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Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study.

ABSTRACT: BACKGROUND: Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. METHODS: We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. RESULTS: Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. CONCLUSION: Tumor suppressor protein SMAR1 might be used as a phenotypic differentiation marker between cancerous and non-cancerous cells.

SUBMITTER: Kaul-Ghanekar R

PROVIDER: S-EPMC2765988 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study.

Kaul-Ghanekar Ruchika R Singh Sandeep S Mamgain Hitesh H Jalota-Badhwar Archana A Paknikar Kishore M KM Chattopadhyay Samit S

BMC cancer 20091002

<h4>Background</h4>Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections.<h4>Methods</h4>We validated knockdown and overexpression of SMA ...[more]

PMID: 19799771

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Project description:BackgroundHighly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1.MethodsImmunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed.ResultsThe expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner.ConclusionsSMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.

Project description:Introduction: Cancer stem cells (CSC), a major culprit of drug-resistant phenotypes and tumor relapse, represent less than 2 % of the bulk of TNBC cells, making them difficult to isolate, study, and thus, limiting our understanding of the pathogenesis of the disease. Current methods for CSC enrichment, such as 3D spheroid culture, genetic modification, and stem cell conditioning, are time consuming, expensive, and unsuitable for high-throughput assays. One way to address these limitations is to use topographical stimuli to enhance CSC populations in planar culture. Physical cues in the breast tumor microenvironment can influence cell behavior through changes in the mechanical properties of the extracellular matrix (ECM). In this study, we used topographical cues on polystyrene films to investigate their effect on the proteome and stemness of standard TNBC cell lines. Methods: The topographical polystyrene-based array was generated using razor printing and polishing methods. Proteome data were analyzed and enriched bioprocesses were identified using R software. Stemness was assessed measuring CD44, CD24 and ALDH markers using flow cytometry, immunofluorescence, detection assays, and further validated with mammosphere assay. EGF/EGFR expression and activity was evaluated using enzyme-linked immunosorbent assay (ELISA), immunofluorescence and antibody membrane array. A dose-response assay was performed to further investigate the effect of surface topography on the sensitivity of cells to the EGFR inhibitor. Results: Surface roughness enriched the CSC population and modulated epidermal growth factor receptor (EGFR) signaling activity in TNBC cells. Enhanced proliferation of MDA-MB-468 cells in roughness correlated with upregulation of the epidermal growth factor (EGF) ligand, which in turn corresponded with a 3-fold increase in the expression of EGFR and a 42% increase in its phosphorylation compared to standard smooth culture surfaces. The results also demonstrated that phenotypic changes associated with topographical (roughness) stimuli significantly decreased the drug sensitivity to the EGFR inhibitor gefitinib. In addition, the proportion of CD44+/CD24-/ALDH+ was enhanced on surface roughness in both MDA-MB-231 and MDA-MB-468 cell lines. We also demonstrated that YAP/TAZ activation decreased in a roughness-dependent manner, confirming the mechanosensing effect of the topographies on the oncogenic activity of the cells. Discussion: Overall, this study demonstrates the potential of surface roughness as a culture strategy to influence oncogenic activity in TNBC cells and enrich CSC populations in planar cultures. Such a culture strategy may benefit high-throughput screening studies seeking to identify compounds with broader tumor efficacy.

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Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study.

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Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study.

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