CD44s mRNA Research Articles

Over Expression of Cancer Stem Cell Marker CD44 and Its Clinical Significance in Patients with Oral Squamous Cell Carcinoma.

Cancer stem cell marker CD44 is a cell-surface glycoprotein which is involved in various cellular functions such as cell-cell interactions, cell adhesion, haematopoiesis and tumour metastasis. The CD44 gene transcription is partly activated by beta-catenin and Wnt signalling pathway, the later pathway being linked to tumour development. However, the role of CD44 in oral squamous cell carcinoma (OSCC) is not well understood. We investigated the expression of CD44 in peripheral circulation, tumour tissues of oral cancer patients and oral squamous cell carcinoma cell lines by ELISA and quantitative (q)-RTPCR. Relative CD44s mRNA expression was significantly higher in peripheral circulation (p = 0.04), tumour tissues (p = 0.049) and in oral cancer cell lines (SCC4, SCC25 p = 0.02, SCC9 p = 0.03). Circulating CD44total protein levels were also significantly (p < 0.001) higher in OSCC patients that positively correlated with increasing tumour load and loco-regional spread of the tumour. The circulating tumour stem cell marker CD44 appears to be a potent indicator of tumour progression and may be useful for developing suitable therapeutics strategies for patients with oral squamous cell carcinoma.

Stress-Response Protein RBM3 Attenuates the Stem-like Properties of Prostate Cancer Cells by Interfering with CD44 Variant Splicing

Stress-response pathways play an important role in cancer. The cold-inducible RNA-binding protein RBM3 is upregulated in several types of cancer, including prostate cancer, but its pathogenic contributions are undetermined. RBM3 is expressed at low basal levels in human fetal prostate or in CD133(+) prostate epithelial cells (PrEC), compared with the adult prostate or CD133-PrEC, and RBM3 is downregulated in cells cultured in soft agar or exposed to stress. Notably, RBM3 overexpression in prostate cancer cells attenuated their stem cell-like properties in vitro as well as their tumorigenic potential in vivo. Interestingly, either overexpressing RBM3 or culturing cells at 32°C suppressed RNA splicing of the CD44 variant v8-v10 and increased expression of the standard CD44 (CD44s) isoform. Conversely, silencing RBM3 or culturing cells in soft agar (under conditions that enrich for stem cell-like cells) increased the ratio of CD44v8-v10 to CD44s mRNA. Mechanistic investigations showed that elevating CD44v8-v10 interfered with MMP9-mediated cleavage of CD44s and suppressed expression of cyclin D1, whereas siRNA-mediated silencing of CD44v8-v10 impaired the ability of prostate cancer cells to form colonies in soft agar. Together, these findings suggested that RBM3 contributed to stem cell-like character in prostate cancer by inhibiting CD44v8-v10 splicing. Our work uncovers a hitherto unappreciated role of RBM3 in linking stress-regulated RNA splicing to tumorigenesis, with potential prognostic and therapeutic implications in prostate cancer.

Abstract 280: Switching of CD44 and hMena mRNA splice variants between the intramucosal lesion and invasive front of early stage human colorectal carcinoma (pT1), and their relationship to the expression of cancer stem cell markers and clinicopathological features.

