CD44 Variant Research Articles

Abstract 1148: Epithelial to mesenchymal transition might be induced via CD44 isoform switch in colorectal cancer

Abstract Backgrounds: The epithelial to mesenchymal transition (EMT) is well known to play a crucial role in cancer invasion and metastasis and has been actively investigated in colorectal cancer (CRC). In recent years, the isoform switch of CD44, which is recognized to be a cancer stem cell marker, has been reported to associate with EMT, especially in breast cancer and hepatocellular carcinoma. However, the clinical impact of CD44 isoform switch to EMT in CRC is still unknown. Methods: E-cadherin, vimentin, CD44 standard (CD44s) and CD44 variant9 (CD44v9) expression were measured in 14 CRC cell lines and 150 CRC patients by real-time PCR. EMT status and CD44 status was determined by calculating vimentin/E-cadherin and CD44s/CD44v9 expression ratio, respectively. The CRC cell lines and patients were divided into two groups based on the EMT and CD44 status. We examined the association between the EMT status and CD44 isoform switch, and analyzed the correlation with clinicopathological factors and prognosis. Finally, we evaluated the effect of CD44 knockdown by siRNA in CRC cell lines by measuring the proliferation, migration and invasion ability. Results: 1. The CRC cell lines were classified into 8 epithelial, 4 mesenchymal and 2 intermediate type. Of these cell lines, CD44s was highly expressed in mesenchymal type cell lines, whereas, CD44v9 was highly expressed in epithelial type cell lines. 2. The 150 CRC patients were divided into 115 epithelial group and 35 mesenchymal group based on vimentin/E-cadherin expression ratio. Mesenchymal group showed significantly poorer survival than epithelial group (5-year survival rate; epithelial: 85.5% vs. mesenchymal: 62.1%, P = 0.009). EMT status was significantly correlated with invasion depth of tumor (P = 0.036), lymphatic vessel involvement (P = 0.034) and histological grade (P = 0.001). 3. High CD44 status group showed poorer prognosis than low CD44 status group (5- year survival rate; low group: 85.0% vs. high group: 62.1%, P = 0.025). There was a significant correlation between EMT status and CD44 status (r = 0.298, P < 0.001). On multivariate analysis, pathological T4 stage (P = 0.011), liver metastasis positive (P = 0.001), high CA19-9 level (≥37) (P = 0.003) and CD44 status (P = 0.001) were independent prognostic factors for CRC patients. 4. In COL-3-JCK, which is identified to be mesenchymal type, CD44 knockdown by siRNA decreased the vimentin expression at protein level. When the functional analysis were performed, CD44 knockdown significantly reduced the abilities of cell proliferation (P < 0.001), migration (P = 0.004) and invasion (P = 0.025). Discussion: EMT was a critical prognostic factor and CD44 isoform switch might contribute to EMT induction in CRC. These results could support a better understanding of the relationship between EMT and cancer stem cell. Therefore, further investigation of the specific splice isoform of CD44 might lead to novel therapeutic intervention for CRC patients. Citation Format: Naoki Mashita, Suguru Yamada, Naoki Iwata, Mitsuro Kanda, Daisuke Kobayashi, Chie Tanaka, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yashuhiro Kodera. Epithelial to mesenchymal transition might be induced via CD44 isoform switch in colorectal cancer. [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 1148. doi:10.1158/1538-7445.AM2014-1148

Abstract 1919: Profiling of cancer cell lines demonstrates a dynamic relationship between epithelial, mesenchymal and cancer stem states

