Treatment resistance is a major clinical challenge in BC management and contributes to poor patient outcomes (35). The results of this study emphasize the important role that cholesterol metabolism plays in BC progression as an adaptive mechanism in response to CDDP treatment. We showed that CDDP alters cholesterol metabolism in luminal A (MCF-7) and TNBC (MDA-MB-231) cell models, and that CDDP together with ATV reduced cell proliferation and cell viability to a greater extent than CDDP alone, especially in MDA-MB-231 cells. In these cells, the combination of CDDP and ATV dramatically altered cellular cholesterol metabolism. These findings indicate that the elevated susceptibility of MDA-MB-231 cells to co-administration of CDDP and ATV, compared to MCF-7 cells, was associated with an increased reliance on CE availability. Moreover, ATV restored CDDP sensitivity in CDDP-resistant MDA-MB-231 cells. Collectively, our data implicate the upregulation of CE storage as an adaptive response that contributes to chemotherapy resistance.
In a recently reported case study, CDDP reduced metastatic legions in heavily pre-treated TNBC when used as monotherapy (36). Despite the initial successful response to platinum-based chemotherapy in many settings, including TNBC, acquirement of resistance occurs in association with many changes in gene expression (37), ultimately resulting in disease relapses. Here, we assessed the role of cholesterol metabolism in the acute and chronic setting using cell culture. Interestingly, we observed that MDA-MB-231 cells were intrinsically more resistant to CDDP than MCF-7 cells, as judged by their metabolic cell viability (Fig. 1). This was the first hint that CDDP possibly acted differentially on cellular metabolism of TNBC cells compared to luminal A BC cells. In this regard, cholesterol metabolism has been targeted in BC treatment as noted by ongoing clinical trials, of which over 40 trials are testing the effects of statins in BC (clinicaltrials.gov). Of these, two are evaluating the benefits of ATV in association with conventional chemotherapy in TNBC patients (NCT03358017; NCT03872388). Nonetheless, whether cholesterol metabolism contributes to the development of chemoresistance towards CDDP in TNBC cells remains unexplored.
In the past decades, many studies have shown that chemotherapeutic drugs can lead to metabolic disorders in patients, including changes in serum lipids and lipoproteins (32, 38). For example, mice treated repeatedly with paclitaxel and CDDP had increased circulating cholesterol levels (39). Similarly, CDDP-resistant ovarian cancer cells exhibit increased expression of key cholesterol metabolism genes, compared to treatment sensitive cells, including sterol regulatory-element binding protein 2, LDLR, and HMGCR, thereby suggesting that upregulated cholesterol metabolism might contribute to CDDP resistance in ovarian cancer (40). While acute CDDP exposure decreased LDLR protein levels in both BC cell lines studied in the present work, a reduction in HMGCR levels was observed only in MDA-MB-231 cells, suggesting the occurrence of cancer cell type-specific changes in cholesterol handling and CE synthesis. Given a trend of increased ACAT-1 expression, concomitantly with higher CE content after CDDP treatment, this enzyme plays an important role in triggering metabolic changes in BC cells, contributing to the high proliferation rate of MDA-MB-231 and different responses to anti-cancer therapy. In fact, elevated CE storage mediated through upregulated ACAT-1 activity may provide growing cells with immediately available building blocks for membrane construction and reduce the need for de novo lipid synthesis (41).
Cancer cells display metabolic reprogramming to better utilize lipids for tumor development and progression (4), targeting these pathways with a combination of low-toxic compounds may become an alternative therapeutic intervention. Emerging evidence suggests that a decrease of CE accumulation via ACAT-1 inhibition significantly improves chemotherapy in melanoma (42) and in prostate cancer cell growth (19, 43). Moreover, reducing fatty acid metabolism by inhibiting fatty acid synthase (FASN), enhanced the response to CDDP and sensitized resistant ovarian and mammary cells to CDDP (44, 45). In BC models, FASN inhibition improved doxorubicin, docetaxel, paclitaxel, and vinorelbine chemotherapy (46, 47). In this study, the reduction of cholesterol esterification in MDA-MB-231 cells following exposure to a combined treatment with CDDP and ATV resulted in the inhibition of all key proteins involved in CE formation (Fig. 6), highlighting the potential significance of improving CDDP response with ATV co-treatment in TNBC.
