Establishing the role of Cholesteryl Ester Transfer Protein (CETP) in breast cancer

Abstract

Hormone responsive breast cancer (BC) is the most common sub-type of BC and relies on steroid hormones for cell proliferation and survival, therefore endocrine therapy, Tamoxifen (TAM), serves as the main therapeutic strategy in treating and preventing the recurrences of hormone receptor positive BC. Despite the use of TAM for over four decades, overcoming drug resistance remains challenging. In our previous research, we investigated the effect of reducing intracellular cholesterol as a possible alternative method to induce cell death in BC cells, due to the fact that steroid hormones are derived from cholesterol and evidently, an increased level of cholesterol is associated with cancer progression. An important protein involved in cholesterol homeostasis is the Cholesteryl Ester Transfer Protein (CETP). CETP maintains cholesterol homeostasis by storing cholesteryl esters (CEs) in lipid droplets. The intracellular accumulation of CEs leads to aggressive cancer development and drug resistance. Therefore, we aimed to investigate whether CETP can possibly be used as a drug-resistance marker in BC. The preliminary results showed that knocking down CETP resulted in increased apoptosis in MCF-7 cells when treated with TAM (by 10-40%) and various other drugs. Furthermore, CETP knock-down with the addition of a cholesterol depleting agent increased apoptosis by 10-fold when compared to the non-transfected MCF-7 cells, possibly due to a decrease in CEs content. Similar results were observed in MDA-MB 231 cells. Therefore, it was concluded that CETP could thus serve as a potential drug-resistance marker in cancer cells, more specifically BC. However, the molecular mechanisms of CETP in the involvement of this resistance have not yet been explored. Therefore, we further looked at several genes that were affected from knocking down CETP in both cell lines using the RT² Profiler™ PCR arrays (Qiagen, Germany; Whitehead Scientific, SA), where each array identifies the expression of 84 genes involved in the human lipoprotein signalling and cholesterol metabolism pathway and 84 genes involved in the human cancer resistance pathway. From the arrays it was found that CETP knock-down, in MCF-7 cells, lowered the expression profiles of genes involved in the cholesterol synthesis and cholesterol uptake pathways, thereby lowering internal cholesterol levels. In addition, there was a shift of gene expression from high levels to low levels in hormone receptors and drug efflux pumps, thus reducing resistance. While in MDA-MB 231 cells, CETP knock-down increased the expression of genes involved in cholesterol efflux and interestingly, there was an increase in estrogen receptor genes, which possibly sensitized these triple negative BC (TNBC) cells to TAM. In addition to the in vitro study, we performed a mice xenograft study, and it was found that the mice injected with CETP knocked down cells had an 86.45% decrease in tumour growth compared to the mice injected with non-transfected cells. Therefore, CETP is a promising drug resistance marker in predicting the effectiveness of cancer treatments and possibly a good target in reducing drug resistance through a decrease in excess cholesterol