Showing papers on "CD44 published in 2005"

Prospective Identification of Tumorigenic Prostate Cancer Stem Cells

Abstract: Existing therapies for prostate cancer eradicates the bulk of cells within a tumor. However, most patients go on to develop androgen-independent disease that remains incurable by current treatment strategies. There is now increasing evidence in some malignancies that the tumor cells are organized as a hierarchy originating from rare stem cells that are responsible for maintaining the tumor. We report here the identification and characterization of a cancer stem cell population from human prostate tumors, which possess a significant capacity for self-renewal. These cells are also able to regenerate the phenotypically mixed populations of nonclonogenic cells, which express differentiated cell products, such as androgen receptor and prostatic acid phosphatase. The cancer stem cells have a CD44+/α2β1hi/CD133+ phenotype, and we have exploited these markers to isolate cells from a series of prostate tumors with differing Gleason grade and metastatic states. Approximately 0.1% of cells in any tumor expressed this phenotype, and there was no correlation between the number of CD44+/α2β1hi/CD133+ cells and tumor grade. The identification of a prostate cancer stem cell provides a powerful tool to investigate the tumorigenic process and to develop therapies targeted to the stem cell.

Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.

Abstract: Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24(-/low) cells, which exclusively retain tumorigenic activity and display stem cell-like properties. However, at present, direct evidence that breast cancer-initiating cells can be propagated in vitro is still lacking. We report here the isolation and in vitro propagation of breast cancer-initiating cells from three breast cancer lesions and from an established breast carcinoma cell line. Our breast carcinoma-derived cultures encompassed undifferentiated cells capable of self-renewal, extensive proliferation as clonal nonadherent spherical clusters, and differentiation along different mammary epithelial lineages (ductal and myoepithelial). Interestingly, cultured cells were CD44+/CD24- and Cx43-, overexpressed neoangiogenic and cytoprotective factors, expressed the putative stem cell marker Oct-4, and gave rise to new tumors when as few as 10(3) cells were injected into the mammary fat pad of SCID mice. Long-term cultures of breast tumorigenic cells with stem/progenitor cell properties represent a suitable in vitro model to study breast cancer-initiating cells and to develop therapeutic strategies aimed at eradicating the tumorigenic subpopulation within breast cancer.

Human Umbilical Cord Perivascular (HUCPV) Cells: A Source of Mesenchymal Progenitors

Abstract: We describe the isolation of a nonhematopoietic (CD45-, CD34-, SH2+, SH3+, Thy-1+, CD44+) human umbilical cord perivascular (HUCPV) cell population. Each HUCPV cell harvest (2-5 x 10(6), depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed alpha-actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibro-blast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 10(10) HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/10(5) CFU-F, which increased to 7.5/10(5) CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10(4) and 1.3/10(4) CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC-/-). This population increased to 95% following passage and cryopreservation (P5). We conclude that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC-/- phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies.

Prevalence of CD44+/CD24−/low Cells in Breast Cancer May Not Be Associated with Clinical Outcome but May Favor Distant Metastasis

Abstract: Purpose: Breast cancer is composed of phenotypically diverse populations of cancer cells. The ability to form breast tumors has been shown by in vitro / in vivo studies to be restricted to epithelial tumor cells with CD44 + /CD24 −/low characteristics. Validation of these findings with respect to detection in clinical samples, prognosis, and clinical relevance is in demand. Experimental Design: We investigated breast cancer tissues for the prevalence of CD44 + /CD24 −/low tumor cells and their prognostic value. The study included paraffin-embedded tissues of 136 patients with and without recurrences. In addition, a breast cancer progression array with normal, carcinoma in situ , and carcinoma tissues was analyzed. We applied double-staining immunohistochemistry for the detection of CD44 + /CD24 −/low cells. Evaluation was by microscopic pathologic inspection and automated image analysis. Results: CD44 + /CD24 −/low cells ranged from 0% to 40% in normal breast and from 0% to 80% in breast tumor tissues. The prevalence of CD44 + /CD24 −/low tumor cells in 122 tumors was ≤10% in the majority (78%) of cases and >10% in the remainder. There was no significant correlation between CD44 + /CD24 −/low tumor cell prevalence and tumor progression. Although recurrences of tumors with high percentages of CD44 + /CD24 −/low tumor cells were mainly distant, preferably osseous metastasis, there was no correlation with the event-free and overall survival. There was no influence on the response to different treatment modalities. Conclusions: Our findings suggest that the prevalence of CD44 + /CD24 −/low tumor cells in breast cancer may not be associated with clinical outcome and survival but may favor distant metastasis.

