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CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy

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CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy Published OnlineFirst June 7, 2016; DOI: 10.1158/1078-0432.CCR-15-3115 Biology of Human Tumors Clinical Cancer Research CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy Shujie Zhao1, Chen Chen1, Katherine Chang1,2, Anand Karnad1,2, Jaishree Jagirdar3, Addanki P. Kumar4,5, and James W. Freeman1,2,5 Abstract Purpose: A subpopulation of pancreatic ductal adenocarci- Results: We identified an invasive cell population that gives rise noma (PDAC) cells is thought to be inherently resistant to to gemcitabine-resistant tumors. These cancer cells express a high chemotherapy or to give rise to tumor cells that become level of CD44 standard isoform and have an EMT phenotype resistant during treatment. Here we determined the role of (CD44s/EMT). In vivo, CD44s/EMT engraft and expand rapidly CD44 expression and its isoforms as a marker and potential and give rise to tumors that express high levels of CD44 isoforms target for tumor cells that give rise to invasive and gemcitabine- that contain multiple exon variants. CD44low-expressing cells resistant tumors. show continued sensitivity to gemcitabine in vivo and knockdown Experimental Design: RT-PCR, Western blotting, and DNA of CD44 in CD44s/EMT cells increases sensitivity to gemcitabine sequencing was used to determine CD44 isoform and expression and decreases invasiveness. levels. Flow cytometry was used to sort cells on the basis of their Conclusions: PDAC cells expressing high levels of CD44s with CD44 expression level. CD44 expression was knocked down a mesenchymal-like phenotype were highly invasive and devel- using shRNA. Tumorigenic properties were determined by clono- oped gemcitabine resistance in vivo. Thus, initial targeting CD44 genic and Matrigel assays, IHC, tumor growth in vivo using or reversing the CD44high phenotype may improve therapeutic luciferase imaging and by tumor weight. response. Clin Cancer Res; 1–13. Ó2016 AACR. Introduction (MET), which is believed to favor tumor engraftment and growth of cancer cells at metastatic sites (3, 5, 6). The phrase adaptive Heterogeneity of cancer cells within tumors and established plasticity explains how cancer cells gain selective growth and cancer cell lines is well known. The impact that this heterogeneity survival capabilities through phenotypic changes in response to on pathobiology and therapy has not been fully investigated. their environment. During the last few years, the role of epithelial Epithelial-to-mesenchymal transition (EMT) and acquired cancer cell plasticity in cancer development has gained more attention; stem cell properties are a major cause of cancer cell heterogeneity however, the mechanisms that initiate and enable phenotypic (1, 2). EMT represents an adaptive plasticity defined as loss of changes are not fully defined. membrane polarity and acquired migratory properties that is CD44, a nonkinase transmembrane receptor that binds hya- characteristic of mesenchymal cells (3). EMT is thought to play luronan (HA), has been found to play a role in the EMT process an important role in early cancer cell dissemination and metas- (7, 8). CD44 is a widely expressed adhesion molecule, con- tasis (3, 4). The development of metastasis is believed to require tributing to cell–cell and cell–matrix adhesion, cell growth, EMT, which enables cancer cells to migrate from their primary differentiation, and trafficking, and is highly expressed on stem tumor sites. However, recolonizing at distant sites may require cells(2,9).CD44isencodedby20exonsandundergoes redifferentiation or a mesenchymal-to-epithelial transition extensive alternative splicing to generate CD44 standard (CD44s) and CD44 variant (CD44v) forms. CD44s consists the first five exons (exons 1–5) and last five exons (exons 16– 1 Department of Medicine, Division of Medical Oncology, University of 20). The CD44 variable exons are typically numbered v1 to v10 Texas Health Science Center at San Antonio, San Antonio, Texas. 2Experimental and Developmental Therapeutics Program, Cancer (v1 is not encoded in human) corresponding to the genomic Therapy and Research Center, San Antonio, Texas. 3Department of exons 6–15 and are alternatively spliced and incorporated Pathology, University of Texas Health Science Center at San Antonio, between the exon 5 and exon 16, either as a single exon variant San Antonio, Texas. 4Department of Urology, University of Texas Health Science Center at San Antonio, San Antonio, Texas. 