Johanne Weberpals

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

BackgroundThe epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.MethodsAgilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.ResultsMorphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.ConclusionsThis work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

The DNA repair proteins BRCA1 and ERCC1 as predictive markers in sporadic ovarian cancer

This study compares Breast Cancer 1 (BRCA1) and excision repair cross complementation group 1 (ERCC1) expression as predictive markers and evaluates the in vitro enhancement of platinum sensitivity using targeted agents in sporadic ovarian cancer (OC). A retrospective study was performed of advanced stage OC patients receiving platinum‐based chemotherapy. BRCA1 and ERCC1 mRNA expression was determined from frozen tissue of 51 patients. Median overall survival (OS) was longer for patients with lower BRCA1 vs. higher BRCA1 (46 vs.33 months, p = 0.03). High BRCA1 was predictive of poorer OS specifically in patients with residual disease (RD) <2 cm (p = 0.03). There was a non‐significant association for patients with lower ERCC1 and RD <2 cm in favor of improved OS and time to progression. Patients who expressed higher levels of both BRCA1 and ERCC1 mRNA had a shorter OS compared to patients with lower levels of either or both transcript (33 vs.46 months, p = 0.04). When Cox proportional modeling was used by representing BRCA1 and ERCC1 mRNA expression as a continuous variable, both emerge as potential predictors of survival. OC cell lines were exposed to chemotherapy in combination with DNA repair pathway inhibitors and cell viability was assessed. In vitro histone deacetylase (HDAC) inhibition increased the sensitivity of A2780s/cp cells to cisplatin and carboplatin but not to taxol, coincident with a significant decrease in BRCA1 and ERCC1 expression, suggesting that this compound directly targets DNA repair. In summary, this study shows that low BRCA1 and ERCC1 expression correlate with improved survival in advanced OC and HDAC inhibition induces synergistic cytotoxicity with platinum in vitro.

Sporadic Epithelial Ovarian Cancer: Clinical Relevance of BRCA1 Inhibition in the DNA Damage and Repair Pathway

Among the most promising pathways for molecular targets in sporadic epithelial ovarian cancer (SEOC) are those involving the BRCA1 protein. Because somatic mutations in BRCA1 are rare in SEOC, it was originally postulated that BRCA1 plays a limited role in the pathogenesis of this disease. However, inactivation of BRCA1 through various mechanisms is a relatively frequent event in ovarian cancer. This is important because BRCA1 is involved in the cellular response to DNA damage and repair and has an essential role in the maintenance of genomic stability. The BRCA1 tumor suppressor protein is known to interact with genes and proteins known collectively as the BRCA1 pathway, and defects in this pathway are believed to be a driving force for cancer progression. As a result, there is compelling evidence to suggest that the dysfunction of BRCA1 may be a central mechanism in all ovarian carcinogenesis, and this has clinical and molecular significance beyond the management of patients with hereditary ovarian cancer. The aim of this review is to evaluate the evidence for BRCA1 dysfunction in SEOC and to link this dysfunction to a defective DNA repair pathway and ultimately the promotion of genomic instability and tumorigenesis. Furthermore, we advocate the continued need to study BRCA1 and its pathway by prospectively correlating clinicopathologic data with molecular aberrations. This will determine whether BRCA1 has relevance as a predictive and prognostic marker in SEOC and whether aberrations in the BRCA1 pathway warrant further study as potential therapeutic targets.

Identification of the IGF1/PI3K/NF κB/ERK gene signalling networks associated with chemotherapy resistance and treatment response in high-grade serous epithelial ovarian cancer

BackgroundResistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression profiling to delineate major deregulated pathways and biomarkers associated with the development of intrinsic chemotherapy resistance upon exposure to standard first-line therapy for ovarian cancer.MethodsThe study cohort comprised 28 patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. All 28 patients had advanced stage, high-grade serous ovarian cancer, and were treated with standard platinum-based chemotherapy. Twelve patient tumours demonstrating relative resistance to platinum chemotherapy corresponding to shorter PFS (< eight months) were compared to sixteen tumours from platinum-sensitive patients (PFS > eighteen months). Whole transcriptome profiling was performed using an Affymetrix high-resolution microarray platform to permit global comparisons of gene expression profiles between tumours from the resistant group and the sensitive group.ResultsMicroarray data analysis revealed a set of 204 discriminating genes possessing expression levels which could influence differential chemotherapy response between the two groups. Robust statistical testing was then performed which eliminated a dependence on the normalization algorithm employed, producing a restricted list of differentially regulated genes, and which found IGF1 to be the most strongly differentially expressed gene. Pathway analysis, based on the list of 204 genes, revealed enrichment in genes primarily involved in the IGF1/PI3K/NF κB/ERK gene signalling networks.ConclusionsThis study has identified pathway specific prognostic biomarkers possibly underlying a differential chemotherapy response in patients undergoing standard platinum-based treatment of serous epithelial ovarian cancer. In addition, our results provide a pathway context for further experimental validations, and the findings are a significant step towards future therapeutic interventions.