May Not Be Associated with Clinical Outcome but May Favor Distant Metastasis

Abstract: Purpose: Breast cancer is composed of phenotypically diverse populations of cancer cells. The ability to form breast tumors has been shown by in vitro/in vivo studies to be restricted to epithelial tumor cells with CD44 + /CD24 /low characteristics. Validation of these findings with respect to detection in clinical samples, prognosis, and clinical relevance is in demand. Experimental Design: We investigated breast cancer tissues for the prevalence of CD44 + /CD24 /low tumor cells and their prognostic value. The study included paraffinembedded tissues of 136 patients with and without recurrences. In addition, a breast cancer progression array with normal, carcinoma in situ, and carcinoma tissues was analyzed. We applied double-staining immunohistochemistry for the detection of CD44 + /CD24 /low cells. Evaluation was by microscopic pathologic inspection and automated image analysis. Results: CD44 + /CD24 /low cells ranged from 0% to 40%

Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells

Abstract: Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

The CD44 receptor interacts with P-glycoprotein to promote cell migration and invasion in cancer.

Abstract: Invasion and metastases of cancer cells and the development of resistance to anticancer therapies are the main causes of morbidity and mortality from cancer. For more than two decades, these two important but not clearly related aspects in the biology of cancer have been extensively studied. Specifically, P-glycoprotein and CD44 have been characterized and are known to be determinants of multidrug resistance (MDR) and metastases. Despite this body of knowledge, few reports have linked the two phenotypes and only recently have there been reasons to suspect a direct connection. In this report, we show that a novel physical and genetic interaction between CD44s and P-glycoprotein is in part responsible for the correlation between MDR and invasive potential in cancer cells. P-glycoprotein-specific substrates that interfere with its function reduced in vitro invasion, migration, and the physical colocalization of CD44s and P-glycoprotein. CD44 expression in sensitive cells promoted the expression of P-glycoprotein and the MDR phenotype. RNA interference of MDR1 inhibited the rate of cell migration. These data indicate that there is a close interaction between CD44 and P-glycoprotein that results in the concurrent expression and modulation of two malignant phenotypes, invasion and MDR.

CD44 Attenuates Metastatic Invasion during Breast Cancer Progression

Abstract: Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation.

Abstract: Human placenta-derived mononuclear cells (MNC) were isolated by a Percoll density gradient and cultured in mesenchymal stem cell (MSC) maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblast-like morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and alpha-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class I major histocompatibility complex (MHC-I), but they did not express MHC-II molecules. Additionally these cells could suppress umbilical cord blood (UCB) lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells (HSC) from UCB to reduce the potential graft-versus-host disease (GVHD) in recipients.

Tumor Cells Deactivate Human Monocytes by Up-Regulating IL-1 Receptor Associated Kinase-M Expression via CD44 and TLR4

Abstract: Although blood monocytes possess significant cytotoxic activity against tumor cells, tumor-infiltrating monocytes are commonly deactivated in cancer patients. Monocytes pre-exposed to tumor cells show significantly decreased expression levels of TNF-α, IL-12p40, and IL-1R-associated kinase (IRAK)-1. Activation of the Ser/Thr kinase IRAK-1 is an important event in several inflammatory processes. By contrast, another IRAK family member, IRAK-M, negatively regulates this pathway, and is up-regulated in cultures of endotoxin-tolerant monocytes and in monocytes from septic patients within the timeframe of tolerance. In this study, we show that IRAK-M expression is enhanced at the mRNA and protein level in human monocytes cultured in the presence of tumor cells. IRAK-M was induced in monocytes upon coculturing with different tumor cells, as well as by fixed tumor cells and medium supplemented with the supernatant from tumor cell cultures. Moreover, blood monocytes from patients with chronic myeloid leukemia and patients with metastasis also overexpressed IRAK-M. Low concentrations of hyaluronan, a cell surface glycosaminoglycan released by tumor cells, also up-regulated IRAK-M. The induction of IRAK-M by hyaluronan and tumor cells was abolished by incubation with anti-CD44 or anti-TLR4 blocking Abs. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates both TNF-α mRNA expression and protein production in human monocytes re-exposed to a tumor cell line. Altogether, our findings indicate that deactivation of human monocytes in the presence of tumor cells involves IRAK-M up-regulation, and this effect appears to be mediated by hyaluronan through the engagement of CD44 and TLR4.

