Podratz, Karl C.

HE4, also known as WFDC2, is a useful biomarker for ovarian cancer when either used alone or in combination with CA125. HE4 is also overexpressed in endometrial cancer (EC), but its function in cancer cells is not clear. In this study, we investigate the role of HE4 in EC progression. An HE4-overexpression system was established by cloning the HE4 prototypic mRNA variant (HE4-V0) into a eukaryotic expression vector. Following transfection, stable clones in two EC cell lines were selected. The effects of HE4 overexpression on cell growth and function were measured with the use of cell proliferation assay, matrigel invasion, and soft agar gel colony formation assays. HE4-induced cancer cell proliferation in vivo was examined in a mouse xenograft model. HE4 overexpression significantly enhanced EC cell proliferation, matrigel invasion, and colony formation in soft agar. Moreover, HE4 overexpression promoted tumor growth in the mouse xenograft model. HE4 overexpression enhanced several malignant phenotypes in cell culture and in a mouse model. These results are consistent with our previous observation that high levels of serum HE4 closely correlate with the stage, myometrial invasion and tumor size in patients with EC. This study provides evidence that HE4 overexpression directly impacts tumor progression in endometrial cancer.

Abstract It is estimated that more than 42,000 new cases and 7,780 deaths will be attributed to endometrial cancer (EC) during 2009. Considering the anticipated number of deaths from EC has nearly tripled since 1987 (2,900), a mandate exists to develop new paradigms focusing on prevention, early detection and treatment. Detailed cataloging of the phenotypic, biochemical and genetic signatures of EC will facilitate the maturation of a risk profile for EC thereby affording patients at risk the opportunity for enrollment in prevention or early therapeutic trials. Endometrioid carcinoma (or type I EC) accounts for approximately 80% of endometrial cancers. Epidemiological studies have provided convincing evidence for the pivotal role of obesity in the etiology of this disease. Adiposity induced hyperinsulinemia and hyperestrogenemia are compounding factors and result in physiological alterations that collectively have been reported to facilitate mitogenesis, stimulate neoplastic growth and inhibit apoptosis. The stimulatory effects of estrogen on the endometrium are well characterized. Furthermore, there is increasing evidence that hyperinsulinemia is more directly involved in carcinogenesis. Insulin functions as a growth factor by increasing endometrial insulin receptors thereby enhancing the growth potential of the endometrium to IGFs which are up regulated by estrogens and considered strong mitogens that stimulate cell proliferation and suppress apoptosis. Insulin also inhibits serum and tissue levels of IGFBP thereby increasing the bioavailability of IGF. While the above metabolic abnormalities facilitate incessant proliferative, the evolutionary mechanisms initiating cell transformation from proliferative endometrium to CAH (complex atypical hyperplasia), the precursor lesion, and EC remain unknown. The potential mechanisms include mutations, gene silencing or inactivation of chromatin or a combination of these processes. Microsatellite instability (MSI), reflecting silencing of one or more mismatch repair genes, has been witnessed in approximately 30% of endometrioid EC and 70% of the MSI cohort correlated with methylation of MLH1. Multiple additional major genetic alterations including inactivation of tumor suppressor genes and mutations in oncogenes have been reported. Mutations in PTEN, which antagonizes the PI3K/AKT pathway, have been reported in as high as 83% of EC and 55% of precursor lesions. PTEN mutations are more frequently associated with MSI+ (60–80%) than with MSI- (24–35%) lesions and have a favorable prognosis; patients with PTEN silencing due to promoter methylation have a poorer prognosis. PIK3CA mutations occurring within the catalytic subunit of PI3K constitutively activate AKT and have been detected in 39% of EC but only in 7% of CAH suggesting a later event than PTEN mutations. Mutations in K-ras and β-catenin have been observed in 10–35% of EC resulting in constitutive activation of the MAPK and β-catenin pathways, respectively. The observations that 42–69% of K-ras mutations occur in MSI+ EC and that the majority are G to A transition mutations suggest the silencing of MGMT as a possible etiology. Gene silencing via methylation of gene promoters is a common phenomenon witnessed in nearly all solid tumors. Methylation of cytosine in CpG islands results in the binding domain proteins recruiting histone deacetylases. The subsequent deacetylation and methylation of histone lysine result in tight histone/DNA interactions which impede transcription. While DNMT1 is necessary for maintenance methylation, DNMT3A and 3B are considered responsible for the de novo methylation at the 5-position of cytosine. Several genes have been reported to be silenced via promoter methylation in EC including MLH1, APC (critical component in the β-catenin pathway), PTEN (as noted above), estrogen receptors and progesterone receptors. Several additional cogent tumor suppressor genes or receptors silenced in other hormone responsive tissues such as ovary, breast and prostate and functional in the four major molecular pathways functioning in the endometrium are candidates for promoter CpG methylation. These include MGMT, IGFBP3, RUNX3, and CDKN2A. We have reported varying levels of DNMT mRNA and protein expression in EC tissue specimens and cell culture; DNMT1 and DNMT3B were up regulated in tissues and cell culture while DNMT2 and 3A did not differ significantly from controls. The addition of estrogen to Ishikawa cells (estrogen receptor positive) resulted in an up regulation of DNMT3B. Considering progesterone receptors are absent in many moderate and essentially all poorly differentiated EC, PRB promoter methylation was assessed in Hec-1B and KLE cells before and after treatment with methyltransferase (MT) and histone deacetylase (HDAC) inhibitors alone or in combination. The PRB promoters in both cell lines were methylated prior to treatment with ADC (aza-deoxycytidine), a methyltransferase inhibitor, and became demethylated following exposure to ADC. PR-B mRNA expression in both HEC-1B and KLE cells increased in response to both MT and HDAC inhibitors with a synergistic effect noted with the combination of ADC and TSA. Western blot confirmed the corresponding PR-B protein expression levels. The effect of TSA on DNMT mRNA expression was analyzed in Ishikawa cells. TSA did not significantly alter DNMT1 or -3A mRNA levels but markedly suppressed DNMT3B mRNA expression. RNA decay curves demonstrated rapid degradation of DMNT3B mRNA following TSA treatment of both Ishikawa and AN3 cells. Recent reports have demonstrated a direct interaction of miRNA 29b with the 3′ untranslated regions of both DNMT3A and DNMT3B in lung cancer and acute myeloid leukemia resulting in downregulation of both DNMT3A and DNMT3B. Further studies of the epigenome in endometrial cancer and other solid tumors are required to test the utility of using methylation markers for diagnostic and prognostic purposes and facilitating strategies in the development of epigenetic therapies. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN02-02.

