Prognostic value and biological function of LRRN4 in colorectal cancer

Abstract

Background: Several nervous and nerve-related biomarkers have been detected in colorectal cancer (CRC) and can contribute to the progression of CRC. However, the role of leucine-rich repeat neuronal 4 (LRRN4), a recently identified neurogenic marker, in CRC remains unclear.

Methods: We examined the expression and clinical outcomes of LRRN4 in CRC from TCGA-COREAD mRNA-sequencing datasets and immunohistochemistry in a Chinese cohort. Furthermore, colony formation, flow cytometry, wound healing assays and mouse xenograft models were used to investigate the biological significance of LRRN4 in CRC cell lines with LRRN4 knockdown or overexpression in vitro and in vivo. In addition, weighted coexpression network analysis, DAVID and western blot analysis were used to explore the potential molecular mechanism.

Results: We provide the first evidence that LRRN4 expression, at both the mRNA and protein levels, was remarkably high in CRC compared to controls and positively correlated with the clinical outcome of CRC patients. Specifically, LRRN4 was an independent prognostic factor for progression-free survival and overall survival in CRC patients. Further functional experiments showed that LRRN4 promoted cell proliferation, cell DNA synthesis and cell migration and inhibited apoptosis. Knockdown of LRRN4 can correspondingly decrease these effects in vitro and can significantly suppress the growth of xenografts. Several biological functions and signaling pathways were regulated by LRRN4, including proteoglycans in cancer, glutamatergic synapse, Ras, MAPK and PI3K. LRRN4 knockdown resulted in downregulation of Akt, p-Akt, ERK1/2 and p-ERK1/2, the downstream of the Ras/MAPK signaling pathway, overexpression of LRRN4 leaded to the upregulation of these proteins.

Conclusions: Our results suggest that LRRN4 could be a biological and molecular determinant to stratify CRC patients into distinct risk categories, and mechanistically, this is likely attributable to LRRN4 regulating several malignant phenotypes of neoplastic cells via RAS/MAPK signal pathways.

Keywords: Colorectal cancer; Leucine-rich repeat neuronal 4; Neurogenic biomarker; Prognosis; RAS/MAPK signal pathways.

Fig. 1

Expression of LRRN4 in CRC tissue with different clinicopathological characteristics from the TCGA-COREAD cohort. A Expression of LRRN4 in CRC and normal colorectal tissues. LRRN4 expression in CRC tissue of different pathological stages (B), lymphatic invasion (C), pathological T stage (D), pathological N stage (E), pathological M stage (F), progression-free survival status (PFS) (G) and overall survival status (OS) (H)

Fig. 2

LRRN4 expression positively correlated with the clinical outcome of CRC patients at both the mRNA and protein levels. A Kaplan–Meier curves of PFS of CRC from TCGA based on the expression of LRRN4 mRNA, B Kaplan–Meier curves of OS of CRC from TCGA based on the expression of LRRN4 mRNA, C Representative IHC images with low expression and high expression of LRRN4 protein. Magnification 200 × , scale bars correspond to 100 μm (left) and magnification 400 × , scale bars correspond to 50 μm (right). Arrows indicate cells with low or high expression of LRRN4. D Patients percent with high or low LRRN4 expression in CRC and normal colorectal tissues. E Kaplan–Meier curves of OS of CRC in a Chinese cohort based on the expression of LRRN4 protein

Fig. 3

LRRN4 is highly expressed in CRC cell lines and LRRN4 promotes cell proliferation. A Expression of LRRN4 in SW480, Caco2, HCT-116, LoVo and HIEC-6 cells. B LRRN4 expression in Caco2 and SW480 cells with constitutive expression of two different LRRN4 shRNAs (kd1 or kd2), and LRRN4 expression in HCT-116 and LoVo cells constitutively expressing LRRN4 open reading frame (oe). C EdU proliferation assay and quantification in Caco2 and SW480 cells with LRRN4 knockdown and in HCT-116 and LoVo cells overexpressing LRRN4. D Colony formation assay of cell clonal proliferation ability in Caco2 and SW480 cells with knockdown of LRRN4 and in HCT-116 and LoVo cells overexpressing LRRN4. Bars represent the means with SEM from at least three independent experiments

Fig. 4

LRRN4 promotes cell DNA synthesis and inhibits apoptosis in CRC cells. A Representative histogram of the gated cells in the G0/G1, S and G2/M phases and quantitative analysis of the S phase proportion in Caco2 and SW480 cells with LRRN4 knockdown. B Representative histogram of the gated cells in the G0/G1, S and G2/M phases and quantitative analysis of the S phase proportion in HCT-116 and LoVo cells overexpressing LRRN4. C Representative plots of YF®647A-Annexin V flow cytometry and DAPI staining experiments and quantitative analysis of Annexin V-positive Caco2 with LRRN4 knockdown. D Representative plots of YF®647A-Annexin V flow cytometry and DAPI staining experiments and quantitative analysis of Annexin V-positive SW480 with LRRN4 knockdown. E Representative plots of YF®647A-Annexin V flow cytometry and DAPI staining experiments and quantitative analysis of Annexin V-positive HCT-116 and LoVo cells overexpressing LRRN4. F Representative images of Western blot analyses of PCNA, Chk1, p-Chk1, Bcl-2, Bcl-xl, caspase 3 and cleaved caspase 3 (c-caspase3) in CRC cells with knockdown or overexpression of LRRN4. Bars represent the means with SEM from at least three independent experiments

Fig. 5

LRRN4 accelerates cell migration. A Representative images of scratched monolayer re-epithelialization and quantitative analysis in Caco₂ and SW480 cells with knockdown of LRRN4. B Representative images of scratched monolayer re-epithelialization and quantitative analysis in HCT-116 and LoVo cells overexpressing LRRN4. Bars represent the means with SEM from at least three independent experiments

Fig. 6

Effects of LRRN4 on Caco2 xenograft tumor growth in SCID mice. A Image of harvested xenograft tumors from SCID mice in each group (Caco2-scr, Caco2-kd1 and Caco2-kd2) at necropsy. B Tumor volume (mm³) of xenografts with Caco2-scr, Caco-kd1 and Caco2-kd2 as measured twice a week. C The histology images of xenograft tumors. Magnification 200 × , scale bars correspond to 100 μm. D Expression of LRRN4 in xenograft tumors (Caco2-scr, Caco2-kd1 and Caco2-kd2). Magnification 200 × , scale bars correspond to 100 μm (left) and magnification 400 × , scale bars correspond to 50 μm (right). *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 7

The GO and KEGG pathways of LRRN4-related genes. A, B Association between gene expression modules and clinicopathologic characteristics and LRRN4 expression. Pearson’s correlation analysis was performed to obtain Pearson’s correlation (p value). C The cellular component (CC), D biological process (BP), E molecular function (MF), and F KEGG pathways of LRRN4-related genes in the magenta module, where p < 0.05 are displayed

Fig. 8

Representative images of western blot analyses of Akt, p-Akt, ERK1/2 and p-ERK1/2 in CRC cells with LRRN knockdown or overexpression