Cancer Letters

Cancer Letters

Volume 476, 28 April 2020, Pages 161-169
Cancer Letters

Original Articles
Laminin 521 enhances self-renewal via STAT3 activation and promotes tumor progression in colorectal cancer

https://doi.org/10.1016/j.canlet.2020.02.026Get rights and content

Highlights

  • Laminin 521 (LN521) promotes self-renewal and invasion in colorectal cancer cells.

  • Integrin α3β1, α6β1 and activation of STAT3 signaling are instrumental to Laminin 521-driven promotion of self-renewal.

  • Laminin alpha-5 expression is correlated with self-renewal frequency in patient-derived colorectal cancer cells.

  • Laminin alpha-5 is detected within cancer stem cell-rich areas in human and mouse liver metastases.

  • High LN521 expression is significantly associated with poor clinical outcome in several colorectal cancer patient cohorts.

  • LN521 contributes to metastasis progression in colorectal cancer by promoting invasion and self-renewal.

Abstract

Remodeling of basement membrane proteins contributes to tumor progression towards the metastatic stage. One of these proteins, laminin 521 (LN521), sustains embryonic and induced pluripotent stem cell self-renewal, but its putative role in cancer is poorly described. In the present study we found that LN521 promotes colorectal cancer (CRC) cell self-renewal and invasion. siRNA-mediated knockdown of endogenously-produced laminin alpha 5, as well as treatment with neutralizing antibodies against integrin α3β1 and α6β1, were able to reverse the effect of LN521 on self-renewal. Exposure of CRC cells to LN521 enhanced STAT3 phosphorylation, and incubation with STAT3 inhibitors Napabucasin and Stattic was sufficient to block the LN521-driven self-renewal increase. Robust expression of laminin alpha 5 was detected in 7/10 liver metastases tissue sections collected from CRC patients as well as in mouse liver metastasis xenografts, in most cases within areas expressing metastasis cancer stem cell markers such as c-KIT and CD44v6. Finally, retrospective analysis of multiple CRC datasets highlighted the significant association between high LN521 mRNA expression and poor clinical outcome in colorectal cancer patients. Collectively our results indicate that high Laminin 521 expression is a frequent feature of metastatic dissemination in CRC and that it promotes cell invasion and self-renewal, the latter through engagement of integrin isoforms and activation of STAT3 signaling.

Introduction

Although clinical outcomes have steadily improved over the last few decades for patients with colorectal cancer, survival remains poor for those who have developed metastatic disease to distant sites such as the liver, lung or peritoneum [1]. Characterizing cellular and molecular processes that drive metastasis progression therefore represents an essential step to better prevent or target metastatic disease. Previous studies have demonstrated that the development of metastases requires tumor cells to display invasive properties, enabling their dissemination from primary sites and transit through the lymphatic and/or blood stream, as well as survival and self-renewing properties that will drive their ability to initiate metastasis growth and colonize the distant site [2,3]. Invasion and self-renewal are both sensitive to microenvironmental cues provided by cellular and non-cellular actors of the microenvironment. Among the latter, extracellular matrix molecules have been described as instrumental modulators of cell motility, invasion, and stemness [4]. In particular, remodeling of basement membrane proteins such as laminins during cancer development is thought to alter interaction of these proteins with receptors located at the surface of cancer cells, thereby leading to phenotypic modifications [5].

Laminins are large multimeric basement membrane proteins, with functional units constituted through the assembly of one alpha, one beta and one gamma isoform [6]. In the present work we analyzed the contribution of laminin alpha 5 (LAMA5), and more specifically of the laminin-521 isoform (LN521, constituted by one alpha 5, one beta-2 and one gamma-1 chain), in the regulation of self-renewal and invasion of colorectal cancer cells. Laminin-521 (LN521) was previously shown to be important in the maintenance of embryonic and induced-pluripotent stem cells [7,8], and we hypothesized that exposure to LN521 could also promote self-renewal in colorectal cancer cells. Since long-term self-renewal is a hallmark of cancer stem cells (CSCs) and contributes to their driving role in cancer progression and poor clinical outcomes [9,10], we also investigated whether LN521 may play a role in regulating phenotypic and clinical features that are classically associated with metastatic development, such as cell migration, invasion and patient survival.

Section snippets

Materials and methods

Immunohistochemical staining, Extreme Limiting Dilution Analysis (ELDA), Protein and RNA quantitation, in vivo liver metastasis induction in immunocompromised mice, as well as analysis of associations between laminin expression and CRC patient survival, were performed as previously described [11,12]. Details for these procedures are provided under Supplemental Materials and Methods.

Laminin-521 promotes self-renewal and invasion of CRC cells

To determine whether exposure to LN521 impacted on the self-renewal of colorectal cancer cells, we first exposed human CRC cells to various laminin isoforms and subsequently resuspended and seeded them into an extreme limiting dilution analysis (ELDA) in suspension. The ability of these cells to generate at least one colonosphere per well under all limiting dilution conditions was quantified and the frequency of self-renewing cells was calculated as described previously [11,13] using the //bioinf.wehi.edu.au/software/elda/

Discussion

In the present study we characterized the promoting role of laminin-521 on colorectal cancer cell self-renewal and invasion and found that endogenous production of the laminin alpha 5 chain contributed to this effect. We demonstrated that integrins α3β1 and α6β1 are likely mediators of the self-renewal promotion induced by laminin alpha 5 and that downstream activation of STAT3 signaling is required for the promotion of self-renewal by LN521. Finally, we also found that colorectal cancer liver

Funding

This work was supported by the National Health and Medical Research Council of Australia [grants number GNT1049561 and GNT1069024].

CRediT authorship contribution statement

Yan Qin: Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing - original draft. Carolyn Shembrey: Investigation, Validation, Writing - review & editing. Jai Smith: Investigation, Validation, Visualization. Sophie Paquet-Fifield: Investigation, Methodology. Corina Behrenbruch: Methodology, Resources. Laura M. Beyit: Investigation. Benjamin N.J. Thomson: Resources. Alexander G. Heriot: Resources, Conceptualization. Yuan Cao: Supervision, Formal analysis, Writing

Declaration of competing interest

The authors have no conflict of interest to declare in relation with this work.

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