is an immediate response to IL-3 deprivation. This is followed by an accumulation of cells in the G1 phase of the cell cycle, and eventual cell death. However, clones of 32D cells harboring an expression vector which constitutively expresses murine c-myc did not down-regulate ODC transcripts in response to IL-3 withdrawal, and they failed to G1 arrest. Moreover, in contrast to control cultures in which the majority of death occurred following G1 arrest, c-myc clones rapidly initiated a program of cell death characteristic of apoptosis following IL-3 deprivation, and their subsequent loss of viability occurred with accelerated kinetics. The premature induction of apoptosis in cells harboring a deregulated c-myc gene suggests that apoptosis may be an important mechanism in the elimination of hematopoietic cells harboring mutations, such as constitutive c-myc expression, which imbalance normal cell cycle regulatory controls.
The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.
Transgenic mice bearing the cellular myc oncogene coupled to the immunoglobulin mu or kappa enhancer frequently develop a fatal lymphoma within a few months of birth. Since the tumours represent represent both immature and mature B lymphocytes, constitutive c-myc expression appears to be highly leukaemogenic at several stages of B-cell maturation. These myc mice should aid study of lymphoma development, B-cell ontogeny and immunoglobulin regulation.
The highly selective, enzyme-activated, irreversible inhibitor of L-ornithine decarboxylase, DL-alpha-difluoromethylornithine,
suppresses the increase in uterine L-ornithine decarboxylase activity associated with early embryogenesis in the mouse and
arrests embryonic development at that stage. Contragestational effects were confirmed in the rat and rabbit. An increase in
L-ornithine decarboxylase activity that leads to a rapid increase in putrescine concentration appears to be essential during
a critical period after implantation for continued mammalian embryonal growth.
Ornithine decarboxylase, a critical regulatory enzyme for polyamine biosynthesis, is highly inducible by growth-promoting stimuli in mouse epidermis but the enzyme level is only transiently elevated due to rapid turnover of the protein. Here we report that constitutive overexpression of the enzyme in the skin of transgenic mice causes several phenotypic abnormalities. Effects observed include development of dermal follicular cysts, excessive skin wrinkling, enhanced nail growth, alopecia, and spontaneous tumor development. These results indicate that up-regulation of polyamine biosynthesis can profoundly disturb skin homeostasis and alter susceptibility to neoplastic development.
The role of the product of the c-myc protooncogene in the regulation of cellular proliferation and differentiation is well established. Recent reports that c-Myc can serve as a sequence-specific transcriptional activator have begun to elucidate the mechanism by which c-Myc exerts such a profound effect on the mitotic status of a cell. To identify a potential target gene for Myc-mediated trans-activation, we examined the regulation of the ornithine decarboxylase (ODC) gene by c-Myc. ODC is the first and rate-limiting enzyme involved in the synthesis of the polyamines and has been shown to be required for entry into and progression through the cell cycle. Using a conditionally active c-Myc-estrogen receptor chimeric protein, we found estrogen-dependent activation of ODC expression and enzymatic activity. The induction of ODC mRNA expression was not dependent upon de novo protein synthesis. These data suggest that one downstream pathway for Myc-directed cell cycle control is the induction of ODC expression.
Constitutive c-myc expression suppresses cell cycle arrest, promotes entry into S phase, and results in the growth factor-independent expression of ornithine decarboxylase (ODC; EC 4.1.1.17). The ODC gene contains a conserved repeat of the Myc binding site, CACGTG, in intron 1. In this report, we demonstrate that c-Myc is a potent transactivator of ODC promoter-reporter gene constructs in fibroblasts that requires the CACGTG repeat. These sites conferred Myc responsiveness on heterologous promoter constructs, suggesting that ODC is regulated by Myc at the level of transcription initiation. Analysis of deletion and point mutants of c-myc revealed that domains required for transactivation of the ODC promoter did not include the leucine zipper of the Myc protein. This suggests that Myc may interact with transcription factors other than Max to transactivate the ODC gene.
The uptake and release of the natural polyamines putrescine, spermidine and spermine by mammalian cells are integral parts of the systems that regulate the intracellular concentrations of these biogenic amines according to needs. Although a general feature of all tissues, polyamine uptake into intestinal mucosa cells is perhaps the most obvious polyamine transport pathway of physiological and pathophysiological importance. Mutant cell lines lacking the ability to take up polyamines from the environment are capable of releasing polyamines. This indicates that uptake and release are functions of two different transport systems. The isolation of a transporter gene from a mammalian cell line is still lacking. Overaccumulation of polyamines is controlled by release and by a feedback regulation system that involves de novo synthesis of antizyme, a well known protein that also regulates the activity of ornithine decarboxylase. Recent work has demonstrated that Ca(2+)-signalling pathways are also involved. Although there is consensus about the importance of polyamine uptake inhibitors in the treatment of neoplastic disorders, a practically useful uptake inhibitor is still missing. However, the attempts to target tumours, and to increase the selectivity of cytotoxic agents by combining them with the polyamine structure, are promising. New, less toxic and more selective anticancer drugs can be expected from this approach.
