BRCA1 185delAG mutation inhibits Akt-dependent, IAP-mediated caspase 3 inactivation in human ovarian surface epithelial cells

BRCA1 mutations have long been associated with altered apoptosis. We have recently reported that caspase 3 activation is increased in human ovarian surface epithelial (OSE) cells expressing a germline N-terminal BRCA1 185delAG mutation. Here, we report increased caspase 3 activity in 185delAG OSE cells associated with decreased expression of cIAP-1 and X-linked inhibitor of apoptosis (XIAP), and decreased ubiquitination of caspase 3. Overexpression of XIAP restored active caspase 3 ubiquitination and lowered levels of caspase 3 activity. Further, the BRCA1 185delAG mutation was associated with reduced levels of phosphorylated Akt1. Transfection with activated Akt1 led to increased cIAP-1 and XIAP levels similar to that seen in BRCA1 185delAG cell lines. Taken together, these data suggest a direct link between the BRCA1 185delAG mutation and alterations in the caspase-mediated apoptotic pathway.     

BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ERalpha signaling. However, many ERalpha-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ERalpha signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ERalpha-negative cells. We previously noticed that both ERalpha-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ERalpha-negative cell lines even exceeded its over-expression level in ERalpha-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ERalpha-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.     

Sustained suppression of Fas ligand expression in cisplatin-resistant human ovarian surface epithelial cancer cells

Although cisplatin derivatives are first line chemotherapeutic agents for the treatment of ovarian epithelial cancer, chemoresistance is a major therapeutic problem. Although the cytotoxic effect of these agents are believed to be mediated through the induction of apoptosis, the role of the Fas/FasL system in chemoresistance in human ovarian epithelial cancer is not fully understood. In the present study, we have used cultures of established cell lines of cisplatin-sensitive human ovarian epithelial tumours (OV2008 and A2780-s) and their resistant variants (C13* and A2780-cp, respectively) to assess the role ofFas/FasL system in the chemo-responsiveness of ovarian cancer cells to cisplatin. Cisplatin was effective in inducing the expression of cell-associated Fas and FasL, soluble FasL and apoptosis in concentration and time-dependent fashion in both cisplatin-sensitive cell lines (OV2008 and A2780-s). In contrast, while cisplatin was effective in increasing cell-associated Fas protein content in C13*, it failed to up-regulate FasL (cell-associated and soluble forms) and induce apoptosis, irrespective of concentration and duration of cisplatin treatment. Concentrated spent media from OV2008 cultures after cisplatin treatment were effective in inducing apoptosis in C13* cells which was partly inhibited by the antagonistic Fas monoclonal antibody (mAb) suggesting that the soluble FasL present in the spent media was biologically active. In the resistant A2780-cp cells, neither Fas nor FasL up-regulation were evident in the presence of the chemotherapeutic agent and apoptosis remained low compared to its sensitive counterpart. Activation of the Fas signalling pathway, by addition to the cultures an agonistic Fas mAb, was equally effective in inducing apoptosis in the cisplatin-sensitive (OV2008) and -resistant variant C13*, although these responses were of lower magnitude compared to that observed with cisplatin in the chemosensitive cells. A significant interaction between cisplatin and agonistic Fas mAb was observed in the apoptotic response in OV2008 and C13* when cultured in the presence of both agents. Immunohistochemistry of human ovarian epithelial carcinomas reveals the presence of Fas in low abundance in proliferatively active cells but in high levels in quiescent ones. Although the expression pattern of FasL in the tumour was similar to that of Fas, the protein content was considerably lower. Taken together, these data suggest that the dysregulation of the Fas/FasL system may be an important determinant in cisplatin resistance in ovarian epithelial cancer cells. Our results are also supportive of the notion that combined immuno- and chemo-therapy (i.e., agonistic Fas mAb plus cisplatin) may provide added benefits in the treatment of both chemo-sensitive and -resistant ovarian tumours.     

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.     

