Shripad Joshi

Transcriptional Activation of ZEB1 by Slug Leads to Cooperative Regulation of the Epithelial–Mesenchymal Transition-Like Phenotype in Melanoma

The E-box-binding zinc finger transcription factors Slug and ZEB1 are important repressors of E-cadherin, contributing to epithelial-mesenchymal transition (EMT) in primary epithelial cancers. Activator or repressor status of EMT transcription factors defines consequences for tumorigenesis. We show that changes in expression levels of Slug in melanoma cell lines lead to concomitant alterations of ZEB1 expression. Electrophoretic mobility shift, luciferase reporter, and chromatin immunoprecipitation assays identified Slug as a direct transcriptional activator at E-boxes of the ZEB1 promoter. Transcriptional activation of ZEB1 was demonstrated to be specific for Slug, as EMT regulators Snail and Twist failed to influence ZEB1 expression. Slug and ZEB1 cooperatively repressed E-cadherin expression resulting in decreased adhesion to human keratinocytes, but promoted migration of melanoma cells. Our results show that the transcriptional activity of ZEB1 is increased by Slug, suggesting a hierarchical organized expression of EMT transcription factors through directed activation, triggering an EMT-like process in melanoma.

The Cadherin Switch in Melanoma Instigated by HGF is Mediated through Epithelial-Mesenchymal Transition Regulators

Dear Sir, A crucial step in melanoma initiation is the progressive loss of E-cadherin expression, followed by an upregulation of N-cadherin in melanoma cells (Hsu et al., 1996; Tang et al., 1994; Hsu et al., 2000). This process is essentially accomplished by epithelial-mesenchymal transition regulators (EMTRs). Representatives of EMTRs are Snail and Slug of the Snail family of zinc finger transcription factors and Twist of the basic helix-loop-helix (bHLH) family (Cano et al., 2000; Yang et al., 2004). Growth factors govern the continuous repression of E-cadherin via EMTRs to facilitate tumor progression (Thiery and Chopin, 1999; Fuxe et al., 2010). Grotegut et al. (2006) demonstrated that HGF induced scattering and motility in epithelial cells is related to Snail upregulation. We have shown previously that overexpression of HGF downregulates E-cadherin in melanocytic cells (Li et al., 2001). Here we report that HGF leads to stage dependent changes in EMTR expression. Protein expression levels of Slug, Snail and Twist were determined over a period of 24h after stimulation with 50ng/ml of rhHGF in three representative melanocytic cell lines. In melanocytes (FOM101) an increase in Slug and Snail expression was observed (Fig. 1A), in WM35 (Fig. S1A) and WM164 (Fig. 1B) melanoma cells, Slug was profoundly downregulated, whereas Twist expression increased in the nucleus and the cytoplasm (Fig. 1B). To simulate the cadherin switch in melanoma, WM164 cells, which express E- and N-cadherin, were transduced with an adenoviral vector encoding the cDNA of HGF (Ad.CMV.rhHGF) or a mock vector control (Ad.CMV.LacZ) at 20pfu/cell. A profound decrease of E-cadherin and an increase of N-cadherin expression was observed (Fig. 1C). Further, to investigate on signaling pathways involved in EMTR regulation after HGF exposure, we used specific inhibitors of MAPK (PD 98059), PI3K (LY 294002), GSK3β (GSK3β-inhibitor VIII and lithium chloride [LiCl]) or NF-κB (BAY 11-7082). In FOM101, addition of BAY 11-7082 completely abolished protein levels of Slug and Snail in the nucleus (Fig. 1D, left panel) and inhibition of GSK3β led to decreased Slug levels (Fig.1D, right panel). In melanoma cells exposure to PD 98059 led to a significant increase in Slug expression, which resulted in a downregulation of E-cadherin in WM164 (Fig. 1E, left panel and S1B, left panel). Like in melanocytes, inhibition of GSK3β decreased Slug levels (Fig. 1E, right panel and S1B, right panel). Inhibition of MAPK, PI3K or GSK3β leads to downregulation of Twist in the nucleus (Fig. 1E, left panel). Figure 1 HGF mediated stage specific changes of EMTR expression in melanocytic cells are dependent on NF-κB, MAPK, PI3K, and GSK3β. Melanocytic cells were either stimulated with rhHGF (50ng/ml) up to 24h (A, B) or transduced with Ad.CMV.hHGF (20pfu/cell) ... Since HGF led to differential expression levels of Slug and Twist in melanoma cell lines, we determined changes in cadherin levels of both EMTRs after silencing in WM164 cells with consequences for adhesion and migration. Silencing of Slug led to a doubling of E-cadherin levels, whereas expression of N-cadherin was significantly downregulated after Twist silencing with minor changes in E-cadherin levels (Fig. 2A). Correspondingly, we observed a highly significant increase in adhesion of WM164 cells to keratinocytes after silencing of Slug (Fig. 2B) and a profound reduction of adhesion to fibroblasts after silencing of Twist, but not Slug (Fig. 2C). Silencing of both EMTRs resulted in a significant reduction of migration, although silencing of Twist showed a more pronounced effect in scratch assays (Fig. 2D,E). Slug overexpression in WM9 and WM164 cells led to a downregulation of E-cadherin but no changes in N-cadherin levels (Fig. S2A), adhesion to keratinocytes, but not fibroblasts, was clearly reduced (Fig. S2B,C) and migration was enhanced (Fig. S2D,E). Taken together, these results demonstrate Slug to be important for the loss of epithelial properties and a predominant effect of Twist on N-cadherin regulation and adhesion to fibroblasts. Figure 2 Twist promotes adhesion of WM164 cells to fibroblasts. Slug (siSlug) and Twist (siTwist) were silenced and expression levels compared to control siRNA (scRNA). (A) Silencing of Slug led to increased E-cadherin levels whereas silencing of Twist resulted ... Based on the differential regulation of Slug and Twist we examined staining patterns in melanocytic tissue by immunohistochemistry. Examination revealed a highly positive staining for Twist in the cytoplasm of nevi but nearly absence in the nucleus (Fig. 2F and S3A,B). In primary as well as in metastatic lesions cytoplasmatic and nuclear staining of Twist was observed (Fig. 2F and S3A). In contrast, Slug was already found in the nucleus and cytoplasm of nevi (Fig. 2F and S3A). These data suggest an inverse nuclear expression of Slug and Twist in the progression of melanoma. Indeed, probing tissue microarrays with anti-Twist antibodies indicated an association with worse patient survival (Hoek et al., 2004). Together we show that the cadherin switch mediated by HGF is accomplished through stage specific changes in expression levels of Snail, Slug and Twist, suggesting a hierarchical activation of Slug and Twist in melanoma progression. Our data further suggest that Twist is driving melanoma conversion after the initial de-coupling from keratinocytes initiating binding to fibroblasts.