In multistage tumorigenesis models, ornithine decarboxylase (ODC) is usually dysregulated at some point during tumor promotion, an early stage of carcinogenesis. To address the question whether constitutive overexpression of ODC would be a sufficient condition for tumor promotion, mice with high levels of ODC expression targeted to epidermal keratinocytes were used in skin tumorigenesis experiments. Transgenic mice with ODC targeted to hair follicle keratinocytes were much more sensitive than littermate controls to initiation with a single low dose of carcinogen; in fact, such mice no longer required treatment with tumor promoters for tumors to develop. Targeting ODC overexpression to both interfollicular and follicular keratinocytes did not further enhance tumor yield. Our results suggest that most, if not all, target cells for chemical carcinogens in the skin reside in hair follicles, and ODC overexpression is sufficient to activate such cells to expand clonally to form epidermal tumors.
The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis--changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER.
The INK4a/ARF locus encodes upstream regulators of the retinoblastoma and p53 tumor suppressor gene products. To compare the impact of these loci on tumor development and treatment response, the Emu-myc transgenic lymphoma model was used to generate genetically defined tumors with mutations in the INK4a/ARF, Rb, or p53 genes. Like p53 null lymphomas, INK4a/ARF null lymphomas formed rapidly, were highly invasive, displayed apoptotic defects, and were markedly resistant to chemotherapy in vitro and in vivo. Furthermore, INK4a/ARF(-/-) lymphomas displayed reduced p53 activity despite the presence of wild-type p53 genes. Consequently, INK4a/ARF and p53 mutations lead to aggressive tumors by disrupting overlapping tumor suppressor functions. These data have important implications for understanding the clinical behavior of human tumors.
The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).
We have shown that ornithine decarboxylase (ODC) overexpression in the skin of TG.AC v-Ha-ras transgenic mice induces the formation of spontaneous skin carcinomas. Treatment of ODC/Ras double transgenic mice with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC enzyme activity, causes a rapid regression of these spontaneous tumors. DFMO treatment led to dramatic decreases in ODC activity and putrescine levels, but v-Ha-ras expression was not affected in the regressed tumors. Moreover, cyclin D1 continued to be strongly expressed in the basal epithelial cells of regressed tumors, and there was no decrease in the proliferative index of these same tumor cells. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling analyses revealed increased DNA fragmentation in DFMO regressed tumors compared with similarly sized spontaneous tumors from ODC/Ras transgenic mice not treated with DFMO. Moreover, the blood vessel count was significantly decreased in regressed tumors within the first four days of DFMO treatment. The decreased vasculature in DFMO regressed tumors was not attributable to altered expression of murine vascular endothelial growth factor (VEGF) isoforms. Elevated levels of ODC activity in the skin of K6/ODC transgenic mice increased the dermal vascularization compared with that in nontransgenic normal littermates. Our results suggest that ODC stimulates an angiogenic factor(s) other than VEGF and/or may play a key role in a cell survival effector pathway of Ras that is independent of a Ras-induced proliferation pathway.
It has been known for > 10 years that there are two alleles of the human ornithine decarboxylase (ODC) gene, defined by a polymorphic PstI RFLP in intron 1. We have sequenced a large portion of each of the two alleles, including some of the 5' promoter region, exon 1, intron 1, and exon 2, and determined that a single nucleotide polymorphism at base +317 (relative to transcription start site) is responsible for the presence or absence of the PstI restriction site. We have developed two genotyping assays, a PCR-RFLP assay and a high-throughput TaqMan-based method, and determined the ODC genotype distribution in >900 North American DNA samples. On the basis of its location between two closely spaced Myc/Max binding sites (E-boxes), we speculated that the single nucleotide polymorphism at base +317 could have functional significance. Results of transfection assays with allele-specific reporter constructs support this hypothesis. The promoter/regulatory region derived from the minor ODC allele (A allele) was more effective in driving luciferase expression in these assays than the identical region from the major allele (G allele). Our results suggest that individuals homozygous for the A allele may be capable of greater ODC expression after environmental exposures, especially those that up-regulate c-MYC expression.
Recent work has discovered the role of the cyclin-dependent kinase (CDK)-binding protein Cks1 in degrading the CDK inhibitor p27(Kip1). This process is essential for DNA replication and is aberrantly enhanced in cancer. Surprisingly, new work indicates that this function of Cks1 is independent of CDKs and links Cks1 with the Skp2 subunit of the SCF ubiquitin ligase.