Abstract Our previous work has demonstrated that exposure of M2 - Tumor Associated Macrophages M2-TAMs) to epithelial cancer cells can induce an Epithelial to Mesenchymal Transition (EMT). The resulting mesenchymal cancer cells exhibit a more aggressive phenotype, which places patients at a higher risk of relapse and death. Although most patients are cured by radical prostatectomy or radiation, it has been demonstrated that the majority of patients have disseminated tumor cells in their bone marrow at the time of primary treatment and approximately 10-15% of these men will eventually suffer a relapse after a period of dormancy. Further characterization of these disseminated cells both at the time of primary treatment and over time, is needed. It is unclear if these cells, especially those destined to eventually proliferate, are “mesenchymal”, “epithelial”, or “cancer stem” cells (CSCs). In this work, we investigate the CSC molecular and gene expression profile across multiple cancer cell lines and cell phenotypes. In part, this was done by utilizing various culturing conditions, such as 3D growth in stem cell media in order to generate sphere-like cultures known as tumorspheres. Flow cytometric analysis was performed based on ALDH, CD44 variant and CD133 expression. This was followed by gene expression analysis and characterization of the resulting tumorspheres and standard 2D cultures. The results demonstrate that there may be a level of plasticity that allows these cancer cells to alter gene expression and shift towards a CSC state and/or that there is a common CSC progenitor that exists within the population which can give rise to both epithelial and mesenchymal cell types. Therefore, furthering our understanding of the dynamic relationship between CSCs, EMT, and the converse mesenchymal to epithelial transition, is critical in developing novel therapeutics and prognostic tools in the field of cancer medicine. Citation Format: James R. Hernandez, Steven M. Mooney, Takumi Shiraishi, James E. Verdone, Calvin A. Harberg, Donald Vindivich, Kenneth J. Pienta. Profiling of cancer cell lines demonstrates a dynamic relationship between epithelial, mesenchymal and cancer stem states. [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 1919. doi:10.1158/1538-7445.AM2014-1919

Abstract 4798: The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation

Abstract Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm initiated in hematopoietic stem cells by expression of the BCR-ABL1 fusion oncogene and its protein product, which enhances ABL1 kinase activity. Targeted BCR-ABL tyrosine kinase inhibitors (TKIs), such as Imatinib, and the second generation TKIs like Nilotinib as well as the dual specific SCR and ABL inhibitor Dasatinib have significantly slowed disease progression by eradicating the bulk of BCR-ABL1 expressing cells in the circulation. However, progression to a therapeutically resistant blast crisis (BC) phase is driven by CD34+CD38+Lin- progenitors that co-opt stem cell properties, such as enhanced self-renewal and survival, albeit in a deregulated manner. These BC leukemia stem cells (BC LSC) harbor enhanced BCL2 and beta-catenin expression. Seminal research shows that a key Wnt/beta-catenin target gene, CD44, plays a vital role in cancer stem cell survival and retention in the malignant microenvironment. Because alternative mRNA splicing in humans generates many CD44 isoforms, we investigated CD44 variant expression and retention of human BC LSC in specific hematopoietic niches. Methods and Results We performed whole transcriptome RNA sequencing (RNA Seq) of FACS sorted progenitors (Lin-CD34+CD38+) from chronic phase (CP)(n=8) and blast crisis (BC)(n=8) CML patients as well as from normal cord blood (CB) (n=7) and adult peripheral blood (NPB)(n=4). A number of CD44 transcript variants were detected: 3, 4 (CD44s), 5, 6, 7, 8 plus additional variants. RNA seq analysis uncovered a higher overall CD44 expression in BC compared to CP progenitors. Notably, one isoform of CD44, variant 3, was highly upregulated in BC compared to CP progenitors and minimally expressed by their normal counterparts. Moreover, BC progenitors that engrafted in RAG2-/-γc-/- harbored CD44v3 expression in the bone marrow and the splenic niche and this differential BC LSC isoform expression was reduced after dasatinib treatment. In addition, BC LSC in the spleen showed a reduction of migration specific markers, such as RHAMM, ICAM-1 and Osteopontin, compared with the bone marrow resident BC LSC. Furthermore, a comparative splice isoform specific qRT-PCR analysis of CD44 variants expression in young versus old bone marrow showed no correlation with aging. In fact, human embryonic stem cells harbored variant 3 expression. Conclusions These data suggest that CD44 transcript variant 3 upregulation occurs following malignant reprogramming of hematopoietic progenitors that enables them to adopt features of an embryonic transcriptional program in select microenvironments. Detection of CD44 variant 3 has the potential to more precisely identify patients at risk for relapse and progression, suggest that combined therapeutic strategies involving a BCR-ABL specific TKI and a CD44 monoclonal antibody may decrease relapse and BC transformation rates. Citation Format: Frida L. Holm, Eva Hellqvist, Cayla Mason, Christian Barrett, Kelly A. Frazer, Anil Sadarangani, Catriona HM Jamieson. The niche specific role of CD44 splice isoform expression in blast crisis leukemia stem cell generation. [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 4798. doi:10.1158/1538-7445.AM2014-4798