Antisense-Mediated Suppression of Hyaluronan Synthase 2 Inhibits the Tumorigenesis and Progression of Breast Cancer

Abstract: The progression of several cancers is correlated with the increased synthesis of the glycosaminoglycan, hyaluronan. Hyaluronan is synthesized at the plasma membrane by various isoforms of hyaluronan synthases (HAS). The importance of HAS2 expression in highly invasive breast cancer was characterized by the antisense inhibition of HAS2 (ASHAS2). The effect of HAS2 inhibition on cell proliferation, migration, hyaluronan metabolism, and receptor status was characterized in vitro, whereas the effect on tumorigenicity and metastasis was established in vivo. HAS2 inhibition resulted in a 24-hour lag in proliferation that was concomitant to transient arrest of 79% of the cell population in G0-G1. Inhibition of HAS2 did not alter the expression of the other HAS isoforms, whereas hyaluronidase (HYAL2) and the hyaluronan receptor, CD44, were significantly down-regulated. ASHAS2 cells accumulated greater amounts of high molecular weight hyaluronan (>10,000 kDa) in the culture medium, whereas mock and parental cells liberated less hyaluronan of three distinct molecular weights (100, 400, and 3,000 kDa). The inhibition of HAS2 in the highly invasive MDA-MB-231 breast cancer cell line inhibited the initiation and progression of primary and secondary tumor formation following s.c. and intracardiac inoculation into nude mice, whereas controls readily established both primary and secondary tumors. The lack of primary and secondary tumor formation was manifested by increased survival times where ASHAS2 animals survived 172% longer than the control animals. Collectively, these unique results strongly implicate the central role of HAS2 in the initiation and progression of breast cancer, potentially highlighting the co-dependency between HAS2, CD44, and HYAL2 expression.

CD44 on LS174T colon carcinoma cells possesses E-selectin ligand activity

Abstract: Metastasis of circulating tumor cells requires a multistep cascade of events initiated by adhesion of tumor cells to the vascular endothelium of involved tissues. This process occurs under the forces of blood flow and is promoted by adhesion molecules specialized to interact under shear conditions. The endothelial molecule E-selectin is a major mediator of these adhesive events, and there is strong evidence that E-selectin receptor-ligand interactions contribute to the formation of metastasis. However, little is known about the identity of E-selectin ligand(s) expressed on cancer cells. To address this issue, we did SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, and blot rolling assays of LS174T, a colon carcinoma cell line known to interact with E-selectin under physiologic flow conditions. Our studies show that LS174T cells express the hematopoietic cell E/L-selectin (HCELL) glycoform of CD44, which functions as a high-affinity E-selectin glycoprotein ligand on these cells. However, in contrast to the HCELL glycoform on human hematopoietic progenitor cells, which expresses carbohydrate-binding determinant(s) for E-selectin primarily on N-glycans of standard CD44, the relevant determinant(s) on LS174T cells is expressed on O-glycans and is predominantly found on variant isoforms of CD44 (CD44v). Our finding that tumor-associated CD44 splice variant(s) express E-selectin ligand activity provides novel perspectives on the biology of CD44 in cancer metastasis.

Chemotaxis towards hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding.

Abstract: The accumulation of the extracellular matrix glycosaminoglycan hyaluronan by tumours and tumour-associated stroma promotes cancer cell invasion and metastasis. Using the Dunn chamber chemotaxis assay, we demonstrate for the first time that high molecular mass hyaluronan acts as a soluble chemoattractant promoting the directional migration of MDA-MB-468 and MDA-MB-231 breast cancer cells. Moreover, chemotaxis towards hyaluronan, but not foetal bovine serum, can be abrogated following treatment of the cells with siRNA oligonucleotides to downregulate CD44 expression. These data indicate that CD44 is the principal receptor mediating this response and that CD44 expression is not a general requirement for cell migration and gradient sensing, rather it elicits a ligand-specific response. However, expression of CD44 alone is not sufficient to drive chemotaxis towards hyaluronan, as NIH-3T3 fibroblasts were unable to respond to a hyaluronan gradient even when transfected with high levels of human CD44. For NIH-3T3 cells to bind exogenous hyaluronan, it was necessary to both increase the level of receptor expression and remove a hyaluronan pericellular matrix. Together, these studies reveal a direct mechanism for promoting cell invasion into the hyaluronan-rich matrix and predict that in the complex multicellular environment in vivo, multiple mechanisms exist to regulate the ability of a cell to respond to a chemotactic hyaluronan gradient.