AbstractPurpose: The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines.Experimental design: Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA1 knocked down clones.Results: Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all seven papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a three- to fourfold increase in the invasive potential. Exogenous expression of HtrA1 in Ark1 and Ark2 cells resulted in three- to fourfold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark2 cells. Immunohistochemical analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (P = 0.016).Conclusions: Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. Clin Cancer Res; 17(3); 427–36. ©2010 AACR.

Abstract Edmonston vaccine strains of measles virus (MV) have shown significant antitumor activity in preclinical models of ovarian cancer. We engineered MV to express the marker peptide carcinoembryonic antigen (MV-CEA virus) to also permit real-time monitoring of viral gene expression in tumors in the clinical setting. Patients with Taxol and platinum-refractory recurrent ovarian cancer and normal CEA levels were eligible for this phase I trial. Twenty-one patients were treated with MV-CEA i.p. every 4 weeks for up to 6 cycles at seven different dose levels (103–109 TCID50). We observed no dose-limiting toxicity, treatment-induced immunosuppression, development of anti-CEA antibodies, increase in anti-MV antibody titers, or virus shedding in urine or saliva. Dose-dependent CEA elevation in peritoneal fluid and serum was observed. Immunohistochemical analysis of patient tumor specimens revealed overexpression of measles receptor CD46 in 13 of 15 patients. Best objective response was dose-dependent disease stabilization in 14 of 21 patients with a median duration of 92.5 days (range, 54–277 days). Five patients had significant decreases in CA-125 levels. Median survival of patients on study was 12.15 months (range, 1.3–38.4 months), comparing favorably to an expected median survival of 6 months in this patient population. Our findings indicate that i.p. administration of MV-CEA is well tolerated and results in dose-dependent biological activity in a cohort of heavily pretreated recurrent ovarian cancer patients. Cancer Res; 70(3); 875–82

