Alison M. Karst

Transformation of the Fallopian Tube Secretory Epithelium Leads to High-Grade Serous Ovarian Cancer in Brca;Tp53;Pten Models

High-grade serous ovarian carcinoma presents significant clinical and therapeutic challenges. Although the traditional model of carcinogenesis has focused on the ovary as a tumor initiation site, recent studies suggest that there may be additional sites of origin outside the ovary, namely the secretory cells of the fallopian tube. Our study demonstrates that high-grade serous tumors can originate in fallopian tubal secretory epithelial cells and also establishes serous tubal intraepithelial carcinoma as the precursor lesion to high-grade serous ovarian and peritoneal carcinomas in animal models targeting the Brca, Tp53, and Pten genes. These findings offer an avenue to address clinically important questions that are critical for cancer prevention and early detection in women carrying BRCA1 and BRCA2 mutations.

Ovarian Cancer Pathogenesis: A Model in Evolution

Ovarian cancer is a deadly disease for which there is no effective means of early detection. Ovarian carcinomas comprise a diverse group of neoplasms, exhibiting a wide range of morphological characteristics, clinical manifestations, genetic alterations, and tumor behaviors. This high degree of heterogeneity presents a major clinical challenge in both diagnosing and treating ovarian cancer. Furthermore, the early events leading to ovarian carcinoma development are poorly understood, thus complicating efforts to develop screening modalities for this disease. Here, we provide an overview of the current models of ovarian cancer pathogenesis, highlighting recent findings implicating the fallopian tube fimbria as a possible site of origin of ovarian carcinomas. The ovarian cancer model will continue to evolve as we learn more about the genetics and etiology of this disease.

Modeling high-grade serous ovarian carcinogenesis from the fallopian tube

High-grade serous ovarian carcinoma (HGSOC) is a lethal disease for which improved screening and treatment strategies are urgently needed. Progress in these areas is impeded by our poor understanding of HGSOC pathogenesis. Most ovarian cancer research is based on the hypothesis that HGSOC arises from ovarian surface epithelial cells. However, recent studies suggest that >50% of high-grade serous carcinomas involving the ovary likely arise from fallopian tube epithelium. Therefore, limiting HGSOC research to modeling based on ovarian surface epithelium alone is inadequate. To address the need for a fallopian tube–based model of HGSOC, we have developed a system for studying human fallopian tube secretory epithelial cell (FTSEC) transformation. Our model is based on (i) immortalization of FTSECs isolated from primary samples of normal, nondiseased human fallopian tubes, (ii) transformation of FTSECs with defined genetic elements, and (iii) xenograft-based tumorigenic assays. We use our model to show that FTSECs immortalized with human telomerase reverse transcriptase (hTERT) plus SV40 large T and small T antigens are transformed by either oncogenic Ras (H-RasV12) or c-Myc expression, leading to increased proliferation, clonogenicity, and anchorage-independent growth. Additionally, we demonstrate that FTSECs remain susceptible to c-Myc–mediated transformation in the absence of viral oncoproteins, by replacing SV40 large T and small T antigens with sh-p53, mutant CDK4 (CDK4R24C), and sh-PP2A-B56γ. Importantly, all transformed FTSECs gave rise to high-grade Müllerian carcinomas that were grossly, histologically, immunophenotypically, and genomically similar to human HGSOC. With this model, we will now be able to assess the transformative effects of specific genetic alterations on FTSECs in order to characterize their respective roles in HGSOC development.