Osteopontin silencing by small interfering RNA suppresses in vitro and in vivo CT26 murine colon adenocarcinoma metastasis.

Abstract: Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for alpha(v)beta3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 +/- 1.49 g versus 1.88 +/- 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.

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.

Abstract: 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 of cell adhesion molecules in oesophageal carcinoma and its prognostic value.

Abstract: Oesophageal carcinoma remains a disease of poor prognosis. Surgical cure rates are compromised by the fact that most patients are diagnosed at a late stage of disease because of the delayed onset of symptoms, by which time metastases and organ infiltration may have already occurred. Thus, invasion and metastases play a key role in influencing patient survival, and the search for novel treatments may therefore hinge on gaining insight into the mechanisms controlling these processes. It has been established that the initial step in the metastatic cascade is the detachment of tumour cells from the primary tumour via dysregulation of normal cell–cell and cell–matrix interactions. Distinct proteins known as cell adhesion molecules (CAMs) mediate these interactions. In recent years, a plethora of information has contributed to the in depth understanding of these molecules. This review provides a brief description of five families of CAMs (cadherins, integrins, CD44, immunoglobulin superfamily, and selectins) and highlights their altered expression in relation both to prognosis and tumour behaviour in squamous cell carcinoma and adenocarcinoma of the oesophagus.

Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12

Abstract: Activated forms of Ras family members are prevalent in many cancers where Ras mutants transduce signals essential for transformation, angiogenesis, invasion and metastasis. As a cancer progression model, we used NIH3T3 cells to explore the mechanism of Ras-induced tumorigenesis. Ras family mutants H-RasV12 and Rit79L strongly induced foci formation, while Rho family mutants RhoA-QL, Rac1-QL and Cdc42-QL were less effective. A comparison of downstream transcriptional targets of Ras and Rho family members using a 26 383 element cDNA microarray revealed that the osteopontin (OPN) gene exhibited the best correlation between magnitude of gene expression change and level of foci formation (r=0.96, P<0.001). In association with H-RasV12- and Rit79L-mediated transformation, foci secreted OPN protein and upregulated the OPN receptor CD44, suggesting the novel initiation of an aberrant OPN-CD44-Rac autocrine pathway. In support of this were the following observations. First, RGD-deficient OPN protein-binding activity was present in H-RasV12-transformed cells but not in control cells, and binding activity was inhibited by the CD44 blocking antibody. Second, foci formation, cell invasion and Rac activity were induced by H-RasV12 and inhibited by the CD44 blocking antibody. Third, foci formation by H-RasV12 was substantially reduced by a short interfering RNA (siRNA) specifically targeting OPN expression for knockdown. Fourth, H-RasV12-mediated transformation was not blocked by the GRGDS peptide, suggesting that OPN effects were not mediated by the integrins. Lastly, OPN knockdown affected the downstream expression of 160 '2nd tier' genes, and at least a subset of these genes appears to be involved in transformation. Indeed, four genes were selected for knockdown, each resulting in a disruption of foci formation and/or invasion. These results underscore the role of aberrant autocrine signaling and transcriptional networking during tumorigenesis.

Hematopoietic progenitor cells and cellular microenvironment: behavioral and molecular changes upon interaction.

Abstract: Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in controlling cell division. Using human hematopoietic progenitor cells (CD34+/CD38-) and a stroma cell line (AFT024) as a model, we have studied the initial behavioral and molecular sequel of this interaction. Time-lapse microscopy showed that CD34+/CD38- cells actively migrated toward and sought contact with stroma cells and 30% of them adhered firmly to AFT024 stroma through the uropod. CD44 and CD34 are colocalized at the site of contact. Gene expression profiles of CD34+/CD38- cells upon cultivation with or without stroma for 16, 20, 48, or 72 hours were analyzed using our human genome cDNA microarray. Chk1, egr1, and cxcl2 were among the first genes upregulated within 16 hours. Genes with the highest upregulation throughout the time course included tubulin genes, ezrin, c1qr1, fos, pcna, mcm6, ung, and dnmt1, genes that play an essential role in reorganization of the cytoskeleton system, stabilization of DNA, and methylation patterns. Our results demonstrate directed migration of CD34+/CD38- cells toward AFT024 and adhesion through the uropod and that upon interaction with supportive stroma, reorganization of the cytoskeleton system, regulation of cell division, and maintenance of genetic stability represent the most essential steps.

CD44 expression is associated with increased survival in node-negative invasive breast carcinoma.

