Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

Background: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. Conclusions: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance

Protein nanotechnology

Cite this entry as: Yaradoddi J.S., Kontro M.H., Ganachari S.V., Sulochana M.B., Agsar D. (2019) Protein Nanotechnology. In: Martínez L., Kharissova O., Kharisov B. (eds) Handbook of Ecomaterials. Springer, Cham Publisher Name: Springer, Cham DOI: https://doi.org/10.1007/978-3-319-68255-6_192 Print ISBN: 978-3-319-68254-9 Online ISBN: 978-3-319-68255-6 First Online: 14 February 2019Medical management should be well-preserved; in particular, a fast, easy, and cheap diagnosis. Sometimes, a RNA and DNA nanobio-based diagnostic may not provide precise data with regard to specific disorders. Therefore, some quantifiable protein information and molecular folding are required for the analysis of such disorders. Proteins at minute concentrations are typically undetectable under normal circumstances nowadays, and can be measured and quantified using protein nanotechnology methods. On the other hand, protein machinery carry out tasks that are unsafe for cell behavior, comprising DNA duplication, intracellular carriage, ion pumps, and cellular motility. They have changed with unbelievable multiplicity, precision, efficacy, and a substantial number of studies in contemporary biology have been intended to expose the vital mechanisms or processes of their primary function. This chapter also emphasizes the recent developments in protein nanotechnology, with a special focus on molecular cytoskeletal motors, dyneins, myosins, and kinesins. They constitute a subcategory of protein machineries; they have distinguished properties and are able to convert biochemical energy to work automatically.Peer reviewe

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion

A low molecular weight heparin inhibits experimental metastasis in mice independently of the endothelial glycocalyx

Contains fulltext : 88997.pdf (publisher's version ) (Open Access)BACKGROUND: Some low molecular weight heparins (LMWHs) prolong survival of cancer patients and inhibit experimental metastasis. The underlying mechanisms are still not clear but it has been suggested that LMWHs (at least in part) limit metastasis by preventing cancer cell-induced destruction of the endothelial glycocalyx. METHODOLOGY/PRINCIPAL FINDINGS: To prove or refute this hypothesis, we determined the net effects of the endothelial glycocalyx in cancer cell extravasation and we assessed the anti-metastatic effect of a clinically used LMWH in the presence and absence of an intact endothelial glycocalyx. We show that both exogenous enzymatic degradation as well as endogenous genetic modification of the endothelial glycocalyx decreased pulmonary tumor formation in a murine experimental metastasis model. Moreover, LMWH administration significantly reduced the number of pulmonary tumor foci and thus experimental metastasis both in the presence or absence of an intact endothelial glycocalyx. CONCLUSIONS: In summary, this paper shows that the net effect of the endothelial glycocalyx enhances experimental metastasis and that a LMWH does not limit experimental metastasis by a process involving the endothelial glycocalyx

Calcium induced skim-milk gelation during heating as affected by pH

International audienceAbstractMilk gels (acid or rennet) are used by dairy industry to produce dairy products such as yogurt and cheese. Enrichment of milk with calcium salts and heat treatment are known to produce “calcium-milk coagulum” as a new type of milk gels, due to reduction of milk protein charges through calcium binding. The combination of heat treatment and calcium addition to milk results in gel structures, but the effect of calcium addition and pH adjustment during heating of milk is still unclear. The role of added calcium and decreasing pH were investigated by addition of calcium chloride (30 mM) to reconstituted skim milk followed by pH adjustment by hydrochloric acid and sodium hydroxide (4.6 < pH < 6.6 investigated), followed by heating at 90 °C for 10 min and overnight storage at 22 °C. In parallel, samples with no addition of calcium chloride were produced under the same conditions. The time and temperature to reach the gelation point, as detected by dynamic measurements of storage modulus (G′), were decreasing as pH decreased without addition of calcium, while calcium addition made gelation time and temperature independent of pH except for pH 4.6. Heat treatment combined with calcium addition was found, using confocal laser microscopy, to provide a fine and dense gel structure for skim milk with higher pH, while at pH lower than 5.6, the gel structure was similar to the structure of acid-induced gels. The last observation helps to establish a pH limit for production of calcium gels