[Show abstract][Hide abstract] ABSTRACT: Despite the strides that immune therapy has made in mediating tumor regression, the clinical effect is often transient, and more durable responses are still needed. The temporary nature of the immune response is attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, T regulatory cells (Treg). Although the depletion of Treg has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. Here, we found that PI3K-Akt pathway inhibitors selectively inhibit Treg with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Treg in comparison to Tconv cells when treated with different Akt and PI3K inhibitors. This effect was observed both in human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Treg both in naïve and tumor-bearing mice. Furthermore, these PI3K-Akt inhibitors lead to a significant therapeutic anti-tumor effect, which was shown to be Treg dependent. In this work, we report using PI3K-Akt pathway inhibitors as potent selective agents for the depletion of suppressive Treg. These inhibitors are shown to enhance the anti-tumor immune response and are therefore promising potential Treg-depletion clinical reagents.
[Show abstract][Hide abstract] ABSTRACT: Studies have shown that a regulatory T cell (Treg) cell decrease accompanied complete regression of tumor growth induced by a Listeria monocytogenes (Lm)-based vaccine expressing a fusion protein consisting of truncated listeriolysin O (LLO) and human papilloma virus (HPV) E7 protein (Lm-LLO-E7). However, how Lm-based vaccine causes Treg decrease remains unclear yet. Using a highly attenuated Lm dal dat ∆actA strain (LmddA)-based vaccine, we report here that the vector LmddA itself was sufficient to induce a decrease in the proportion of Treg cells by preferentially expanding CD4+FoxP3- T cells and CD8+ T cells, by a mechanism dependent on and directly mediated by LLO. Episomal expression of a nonhemolytic truncated LLO in Lm (LmddA-LLO) significantly augmented the expansion, thus decreasing Treg frequency to a lower degree. While adoptive transfer of Tregs compromised the anti-tumor efficacy of LmddA-LLO-E7 vaccine, a combination of LmddA-LLO and an Lm-based vaccine expressing E7 protein (Lm-E7) induced complete regression against established TC-1 tumors. An Lm recombinant replacing LLO with perfringolysin O (PFO), allowing exit from the phagolysosome but without LLO, confirmed that the adjuvant effect was dependent on LLO itself. These results suggest that LLO may serve as a promising adjuvant by preferentially inducing CD4+FoxP3- T cell and CD8+ T cell expansion, thus improving the ratio of Tregs to CD4+FoxP3- T cells and to CD8+ T cells and favoring immune responses to eradicate tumor.
[Show abstract][Hide abstract] ABSTRACT: Programmed death receptor 1 (PD-1) is an important signaling molecule often involved in tumor-mediated suppression of activated immune cells. Binding of this receptor to its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), attenuates T cell activation, reduces IL-2 and IFN-γ secretion, decreases proliferation and cytotoxicity, and induces apoptosis. B7-DC-Ig is a recombinant protein that binds and targets PD-1. It is composed of an extracellular domain of murine B7-DC fused to the Fc portion of murine IgG2a. In this study, we demonstrate that B7-DC-Ig can enhance the therapeutic efficacy of vaccine when combined with cyclophosphamide. We show that this combination significantly enhances Ag-specific immune responses and leads to complete eradication of established tumors in 60% of mice and that this effect is CD8 dependent. We identified a novel mechanism by which B7-DC-Ig exerts its therapeutic effect that is distinctly different from direct blocking of the PD-L1-PD-1 interaction. In this study, we demonstrate that there are significant differences between levels and timing of surface PD-1 expression on different T cell subsets. We found that these differences play critical roles in anti-tumor immune effect exhibited by B7-DC-Ig through inhibiting proliferation of PD-1(high) CD4 T cells, leading to a significant decrease in the level of these cells, which are enriched for regulatory T cells, within the tumor. In addition, it also leads to a decrease in PD-1(high) CD8 T cells, tipping the balance toward nonexhausted functional PD-1(low) CD8 T cells. We believe that the PD-1 expression level on T cells is a crucial factor that needs to be considered when designing PD-1-targeting immune therapies.
The Journal of Immunology 07/2012; 189(5):2338-47. DOI:10.4049/jimmunol.1103085 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.
PLoS ONE 01/2012; 7(1):e29079. DOI:10.1371/journal.pone.0029079 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients.
PLoS ONE 07/2011; 6(7):e21121. DOI:10.1371/journal.pone.0021121 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.
Cancer cell 08/2010; 18(2):185-97. DOI:10.1016/j.ccr.2010.06.016 · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. Here, we identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors. Independent evaluation confirmed the association of a prognostic gene microfibril-associated glycoprotein 2 (MAGP2) with poor prognosis, whereas in vitro mechanistic analyses demonstrated its ability to prolong tumor cell survival and stimulate endothelial cell motility and survival via the alpha(V)beta(3) integrin receptor. Increased MAGP2 expression correlated with microvessel density suggesting a proangiogenic role in vivo. Thus, MAGP2 may serve as a survival-associated target.