Abstract: Purpose: CD44 is a multifunctional cell surface receptor with many known splice variants, some of which have been reported to play a role in tumor progression. The purpose of this study was to evaluate the prognostic significance of CD44 isoforms in early-stage, lymph node–negative invasive breast carcinoma. Experimental Design: Immunohistochemical staining for CD44 isoforms was done on archival paraffin tissue sections of invasive breast carcinoma from a cohort of lymph node–negative patients who received no adjuvant tamoxifen or chemotherapy and who had a mean clinical follow-up period of 15 years. Immunohistochemical staining was done with antibodies to CD44s, the standard isoform of CD44, and to isoforms containing variant exon 6 (CD44v6); levels of staining were correlated with clinical outcome data. Results: There was a trend towards increased disease-free survival for patients whose tumors had high anti-CD44s positivity ( P = 0.05), and a significant association was observed between anti-CD44s positivity and disease-related survival ( P = 0.04). Expression of CD44v6 isoforms did not correlate with clinical outcome. Conclusion: CD44 expression, as assessed by immunohistochemical staining with anti-CD44s, may be a favorable prognostic factor in patients with node-negative invasive breast carcinoma.

Osteopontin Is Overexpressed in Human Papillary Thyroid Carcinomas and Enhances Thyroid Carcinoma Cell Invasiveness

Abstract: Context: The transmembrane glycoprotein CD44v6 is overexpressed in most papillary thyroid carcinomas (PTC). We previously reported that osteopontin (OPN), a secreted glycoprotein that functions as a ligand for CD44v6, is overexpressed in thyrocytes transformed by the RET/PTC oncogene. Objective: In this study we asked whether OPN is overexpressed in human PTC samples, and whether its expression correlates with clinical and histological features of the tumors. Furthermore, we wanted to establish the functional role of the CD44-OPN axis in thyroid tumorigenesis. Design: Thyroid samples from 117 patients who had undergone surgical resection of the thyroid gland for benign or malignant lesions were collected. OPN and CD44 expressions were evaluated by immunohistochemistry with specific monoclonal antibodies. OPN expression was correlated with different PTC histological variants, lymph node metastasis, and PTC size. Results: In this study we show that OPN is overexpressed in human PTCs with respect to normal thyroid tissue, follicular adenomas, and multinodular goiters (P 0.05). The prevalence and intensity of OPN stainingweresignificantlycorrelatedwiththepresenceoflymphnode metastases (P 0.0091) and tumor size (P 0.0001). We also show that treatment of human PTC cells with recombinant exogenous OPN stimulated Matrigel invasion and activated the ERK and V-AKT murine thymoma viral oncogene homolog 1/protein kinase B; signaling pathways. Blockage of anti-CD44 antibodies prevented these effects. Conclusions: Given its prevalence and its correlation with aggressive features of human PTCs, we suggest that OPN might be used as a diagnostic and prognostic marker for these tumors. Furthermore, given the role of the OPN-CD44v6 axis in PTC cells, we suggest that CD44 and/or OPN may be molecular targets for therapeutic intervention in aggressive PTCs. (J Clin Endocrinol Metab 90: 5270–5278, 2005)

Engagement of CD44 modulates cyclooxygenase induction, VEGF generation, and proliferation in human vascular endothelial cells.

Abstract: CD44 is a receptor for hyaluronic acid and is found on the surface of hematopoetic cells and in mesenchymal tissue. It is also expressed on endothelial cells (EC). Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins in EC. Here we show that engagement of CD44 with signaling monoclonal antibodies (mAbs) or its natural ligand hyaluronic acid induces COX-2 and prostacyclin (PGI2) formation in human EC. This induction was blocked by mAbs that have been shown to inhibit CD44-mediated intracellular signaling. COX-1 induction was not observed after CD44 ligation. CD44-stimulated COX-2 activation/PGI2 production was accompanied by the production of the potent endothelial mitogen, vascular endothelial growth factor (VEGF) and was inhibited by a neutralizing VEGF antibody. Moreover, this COX-2 induction was also associated with an increase in EC proliferation that was inhibited by the blocking anti-CD44 mAbs and a COX-2-specific inhibitor. This is the first study to show that engagement of CD44 with mAbs or its natural ligand induces COX-2, generates VEGF, and thus leads to an increase in EC proliferation. Results from this study may have important and widespread implications for the development of novel therapeutic agents for modulating blood vessel growth during ischemic heart disease, during inflammation, or around solid tumors.