Jessica A. Beach

A Collagen-Remodeling Gene Signature Regulated by TGF-β Signaling Is Associated with Metastasis and Poor Survival in Serous Ovarian Cancer

Purpose: To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer. Experimental Design: Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively. Results: We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice. Conclusion: Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target. Clin Cancer Res; 20(3); 711–23. ©2013 AACR.

Succinate dehydrogenase inhibition leads to epithelial-mesenchymal transition and reprogrammed carbon metabolism

BackgroundSuccinate dehydrogenase (SDH) is a mitochondrial metabolic enzyme complex involved in both the electron transport chain and the citric acid cycle. SDH mutations resulting in enzymatic dysfunction have been found to be a predisposing factor in various hereditary cancers. Therefore, SDH has been implicated as a tumor suppressor.ResultsWe identified that dysregulation of SDH components also occurs in serous ovarian cancer, particularly the SDH subunit SDHB. Targeted knockdown of Sdhb in mouse ovarian cancer cells resulted in enhanced proliferation and an epithelial-to-mesenchymal transition (EMT). Bioinformatics analysis revealed that decreased SDHB expression leads to a transcriptional upregulation of genes involved in metabolic networks affecting histone methylation. We confirmed that Sdhb knockdown leads to a hypermethylated epigenome that is sufficient to promote EMT. Metabolically, the loss of Sdhb resulted in reprogrammed carbon source utilization and mitochondrial dysfunction. This altered metabolic state of Sdhb knockdown cells rendered them hypersensitive to energy stress.ConclusionsThese data illustrate how SDH dysfunction alters the epigenetic and metabolic landscape in ovarian cancer. By analyzing the involvement of this enzyme in transcriptional and metabolic networks, we find a metabolic Achilles’ heel that can be exploited therapeutically. Analyses of this type provide an understanding how specific perturbations in cancer metabolism may lead to novel anticancer strategies.

Reversing Platinum Resistance in High-Grade Serous Ovarian Carcinoma: Targeting BRCA and the Homologous Recombination System

Resistance to platinum chemotherapy is one of the main factors driving ovarian cancer mortality, and overcoming platinum resistance is considered one of the greatest challenges in ovarian cancer research. Genetic and functional evidence points to the homologous recombination (HR) DNA repair system, and BRCA1 and BRCA2 in particular, as main determinants of response to platinum therapy. BRCA-mutant ovarian cancers are especially sensitive to platinum, associated with better survival, and amenable to poly ADP ribose polymerase inhibitor treatment. Here, we discuss a therapeutic concept that seeks to disrupt HR capacity via targeting of BRCA1 and BRCA2 functionality in order to reverse platinum resistance in BRCA-proficient high-grade serous ovarian cancers (HGSOC). We review the molecular signaling pathways that converge on BRCA1 and BRCA2, their activation status in ovarian cancer, and therapeutic options to modulate BRCA function. Several recent publications demonstrate efficient chemosensitization of BRCA-proficient cancers by combining targeted therapy with standard platinum-based agents. Due to its inherent genomic heterogeneity, molecularly defined subgroups of HGSOC may require different approaches. We seek to provide an overview of available agents and their potential use to reverse platinum resistance by inhibiting the HR system, either directly or indirectly, by targeting oncogenic activators of HR.

Suboptimal cytoreduction in ovarian carcinoma is associated with molecular pathways characteristic of increased stromal activation

OBJECTIVE Suboptimal cytoreductive surgery in advanced epithelial ovarian cancer (EOC) is associated with poor survival but it is unknown if poor outcome is due to the intrinsic biology of unresectable tumors or insufficient surgical effort resulting in residual tumor-sustaining clones. Our objective was to identify the potential molecular pathway(s) and cell type(s) that may be responsible for suboptimal surgical resection. METHODS By comparing gene expression in optimally and suboptimally cytoreduced patients, we identified a gene network associated with suboptimal cytoreduction and explored the biological processes and cell types associated with this gene network. RESULTS We show that primary tumors from suboptimally cytoreduced patients express molecular signatures that are typically present in a distinct molecular subtype of EOC characterized by increased stromal activation and lymphovascular invasion. Similar molecular pathways are present in EOC metastases, suggesting that primary tumors in suboptimally cytoreduced patients are biologically similar to metastatic tumors. We demonstrate that the suboptimal cytoreduction network genes are enriched in reactive tumor stroma cells rather than malignant tumor cells. CONCLUSION Our data suggest that the success of cytoreductive surgery is dictated by tumor biology, such as extensive stromal reaction and increased invasiveness, which may hinder surgical resection and ultimately lead to poor survival.