Abstract Background:Epithelial-Mesenchymal Transition (EMT) is a key process involved in both tumor metastasis and cancer stem cell development. Recent studies have indicated that regulation at the level of alternative splicing constitutes a critical mechanism that controls EMT and cancer progression (Warzecha CC et al. Mol Cell 33:591,2009). The aim of this study was to clarify the mRNA expression statuses of CD44 and hMena splice variants and colon cancer stemness markers in pT1 stage colorectal carcinoma (CRC). Materials and methods:The mRNA expression of CD44 and hMena splice variants as well as colorectal cancer stemness markers, such as ALDH1, CD133 and CD26, were analyzed in 58 tumors of early CRC featuring submucosal invasion (pT1). The samples were obtained from endoscopically resected specimens between 2006 and 2010 at the Asahi General Hospital. The mRNAs were extracted from the mucosal lesion and invasive front of each CRC specimen using laser-captured microdissection then analyzed by semi-quantitative real-time PCR. The correlations between expression of these mRNAs and clinicopathological factors were also investigated. This study was approved by institutional review boards. Results:Significantly higher CD44s mRNA levels were predominantly expressed in the samples from invasive fronts when compared with those measured from intramucosal lesion, while CD44v mRNA levels were significantly higher in the samples from intramucosal lesions than of those from invasive fronts (P=0.0004 and 0.001, respectively). The hMena+11a mRNA levels in the mucosal lesions were higher than those measured from the invasive front (p=0.006). The mRNA levels of hMenaINV, in contrast, showed the reversed expression pattern. Thus, cell-type specific expression of epithelial and mesenchmal isoforms of hMena is achieved through tight regulation of mutually exclusive exons hMena+11a and MenaINV, respectively. The mRNA levels of ALDH1, CD133 and CD26 were low and did not differ between samples from the intramucosal area and invasive front. However, ALDH1 transcript levels increased at the area of subserosal invasion of advanced CRCs. Notably, higher expression levels of hMenaINV mRNAs in samples extracted from invasive fronts were associated with tumor budding grade (P=0.008). Discussion:Our study indicates that the expression of CD44s and MenaINV may reflect the EMT better than the cancer stem cell markers during the initial submucosal invasion of CRC. In summary, the expression of CD44 and hMena splice variants could be potentially useful biomarkers for evaluating the malignant potential of CRC. Citation Format: Hiroshi Yoshida, Akihiro Toyoda, Noriyuki Tanaka, Wataru Masuda, Motoo Kitagawa, Yoshio Suzuki, Kenichi Harigaya. Switching of CD44 and hMena mRNA splice variants between the intramucosal lesion and invasive front of early stage human colorectal carcinoma (pT1), and their relationship to the expression of cancer stem cell markers and clinicopathological features. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 280. doi:10.1158/1538-7445.AM2013-280

Abstract 3319: CD44 standard isoform regulates the mesenchymal phenotype in hepatocellular carcinoma cells, and associates with a poor prognosis in patients.

Abstract Purpose: Cancer stem cells (CSCs) in many tumors have been identified using CD44, either individually or in combination with other markers and are involved in tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is known to induce cancer invasion, metastasis, and CSCs properties. However, the underlying molecular mechanisms which integrate EMT and CSCs properties remain largely unknown. In this study, we test the hypothesis that CSCs markers play an important role in regulating EMT in hepatocellular carcinoma (HCC). Experimental Design: The relationship between the CSCs markers and EMT markers was investigated in HCC cell lines. The EMT markers, invasive phenotype, the relevant transcriptional factors and signaling pathways induced by CD44 were examined in vitro. The clinicopathological importance of CD44 standard isoform (CD44s) expression was analyzed by an immunohistochemical analysis, and the correlation of CD44s and EMT markers was determined in 150 patients with hepatocellular carcinoma. Results: HCC cell lines with high CD44 expression showed decreased E-cadherin expression and increased vimentin expression. CD44s mRNA was dominant form of CD44 mRNA present in HCC cell lines with the mesenchymal phenotype. Overexpression of CD44s in HCC cells induced the mesenchymal phenotype and increased tumor cell invasion, whereas knockdown of CD44s in HCC cells led to loss the mesenchymal phenotype and decreased tumor cell invasion. CD44s expression was associated with E-cadherin low expression (P=0.039) and vimentin high expression (P&amp;lt;0.001). CD44s expression was related to a large tumor size (P=0.003), multiple tumors (P=0.032), and poor tumor differentiation (P=0.020), and a shorter disease-free (P=0.023) and overall survival (P=0.013) in patients with HCC. Conclusions: our study will be valuable for understanding the relationship between CSCs and EMT, and the identification of new treatment targets for future HCC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3319. doi:1538-7445.AM2012-3319

Inhibition of the proliferation of human periodontal ligament fibroblasts by hyaluronidase

