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Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma.

ABSTRACT: Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines. Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS). Results: Both CD44v^high/ALDH^high and CD44v^high/ALDH^low cells were enriched in cells with CSC characteristics, with the CD44v^high/ALDH^low cells being more proliferative and more resistant to chemotherapeutics, whereas CD44v^high/ALDH^high cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44v^high/ALDH^low cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44v^high/ALDH^high and CD44v^high/ALDH^low cells. CD44v^high expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression. Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44v^high/ALDH^low cells and others to CD44v^high/ALDH^high cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

SUBMITTER: Nishino M

PROVIDER: S-EPMC5556640 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma.

Nishino Makoto M Ozaki Mari M Hegab Ahmed E AE Hamamoto Junko J Kagawa Shizuko S Arai Daisuke D Yasuda Hiroyuki H Naoki Katsuhiko K Soejima Kenzo K Saya Hideyuki H Betsuyaku Tomoko T

Journal of Cancer 20170701 10

<b>Background:</b> Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines. <b>Methods:</b> W ...[more]

PMID: 28819374

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Project description:The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential.The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44(+) cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44(+) cells consisted of mixed CD44(+) and CD44(-) cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44(+) and CD44(+) cells derived from CD44(+)-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44(-) cells. Furthermore, freshly sorted CD44(+) cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44(-) cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas.Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.

Project description:Manganese superoxide dismutase (MnSOD) promotes invasive and migratory activities by upregulating Forkhead box protein M1 (FoxM1) expression. The present study investigated whether modulation of MnSOD and FoxM1 expression was responsible for the antitumor effects of genistein on cancer stem-like cells (CSLCs) derived from non-small cell lung cancer cells (NSCLCs). Spheroids prepared from H460 or A549 cells were defined as lung cancer stem-like cells (LCSLCs) and were treated with genistein. The Cell Counting Kit-8 assay was performed to assess human lung fibroblast IMR-90 cell proliferation, as well as NSCLC H460 and A549 cell proliferation following treatment with genistein. MnSOD, FoxM1, cluster of differentiation (CD)133, CD44, BMI1 proto-oncogene, polycomb ring finger (Bmi1) and Nanog homeobox (Nanog) protein expression levels were examined via western blotting. The sphere formation assay was conducted to evaluate LCSLC self-renewal potential, and LSCLC migratory and invasive activities were analyzed using the wound healing and Transwell invasion assays, respectively. Knockdown and overexpression of MnSOD and FOXM1 via short hairpin-RNA or cDNA transfection were performed. The results indicated that genistein (80 and 100 µM) suppressed H460 and A549 cell viability compared with IMR-90 cells. Sub-cytotoxic concentrations of genistein (20 and 40 µM) inhibited sphere formation activity and decreased the protein expression levels of CD133, CD44, Bmi1 and Nanog in LCSLCs compared with the control group. Genistein also suppressed the migratory and invasive activities of LCSLCs compared with the control group. MnSOD and FoxM1 overexpression antagonized the effects of genistein (40 µM), whereas MnSOD and FoxM1 knockdown enhanced the inhibitory effects of genistein (20 µM) on CSLC characteristics of LCSLCs. Overall, the results suggested that genistein suppressed lung cancer cell CSLC characteristics by modulating MnSOD and FoxM1 expression levels.

Dataset Information

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma.

Publications

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma.

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