Breast cancer 1 (BRCA1) protein expression as a prognostic marker in sporadic epithelial ovarian carcinoma: an NCIC CTG OV.16 correlative study

BACKGROUND Breast cancer 1 (BRCA1) protein inactivation in sporadic ovarian carcinoma (OC) is common and low BRCA1 expression is linked with platinum sensitivity. The clinical validation of BRCA1 as a prognostic marker in OC remains unresolved. PATIENTS AND METHODS In 251 patient samples from the NCIC CTG clinical trial, OV.16, BRCA1 protein expression was determined by immunohistochemistry. RESULTS For all patients, when BRCA1 score was analyzed as a continuous variable, there was no significant correlation between BRCA1 protein expression and progression-free survival (PFS) [adjusted hazard ratio (HR) = 1.15 (0.96-1.37), P = 0.12] or response rate [HR = 0.89 (0.70-1.12), P = 0.32]. In the 116 patients with minimal residual disease (RD), higher BRCA1 expression correlated significantly with worse PFS [HR = 1.40 (1.04-1.89), P = 0.03]. Subgroup analysis divided patients with minimal RD into low (BRCA1 ≤2.5) and high (BRCA1 >2.5) expression groups. Patients with low BRCA1 expression had a more favorable outcome [median PFS was 24.7 and 16.6 months in patients with low and high BRCA1, respectively; HR = 0.56 (0.35-0.89), P = 0.01]. CONCLUSIONS This study suggests that BRCA1 protein is a prognostic marker in sporadic OC patients with minimal RD. Further research is needed to evaluate BRCA1 as a predictive biomarker and to target BRCA1 expression to enhance chemotherapeutic sensitivity.

BRCA1 as a Therapeutic Target in Sporadic Epithelial Ovarian Cancer

In sporadic epithelial ovarian cancer (EOC), the inactivation of BRCA1 through various mechanisms is a relatively common event. BRCA1 protein dysfunction results in the breakdown of various critical pathways in the cell, notably, the DNA damage response and repair pathway. Tumors from patients with BRCA1 germline mutations have an increased sensitivity to DNA damaging chemotherapeutic agents, such as cisplatin, due to defective DNA repair. Thus, inhibiting BRCA1 in sporadic EOC using novel targeted therapies is an attractive strategy for the treatment of advanced or recurrent EOC. Several classes of small molecule inhibitors that affect BRCA1 have now been tested in preclinical and clinical studies suggesting that this is a rational therapeutic approach. The aim of this paper is to provide an understanding of how BRCA1 has evolved into a promising target for the treatment of sporadic disease and to outline the main potential small molecule inhibitors of BRCA1 in EOC.

A phase II study of single-agent RO4929097, a gamma-secretase inhibitor of Notch signaling, in patients with recurrent platinum-resistant epithelial ovarian cancer: A study of the Princess Margaret, Chicago and California phase II consortia

PURPOSE A phase II study was performed to evaluate the efficacy and safety of single-agent RO4929097 (a gamma-secretase inhibitor) in patients with recurrent platinum-resistant ovarian cancer. EXPERIMENTAL DESIGN Women with progressive platinum-resistant ovarian cancer treated with ≤2 chemotherapy regimens for recurrent disease were enrolled in this trial. Patients received oral RO4929097 at 20 mg once daily, 3 days on/4 days off each week in a three week cycle. The primary endpoint was progression-free survival (PFS) rate at the end of 4 cycles. Secondary objectives included assessment of the safety of RO4929097 and exploration of molecular correlates of outcome in archival tumor tissue and serum. RESULTS Of 45 patients enrolled, 40 were evaluable for response. Thirty-seven (82%) patients had high-grade ovarian cancer. No objective responses were observed. Fifteen patients (33%) had stable disease as their best response, with a median duration of 3.1 months. The median PFS for the whole group was 1.3 months (1.2-2.5). Treatment was generally well tolerated with 10% of patients discontinuing treatment due to an adverse event. In high grade serous ovarian cancer patients, the median PFS trended higher when the expression of intracellular Notch (NICD) protein by immunohistochemistry was high versus low (3.3 versus 1.3 months, p=0.09). No clear relationship between circulating angiogenic factors and PFS was found despite a suggestion of an improved outcome with higher baseline VEGFA levels. CONCLUSIONS RO4929097 has insufficient activity as a single-agent in platinum-resistant ovarian cancer to warrant further study as monotherapy. Future studies are needed to explore the potential for cohort enrichment using NICD expression.

