Cancer Letters

Cancer Letters

Volume 424, 28 June 2018, Pages 9-18
Cancer Letters

Original Articles
The intestinal stem cell regulating gene ASCL2 is required for L1-mediated colon cancer progression

https://doi.org/10.1016/j.canlet.2018.03.022Get rights and content

Highlights

  • L1 induces the transcription factor and intestinal stem cell determinant Ascl2.

  • The mechanism involves increased β-catenin-Lef-1/TCF transactivation.

  • The induction of Ascl2 expression by L1 is required for colon cancer progression.

  • L1 and Ascl2 co-localize at the invasive edge of human colon cancer tissue.

Abstract

Aberrant Wnt/β-catenin signaling is a common event during human colorectal cancer (CRC) development. Previously, we characterized members of the L1 family of cell adhesion receptors as targets of β-catenin-LEF1/TCF transactivation that are expressed at the invasive CRC tissue edge. Overexpression of L1 in CRC cells confers enhanced motility, tumorigenesis and liver metastasis. We identified several downstream targets of L1-mediated signaling that are considered key intestinal stem cell signature genes. Here, we investigated the involvement of ASCL2, a Wnt target gene and key determinant of intestinal stem cell state, in L1-mediated CRC progression. In L1 overexpressing CRC cells we found an increase in ASCL2, a decrease in E-cadherin and accumulation of nuclear β-catenin, β-catenin-LEF1/TCF transactivation and target gene expression. The increase in ASCL2 by L1 overexpression enhanced ASCL2 target gene expression, conferred increased motility, tumorigenesis and metastasis, similar to L1 overexpression. Suppression of ASCL2 in cells expressing L1 blocked these tumorigenic properties. In human CRC tissue, ASCL2 was detected in the nuclei of cells at invasive areas of the tumor that also expressed L1. The results suggest that increased ASCL2 expression is a critical step in L1-mediated CRC progression.

Introduction

In multicellular organisms, cell and tissue morphogenesis are tightly regulated by the adhesive interactions between neighboring cells. Coordination between cell-cell adhesion-mediated signaling and gene expression is a characteristic feature of normal tissue homeostasis. Changes in this coordination are often detected in the later phases of cancer development, especially during the invasion and metastasis of cancer cells to distant organs. In our studies on the molecular basis of the changes in adhesion-mediated signaling that occur during cancer development, we focused on the Wnt/β-catenin pathway. β-catenin is a key determinant of cell-cell adhesion linking adhesion receptors to the cytoskeleton [1]. In addition, β-catenin also transduces the Wnt signal into the nucleus where it serves as a co-transcriptional activator (together with LEF1/TCF) of Wnt target genes [1,2]. The Wnt/β-catenin pathway is highly conserved during evolution as a central regulator of tissue homeostasis by regulating the stem cell compartment and hyperactivation of Wnt signaling is a hallmark of colorectal cancer (CRC) development [[2], [3], [4]]. In previous studies, we detected members of the L1 family of cell adhesion receptors (L1 and Nr-CAM) as novel target genes of Wnt/β-catenin signaling that are aberrantly activated during CRC progression [5,6]. L1 is mostly known for its multiple roles in adhesion and cell motility-related processes in the developing nervous system, including neurite elongation, axonal growth, fasciculation and path finding [7]. Inherited mutations in L1 are associated with severe brain developmental diseases including X-linked hydrocephalus, MASA syndrome and L1 syndrome [8,9]. We showed that L1 is induced in CRC cells at the invasive front of human CRC tissue [6] and its expression in CRC cells confers enhanced proliferation in the absence of serum, increased motility, invasion and liver metastasis [6,10,11]. Gene array analysis of human CRC tissue and L1-transfected human CRC cell lines revealed a number of genes induced by L1 via an L1-ezrin-NF-κB signaling pathway [12], including IGFBP2 [13], SMOC2 [14] and CD10 [15], or a pathway involving STAT-1 in the case of CLU [16]. Interestingly, we found that some of these L1-induced genes in CRC cells are also preferentially localized in the intestinal stem cell compartment [13,14,16], at the bottom of normal colonic crypts, and were identified as intestinal stem cell signature genes [17,18]. In the normal intestine and colon, the pit-like recessions of the epithelium, known as crypts, contain a small population of stem cells at the crypt bottom, and these cells are characterized by specifically expressing the LGR5 gene (a Wnt target gene) [19,20]. The Lgr5+ stem cells generate all types of intestinal cell lineages in the mouse, and an inducible activation of Wnt signaling in stem cells leads to adenoma formation by Lgr5+ stem cells, strongly implicating that these cells are responsible for the initiation of CRC development [21]. In this study, we examined whether the Wnt target gene and transcription factor Achete scute-like 2 (ASCL2), that is a key regulator of stemness and is exclusively expressed in Lgr5+ intestinal stem cells [22,23], is involved in L1-mediated CRC progression.

