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