As the condition seen in chronic smokers [13,

As an additional carcinogenic trigger to induce transformation, we selected BP as a procarcinogen after testing the metabolic competence and cytotoxicity of the vimentin expressing and its vector control cells (Fig. 2A and B). BP was used as a carcinogen mainly because: 1. It is mutagenic and carcinogenic in animal models and cell lines. 2. It simulates the condition seen in chronic smokers 13, 22. 3. The parental cell line DOK was derived from a heavy smoker 11. Visible colonies appeared on soft agar after 35 weeks of carcinogen exposure, marking the attainment of an in vitro transformed state. Interestingly, BP treated vimentin expressing cells demonstrated an approximate 2-fold increase in the number of colonies formed on soft agar as compared to its treated vector control cells . The increased transformation efficiency seen in BP treated vimentin expressing cells may be attributed to its pre-acquired molecular level changes in EMT and stemness related regulators. Similar findings were reported by Tellez et al. They showed induction of a dedifferentiation program characterized by EMT and stemness related changes, which occurs early in the process of transformation. These changes were shown to contribute to the process of cancer intiation to finally achieve transformation 23. Although, in this report, the changes in EMT and stemness markers were acquired by an epigenetic mechanism, in our study, the likelihood of vimentin, a cytoskeletal protein, exerting a direct epigenetic regulation is remote. In this context, we assume the possibility of vimentin targeting some of its interactors or intermediates to subsequently bring about the transcriptional regulation of EMT and stemness related proteins .Next we wanted to understand the phenotypic changes associated with the in vitro transformed vimentin expressing cells. BP treated vimentin positive cells showed a significant increase in its in vitro invasive potential, although no significant difference was seen in the migration and proliferation potential of the two clones . Exogenously expressed vimentin levels remained unchanged till the end of BP treatment (Supplementary Fig. S4A and B). Surprisingly, neither BP treated vimentin expressing nor its vector control cells showed endogenous induction of vimentin expression, which suggests that perhaps it occurs very late in the process of transformation and further treatment with BP for several weeks, might be required to achieve this stage. The E-cadherin expression remained downregulated while expression of Oct-4, Sox2 and Nanog showed significant increase in BP treated vimentin positive cells (Fig. 3F, Supplementary Fig. S5A).  Additionally, BP treated vimentin positive cells showed no difference in the levels of p53, Ras, ?-catenin and EMT regulators (Snail, Slug, Twist), while a decline was seen in the protein level of involucrin (Supplementary Fig. S5A-C). Interestingly, tumorigenic potential of BP treated vimentin positive cells was significantly higher, as reflected by the increase in volume of subcutaneous tumors, in NOD-SCID mice. On the other hand control cells failed to form tumors and in one case they formed a cyst . Furthermore, IHC analysis confirmed the differential levels of vimentin between the vimentin positive and its control group