Tumour-Derived Laminin α5 (LAMA5) Promotes Colorectal Liver Metastasis Growth, Branching Angiogenesis and Notch Pathway Inhibition

Abstract

Hepatic metastatic growth is dependent upon stromal factors including the matrisomal proteins that make up the extracellular matrix (ECM). Laminins are ECM glycoproteins with several functions relevant to tumour progression including angiogenesis. We investigated whether metastatic colon cancer cells produce the laminins required for vascular basement membrane assembly as a mechanism for the promotion of angiogenesis and liver metastasis growth. qPCR was performed using human-specific primers to laminin chains on RNA from orthotopic human colorectal liver metastases. Laminin α5 (LAMA5) expression was inhibited in colon cancer cells using shRNA. Notch pathway gene expression was determined in endothelia from hepatic metastases. Orthotopic hepatic metastases expressed human laminin chains α5, β1 and γ1 (laminin 511), all of which are required for vascular basement membrane assembly. The expression of Laminin 511 was associated with reduced survival in several independent colorectal cancer cohorts and angiogenesis signatures or vessel density significantly correlated with LAMA5 expression. Colorectal cancer cells in culture made little LAMA5, but its levels were increased by culture in a medium conditioned by tumour-derived CD11b⁺ myeloid cells through TNFα/NFκB pathway signalling. Down-regulation of LAMA5 in cancer cells impaired liver metastatic growth and resulted in reduced intra-tumoural vessel branching and increased the expression of Notch pathway genes in metastasis-derived endothelia. This data demonstrates a mechanism whereby tumour inflammation induces LAMA5 expression in colorectal cancer cells. LAMA5 is required for the successful growth of hepatic metastases where it promotes branching angiogenesis and modulates Notch signalling.

Keywords: Notch; angiogenesis; laminin; liver metastasis; matrisome; microenvironment.

Figure 1

Laminin 511 is expressed by colon cancer cells in metastatic tumours and is associated with adverse outcomes in colorectal cancer. (A) Immunofluorescence staining for laminin (green) in orthotopic liver metastases developed using HT29, HCT116 or LoVo human colon cancer cell lines or in a human liver metastasis resection specimen. Counterstained with DAPI. M indicates metastasis and the white border demarcates the metastasis–liver interface. Scale bar represents 100 μm. (B) Immunofluorescence staining of HT29 orthotopic liver metastasis stained for the indicated markers using non species-specific antibodies. Scale bar represents 30 μm. (C) mRNA profile of all known laminin and collagen type IV chain genes in RNA extracted from orthotopic HT29 (green), HCT116 (blue) and LoVo (pink) liver metastases using human-specific primers. (D) Oncomine^TM data demonstrating mRNA expression of LAMA5, LAMB1 and LAMC1 in colon cancer relative to normal colon from multiple independent GSE datasets. (E) Expression of the indicated laminin chains in normal colon, primary colon cancer, normal liver and liver metastases through analysis of data from GSE41258. Error bars are the SEMs compared with Students t-test. (F) Kaplan–Meier curves (Log-rank comparison) showing cancer-specific or absolute survival in data generated from publicly available gene array datasets. Patient groups are defined by over-expression of LAMA5, LAMB1 and LAMC1 (red) or normal expression of these genes (blue). * 0.01 < p ≤ 0.05, *** p ≤ 0.001.

Figure 1

Laminin 511 is expressed by colon cancer cells in metastatic tumours and is associated with adverse outcomes in colorectal cancer. (A) Immunofluorescence staining for laminin (green) in orthotopic liver metastases developed using HT29, HCT116 or LoVo human colon cancer cell lines or in a human liver metastasis resection specimen. Counterstained with DAPI. M indicates metastasis and the white border demarcates the metastasis–liver interface. Scale bar represents 100 μm. (B) Immunofluorescence staining of HT29 orthotopic liver metastasis stained for the indicated markers using non species-specific antibodies. Scale bar represents 30 μm. (C) mRNA profile of all known laminin and collagen type IV chain genes in RNA extracted from orthotopic HT29 (green), HCT116 (blue) and LoVo (pink) liver metastases using human-specific primers. (D) Oncomine^TM data demonstrating mRNA expression of LAMA5, LAMB1 and LAMC1 in colon cancer relative to normal colon from multiple independent GSE datasets. (E) Expression of the indicated laminin chains in normal colon, primary colon cancer, normal liver and liver metastases through analysis of data from GSE41258. Error bars are the SEMs compared with Students t-test. (F) Kaplan–Meier curves (Log-rank comparison) showing cancer-specific or absolute survival in data generated from publicly available gene array datasets. Patient groups are defined by over-expression of LAMA5, LAMB1 and LAMC1 (red) or normal expression of these genes (blue). * 0.01 < p ≤ 0.05, *** p ≤ 0.001.