Cancer cell 12/2009; 16(6):521-32. DOI:10.1016/j.ccr.2009.10.018 · 23.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.
American Journal Of Pathology 09/2009; 175(2):592-604. DOI:10.2353/ajpath.2009.080461 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently used RNA interference to show that a negative correlation of L-asparaginase (L-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and L-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, L-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of L-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical L-ASP activity in ovarian cancer.
Molecular Cancer Therapeutics 11/2008; 7(10):3123-8. DOI:10.1158/1535-7163.MCT-08-0589 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the existence of morphologically indistinguishable disease, patients with advanced ovarian tumors display a broad range of survival end points. We hypothesize that gene expression profiling can identify a prognostic signature accounting for these distinct clinical outcomes. To resolve survival-associated loci, gene expression profiling was completed for an extensive set of 185 (90 optimal/95 suboptimal) primary ovarian tumors using the Affymetrix human U133A microarray. Cox regression analysis identified probe sets associated with survival in optimally and suboptimally debulked tumor sets at a P value of <0.01. Leave-one-out cross-validation was applied to each tumor cohort and confirmed by a permutation test. External validation was conducted by applying the gene signature to a publicly available array database of expression profiles of advanced stage suboptimally debulked tumors. The prognostic signature successfully classified the tumors according to survival for suboptimally (P = 0.0179) but not optimally debulked (P = 0.144) patients. The suboptimal gene signature was validated using the independent set of tumors (odds ratio, 8.75; P = 0.0146). To elucidate signaling events amenable to therapeutic intervention in suboptimally debulked patients, pathway analysis was completed for the top 57 survival-associated probe sets. For suboptimally debulked patients, confirmation of the predictive gene signature supports the existence of a clinically relevant predictor, as well as the possibility of novel therapeutic opportunities. Ultimately, the prognostic classifier defined for suboptimally debulked tumors may aid in the classification and enhancement of patient outcome for this high-risk population.
Cancer Research 08/2008; 68(13):5478-86. DOI:10.1158/0008-5472.CAN-07-6595 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is a major health problem for women in the United States. Despite evidence of considerable heterogeneity, most cases of ovarian cancer are treated in a similar fashion. The molecular basis for the clinicopathologic characteristics of these tumors remains poorly defined. Whole genome expression profiling is a genomic tool, which can identify dysregulated genes and uncover unique sub-classes of tumors. The application of this technology to ovarian cancer has provided a solid molecular basis for differences in histology and grade of ovarian tumors. Differentially expressed genes identified pathways implicated in cell proliferation, invasion, motility, chromosomal instability, and gene silencing and provided new insights into the origin and potential treatment of these cancers. The added knowledge provided by global gene expression profiling should allow for a more rational treatment of ovarian cancers. These techniques are leading to a paradigm shift from empirical treatment to an individually tailored approach. This review summarizes the new genomic data on epithelial ovarian cancers of different histology and grade and the impact it will have on our understanding and treatment of this disease.
Cell Research 06/2008; 18(5):538-48. DOI:10.1038/cr.2008.52 · 12.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IntroductionExpression Profiling of Ovarian CancerDefining the Biologic Relationship among Ovarian Tumors of Varying GradeDefining the Biologic Relationship among Ovarian Cancers of Different HistologiesComplexities of Whole-Genome ProfilingIn Silico Pathway IdentificationNoiseReferences
Pathway Analysis for Drug Discovery: Computational Infrastructure and Applications, 04/2008: pages 149 - 164; , ISBN: 9780470399279
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is the most lethal gynecologic cancer, accounting for over 16,000 deaths. In the US annually (1). The poor prognosis of this disease is due to the lack of reliable screening tools, the late stage of disease at the time of diagnosis, the high rate of recurrence of the disease, and the poor response to chemotherapy in the recurrent setting. Patients who present with stage I disease have over 90% 5-year survival, while those diagnosed with stage III disease have less than 20% 5-year survival (2). However, only 25% of patients with ovarian cancer are diagnosed with stage I disease (1). Despite new chemotherapeutic regimens and radical surgical debulking procedures, only minimal improvement in overall survival has been appreciated in the last several decades.
Epithelial ovarian cancer encompasses four major histotypes: papillary serous, endometrioid, mucinous, and clear cell. These histotypes resemble various müllerian cell types, with serous tumors resembling Fallopian tube, endometrioid tumors resembling uterine endometrium, mucinous tumors resembling the endocervix, and clear cell tumors resembling endometrial glands during pregnancy (2). Tumors are graded from 1 to 3, with grade 3 being the most poorly differentiated. Tumors of low malignant potential (LMP) display the atypical cellular features of cancer, but do not invade into the ovarian stroma (2).
