LATS1 exerts tumor suppressor functions via targeting Gli1 in colorectal cancer

Abstract

Background: The Hippo pathway's primary kinase component, large tumor suppressor 1 (LATS1), has been hypothesized as a tumor suppressor in a variety of cancers. LATS1's biological effects on colorectal cancer (CRC) are yet to be determined. Methods: The analysis of LATS1 mRNA expression in CRC was conducted using public databases from the Gene Expressing Profiling Interactive Analysis database (GEPIA). Investigation for the expression of LATS1 protein in 102 CRC tumor tissues and 57 normal tissues was performed using immunohistochemistry (IHC) analysis. In vitro genetic manipulation was used to explore the potential role and mechanism of LATS1 in the regulation of proliferation and migration of CRC cells. Results: LATS1 was found to be considerably downregulated in CRC tissues, with much lower levels in individuals with bigger tumors of size (≥5 cm), deeper invasion (T3-4), positive lymph node metastasis (LNM), and advanced tumor-node-metastasis (TNM) stage (III-IV). As exhibited by clinical data analysis, LATS1 loss was significantly associated with TNM and LNM staging in CRC patients. Furthermore, our in vitro investigations revealed that LATS1 depletion increased CRC cell proliferation and migration in HCT116 cells, whereas overexpressing LATS1 had the opposite effect in SW620 cells. LATS1 suppressed the expression of glioma-associated oncogene-1 (Gli1), and LATS1's tumor-suppressive actions in CRC are dependent on Gli1. Moreover, LATS1 could modulate Yes-associated protein 1 (YAP1) expression and mTOR activation in CRC cells. Conclusion: Our findings identify the LATS1 as a unique Gli1 regulator in CRC cell migration and proliferation, and suggest that LATS1 may serve as a potential therapeutic target for CRC.

Keywords: Gli1; LATS1; colorectal cancer; migration; proliferation.

Figure 1

LATS1 expression in human CRC tissues. (A) Data from the GEPIA database showed that LATS1 mRNA was downregulated in CRC tumor tissues (T) compared to normal controls (N), COAD colon adenocarcinoma, READ rectal adenocarcinoma. (B) IHC staining for LATS1 in CRC tissues and peritumoural normal tissues. (C) The IHC score of LATS1 in CRC tissue samples (T) and normal tissues (N). (D-I) LATS1 expression in CRC tumor tissues of different tumor size (D), depth of invasion (E), lymph node metastasis (F), TNM stage (G), tumor location (H) and degree of differentiation (I). NS, nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

LATS1 expression in human CRC cell lines. (A-B) Western blot (A) and qRT-PCR (B) analysis of LATS1 expression in CRC cell lines (HCT8, HCT116, LoVo, RKO, SW480, SW620, CaCo2 and DLD-1). (C-D) Western blot (C) and qRT-PCR (D) analysis of LATS1 expression in HCT116 and SW480 cells stably transfected with control-shRNA (NC) or shRNA against LATS1 (KD). (E-F) Western blot (E) and qRT-PCR (F) analysis of LATS1 expression in SW620 and CaCo2 cells stably transfected with empty vector (VEC) or plasmids encoding human LATS1 (OE). ***P < 0.001.

Figure 3

LATS1 plays tumor-suppresive roles in CRC. (A) Colony formation assay in HCT116 cells stably transfected with control-shRNA (NC) or shRNA against LATS1 (KD). (B) Colony formation assay in SW620 cells stably transfected with empty vector (VEC) or plasmids encoding human LATS1 (OE). (C) Transwell assay in HCT116 cells (NC vs. KD). (D) Transwell assay in SW620 cells (VEC vs. OE). **P < 0.01.

Figure 4

LATS1 inhibits CRC cell proliferation and migration in a Gli1-dependent manner. (A) Western blot analysis of Gli1 expression in HCT116 and SW480 cells stably transfected with control-shRNA (NC) or shRNA against LATS1 (KD). (B) Western blot analysis of Gli1 expression in SW620 and CaCo2 cells stably transfected with empty vector (VEC) or plasmids encoding human LATS1 (OE). (C) Western blot analysis of Gli1 expression in LATS1-depleted HCT116 cells with or without Gli1 siRNA treatment. (D) Colony formation assay in LATS1-depleted HCT116 cells with or without Gli1 siRNA treatment. (E) Transwell assay in LATS1-depleted HCT116 cells with or without Gli1 siRNA treatment. NS, nonsignificant, **P < 0.01, ***P < 0.001.

Figure 5

LATS1 regulates YAP1 expression and mTOR signaling pathway. (A) Western blot analysis of the indicated proteins in HCT116 and SW480 cells stably transfected with control-shRNA (NC) or shRNA against LATS1 (KD). (B) Western blot analysis of the indicated proteins in SW620 and CaCo2 cells stably transfected with empty vector (VEC) or plasmids encoding human LATS1 (OE). *P < 0.05, **P < 0.01.