To characterize molecular changes accompanying the stepwise progression to breast cancer and to identify functional target pathways, we performed miRNA and RNA sequencing using MCF10A cell lines based model system that replicates the multi-step progression involving normal, preneoplastic, ductal carcinoma in situ, and invasive carcinoma cells, where the carcinoma most resemble the basal-like subgroup of human breast cancers. These analyses suggest that 70% of miRNA alterations occurred during the initial progression from normal to a preneoplastic stage. Most of these early changes reflected a global upregulation of miRNAs. This was consistent with a global increase in the miRNA-processing enzyme DICER, which was upregulated as a direct result of loss of miRNA let-7b-5p. Several oncogenic and tumor suppressor pathways were also found to change early, prior to histologic stigmata of cancer. Our finding that most genomic changes in the progression to basal-like breast cancer occurred in the earliest stages of histologic progression has implications for breast cancer prevention and selection of appropriate control tissues in molecular studies. Furthermore, in support of a functional significance of let-7b-5p loss, we found its low levels to predict poor disease-free survival and overall survival in breast cancer patients.

Background Although targeting of the cholesterol pathway by statins prevents breast cancer development in mouse models, efficacy is not absolute. Therefore, the goal of this study is to investigate if the upregulation in the cholesterol biosynthesis pathway genes associates with response to statin chemoprevention and may potentially be used as response biomarkers. Methods Expression of cholesterol biosynthesis pathway genes was initially derived from the RNA sequencing of MCF10A cell line- based breast cancer progression model system and subsequently validated by quantitative PCR assay. Response to fluvastatin was assessed in vitro using the MCF10A cell line model system, including a statin resistant cell line that was generated (MCF10.AT1-R), and measured using colony forming assays. In vivo efficacy of statin for chemoprevention was assessed in the SV40C3 TAg mouse model. Mammary tumors were identified by histologic analysis of the mammary glands. Mammary glands without histologic evidence of high-grade lesions (in situ and/or invasive carcinoma) were considered responsive to statin treatment. Results We found more than 70% of a published multi-gene fluvastatin resistance signature to be significantly upregulated during breast cancer progression and inversely correlated with statin inhibition of cellular growth and proliferation. This inherent statin resistance gene signature was also largely shared with the signature of acquired resistance to fluvastatin in MCF10.AT1-R cell line model of acquired statin resistance. These inherent resistance genes and genes exclusive to acquired statin resistance map to steroid-, and terpenoid backbone- biosynthesis pathway. We found upregulation of ~ 80% of cholesterol biosynthesis pathway genes in the tumor bearing mammary glands of SV40 C3TAg transgenic mouse model of TNBC, suggesting the involvement of cholesterol biosynthesis pathway in resistance to statin chemoprevention in vivo. A panel of 13-genes from the pathway significantly associated with response to statin treatment, as did the expression level of HMGCR alone in a mouse model of breast cancer suggesting their utility to predict the efficacy of statin chemoprevention. Conclusions High basal level, or restorative upregulation, in the cholesterol biosynthesis pathway genes appear to be strongly associated with resistance to statin chemoprevention for breast cancer and may serve as a biomarker to tailor statin treatment to individuals who are most likely to benefit.

Purpose Primary prevention of hormonally insensitive breast cancers remains an important clinical need and repurposing existing low-toxicity drugs represents a low-cost, efficient strategy for meeting this goal. This study targeted the cholesterol pathway using fluvastatin, a cholesterol-lowering drug, and aspirin, an AMPK activator that acts as a brake in the cholesterol pathway, in a transgenic mouse model of triple-negative breast cancer (TNBC). Methods Using SV40C3 TAg mice, the efficacy and mechanism of fluvastatin, aspirin, or both in combination were compared with vehicle alone. Results Sixteen-weeks of fluvastatin treatment resulted in significant delay in onset of tumors (20 weeks vs. 16.8 weeks in vehicle treatment, p = 0.01) and inhibited tumor incidence and tumor multiplicity by 50% relative to the vehicle control. In animals that developed tumors, fluvastatin treatment inhibited tumor weight by 75% relative to vehicle control. Aspirin alone did not significantly affect tumor latency, tumor incidence or tumor burden compared to vehicle control. Fluvastatin and aspirin in combination delayed the onset of tumors but failed to inhibit tumor incidence and tumor multiplicity. The growth-inhibitory effects of fluvastatin were mediated through increased FAS/FASL mediated apoptotic cell death that was characterized by increased cleaved PARP and driven in part by depletion of an isoprenoid, geranyl geranyl pyrophosphate (GGPP). Conclusions In line with NCI’s emphasis to repurpose low-toxicity drugs for prevention of cancer, fluvastatin was effective for prevention of TNBC and warrants further clinical testing. Aspirin did not provide chemopreventive benefit.

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Introduction: Primary prevention of breast cancer that is effective at preventing both HR- and HR+ tumors, remains an important clinical need and repurposing of existing low toxicity drugs represents a low cost, efficient strategy for meeting this goal. Cholesterol pathway activation is associated with poor prognosis across all subtypes of breast cancer patients. Relevant to breast cancer prevention, the breast epithelium in women with atypical hyperplasia, a known high risk state, is known to have increase in cholesterol levels and an increase in oxidative products of cholesterol. Additionally, cholesterol pathway genes such as HMG Co A reductase (HMGCR) are deregulated in transformed cells and drive the initiation and progression of breast cancer. These observations suggest a role for the cholesterol pathway early in tumorigenesis, thus making it an attractive target for prevention. Methods: Using SV40C3 tag mice, a transgenic mouse model of TNBC, we targeted the cholesterol pathway by fluvastatin. Fluvastatin is a cholesterol lowering drug that blocks the cholesterol pathway activation through inhibition of HMGCR, a rate limiting enzyme of the pathway. Ten mice each were treated with fluvastatin (10mg/kg body weight/ day) alone, in combination with aspirin, AMPK activator, or vehicle control for 16 weeks starting at the age of about 6 weeks. Starting at the age of 10 weeks, mice were regularly examined for the presence of mammary tumors and any palpable mass of 0.3mm or bigger was considered as a lesion. At the end of 16 weeks of treatment, all mice were euthanized and mammary glands were excised, weighed and divided in 2 parts- one was fixed in formalin for any histochemical analyses and the other half was snap frozen for RNA extractions. Results: Fluvastatin treatment for 16 weeks reduced the tumor incidence and average tumor burden by 50%. Additionally, in animals that developed tumors, fluvastatin delayed the onset of palpable tumors by 2.5 weeks and inhibited tumor size, as indicated by a 4- fold lesser tumor weight in the fluvastatin treated group relative to vehicle control group. To our surprise, the combo group (fluvastatin and aspirin) did not inhibit tumor incidence or size. We found the growth inhibitory effects of fluvastatin to be mediated by increased programmed cell death. Conclusions: In line with NCI's emphasis to repurpose low toxicity drugs for prevention of cancer, our pre-clinical data supports efforts to test the efficacy of fluvastatin for prevention of TNBC in clinical trials. Citation Format: Anjana Bhardwaj, Matthew D. Embury, Raniv D. Rojo, Constance Albarracin, Isabelle Bedrosian. Fluvastatin inhibits the development of breast cancer in SV40C3Tag mouse model of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 20.