Browsing by Subject "ovarian cancer"

Ovarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of ovarian cancer related deaths. Despite the clinical significance of OC omental metastases, the precise molecular mechanisms which drive this phenomenon have not been well characterized, making the resulting aggressive phenotype even more puzzling. Recent evidence has highlighted the importance of metabolic reprograming in driving this tumoral behavior, with OC metastases adapting to utilize nutrients available in the metastatic niche to rapidly proliferate. To better understand the metabolic changes that underlie the aggressive nature of OC, we undertook a broad investigation to better characterize metabolic reprogramming in ovarian cancer, with a focus on omental metastasis and chemoresistance. Firstly, we sought to expand the arsenal of tools used to study OC metabolism. In particular, we were interested in using organoids, self-organizing, expanding 3D cultures derived from stem cells, to study OC. Using tissue derived from patients, these miniaturized models have been shown to recapitulate various aspects of patient physiology and disease phenotypes including genetic profiles and drug sensitivities. However, as metabolism modeling in these 3D cultures remains yet unexplored, we aimed to introduce genetically encoded, fluorescent biosensors as robust tools to interrogate metabolism in this context. In Chapter 2, we detail our investigation in which we transfected plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n=8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC. Secondly, in Chapter 3, we focused on characterizing the role of the pentose phosphate pathway (PPP), a metabolic pathway responsible for producing nucleotide pentose precursors through a nonoxidative series of reactions and the reducing equivalent NADPH through a distinct oxidative branch. Using computational analysis of gene expression data, metabolomics analysis, and biochemical approaches, we observed upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, of metastatic OC cells in the omentum compared to primary OC tumors. We established these increases coincided with increased oxidative stress experienced by OC cells in the omental microenvironment, using both established oxidative stress assays and genetically encoded biosensors; and sought to understand if the PPP was an important cellular mechanism to compensate for this metabolic pressure. Indeed, both shRNA-mediated and pharmacological inhibition of G6PD, the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting this adaptive metabolic dependency is important for OC omental metastasis. This work collectively illustrates the importance of characterizing OC metabolism and supports future efforts to develop tools to more effectively investigate and target aspects of metabolic reprogramming in OC which drive metastasis and chemoresistance.

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MAL (myelin and lymphocyte protein), has been implicated in several malignancies including esophageal, gastric, and cervical cancers. We have demonstrated that the MAL protein is expressed in the normal breast epithelium, and aberrantly expressed in breast cancer. Bisulfite sequencing of the MAL promoter CpG island revealed hypermethylation in breast cancer cell lines and 69% of primary tumors analyzed compared with normal breast epithelial cells. Differential methylation between normal and cancer DNA was confined to the proximal promoter region. In a subset of breast cancer cell lines, promoter methylation correlated with transcriptional silencing that was reversible with the methylation inhibitor decitabine. Furthermore, exogenous expression of MAL in breast cancer cell lines resulted in decreased cell proliferation, motility, reduced cell invasion through Matrigel and suppressed anchorage-independent growth in soft agar. In a cohort of 122 primary breast tumors, immunohistochemical analysis revealed that the MAL protein was an independent predictor of benefit from adjuvant chemotherapy. Moreover, overexpression of MAL in triple-negative MDA-MB-468 and BT20 breast cancer cell lines was sufficient to confer sensitivity to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibition and was associated with reduced phosphatidylinositol-3 kinase (PI3K)/Akt signaling. Immunohistochemistry studies conducted on 144 late-stage serous ovarian cancers showed that MAL expression was a significant predictor of survival. Knockdown of MAL expression in the SKOV8 ovarian cancer cell line reduced cell proliferation and resulted in increased sensitivity to the chemotherapeutic drug carboplatin. Thus, we have identified the MAL gene as a novel epigenetically regulated gene in breast cancer with implications for response to chemotherapy in both breast and ovarian cancer. Furthermore, we have shown that the MAL protein has predictive and prognostic value in breast and ovarian cancers, respectively.