Biological Effects Of CD44in Tumor

Background:Breast cancer is the most common malignant disease among tumors in women. In these patients, it is not the primary tumor, but its metastases at distant sites are the main causes of death. Improving our understanding of the molecular mechanisms of the metastatic process might also improve clinical management of the disease. CD44proteins, members of cell-adhesion molecules family, are class I transmembrane glycoproteins encoded by a single, highly conserved gene. CD44is expressed on the surface of most vertebrate cells and tissues including breast cancer. The isoforms of CD44can be generated by alternative spliced and post-translation modifications. CD44proteins play very important roles in many cellular physiological and pathological processes though interacting with a range of cell-surface receptors. A lot of evidences implicate that CD44is up-regulated in sorts of tumor cells and participates in the regulation of growth, invasion, migration and homing of hematopoietic stem cells, and so on. CD44also is a molecular marker of cancer stem cells, and so the regulation of CD44gene expression is a complex process, which differs depending on tumor cell types and growth condition. CD44is among those genes most highly associated with ER-breast tumors for breast cancer progression, suggesting that CD44is a new therapeutic target for the prevention and treatment of ER negative breast cancer. Na+/H+exchanger1(NHE1), belonging to Na+/H+exchangers family and expressed ubiquitously in mammal cells, is an integral membrane protein and higher expressed in tumor cells. The primary regulator of pH; and pHe is known to be the Na+/H+exchanger1(NHE1), which can catalyze the extrusion of intracellular proton ions in exchange for extracellular sodium ions. Thus, NHE1activation may induce extracellular acidification and intracellular alkalinization, and this acid-base microenvironment homeostasis is thought to be critical to activation of extracellular proteases and degradation of the ECM resulting in motility and invasion of the tumor cell. Accumulated lines of evidences have demonstrated that the alkaline pHi in tumor cells has recently been associated with the higher expression level of vascular endothelial growth factor, which plays an important role in the induction of neovascularization and promotes tumor growth and metastatic potential. Except for the regulation of pHi, NHE1can act as a scaffold protein to participate in the intracellular signal transduction and regulate kinds of physiological and pathological processes. However, whether NHE1participates in the CD44mediated the metastasis of breast cancer cells and whether there is a direct regulating correlation between CD44and NHE1in breast cancer cells are not clear.Objective:To investigate the biological roles and molecular mechanism of CD44and NHE1in invasion and migration of MDA-MB-231cells and MCF-7cells; to elucidate the direct regulation model between CD44and NHE1.Methods:The expressions of CD44and NHE1in two breast cancer cell lines MDA-MB-231cells and MCF-7cells were detected by quantitative real-time PCR and western blotting, and then confirmed that there is a positive correlation between CD44and NHE1. The invasion and migration of breast cancer cells after treatment with CD44shRNA were detected by transwell experiment and wound-healing assay. The changes of NHE1expression in CD44silenced breast cancer cells were examined by quantitative real-time PCR, western blotting and immunofluorescence assay. The CD44was over-expressed in MCF-7cells and NHE1silenced MDA-MB-231cells to investigate the effects of CD44expression and biological roles on metastasis of breast cancer cells and NHE1expression. The quantitative real-time PCR and western blotting were used to determine the change of expression and activities of MMPs. Cariporide, a specific inhibitor of NHE1, was used to test the effects of NHE1inhibition on metastasis of breast cancer cells and the expression of MMPs. PD98059, a specific inhibitor of ERK1/2, was used to confirm the effects of signaling pathways on the process of CD44regulating metastasis.Results:The distinctive expressions of CD44were confirmed in different tumor cell lines. Comparing with MCF-7cells which exhibit a less pronounced metastatic and invasive phenotype, the expressions of CD44and NHE1were higher in MDA-MB-231cells and CD44may have a positive correlation with NHE1correlating with the invasive status of cell lines. The metastasis of MDA-MB-231cells significantly decreased when CD44expression was silenced and the expressions of NHE1and MMPs were markedly decreased. Consistent with the effects of CD44interference, NHE1down-regulation caused obviously decreased metastasis of MDA-MB-231cells and MMPs expressions. When over-expressed the level of CD44in MCF-7cells and NHE1silenced MDA-MB-231cells,the down-regulated expressions of NHE1were recovered by CD44over-expression, and the reduced metastatic abilities of MDA-MB-231cells caused by NHE1down-regulation were also significantly improved. The invasive and metastatic abilities were noticeably reduced pretreatment with Cariporiede or PD98059. However, when the two specific inhibitors were simultaneously used, the migration and invasion of MDA-MB-231cells reduced much more compared with either