Figure 2

Laminin 511 expression in primary and metastatic colon cancers is associated with angiogenesis. (A) Immunofluorescence staining of HT29 (top) and LoVo (bottom) orthotopic liver metastases and subcutaneous tumours stained for LAMA5 (human-specific antibody, h-LAMA5, green), collagen type IV (red) and CD31 (white). Scale bars represent 20 μm. (B) Gene Set Enrichment Analysis (GSEA) for enrichment of a 20-gene signature regulating angiogenesis in TCGA colon cancers over-expressing laminin LAMA5, LAMB1 and LAMC. (C) Immunofluorescence staining of archival human primary colon cancer and liver metastasis resection specimens stained for LAMA5 (green) and CD31 (red). Scale bars represent 40 μm. (D) Immunofluorescence staining of archival liver metastasis resection specimens stained for LAMA5 and LAMB1 (both green) and CD31 (red). Scale bars represent 40 μm. (E) Immunofluorescence staining of human liver metastases from a patient with low microvessel density (top row) and one with high microvessel density (lower row) stained for LAMA5 (green) and von Willebrand factor (red). Scale bars represent 100 μm. (F) Scatter plot of the percentage of liver metastasis area staining positive for LAMA5 (x) vs microvessel density (y) determined by measuring the percentage area of the tumour staining positive for von Willebrand factor. Data shown for liver metastases from 30 patients with at least five tumour areas quantified per patient (Spearman’s rank correlation).

Figure 3

Laminin 511 expression is promoted by myeloid cell activity. (A) GSEA for enrichment of gene signatures reported to define specific immune cell infiltrates (immunomes) in TCGA colon cancers over-expressing LAMA5, LAMB1 and LAMC1. (B) Immunofluorescence staining for LAMA5 in HT29 liver metastases from control mice (top) and those treated with anti-Ly6G (1A8), an antibody that depletes neutrophils (lower). Scale bar represents 20 μm. (C) Quantification of the percentage of the liver metastatic area staining positive for LAMA5 in HT29 or HCT116 tumour-bearing control mice (green), or those depleted of neutrophils (pink). A minimum of five mice per group were included with at least three tumour areas quantified per mouse. (D) LAMA5 expression in cultured murine and human colon cancer cells after 24 h of culture in naïve or liver metastasis-derived CD11b⁺ myeloid cell-conditioned media. Error bars generated from biological triplicates. (E) Semi-quantitative analysis of the concentration of various cytokine proteins in the conditioned media from naïve (grey) or liver metastasis-derived (clear) CD11b⁺ myeloid cells determined using a commercially available antibody-based protein array. Error bars generated from biological triplicates. (F) LAMA5 expression in cultured murine and human colon cancer cells or the murine endothelial cell line 2H11, 24 h after stimulation with PBS (grey) or 100 ng/mL TNFα (clear). Error bars generated from biological triplicates. (G) Flow cytometry of NFκB-GFP reporter HT29 cells 24 h following treatment with PBS (top) or 100 ng/mL TNFα (below) demonstrating an increase in GFP expression in the later. (H) Percentage GFP⁺ HT29, HCT116 or LoVo cells 24 h following treatment with increasing concentrations of TNFα to a maximum concentration of 100 ng/mL. Error bars generated from biological triplicates. (I) Percentage GFP⁺ HCT116 or LoVo cells 24 h following treatment with TNFα and/or the NFκB inhibitor ML120B as indicated. (J) LAMA5 expression in HCT116 cells treated with 100 ng/mL TNFα with (grey) or without (green) 40 μm ML120B. Error bars generated from biological triplicates. Student’s t-test throughout. p < 0.05 considered significant. * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, *** p ≤ 0.001, ns: not significant.

Figure 4

Inhibition of tumour-derived LAMA5 inhibits the growth of colorectal liver metastases and promotes endothelial Notch signalling. (A) Western blotting for LAMA5 in Hct-116 and HT29 colon cancer cell lines transfected with non-targeting control (ctrl) or one of two LAMA5 shRNA constructs (sh1 or sh2). (B) Liver metastasis growth in Hct-116 ctrl and Hct-116-sh1 or Hct-116-sh2 cell lines. (C) Liver metastasis growth in HT29ctrl and HT29-sh1 or HT29-sh2 cell lines. (D) Immunofluorescence staining for the human LAMA5 protein in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. Scale bars represent 40 µm. (E) Immunofluorescence staining for CD31 in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. Scale bar represents 40 µm. (F) Quantification of total CD31⁺ area, total and average vessel length and vessel junction density in liver metastases developed using HT29ctrl or HT29-sh2 cell lines. (G) Immunofluorescence staining for vessel perfusion (tail vein injection of anti-CD31) in red and endothelial cells (immunohistochemistry for CD31) in green in HT29ctrl and HT29-sh2 liver metastases. Scale bars represent 40 µm. (H) Quantification of the percentage of perfused vessels in liver metastases developed using HT29ctrl and HT29-sh2 cell lines. (I) RNA expression of endothelial and immune cell genes in endothelial (CD31⁺) and non-endothelial (CD31^neg) cells MACS-separated from liver metastases generated in mice using HT29ctrl or HT29-sh2 cell lines. (J) Endothelial cell expression of genes involved in the regulation of angiogenesis presented as fold-change in expression in CD31⁺ cells MACS-separated from HT29-sh2 metastases relative to those from HT29ctrl metastases. (K) Western blotting for Hey2 protein in lysates derived from hepatic metastases developed using HT29ctrl or HT29-sh2 cell lines. (L) Blotting for LAMA5 in matrices derived from HT29 control and shLAMA5 cells following 7-day serum-starved culture with and without TNFα supplementation at 100 ng/mL. (M) Representative immunocytochemistry images of 2H11 endothelia following culture on matrices from (L) stained for the indicated Notch pathway proteins. (N,O) Quantification of HEY2 and NCID nuclear staining intensity in endothelia from (M). Analysis performed on at least 40 cells across experiments in triplicate. Student’s t-test throughout. p < 0.05 considered significant. * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, *** p ≤ 0.001, ns: not significant.