Original ArticlesThe intestinal stem cell regulating gene ASCL2 is required for L1-mediated colon cancer 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)
- et al.
Linking colorectal cancer to Wnt signaling
Cell
(2000) - et al.
L1-mediated axon outgrowth occurs via a homophilic binding mechanism
Neuron
(1989) - et al.
Errors in corticospinal axon guidance in mice lacking the neural cell adhesion molecule L1
Curr. Biol.
(1998) - et al.
Mutations in the cell adhesion molecule L1 cause mental retardation
Trends Neurosci.
(1995) - et al.
The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse
Cell Stem Cell
(2011) The intestinal crypt, a prototype stem cell compartment
Cell
(2013)- et al.
Transcription factor achete scute-like 2 controls intestinal stem cell fate
Cell
(2009) - et al.
Ascl2 acts as an R-spondin/Wnt-responsive switch to control stemness in intestinal crypts
Cell Stem Cell
(2015) - et al.
A positive regulatory loop between a Wnt-regulated non-coding RNA and ASCL2 controls intestinal stem cell fate
Cell Reports
(2016) - et al.
The cadherin-catenin adhesion system in adhesion, signaling and cancer
J. Clin. Invest
(2002)
Stem cell signaling: an integral program for tissue renewal and regeneration: Wnt signaling and stem cell control
Science
Wnt signaling in cancer stem cells and colon cancer metastasis, F1000
Res.
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.
L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers
J. Cell Biol.
Expression of L1-CAM and ADAM10 in human colon cancer cells induces metastasis
Cancer Res.
The Wnt target gene L1 in colon cancer invasion and metastasis
Cancers
Nuclear factor-kappaB signaling and ezrin are essential for L1-mediated metastasis of colon cancer cells
J. Cell Sci.
Global analysis of L1-transcriptomes identified IGFBP-2 as a target of ezrin and NF-kappaB signaling that promotes colon cancer progression
Oncogene
Induction of the intestinal stem cell signature gene SMOC-2 is required for L1-mediated colon cancer progression
Oncogene
A point mutation in the extracellular domain of L1 blocks its capacity to confer metastasis in colon cancer cells via CD10
Oncogene
Cited by (19)
TET2–BCLAF1 transcription repression complex epigenetically regulates the expression of colorectal cancer gene Ascl2 via methylation of its promoter
2022, Journal of Biological ChemistryCitation Excerpt :It is important to identify the regulatory mechanisms and signaling pathways involved in CRC progenitor cells to develop novel reagents to target the refractory CRC progenitor cell population (45). Ascl2 can be regulated by the hpo/Mst signaling pathway, as well as the Tssc3, HIF 1α and 2α, L1 family of cell adhesion receptors, and through Ascl2 autoregulation (9, 46–50). Although it was reported that aberrant upregulation of Ascl2 by promoter demethylation promotes the growth and resistance to 5-fluorouracil of gastric cancer cells (51), there is no available report about the epigenetic regulatory mechanism regulating abnormal expression of Ascl2 in CRC cells.
Zymosan-A promotes the regeneration of intestinal stem cells by upregulating ASCL2
2022, Cell Death and Disease
- ☆
This study was supported by grants from the Israel Science Foundation and the Israel Cancer Research Fund