Section snippets

Cell culture, transfections, cell proliferation and motility assays

The cell lines HEK 293T, LS 174T, DLD-1 and HCT 116 were obtained from ATTC (Biological Industries, Beit Haemek, Israel) and grown as described [10]. LS 174T-L1, DLD-1-L1, HCT 116-L1 cells were maintained in medium containing neomycin (800 μg/ml) and LS 174T-L1+shAscl2 and LS 174T-L1+shp65 cells were cultured in medium containing both neomycin (800 μg/ml) and puromycin (10 μg/ml). LS 174T-Ascl2 cells were maintained in medium containing zeocin (50 μg/ml). Transient transfection of HEK 293T

L1 induces the expression of ASCL2 in CRC cells and the suppression of ASCL2 in cells transfected with L1 reduces their growth, motile, tumorigenic and metastatic abilities

To determine whether the expression of ASCL2 is affected by L1 in human CRC cells, the levels of ASCL2 RNA and protein were determined in various human CRC cell lines (LS 174T, HCT 116 and DLD-1) stably overexpressing either L1, or the empty vector (pcDNA3). The results shown in Fig. 1A, demonstrate that ASCL2 RNA levels were increased between 8 and 17-fold in LS 174T CRC cell clones stably transfected with L1. The levels of ASCL2 protein were also elevated in nuclear extracts of such

Discussion

In the present study, we have shown that ASCL2, a transcription factor and Wnt target gene that regulates intestinal stem cells [22,23], is elevated during L1-mediated induction of CRC cell invasion and metastasis. Suppression of endogenous ASCL2 levels in CRC cells overexpressing L1, blocked the tumorigenic properties conferred by L1 in such cells. Forced expression of ASCL2 (on its own) in CRC cells induced increased motility, tumorigenesis and liver metastasis. The induction of ASCL2 by L1

Conclusion

We have shown that ASCL2 is induced during L1-mediated CRC progression by a mechanism involving downregulation of E-cadherin-mediated adhesion and increased β-catenin-LEF/TCF transactivation of target genes. Future studies should be directed towards targeting the levels of ASCL2 in CRC aiming to suppress the development of the invasive-metastatic phenotype.

Declarations of interest

None.

Conflicts of interest

The authors declare no conflict of interest.

References (40)

  • H. Clevers et al.

    Stem cell signaling: an integral program for tissue renewal and regeneration: Wnt signaling and stem cell control

    Science

    (2014)
  • S. Basu et al.

    Wnt signaling in cancer stem cells and colon cancer metastasis, F1000

    Res.

    (2016)
  • M.E. Conacci-Sorrell et al.

    Nr-CAM is a target gene of the beta-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis

    Genes Dev.

    (2002)
  • N. Gavert et al.

    L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers

    J. Cell Biol.

    (2005)
  • N. Gavert et al.

    Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis

    Cancer Res.

    (2007)
  • G. Haase et al.

    The Wnt target gene L1 in colon cancer invasion and metastasis

    Cancers

    (2016)
  • N. Gavert et al.

    Nuclear factor-kappaB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells

    J. Cell Sci.

    (2010)
  • A. Ben-Shmuel et al.

    Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-kappaB signaling that promotes colon cancer progression

    Oncogene

    (2013)
  • A. Shvab et al.

    Induction of the intestinal stem cell signature gene SMOC-2 is required for L1-mediated colon cancer progression

    Oncogene

    (2016)
  • G. Haase et al.

    A point mutation in the extracellular domain of L1 blocks its capacity to confer metastasis in colon cancer cells via CD10

    Oncogene

    (2016)
  • Cited by (19)

    View all citing articles on Scopus

    This study was supported by grants from the Israel Science Foundation and the Israel Cancer Research Fund

    View full text