BMAL1 promotes colorectal cancer cell migration and invasion through ERK- and JNK-dependent c-Myc expression

Abstract

Background: Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT-like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC.

Methods: The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial-to-mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK-8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo.

Results: We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial-to-mesenchymal transition (EMT) pathway and induce the β-catenin release further promotes the expression of oncogene c-Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK-signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c-Myc in vitro. Taken together, these results suggested that the BMAL1/ c-Myc-signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK-dependent pathway.

Conclusions: Our study showed that BMAL1 promotes CRC metastasis through MAPK-c-Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

Keywords: BMAL1; EMT; MAPK signaling pathway; c-Myc; colorectal cancer.

FIGURE 1

High BMAL1 expression was significantly associated with poor prognosis in CRC. (A) Patients in The Cancer Genome Atlas (TCGA) and Sir Run Run Shaw cohorts (n = 84) were stratified according to the BMAL1 gene expression signature. A total of 58.3% (49/84) CRC tissues were BMAL1 overexpression. Kaplan–Meyer overall survival curves of CRC with different BMAL1 genotypes (p < 0.05). (B) Representative photographs showing immunohistochemical expression of BMAL1 in the adjacent normal tissue (0 = negtive), colorectal cancer and the lymph node. 40× & 200× magnification; 1 = mild, 2 = moderate, 3 = strong.

FIGURE 2

BMAL1 affects colorectal cancer cells' growth. (A) Representative western blot analysis of BMAL1 and Ki‐67 protein expression in transfected cell lines. (B) SW480 cells transfected with BMAL1 and shBMAL1 were subjected to the CCK‐8 assay after transfection. (C, D) Stably transfected RKO and SW480 cells were seeded onto 6‐well plates. The number of colonies was counted on the 10th day after seeding. (E) Western blots were quantified by ImageJ densitometric analysis and normalized to controls. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 3

Migration activity was regulated by BMAL1. (A, C) Wound‐healing assay showed that migration activity was enhanced by BMAL1. BMAL1 silencing suppressed the migration ability of SW480 cells. (B, D) Transwell assays showed that BMAL1 enhanced the migration and invasion potential of CRC. BMAL1 silencing decreased the migration and invasion of RKO and SW480 cells. (E, G) Western blot assay showed that BMAL1 significantly increased the expression of β‐catenin, N‐cadhrein, vimentin but inhibited E‐cadhrein expression. (F, H) Western blots were quantified by ImageJ densitometric analysis and normalized to controls. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 4

BMAL1 regulates c‐Myc expression. (A) BMAL1 activated c‐Myc protein expression in RKO/BMAL1 and SW480/BMAL1 cells, which detected by western blot. (B) Western blots were quantified by ImageJ densitometric analysis and normalized to controls. Data are expressed as mean ± SD (n = 3). (C) Immunohistochemical detection of c‐Myc in representative tumor samples, the adjacent normal tissue and lymph node from CRC of the indicated genotypes. 40× & 200× magnification. 1 = mild, 2 = moderate, 3 = strong. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 5

MAPK‐signaling pathway is involved in BMAL1‐induced migration. (A) BMAL1 activated MAPK‐signaling pathway in RKO/BMAL1 and SW480/BMAL1 cells. The related proteins in MAPK‐signaling pathway were detected by western blot. BMAL1 actived the RAF, P‐MEK, P‐ERK, JNK expression, while the P38 was not affected. (B) Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 6

BMAL1 modulates the c‐Myc expression through ERK‐ and JNK‐dependent signaling pathway. (A) Inhibiting the MAPK pathway suppressed c‐Myc but increased E‐cadhrein expression in vitro using ERK1/2 (APExBIO PD98059, 10 μM) and JNK inhibitor (SP600125, 10 μM). (B) MAPK pathway inhibition markedly decreased the protein expression of c‐Myc. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 7

BMAL1 promotes tumorigenesis and tumor metastasis in mice. (A) BMAL1 knockdown significantly inhibited CRC proliferation in vivo. Cancer cells were injected into the nude mice. The tumors were resected from mice after 35 days. (B) At the indicated times, subcutaneous tumor size (mm) was measured with calipers (mean ± SD, n = 4). (C) Immunohistochemistry staining of BMAL1 and E‐cadhrein in xenograft tissues. 40× & 200× magnification. 1 = mild, 2 = moderate, 3 = strong. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 8

BMAL1 promotes colorectal cancer cells proliferation and migration via BMAL1/MAPK/c‐Myc pathway. This figure was created with BioRender.com.