TNBC is the most malignance type of breast cancer with high tolerance, high metastasis rate, and high recurrence rate, and is still lack of effective treatment. With the advances in cancer research, the role of cholesterol in tumorigenesis is getting more and more attention. Compared with ER+ breast cancers, TNBC had relatively high cholesterol content and genes related to cholesterol synthesis pathway based on 2000 patient samples35. Statins, which inhibit HMGCR to suppress cholesterol synthesis, were reported to inhibit TNBC proliferation, especially in TP53 mutant cells36. Statins can induce TNBC apoptosis, ferroptosis and autophagy to limit the proliferation and metastasis of TNBC36–38. Mechanistical study implied that cholesterol can act as an endogenous ligand of the estrogen-related receptor alpha (ERRα) which promotes metabolic reprogramming to augment aerobic glycolysis and NADPH levels39.
β-CD and their derivatives were mainly used as drug carriers. Among them, HP-β-CD is able to effectively modulate the abnormal accumulation of cholesterol caused by abnormal intracellular trafficking in Niemann-Pick Type C1 disease, a lysosomal storage disorder40. However, the role and underlying mechanisms of HP-β-CD on TNBC were unclear. Herein, we demonstrated that HP-β-CD is able to inhibit the growth and proliferation of TNBC by reducing intracellular cholesterol levels in vitro. Consistent with it, the cholesterol content in tumor tissue was decreased, while ABCA1 and ABCG1 expression and circulating HDL-C level were elevated, implying that HP-β-CD enhanced the reverse cholesterol transport in mice. We also examined that HP-β-CD did not affect the expression of HMGCR, SREBP-1c and SREBP2, suggesting HP-β-CD had no effect on cholesterol synthesis.
HP-β-CD also suppressed expression of Ki-67, cell proliferation marker; BCL-2, apoptosis inhibitor, and CCND2, cell cycle regulatory protein in tumor tissues. Consistent with the reduced genes expression above, HP-β-CD induced more cancer cells arrested in G0/G1 phase. Although it is not clear how these genes are regulated, it is reasonable to believe, based on the results of experiments in cells and mice, that changes in these genes are strongly associated with cholesterol lowering. Interestingly, we also demonstrated that HP-β-CD inhibited TGF-β-mediated EMT and tumor metastasis to the liver and lungs. The similar results were observed in melanoma metastasis model mice. The number of black nodules formed in the lungs was significantly lower in the HP-β-CD administration group, suggesting that there may be a broad spectrum of tumor suppressive effects of HP-β-CD.
Immunotherapy plays an important role in both scientific research and the clinic. Ma et al. demonstrated that cholesterol accumulation promotes exhaustion of CD8+ T cells and enhances the expression of immune checkpoint molecules through ER stress and promotes an immunosuppressive environment, which in turn promotes tumor development20. In the current study, we determined HP-β-CD significantly elevated the infiltrated CD3+ T cells in the TME. Further analysis demonstrates that HP-β-CD treatment increased the number of CD8+ T cells and decreased the number of PD1+CD8+ T cells in the TME compared with the control group. The total T cell population was also upregulated by HP-β-CD in peripheral blood, lymph node and spleen. Interestingly, IFN-β but not IFN-γ was significantly increased by HP-β-CD, implying the tumor-killing effect of CD8+ T cells was enhanced by HP-β-CD. In line with previous study, we demonstrated that HP-β-CD could attenuate ER stress and expression of immune checkpoint molecules. We speculate that HP-β-CD improves the exhaustion of immune cells caused by the excessive accumulation of cholesterol, and simultaneously improves the whole-body immune level by regulating the proliferation and differentiation of T cells, thus increasing the recruitment of T cells to the TME and achieving a tumor suppressive effect. Indeed, HP-β-CD-increased the number of CD3+ and CD8+ T cells in tumors was blocked in high cholesterol diet-fed mice.
Tumor associated macrophages are recruited to the TME mainly through the CCL2-CCR2 signaling pathway33. An increasing number of studies have shown that TAMs have a series of functions including supporting the proliferation, invasion, metastasis of tumor cells and promoting tumor development, which are highly correlated with the poor prognosis of cancer patients41. We found that the number of TAMs was significantly reduced, and the expression of chemokines CCL2 and CCL5 was significantly inhibited by HP-β-CD treatment. Previous studies have shown that TNF-α can induce CCL2 expression through the MAPK signaling pathway42. We determined that the levels of TNF-α, CCL2, p-NF-κB and p-p38MAPK were all significantly decreased after HP-β-CD treatment, and the above results suggest that HP-β-CD may also exert antitumor effects by decreasing TNF-α-p38MAPK signaling pathway, thereby inhibiting the CCL2-CCR2 signaling pathway to reduce the recruitment of TAMs to the tumor microenvironment.
In conclusion, we determined that HP-β-CD can inhibit TNBC proliferation and migration by reducing intracellular cholesterol levels in tumor cells. Additionally, we determined that HP-β-CD reduced cholesterol level by inducing ABCA1 and ABCG1 expression to enhance reversed cholesterol transport but not limiting cholesterol synthesis. HP-β-CD also promoted T cell proliferation and recruitment to the TME via reducing ER stress and immune checkpoint protein expression and improved CD8+ T cell exhaustion. Additionally, HP-β-CD inhibited TAMs accumulation in the TME through reducing TNF-α-p38MAPK-CCL2-CCR2 signaling pathway. Future studies will focus on exploring the specific mechanisms by which cholesterol levels affect T cell proliferation as well as immune depletion, and validating the signaling pathways by which cyclodextrins inhibit the recruitment of tumor associated macrophages. Altogether, our data highlight HP-β-CD may be a potential general antitumor clinical drug.