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Human epidermal growth factor receptor 2 (HER2)/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor-initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified that surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers and CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu transgenic mice, indicating that aberrant luminal progenitors originated in Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49fhighCD61high-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased transforming growth factor-β (TGFβ) signaling and exhibited gene expression signatures of stemness, TGFβ signaling and epithelial-to-mesenchymal transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin-β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu transgenic mice, unraveled their properties and identified the cooperative integrin-β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs. [ABSTRACT FROM AUTHOR]

Keratins (K) are epithelia specific intermediate filament (IF) proteins expressed in a differentiation-dependent and tissue-specific manner. Twenty eight different polypeptides of human keratins have been identified. Keratins are subdivided into two subfamilies, i.e. acidic - with low molecular weights and basic - with high molecular weights, and are always expressed in pairs comprising one member of each subfamily. The keratin pair of 8 and 18 is predominantly expressed by all simple epithelia. This pair is known to be aberrantly expressed by a variety of squamous cell carcinomas. FBM is an immortalized but non-transformed cell line derived in our laboratory from the buccal mucosa of a human fetus. To understand the role of K8/18 in malignant transformation of stratified epithelial cells, full-length keratin 8 gene was transfected in FBM cells. Transfection of K8 gene in these cells resulted in the formation of K8/18 filaments and concomitant malignant transformation of the cells. These cells when injected subcutaneously in nude mice, produced tumors at the site of injection. Three tumor cell lines have been established from three tumors derived from three different K8 transected FBM clones. These cell lines are being further characterized. Keratin expression pattern, cell doubling time, soft agar colony forming assay, cell cycle analysis using flow cytometry, growth supplement independence and nude mice injections were carried out. Analysis of keratin expression based on 2-D gel electrophoresis followed by western blot has demonstrated expression of keratins 1, 5, 7, 10 in one of the cell lines. Keratin 8/18 expression was seen in all the cell lines similar to transfected clones. These cell lines showed reduced doubling time compared to parental K8 transfected clones, anchorage independent growth and tumors in nude mice. These cells also showed polyploidy. These cell lines will be useful to study different aspects of oral carcinogenesis, including chemoprevention. [ABSTRACT FROM AUTHOR]

Cytokeratins (CK) are intermediate filament proteins found in epithelia and are expressed in a tissue-specific and paired manner. CK pairs comprise of one member each from acidic and basic subfamilies. Oral epithelium is stratified and is an excellent example to illustrate tissue-specific CK expression. e.g. non keratinizing buccal mucosa (BM) expresses CK 4, 5, 13 and 14 while keratinizing dorsal tongue expresses CK 1, 2, 5, 6, 10, 14 and either 16 or 17. Our group has been working on CK expression in human oral pre-cancer and cancer, and we have shown changes in CK expression during oral carcinogenesis. Some of the common changes were non-expression of CK 5 and aberrant expression of cytokeratin 1, 8 and 18. Both retrospective and prospective studies using Immunohistochemistry and RT-PCR are in progress in our laboratory for prognostication of human oral cancer. Only 20% of the samples studied exhibit simultaneous aberrant expression of CK 8, 18 and non-expression of CK 5 together. These results are being correlated with the clinical parameters of the patients. CK-associated proteins like vimentin, a6 and β4 integrins are also being analyzed for their expression patterns. An experimental murine model is being used to analyze sequential stages in tongue and buccal mucosal carcinogenesis. CK and associated proteins like desmoplakin and α6 and β4 integrins are being analyzed at various stages of oral carcinogenesis. Our preliminary experiments have shown alteration in CK as well as desmoplakin expression at various stages of lingual carcinogenesis. Our in vitro studies on role of CK 8 and 18 in malignant transformation of stratified epithelial cells have shown that this CK pair contributes to the malignant transformation of stratified epithelial cells. Thus, in conclusion, it appears that CK and their associated proteins can possibly be used as prognostic markers in oral cancer, and CK 8, 18 play an active role in the transformation of stratified epithelia. [ABSTRACT FROM AUTHOR]