Hyaluronan (HA) exists in various living tissues as one of the major matrix macromolecules, and is well known to play an integral role in cell differentiation and proliferation. The present study was conducted to elucidate whether or not the proliferation of periodontal ligament (PDL) cells are affected specifically by the degradation of HA by hyaluronidasze (HAase). Human PDL fibroblasts were isolated and cultured with and without 15–150 U/ml bovine testicular HAase from 1 to 11 days after seeding. The cells were also cultured with anti-CD44 antibody of 2 μg/ml. For the control against the anti-CD44 antibody treatment, 2 μg/ml IgG was used. The HA-dependent pericellular matrix was visualized by particle-exclusion assay. The number of cells was counted by MTT assay during the proliferation. The mRNA levels of HA synthases (HASs), HAases (HYALs) and CD44s were examined by a quantitative real-time PCR analysis. The cell proliferation was inhibited by the treatment with HAase and anti-CD44 antibody in cultured PDL fibroblasts. HASs mRNAs were down-regulated, whereas HYALs mRNAs were up-regulated significantly by the treatment with HAase and anti-CD44 antibody. The CD44s mRNA level exhibited no significant changes. These results suggest that HA may contribute to modulate the proliferation of cultured human PDL cells through a CD44-mediated mechanism.

Expression of CD44s mRNA in gastric carcinoma

To study the expression of CD44s mRNA in the occurrence, development and invasion of gastric carcinoma (GC). The expressions of CD44s mRNA in 66 cases of GC, 25 cases of superficial gastritis and 25 cases of atypical hyperplasia were examined by in situ hybridization (ISH). There was no expression of CD44s mRNA in the group of superficial gastritis; the positive rate was 20%(5/25) in the group of atypical hyperplasia and 62.12%(41/66) in the group of gastric carcinoma. The positive rate in poor differentiation group was significantly higher than that in well differentiation group (P<0.05), and the positive rate of lymph node metastasis group was significantly higher than that in negative lymph node metastasis group(P<0.05). The expression of CD44s mRNA was related to cell differentiation degree and lymph node metastasis, the activation of CD44s gene was related to strong invasion of cancer cells and poor prognosis.

Enhanced cell surface CD44 variant (v6, v9) expression by osteopontin in breast cancer epithelial cells facilitates tumor cell migration: Novel post-transcriptional, post-translational regulation

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein that functions both as a cell attachment and chemotactic factor. Elevated expression of OPN confers enhanced metastatic ability on transformed cells, suggesting that OPN may contribute to the malignant progression of tumors. Migration of mammary carcinoma cells is stimulated by OPN via interactions with integrins and CD44 cell surface receptors. We hypothesized that OPN modulates specific CD44 isoform expression to facilitate breast cancer cell migration. The 21NT tumorigenic human breast cancer cell line was examined for regulation of CD44 expression at both the mRNA and protein levels in response to an engineered increase in OPN expression under CMV promoter control. Significant up-regulation of CD44s isoform mRNA expression was observed, but no change in CD44v6, v8, v9 or v10 mRNA levels. While there were elevated levels of CD44s, v6 and v9 protein at the cell surface, at the level of total cellular protein only CD44s and v6 were markedly increased. This suggests that OPN can regulate CD44 expression at both transcriptional and post-transcriptional (both amount and localization of protein) levels. To validate the functional consequence of OPN regulation of CD44 expression, we demonstrate that OPN-mediated cell migration was reduced by exposure to a anti-pan CD44 antibody, and to anti-CD44v6 and anti-CD44v9 function-blocking antibodies. Our data provide evidence that in 21NT cells OPN enhances CD44s mRNA expression, increases cell surface expression of CD44 variant forms without a change in mRNA levels, and stimulates cell migration.

