A Necessary Role for Increased Biglycan Expression during L1-Mediated Colon Cancer Progression

Abstract

Aberrant activation of Wnt/β-catenin signaling and downstream β-catenin-TCF target genes is a hallmark of colorectal cancer (CRC) development. We identified the immunoglobulin-like cell adhesion receptor L1CAM (L1) as a target of β-catenin-TCF transactivation in CRC cells. Overexpression of L1 in CRC cells confers enhanced proliferation, motility, tumorigenesis, and liver metastasis, and L1 is exclusively localized at invasive areas of human CRC tissue. Several genes are induced after L1 transfection into CRC cells by a mechanism involving the L1-ezrin-NF-κB pathway. We conducted a secretomic analysis of the proteins in the culture medium of L1-overexpressing CRC cells. We detected a highly increased level of biglycan, a small leucine-rich ECM component, and a signaling molecule. We found that induction of biglycan is required for the cellular processes conferred by L1, including enhanced proliferation, motility, tumorigenesis, and liver metastasis. The suppression of endogenous biglycan levels or a point mutation in the L1 ectodomain that regulates cell–cell adhesion mediated by L1 blocked the enhanced tumorigenic properties conferred by L1. The mechanism of biglycan induction by L1 involves the L1-NF-κB pathway. Blocking NF-κB signaling in L1 expressing cells suppressed the induction of biglycan and the tumorigenic properties conferred by L1. Biglycan expression was undetectable in the normal colonic mucosa, but expressed at highly increased levels in the tumor tissue, especially in the stroma. The therapeutic strategies to target biglycan expression might provide a useful approach for CRC treatment in L1-overexpressing tumors.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1

“…RNA isolation from cells was performed using the Bio-Tri reagent (Bio-Lab, Jerusalem, Israel), in accordance with the manufacturer’s protocol. First-strand cDNA synthesis from the isolated RNA was carried out using a SuperScript™ II Reverse Transcriptase kit (Thermo Fisher Scientific, Waltham, MA, USA), in the manner suggested by the manufacturer [ 13 ]. Supplementary Table S1 describes the primers used for the amplification of target genes.…”

“…Five-week-old male athymic nude mice (Foxn-1u) were used to determine subcutaneous tumor growth, as described in [ 4 , 13 ]. In summary, 3 × 10 6 cells suspended in 100 µL of PBS were injected at different sites into the flanks of the mice [ 5 ].…”

“…Metastasis was determined by examining the ability of CRC cells to migrate from the spleen to the liver in athymic male nude mice after injecting 3 × 10 6 cells suspended in 20 µL of PBS into the distal tip of the spleen, as described in [ 4 , 13 ]. To anesthetize the mice, Xylazine and Ketamine were injected into the peritoneum during the process.…”

“…A key downstream signaling mechanism involved in L1-mediated tumorigenesis is an L1–ezrin–NF-κB pathway that includes the activation of several genes which, when expressed in CRC cells, can mimic the effects conferred by L1 overexpression [ 5 , 6 ]. The identification of the pro-tumorigenic properties of such genes by transcriptomic, proteomic, and secretomic analyses demonstrated their increased levels after L1 expression in CRC cells [ 1 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. Since tumor progression includes, in addition to the induction of pro-tumorigenic genes, the downregulation of tumor suppressor genes, we wished to investigate the possibility that the L1-mediated CRC progression also involves the inhibition of tumor suppressors.…”

ZNF133 is a potent suppressor in breast carcinogenesis through dampening L1CAM, a driver for tumor cell invasion

“…The control of L1CAM expression in cancers is complex and is affected by both transcriptional and epigenetic mechanisms. Positive regulation of L1CAM was reported for the transcription factors β-catenin and SLUG in colon, endometrial and pancreatic cancer cells (19,24), and negative regulation of L1CAM was found for the AR and REST in triple-negative breast cancers and small cell lung cancer (25,26).…”