Robert M. Wenham

The Molecular Biology of Endometrial Cancers and the Implications for Pathogenesis, Classification, and Targeted Therapies

BACKGROUND Understanding and identifying molecular biology and genetics of endometrial cancer are central to the development of novel therapies. This article reviews the molecular basis for genesis of endometrial cancer with regard to pathogenesis, classification, and implications for targeted therapies. METHODS Genes and cellular pathways that may have an important role in endometrial cancers, both endometrioid and nonendometrioid cancers, are identified. Recently studied drugs and potential future drugs that target some of these genes and pathways are reviewed. RESULTS The most frequent genetic alteration of endometrioid endometrial cancer is PTEN. PI3CA and K-ras mutations are less common but are often associated with PTEN. Alterations in MLH1 and MSH6 are documented with microsatellite instability. Beta-catenin has a minor but significant association. Conversely, p53 mutation is more often associated with nonendometrioid cancer; others being inactivation of p16 and/or overexpression of HER-2/neu. Absence of E-cadherin is more often than not present in nonendometrioid cancers and is associated with poor prognosis. Novel agents that target the AKT-PI3K-mTOR pathway and those that inhibit epidermal growth factor receptor (EGFR), vascular endothelial growth factors (VEGF), fibroblast growth factor receptor 2 (FGFR2), and folate receptors are currently being investigated. CONCLUSIONS Novel targeted agents, either alone or in combination with cytotoxic agents, may result in superior treatment for patients.

MicroRNAs and their target messenger RNAs associated with endometrial carcinogenesis.

OBJECTIVE Recent advances in gene expression technology have provided insights into global messenger RNA (mRNA) expression changes associated with endometrial cancer development. However, the post-transcriptional events that may also have phenotypic consequences remain to be completely delineated. MicroRNAs (miRNAs) are small non-coding RNA transcripts, that influence cell function via modulation of post-transcriptional activity of multiple target mRNA genes. Although recent reports suggest that miRNAs may influence human cancer development, their role in endometrial carcinogenesis remains to be described. METHODS We measured expression of 335 unique human miRNAs in 61 fresh-frozen endometrial specimens, including 37 endometrial cancers, 20 normal endometrium, and 4 complex atypical hyperplasia samples. In parallel, expression of 22,000 mRNA genes was analyzed using the Affymetrix Human U133A GeneChips in 29 of the endometrial samples, including 20 endometrial carcinomas and 9 normal endometrial samples. Differentially expressed mRNAs, miRNAs, and predicted miRNA-mRNA targets were integrated and evaluated for representation of relevant functional biologic pathways. RESULTS Thirteen miRNAs (p<0.02) and 90 mRNAs (FDR; 0%) were identified to be associated with endometrial cancer development. Twenty-six of the 90 (29%) differentially expressed mRNAs are Sangar-database predicted mRNA targets of the 13 miRNAs. Pathway analysis demonstrates significant involvement of these 26 mRNA genes in processes including cell death, growth, proliferation, and carcinogenesis. CONCLUSION We have identified miRNAs and mRNAs associated with endometrial cancer development. Further, our strategy of integrating miRNA/mRNA data may also aid in the identification of important biologic pathways and additional unique genes that have importance in endometrial pathogenesis.

MicroRNAs and their target messenger RNAs associated with ovarian cancer response to chemotherapy

OBJECTIVE Few successful therapeutic options exist for patients with recurrent ovarian cancer (OVCA). This is due in part to an incomplete understanding of the molecular determinants of chemotherapy-response. Recently, it has been shown that microRNAs (miRNAs) influence messenger-RNA (mRNA) post-transcriptional control and can contribute to human carcinogenesis. The objective of the current study was to explore the role of miRNAs, and their predicted mRNA targets, in OVCA in-vitro response to chemotherapy. METHODS The expression of 335 unique miRNAs was measured in 16 OVCA cell lines. In parallel, the sensitivity of these cell lines to 6 commonly used chemotherapeutic agents (cisplatin, doxorubicin, topotecan, paclitaxel, docetaxel, and gemcitabine) was evaluated by in-vitro cell proliferation assay. MiRNAs associated with cell line drug response were identified by linear regression analysis, and their predicted mRNA targets subject to functional biologic pathway analyses. RESULTS Twenty-seven miRNAs were found to be associated with response to the one or more of the 6 salvage chemotherapies tested (p<0.05). Predicted targets of these miRNAs included 52 mRNAs, previously reported to be associated with chemo-responsiveness, and which are also involved in functional biologic pathways that influence cancer cell cytotoxicity, carcinogenesis, cell mitosis, p53 signaling, and tumor cell growth and invasion. CONCLUSION We have identified miRNAs and their predicted target mRNAs associated with ovarian cancer cell response to chemotherapeutic agents. Our strategy of integrating miRNA and mRNA data may aid in the characterization of important molecular pathways associated with OVCA chemo-response.