To directly evaluate the role of increased ornithine decarboxylase (ODC) and polyamines in mouse skin carcinogenesis, we used bovine keratin 5 (K5) and keratin 6 (K6) promoter elements to direct the expression of antizyme (AZ) to specific skin cell populations. AZ is a multifunctional regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. K5-AZ mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 0 and 24 h exhibit increases in epidermal and dermal ODC activity that are reduced in magnitude. K6-AZ mice treated similarly do not show any increased ODC activity or protein after a second application due to TPA-induced expression of AZ protein. Epidermal and dermal polyamine content, particularly spermidine, is reduced in untreated K5-AZ mice and TPA-treated K5-AZ and K6-AZ mice. Susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis was also investigated for two K6-AZ transgenic lines [K6-AZ(52) and K6-AZ(18)] and a single K5-AZ line. K6-AZ(52) mice had a substantial delay in tumor onset and a >80% reduction in tumor multiplicity compared with normal littermates. K6-AZ(18) and K5-AZ mice also developed fewer papillomas than littermate controls (35% and 50%, respectively), and the combination of these lines to produce double transgenic animals yielded an additive decrease (70%) in tumor multiplicity. These mice demonstrate for the first time that AZ suppresses tumor growth in an animal cancer model and provide a valuable model system to evaluate the role of ODC and polyamines in skin tumorigenesis.
N(1),N(11)-Diethylnorspermine (DENSPM) is a polyamine analogue with clinicalrelevance as an experimental anticancer agent and the ability to elicit a profound apoptotic response in certain cell types. Here, we characterize the polyamine effects and apoptotic signaling events initiated by treatment of SK-MEL-28 human melanoma with 10 microM DENSPM. Maximal induction of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine pool depletion were seen by 16 h, whereas early apoptosis was first apparent at 36 h. Intermediate events related to apoptotic signaling were sought between 16 and 36 h. A loss of mitochondrial transmembrane potential (Deltapsi(m)) beginning at 24 h was followed by the release of cytochrome c into the cytosol at 30 h. Loss of mitochondrial integrity was accompanied by caspase-3 activation and poly(ADP-ribose) polymerase digestion from 30 to 36 h. The caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells resistant to analogue-induced caspase-3 activation and reduced the apoptotic response in a dose-dependent manner. Because polyamine reduction achieved by inhibitors of polyamine biosynthesis inhibited growth but did not cause apoptosis, we looked for alternative polyamine-related events, focusing on induction of SSAT. Three DENSPM analogues that differentially induced SSAT activity but similarly depleted polyamine pools revealed a close correlation between enzyme induction and cytochrome c release, caspase activation, and apoptosis. Dose-dependent inhibition of polyamine oxidase, an enzyme that oxidizes acetylated polyamines generated by SSAT and releases toxic by-products such as H(2)O(2) and aldehydes, prevented cytochrome c release, caspase activation, and apoptosis. Taken together, the findings indicate that DENSPM-induced apoptosis is at least partially initiated via massive induction of SSAT and related oxidative events and subsequently mediated by the mitochondrial apoptotic signaling pathway as indicated by cytochrome c release and caspase activation.
Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme
which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation,
transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring
a disrupted ODC gene.ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation,
loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable
of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly,
analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required
for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development,
and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.
To explore the role of c-Myc in carcinogenesis, we have developed a reversible transgenic model of pancreatic beta cell oncogenesis using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature beta cells induces uniform beta cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes beta cell mass. Thus, the oncogenic potential of c-Myc in beta cells is masked by apoptosis. Upon suppression of c-Myc-induced beta cell apoptosis by coexpression of Bcl-x(L), c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and beta cell apoptosis. Our data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions.
Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.
Pharmacological inactivation of oncogenes is being investigated as a possible therapeutic strategy for cancer. One potential drawback is that cessation of such therapy may allow reactivation of the oncogene and tumor regrowth. We used a conditional transgenic mouse model for MYC-induced tumorigenesis to demonstrate that brief inactivation of MYC results in the sustained regression of tumors and the differentiation of osteogenic sarcoma cells into mature osteocytes. Subsequent reactivation of MYC did not restore the cells' malignant properties but instead induced apoptosis. Thus, brief MYC inactivation appears to cause epigenetic changes in tumor cells that render them insensitive to MYC-induced tumorigenesis. These results raise the possibility that transient inactivation of MYC may be an effective therapy for certain cancers.
Germline TP53 mutations are responsible for the large majority of classic LFS families, and a smaller proportion of LFL families. In some of the families shown to have no germline TP53 mutation, germline hChk2 mutations have been described. In some cases the functional consequences of the latter have been demonstrated, although there are still relatively few reports of such mutations. Due to the paucity of families currently described with hChk2 mutations, it is not possible to reach any conclusions concerning the phenotypic/clinical differences between the two types of germline mutation. At least one family with a germline hChk2 mutation is a classic LFS family, whereas others are LFL, variant-LFS, or phenotypically suggestive of LFS. However, there is still a significant number of LFS/LFL families for which no underlying genetic determinant has been identified. It will be fascinating to see what genetic defects are responsible, and whether they involve additional components of DNA damage recognition, repair, or cell cycle checkpoint pathways.
Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Emu-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Emu-Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27(Kip1) and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27(Kip1).