ALKAL1 gene silencing prevents colorectal cancer progression via suppressing Sonic Hedgehog (SHH) signaling pathway

Abstract

Anaplastic lymphoma kinase (ALK) has been described in a range of human cancers and is involved in cancer initiation and progression via activating multiple signaling pathways, such as the PI3K-AKT, CRKL-C3G, MEKK2/3-MEK5-ERK5, JAK-STAT and MAPK signal pathways. Recently ALK and LTK ligand 1 (ALKAL1) also named "augmentor-β" or "FAM150A" is identified as a potent activating ligands for human ALK that bind to the extracellular domain of ALK. However, due to its poor stability, the mechanisms of ALKAL1 underlying the tumor progression in the human cancers including colorectal cancer have not been well documented. Herein, ALKAL1 expression was evaluated by RNA sequencing datasets from The Cancer Genome Atlas (TCGA) of 625 cases colorectal cancer, immunohistochemical analysis of 377 cases colorectal cancer tissues, and Western blotting even Real-time PCR of 10 pairs of colorectal cancer tissues and adjacent normal tissues, as well as 8 colorectal cancer cell lines. Statistical analysis was performed to explore the correlation between ALKAL1 expression and clinicopathological features in colorectal cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between ALKAL1 expression and overall survival. In vitro and in vivo assays were performed to assess the biological roles of ALKAL1 in colorectal cancer. Gene set enrichment analysis (GSEA), Western blotting and luciferase assays were used to identify the underlying signal pathway involved in the tumor progression role of ALKAL1. As a result, we showed that ALKAL1 was upregulated in colorectal cancer tissues and cell lines. Upregulation of ALKAL1 correlated with tumor malignancy and poor prognosis in colorectal cancer. ALKAL1 silencing inhibited tumorigenesis, metastasis and invasion of colorectal cancer cells, and inhibited SHH signaling pathway, which is essential for ALKAL1 induced migration. Our findings reveal a new mechanism by which ALKAL1 participates in colorectal cancer migration and invasion via activating the SHH signaling pathway.

Keywords: ALKAL1; SHH signaling pathway; colorectal cancer; metastasis.

Figure 1

ALKAL1 is upregulated in colorectal cancer tissues and cell lines. A-D) ALKAL1 expression levels was markedly upregulated in colorectal cancer tissues as assessed by analyzing TCGA colorectal cancer miRNA sequencing datasets. (E) Real-time PCR analysis of ALKAL1 in 10 primary colorectal cancer tissues compared with the matched adjacent normal tissues (ANT). (F) ALKAL1 protein expression in 10 paired CRC and adjacent normal tissues (ANT) from the same patient were detected by Western blot. α-tubulin was used as the loading control. (G) ALKAL1 mRNA expression was determined by RT-PCR in selected 8 colorectal cancer cell lines (Caco-2, DLD-1, HCT-8, LS 174T, RKO, SW480, SW620 and T84). (H) ALKAL1 protein expression was determined by Western blot in selected 8 colorectal cancer cell lines (Caco-2, DLD-1, HCT-8, LS 174T, RKO, SW480, SW620 and T84).

Figure 2

Upregulation of ALKAL1 correlates with tumor malignancy and poor prognosis in colorectal cancer. (A) Representative photomicrographs of immunohistochemical analysis for ALKAL1 expression at various pathological stages, including benign, precancerous, and tumor stages I to IV. ALKAL1 was stained in brown. (B) Distribution of ALKAL1 immunostaining index across a variety of pathological stages of colorectal cancer. High expression of ALKAL1 was observed in 240/353 colorectal cancer tissue samples. (C-E) High expression of ALKAL1 was strong positive correlated with tumor classification, node classification and pathological stages. (F) Kaplan-Meier survival analysis revealed that patients with high ALKAL1 expression had poor overall survival. (G) Kaplan-Meier analysis of overall survival curves of colorectal cancer patients datasets from TCGA.

