Elsevier

Biochimie

Volume 174, July 2020, Pages 107-116
Biochimie

Knockdown of the α5 laminin chain affects differentiation of colorectal cancer cells and their sensitivity to chemotherapy

https://doi.org/10.1016/j.biochi.2020.04.016Get rights and content

Highlights

  • Knockdown of the α5 laminin chain induced partial dedifferentiation of HT29 cells.

  • Dedifferentiation of HT29 cells was due to Wnt and mTORC1 signaling changes.

  • Knockdown of the α5 laminin chain was associated with ER-stress activation.

  • ER-stress facilitated sensitivity of HT29 cells to 5-fluorouracil.

Abstract

The interaction of tumor cells with the extracellular matrix (ECM) may affect the rate of cancer progression and metastasis. One of the major components of ECM are laminins, the heterotrimeric glycoproteins consisting of α-, β-, and γ-chains (αβγ). Laminins interact with their cell surface receptors and, thus, regulate multiple cellular processes. In this work, we demonstrate that shRNA-mediated knockdown of the α5 laminin chain results in Wnt- and mTORC1-dependent partial dedifferentiation of colorectal cancer cells. Furthermore, we showed that this dedifferentiation involved activation of ER-stress signaling, pathway promoting the sensitivity of cells to 5-fluorouracil.

Introduction

Laminins is a family of cell adhesion glycoproteins capable to assemble into αβγ trimers, which are major components of the basement membranes [1]. The interaction of laminins with cells is mediated mainly by binding of C-terminal globular domains of their α chains to various laminin-specific cell surface receptors, including integrins, Lutheran protein, 67 kDa laminin receptor, α-dystroglycan and some others, which leads to transmitting downstream signals relevant for the maintenance of both normal and tumor cells functioning. Besides, laminins play various roles in certain stages of the metastasis process [2]. Moreover, for many tumor types, the expression levels of individual laminin chains have been shown to carry prognostic significance [3]. For instance, in case of colorectal cancer, an increase in the ratio of expression levels of genes encoding α4 and α5 laminin chains (LAMA4/LAMA5) was associated with a poor prognosis [4]. Importantly, the changes in the laminin composition may both affect physical properties of basement membrane (for example, by forming denser and more rigid polymer “network” due to increased content of the α5 chain [2]), and tumor cell properties due to formation of more tight intercellular contacts [5]. Finally, changes in the expression profile of laminin chains have been also observed during epithelial-mesenchymal transition (EMT) [2,6,7]. Accordingly, α5 laminin chain has been previously shown to participate in the morphogenesis and differentiation of the epithelium in mouse small intestine [8].

Under standard culture conditions, human colorectal cancer cells HT29 are non-polarized, but carry a potential to differentiate. These cells form so-called “flat-foci” characterized predominantly by the epithelial phenotype, although some mesenchymal traits are also preserved [9]. Another feature of HT29 line is its heterogeneity, since it is comprised of cells producing a mucin-like matrix, as well as other the cells capable to differentiate into enterocytes of the small intestine [10]. Interestingly, the differentiation of HT29 cells is reversible, indicating their plasticity (the ability to change properties along the axis of “undifferentiated — fully differentiated cells”) - the property which contributes to metastatic spread of tumor cells [[11], [12], [13]]. Extracellular matrix (ECM) plays a substantial role in this process [[14], [15], [16]].

In this work, we aimed to investigate the effects of α5 laminin chain knockdown in HT29 colon cancer cells and observed the changes in the transcriptome and proteome profiles pointing at their partial dedifferentiation after the α5 laminin chain knockdown which was accompanied by an increase in the sensitivity to 5-fluorouracil.

Section snippets

Cell culture and generation of LAMA5 knockdown HT-29 cells

The routine culturing of the colorectal adenocarcinoma HT-29 cells as well as cells transduced by lentiviral particles containing either LAMA5 shRNA or control scrambled shRNA was performed as described previously [7]. The HT-29 cells were additionally tested for mycoplasma contamination using the MycoReport PCR kit (Evrogen, Russia). DNA oligonucleotides containing the LAMA5 shRNA sequences (shLAMA5) flanked by BamHI and EcoRI sites (Table 1) were chemically synthesized by Evrogen (Russia).

Knockdown of LAMA5 gene decrease the proliferation of HT29 cells

The HT29 cell line is a standard model in colorectal cancer research. We have transduced the HT29 cells by lentiviral constructs encoding LAMA5 gene shRNAs. Out of three different shRNAs used (Table 1), only one (shLAMA5#3) mediated significant downregulation of LAMA5 mRNA expression (1.7-fold decrease, Fig. 1A). To evaluate the efficiency of the LAMA5 knockdown on a protein level, both the intracellular and basement membrane fractions of laminin α5 chain protein were quantified. In HT-29 cells

Discussion

Malignant transformation of human tissues is initiated by genetic aberrations, whereas the rate of disease progression and the metastatic process highly dependent on the interaction of tumor cells with their cellular and non-cellular microenvironment [15]. In particular, tumors actively remodel ECM, which then promotes pathogenesis and compromises treatment efficacy. Further, ECM interaction with integrins elicits the signaling essential for the maintenance and differentiation of adult stem

Funding

The study was supported by the Russian Science Foundation (project 17-14-01338).