Sphingosine kinase 1 is required for TGF-β mediated fibroblastto- myofibroblast differentiation in ovarian cancer.

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

ADAM12 is a prognostic factor associated with an aggressive molecular subtype of high-grade serous ovarian carcinoma.

ADAM metallopeptidase domain 12 (ADAM12) is a promising biomarker because of its low expression in normal tissues and high expression in a variety of human cancers. However, ADAM12 levels in ovarian cancer have not been well characterized. We previously identified ADAM12 as one of the signature genes associated with poor survival in high-grade serous ovarian carcinoma (HGSOC). Here, we sought to determine if high levels of the ADAM12 protein and/or messenger RNA (mRNA) are associated with clinical variables in HGSOC. We show that high protein levels of ADAM12 in banked preoperative sera are associated with shorter progression-free and overall survival. Tumor levels of ADAM12 mRNA were also associated with shorter progression-free and overall survival as well as with lymphatic and vascular invasion, and residual tumor volume following cytoreductive surgery. The majority of genes co-expressed with ADAM12 in HGSOC were transforming growth factor (TGF)β signaling targets that function in collagen remodeling and cell-matrix adhesion. In tumor sections, the ADAM12 protein and mRNA were expressed in epithelial cancer cells and surrounding stromal cells. In vitro data showed that ADAM12 mRNA levels can be increased by TGFβ signaling and direct contact between epithelial and stromal cells. High tumor levels of ADAM12 mRNA were characteristic of the mesenchymal/desmoplastic molecular subtype of HGSOC, which is known to have the poorest prognosis. Thus, ADAM12 may be a useful biomarker of aggressive ovarian cancer for which standard treatment is not effective.

Abstract B09: Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer

Sphingosine kinase-1 (SPHK1) is an enzyme that catalyzes the formation of the prosurvival second messenger sphingosine-1-phosphate (S1P) from the proapoptotic lipid sphingosine. The balance between sphingosine and S1P forms a sphingolipid rheostat that is primed for cell death when the balance shifts towards sphingosine/ceramide or to cell survival when S1P levels are increased. Elevated levels of S1P have been observed in several cancers, including ovarian cancer where it is elevated in both the ascites and serum of patients. Correspondingly, high expression of SPHK1 has also been described in multiple cancer types and has been linked to disease progression. Here we report that SPHK1 is overexpressed in a subset of epithelial ovarian cancers (EOC) and correlates with poor progression-free survival. Overexpression and knockdown of SPHK1 in multiple human EOC cell lines modulates in vitro cell proliferation, anchorage-independent growth, and chemosensitivity. In mouse xenograft studies, intraperitoneal administration of a SPHK1-specific inhibitor (SKI-5c) decreases tumor size, indicating that SPHK1 may be a potential therapeutic target in EOC. S1P is a secreted factor that has been shown to influence the tumor microenvironment of other cancers; however its role in EOC has not been fully established. Using a co-culture model, we show that overexpression of SPHK1 in EOC cells stimulates the transition of normal ovarian stromal fibroblasts to myofibroblasts, and enhances stromal SPHK1 expression. In a reciprocal manner, overexpression of SPHK1 in stromal fibroblast results in increased expression of MMP2 and MMP9 in EOC cells, which may contribute to a more invasive phenotype. Interestingly, stromal SPHK1 expression also enhanced expression of SPHK1 and the S1P receptor (S1PR1) in the EOC cells. Biostatistical analysis suggests a significant correlation between SPHK1 expression in EOC cells and the expression of several extracellular matrix genes (FN1, POSTN, VCAN), which have been shown to have role in the EOC tumor microenvironment. Overall, these results suggest that SPHK1 is a critical regulator of ovarian tumor cell proliferation and survival, and a mediator of tumor-stroma interaction. Citation Format: Jessica A. Beach, Paul-Joseph Aspuria, Dong-Joo Cheon, Maricel C. Gozo, Beth Y. Karlan, Sandra Orsulic. Sphingosine kinase 1 (SPHK1) is a novel mediator of tumor-stroma interaction in ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B09. doi:10.1158/1538-7445.CHTME14-B09

Suboptimal Cytoreduction in Ovarian Carcinoma Is Associated With Molecular Pathways Characteristic of Increased Stromal Activation

Author(s): Liu, Zhenqiu; Beach, Jessica A; Agadjanian, Hasmik; Jia, Dongyu; Aspuria, Paul-Joseph; Karlan, Beth Y; Orsulic, Sandra

Abstract B64: Suboptimal cytoreduction in ovarian carcinoma is associated with molecular pathways characteristic of increased stromal activation.

