[Show abstract][Hide abstract] ABSTRACT: The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci 1 . Recent work suggested that rather than up-and downregulating selected groups of genes 1–3 , Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription 4,5 . However, the available data did not readily discrim-inate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunopre-cipitation and RNA expression profiles during B-cell lymphoma-genesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations 6 , we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') 4,5 when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) 7,8 or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amp-lification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome 4,5,9–11
[Show abstract][Hide abstract] ABSTRACT: Myc is a cellular oncogene frequently deregulated in cancer that has the ability to stimulate cellular growth by promoting a number of proliferative and pro-survival pathways. Here we will focus on how Myc controls a number of diverse cellular processes that converge to ensure processivity and robustness of DNA synthesis, thus preventing the inherent replicative stress responses usually evoked by oncogenic lesions. While these processes provide cancer cells with a long-term proliferative advantage, they also represent cancer liabilities that can be exploited to devise innovative therapeutic approaches to target Myc overexpressing tumors. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology, edited by Dr. Giovanni Perini and Dr. Barbara Majello.
[Show abstract][Hide abstract] ABSTRACT: A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. CcnE1(-/-) mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, CcnE1(-/-) HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in CcnE1(-/-) animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.
[Show abstract][Hide abstract] ABSTRACT: Activation of oncogenes is generally associated with the induction of DNA damage response (DDR) signaling, which acts as a barrier to tumor progression. In this review we will present an overview of the DDR associated with oncogenic activation of Myc, with special focus on two opposite and paradoxical aspects of this response: (1) the role of the Myc-induced DDR in tumor suppression; (2) its role in dampening Myc-induced replication stress, thereby protecting the viability of prospective cancer cells. These opposing effects on cancer progression are controlled by two different branches of DDR signaling, respectively ATM/CHK2 and ATR/CHK1. Indeed, while ATM activity constitutes a barrier to malignant transformation, full activation of ATR and CHK1 is essential for tumor maintenance, providing important opportunities for therapeutic intervention. Thus, the Myc-induced DDR acts as a double-edged sword in tumor progression.
[Show abstract][Hide abstract] ABSTRACT: Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.
[Show abstract][Hide abstract] ABSTRACT: p53 is the central regulator of cell fate following genotoxic stress and oncogene activation. Its activity is controlled by several posttranslational modifications. Originally defined as a critical layer of p53 regulation in human cell lines, p53 lysine methylation by Set7/9 (also called Setd7) was proposed to fulfill a similar function in vivo in the mouse, promoting p53 acetylation, stabilization, and activation upon DNA damage (Kurash et al., 2008). We tested the physiological relevance of this circuit in an independent Set7/9 knockout mouse strain. Deletion of Set7/9 had no effect on p53-dependent cell-cycle arrest or apoptosis following sublethal or lethal DNA damage induced by radiation or genotoxic agents. Set7/9 was also dispensable for p53 acetylation following irradiation. c-myc oncogene-induced apoptosis was also independent of Set7/9, and analysis of p53 target genes showed that Set7/9 is not required for the p53-dependent gene expression program. Our data indicate that Set7/9 is dispensable for p53 function in the mouse.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic Initiation Factor 6 (eIF6) controls translation by regulating 80S subunit formation. eIF6 is overexpressed in tumors. Here, we demonstrate that eIF6 inactivation delays tumorigenesis and reduces tumor growth in vivo. eIF6(+/-) mice resist to Myc-induced lymphomagenesis and have prolonged tumor-free survival and reduced tumor growth. eIF6(+/-) mice are also protected by p53 loss. Myc-driven lymphomas contain PKCβII and phosphorylated eIF6; eIF6 is phosphorylated by tumor-derived PKCβII, but not by the eIF4F activator mTORC1. Mutation of PKCβII phosphosite of eIF6 reduces tumor growth. Thus, eIF6 is a rate-limiting controller of initiation of translation, able to affect tumorigenesis and tumor growth. Modulation of eIF6 activity, independent from eIF4F complex, may lead to a therapeutical avenue in tumor therapy.
Cancer cell 06/2011; 19(6):765-75. DOI:10.1016/j.ccr.2011.04.018 · 23.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16(INK4a) and p21(Cip1), and caused senescence in cells lacking Cdk2, but not in Cdk2-proficient cells. We show here that suppression of Myc-induced senescence by Cdk2 does not occur through phosphorylation of its purported substrate residue in Myc (Ser 62). Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescencesuppressing activities were critical for tumor progression, as deficiency in either Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFβ promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicited, since it positively regulated Wrn, Telomerase and Cdk2 activity. Furthermore, Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.
[Show abstract][Hide abstract] ABSTRACT: Activated oncogenes induce compensatory tumour-suppressive responses, such as cellular senescence or apoptosis, but the signals determining the main outcome remain to be fully understood. Here, we uncover a role for Cdk2 (cyclin-dependent kinase 2) in suppressing Myc-induced senescence. Short-term activation of Myc promoted cell-cycle progression in either wild-type or Cdk2 knockout mouse embryo fibroblasts (MEFs). In the knockout MEFs, however, the initial hyper-proliferative response was followed by cellular senescence. Loss of Cdk2 also caused sensitization to Myc-induced senescence in pancreatic beta-cells or splenic B-cells in vivo, correlating with delayed lymphoma onset in the latter. Cdk2-/- MEFs also senesced upon ectopic Wnt signalling or, without an oncogene, upon oxygen-induced culture shock. Myc also causes senescence in cells lacking the DNA repair protein Wrn. However, unlike loss of Wrn, loss of Cdk2 did not enhance Myc-induced replication stress, implying that these proteins suppress senescence through different routes. In MEFs, Myc-induced senescence was genetically dependent on the ARF-p53-p21Cip1 and p16INK4a-pRb pathways, p21Cip1 and p16INK4a being selectively induced in Cdk2-/- cells. Thus, although redundant for cell-cycle progression and development, Cdk2 has a unique role in suppressing oncogene- and/or stress-induced senescence. Pharmacological inhibition of Cdk2 induced Myc-dependent senescence in various cell types, including a p53-null human cancer cell line. Our data warrant re-assessment of Cdk2 as a therapeutic target in Myc- or Wnt-driven tumours.
