Kenneth P. Nephew

Epigenetic Resensitization to Platinum in Ovarian Cancer

Preclinical studies have shown that hypomethylating agents reverse platinum resistance in ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining study, we tested the clinical and biologic activity of low-dose decitabine administered before carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate (RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood mononuclear cells and tumors. The number of demethylated genes was greater (P < 0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244 genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included cytokine-cytokine receptor interactions, drug transporters, and mitogen-activated protein kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis (P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and HOXA11 in tumors positively correlated with PFS (P < 0.05). Together, the results of this study suggest that low-dose decitabine altered DNA methylation of genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resulting in a high RR and prolonged PFS.

Epithelial ovarian cancer experimental models

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge, and many approaches used to study other solid tumors (for example, lung, colon and breast) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment (TME). This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis and chemoresistance. As these new approaches more accurately recapitulate the complex TME, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.

Epigenetic targeting of ovarian cancer stem cells

Emerging results indicate that cancer stem-like cells contribute to chemoresistance and poor clinical outcomes in many cancers, including ovarian cancer. As epigenetic regulators play a major role in the control of normal stem cell differentiation, epigenetics may offer a useful arena to develop strategies to target cancer stem-like cells. Epigenetic aberrations, especially DNA methylation, silence tumor-suppressor and differentiation-associated genes that regulate the survival of ovarian cancer stem-like cells (OCSC). In this study, we tested the hypothesis that DNA-hypomethylating agents may be able to reset OCSC toward a differentiated phenotype by evaluating the effects of the new DNA methytransferase inhibitor SGI-110 on OCSC phenotype, as defined by expression of the cancer stem-like marker aldehyde dehydrogenase (ALDH). We demonstrated that ALDH(+) ovarian cancer cells possess multiple stem cell characteristics, were highly chemoresistant, and were enriched in xenografts residual after platinum therapy. Low-dose SGI-110 reduced the stem-like properties of ALDH(+) cells, including their tumor-initiating capacity, resensitized these OCSCs to platinum, and induced reexpression of differentiation-associated genes. Maintenance treatment with SGI-110 after carboplatin inhibited OCSC growth, causing global tumor hypomethylation and decreased tumor progression. Our work offers preclinical evidence that epigenome-targeting strategies have the potential to delay tumor progression by reprogramming residual cancer stem-like cells. Furthermore, the results suggest that SGI-110 might be administered in combination with platinum to prevent the development of recurrent and chemoresistant ovarian cancer.

Expression of Estrogen Receptor Coactivators in the Rat Uterus

Abstract Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17β or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17β, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Dose-dependent effects of 4-hydroxytamoxifen, the active metabolite of tamoxifen, on estrogen receptor-α expression in the rat uterus

Tamoxifen, a selective estrogen receptor modulator, has agonist or antagonist activity, depending on the target tissue. The estrogen-like agonist effects of tamoxifen in the uterus are mediated primarily by 4-hydroxytamoxifen (4OH), the major active metabolite. Tamoxifen, 4OH and estradiol-17&bgr; (E2) all bind to estrogen receptors (ER&agr; and ER&bgr;), but with different affinities, suggesting that these ligands are capable of producing differential in vivo effects on the uterus. However, differences in short-term effects of tamoxifen, 4OH and E2 on the uterus have not been compared in the rat in vivo. Thus, we treated adult, ovariectomized rats (225–250u2009g) with vehicle (sesame oil), tamoxifen (1u2009mg/kg body weight), 4OH (0.01, 0.1 or 1.0u2009mg/kg body weight), E2 (40u2009μg/kg body weight), estradiol valerate (a long-lasting estrogen; 40u2009μg/kg body weight) or ICI 182,780 (a pure anti-estrogen; 1u2009mg/kg body weight). Animals were sacrificed at 0, 3, 6, 12 or 24u2009h post-injection, and protein and mRNA levels for ER&agr; and two estrogen-regulated early response genes, c-fos and jun-B, were examined. Administration of E2 and 4OH (1u2009mg/kg body weight dose) resulted in down-regulation of uterine ER&agr; protein in the uterine luminal and glandular epithelium by 6u2009h post-treatment. In contrast, no change in ER&agr; level was observed after treatment with tamoxifen. Rapid (by 3u2009h) and transient increases in c-fos and jun-B mRNA levels were observed after E2 treatment; however, c-fos and jun-B induction by 4OH was highly dose dependent, and higher 4OH doses induced rapid but persistent proto-oncogene expression in vivo. Our results demonstrate that tamoxifen and its major metabolite have differential effects on uterine gene expression, and 4OH is highly estrogenic in the rat uterus.

Strain differences in tamoxifen sensitivity of Sprague-Dawley and Fischer 344 rats.