MicroRNA MiR-214 regulates ovarian cancer cell stemness by targeting p53/Nanog.

Background: Ovarian cancer stem cells (OCSC) play a critical role in chemoresistance and relapse. Results: Expression of miR-214 induces, whereas knockdown of miR-214 decreases, OCSC and Nanog. MiR-214 targets p53, a repressor of Nanog. Conclusion: miR-214 targets p53 to induce OCSC and Nanog. Significance: MiR-214 is a target for OCSC. Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.

First-in-human trial of a poly(ADP)-ribose polymerase (PARP) inhibitor MK-4827 in advanced cancer patients with antitumor activity in BRCA-deficient tumors and sporadic ovarian cancers (soc).

3102 Background: MK4827 is an orally active, PARP 1/2 inhibitor with nanomolar potency. It induces selective synthetic lethality in homologous recombination (HR) repair deficient tumors with BRCA1/2 loss and in tumor cell lines with non-BRCA-related HR defects, supporting broad clinical application. A phase I study was undertaken to determine the toxicity and tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) profiles, and preliminary anti-tumor activity. METHODS Patients (p) with advanced solid tumors enriched for BRCA-mutation carriers (BRCA-MC) and non-BRCA HR defects received once daily, escalating doses of MK4827 in cohorts of 3-6 p. Dose escalation was guided by toxicity, PK and PD data. RESULTS 60 p (M13, F47; median age 56 yr; 21 BRCA-MC) were treated at 10 dose levels from 30mg to 400mg on days 1-21 of a 28 day cycle (C) in C1, followed by continuous dosing. 20 additional p with soc were enrolled at the MTD. Prior systemic treatments were ≤2 (23p), ≥3 (17p), and ≥4 (40p). Overall, DLT was observed in 4p: grade (G) 3 fatigue in 1/6p at 30mg, reversible G3 pneumonitis in 1/6p at 60mg, and reversible G4 thrombocytopenia in 2/6p treated at 400mg. The MTD was established at 300mg. Other MK4827 related G1-2 reversible adverse events included fatigue, anorexia, nausea and myelosuppression. Dose proportional PK was observed with a mean t1/2 of 40 hours (range 37-42 hr). PD studies confirmed PARP inhibition in peripheral blood mononuclear cells at doses of ≥80 mg. Antitumor activity was observed in both sporadic and BRCA-MC cancers. In total, there have been 12p with partial responses (PR) (10 ovarian [7 BRCA-MC; 3 soc], 2 breast, 10/12 BRCA-MC cancers, 4/12 with ongoing treatment), and 8p with stable disease (SD) (4 ovarian [2 BRMC-MC], 2 NSCLC, 2/4 BRCA-MC) ≥120 days. PRs have ranged from 86(+)-483 days and SD from 18(+)-354 days. CONCLUSIONS MK-4827 was well tolerated, had linear PKs, evidence of target modulation, and promising antitumor activity in both BRCA-MC and sporadic cancer. Specific cohort expansions in other nonhereditary tumors enriched for HR defects is ongoing. Updated safety and response data will be provided.

