David Pépin | Harvard Medical School (original) (raw)

Papers by David Pépin

Molecular Endocrinology, Jul 2, 2013

Luteinization is a complex process, stimulated by gonadotropins, that promotes ovulation and deve... more Luteinization is a complex process, stimulated by gonadotropins, that promotes ovulation and development of the corpus luteum through terminal differentiation of granulosa cells. The pronounced expression of the mammalian imitation switch (ISWI) genes, SNF2H and SNF2L, in adult ovaries prompted us to investigate the role of these chromatin remodeling proteins during follicular development and luteinization. SNF2H expression is highest during growth of preovulatory follicles and becomes less prevalent during luteinization. In contrast, both SNF2L transcript and SNF2L protein levels are rapidly increased in granulosa cells of the mouse ovary 8 h after human chorionic gonadotropin treatment, and continue to be expressed 36 h later within the functional corpus luteum. We demonstrate a physical interaction between SNF2L and the progesterone receptor A isoform, which regulates progesterone receptor-responsive genes required for ovulation. Moreover, chromatin immunoprecipitation demonstrated that, after gonadotropin stimulation, SNF2L is associated with the proximal promoter of the steroidogenic acute regulatory protein (StAR) gene, a classic marker of luteinization in granulosa cells. Interaction of SNF2L with the StAR promoter is required for StAR expression, because small interfering RNA knockdown of SNF2L prevents the activation of the StAR gene. Our results provide the first indication that ISWI chromatin remodeling proteins are responsive to the LH surge and that this response is required for the activation of the StAR gene and the overall development of a functional luteal cell. (Molecular Endocrinology 20: [2406][2407][2408][2409][2410][2411][2412][2413][2414][2415][2416][2417] 2006)

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue spec... more CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemi-cal (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in asci-tes fluid, may be indicative of a more metastatic disease and worse prognosis.

Proceedings of the National Academy of Sciences, 2015

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurr... more To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.

Technology (Elmsford, N.Y.), 2013

Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro... more Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenita...

Clinical Cancer Research, 2013

Oncotarget, Jan 15, 2014

Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and... more Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by LDE225 restores and increases melanoma cells' sensitivity to BRAF-I. These effects are mediated by PDGFRα down-regulation and by ERK and AKT inhibition. The clinical relevance of these data is indicated by t...

Cytometry Part A, 2014

Poly (ADP-Ribose) polymerase-1 (PARP-1) is involved in the DNA repairing system by sensing and si... more Poly (ADP-Ribose) polymerase-1 (PARP-1) is involved in the DNA repairing system by sensing and signaling the presence of DNA damage. Inhibition of PARP-1 is tested in combination with DNA damaging agents such as topoisomerase I inhibitors or ionizing radiations (RT) for the treatment of glioblastoma (GBM). Disruption of p53, widely prevalent in GBMs, plays a major role in DNA repairing system. The current study investigates whether p53 activity has an effect on the sensitivity of human GBM cells to PARP-1 inhibitors in combination with topoisomerase I inhibitor topotecan (TPT) and/or RT. Human GBM cell lines carrying a different functional status of p53 were treated with PARP-1 inhibitor NU1025, in combination with TPT and/or RT. Cytotoxic effects were examined by analyzing the antiproliferative activity, the cell cycle perturbations, and the DNA damage induced by combined treatments. PARP inhibition enhanced the antiproliferative activity, the cell cycle perturbations and the DNA damage induced by both TPT or RT in GBM cells. These effects were influenced by the p53 activity: cells carrying an active p53 were more sensitive to the combination of PARP inhibitor and RT, while cells carrying an inactive p53 displayed a higher sensitivity to the combination of PARP inhibitor and TPT. Our study suggests that p53 activity influences the differential sensitivity of GBM cells to combined treatments of TPT, RT, and PARP inhibitors.

Trends in Endocrinology & Metabolism, 2007

Chromatin has emerged as an important regulator of gene expression, interposed between cell signa... more Chromatin has emerged as an important regulator of gene expression, interposed between cell signaling pathways and transcriptional machinery. It participates in transmitting extra-and intra-cellular signals that coordinate ovarian events: ovarian follicle development, the meiotic maturation of the oocyte that precedes ovulation, and the ovulatory process and consequent luteinization. Recent evidence from model organisms and mammals suggests that chromatin signaling is achieved, in part, by imitation switch (ISWI) ATP-dependent chromatin-remodeling complexes. This review highlights a role for complexes containing the ISWI ATPase sucrose nonfermenting-2h (Snf2h) in proliferation in somatic and germ cells and also in meiosis in germ cells. Moreover, complexes containing the Snf2l ATPase dictate the differentiation of somatic cells and act in the induction of the terminal phases of meiosis in the oocyte.

Proceedings of the National Academy of Sciences, 2012

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It ha... more Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclindependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics. chemotherapy with cisplatin | pluripotency factors

PloS one, 2011

Human tissue kallikreins (KLKs) are members of a multigene family of serine proteases aberrantly ... more Human tissue kallikreins (KLKs) are members of a multigene family of serine proteases aberrantly expressed in many cancer types. In ovarian cancer, 12 KLKs are upregulated, and of those KLK5, 6 and 10 have been the focus of investigations into new diagnostic and prognostic biomarkers. However, little is known about the contributions of KLK5, 6 and 10 to ovarian cancer pathophysiology.In this study, a panel of 13 human ovarian cancer cell lines was screened by ELISA for secretion of KLK5, 6, 8, 10, 13, and 14. The ES-2 cell line, devoid of these kallikreins, was transfected with expression vectors of KLK5, 6 and 10 individually or in pairs. Co-expression of KLK5, 6 and 10 was correlated with lessened aggressivity of ovarian cancer cell lines as defined by reduced colony formation in soft agar and tumorigenicity in nude mice. ES-2 clones overexpressing KLK5, 10/5, 10/6, 5/6 made significantly fewer colonies in soft agar. When compared to control mice, survival of mice injected with ES...

Biology of Reproduction, 2013

Imitation switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that al... more Imitation switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that alter nucleosome positioning by hydrolyzing ATP to regulate access to DNA. In mice, there are two paralogs, SNF2-homolog (SNF2H) and SNF2-like (SNF2L), which participate in different complexes and have contrasting patterns of expression. Here we investigate the role of SNF2L in ovaries by characterizing a mouse bearing an inactivating deletion of exon 6 that disrupts the ATPase domain. Snf2l mutant mice produce significantly fewer eggs than control mice when superovulated. Gonadotropin stimulation leads to a significant deficit in secondary follicles and an increase in abnormal antral follicles. Mutant females also failed to induce fibrinogen-like 2 (Fgl2) in response to human chorionic gonadotropin (hCG) stimulation, while overexpression of SNF2L was sufficient to drive its expression in granulosa cells. SNF2L was also shown to directly interact with the nuclear receptor co-activator flightless I (FLI-I) as shown by immunoprecipitation. These results begin to establish a role for SNF2L in the precise coordination of gene expression in granulosa cells during folliculogenesis and its broader implications in fertility.

Molecular Endocrinology, 2006