The forgotten woman's cancer: vulvar squamous cell carcinoma (VSCC) and a targeted approach to therapy

BACKGROUND The incidence of vulvar squamous cell carcinoma (VSCC) has been on the rise since the 1990s. There has been no new treatment for advanced and recurrent disease in decades, with most women succumbing to the disease. Despite two distinct etiologies of VSCC, human papillomavirus (HPV)-associated and HPV-independent disease, there is no difference in therapeutic options. METHODS A literature review was carried out by searching EMBASE and Medline databases between January 1990 and March 2016 by pairing the keywords of vulvar carcinoma, vulva cancer, vulvar and vulva with molecular markers involved in the cell cycle, apoptosis and angiogenesis. Molecular targets of prognostic significance were identified and targeted agents of therapeutic relevance to both HPV-independent and HPV-associated VSCC were then reviewed. RESULTS Recent advances in our understanding of the molecular biology of VSCC provide insight into the future management of VSCC with molecular targeted therapies. Aberrant cell cycle activity is common in both HPV-associated and HPV-independent VSCC and is characterized by overexpression of p53, Rb and cyclin D1, supporting targeting of these protein products and their downstream pathways. Extracellular regulators of cellular activity, such as EGFR, as well as inhibitors of angiogenesis are being clinically evaluated in VSCC. HPV-independent VSCC is characterized by actionable mutations, including PI3K, CDKN2A and PTEN. In HPV-associated disease, therapeutic vaccines targeting the E6 and E7 HPV oncogenes and immune-based therapies are under investigation. CONCLUSION There has been a paucity of clinical trials in recent years in this neglected womens cancer. Directed therapy against cell cycle regulatory molecules and extracellular proteins and the inhibition of angiogenesis are of broad therapeutic relevance in VSCC. Therapeutic strategies that target actionable mutations should be explored. In HPV-associated VSCC, novel treatments that exploit the virology of HPV and/or enhance the host immune response merit further study.

The effect of the histone deacetylase inhibitor M344 on BRCA1 expression in breast and ovarian cancer cells

BackgroundThe inhibition of Br east Ca ncer 1 (BRCA1) expression sensitizes breast and ovarian cancer cells to platinum chemotherapy. However, therapeutically relevant agents that target BRCA1 expression have not been identified. Our recent report suggested the potential of the histone deacetylase (HDAC) inhibitor, M344, to inhibit BRCA1 expression. In this study, we further evaluated the effect of M344 on BRCA1 mRNA and protein expression, as well as its effect on cisplatin-induced cytotoxicity in various breast (MCF7, T-47D and HCC1937) and ovarian (A2780s, A2780cp and OVCAR-4) cancer cell lines.ResultsWith the addition of M344, the platinum-sensitive breast and ovarian cancer cell lines that displayed relatively high BRCA1 protein levels demonstrated significant potentiation of cisplatin cytotoxicity in association with a reduction of BRCA1 protein. The cisplatin-resistant cell lines, T-47D and A2780s, elicited increased cytotoxicity of cisplatin with M344 and down regulation of BRCA1 protein levels. A2780s cells subjected to combination platinum and M344 treatment, demonstrated increased DNA damage as assessed by the presence of phosphorylated H2A.X foci in comparison to either treatment alone. Using Chromatin Immunoprecipitation, A2780s and MCF7 cells exposed to M344 alone and in combination with cisplatin, did not demonstrate enhanced acetylated Histone 4 at the BRCA1 promoter, suggesting an indirect effect on this promoter.ConclusionsThe enhanced sensitivity of HDAC inhibition to platinum may be mediated through a BRCA1-dependent mechanism in breast and ovarian cancer cells. The findings of this study may be important in the future design of clinical trials involving HDAC inhibitors using BRCA1 as a tumour biomarker.

A rare case of ectopic adrenocorticotropic hormone syndrome in small cell carcinoma of the vagina: a case report.

Objective. To describe ectopic adrenocorticotropic hormone (ACTH) syndrome (EAS) to increase awareness of this condition when treating patients with gynecological small cell carcinoma (SmCC). Materials and Methods. This is a review of a 61-year-old woman with primary vaginal SmCC and an atypical presentation of Cushing syndrome. This case describes the molecular rationale, caveats in clinical presentation, pathological diagnosis, and management options for patients with this rare syndrome. Results. After treatment with primary chemotherapy, the patient presented acutely with delirium and metabolic disturbances. This was associated with elevated ACTH and gastrin levels without any tumor staining for ACTH. The patient was initially managed with ketoconazole to control the EAS. Complications developed related to her Cushing syndrome, and she succumbed to complications of her disease. Conclusions. Vaginal SmCC complicated by EAS is a rare paraneoplastic syndrome, and this case history outlines the management options for patients with this condition and reviews the pertinent literature.