The clinical characteristics of ovarian tumors vary according to histology and grade. Although clear cell ovarian cancer usually presents with earlier stage of the disease, there is a higher rate of recurrence among these patients. In fact, the 5-year survival for patients with stage I of clear cell ovarian cancer is only 60% (2). Late stage clear cell cancer also carries a worse prognosis when compared with papillary serous cancer, with an overall median survival of 12 months compared with 22 months (3). Mucinous cancers have a poorer response to chemotherapy and worse survival than the other epithelial ovarian histotypes (4). Patients with endometrioid ovarian cancers tend to have a better prognosis. Higher tumor grade correlates with poorer prognosis. Patients with LMP tumors have a 5-year survival over 95%. Among patients with early stage of disease, the survival drops down from 97% for patients with grade I tumors to 50% for those with grade III tumors (5).
Advances in Experimental Medicine and Biology 02/2008; 622:23-33. DOI:10.1007/978-0-387-68969-2_3 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is complex disease composed of different histological grades and types. However, the underlying molecular mechanisms involved in the development of different phenotypes remain largely unknown. Epidemiological studies identified multiple exogenous and endogenous risk factors for ovarian cancer development. Among them, an inflammatory stromal microenvironment seems to play a critical role in the initiation of the disease. The interaction between such a microenvironment, genetic polymorphisms, and different epithelial components such as endosalpingiosis, endometriosis, and ovarian inclusion cyst in the ovarian cortex may induce different genetic changes identified in the epithelial component of different histological types of ovarian tumors. Genetic studies on different histological grades and types provide insight into the pathogenetic pathways for the development of different disease phenotypes. However, the link between all these genetic changes and the etiological factors remains to be established.
[Show abstract][Hide abstract] ABSTRACT: Differentially Expressed Nucleolar TGF-beta1 Target (DENTT) is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) superfamily whose mRNA is induced by TGF-beta1 in TGF-beta1-responsive human lung cancer cells. Monkey DENTT mRNA contains a 2085-bp open reading frame that encodes a predicted polypeptide of 695 amino acids with five nuclear localization signals, two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. RT-PCR amplification and Western blot analyses showed DENTT mRNA and protein in adult monkey tissues, including the adrenal gland, cerebral cortex, and ovary. Immunohistochemical staining showed that numerous neurons were intensely immunoreactive for DENTT, as were anterior pituitary secretory cells, thyroid follicular cells, and smooth muscle cells of arteries and lung bronchial walls. DENTT expression was also prominent in monkey bronchiolar-alveolar adenomas and cell lines. While the addition of TGF-beta1 or retinoic acid to monkey normal lung bronchial 12MBr6 cells and human lung cancer NCI-H727 cells increased DENTT protein production, TGF-beta1 together with retinoic acid resulted in a more sustained increase in DENTT production than with TGF-beta1 or retinoic acid alone. Transient transfection studies showed that ectopic DENTT expression significantly increased TGF-beta1-responsive 3TP-Lux and CAGA12-Lux reporter transcription in 12MBr6 and NCI-H727 cells with TGF-beta1 addition, while ectopic DENTT expression had no significant effect on the transcription of a retinoic acid-responsive element reporter in the presence of retinoic acid or TGF-beta1. These findings suggest new possibilities for DENTT as a TGF-beta1-regulated, but not a retinoic acid-regulated member of the TTSN superfamily in primate physiology.
[Show abstract][Hide abstract] ABSTRACT: Adrenomedullin (AM) is a 52 amino acid peptide involved in the pathophysiology of several human diseases. Here we show the gene structure, organ distribution, and regulated expression of AM in monkey. The monkey AM (mAM) gene is located on the short arm of chromosome 9 and it codes for a 185 amino acid preprohormone, which contains two amidated peptides identical to the human AM and proadrenomedullin N-terminal 20 peptide. The promoter region of the mAM gene contains a variety of transcription factor binding motifs. mAM is widely expressed throughout many organs as shown by real-time PCR and immunohistochemical techniques, and we have found similar levels of circulating plasma AM in monkeys and humans. A significant upregulation of the mAM mRNA was observed in monkey cells exposed to low oxygen tension conditions, TGF-beta1, all-trans-retinoic acid, and dexamethasone. Our collective data show a high degree of homology between mAM and hAM, which renders the monkey an attractive animal model for future pharmacological and pre-clinical studies targeting AM.
Biochemical and Biophysical Research Communications 10/2004; 321(4):859-69. DOI:10.1016/j.bbrc.2004.07.032 · 2.30 Impact Factor