Availability of data and materials

The microarray datasets generated and analyzed during the current study are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7754.

Author contributions

Conception and design of the experiments: A. Tonevitsky, S. Rodin, D. Maltseva; collection of the data: D. Maltseva, M. Raygorodskaya, E. Knyazev, V. Zgoda, O. Tikhonova; proteome data processing: S. Nikulin; analysis and interpretation of the data: D. Maltseva, S. Zaidi, A. Baranova, A. Tonevitsky; writing—original draft preparation: D. Maltseva; critically revising the article: A. Baranova, S. Rodin, A. Turchinovich, A. Tonevitsky. All authors have read and agreed to the published version of

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgments

The authors gratefully acknowledge the assistance of the “Human Proteome” Core Facility (Institute of Biomedical Chemistry, Moscow, Russia) in proteomic data generation.

References (75)

  • K. Maiese

    Novel stem cell strategies with mTOR

    Mol. Med. MTOR

    (2016)
  • A. Philpott et al.

    Lineage selection and plasticity in the intestinal crypt

    Curr. Opin. Cell Biol.

    (2014)
  • K. Kratochvílová et al.

    The role of the endoplasmic reticulum stress in stemness, pluripotency and development

    Eur. J. Cell Biol.

    (2016)
  • J.A. Makarova et al.

    Intracellular and extracellular microRNA: an update on localization and biological role

    Prog. Histochem. Cytochem.

    (2016)
  • A. Pozzi et al.

    The nature and biology of basement membranes

    Matrix Biol.

    (2017)
  • S.F. Parsons et al.

    Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind α5 chain-containing human laminin with high affinity

    Blood

    (2001)
  • R. Nishiuchi et al.

    Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant ??3??1, ??6??1, ??7??1 and ??6??4 integrins

    Matrix Biol.

    (2006)
  • L. Seguin et al.

    Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance

    Trends Cell Biol.

    (2015)
  • D. Senft et al.

    UPR, autophagy, and mitochondria crosstalk underlies the ER stress response

    Trends Biochem. Sci.

    (2015)
  • A.Y. Choo et al.

    Mind the GAP: Wnt steps onto the mTORC1 train

    Cell

    (2006)
  • T.K. Noah et al.

    Intestinal development and differentiation

    Exp. Cell Res.

    (2011)
  • J.-E. Oh et al.

    α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

    Exp. Cell Res.

    (2009)
  • V. Rausch et al.

    The Hippo pathway, YAP/TAZ, and the plasma membrane

    Trends Cell Biol.

    (2020)
  • A. Takaguri et al.

    The protective role of YAP1 on ER stress-induced cell death in vascular smooth muscle cells

    Eur. J. Pharmacol.

    (2017)
  • A. Domogatskaya et al.

    Functional diversity of laminins

    Annu. Rev. Cell Dev. Biol.

    (2012)
  • D.V. Maltseva et al.

    Laminins in metastatic cancer

    Mol. Biol.

    (2018)
  • V.V. Galatenko et al.

    Cumulative prognostic power of laminin genes in colorectal cancer

    BMC Med. Genom.

    (2018)
  • I. Pastushenko et al.

    Identification of the tumour transition states occurring during EMT

    Nature

    (2018)
  • D.V. Mal’tseva et al.

    Effects of laminins 332 and 411 on the epithelial—mesenchymal status of colorectal cancer cells

    Bull. Exp. Biol. Med.

    (2019)
  • Z.X. Mahoney et al.

    Laminin alpha 5 influences the architecture of the mouse small intestine mucosa

    J. Cell Sci.

    (2008)
  • D.V. Maltseva et al.

    Epithelial to mesenchymal transition marker in 2D and 3D colon cancer cell cultures in the presence of laminin 332 and 411

    Mol. Biol.

    (2019)
  • C.L. Chaffer et al.

    A perspective on cancer cell metastasis

    Science

    (2011)
  • J.L. Leight et al.

    Extracellular matrix remodeling and stiffening modulate tumor phenotype and treatment response

    Annu. Rev. Cell Biol.

    (2017)
  • D.V. Maltseva et al.

    The effect of laminins on chemoresistance of colorectal cancer cells

    Russ. Chem. Bull.

    (2018)
  • N.A. Krainova et al.

    Evaluation of potential reference genes for qRT-PCR data normalization in HeLa cells

    Appl. Biochem. Microbiol.

    (2013)
  • M.Y. Shkurnikov et al.

    Role of L1CAM in the regulation of the canonical Wnt pathway and class I MAGE Genes

    Bull. Exp. Biol. Med.

    (2016)
  • M.Y. Shkurnikov et al.

    Expression of stroma components in the lymph nodes affected by prostate cancer metastases

    Mol. Biol.

    (2018)
  • Cited by (0)

    View full text