Abstract 3598: Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation

Abstract Background: CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor. We have reported the functional role of CD44 variant isoform in the maintenance of low reactive oxygen species (ROS) levels in gastrointestinal cancer cells. While, microRNA (miRNA) is involved in the pathogenesis of many cancers and the networks of miRNA regulate CSCs properties. However, the mechanism underlying the miRNA in the regulation of CSC marker is mostly unrevealed. Objectives: The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop newmolecular targets in gastrointestinal cancer. Methods and Results: To investigate the mechanism in the regulation of CD44, We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 down-regulation and CD44 up-regulation in 63 patients with surgically-resected gastric cancer. Conclusions: Macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer. Citation Format: Takatsugu Ishimoto, Naoya Yoshida, Hidetaka Sugihara, Daisuke Izumi, Keisuke Miyake, Hiroshi Sawayama, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Hideo Baba. Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation. [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 3598. doi:10.1158/1538-7445.AM2014-3598

Abstract 4967: ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell

Abstract Cancer metastasis is established by the seeding and successful colonization of stem-like cancer cells which show tumor-initiating ability at distant organs. CD44 is a cell surface marker protein which is highly expressed in stem-like cells of several human cancers including breast cancer. CD44 exists in various isoforms produced through alternative splicing of mRNA. Increased expression of CD44 variant isoform (CD44v) has been shown to correlate with metastatic potential and aggressive clinical behavior of various human cancers. We recently showed that CD44v contributes to reactive oxygen species (ROS)-defense in cancer cells by enhancing cystine uptake by the cystine transporter xCT. However, the functional relevance of CD44v and xCT in the metastatic spread of stem-like cancer cells has remained elusive. In the present study, we show that metastatic breast cancer 4T1 cells consist of CD44v+ and CD44v- cells. The orthotopic transplantation of CD44v+ population but not that of CD44v-, in mice resulted in efficient lung metastasis. We found that CD44v expressing metastatic cancer cells highly express xCT and thereby maintain high reduced glutathione (GSH) level. A specific xCT inhibitor sulfasalazine suppressed lung metastatsis of CD44v+ cells. Alternative splicing of CD44mRNA in such metastatic cancer cells is regulated by Epithelial Splicing Related Protein1 (ESRP1) and short hairpin RNA (shRNA) targeting ESRP1 switched isoform from CD44v to CD44 standard (CD44s), leading to the suppression of lung metastasis. Furthermore, we found that ESRP1 expression is regulated epigenetically through histone modification by performing chromatin immunoprecipitation (ChIP) sequencing analysis of the ESRP1 locus in CD44v+ and CD44v- cells. These findings establish a novel role of ESRP1-regulated alternative splicing program in the regulation of antioxidant status of stem-like cancer cells which is crucial for the metastatic ability. Citation Format: Kenji Tsuchihashi, Osamu Nagano, Toshifumi Yae, Takatsugu Ishimoto, Takeshi Motohara, Momoko Yoshikawa, Go J Yoshida, Takeyuki Wada, Takashi Masuko, Kaoru Mogushi, Hiroshi Tanaka, Tsuyoshi Osawa, Yasuharu Kanki, Takashi Minami, Hiroyuki Aburatani, Mitsuyo Ohmura, Akiko Kubo, Makoto Suematsu, Kazuhisa Takahashi, Eishi Baba, Koichi Akashi, Hideyuki Saya. ESRP1 regulated alternative splicing of CD44mRNA enhances lung colonization of metastatic cancer cell. [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 4967. doi:10.1158/1538-7445.AM2014-4967

Brain metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression through epigenetic regulation of the spliceosome.

Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6-positive MBM migration is reduced by blocking with a CD44v6-specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM-specific factor with theranostic utility for stratifying patients.

Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.

BackgroundCancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs.MethodsThe human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed.ResultsPoorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells.ConclusionOur novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-722) contains supplementary material, which is available to authorized users.

Induction of Cd44 Variant 9-Expressing Cancer Stem Cells Attenuates the Efficacy of Chemoradioselection and Worsens the Prognosis of Patients with Advanced Head and Neck Cancer

ABSTRACT Aim: At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30-40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chemoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC). Methods: Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies. Results: The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (p = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among 5 candidate factors (T, N, stage, response to CCRT, and CD44v9), CD44v9 positivity alone was significantly correlated with the poor prognosis (HR: 3.140, 95% CI: 1.230-8.017, p = 0.0167). Furthermore, the survival rate of the CD44v9-induced group was significantly (p = 0.04) worse than the CD44v9-non-induced group. Conclusions: CRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. The addition of an xCT (a coupling molecule of CD44v9) inhibitor (e.g., sulfasalazine) to chemoradioselection may provide a new approach for clinically feasible CSC-targeted therapy in HNSCC, improving the efficacy of chemoradioselection and consequent organ preservation and survival. Disclosure: All authors have declared no conflicts of interest.

Prognostic value of CD44 variant exon 6 expression in non-small cell lung cancer: a meta-analysis.

CD44v6 (CD44 variant exon 6) is the chief CD44 variant isoform regulating tumor invasion, progression, and metastasis. The prognostic value of CD44v6 expression in non small cell lung cancer (NSCLC) has been evaluated in many studies, but the results have remained controversial. Thus, we performed a meta- analysis of currently available studies to investigate the prognostic value of CD44v6 expression in NSCLC patients and the relationship between the expression of CD44v6 and clinicopathological features. Two independent reviewers searched the relevant literature in Pubmed, Medline and Embase from 1946 to January 2014. Overall survival (OS) and various clinicopathological features were collected from included studies. This meta-analysis was accomplished using STATA 12.0 and Revman 5.2 software. Pooled hazard ratios (HRs) with 95% confidence intervals (95%CIs) were calculated to estimate the effects. A total of 921 NSCLC patients from ten studies met the inclusion criteria. The results showed that CD44v6 high expression was a prognostic factor for poor survival (HR=1.91, 95%CI=1.12-3.26, p<0.05). With respect to clinicopathological features, CD44v6 high expression was related to histopathologic type (squamous cell carcinoma versus adenocarcinoma: OR=2.72, 95%CI=1.38-5.38, p=0.004), and lymph node metastasis (OR=3.02, 95%CI=1.93-4.72, p<0.00001). Our results suggested CD44v6 high expression as a poor prognostic factor for NSCLC, and CD44v6 expression is associated with lymph node metastasis and histopathologic type. Therefore, CD44v6 expression can be used as a novel prognostic marker in NSCLC cases.

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population.

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

Genetic association of osteopontin (OPN) and its receptor CD44 genes with susceptibility to Chinese gastric cancer patients.

