Cholesterol metabolism in breast cancer: Prognostic factors and optimizing treatment

AbstractBreast cancer (BC) is the most common cancer among women in Sweden and worldwide. Adjuvant endocrine therapies are effective, yet 20-30% patients experience disease relapse. Altered cholesterol metabolism is an emerging hallmark of breast cancer proliferation and endocrine therapy resistance. Studies have shown that the use of cholesterol-lowering medications (statins) reduces the risk of disease recurrence in a subset of patients. However, the patterns of breast cancer recurrence remains poorly understood, and treatment predictive biomarkers are needed. 27-hydroxycholesterol (27HC), an oxysterol synthesized from cholesterol by the enzyme CYP27A1, is a selective estrogen receptor (ER) modulator. It has been shown that 27HC is an important mediator that links cholesterol and breast cancer pathology. Aims: The main aims of this thesis were i) to explore the molecular features associated with the heterogeneous response of BC cells to statin treatment alone or in combination with endocrine therapy, ii) investigate the patterns of BC recurrence in patients with post-diagnosis statin use, and iii) characterize CYP27A1 expression in primary BC in relation to tumor pathological features and prognosis. Methods: In papers I and V, BC cell lines were treated with statins and/or endocrine treatments to study its impact on cell growth, lipid metabolism, endocrine signaling, and other metabolic pathways using a combination of global transcriptional profiling and molecular assays. In paper II, the prognostic impact of statin use on disease recurrence was investigated in the Malmö Diet and Cancer Study Cohort. Disease recurrence rates were compared between post-diagnosis statin users versus non users using Cox regression models to compute crude and adjusted hazard ratios. In paper III and IV, the prognostic impact of CYP27A1 expression, as measured by immunohistochemistry and/or RNAscope, was evaluated in three independent cohorts of BC patients. In paper IV, the impact of 27HC on BC cell proliferation was measured under lipid depleted conditions.Results: Statin treatment induced a dose- and time-dependent accumulation of intracellular lipid droplets and significantly upregulated the expression of Stearoyl-CoA desaturase—a key regulator of lipid synthesis in statin-insensitive cell lines. Statin use was associated with a reduced risk of distant BC recurrence, but no association between loco-regional recurrence was found. CYP27A1 is a prognostic biomarker with differential impact based on patients’ menopausal status. High CYP27A1 protein expression was independently associated with aggressive tumor characteristics and predicted for inferior overall survival for postmenopausal patients with ER+ BC. Elevated expression of CYP27A1, both at the protein and transcript levels, was associated with high histological grade and high Ki67 expression and was independently prognostic for superior survival outcomes in premenopausal BC patients with node-negative disease. BC cells have heterogeneous response to statin and endocrine combination therapy. Long-term atorvastatin treatment upregulated ER expression in tamoxifen-resistant MCF7 cells while the expression of cyclin D1, E cadherin, and Snail were downregulated in endocrine therapy-resistant MCF7 cells treated with atorvastatin. The gene expression of CYP27A1 was downregulated upon addition of atorvastatin in long-term estrogen-deprived MCF7 cells.Conclusions: Taken together, our results indicate that statins induce heterogeneous anti-proliferative and transcriptional activity in BC cell lines. Statins reduce distant disease recurrence in a subgroup of BC patients. High CYP27A1 is a poor prognostic marker in post-menopausal BC patients. Long term statin and estrogen deprivation reduced CYP27A1 expression in MCF7 cell line. Further studies to assess the clinical utility of CYP27A1 as a treatment predictive marker for statin use are warranted