IGF1 (CA)19 Repeat and IGFBP3 -202 A/C Genotypes and the Risk of Prostate Cancer in Black and White Men

We investigated the relationship between the insulin-like growth factor-1 (IGF1) cytosine-adenine repeat (CA)19 polymorphism located upstream of the genes transcription start site, the insulin-like growth factor binding protein-3 (IGFBP3) −202 A/C promoter region polymorphism, and prostate cancer risk in Black and White men. Study subjects were U.S. veterans ages 41 to 75 years identified at the Durham Veterans Administration Medical Center over a 2.5-year period. Controls (n = 93) were frequency matched to cases (n = 100) based on race (Black or White) and age. Multivariable unconditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the associations between the polymorphisms and prostate cancer risk. For Blacks and Whites combined, an inverse association between prostate cancer and being homozygous for the most common IGF1 repeat allele, (CA)19, (adjusted OR, 0.3; 95% CI, 0.1-0.7) was observed. Similar associations were noted for both Blacks (OR, 0.2; 95% CI, 0.0-0.8) and Whites (OR, 0.4; 95% CI, 0.1-1.6) separately. No statistically significant associations between the IGFBP3 C allele and prostate cancer were noted for Blacks (adjusted OR, 2.3; 95% CI, 0.8-6.2) or Whites (OR, 1.0; 95% CI, 0.3-3.1). The prevalence of the homozygous IGF1 (CA)19 genotype was much lower in Black controls (21%) than White controls (46%), which may, in part, explain the increased prostate cancer incidence in Black versus White men. Further research is needed to confirm these findings.

Gedunin, a novel natural substance, inhibits ovarian cancer cell proliferation.

The discovery of more active therapeutic compounds is essential if the outcome for patients with advanced-stage epithelial ovarian cancer is to be improved. Gedunin, an extract of the neem tree, has been used as a natural remedy for centuries in Asia. Recently, gedunin has been shown to have potential in vitro antineoplastic properties; however, its effect on ovarian cancer cells is unknown. We evaluated the in vitro effect of gedunin on SKOV3, OVCAR4, and OVCAR8 ovarian cancer cell lines proliferation, alone and in the presence of cisplatin. Furthermore, we analyzed in vitro gedunin sensitivity data, integrated with genome-wide expression data from 54 cancer cell lines in an effort to identify genes and molecular pathways that underlie the mechanism of gedunin action. In vitro treatment of ovarian cancer cell lines with gedunin alone produced up to an 80% decrease in cell proliferation (P < 0.01) and, combining gedunin with cisplatin, demonstrated up to a 47% (P < 0.01) decrease in cell proliferation compared with cisplatin treatment alone. Bioinformatic analysis of integrated gedunin sensitivity and gene expression data identified 52 genes to be associated with gedunin sensitivity. These genes are involved in molecular functions related to cell cycle control, carcinogenesis, lipid metabolism, and molecular transportation. We conclude that gedunin has in vitro activity against ovarian cancer cells and, further, may enhance the antiproliferative effect of cisplatin. The molecular determinants of in vitro gedunin response are complex and may include modulation of cell survival and apoptosis pathways.

Matrix Metalloproteinase-1 Gene Promoter Polymorphism and Risk of Ovarian Cancer:

Objective: It has been suggested that the 2G allele of a guanine insertion-deletion promoter polymorphism in the promoter of the matrix metalloproteinase-1 (MMP1) gene may increase susceptibility to ovarian cancer. The 2G allele also has been associated with increased MMP1 expression. We investigated the relationship between the MMP1 polymorphism and ovarian cancer risk in a large population-based, case-control study. Methods: The MMP1 promoter polymorphism was examined in white blood cell DNA from 311 cases and 387 age-and race-matched controls using a radiolabeled polymerase chain reaction assay. In addition, genotyping of the MMP1 polymorphism performed in 42 advanced-stage invasive serous ovarian cancers was compared to their mean relative MMP1 expression from Affymetrix microarrays. Results: The 2G allele frequency did not differ significantly between cases (0.49) and controls (0.48), and the distribution of genotypes was in Hardy-Weinberg equilibrium. Using 1G homozygotes as the reference group, neither 2G homozygotes (odds ratio 1.1, 95% confidence interval 0.7-1.7) nor heterozygotes plus 2G homozygotes (odds ratio 0.9, 95% confidence interval 0.7-1.3) had an increased risk of ovarian cancer. There was also no relationship between MMP1 genotype and histologic grade, histologic type, stage, or tumor behavior (borderline versus invasive). The mean MMP1 expression was twice as high in 2G homozygotes relative to 1G homozygotes, but this difference was not statistically significant. Conclusion: The reported association between the MMP1 promoter polymorphism and ovarian cancer risk was not supported by our data. There was a suggestion that the 2G allele may be associated with higher MMP1 expression, and this finding is worthy of further investigation.

