In the present study, we examined the role of HDL and its receptor, SR-BI, in breast cancer development and progression. We found that HDL3 stimulates migration and activates signaling pathways such as MAPK and PI3K in two breast cancer cell lines. Inhibiting selective HDL-cholesteryl ester uptake by knocking down or pharmacologically inhibiting SR-BI resulted in an attenuation of cell-signaling events induced by HDL. Additionally, loss of SR-BI resulted in decreased proliferation, migration, and tumor growth of MDA-MB-231 cells. These findings suggest that regulating cholesterol metabolism and cellular signaling pathways via SR-BI may be linked and may additionally identify new targets associated with tumor progression.
HDL, signal transduction, and cellular migration
HDL has a well-established role in the etiology of atherosclerosis, particularly in reverse cholesterol transport, whereby HDL removes excess cholesterol molecules from peripheral tissues and returns them to the liver for excretion or recycling [
40]. In addition, HDL functions in a number of other cellular processes, including inhibition of apoptosis in macrophages [
41], induction of migration in endothelial cells [
24], and the initiation of cell-signaling events in multiple cell types [
22,
23,
42]. Although clinical studies have suggested that plasma HDL levels may be correlated with increased breast cancer risk [
5,
7‐
9,
12], the mechanisms by which HDL exerts its effect have yet to be elucidated. HDL has been shown to activate Erk1/2 in fibroblasts [
23,
42], Chinese hamster ovary cells [
22], endothelial cells [
23], and prostate cancer cells [
35,
43]. Studies have also shown that it can activate Akt in endothelial [
23] and prostate cancer cells [
35,
43]. Interestingly, the activation of Erk1/2 [
44] and Akt [
39,
45] has been implicated in several human cancers, including breast cancer.
In the present study, we established a role for HDL as a mediator of signal transduction in two breast cancer cell lines. Consistent with the results obtained in other cell types, we found that, in both MCF7 and MDA-MB-231 cells, incubation with HDL3 induces a rapid activation of both Erk1/2 and Akt signaling pathways. These novel findings in breast cancer suggest that HDL may regulate various signaling pathways and may therefore alter tumor progression.
In the present study, we found that HDL can induce migration of two breast cancer cell lines, MCF7 and MDA-MB-231, suggesting that HDL may play a role in the early stages of metastasis. This finding is consistent with previous studies showing that HDL can stimulate migration of endothelial cells [
24,
46]. Interestingly, the observed migration was shown to be mediated by SR-BI [
24]. By contrast, a recent study reported that HDL inhibits migration of MDA-MB-231 in Boyden chamber assays [
14]. However, in this study, the investigators used serum as the chemoattractant, and HDL was added to the upper chamber, thereby measuring the ability of HDL to prevent cellular migration induced by serum. By contrast, our method allows the analysis of the role of HDL in the regulation of cellular migration and therefore allows a direct measurement of the capability of HDL to induce migration of MDA-MB-231 cells. As a result, our results indicate that HDL may play a role in the pathogenesis of breast cancer, especially in the later stages.
SR-BI, signal-transduction regulation, and tumor formation
SR-BI has been implicated as a mediator of several cell-signaling events in the context of atherosclerosis [
22,
23,
25‐
27]. Previous studies have shown that HDL binding to SR-BI and subsequent lipid transfer are sufficient to activate Src, which subsequently activates the PI3K/Akt and MAPK pathways [
25,
27]. In endothelial cells, one of the downstream effectors of Akt is eNOS, which catalyzes the production of NO. The results obtained in the present study are consistent with the hypothesis that SR-BI may also play a role in signal transduction in the context of cancer. In agreement with this hypothesis, upon knockdown or pharmacologic inhibition of SR-BI in MDA-MB-231 cells, Akt activation was significantly reduced, suggesting that SR-BI may be mediating this response. In addition, downregulation of SR-BI was accompanied by a reduction of total cholesterol levels in MDA-MB-231 cells. These results are consistent with reports that indicate that the cholesterol flux mediated by SR-BI plays a role in the regulation of signal-transduction initiation [
26]. In our model, decreased total cholesterol levels may represent a reduction in SR-BI-mediated cholesterol flux and therefore significantly reduce signal-transduction activation. SR-BI also binds LDL, which can, like HDL, promote the cellular entry of cholesteryl ester. Although LDL, may promote the entry of cholesteryl ester via SR-BI, it is not sufficient to induce migration of breast cancer cells, and it does not appear to alter Akt activation (Figure
2C, D). Taken together, our data suggest that both cholesteryl ester entry via HDL-SR-BI and Akt activation are required for cellular proliferation and migration, and, eventually, tumor growth.
Activation of the PI3K/Akt pathway promotes growth, survival, and proliferation [
45] and has been implicated in a variety of human cancers [
39]. Importantly, Akt is aberrantly hyperactivated in approximately 40% of breast cancers [
39]. We observed a reduction in proliferation and migration in the SR-BI-knockdown cells compared with control cells in association with reduced Akt activation. These results suggest that SR-BI may mediate the activation of Akt and its downstream effects in the presence of HDL. Mechanistically, we showed that the inhibition of the PI3K/Akt pathway results in significantly reduced proliferation of shCTL MDA-MB-231 cells, similar to the reduction in proliferation observed in shSRBI MDA-MB-231 cells. Importantly, no further reduction in proliferation of shSRBI MDA-MB-231 cells was detected upon inhibition of the PI3K/Akt pathway. Taken together, these data suggest that reduced Akt activation observed in the shSRBI MDA-MB-231 cells may be responsible for reduced proliferation of these cells compared with shCTL MDA-MB-231 cells.
Previous studies suggested a role for SR-BI in the etiology of breast cancer. Cao
et al. [
30] showed that expression of SR-BI is increased in human breast tumors compared with the normal surrounding tissue. They also demonstrated that recombinant expression of a mutant form of SR-BI, which lacked the carboxyl-terminal tail of the protein, could inhibit proliferation of breast cancer cells. Their study further suggested that this effect was possibly due to reduced Akt activation. Our study is the first to demonstrate directly that Akt activation is reduced when SR-BI is knocked down or pharmacologically inhibited. Furthermore, in agreement with the previously mentioned study [
30], we showed that proliferation of MDA-MB-231 cells was significantly inhibited by downregulation of SR-BI protein levels and by pharmacologic inhibition of SR-BI. In addition, we observed that SR-BI knockdown inhibits migration. This finding may suggest a role for SR-BI in the initiation of metastasis. Finally, we demonstrated that knockdown of SR-BI in MDA-MB-231 cells can lead to reduced tumor growth
in vivo accompanied by increased activation of Erk1/2 and Akt, and an increase in cellular apoptosis. In MCF7 cells, knockdown of SR-BI also led to reduced xenograft tumor growth.
Previous studies have shown that Akt can inhibit apoptosis through a variety of mechanisms, including the phosphorylation of BAD, thus preventing cytochrome
c release from mitochondria and the direct inhibition of the caspase activation cascade [
39]. Increased cellular cholesterol levels have been shown to increase Akt activation and decrease apoptosis in prostate cancer cells [
47]. Consistent with these observations, a recent study showed that inhibition of xenograft tumor growth could be achieved with colon cancer cells that re-express the ATP-binding cassette transporter A1 (ABCA1) [
48]. ABCA1 is a lipid transporter that mediates the efflux of cellular cholesterol to lipid-free apolipoprotein A-I [
49]. Furthermore, in this study, re-expression of ABCA1 resulted in decreased mitochondrial cholesterol content and increased release of cytochrome
c, which ultimately led to increased apoptosis. Our work also indicated that SR-BI knockdown can significantly reduce apoptosis in xenograft tumors, as shown by TUNEL staining. Consequently, a reduction in the levels of cellular cholesterol content may be responsible, at least in part, for the decreased apoptosis observed in our model. Taken together, these data also suggest an important role for cholesterol in the regulation of cellular signaling pathways and tumor formation. Importantly, excess cellular cholesterol accumulates in the form of esterified cholesterol. Previous works and ours suggest that the accumulation of esterified cholesterol may lead to a modification of signaling pathways associated with proliferation and migration in tumors. Consistent with this hypothesis, increasing cellular esterified cholesterol levels have been shown to induce cellular proliferation and enhance invasiveness of tumor cell lines [
50]. Conversely, the inhibition of cholesterol esterification has been shown to have the reverse effect [
51,
52].