(1) To investigate associations between single nucleotide polymorphisms (SNPs) in osteopontin (OPN) and its receptor-cluster of differentiation 44 (CD44) genes and gastric cancer susceptibility. (2) To explore the correlation of OPN and CD44 expression of gastric cancer. We detected 26 SNPs of the genes in gastric cancer patients from the Chinese Han population by Sequenom technique and performed expression of OPN in combination with CD44 in 243 tissues samples of the cases by tissue microarray and immunohistochemistry (IHC). We found that the minor alleles of OPN rs4754C>T and OPN rs9138C>A remained strongly associated with decreased gastric cancer risk (P=1.53×10(-4), odds ratio (OR) 0.642, 95% confidence interval (CI) 0.511-0.808 and P=1.59×10(-4), OR 0.642, 95%CI 0.510-0.809). OPN variant rs1126772A>G and CD44 variant rs353639A>C significantly contributed to elevated risk of gastric cancer (P=0.042, OR 1.279, 95% CI 1.008-1.622 and P=0.047, OR 1.334, 95% CI 1.003-1.772). Haplotypes of OPN and CD44 variants significantly influenced risk of gastric cancer. Clinical data indicated that rs4754 and rs9138 of OPN were significantly associated with smoking (P=0.029, OR 0.343, 95% CI 0.127-0.926 and P=0.029, OR 0.343, 95%CI 0.127-0.926) and OPN rs1126772 revealed associations with tumor-node-metastasis (TNM) stage (P=0.025, OR 1.765, 95% CI 1.073-2.905) and tumor differentiation (P=0.031, OR 1.722, 95% CI 1.049-2.825). OPN expression was observed in 133 of the 243 cases (54.7%) by IHC and was correlated with serosa invasion (P=0.013), TNM stage (P=0.003) and lymph node metastasis (P=0.002). CD44 expression was found in 92 of the 243 cases (37.9%) and was associated with tumor size (P=0.005) and lymph node metastasis (P=0.023), respectively. The OPN expression displayed a positive association with CD44 (P=0.01, r s=0.164). We found that the polymorphisms rs4754, rs9138 and rs1126772 of OPN gene and rs353639 of CD44 gene were significantly associated with gastric cancer. Our IHC data indicated that interaction of OPN and CD44 protein would promote progression and metastasis of gastric cancer.

Effectiveness of plasma treatment on gastric cancer cells.

Treatment of peritoneal carcinomatosis arising from gastric cancer remains a considerable challenge. In recent years, the anticancer effect of nonequilibrium atmospheric pressure plasma (NEAPP) has been reported in several cancer cell lines. Use of NEAPP may develop into a new class of anticancer therapy that augments surgery, chemotherapy, and radiotherapy. Gastric cancer cells were assessed for changes in cell morphology and rate of proliferation after treatment with NEAPP-exposed medium (PAM). To explore the functional mechanism, caspase3/7, annexin V, and uptake of reactive oxygen species (ROS) were evaluated, along with the effect of the ROS scavenger N-acetylcysteine (NAC). PAM treatment for 24h affected cell morphology, suggestive of induction of apoptosis. PAM cytotoxicity was influenced by the time of exposure to PAM, the type of cell line, and the number of cells seeded. Cells treated with PAM for 2h demonstrated activated caspase3/7 and an increased proportion of annexinV-positive cells compared with untreated cells. Additionally, ROS uptake was observed in PAM-treated cells, whereas NAC reduced the cytotoxicity induced by PAM presumably through reduction of ROS uptake. Furthermore, CD44 variant 9, which reportedly leads to glutathione synthesis and suppresses stress signaling of ROS, was overexpressed in PAM-resistant cells. PAM treatment induced apoptosis of gastric cancer cells through generation and uptake of ROS. Local administration of PAM could develop into an option to treat peritoneal carcinomatosis.

Immunophenotypic features of metastatic lymph node tumors to predict recurrence in N2 lung squamous cell carcinoma.