inhibitor. And so the ERK1/2inhibitor PD98059has a synergistic effect with Cariporide.Conclusions:Taken together, these data indicate that CD44has a positive correlation with NHE1in breast cancer cells and CD44can regulate the expression of NHE1to regulate the metastasis of breast cancer cells. During these processes, MMPs and ERK1/2is indispensable for CD44-mediated the metastasis of breast cancer cells. Our findings provide a theoretical basis that simultaneously targeting to CD44and NHE1may be novel therapeutic strategies for treating breast cancer. Background:Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with an incidence of one or two cases per100,000adults and accounts for15%of newly diagnosed cases of leukemia in adults. The chromic myeloid leukemia is a stem-cell disorder and affects mainly elderly people. The disease course of CML is triphasic, starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Central to the pathogenesis of CML is derived from the relocation of the portion of Abelson murine leukemia (ABL) gene from chromosome9to the portion of breakpoint cluster region (BCR) gene locus on chromosome22, which results in expression of an oncoprotein, termed BCR-ABL. BCR-ABL is a constitutively active tyrosine kinase that promotes growth and replication through downstream signaling pathways such as RAS, RAF, JUN kinase, MYC and STAT. This influences leukemogenesis by creating a cytokine-independent cell cycle with aberrant apoptotic signals in response to cytokine withdrawal. Firstly, drug therapy for CML was limited to nonspecific agents such as busulfan, hydroxyurea, and interferon-alfa (INF-a). INF-a led to regression of the disease and improved survival but was hampered by a multitude of toxicities. Allogeneic stem cell transplantation (AlloSCT) was a curative intervention but carried with it a high risk of morbidity and mortality. Further, alloSCT is only an option for patients with excellent performance status and an appropriate stem cell donor. Small molecule tyrosine kinase inhibitors (TKIs) were developed to exploit the presence of the aberrantly expressed BCR-ABL protein in CML cells. This targeted approach was found to dramatically alter the natural history of the disease and improve10-year overall survival of patients with chronic myeloid leukemia. A minority of CML patients in chronic phase and a substantial proportion in advanced disease phases are either initially refractory to imatinib treatment or lose imatinib sensitivity from nonspecific multidrug resistance to tyrosine kinase inhibitor over time and experience relapse. So it is indispensable to investigate the molecular mechanism of pathogenesis of CML.Objective:To investigate the biological roles and molecular mechanism of CD44in proliferation of K562chronic myeloid leukemia cells; to elucidate the effects of CD44down-regulation on cell cycles and apoptosis of K562cells; to study the direct signaling pathway mediated by CD44in pathogenesis of CML and provide the targets for CML therapy.Methods:The CD44expressions in patients with different leukemia were detected by RT-PCR and quantitative real-time PCR. And the expression of CD44in K562cells examined by quantitative real-time PCR and western blotting were compared with that in normal volunteers. So we confirmed that there is a positive correlation between CD44expression and the pathogenesis of CML. The expression of CD44in K562cells was silenced and the changes of CD44expression were examined by quantitative real-time PCR, western blotting and immunofluorescence assay. The proliferation of CD44silenced K562cells were detected by MTT experiment. After that, the change of cell cycle and apoptosis of K562cells were measured by flow cytometry. Then, the mRNA and protein expressions of p21, cyclin D1and β-catenin were detected by quantitative real-time PCR and western blotting. The nuclear accumulations of β-catenin were examined by immunofluorescence assay.Results:The distinctive expression of CD44was confirmed in patients with different leukemia and the expression of CD44in K562cells were higher compared with that in healthy volunteers. The expression of CD44in K562cells was decreased when trasnfected with CD44shRNA plasmids. And the down-regulation of CD44significantly decreased the proliferation of K562cells. The Go/G1arrest of cells cycle in K562cells were the major causes for the decreased proliferation. Meanwhile the apoptosis of K562cells was not changed. The Wnt/β-catenin signaling pathway was inhibited by CD44down-regulation and the expression of p-β-catenin was markedly increased. The nuclear accumulations of β-catenin in K562cells prominently decreased after treatment with CD44shRNA.Conclusion:Taken together, these data indicate that CD44has a positive correlation with proliferation of K.562cells and CD44down-regulation can induce the Go/G1arrest of cells cycle to decrease the proliferation of K562cells. During these processes, Wnt/β-catenin signaling pathway is indispensable for CD44-mediated the proliferation of K562cells. The down-regulation of CD44can increase the phosphorylation of β-catenin and decrease the nuclear accumulation of β-catenin, which inhibit the proliferation of K562cells. Our findings provide a theoretical basis that targeting to CD44may be a novel therapeutic strategy for treating CML.