Abstract The use of histone deacetylase (HDAC) inhibitors has shown promise for a variety of malignancies. In this investigation, we define the activity of this class of inhibitors in combination with traditional cytotoxic chemotherapy in endometrial cancer cells. Significant reductions in growth were observed in Ark2 and KLE endometrial cancer cells following treatment with paclitaxel, doxorubicin, carboplatin, or the HDAC inhibitor trichostatin A (TSA). However, only combined treatment with TSA/paclitaxel caused synergistic inhibition of cell growth. This combination also resulted in significant changes in cell morphology. Using cell cycle analysis, nuclear staining, and Western blot analysis for poly(ADP-ribose) polymerase and caspase-9 degradation products, TSA/paclitaxel showed the most dramatic activation of the apoptotic cascade. These effects were also observed when the HDAC inhibitors HDAC inhibitor-1 or oxamflatin were substituted for TSA. The anticancer properties of paclitaxel are known to result in part from inhibition of microtubule depolymerization, which results in apoptosis. We show that TSA administration also stabilizes microtubules via α-tubulin acetylation. Furthermore, using Western blot and immunohistochemical analysis, treatment with TSA/paclitaxel led to a significant increase in acetylated tubulin and microtubule stabilization. These effects were confirmed in a mouse xenograft model. Moreover, TSA/paclitaxel resulted in a 50% reduction in tumor weight compared with either agent alone. This study provides in vivo evidence of nonhistone protein acetylation as one possible mechanism by which HDAC inhibitors reduce cancer growth. The TSA/paclitaxel combination seems to hold promise for the treatment of serous endometrial carcinoma and other malignancies with limited sensitivity to paclitaxel. [Mol Cancer Ther 2006;5(11):2767–76]

Abstract Lapatinib (GW572016) is a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases. Here, we explore the therapeutic potential of lapatinib by testing its effect on tumor cell growth in a panel of 31 characterized human breast cancer cell lines, including trastuzumab-conditioned HER-2-positive cell lines. We further characterize its activity in combination with trastuzumab and analyze whether EGFR and HER-2 expression or changes induced in the activation of EGFR, HER-2, Raf, AKT, or extracellular signal-regulated kinase (ERK) are markers of drug activity. We report that concentration-dependent antiproliferative effects of lapatinib were seen in all breast cancer cell lines tested but varied significantly between individual cell lines with up to 1,000-fold difference in the IC50s (range, 0.010-18.6 μmol/L). Response to lapatinib was significantly correlated with HER-2 expression and its ability to inhibit HER-2, Raf, AKT, and ERK phosphorylation. Long-term in vivo lapatinib studies were conducted with human breast cancer xenografts in athymic mice. Treatment over 77 days resulted in a sustained and significant reduction in xenograft volume compared with untreated controls. For the combination of lapatinib plus trastuzumab, synergistic drug interactions were observed in four different HER-2-overexpressing cell lines. Moreover, lapatinib retained significant in vitro activity against cell lines selected for long-term outgrowth (>9 months) in trastuzumab-containing (100 μg/mL) culture medium. These observations provide a clear biological rationale to test lapatinib as a single agent or in combination with trastuzumab in HER-2-overexpressing breast cancer and in patients with clinical resistance to trastuzumab. (Cancer Res 2006; 66(3): 1630-9)

Abstract Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecologic cancers in the United States. Because women who are diagnosed with early stage disease have a better prognosis than women diagnosed with late stage disease, early detection represents a potentially practical approach to reduce the mortality associated with EOC. Unfortunately, no single screening test has proven to be effective for this purpose, and a valid and feasible screening program to detect early stage EOC in the general population has not yet been devised. Consequently, research has focused on coupling two or more screening modalities to improve program validity and feasibility. Serum cancer antigen 125 (CA125) and a soluble isoform of the epidermal growth factor receptor (p110 sEGFR) have been studied individually as biomarkers of ovarian cancer. In this study, we compare serum CA125 levels and sEGFR concentrations in women with EOC to women with benign gynecologic conditions of ovarian and non-ovarian origin. We show that serum sEGFR concentrations are lower in patients with EOC than in women with benign gynecologic conditions, whereas serum CA125 levels are higher in patients to EOC compared with women with benign gynecologic conditions. These data also reveal that age and serum sEGFR concentrations modify the association between CA125 levels and EOC versus benign gynecologic disease. Hence, age- and sEGFR-dependent CA125 cutoff thresholds improve the ability of CA125 to discern EOC patients from women with benign ovarian tumors and non-ovarian gynecologic conditions. Our analyses show that parallel testing with fixed sEGFR and CA125 cutoff thresholds optimizes sensitivity to detect EOC, whereas serial testing with age- and sEGFR-dependent CA125 cutoff thresholds optimizes test specificity, and overall accuracy to discern patients with EOC from women with benign ovarian and non-ovarian gynecologic conditions. The combined use of serologic sEGFR and CA125, thus, has improved utility for screening and diagnosing EOC, which may increase the positive predictive value of a multimodal screening program that incorporates these biomarkers to detect and subsequently differentiate benign from malignant ovarian tumors.

Abstract It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from ∼4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.