5Research or as a combination of several exons variant (7, 10). Functional and Development, Audie Murphy Veterans Administration Hospital, significance of CD44s and CD44v are not fully understood. San Antonio, Texas. Recent studies show aberrant levels and variant forms of CD44 Corresponding Author: James W. Freeman, Department of Medicine, Division of are expressed in a variety of tumor types including PDAC. High Medical Oncology, University of Texas Health Science Center at San Antonio, level of CD44 is thought to be associated with poor prognosis 7703 Floyd Curl Drive, San Antonio, TX 78229-3900; Phone: 210-567-5298; Fax: ofcancerandCD44v6andv3arereportedtoberelated 210-567-6687; E-mail: [email protected]. to cancer metastasis (9, 11–17). Studies show that CD44v6 doi: 10.1158/1078-0432.CCR-15-3115 expression is largely restricted to the advanced stages of tumor Ó2016 American Association for Cancer Research. progression and is more prevalent in metastatic than in www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 7, 2016; DOI: 10.1158/1078-0432.CCR-15-3115 Zhao et al. cells may show further plasticity by undergoing a CD44 isoform Translational Relevance change and a mesenchymal-to-epithelial (MET) switch. Chemo- Progression of PDAC after initial response to gemcitabine therapy or other environmental stresses may induce a CD44high treatment is attributed to the expansion of a subpopulation of -expressing cell population that promotes cell survival and inherently resistant tumor cells. It is important to understand increases invasiveness. Thus, targeting CD44high-expressing cells the biology of this resistant subpopulation of PDAC cells and may be an important strategy for increasing the response to to develop strategies for targeting these cells to prevent tumor chemotherapy. regrowth and progression. We found that gemcitabine treat- ment led to expansion of cells that express CD44. Here we Materials and Methods show the separation of a subpopulation of PDAC cells based Cell culture and materials on high expression level of CD44. This cell population Human pancreatic ductal adenocarcinomas (PDAC) cell lines expressed a high level of CD44 standard isoform, was mes- BxPC-3, Capan-2, MIA PaCa-2, CFPAC-1, Panc-1, AsPC-1 were enchymal-like, highly invasive, and gave rise to gemcitabine- from ATCC, UK Pan-1 was established in our laboratory (29), and resistant tumors that exhibited a CD44 isoform switch with HPNE (hTERT-immortalized human pancreas nestin expressing expression of multi-exon isoform variants of CD44. Knock- cell line) was obtained from Michel M. Ouellette (University of down of CD44 in CD44high cell resulted in cells that were more Nebraska Medical Center, Omaha, NE; ref. 30). Cells were main- sensitive to gemcitabine and less invasive. These studies sug- tained in medium as recommended and supplemented with 10% gest that high CD44 expression is a marker for gemcitabine FBS in a 37C, 5% CO incubator. The plasmid shCD44-2 pRRL resistance in PDAC and that targeting CD44high-expressing 2 was a gift from Bob Weinberg (Addgene plasmid # 19123), which cells may provide a therapeutic strategy for improving survival. targets all isoforms of human CD44 (31). PMMP-luciferase plas- mid was from Dr. Ricardo Aguiar (University of Texas Health Science Center, San Antonio, TX). The plasmids were transfected into human embryonic kidney 293T packaging cells (ATCC) using FuGENE 6 (Roche Applied Science) according to the manufac- nonmetastatic colorectal adenocarcinomas (18). Elevated turer's protocol. Cells were then infected with the viral medium expression of CD44v6 in pancreatic cancer appears to play a from the packaging cells 48 hours after transfection. The CD44 functional role in metastasis (13, 19). CD44 isoform switching knockdown cells were selected by GFP cell sorting (Flow Cyto- fromavariantformtoastandardformduringtheEMTprocess metry Core of University of Texas Health Science Center at San has been reported and CD44s is correlated to an EMT pheno- Antonio, TX). The luciferase-expressing clones were selected with type (1, 20, 21). We previously showed that treating a PDAC 300 mg/mL of G418. CD44 levels were determined by Western cell line BxPC-3 with gemcitabine induces cells to undergo an blotting analysis and luciferase expression was determined using EMT, to become more invasive and to show a phenotypic luciferase assay kit (Promega). Gemcitabine-HCl for Injection was switch from expressing mainly low levels of CD44v to expres- manufactured by Eli Lily and was dissolved in sterile saline (0.9% sing high levels of CD44s (22). This EMT/CD44s phenotype sodium chloride) at a concentration of 20 mg/mL. reverses after multiple passages following removal of gemcita- bine, suggesting that PDAC cells respond to chemotherapy by FACS and ALDEFLUOR assay adaptive plasticity and that CD44 may play a role in this Subconfluent cells were dissociated with trypsin and washed process.
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