Investigating the mechanism of obesity-associated estrogen receptor negative breast cancer to identify novel agents for chemoprevention

The United States is undergoing an obesity epidemic as nearly 40% of adults are obese. This number is only expected to increase. Evidence over the last two decades demonstrates the obesity public health crisis has devastating impacts on cancer incidence, specifically breast cancer. As a breast cancer risk factor, elevated body mass index (BMI) is associated primarily with postmenopausal, estrogen receptor (ER) positive (ER+) breast cancer. However, BMI does not describe adipose tissue distribution. Evidence suggests not all adipose tissue is "unhealthy", nor is adipose tissue universally associated with breast cancer risk. Now we are learning that central obesity reflects accumulation of a type of adipose tissue that is particularly harmful because it is a strong predictor of pre-menopausal ER negative (ER-) breast cancers: triple negative breast cancer (TNBC) and HER2+ER-. Currently, there are limited pharmacological means that target ER- breast cancer including PARP inhibitors and anti-PD-L 1 inhibitors. Furthermore, TNBC, a subtype lacks the ER, is an aggressive form of breast cancer that affects 10-20% of all patients. These patients would greatly benefit from preventative agents. Selective estrogen receptor modulators (SERMs), like tamoxifen, effectively reduce the risk of ER+ breast cancers by 50%. Unfortunately, SERMS are ineffective against ER- breast cancers. Because the mechanisms of how obesity promotes ER- breast cancer are unknown, thus developing targeted prevention strategies is difficult. Therefore, the objective of this dissertation is to determine the mechanisms of visceral obesity driven ER- breast cancer and use this mechanism to identify chemopreventive compounds.Literature suggests fibroblast growth factor 2 (FGF2) and its main receptor fibroblast growth factor receptor 1 (FGFR1) could be a potential mechanism of obesity-promoted pre-menopausal ER- breast cancer. I previously demonstrated that one ER- mammary epithelial cell line undergoes malignant transformation when treated with factors from visceral adipose tissue (VAT). In addition to the Bernard lab's studies on FGF2-FGFR1 mediated malignant transformation, another group recently identified FGFR1 activation as a primary pathway for obesity-associated progression of ER+ breast cancer after estrogen deprivation. This exciting discovery also implicates FGFR1 signaling as a primary node of breast cancer progression independent of estrogen signaling. Together, this suggests FGFR1 activation in the context of obesity may contribute to both malignant transformation and progression. Therefore, I hypothesize FGF2/FGFR1 activation is a critical mechanism in VAT-associated breast epithelial cell transformation and is a potential target for chemoprevention.Herein, I have demonstrated that VAT and FGF2 transforms ER- breast epithelial cells and this is prevented/attenuated by a selective FGFR1 inhibitor. This revealed FGF2/FGFR1 as a critical signaling mechanism that I utilized to develop a target-based, phenotypic transformation high throughput screen (HTS) to identify chemopreventive compounds that prevent/attenuate FGF2-stimulated transformation. With this assay, fluvastatin was identified as the lead candidate for chemoprevention. Fluvastatin inhibits HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway. Interestingly, there is no known mechanistic connection between FGF2/FGFR1 signaling and the mevalonate pathway. Endeavoring to establish a potential connection, my data revealed that factors from VAT upregulated protein expression of mevalonate pathway enzymes such as HMG-CoA synthase 1 (HMGCS1), farnesyltransferase (FNTA), squalene synthesis (FDFT1), and HRas. Furthermore, a selective FGFR1 inhibitor effectively prevented this VAT-induced upregulation of these enzymes. This suggests that FGF2/FGFR1 influences the mevalonate pathway. Products of the mevalonate pathway include cholesterol and isoprenoids like farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These isoprenoids could be important in transformation as GTP binding proteins (GTPase) like oncogenic Ras require FPP to undergo prenylation, a process that is necessary for oncogenic Ras activation. While previous literature has demonstrated HRas activation stimulates transformation of ER- breast epithelial cells, my data did not show that HRas activation as a critical step in FGF2/VAT stimulated transformation. Further studies are needed to identify exactly how FGF2/FGFR1 and the mevalonate pathways are interconnected. Overall, these studies implicate the mevalonate pathway in FGF2/FGFR1 signaling and suggest that components of the pathway may serve as targets for prevention.