Why some women are at increased risk for the development of endometrial carcinoma while taking the antiestrogen tamoxifen (Tam) for breast cancer treatment or prevention is unknown. Various strains of rodents display differences in sensitivity to compounds with estrogenic activity, but whether differences in Tam sensitivity exist in rodent strains has not been investigated. In the present study, we investigated whether rat strain differences in reproductive tract sensitivity to Tam and estrogen exist between Fischer 344 (F344) and Sprague Dawley (SD) rats. Immature (21–23 day;n= 6/group), ovariectomized F344 and SD rats were treated with vehicle (control), 17 &bgr;-estradiol (E2) [1×10−6 to 1.0u2009μ g/kg body weight (BW)] or 4-OH tamoxifen (4-OHT) (1×10−4 to 10u2009mg/kg BW) for 2 days and then sacrificed on day 3. Reproductive tracts were collected, weighed, and examined for changes in histomorphology and expression of ER &agr; and nuclear receptor co-regulators (SRC1, p300, CARM1, GRIP1, SPA, REA and Uba3). Treatment with E2 (1×10−5u2009μ g/kg BW) increased (p <0.05) uterine epithelial cell height in F344 but not SD rats, demonstrating increased sensitivity of the F344 strain to E2. Conversely, treatment with 1×10−3u2009mg/kg BW 4-OHT increased (p <0.05) uterine weight and epithelial cell height in SD but not F344 rats, demonstrating that the SD strain is more sensitive to the antiestrogen. Northern and Western blot and immunohistochemical analysis revealed that ER &agr; expression levels in the SD and F344 uterus were not different. Expression of receptor co-regulators was higher in the uterus compared to the vagina regardless of strain and higher CARM1 expression was seen in SD uterus compared to F344 rats. Understanding differences in Tam sensitivity may help us to better understand why some women develop endometrial cancer while taking Tam and be beneficial in treatment decisions for breast cancer patients.

Epigenetics and ovarian cancer.

Epigenetics is a broad term that refers to all stably heritable alterations in gene expression that occur without changes in DNA base sequence. Epigenetic phenomena include deoxycytosine methylation, histone protein modifications, nucleosome position effects on DNA, and gene regulation by noncoding RNA molecules (Fig. 6.1); the overall epigenetic state corresponding with a specific cell phenotype is referred to as an ‘‘epigenome.’’ The Human Genome Project, completed in 2003, has provided a wealth of data regarding the relationship of DNA sequence to human health, and one interesting outcome of that project was the observation that humans possess far fewer genes than previously predicted. That vast underestimation of human genes suggested amuch greater role for phenotype-specific gene regulation by other mechanisms, including epigenetic modifications. Consequently, a Human Epigenome Project, an international public/private consortium, has now been initiated to provide a set of normal, reference epigenomes to permit study of the role of epigenetics in biological processes such as differentiation, proliferation, and various disease states, including cancer. To date, the best-studied epigenetic process is methylation of deoxycytosine, usually located within the dinucleotide CpG, and catalyzed by enzymes known as DNA methyltransferases (DNMTs). DNA methylation is strongly correlated with gene silencing, and in normal cells, heterochromatin and the inactive female X chromosome are extensively methylated. This modification also occurs within repeat elements, where it is believed to silence potentially harmful transposable elements. Aside from heterochromatin and repeats, distinct regions, usually within 50 untranslated regions and the first exons of genes, are normally protected from this modification. It is now well established, however, that these localized ‘‘CpG islands’’ become increasingly hypermethylated during tumor advancement

Stranded Whole Transcriptome RNA-Seq for All RNA Types

Stranded whole transcriptome RNA‐Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to, miRNA (microRNA), piRNA (Piwi‐interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non‐coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA), and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications.

Empirical bayes model comparisons for differential methylation analysis

A number of empirical Bayes models were developed to investigate the differential methylation analysis. However, it is not clear which empirical Bayes model performs best in differential methylation analysis. In this paper, five empirical Bayes models were constructed and applied to the differential methylation analysis of A2780 cells between before and after 1, 3, and 5 round of cisplatin treatment. The log-normal model with the background variance showed the lowest minimized negative log-likelihood. It inferred increasing number of differentially methylated loci from 1 to 3 to 5 rounds of cisplatin treatment on the A2780 cells, which was consistent to cisplatin resistant IC50 data. Among differentially methylated loci selected from each empirical model, three time dependent methylation patterns were defined: stochastic hypomethylation, stochastic hypermethylation, and random methylation. If the empirical Bayes model of the DNA methylation assumed log-normal distribution, both stochastically hypomethylated loci and stochastically hypermethylated loci were enriched with a number of transcription factor binding sites. Almost no TFBS enrichment was observed if the gamma distribution was assumed in the empirical Bayes model. In summary, by comparing the performances of the differential methylation analysis and the TFBS enrichment analysis, log-normal distribution is a better statistical assumption than the gamma distribution in the empirical Bayes model.