TGF‐β1 and TNF‐α differentially regulate Twist1 mediated resistance towards BRAF/MEK inhibition in melanoma

Resistance to BRAF and MEK inhibition is a common phenomenon in melanoma. Cytokines and transcription factors have been attributed to contribute to the loss of sensitivity towards these inhibitors. Here, we show that transforming growth factor (TGF)‐β1 if combined with PLX4032, a BRAF inhibitor, or GSK1120212, a MEK inhibitor, substantially increased cell death in BRAF‐mutant melanoma cell lines. This increase was based on the combined regulatory decrease in Twist1, an antiapoptotic protein. Overexpression or silencing of Twist1 attenuated or aggravated induction of apoptosis through PLX4032 or GSK1120212, respectively. Exposure to tumour necrosis factor (TNF)‐α, however, led to increased Twist1 levels and oppositional decrease in cell death if exposed to PLX4032 or GSK1120212. This increase in drug resistance again depended on Twist1 levels. Our studies suggest that Twist1 as a common downstream target of multiple signalling cascades plays a crucial role in mediating drug resistance to BRAF‐ and MEK‐targeted molecular inhibitors.

Gα13 mediates human cytomegalovirus-encoded chemokine receptor US28-induced cell death in melanoma

US28, a constitutively active G‐protein‐coupled receptor encoded by the human cytomegalovirus, leads to mechanistically unknown programmed cell death. Here we show that expression of wild‐type US28 in human melanoma cells leads to apoptotic cell death via caspase 3 activation along with reduced cell proliferation. Reduced tumor growth upon US28 expression was observed in a xenograft mouse model. The signaling mute US28R129A showed a reduced antiproliferative effect. On evaluating different G‐proteins coupled to US28 for signal transduction, Gα13 was identified as the main G‐protein executing the apoptotic effect. Silencing of Gα13 but not Gαq resulted in a substantial increase in cell survival. Overexpression of Gα13 but not Gαq and their GTPase deficient forms Gα13Q226L and GαqQ209L, respectively, confirmed the requirement of Gα13 for US28 mediated cell death. Increasing expression of Gα13 alone induced cell death underscoring its relay function for US28 mediated decreased cell viability. Further reduced expression of Gα13 in melanoma cell lines isolated from advanced lesions and melanoma tissue was observed. These findings identified Gα13 as crucial for US28‐induced cell death, substantiating that the effect of US28 on cell fate depends on preferred G‐protein binding.