Suboptimal cytoreductive surgery in advanced epithelial ovarian cancer (EOC) is associated with poor survival but it is unknown if poor outcome is due to the intrinsic biology of unresectable tumors or insufficient surgical effort resulting in residual tumor-sustaining clones. Our objective was to identify the potential molecular pathway(s) and cell type(s) that may be responsible for suboptimal surgical resection. By comparing gene expression in optimally and suboptimally cytoreduced patients, we identified a gene network associated with suboptimal cytoreduction and explored the biological processes and cell types associated with this gene network. We show that primary tumors from suboptimally cytoreduced patients express molecular signatures that are typically present in a distinct molecular subtype of EOC characterized by increased stromal activation and lymphovascular invasion. Similar molecular pathways are present in EOC metastases, suggesting that primary tumors in suboptimally cytoreduced patients are biologically similar to metastatic tumors. We demonstrate that the suboptimal cytoreduction network genes are enriched in reactive tumor stroma cells rather than malignant tumor cells. Our data suggest that the success of cytoreductive surgery is dictated by tumor biology, such as extensive stromal reaction and increased invasiveness, which may hinder surgical resection and ultimately lead to poor survival. Citation Format: Zhenqiu Liu, Jessica A. Beach, Hasmik Agadjanian, Dongyu Jia, Paul-Joseph Aspuria, Beth Y. Karlan, Sandra Orsulic. Suboptimal cytoreduction in ovarian carcinoma is associated with molecular pathways characteristic of increased stromal activation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B64.

Abstract A39: COL11A1 is a regulator of tumor microenvironment and a biomarker of metastatic progression

As major constituents of the extracellular matrix (ECM), collagens affect numerous biological processes including cell differentiation, proliferation, migration, and tumorigenesis. Collagen type XI alpha 1 (COL11A1) encodes one of the alpha chains of type XI minor fibrillar collagen, which is normally expressed in cartilage. We and others have detected aberrant expression of COL11A1 in several types of cancer. Increased levels of COL11A1 are associated with more invasive and metastatic tumors, abundant cancer-associated desmoplastic stroma, and poor clinical outcome. However, the functional significance of COL11A1 expression in cancer remains poorly understood. Previously, we identified COL11A1 as one of ten signature genes associated with poor survival in serous ovarian cancer. Furthermore, we identified COL11A1 as the most significantly upregulated metastasis gene in matched pairs of primary and metastatic tumors. In situ hybridization and immunohistochemistry analyses showed that COL11A1 expression patterns correlate with disease progression in clinical samples; with low, intermediate, and high levels of COL11A1 observed in primary tumors, concurrent metastases, and recurrent/persistent metastases, respectively. COL11A1 was mainly expressed in the intra/peri-tumoral stromal cells. Endothelial cells and other stromal cells >1 mm from the tumor epithelial cells were negative for COL11A1. Although COL11A1 expression was predominantly confined to stromal cells, rare clusters of tumor epithelial cells were also positive. Taken together, these data suggest that COL11A1 is a biomarker of metastatic progression in serous ovarian cancer and a biomarker of cancer-associated desmoplastic stroma. Functionally, we hypothesize that COL11A1 expression may contribute to poor patient survival through several mechanisms. Knockdown of COL11A1 in the human ovarian cancer cell line A2780 resulted in a significant decrease in cell migration in vitro, and diminished invasion and metastasis in mouse xenografts. In the clinical setting, the development of chemoresistance is one of the most significant obstacles to effective therapy. To this end, we showed that expression of COL11A1 in cisplatin resistant A2780 cells is 100-fold higher in comparison to the cisplatin sensitive parental cell line. In addition, we observed a significant decrease in the ALDH+ cancer stem cell (CSC) population after COL11A1 knockdown in A2780 cells, implying that COL11A1 may contribute to cisplatin resistance through modulation of the CSC population. Our current research focuses on the role and mechanism by which COL11A1 expressed by cancer-associated stroma influence cisplatin resistance and the CSC population. At this meeting, we will present our most current data on COL11A1 as a critical player in chemoresistance and metastasis in ovarian cancer. Citation Format: Dong-Joo Cheon, Jessica Beach, Ann Walts, Beth Karlan, Sandra Orsulic. COL11A1 is a regulator of tumor microenvironment and a biomarker of metastatic progression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A39. doi:10.1158/1538-7445.CHTME14-A39