Proteome changes in ovarian epithelial cells derived from women with BRCA1 mutations and family histories of cancer

Malignant transformation of the ovarian surface epithelium (OSE) accounts for most ovarian carcinoma. Detection of preneoplastic changes in the OSE leading to overt malignancy is important in prevention and management of ovarian cancer. We identified OSE proteins with altered expression derived from women with a family history (FH) of ovarian and/or breast cancer and mutations in the BRCA1 tumor suppressor gene. Proteins from SV-40-transformed FH-OSE cell lines and control OSE lines derived from women without such histories (non-family history) were separated by two-dimensional PAGE. Gels were analyzed, a protein data base was created, and proteins were characterized according to their molecular weight, isoelectric point, and relative abundance. Mass spectrometry was performed on tryptic protein digests, and data bases were searched for known proteins with the same theoretical tryptic peptide masses. Several proteins showed altered expression in the FH-OSE cells. Beta-tubulin and to a lesser extent ubiquitin carboxyl-terminal hydrolase and glyoxalase 1 appeared to be up-regulated. In contrast, proteins suppressed in FH lines include the 27-kDa heat shock protein, translationally controlled tumor protein, and several proteins associated with actin modification such as actin prepeptide, F-actin capping protein alpha subunit, and cofilin. Sequencing of several cofilin gel spots revealed phosphorylation of serine 3, a post-translational modification associated with decreased actin binding and cytoskeletal reorganization. Two-dimensional Western blots probed with cofilin antibody showed multiple protein spots with isoelectric points of 6-9 pH units. Blots of one-dimensional gels showed a significant reduction in cofilin expression in three FH lines when compared with three non-family history lines (p < or = 0.05). Identification of these and other OSE proteins may be useful in detecting changes suggestive of increased risk of developing preneoplastic disease and defining the possible role(s) of the BRCA1 gene in regulation of OSE cell function.     

In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures.
Materials and methods:  OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining.
Results:  On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues.
Conclusions:  Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers.     

Role of BRCA1 and BRCA2 gene mutations in epithelial ovarian cancer in Indian population: a pilot study

Ovarian cancer is a silent killer as most patients have non-specific symptoms and usually present in advanced stage of the disease. It occurs due to certain genetic alterations and mutations namely founder mutations, 187delAG and 5385insC in BRCA1 and 6174delT in BRCA2 which are associated with specific family histories. These highly penetrant susceptibility genes responsible for approximately half of families containing 2 or more ovarian cancer cases account for less than 40% of the familial excess malignancy risk. The remaining risk may be due to single nucleotide polymorphisms (SNPs) which are single base change in a DNA sequence with usual alternatives of two possible nucleotides at a given position. Preliminary study involving 30 women with histologically proven epithelial ovarian cancer was conducted and their detailed genetic analysis was carried out. Regions of founder mutations on BRCA1 and BRCA2 were amplified and sequenced using primers designed based on 200 bp upstream and downstream regions of the mutation sites. Five sequence variants in BRCA1 were identified of which three novel sequence variants were found in 23 patients while in BRCA2, one novel sequence variant was found. The three founder mutations 187delAG, 5385insC in BRCA1 and 6174delT in BRCA2 were not seen in any of the subjects.     

Growth of normal human ovarian surface epithelial cells in reduced-serum and serum-free media

Summary The human ovarian surface epithelium (OSE) is believed responsible for over 85% of ovarian cancers, yet little is known about the normal biology of these cells. To date, culture of OSE has only been reported in media with high serum supplements. We have developed two media, one with less than 1% of serum (OSEM-1) and the other comprised of highly purified and defined materials (OSEM-2), which allow us to study OSE under relatively defined conditions. By substituting 0.05% of Pedersen’s fetuin for 15% fetal bovine serum (FBS) with Medium 199/MCDB105 basal medium, the cell numbers reached 50 to 60% of those in the presence of 15% FBS over 7 days. However, over several weeks, the total number of population doublings achieved were comparable to those in 15% FBS. Addition of insulin, transferrin, ethanolamine, lipoic acid, and phosphatidylcholine to the medium with Pedersen’s fetuin (OSEM-1) enhanced growth up to 20% more than in their absence. Supplementation of M199/105 with highly purified (>99%) fetuin, alpha₂-macroglobulin, and hydrocortisone resulted in a defined medium (OSEM-2) that permitted 1 to 2 doublings/7 days. In addition, cells maintained a more normal, epithelial-like morphology in culture for a longer period in the presence of Pedersen’s or purified fetuin than in M199/105/15% FBS, thus increasing the number of morphologically normal cells available for experimentation. Addition of 0.05% Pedersen’s fetuin to M199/105 in the presence of 6 to 8% FBS resulted in levels of growth equivalent to those in M199/105/15% FBS alone. We are now able to study the effects of various compounds on the growth and differentiation of OSE under defined conditions, and have reduced the requirement for FBS to produce large numbers of OSE cells.     

Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer

Differentiated mammary epithelium shows apicobasal polarity, and loss of tissue organization is an early hallmark of breast carcinogenesis. In BRCA1 mutation carriers, accumulation of stem and progenitor cells in normal breast tissue and increased risk of developing tumors of basal-like type suggest that BRCA1 regulates stem/progenitor cell proliferation and differentiation. However, the function of BRCA1 in this process and its link to carcinogenesis remain unknown. Here we depict a molecular mechanism involving BRCA1 and RHAMM that regulates apicobasal polarity and, when perturbed, may increase risk of breast cancer. Starting from complementary genetic analyses across families and populations, we identified common genetic variation at the low-penetrance susceptibility HMMR locus (encoding for RHAMM) that modifies breast cancer risk among BRCA1, but probably not BRCA2, mutation carriers: n = 7,584, weighted hazard ratio (_wHR) = 1.09 (95% CI 1.02–1.16), p_trend = 0.017; and n = 3,965, _wHR = 1.04 (95% CI 0.94–1.16), p_trend = 0.43; respectively. Subsequently, studies of MCF10A apicobasal polarization revealed a central role for BRCA1 and RHAMM, together with AURKA and TPX2, in essential reorganization of microtubules. Mechanistically, reorganization is facilitated by BRCA1 and impaired by AURKA, which is regulated by negative feedback involving RHAMM and TPX2. Taken together, our data provide fundamental insight into apicobasal polarization through BRCA1 function, which may explain the expanded cell subsets and characteristic tumor type accompanying BRCA1 mutation, while also linking this process to sporadic breast cancer through perturbation of HMMR/RHAMM.     

BRCA1 negatively regulates formation of autophagic vacuoles in MCF-7 breast cancer cells

In recent years, the function of different tumour suppressors in the regulation of macroautophagy has been studied. We show here that BRCA1, unlike other tumour suppressors, negatively regulates formation of autophagosomes and lysosomal mass under conditions of both basal and enhanced autophagy. In MCF-7 breast cancer cells, increased formation of autophagic vacuoles after inactivation of BRCA1 by siRNAs is associated with an increase in reactive oxygen species, such as superoxide anion and hydrogen peroxide. This allows one to propose an antioxidant function for BRCA1 and suggests that dysfunctional mitochondria and the generated reactive oxygen species excess could explain the increased macroautophagy observed in the absence of BRCA1. In addition, a quick decrease in BRCA1 levels occurs when MCF-7 cells are switched to a nutrient-poor environment that stimulates macroautophagy and that is also reminiscent of certain phases of tumour growth. Inhibition of BRCA1 synthesis has an important role in this reduction, while there are almost no changes in BRCA1 degradation by lysosomes and proteasomes. Therefore, BRCA1 produces macroautophagy inhibition by reducing the formation of autophagic vacuoles, and this, together with the other results presented here, shows new functional aspects of BRCA1 that could help to clarify the role of autophagy in cancer development.     

Organotypic culture of human ovarian surface epithelial cells: A potential model for ovarian carcinogenesis

Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis. The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2 fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and perhaps malignant transformation of HOSE cells.     

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.     

Ubiquitination and proteasome-mediated degradation of BRCA1 and BARD1 during steroidogenesis in human ovarian granulosa cells

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.