A phase II trial of docetaxel and bevacizumab in recurrent ovarian cancer within 12 months of prior platinum-based chemotherapy.

OBJECTIVES The efficacy and safety of bevacizumab and docetaxel were evaluated in women who developed recurrent epithelial ovarian, fallopian, or peritoneal cancer within 12 months of platinum-based therapy. METHODS Patients received docetaxel (40 mg/m(2)) on days 1 and 8 and bevacizumab (15 mg/kg) on day 1 of a 21-daycycle. Primary endpoint was 6-month progression-free survival (PFS). RESULTS Forty-one patients were evaluable for PFS and 38 for best response; 46% had platinum-free intervals (PFI) of <6 months and 54% between 6 and 12 months. The 6-month PFS was 43.9% (95% confidence interval (CI(95%))=28.6-58.2%). Median PFS (months) was 5.2 (CI(95%)=4.4-7.2) for all patients, 6.2 (CI(95%)=4.1-7.4) for patients with PFI <6 months, and 5.1 (CI(95%)=3.0-7.2) for those with PFI ≥ 6 months. Twenty-two patients showed overall response (CR+PR) (57.9%; CI(95%)=40.8-73.7%), and 32 showed clinical benefit (CR+PR+SD) (84.2%; CI(95%)=68.8-94.0%). For those with complete or partial responses, median duration of response was 4.8 months (0.7-14.5). Median overall survival was 12.4 months (CI(95%)=10.0-21.9). The most common grade 3/4 adverse events (AEs) were neutropenia (14.6% of patients), followed by leukopenia, fatigue, metabolic, and gastrointestinal, with 66% showing any grade 3/4 toxicity. Most common AEs of any grade were gastrointestinal (93%), fatigue (73%), and pain (73%). Four (10%) patients developed hypertension, 1 a gastrointestinal perforation, and another a colovesicular fistula. CONCLUSIONS Bevacizumab and docetaxel administered in patients with recurrent ovarian cancer is an active regimen without new unanticipated toxicities. This combination should be an option for further study or clinical use in recurrent ovarian cancer.

A novel c-Met inhibitor, MK8033, synergizes with carboplatin plus paclitaxel to inhibit ovarian cancer cell growth.

Elevated serum levels of hepatocyte growth factor (HGF) and high tumor expression of c-Met are both indicators of poor overall survival from ovarian cancer (OVCA). In the present study, we evaluated the role of the HGF signaling pathway in OVCA cell line chemoresistance and OVCA patient overall survival as well as the influence of HGF/c-Met signaling inhibition on the sensitivity of OVCA cells to combinational carboplatin plus paclitaxel therapy. The prevalence of the HGF receptor, c-Met, was determined by immunohistochemistry in primary OVCA samples (n=79) and OVCA cell lines (n=41). The influence of the c-Met-specific inhibitor MK8033 on OVCA cell sensitivity to combinations of carboplatin plus paclitaxel was examined in a subset of OVCA cells (n=8) by CellTiter-Blue cell viability assays. Correlation tests were used to identify genes associated with response to MK8033 and carboplatin plus paclitaxel. Identified genes were evaluated for influence on overall survival from OVCA using principal component analysis (PCA) modeling in an independent clinical OVCA dataset (n=218). Immunohistochemistry analysis indicated that 83% of OVCA cells and 92% of primary OVCA expressed the HGF receptor, c-Met. MK8033 exhibited significant anti-proliferative effects against a panel of human OVCA cell lines. Combination index values determined by the Chou-Talalay isobologram equation indicated synergistic activity in combinations of MK8033 and carboplatin plus paclitaxel. Pearsons correlation identified a 47-gene signature to be associated with MK8033-carboplatin plus paclitaxel response. PCA modeling indicated an association of this 47-gene response signature with overall survival from OVCA (P=0.013). These data indicate that HGF/c-Met pathway signaling may influence OVCA chemosensitivity and overall patient survival. Furthermore, HGF/c-Met inhibition by MK8033 represents a promising new therapeutic avenue to increase OVCA sensitivity to carboplatin plus paclitaxel.