Multiple mechanisms have been associated with the acquisition of a chemoresistant phenotype by cancer cells, including drug inactivation, drug efflux, apoptosis suppression, increased DNA repair (36). However, the role of CE metabolism in CDDP resistance in BC cells remains unclear. Results presented here suggest that not only de novo cholesterol synthesis but also exogenous cholesterol, delivered through the uptake of LDL, can contribute to increased CE storage and consequently, resistance to CDDP. Hence, this correlates with elevated LDLR accelerating TNBC tumor growth in hyperlipidemic mouse models (7), and elevated plasma cholesterol levels contributing to drug resistance (48). Notably, BC patients with obesity had worse clinical outcomes in response to paclitaxel and carboplatin treatment than non-obese patients (49). This suggests that the dyslipidemia often associated with obesity, including increased circulating levels cholesterol and CE, can play a critical role in the tumor microenvironment, and influence cell behavior and response to CDDP.
We have previously reported that MCF-7 and MDA-MB-231 cells differ in their intracellular handling of fatty acids and in response to palmitate-induced apoptosis (22). Here, we extend these observations and report that these cells also differ in the levels of ACAT-1, LDLR, and HMGCR (Fig. 5A). Moreover, BC cells have distinct CE etiology so that most of the CE supply in TNBC models like MDA-MB-231 requires elevated ACAT-1 activity and overexpression of LDLR while, in MCF-7, cholesterol and CEs are mostly provided by the cholesterol synthesis route, benefiting only from elevated HMGCR activity. Cholesterol homeostasis is fine-tuned and regulated by feedback mechanisms that allow elevated free cholesterol levels to activate ACAT-1 to catalyze its esterification and storage, while simultaneously inhibiting HMGCR (50). Indeed, free cholesterol levels were substantially higher in MDA-MB-231 compared to MCF-7 cells (Figure S7). Also, our findings suggest a shift towards cholesterol production by TNBC cells under cholesterol deprivation, allowing them to recover efficiently from this condition. We speculate that elevated ACAT-1 levels may allow MDA-MB-231 cells to recover more rapidly from stress, such as drug treatment or cholesterol deprivation. As TNBC cells express elevated ACAT-1 levels, which is coupled to a higher growth rate, we speculate that increased amounts of CE generated by ACAT-1 and stored in lipid droplets are then available for hydrolysis to produce cholesterol, reducing the pool of intracellular CE, and sustaining cell proliferation.
The regulation of cholesterol homeostasis in the various BC subtypes and its contribution to resistance against therapy remains to be fully understood. Previous studies conducted in tumor tissue samples reported a positive correlation between CE content and clinicopathological parameters, such as the occurrence of high-grade tumors (51). Indeed, TNBC specimen expressed higher levels of LDLR and ACAT-1 and displayed elevated intratumoral CE levels compared to luminal A BC (51). However, in these studies, HMGCR mRNA levels were comparable between the BC subtypes. Furthermore, previous microarray studies also demonstrated that basal-like BC exhibit elevated ACAT-1 levels (52, 53), thus corroborating our results. Nonetheless, our data point to the fact that ACAT-1 expression is greater in MDA-MB-231 chronically exposed to CDDP (MDACR), which might reflect events that occur in response to anti-cancer treatments. Remarkably, as shown here, ATV re-sensitized MDACR cells to CDDP. Fromigué et al (27) suggested that high doses of ATV increased drug sensitivity through the modulation of matrix metalloprotease 2 in osteosarcoma cells. On the other hand, Guo and collaborators (28) showed that liver cancer cells (Huh-7) became more sensitive to CDDP in the presence of 100 µM ATV. Here, we report a correlation between the modulation of ACAT-1 expression upon exposure to CDDP in combination with ATV in MDA-MB-231 cells. Moreover, we demonstrate the importance of ACAT-1 and cholesterol availability in the acquisition of chemoresistance to CDDP in a TNBC cell model. Considering that drastic downregulation of ACAT-1 correlated with the enhanced anti-tumor activity of CDDP in combination with ATV in MDA-MB-231, one can speculate that restoration of CDDP activity observed in the presence of ATV in CDDP-resistant TNBC also results from ACAT-1 downregulation and reduced CE availability.