Patients with mediastinal lymph node metastasis (N2) in squamous cell carcinoma (SqCC) of the lung have poor prognosis after surgical resection of the primary tumor. The aim of this study was to clarify predictive factors of the recurrence of pathological lung SqCC with N2 focusing on the biological characteristics of both cancer cells and cancer-associated fibroblasts (CAFs) in primary and metastatic lymph node tumors. We selected 64 patients with pathological primary lung N2 SqCC who underwent surgical complete resection and investigated the expressions of four epithelial–mesenchymal transition-related markers (caveolin, clusterin, E-cadherin, ZEB2), three cancer stem cell-related markers (ALDH-1, CD44 variant6, podoplanin) of cancer cells, and four markers of CAFs (caveolin, CD90, clusterin, podoplanin) in both primary and matched metastatic lymph node tumors in the N2 area. In the primary tumors, the expressions of all the examined molecules were not related to recurrence. However, in the metastatic lymph node tumors, high clusterin and ZEB2 expressions in the cancer cells and high podoplanin expression in the CAFs were significantly correlated with recurrence (P = 0.03, 0.04, and 0.007, respectively). In a multivariate analysis, only podoplanin expression in the CAFs in metastatic lymph node tumors was identified as a significantly independent predictive factor of recurrence (P = 0.03). Our study indicated that the immunophenotypes of both cancer cells and CAFs in metastatic lymph node tumors, but not primary tumors, provide useful information for predicting the recurrence of pathological N2 lung SqCC.

Evaluation of CD44 variant expression in oral, head and neck squamous cell carcinomas using a triple approach and its clinical significance.

Cancer stem cells possess the qualities of self-renewal, tumorigenesis and the ability to recapitulate a heterogeneous tumor. Our group was the first to isolate head and neck squamous cell carcinoma (HNSCC) stem cells using the cell surface marker CD44. CD44 is a trans-membrane glycoprotein with a multitude of key-functions that regulate cancer cell proliferation and metastasis. The variety of CD44 functions is due to tissue-specific patterns of glycosylation of the extracellular portion, and to the multiple protein isoforms (CD44 variants, CD44v) generated by alternative splicing. This study investigates the expression pattern of CD44 variants in HNSCC. Ten cell lines from the most common HNSCC locations and representative of various clinical outcomes were assayed by quantitative realtime PCR, flow cytometry and immunofluorescence comparatively with normal oral keratinocytes. The CD44 v4 and v6 were exclusively abundant in HNSCC while the isoform v1,2 was expressed in normal oral keratinocytes. Of interest, the highest level of CD44v6 expression was detected in advanced metastatic HNSCC, suggesting a link between CD44v6 expression and HNSCC metastasis, while the highest CD44v4 was detected in a stage IV HNSCC refractory to chemotherapy which developed recurrence. Oral-derived HNSCC expressed the highest CD44v4 and v6, and levels corresponded with staging, showing also an increasing tendency with recurrence and metastasis. CD44v were detected predominantly in smaller cells (a characteristic that has been associated with stem cell properties) or cells with mesenchymal morphology (a characteristic that has been associated with the migratory and invasive potential of epithelial tumor cells), suggesting that CD44v differential expression in HNSCC may be representative of the morphological changes inherent during tumor progression towards a more aggressive potential, and thus contributing to the individual tumor biology. The mechanism of CD44 variant involvement in HNSCC progression and metastasis is under investigation.

Epithelial to mesenchymal transition might be induced via CD44 isoform switching in colorectal cancer.

Isoform switching of CD44 is associated with epithelial to mesenchymal transition (EMT) in several cancers; however, the clinical implications of this remain unclear for colorectal cancer (CRC). We measured expression levels of E-cadherin, vimentin, CD44 standard (CD44s) and CD44 variant 9 (CD44v9) transcripts in 14 CRC cell lines and 150 CRC patients. We determined EMT and CD44 status by calculating vimentin/E-cadherin and CD44s/CD44v9 expression ratios, respectively. Associations between EMT status and CD44 isoform switching, and between clinicopathological factors and prognosis were analyzed. CD44s was highly expressed in mesenchymal-type cell lines, while CD44v9 was highly expressed in epithelial-type cell lines. CD44 knockdown resulted in decreased levels of vimentin expression, and significantly reduced proliferation, migration and invasion of cells. In CRC patients, the mesenchymal group and the high CD44 status group exhibited significantly poorer survival than that for the epithelial group (5-year survival; 62.1% vs. 85.5%, P = 0.0085) and the low CD44 status group (5-year survival; 66.1% vs. 85.0%, P = 0.0251). On multivariate analysis, CD44 status was an independent prognostic factor. The status of EMT and CD44 is a critical prognostic factor, with CD44 isoform switching a possible trigger for EMT in CRC.