Figure 3

ALKAL1 silencing is not associated with colorectal cancer cell proliferation. (A) Real-time PCR analysis of ALKAL1 expression in ALKAL1 silencing RKO and SW480 cell lines by endogenously knocking down ALKAL1 with retrovirus (sh.#1 and sh.#2) infection. (B) Western blot analysis of ALKAL1 expression in ALKAL1 silencing RKO and SW480 cell lines by endogenously knocking down ALKAL1 with retrovirus (sh.#1 and sh.#2) infection. (C-D) CCK-8 analysis of RKO and SW480 cells proliferation. (E) Plate colony formation analysis of RKO and SW480 cell by endogenously knocking down ALKAL1 with retrovirus (sh.#1 and sh.#2) infection. (F) Cell cycle analysis with PI staining analysis of RKO and SW480 cells by endogenously knocking down ALKAL1 with retrovirus (sh.#1 and sh.#2) infection. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05.

Figure 4

ALKAL1 silencing inhibits colorectal cancer cell tumorigenesis. (A) Anchorage-independent growth assay analysis of the anchorage-independent growth ability of ALKAL1-silenced colorectal cancer cells. (B) Representative images of excised tumors after injection of SW620 cells transduced with ALKAL1-silencing shRNA or vector control. (C) Tumor volumes were measured every week after the seventh day post injection (n = 6, p < 0.05). (D) Tumor weights were determined after tumor-bearing mice were sacrificed (n = 6, p < 0.05). (E) HE staining analysis of tumor from tumor-bearing mice. (F-H) Representative images of Ki67, vimentin and caspase 3 cleavage immunostaining in xenograft tumors that transduced with ALKAL1-silencing shRNA or vector control. Magnification: 100× for the upper panel, and 400× for the lower panel.

Figure 5

ALKAL1 silencing inhibits metastasis and invasion of colorectal cancer cells. (A) Wound healing and migration assay analysis of the migration ability of ALKAL1 silencing colorectal cancer cells. (B-C) Transwell cell invasion and migration assay analysis of the invasion and migration ability of ALKAL1 silencing colorectal cancer cells. (D) Real-time PCR analysis of some selected gene (ROCK1, ROCK2, TESK1, PDXP, LIMK1, LIMK2, HSP90AA1, CFL1, CFL2, MMP2, MMP3, MMP7, MMP9, TIMP1, TIMP2, TIMP3 and TIMP4 ) expression that related to cell movement and invasion in ALKAL1 silencing RKO and SW480 cell lines. (E) Western blot analysis of ROCK1, LIMK1, MMP3, and MMP9 expression in ALKAL1 silencing RKO and SW480 cell lines.

Figure 6

(A) ALKAL1 silencing inhibits SHH signaling pathway. A gene set enrichment analysis (GSEA) of ALAK1 expression against SHH signaling pathway. (B) ALKAL1 high expression significantly and positively correlated with PTCH protein levels from TCGA. (C) Western blot analysis of nuclear and total level of GLI1 in ALKAL1 silencing RKO and SW480 cell lines. (D-E) Luciferase assay analysis of GLI1 dependent luciferase activity and the expression levels of multiple downstream genes in ALKAL1 silencing RKO and SW480 cells. (F) Western blot analysis of PTCH and GLI1 in subcutaneous xenografts of nude mice.

Figure 7

SHH signaling pathway activation is essential for ALKAL1 induced migration. (A) Wound healing and migration assay analysis of the role of SHH signaling pathway activation in ALKAL1 induced migration using SHH signaling pathway agonist purmorphamine (Purm). (B-C) Transwell cell invasion and migration assay analysis of the invasion and migration ability in ALKAL1 induced migration using SHH signaling pathway agonist purmorphamine (Purm).

Figure 8

Hypothetical model illustrating that ALKAL1 plays an important role in colorectal cancer migration and invasion via activating SHH signaling pathway.