Expression and Glycosylation with Polylactosamine of CD44 Antigen on Macrophages During Follicular Atresia in Pig Ovaries1

Macrophages are essential in cleaning up apoptotic debris during follicular atresia. However, the key factors of this process are still unclear. In the present study, we evaluated CD44 mRNA, CD44 protein, and CD44 antigen glycosylation on macrophages during follicular atresia in the pig. Atresia was classified into five stages: stage I, healthy follicles; stage II, early atretic follicles having apoptotic granulosa cells with an unclear basement membrane; stage III, progressing atretic follicles having apoptotic granulosa cells completely diffused from the basement membrane; stage IV, late atretic follicles with increasing lysosomal activity; and stage V, disintegrated atretic follicles having collapsed theca cells and strong lysosomal activity. Immunohistological analysis showed that macrophages expressing CD44 invaded the inside of stage III follicles, accompanied by a collapse of basement membrane. Semiquantitative RT-PCR showed that only mRNA of the CD44 standard isoform (CD44s) was present in inner cells of follicles, and not any CD44 variant isoform (CD44v) mRNAs. The amount of CD44s mRNA was increased at stage III. Western blot and lectin blot analyses showed that CD44 was markedly expressed at stage III and glycosylated with polylactosamine at the same time. After macrophages invaded atretic follicles at stages III-V, the CD44 expressed on macrophages was glycosylated with polylactosamine. The lysosomal activity began to increase at stage IV, and reached the highest level at stage V. Increased CD44s protein and posttranslational modification of CD44 with polylactosamine on macrophages from stage III could be involved in the cleaning up apoptotic granulosa cells.

Clinicopathological associations of CD44 mRNA and protein expression in primary breast carcinomas.

The purpose of this study was to examine the occurrence of CD44 isoforms in breast carcinomas and their role in predicting clinical outcome. Shock-frozen tumour tissues from 110 patients with breast carcinoma were examined by immunohistochemistry using antibodies directed against CD44s, v5, v6, v7 and v3-10. In addition, 80 of these tumours were available for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD44s and CD44v6. Immunohistochemically, the positive tumours showed cytoplasmic and/or membranous staining with all antibodies. Staining results did not correlate with histological subtype, lymph node status, status of steroid receptors, tumour size or age. Neither was any correlation found for overall and disease-free survival. Quantitative real-time RT-PCR of CD44s and CD44v6, however, revealed that expression of CD44v6 mRNA was significantly associated with lower pathological grade (Pearson chi(2) test P = 0.009; linear-by-linear association P = 0.003). Linear-by-linear association between CD44s mRNA expression and lower pathological grade was also seen (P = 0.02). Survival analysis with the Kaplan-Meier method demonstrated that increased CD44s mRNA expression was significantly associated with both disease-free survival and overall survival (P = 0.0185 and P = 0.0344, respectively). A similar trend for CD44v6 mRNA expression was seen in these cases, but the difference was not significant. Quantitative real-time RT-PCR revealed clinical correlations of CD44s and CD44v6 mRNA expression in breast carcinomas while immunohistochemistry for the protein expression of CD44s and other CD44 variants did not. This contradictory result merits further studies concerning the clinical impact of CD44 molecules in breast carcinomas.

CD44 Expression in Fetal Rat Bone:In Vivoandin VitroAnalysis

Knowledge of the expression pattern of the cell surface glycoprotein CD44 in the development and differentiation of bone is limited. We investigated CD44 expression (a) in bone sections of 21-day-old fetal rat calvaria (RC), metatarsals, and tibiae, (b) in primary cultures of RC cells undergoing differentiationin vitro,and (c) in three rat osteosarcoma cell lines: ROS 17/2.8, UMR 106.01, and UMR 106.06. By immunocytochemistry, Western, Northern, and reverse transcription polymerase chain reaction analyses, we found that osteoblastic cells express the “hematopoietic” or “standard” CD44 (CD44s) isoform. Osteoblastic cellsin vivoandin vitrostained at all detectable stages of differentiation, but intercellular heterogeneity of CD44s staining was evident, with lesser staining in preosteoblastic cells and greater staining in mature osteoblasts and osteocytes. As cells in RC cultures differentiated and formed bonein vitro,CD44s mRNA and protein levels as measured on immunoblots were invariant. All three osteosarcoma cell lines expressed CD44s mRNA and protein. The synthetic glucocorticoid dexamethasone, which stimulates osteogenesis in RC cellsin vitroand regulates a number of osteoblast-associated genes, had no apparent effect on either CD44s protein or mRNA levels. The widespread presence of CD44s in osteoblastic cells at various maturational stages suggests that further analyses will be required to determine what role CD44s may play in osteogenesis and in bone tissue organization.