CD44s Downregulation Research Articles

The novel interplay between CD44 standard isoform and the caspase-1/IL1B pathway to induce hepatocellular carcinoma progression

Accumulating data indicate caspase-1 (CASP1), one of the inflammatory caspases, promotes hepatocellular carcinoma (HCC) progression in tumor proliferation, invasion, EMT phenotype and sorafenib resistance. However, the molecular basis of regulating caspase-1 expression and caspase-1/IL1B (interleukin-1β) pathway in HCC remains unclear. Here, we demonstrated the novel interplay between caspase-1/IL1B activation and cluster differentiation 44 standard isoform (CD44s) in HCC. In this study, we observed that CD44s is responsible for caspase-1/IL1B activation both in HCC tissues and five HCC cell lines. In normoxia conditions, CD44s knockdown repressed the activation of caspase-1/IL1B via stimulating AMPK-mediated autophagy. Moreover, our data suggested that p62-induced autophagic degradation of caspase-1 accounted for caspase-1/IL1B inactivation in CD44s deficient cells. Administration of recombinant human IL1B could rescue impaired proliferation, invasion, and EMT phenotype in CD44s deficient HCC cells. Lastly, hypoxia-mediated caspase-1/IL1B overexpression could be abolished by CD44s downregulation through decreasing HIF1A and enhancing autophagic activity. Overall, targeting CD44s is a novel inhibitory mechanism of caspase-1/IL1B expression, both in normoxia and hypoxia conditions.

Comparative expression of immunohistochemical biomarkers in cribriform and pattern 4 non-cribriform prostatic adenocarcinoma

The morphology of Gleason 4 prostate cancer (PCa) can be subdivided into cribriform and non-cribriform patterns. A large body of evidence has shown that pattern 4 cribriform PCa (especially non-glomeruloid type) is associated with adverse pathologic features and clinical outcomes compared with non-cribriform pattern 4 PCa. The underlying mechanisms for the aggressiveness of cribriform PCa are not fully understood. The aim of this study is to compare the immunohistochemical expression of various biomarkers and to determine the potential proteins that may account for their biologic and clinical differences. A total of 14 biomarkers were studied. The number of non-glomeruloid cribriform PCa cases studied for each biomarker ranged from 18 to 74 and the number of non-cribriform pattern 4 PCa studied for each biomarker ranged from 29 to 112. We demonstrated that, compared with non-cribriform Gleason pattern 4 PCa, EGFR was significantly upregulated and standard CD44 (CD44s) was significantly downregulated in cribriform PCa; no significant differences were found in the expression of AR, NKX3.1, ERG, EZH2, p53, Rb, C-Myc, BCL2, p16, CyclinD1, Her2/Neu, and Synaptophysin between these two groups of pattern 4 PCa. The study also showed, compared to non-cribriform PCa, cribriform PCa presented with significantly higher serum PSA and more advanced tumor stage. The significant overexpression of EGFR and downregulation of CD44s in non-glomeruloid cribriform PCa may, at least, partly explain the unfavorable pathology and clinical results for this growth pattern. Given that EGFR targeted inhibitors are now available, the findings may also have significant therapeutic implications.

Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) patients with active hepatocyte growth factor (HGF)/c-Met signaling have a significantly worse prognosis. c-Met, a high affinity receptor for HGF, plays a critical role in cancer growth, invasion and metastasis. c-Met and CD44 have been utilized as cell surface markers to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers including HCC. In this work, we examine the complex relationship between c-Met and CD44s (standard form), and investigate the specific role of CD44s as a tumor initiator and stemness marker in HCC.MethodsGene and protein expression assays were utilized to investigate the relationship between CD44s and c-Met in HCC cell lines. Tumor-sphere assays and in vivo tumor assays were performed to investigate the role of CD44+ cells as TISCs. Student’s t-test or one-way ANOVA with Tukeys post-hoc test was performed to test for differences amongst groups with a p < .05 as significant.ResultsIn an immunohistochemical and immunoblot analysis of human HCC samples, we observed that more than 39% of human HCC samples express c-Met and CD44. To study the relationship between c-Met and CD44, we used MHCC97-H cells, which are CD44+/c-Met+. The knockdown of c-Met in MHCC97-H cells decreased CD44s, reduced TISC characteristics and decreased tumorsphere formation. Furthermore, we demonstrate that the inhibition of PI3K/AKT signaling decreased CD44s expression and subsequently decreased tumorsphere formation. The down-regulation of CD44s leads to a significant loss of a TISC and mesenchymal phenotype. Finally, the down-regulation of CD44s in MHCC97-H cells decreased tumor initiation in vivo compared with the scrambled control.ConclusionsIn summary, our data suggest that CD44s is modulated by the c-Met-PI3K-AKT signaling cascade to support a mesenchymal and TISC phenotype in HCC cells. Moreover, c-Met could be a potential therapeutic drug for targeting HCC cells with TISC and mesenchymal phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1166-4) contains supplementary material, which is available to authorized users.

