Aberrant expression of vimentin predisposes oral premalignant lesion derived cells towards transformation.

OBJECTIVE: We have previously reported the aberrant expression of vimentin in human oral premalignant lesions and a 4-Nitroquinoline 1-oxide (4NQO) model of rat lingual carcinogenesis. Hence, we wanted to understand whether the expression of vimentin in early stage contributes to the process of transformation. STUDY DESIGN: Vimentin was stably expressed in oral premalignant lesion derived cells (vimentin negative) and various transformation related phenotypic assays were performed. Since vimentin alone failed to transform the cells, an additional carcinogenic stimulus benzo[a]pyrene (BP) was used. Concomitantly, immunohistochemistry (IHC) was performed on oral leukoplakia and tumor tissues for studying the expression of vimentin and E-cadherin. RESULTS: Exogenous expression of vimentin led to the appearance of EMT and stemness-related signatures. Further, upon BP treatment, vimentin expressing clones showed an increase in vitro and in vivo transformation efficiency. Importantly, high vimentin-low E-cadherin expression significantly correlated with the grade of dysplasia, as also with the lymph node metastasis in oral tumors. CONCLUSION: Our study suggests that the expression of vimentin in early stages may be beneficial, although not sufficient to achieve transformation. Further, high vimentin-low E-cadherin expression, if validated in more number of early oral lesions, may prove useful in the identification of high risk human premalignant lesions.

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

Keratin 5/14­mediated cell differentiation and transformation are regulated by TAp63 and Notch­1 in oral squamous cell carcinoma­derived cells.

Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)­derived cells, there was an increase in intracellular Notch­1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14­knockdown background. We observed that there was a decrease in the expression of Notch­1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in ß­catenin signaling in K14­downregulated cells. Notably, when TAp63 was downregulated in K14­knockdown cells, there was increase in non­phospho ß­catenin levels. Hence, this study indicates that TAp63 plays an important role in K14­downregulated cells possibly by regulating the Notch­1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch­1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.