CD44v10, osteopontin and lymphoma growth retardation by a CD44v10-specific antibody.

Blockade of CD44 is considered a therapeutic option for the elimination of leukemia-initiating cells. However, the application of anti-panCD44 can be burdened by severe side effects. We determined whether these side effects could be avoided by replacing anti-panCD44 with CD44 variant isoform (CD44v)-specific antibodies in CD44v-positive hematological malignancies using the EL4 thymoma and CD44v10-transfected EL4 (EL4-v10) as models. Subcutaneous growth of EL4 and EL4-v10 was equally well inhibited by the anti-panCD44 and anti-CD44v10 antibodies, respectively. Ex vivo analysis indicated that natural killer cytotoxicity and antibody-dependent cellular cytotoxicity were the main effector mechanisms. Under local inflammation, the efficacy of anti-CD44v10 prolonged the survival time twofold compared with untreated, EL4-v10 tumor-bearing mice, and this was due to inflammation-induced expression of osteopontin (OPN). A high level of OPN in EL4-v10 tumors supported leukocyte recruitment and tumor-infiltrating T-cell activation. Taken together, in hematological malignancies expressing CD44v, anti-panCD44 can be replaced by CD44v-specific antibodies without a loss in efficacy. Furthermore, CD44v10-specific antibodies appear particularly advantageous in cutaneous leukemia therapy, as CD44v10 binding of OPN drives leukocyte recruitment and activation.

Prognostic significance of CD44 variant 2 upregulation in colorectal cancer.

Background:CD133 and CD44 are putative cancer stem cell (CSC) markers in colorectal cancer (CRC). However, their clinical significance is currently unclear. Here, we evaluated primary CRC cell isolates to determine the significance of several CSC markers, including CD133 and CD44, as predictors of tumourigenesis and prognosis.Methods:CD133- and CD44-positive cells from fresh clinical samples of 77 CRCs were selected by flow cytometric sorting and evaluated for tumourigenicity following subcutaneous transplantation into NOD/SCID mice. Cancer stem cell marker expression was examined in both xenografts and a complementary DNA library compiled from 167 CRC patient samples.Results:CD44+, CD133+ and CD133+CD44+ sub-populations were significantly more tumourigenic than the total cell population. The clinical samples expressed several transcript variants of CD44. Variant 2 was specifically overexpressed in both primary tumours and xenografts in comparison with the normal mucosa. A prognostic assay using qRT–PCR showed that the CD44v2high group (n=84, 5-year survival rate (5-OS): 0.74) had a significantly worse prognosis (P=0.041) than the CD44v2low group (n=83, 5-OS: 0.88).Conclusions:CD44 is an important CSC marker in CRC patients. Furthermore, CRC patients with high expression of CD44v2 have a poorer prognosis than patients with other CD44 variants.

Prognostic value of CD44 and CD44v6 expression in patients with non-small cell lung cancer: meta-analysis.