Abstract 1147: CD44s is required for EMT and stemness properties via ZEB1 activation

Abstract Invasion and metastasis of carcinomas are often promoted by the aberrant epithelial to mesenchymal transition (EMT) program at the invasive front of the tumor. The transcription factor ZEB1 is a crucial promotor of metastasis formation by inducing EMT and tumor initiating capacity. CD44, a cell surface protein, is used as marker to identify tumor initiating cells in many cancer types. It is involved in cell-cell interactions, cell adhesion and migration. Through alternative splicing, controlled by the epithelial splicing factor ESRP1, CD44 is differentially regulated during EMT, resulting in a switch from the variable exon-containing CD44v isoforms to the standard isoform CD44s, which is devoid of all CD44 variable exons. We examine the reciprocal interaction of CD44s and ZEB1 in their function as tumor initiation markers. CD44s and ZEB1 are co-expressed in poorly differentiated human cancer cells.Transient down regulation of CD44s results in decrease of ZEB1, which in turn leads to reduced sphere formation ability. Likewise, inhibition of CD44s increases ESRP1. Knockdown of ESRP1 decreases expression of CD44v isoforms, increases CD44s and ZEB1 and the sphere forming capacity. Additional knockdown of CD44s reduces the sphere formation. Furthermore, ZEB1 inhibits ESRP1 expression, which leads to upregulation of CD44s. In summary we identified a positive feedback loop maintaining stemness and mesenchymal features in cancer cells: CD44s activates expression of ZEB1, which suppresses transcription of the splicing factor ESRP1, resulting in further increase in CD44s expression. Citation Format: Bogdan-Tiberius Preca, Karolina Bajdak, Jessica Pfannstiel, Thomas Brabletz. CD44s is required for EMT and stemness properties via ZEB1 activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1147. doi:10.1158/1538-7445.AM2014-1147

Restoration of CD44S in non-small cell lung cancer cells enhanced their susceptibility to the macrophage cytotoxicity

CD44 is a cell surface receptor for osteopontin (OPN) and hyaluronate. Transformation of normal tissue to a variety of cancers has been demonstrated to be associated with alterations of CD44 isoform expression. However, few reports have paid attention on differences in CD44S expression between non-small cell lung cancer (NSCLC) and adjacent normal lung tissue. In this study, we demonstrate that CD44S expression is down-regulated in NSCLC tissue when compared with paired normal lung tissue. To investigate the role of CD44S down-regulation in NSCLC cells, we reintroduced the CD44S back into the NSCLC cell line, H322 cells, which originally lack CD44S expression. The cytotoxicity by activated macrophage (RAW264.7 cells stimulated with lipopolysaccharide and interferon-γ) against the H322 cells transfected with the CD44S gene (H322ΔS) is more prominent than that against the H322 control transfectants (H322Δneo). The enhanced susceptibility of H322ΔS cells to the activated macrophage cytotoxicity appears to be mediated by the interaction between CD44S expression on H322ΔS cells and OPN produced by activated macrophages since it is completely blocked by either anti-OPN or anti-CD44 antibody. Moreover, H322ΔS cells are attracted toward OPN produced by activated RAW264.7 cells to a much greater extent than H322Δneo cells. These findings suggest that CD44S down-regulation in NSCLC cells may confer a protective advantage of allowing escape from tumoricidal effector cells including activated macrophages of the host.

Co-downregulation of cell adhesion proteins α- and β-catenins, p120CTN, E-cadherin, and CD44 in prostatic adenocarcinomas

Cell adhesion molecule expression has been linked to disease outcome in prostatic adenocarcinomas (PACs). We evaluated the coordinated expression of catenin-related proteins, E-cadherin, N-cadherin, and CD44s in PACs. Archival sections from 112 PACs were immunostained by an automated method (Ventana Medical Systems, Tucson, AZ) using monoclonal antibodies to α- and β-catenins, p120CTN, E-cadherin, N-cadherin, and CD44s proteins. Immunoreactivity was semiquantitatively scored, and results were evaluated for association between these markers. Staining results were also correlated with tumor grade, stage, ploidy, preoperative serum PSA, and postoperative biochemical disease recurrence. Decreased expression of α- and β- catenins, p120CTN, E-cadherin, and CD44s proteins (range, 5% to 49%) was noted in PACs, and downregulation of each of these proteins correlated with high tumor grade (P = .02 to .0001). Although loss of E-cadherin and p120CTN each correlated with stage (E-cadherin, P = .02; p120CTN, P = .02) and ploidy (E-cadherin, P = .0001; p120CTN, P = .004), downregulation of CD44s correlated with ploidy (P = .002), serum PSA (P = .005), and postoperative disease recurrence (P = .02). N-cadherin was positive in only 5% of PACs and did not correlate with any prognostic parameters. α-Catenin downregulation correlated with decreased expression of E-cadherin (P = .0001). Additionally, decreased expression of each of these 2 proteins respectively correlated with loss of β-catenin (P = .0001 and .004), p120CTN (P = .005 and .001), and CD44s (P = .008 and .01). β-Catenin expression levels correlated with p120CTN (P = .01). A trend for co-downregulation of CD44s and p120CTN and of CD44s and β-catenin was observed. In conclusion, the significant association between decreased expression of various members of the CAM family of proteins supports their collective role in mediating cell–cell adhesion. Altered expression of these proteins may be of prognostic value in patients with prostate cancer. HUM PATHOL 32:849-855. Copyright © 2001 by W.B. Saunders Company

CD44s expression in human colon carcinomas influences growth of liver metastases.