[Show abstract][Hide abstract] ABSTRACT: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study, we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab, a compound used for the targeted therapy of breast cancer.
Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed.
In HER2-overexpressing carcinoma cell lines, cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling, as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines, as indicated by the significant decrease in SFE and the loss of serial transplantability, following treatment of HER2-overexpressing xenotransplants.
Here, we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression, thereby representing a critical survival pathway of tumor-initiating cells.
Clinical Cancer Research 04/2009; 15(6):2010-21. DOI:10.1158/1078-0432.CCR-08-1327 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In some HER2-positive breast tumors, cell surface overexpression of HER2 is not associated with gene amplification but may instead rest in altered gene transcription, half-life, or recycling of the oncoprotein. Here, we show that HER2 overexpression in HER2 2+ carcinomas is associated with neither an increase in gene transcription nor a deregulation in the ubiquitin-dependent pathways, but instead seems to be regulated by protein kinase Calpha (PKCalpha) activity. The stimulation of PKCalpha up-regulated HER2 expression, whereas PKCalpha inhibition by pharmacologic treatments and PKCalpha-specific small interfering RNA led to a dramatic down-regulation of HER2 levels only in breast cancer cells HER2 2+. Consistent with the in vitro data, our biochemical analysis of HER2 2+ human primary breast specimens revealed significantly higher levels of phosphorylated PKCalpha compared with HER2-negative tumors. Inhibition of HER2 activation by the tyrosine kinase inhibitor lapatinib led to decreased levels of PKCalpha phosphorylation, clearly indicating a cross-talk between PKCalpha and HER2 molecules. These data suggest that HER2 overexpression in HER2 2+ carcinomas is due to an accumulation of the recycled oncoprotein to the cell surface induced by activated PKCalpha.
Cancer Research 07/2007; 67(11):5308-17. DOI:10.1158/0008-5472.CAN-06-3936 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although mitosis is a general physiologic process, cancer cells are unusually sensitive to mitotic inhibitors. Therefore, there is an interest in the identification of novel mitotic inhibitors. Here, we report the novel discovery of the SIL gene as a regulator of mitotic entry and cell survival. The SIL gene was cloned from leukemia-associated chromosomal translocation. It encodes a cytosolic protein with an unknown function and no homology to known proteins. Previously, we observed an increased expression of SIL in multiple cancers that correlated with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. Here, we show that SIL is important for the transition from the G(2) to the M phases of the cell cycle. Inducible knockdown of SIL in cancer cells in vitro delayed entrance into mitosis, decreased activation of the CDK1 (CDC2)-cyclin B complex, and induced apoptosis in a p53-independent manner. SIL is also essential for the growth of tumor explants in mice. Thus, SIL is required for mitotic entry and cancer cell survival. Because increased expression of SIL has been noted in multiple types of cancers and correlates with metastatic spread, it may be a suitable target for novel anticancer therapy.
Cancer Research 06/2007; 67(9):4022-7. DOI:10.1158/0008-5472.CAN-07-0064 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.
[Show abstract][Hide abstract] ABSTRACT: Sil (SCL interrupting locus) was cloned from the most common chromosomal rearrangement in T-cell acute lymphoblastic leukemia. It is an immediate early gene whose expression is associated with cell proliferation. Sil protein levels are tightly regulated during the cell cycle, reaching peak levels in mitosis and disappearing on transition to G1. A recent study found Sil to be one of 17 genes whose overexpression in primary adenocarcinomas predicts metastatic spread. We hypothesized that Sil might have a role in carcinogenesis. To address this question, we utilized several approaches. Using a multitumor tissue array, we found that Sil protein expression was increased mostly in lung cancer, but also at lower levels, in a subset of other tumors. Microarray gene expression analysis and immunohistochemistry of lung cancer samples verified these observations. Sil gene expression in lung cancer correlated with the expression of several kinetochore check-point genes and with the histopathologic mitotic index. These observations suggest that overexpression of the Sil gene characterizes tumors with increased mitotic activity.
[Show abstract][Hide abstract] ABSTRACT: The Sil gene encodes a cytosolic protein required for mouse embryonic midline and left/right axial development. Based on the phenotype of Sil mutant embryos, we hypothesized that Sil may be required for the activity of Sonic Hedgehog (Shh), a secreted signaling molecule also critically important for the development of the embryonic axes and found mutated in multiple types of cancer. Here we tested the genetic interaction between Sil and the Shh pathway by generating and analyzing embryos carrying mutations in both Sil and Patched (Ptch), a Shh receptor that normally inhibits the signaling pathway in the absence of ligand and when mutated leads to constitutive activation of the pathway. We find that Sil(-/-) Ptch(-/-) embryos do not activate the Shh pathway and instead have a phenotype indistinguishable from Sil(-/-) embryos, in which there is a loss of activity of Shh. These results provide genetic evidence that Sil is an essential component of the Shh response, acting downstream to Ptch.