Mechanistic insights into cholesterol depletion in an in vivo model of pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a silent global burden with a poor survival rate and highly drug resistant. It is believed that its altered cholesterol metabolism, which results from metabolic changes in PDAC, may influence treatment response. Cholesterol depleting agents, also known as cyclodextrins, provide a novel therapeutic strategy to improve treatment response and potentially overcome drug resistance in PDAC. Using 2-hydroxypropyl-β-cyclodextrin (HPβCD) to deplete cholesterol has shown promising results in in vitro PDAC studies. In our lab, HPβCD alone, and the combination of HPβCD with chemotherapeutic drugs has shown improved treatment response from in vivo studies for triple negative breast cancer and colorectal cancer, respectively. Nevertheless, no study has yet examined the impact of using HPβCD in conjunction with chemotherapy on the cholesterol homeostasis and treatment response of PDAC in vivo. Therefore, the aim of this study was to investigate the effects of adjuvant cholesterol depletion, with HPβCD, on the expression of key proteins in cholesterol metabolism and treatment response from cryopreserved PDAC tumour tissue. A PDAC mice xenograft served as the in vivo model for this study. Mice tumours were subjected to treatments consisting of mono- and combination therapies of Gemcitabine, 5-Fluorouracil, and HPβCD. Following successful completion of treatment regimens, tumours were excised and volume was measured. Whole protein was extracted from snap-frozen PDAC mice tumours and quantified using BCA assay, followed by Western blots to quantify the relative protein expression of key proteins involved in cholesterol homeostasis. 5-Fluorouracil monotherapy and its combination with HPβCD significantly reduced tumor volume to a greater extent than Gemcitabine and HPβCD therapies. ACAT1 expression indicates that Gemcitabine upregulated cholesterol storage, which was mitigated by HPβCD, whereas 5-Fluorouracil did not induce a cellular dependence on cholesterol storage. The combination of HPβCD with both Gemcitabine and 5-Fluorouracil reduced cellular dependence on cholesterol synthesis and uptake, as evidenced by decreased HMGCR and LDLR expression. Notably, HPβCD activated the SREBP2 negative feedback loop without increasing cholesterol uptake or synthesis. Furthermore, HPβCD enhanced cholesterol export via ABCG1, whether utilized alone or in combination. These findings elucidate the complex relationship between cholesterol homeostasis and drug response. Further investigation of cholesterol depletion using HPβCD, either as monotherapy or combination therapy in PDAC, is essential to comprehensively understand the mechanisms of drug resistance.