CD44 is a family of cell-surface adhesion molecules which exist in several isoforms arising from mRNA alternative. Malignant transformation of colonic mucosa is associated with alterations in CD44 expression, which result in up-regulation of high-molecular-weight CD44 isoforms and down-regulation of CD44s. We have demonstrated that stable transfection of CD44s into colon-carcinoma cell lines reduces their tumorigenicity. To understand the influence of CD44s expression on the metastatic potential of human colon carcinomas, we measured the ability of several different CD44s-transfected colon carcinomas to establish experimental liver metastases following splenic inoculation in mice. We observed that introduction of CD44s into 2 different human colon carcinoma cell lines, HT29 and KM12C6, resulted in reduced growth of liver metastases by as much as 75%. To explore the relationship between hyaluronate adhesion and metastasis, we transfected HT29 cells with cDNA encoding a mutant CD44s that does not bind to hyaluronate. HT29 transfectants expressing this mutant CD44s demonstrate an 84% reduction in growth of liver metastases, despite minimal binding to hyaluronate by the mutant CD44s. In concert, these results indicate that CD44s down-regulation, which occurs with malignant transformation of colonic mucosa, is associated with enhanced growth of experimental liver metastases. Consequently, the functional consequences of CD44s down-regulation in colon carcinomas may be just as significant as the consequences of up-regulation of other CD44 isoforms.

INTERACTION BETWEEN CD44 AND HYALURONIC ACID REGULATES HUMAN PROSTATE CANCER DEVELOPMENT

Purpose This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. Materials and Methods We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. Results The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. Conclusions These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.

Expression of CD44 variant proteins in adenocarcinoma of Barrett's esophagus and its relation to prognosis.

None of the commonly used staging criteria accurately determine the prognosis of a patient with adenocarcinoma of Barrett's esophagus. The authors therefore assessed the expression pattern and prognostic impact of CD44 standard and CD44 isoforms CD44v4, v5,v6,v7, and v10 in adenocarcinoma of Barrett's esophagus. Specimens from 41 patients with adenocarcinoma of Barrett's esophagus who underwent esophageal resection were embedded in paraffin and studied immunohistochemically to determine the expression of CD44 splice variants. Histomorphologic parameters and survival time were not known at the time of the investigation. Correlations between favorable clinical or histomorphologic parameters and CD44s or any of the split variants could not be established. Down-regulation of CD44s and the split variant v10 was significantly correlated with pT classification. Furthermore, down-regulation of CD44v10 and up-regulation of CD44v7 were significantly correlated with ploidy. There was a significant correlation between CD44s and split variants in tumorous and nontumorous tissue from the same patient. Down-regulation of CD44s and CD44v4 had a significant influence on prognosis in that it was associated with shortened life expectancy. Multivariate analysis revealed that the expression of CD44v4 was an independent factor in prognosis. The results obtained for this small patient sample suggest that CD44v4 is a new independent prognostic parameter for adenocarcinoma of Barrett's esophagus that can be determined preoperatively by biopsy. It may therefore be helpful in planning therapy by allowing the identification of patients who may benefit from esophageal resection as well as those who are at high risk for morbidity and mortality even when the tumor is otherwise resectable. Further studies of larger patient samples are required to validate the results of the current study.

Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas.

To test whether p34cdc2 overexpression, CD44s downregulation, and HER-2/neu amplification correlate with disease recurrence after radical prostatectomy, and to evaluate a possible biologic association between p34cdc2 and HER-2/neu expression. Immunohistochemical (IHC) detection of both p34cdc2 cyclin-dependent kinase (CDK) and CD44s expression and fluorescence in situ hybridization (FISH)-based analysis of HER-2/neu gene status were performed on formalin-fixed, paraffin-embedded sections of 106 prostatic adenocarcinomas (PACs). Findings were correlated with Gleason grade, pathologic stage, DNA ploidy, and postsurgical biochemical disease recurrence. CDK overexpression correlated with tumor grade (P = .001), DNA ploidy (P = .001), pathologic stage (P = .04), and disease recurrence (P = .01). CD44s downregulation correlated with grade (P = .03), ploidy (P = .01), and recurrence (P = .02). HER-2/neu amplification correlated with grade (P = .001), ploidy (P = .001), and recurrence (P = .01). On multivariate analysis, CDK overexpression independently predicted recurrence (P = .001) after prostatectomy. CDK expression correlated with HER-2/neu status with 32 of 65 (49%) tumors that overexpressed CDK and showed concomitant HER-2/neu amplification (P = .04). This study showed that p34cdc2, CD44s, and HER-2/neu are variably expressed or amplified in prostatic carcinoma and that such alteration may affect tumor behavior. In addition, CDK overexpression and HER-2/neu amplification may be biologically related.