We sought to clarify the prognostic value of CD44 in survival of patients with non-small cell lung cancer (NSCLC). We performed a meta-analysis of relevant literature to aggregate the available survival results, using studies published in English until March 2014. Eligible studies dealt with CD44, CD44 standard form (CD44s) and CD44 variant 6 (CD44v6), assessment in NSCLC patients on primary lesions and reported survival data according to CD44 and CD44 isoforms expression. We aggregated 10 trials (5 trials for CD44v6, 3 trials for CD44, and 2 trials for CD44s) comprising 1,074 patients, in this meta-analysis. The combined hazard ratio (HR) with CD44v6 and CD44s was 2.39 (95 % confidence interval (CI) 1.69-3.37) and 1.64 (95 % CI 1.06-2.52), respectively. It associated high CD44v6 and CD44s expression with poor survival in NSCLC patients. However, CD44 overexpression did not significantly correlate with survival in patients with NSCLC (HR 1.44; 95 % CI 0.72-2.89). Our meta-analysis shows that CD44v6 and CD44s overexpression indicates poor prognosis for NSCLC patients. However, the high CD44 expression is not significantly correlated with survival for patients with NSCLC.

Sa1900 CD44v9 Results in Resistance to Trastuzumab via Induction of ROS Resistance

Background: Twenty percent of advanced gastric cancers are HER2-positive. Trastuzumab(Tmab), a monoclonal antibody that interferes with the HER2 receptor, combined with cisplatin and a fluoropyrimidine has been shown to increase survival in HER2-positive gastric cancer. Tmab induces reactive oxygen species (ROS) in lung adenocarcinoma in vitro, and may have similar effects in other cancer types as well. CD44 variant 9 (CD44v9) expression is associated with a poor prognosis in gastric adenocarcinomas. The CD44v9 isoform interacts with and stabilizes xCT, a glutamate-cystine transporter, resulting in increased intracellular levels of reduced glutathione (GSH), and increased defense against ROS. The relationship between CD44v9 expression and HER2 status in the response of gastric cancer to various treatments, including Tmab has not been reported. The present study was conducted to investigate the role of CD44v9 in the effectiveness of Tmab treatment on gastric adenocarcinomas. We hypothesize that CD44v9 causes resistance to Tmab via induction of ROS resistance, and targeting CD44v9 may increase effectiveness of Tmab therapy. Methods: NCI-N87(high HER2), a gastric cancer cell line, was used for this study. Anti-HER2 siRNA was used to knockdown HER2 expression in NCI-N87 cells. The pRc/CMV expressing plasmid encoding human CD44v9 was transfected into NCI-N87 cells (CD44v9-NCI-N87). N-acetyl cysteine (NAC) (15mM) was used as a ROS scavenger for 72h in vitro. Cells were incubated for 72h with different concentrations of Tmab (a range of doses between 0, 10, 50 μg/ml), and cell viability was determined by MTS assay. Intracellular GSH levels were measured by luminescent based assay. Intracellular ROS were measured by flow cytometry with CM-H2DCFDA. Cells were cultured for 24h with different concentrations of Tmab (a range of doses between 0, 40, 80, 120, 200 μg/ml) in this assay. Results: Tmab caused a dose-dependent decrease in cell viability in NCI-N87 cells(10μg/ml 88.2%(p=0.015), 50μg/ml 73.3%(p<0.001)), but did not in anti-HER2 siRNA treated NCI-N87(10μg/ml 99.0%(p=0.879), 50μg/ml 93.0%(p= 0.495) and CD44v9-NCI-N87(10μg/ml 104.4%(p=0.442), 50μg/ml 97.3%(p=0.623)). Tmab increased intracellular ROS level in NCI-N87 cells (p=0.007), but did not in anti-HER2 siRNA treated NCI-N87 and CD44v9-NCI-N87. Intracellular GSH levels were significantly higher in CD44v9-NCI N87 than NCI-N87 (p=0.004). NAC decreased Tmab-induced cell death in NCI-N87. These data indicated that CD44v9 is associated with increased intracellular GSH level, and may protect against Tmab induced ROS damage and cell death. Conclusion: Knock down of CD44v9 expression in HER2 positive gastric cells restores Tmab sensitivity through the restoration of ROS mediated cell death.

CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers.