Statins inhibit tumor progression via an enhancer of zeste homolog 2-mediated epigenetic alteration in colorectal cancer

Abstract

While statin intake has been proven to reduce the risk of colorectal cancer (CRC), the mechanism of antitumor effects and clinical significance in survival benefits remain unclear. Statin-induced antiproliferative effects and its underlying mechanism were examined using six CRC cell lines. Statins except pravastatin showed antiproliferative effects (simvastatin ≥ fluvastatin > atorvastatin) even though both of simvastatin and pravastatin could activate mevalonate pathways, suggesting the statin-mediated antiproliferative effects depended on non-mevalonate pathway. Indeed, statin induced p27(KIP1) expression by downregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2), which acts as an epigenetic gene silencer. Additionally, the use of simvastatin plus classII histone deacetylase (HDAC) inhibitor (MC1568) induced further overexpression of p27(KIP1) by inhibiting HDAC5 induction originated from downregulated EZH2 in CRC cells and synergistically led to considerable antiproliferative effects. In the clinical setting, Statin intake (except pravastatin) displayed the downregulated EZH2 expression and inversely upregulated p27(KIP1) expression in the resected CRC by immunohistochemical staining and resulted in the significantly better prognoses both in overall survival (p = 0.02) and disease free survival (p < 0.01) compared to patients without statin intake. Statins may inhibit tumor progression via an EZH2-mediated epigenetic alteration, which results in survival benefits after resected CRC. Furthermore, statin plus classII HDAC inhibitor could be a novel anticancer therapy by their synergistic effects in CRC.

Keywords: EZH2; HDAC; colorectal cancer; p27; statins.

Figure 1

Statins-induced antiproliferative effects against CRC cell lines in growth assay. (a) Pravastatin: 0, 5, 10, 15 μM of pravastatin was added to the cells for 5 days. Growth suppression was not observed in all six CRC cell line examined. (b) Simvastatin: 0, 5, 10, 15 μM of simvastatin was added to the cells for 5 days. Growth suppression was observed in all six CRC cell line examined. (c) Fluvastatin: 0, 5, 10, 15 μM of fluvastatin was added to the cells for 5 days. Growth suppression was observed in all six CRC cell line examined similar to simvastatin. (d) Atorvastatin: 0, 5, 10, 15 μM of atorastatin was added to the cells for 5 days. Growth suppression was observed in all six CRC cell line examined but weak compared to simvastatin and fluvastatin. The vertical scale is the measured OD detected using cell counting kit.

Figure 2

Statin-mediated activation of mevalonate pathway, and antiproliferative effects by statin plus 5-FU in CRC cell line. (a) Growth assay of simvastatin 5 μM, 5-FU 10 nM and combination (simvastatin 5 μM plus 5-FU 10 nM). The drugs were added to the cells for 5 days, and the living cells were counted. The antieffect of both simvastatin and 5-FU was observed by combination in all six CRC cell lines examined but no synergistic effect was observed. The vertical scale is the measured OD detected using cell counting kit. (b) Growth assay of pravastatin 5 μM, 5-FU 10 nM and combination (pravastatin 5 μM plus 5-FU 10 nM). The drugs were added to the cells for 5 days, and the living cells were counted. Only the antitumor effect of 5-FU was observed in all six CRC cell lines examined. The vertical scale is the measured OD detected using cell counting kit. (c) Western blotting of nuclear fraction to determine nuclear SREBP2 (SP2) expression by statins. Nuclear protein was extracted 48 hr after statin was added at various doses (0, 5, 10, 15 μM) to the cells. SP2 (55kDa) was increased by both pravastatin (left lane) and simvastatin (right lane) in SW620 but not in other CRC cell lines. Histone1 was used for loading control of nuclear protein.

Figure 3

Screening of epigenetic molecules regulated by statins. (a) RT-PCR of HDAC1-10, EZH2. Total RNA of colon cancer cell line DLD1 was extracted 24 hr after various dose of simvastatin was added to the cells (0, 5, 10, 15 μM). Suppression of HDAC3, EZH2 and induction of HDAC5, HDAC7 was observed. Lane 1: 0 μM, lane 2: 5 μM, lane 3: 10 μM, lane 4: 15 μM, lane 5: negative control. GAPDH was used for control. (b) Western blotting of epigenetic molecules. The whole cell lysate was extracted 48 hr after various dose of pravastatin or simvastatin was added to the cells (0, 5, 10, 15 μM). HDAC5 (124 kDa) induction and EZH2 (91kDa) suppression was observed in all six CRC cell lines by simvastatin but not by pravastatin. β-Actin (42 kDa) was used for loading control.

Figure 4

Statin-induced p27 ^KIP1 protein expression and synergistically effects by epigenetic drug of class II HDAC inhibitor (MC1568). (a) Western blotting of p27 by various dose of simvastatin. The whole cell lysate was extracted 48 hr after various dose of simvastatin was added to the cells (0, 5, 10, 15 μM). p27 was induced by simvastatin in all six CRC cell lines examined. (b) Synergistic effect by combination of simvastatin and MC1568. Simvastatin 5 μM and/or MC1568 20 μM was added to the cells 24 hr after seeding the cells. The drugs were changed daily, and the living cells were counted after 5 days. Combination of simvastatin and MC1568 showed synergistic antitumor effect in all six CRC cell lines examined. The vertical scale is the measured OD detected using cell counting kit. (c) Western blotting of p27 by simvastatin 5 μM, MC1568 20 μM and combination (simvastatin 5 μM plus MC1568 20 μM). The whole cell lysate was extracted 24 hr after drugs were added. Combination of simvastatin and MC1568 induced high level of p27 compared to simvastatin or MC1568 alone in all six CRC cell lines examined. Lane 1 is control. Lane 2 is simvastatin 5 μM. Lane 3 is MC1568 20 μM. Lane 4 is combination of simvastatin 5 μM plus MC1568 20 μM. β-Actin (42 kDa) was used for loading control. (d) Suppression of EZH2 by two kinds of siRNA. Left line is siRNA-1, right line in siRNA-2. Lane 1 is control siRNA, Lane 2 is EZH2 siRNA. EZH2 was suppressed by both siRNA and in all CRC cell line. Induction of HDAC5 and p27 was observed in all CRC cell line. β-Actin (42 kDa) was used for loading control.

Figure 5

Immunohistochemical staining of EZH2 and p27^KIP1 and prognostic analyses in CRC patients with or without statin intakes. (a, b) The specimens derived from patients without statin intake (a) and pravastatin intake (b) showed upregulated EZH2 expression and downregulated p27^KIP1 expression. (c) The specimens derived from patients derived from simvastatin intake displayed heterogeneous expressions including upregulated p27 corresponding to downregulated EZH2. The representative findings were shown. (d) Statin-derived survival benefits in R0 resected CRC patients. Left: Kaplan-Meier plot of OS of all patients with statin (+) group (n = 61) and statin (−) group (n = 681). Right: Kaplan-Meier plot of DFS of all patients with statin (+) group (n = 61) and statin (−) group (n = 681).