To investigate whether hyaluronan (HA) and CD44 (hereinafter HA-CD44) promotes head and neck squamous cell carcinoma (HNSCC) chemotherapy resistance and whether HA-CD44 promotes epidermal growth factor receptor (EGFR)-mediated oncogenic signaling to alter chemotherapy sensitivity in HNSCC. Hyaluronan, a glycosaminoglycan component of the extracellular matrix, is a ligand for the transmembrane receptor CD44, which acts through multiple signaling pathways to influence cellular behavior. We recently determined that HA-CD44 promotes phospholipase C-mediated calcium signaling and cisplatin resistance in HNSCC.
Cell line study.
Tumor cell growth with various chemotherapeutic drugs (methotrexate, doxorubicin hydrochloride, adriamycin, and cisplatin) was measured in the presence or absence of HA and other inhibitors of the EGFR-mediated signaling pathway. Immunoblotting was used to study EGFR signaling. Migration assays provided one measure of tumor progression.
The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 2-fold reduced ability of methotrexate and an 8-fold reduced ability of adriamycin to cause HNSCC cell death. Immunoblotting studies demonstrated that HA can promote an association between CD44 and EGFR as well as CD44-dependent activation of EGFR-mediated signaling. Migration assays demonstrated that HA-CD44 can promote tumor migration with EGFR signaling. The presence of AG1478, an EGFR inhibitor, and U0126, an extracellular signal-regulated kinase inhibitor, inhibited HA-mediated tumor growth, migration, and chemotherapy resistance.
Our results indicate that HA promotes CD44/EGFR interaction, EGFR-mediated oncogenic signaling, and chemotherapy resistance in HNSCC. Perturbation of HA-CD44-mediated signaling may be a promising and novel strategy to treat HNSCC.
"CD44 expression potentially influences stem cell behaviour by a wide range of mechanisms  and interaction with hyaluronan, its principal ligand, influences several signalling pathways ,  and initiates signalling functions associated with nuclear translocation . The abrogation of tumour growth resulting from CD44 inhibition, either in vivo or in vitro, indicates its functional significance ,  and differential patterns of expression of CD44 isoforms can similarly affect cellular properties . "
[Show abstract][Hide abstract] ABSTRACT: CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.
PLoS ONE 02/2013; 8(2):e57314. DOI:10.1371/journal.pone.0057314 · 3.23 Impact Factor
"produced by myofibroblasts  were found to stimulate drug resistance in drug-sensitive cancer cells, whereas disruption of endogenous hyaluronan-induced signalling suppresses cellular resistance to several drugs including doxorubicin, taxol, vincristine and methotrexate . Various reports demonstrate that hyaluronan and CD44 promote drug resistance in a variety of cancer cell types, including breast, lung, pancreas and head and neck carcinomas     . Accordingly, cleavage of hyaluronan by hyaluronidase treatment has been reported to enhance the action of various chemotherapeutic drugs . "
[Show abstract][Hide abstract] ABSTRACT: Tumors irrespective of their origin are heterogenous cellular entities whose growth and progression greatly depend on reciprocal interactions between genetically altered (neoplastic) cells and their non-neoplastic microenvironment. Thus, microenvironmental factors promote many steps in carcinogenesis, e.g. proliferation, invasion, angiogenesis, metastasis and chemoresistance. Drug resistance, either intrinsic or acquired, essentially limits the efficacy of chemotherapy in many cancer patients. To some extent, this resistance is maintained by reduced drug accumulation, alterations in drug targets and increased repair of drug-induced DNA damage. However, the pivotal mechanism by which tumor cells elude the cytotoxic effect of chemotherapeutic drugs is their efficient protection from induction and excecution of apoptosis. It is meanwhile well established that cellular and non-cellular components of the tumoral microenvironment, e.g. myofibroblasts and extracellular matrix (ECM) proteins, respectively, contribute to the anti-apoptotic protection of tumor cells. Cellular adhesion molecules (e.g. L1CAM or CD44), chemokines (e.g. CXCL12), integrins and other ECM receptors which are involved in direct and indirect interactions between tumor cells and their microenvironment have been identified as suitable molecular targets to overcome chemoresistance. Accordingly, several therapeutic strategies based on these targets have been already elaborated and tested in preclinical and clinical studies, including inhibitors and blocking antibodies for CD44/hyaluronan, integrins, L1CAM and CXCL12. Even though these approaches turned out to be promising, the upcoming challenge will be to prove the efficacy of these strategies in improving treatment and prognosis of cancer patients.
Current pharmaceutical biotechnology 05/2011; 13(11):2259-72. DOI:10.2174/138920112802501999 · 2.51 Impact Factor
"On the other hand, HA has been shown to contribute to the EGFR pathway via HA-CD44 interaction. HA-CD44 complexes colocalize and potentially transactivate the EGF receptor leading to phosphorylation of ERK1 and ERK2 in glioblastoma cell lines  and to increase tumour growth, migration and resistance to a variety of chemotherapeutic drugs such as methotrexate, doxorubicin, adriamycin and cisplatin in head and neck cancer . In line with this, reduction of HA synthesis by 4-MU enhances the anticancer activity of gemcitabine in pancreatic cancer cells . "
[Show abstract][Hide abstract] ABSTRACT: Oesophageal cancer is a highly aggressive tumour entity with at present poor prognosis. Therefore, novel treatment options are urgently needed. Hyaluronan (HA) is a polysaccharide present in the matrix of human oesophageal squamous cell carcinoma (ESCC). Importantly, in vitro ESCC cells critically depend on HA synthesis to maintain the proliferative phenotype. The aim of the present study is (1) to study HA-synthase (HAS) expression and regulation in human ESCC, and (2) to translate the in vitro results into a mouse xenograft model of human ESCC to study the effects of systemic versus tumour targeted HAS inhibition on proliferation and distribution of tumour-bound and stromal hyaluronan.
mRNA expression was investigated in human ESCC biopsies by semiquantitative real-time RT PCR. Furthermore, human ESCC were xenografted into NMRI nu/nu mice. The effects on tumour progression and morphology of 4-methylumbelliferone (4-MU), an inhibitor of HA-synthesis, and of lentiviral knock down of HA-synthase 3 (HAS3), the main HAS isoform in the human ESCC tissues and the human ESCC cell line used in this study, were determined. Tumour progression was monitored by calliper measurements and by flat-panel detector volume computed tomography (fpVCT). HA content, cellular composition and proliferation (Ki67) were determined histologically.
mRNA of HAS isoform 3 (HAS3) was upregulated in human ESCC biopsies and HAS3 mRNA was positively correlated to expression of the epidermal growth factor (EGF) receptor. EGF was also proven to be a strong inductor of HAS3 mRNA expression in vitro. During the course of seven weeks, 4-MU inhibited progression of xenograft tumours. Interestingly, remodelling of the tumour into a more differentiated phenotype and inhibition of cell proliferation were observed. Lentiviral knockdown of HAS3 in human ESCC cells prior to xenografting mimicked all effects of 4-MU treatment suggesting that hyaluronan produced by ESCC is accountable for major changes in tumour environment in vivo.
Systemic inhibition of HA-synthesis and knockdown of tumour cell HAS3 cause decreased ESCC progression accompanied by tumour stroma remodelling and may therefore be used in novel approaches to ESCC therapy.
Molecular Cancer 03/2011; 10(1):30. DOI:10.1186/1476-4598-10-30 · 4.26 Impact Factor
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