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4

Tumor-penetrating siRNA nanocomplexes credential ID4 as a therapeutic oncogene target in human ovarian cancer. Nanotechnology Sets Sights on Ovarian Tumors In the world of anticancer research, targeting tumor cells is one challenge; penetrating the cells to deliver therapeutics is another. The combination of specific targeting and efficient delivery is the clinical holy grail, wherein optimization of this approach could lead to highly effective cancer therapy in humans. Ren et al. have now developed a nanotechnology platform that allows for just that: targeted intracellular delivery of RNA-based therapeutics to ovarian cancer cells, which halts the oncogenic activity of a potent gene, in this case ID4. In a screen of overexpressed and essential genes in human ovarian cancer, the authors first identified a potential oncogene, ID4. They then confirmed ID4 tumorigenicity and mechanism in vitro in cell lines. After confirming that ID4 was an oncogene, Ren et al. reasoned that “silencing” the gene using small interfering RNA (siRNA) would prevent tumor growth in vivo. The trick was to make sure the siRNA could cross the cell membrane to exert its silencing effects. To accomplish this, the authors designed a tumor-penetrating nanocomplex (TPN) that could not only bind a protein overexpressed on the surface of human cancer cells but also pass through the membrane via a cell-penetrating peptide. Once inside the cells, the TPN could release the siRNA directed against ID4. Tumor homing was confirmed in mouse models of human melanoma and ovarian cancer. In mice harboring subcutaneous ovarian tumors, TPN/siRNA decreased ID4 expression by up to 90% and suppressed tumor growth by 82%. In mice bearing disseminated intra-abdominal tumors, TPN/siRNA allowed 80% of the animals to live 60 or more days. Control treatments did not prevent tumor growth in either study, and the TPN/siRNA therapy did not elicit any immunogenic side effects. Locked and loaded with siRNA, these TPNs are ready to target and kill cancer cells. The authors envision this to be a platform for credentialing oncogenes and for validating RNA interference in preclinical models before development of therapeutics. However, before moving this TPN/siRNA approach to patients, some additional preclinical optimization is necessary, including pharmacokinetics, testing in human cancer models, and increasing siRNA efficiency at lower doses. The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) that comprised small interfering RNA (siRNA) complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We used TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor–bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

PUMA expression is significantly reduced in human cutaneous melanomas

Cutaneous malignant melanoma is an aggressive form of skin cancer, characterized by strong chemoresistance and poor patient prognosis. The molecular mechanisms underlying its resistance to chemotherapy remain unclear but are speculated to involve the dysregulation of apoptotic pathways. In this study, we sought to determine whether PUMA (p53 upregulated modulator of apoptosis) contributes to human melanoma formation, tumor progression, and survival. We used tissue microarray and immunohistochemistry to examine PUMA expression in 107 primary melanomas, 51 metastatic melanomas, and 64 dysplastic nevi. Here we report that PUMA expression is significantly weaker in primary melanomas compared to dysplastic nevi (P<0.0001), and is further reduced in metastatic melanomas compared to primary tumors (P=0.001). We show that weak PUMA expression in melanoma correlates with poorer overall and disease-specific 5-year survival (P<0.005 and P<0.001, respectively) of melanoma patients and that PUMA expression in tumor tissue is an independent predictor of both overall and disease-specific 5-year survival (P=0.05). Additionally, we show that exogenous PUMA expression in human melanoma cell lines (both wild type and mutant p53) results in significant apoptotic cell death. Our results suggest that PUMA expression may be an important prognostic marker for human melanoma and that adenoviral delivery of PUMA sensitizes melanoma cells to apoptosis.

Cyclin E1 deregulation occurs early in secretory cell transformation to promote formation of fallopian tube derived high-grade serous ovarian cancers.

The fallopian tube is now generally considered the dominant site of origin for high-grade serous ovarian carcinoma. However, the molecular pathogenesis of fallopian tube-derived serous carcinomas is poorly understood and there are few experimental studies examining the transformation of human fallopian tube cells. Prompted by recent genomic analyses that identified cyclin E1 (CCNE1) gene amplification as a candidate oncogenic driver in high-grade serous ovarian carcinoma, we evaluated the functional role of cyclin E1 in serous carcinogenesis. Cyclin E1 was expressed in early- and late-stage human tumor samples. In primary human fallopian tube secretory epithelial cells, cyclin E1 expression imparted malignant characteristics to untransformed cells if p53 was compromised, promoting an accumulation of DNA damage and altered transcription of DNA damage response genes related to DNA replication stress. Together, our findings corroborate the hypothesis that cyclin E1 dysregulation acts to drive malignant transformation in fallopian tube secretory cells that are the site of origin of high-grade serous ovarian carcinomas.

Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance.

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Nuclear factor kappa B subunit p50 promotes melanoma angiogenesis by upregulating interleukin‐6 expression

Nuclear factor kappa B (NF‐κB) signaling is deregulated in many tumor types, resulting in aberrant expression and/or activation of NF‐κB transcriptional complexes. We have previously reported that nuclear expression of the NF‐κB subunit p50 is strongly correlated with melanoma progression and poor 5‐year patient survival. In this study, we used cDNA microarray to analyze the gene expression profiles of melanoma cells overexpressing NF‐κB p50. We found that NF‐κB p50 expression strongly induced interleukin‐6 (IL‐6) upregulation in melanoma cells at both the transcriptional and translational levels and that IL‐6 production by melanoma cells enhanced the growth of endothelial cells in vitro. Expression of activating transcription factor 3 (ATF3), a negative regulator of IL‐6 gene transcription, inhibited p50‐mediated IL‐6 upregulation. Knockdown of p50 expression using lentiviral‐based shRNA abrogated IL‐6 induction in melanoma cells and inhibited its effects on endothelial cell growth. Finally, we used an in vivo matrigel plug assay to show that NF‐κB p50 overexpression promotes angiogenesis, while silencing NF‐κB p50 inhibits blood vessel formation. Our results demonstrate for the first time that the NF‐κB p50 subunit mediates melanoma angiogenesis by specifically upregulating IL‐6, highlighting a novel and important role for the NF‐κB p50/IL‐6 signaling axis in melanoma progression.

Role of p53 Up-regulated Modulator of Apoptosis and Phosphorylated Akt in Melanoma Cell Growth, Apoptosis, and Patient Survival

Malignant melanoma is an aggressive and chemoresistant form of skin cancer characterized by rapid metastasis and poor patient prognosis. The development of innovative therapies with improved efficacy is critical to treatment of this disease. Here, we show that aberrant expression of two proteins, p53 up-regulated modulator of apoptosis (PUMA) and phosphorylated Akt (p-Akt), is associated with poor patient survival. Using tissue microarray analysis, we found that patients exhibiting both weak PUMA expression and strong p-Akt expression in their melanoma tumor tissue had significantly worse 5-year survival than patients with either weak PUMA or strong p-Akt expression alone (P < 0.001). Strikingly, no patients exhibiting strong PUMA expression and weak p-Akt expression in primary tumor tissue died within 5 years of diagnosis. We propose a two-pronged therapeutic strategy of (a) boosting PUMA expression and (b) inhibiting Akt phosphorylation in melanoma tumor tissue. Here, we report that a recombinant adenovirus containing human PUMA cDNA (ad-PUMA) efficiently inhibits human melanoma cell survival in vitro, rapidly induces apoptosis, and dramatically suppresses human melanoma tumor growth in a severe combined immunodeficient mouse xenograft model. In melanoma cells strongly expressing p-Akt, we show that Akt/protein kinase B signaling inhibitor-2 (API-2; a small-molecule Akt inhibitor) reduces cell survival in a dose- and time-dependent manner and enhances ad-PUMA-mediated growth inhibition of melanoma cells. Finally, we show that, by combining ad-PUMA and API-2 treatments, human melanoma tumor growth can be inhibited by >80% in vivo compared with controls. Our results suggest that a strategy to correct dysregulated PUMA and p-Akt expression in malignant melanoma may be an effective therapeutic option.

Primary culture and immortalization of human fallopian tube secretory epithelial cells

Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages.