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- Materials and Methods
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Background: Alcohol consumption is associated with the risk of progressive cancers including colon and breast cancer. The mechanisms for the alcohol-induced aggressive behavior of these epithelial cancer cells have not been fully identified. Epithelial–mesenchymal transition (EMT) is a developmental program recently shown to play a role in cancer progression and metastases. We hypothesized that alcohol might promote cancer progression by inducing EMT in cancer cells and tested this hypothesis by assessing alcohol-stimulated changes in phenotypic markers of EMT as well as the EMT transcription factor Snail and its related cell signaling.
Methods: Colon and breast cancer cell lines and a normal intestinal epithelial cell line were tested as well as colonic mucosal biopsy samples from alcoholic subjects. Cells were treated with alcohol and assessed for EMT-related changes using immunofluorescent microscopy, western blotting, reporter assays, RT-PCR, and knockdown of Snail with siRNA.
Results: We show alcohol upregulated the signature EMT phenotypic marker vimentin as well as matrix metalloprotease (MMP)-2, MMP-7, and MMP-9 and cell migration in colon and breast cancer cells—all characteristics of EMT. Alcohol also stimulated nuclear localization of Snail phosphorylated at Ser246, transcription from a Snail reporter plasmid, and Snail mRNA expression by RT-PCR. Snail siRNA knockdown prevented alcohol-stimulated vimentin expression. In vivo, Snail expression was significantly elevated in colonic mucosal biopsies from alcoholics. Also, we found alcohol stimulated activation of epidermal growth factor receptor (EGFR) signaling and an EGFR inhibitor blocked alcohol-induced cell migration and Snail mRNA expression.
Conclusions: Collectively, our data support a novel mechanism for alcohol promoting cancer progression through stimulating the EMT program in cancer cells via an EGFR-Snail mediated pathway. This study reveals new pathways for alcohol-mediated promotion of cancer that could be targeted for therapy or prevention of alcohol-related cancers.
Alcohol (ethanol) consumption is a risk factor for development as well as progression of many cancers (Boffetta and Hashibe, 2006; Seitz and Stickel, 2007). Chronic alcohol consumption is estimated to be directly responsible for about 4% of all cancers worldwide (Boffetta and Hashibe, 2006). Cancers promoted by alcohol include cancers of the upper aerodigestive tract (oropharynx, larynx, esophagus), liver, breast, colon, and rectum (Boffetta and Hashibe, 2006; Seitz and Stickel, 2007). The mechanisms underlying alcohol promotion of these cancers are not certain. However, several distinct alcohol-induced cancer promoting mechanisms have been postulated. These include alcohol mutagenic effects, changes in retinoic acid, folate, or estrogen metabolism, and oxidative stress (Seitz and Stickel, 2007). But, none has been shown to fully explain the mechanism of alcohol-induced aggressive behavior of cancer cells that ultimately results in metastasis, cancer occurrence, and poor outcome.
One cellular pathway involved in cancer progression that has received much recent attention is epithelial–mesenchymal transition (EMT) (Hugo et al., 2007; Thiery, 2002). EMT is a cellular program important during normal development (Nieto, 2002). However, recent studies have shown its importance in cancer progression and metastases (Hugo et al., 2007; Thiery and Sleeman, 2006; Turley et al., 2008). During EMT, epithelial cells lose tight junctions and acquire a more migratory and invasive (mesenchymal) phenotype. These changes include downregulation of junctional proteins including E-cadherin and expression of mesenchymal proteins like vimentin. In addition cells express matrix metalloprotease (MMP) enzymes that dissolve the extracellular matrix and promote cell migration and cancer cell invasion and metastases (Egeblad and Werb, 2002; Thiery and Sleeman, 2006). One key transcription factor regulating EMT is Snail (Nieto, 2002). Others include Snail2, ZEB1/2, and Twist with WNT/β-catenin signaling also being a critical component of EMT (Peinado et al., 2007; Thiery and Sleeman, 2006). Signaling through several growth factor receptors including the epidermal growth factor receptor (EGFR) can induce EMT and Snail expression (Ackland et al., 2003; Peinado et al., 2007).
Because of the important role of EMT in cancer progression, we hypothesized that alcohol abuse may worsen the outcome of epithelial-derived cancers such as colon and breast cancer, by stimulating EMT in cancer cells and thus promoting cancer progression and metastases. The aim of our study was to test our hypothesis by determining if alcohol, at relevant in vivo levels, stimulates key phenotypic and functional signatures of EMT associated with cancer progression in colon and breast cancer cell lines as well as in a nontransformed (normal) intestinal epithelial cell line. We also sought to investigate the signaling mechanism of alcohol-induced promotion of EMT. To this end, we determined the effects of alcohol on activation and mRNA expression of the key EMT Snail transcription factor as well as upstream signaling pathways related to EGFR signaling that might be turning on Snail and EMT. Also, in order to determine the potential relevance of our in vitro findings, we measured Snail mRNA expression in sigmoid colon mucosal samples from alcoholic subjects.
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- Materials and Methods
- Conflict of interest
In the current study, we provide evidence that alcohol induces EMT in cancer cells and this alcohol effect is mediated through Snail activation. EMT is an active focus of current cancer research with a large body of in vitro, animal, and patient evidence strongly supporting a key role for EMT in cancer progression and metastases (Hugo et al., 2007; Thiery, 2002; Thiery and Sleeman, 2006; Turley et al., 2008; Weinberg, 2008). In addition to breast and colon cancer, evidence supports a role for EMT in oral, nasopharyngeal, esophageal, gastric, rectal, cervical, ovarian, thyroid, pancreatic, and prostate cancer to name only a partial list (Hugo et al., 2007; Natalwala et al., 2008; Turley et al., 2008). Recent animal experiments demonstrated direct in vivo evidence for EMT in breast cancer progression (Trimboli et al., 2008). In colon cancer, it has been shown that EMT (vimentin) is associated with early adenoma progression in the APCMin mouse model (Chen et al., 2008). We now, for the first time, provide compelling evidence to support our hypothesis that alcohol promotes breast and colon cancer migration by stimulating the EMT program in cancer cells.
Several prior studies with alcohol in different cell types are relevant to our findings. Two studies describe EMT changes by alcohol treatment in an immortalized keratinocyte cell line (9 weeks alcohol treatment) (Chamulitrat et al., 2003) or a breast epithelial cell line (7 days of alcohol treatment) (Robson et al., 2006). Specific mechanisms for EMT were not investigated in either study. In other studies alcohol was found to stimulate cell migration of breast cancer cells (Ke et al., 2006) and endothelial cells (Morrow et al., 2008). Alcohol has also been shown to both activate and inhibit TACE-mediated processing of TNF-α (Zhao et al., 2003, 2004) and to inhibit EGFR signaling or enhance ErbB2 cell migration (Ma et al., 2003, 2005).Thus, although limited in their investigation of possible mechanisms, these previous studies support our hypothesis for induction of EMT characteristics by alcohol stimulation.
In the present study, we investigated the effects of alcohol in breast and colon cancer cell lines because both of these cancers are promoted by alcohol use. Our data show that alcohol stimulates expression of the signature mesenchymal protein vimentin in breast and colon cancer cells. We also show that Snail knockdown inhibits this alcohol-induced vimentin expression in Caco-2 cells. Epithelial cells express cytokeratin intermediate filaments, while these filaments are composed of vimentin in mesenchymal cells. (De Wever et al., 2008). Vimentin is the most widely used and well validated signature phenotypic marker of EMT (De Wever et al., 2008; Turley et al., 2008). Vimentin expression has been identified in several studies of breast, colon, and other cancers as being associated with increased invasiveness and poor prognosis (De Wever et al., 2008).
Our data for both breast and colon cancer cells shows that alcohol stimulates nuclear localization and phosphorylation of Snail at Ser246, probably by PAK1(Yang et al., 2005). In addition we show alcohol stimulates mRNA expression from a Snail reporter plasmid and Snail mRNA expression in colon and breast cancer cell lines. Snail has been called a master regulator of EMT (Peinado et al., 2007). A hierarchical model of transcriptional regulation of EMT involves other key transcription factors that have been identified including Snail2, ZEB1/2, and Twist but places Snail at the top of this hierarchy (Peinado et al., 2007). Overexpression of Snail alone is sufficient to induce EMT in cell lines in vitro (Cano et al., 2000; Julien et al., 2007) and recently to generate possible cancer stem cells (Mani et al., 2008). In transgenic mice, chronic Snail activation results in multiple epithelial and mesenchymal tumors (Perez-Mancera et al., 2005). In another mouse model, Snail expression determined breast cancer invasiveness and recurrence (Moody et al., 2005). Overexpression of Snail has also been shown to correlate closely with invasiveness and recurrence of breast and colon cancer as well as other cancers in patient studies (Blanco et al., 2002; Natalwala et al., 2008; Roy et al., 2005). We now show, for the first time, that alcohol also promotes EMT in cancer cells through activation of Snail. We propose that Snail-mediated promotion of EMT in epithelial cancer cells by alcohol may be one mechanism for alcohol promotion of cancer progression and poor outcome of colon and breast cancers in alcoholics.
Relevant to our MMP data, Snail has been shown to directly stimulate MMP-2 and MMP-9 expression (Jorda et al., 2005; Yokoyama et al., 2003). Knockdown of Snail in a mouse tumor model resulted in fewer tumors at least in part due to reduced MMP-9 (Olmeda et al., 2007). MMP-3 or MMP-9 can also further promote EMT via Snail (Radisky et al., 2005). In addition, MMP-7 is a target of β-catenin/TCF signaling which is another pathway important in EMT also tied to Snail (Brabletz et al., 1999; Peinado et al., 2007). Increased Snail activity can stimulate β-catenin indirectly by repressing E-cadherin or directly by interacting with β-catenin (Stemmer et al., 2008). Our data supports a mechanism in which alcohol stimulation of activated Snail nuclear localization as well as increased Snail mRNA expression results in promoting EMT and cancer progression by increasing expression of vimentin as well as MMP-2, MMP-7, and MMP-9 in cancer cells.
Experiments in the APCMin mouse model of colon cancer also support our hypothesis for alcohol promoting cancer progression through stimulation of Snail and EMT. Snail is overexpressed in human colon cancer (Roy et al., 2005) and adenoma formation in APCMin mice is inhibited by Snail knockdown (Roy et al., 2004). Significantly, adenoma formation in these mice is enhanced by drinking alcohol (Roy et al., 2002) and recently this same adenoma formation in APCMin mice was shown to occur by an EMT-dependent mechanism with early expression of vimentin (Chen et al., 2008).
Our data suggest that alcohol stimulation of EGFR signaling may promote activation of Snail and EMT. Abnormalities in EGFR signaling or expression are associated with many human cancers (Hynes, 2007). Several different cell signaling pathways have been shown to stimulate induction of EMT including signaling by receptor tyrosine kinases such as the EGFR (Peinado et al., 2007; Thiery and Sleeman, 2006). Chronic stimulation with EGF can result in activation of Snail and EMT (Ackland et al., 2003; Peinado et al., 2007). Blocking EGFR signaling inhibits alcohol-stimulated Snail mRNA expression in our study and Snail mediated colon cancer metastases in mice (Mann et al., 2006). EMT in cervical cancer also correlates with EGFR and Snail overexpression (Lee et al., 2008). Our data suggest alcohol activates EGFR signaling through TACE-mediated transactivation. TACE-mediated EGFR transactivation promotes EMT-mediated cancer progression in breast epithelial cells (Kenny and Bissell, 2007). Our data also show alcohol-stimulated ERK1/2 activation through the EGFR. Overexpression of ERK1/2 also promotes EMT and promoter analysis of Snail reveals a key stimulatory role for ERK1/2 (Barbera et al., 2004; Schramek et al., 2003). In addition, our data show that alcohol stimulates phosphorylation/activation of Snail at Ser246 probably by PAK1 (Yang et al., 2005). EGFR signaling has also been shown to activate PAK1 (Kumar et al., 2006). In further support of a role for EGFR-EMT signaling in our model, an EGFR inhibitor also inhibited alcohol stimulated cell migration in our study. Therefore data from others as well as our own data support a role for alcohol-stimulation of EGFR signaling in activation of Snail nuclear localization and mRNA expression and subsequent promotion of EMT.
It should also be noted that several epidemiological studies suggest that alcohol abuse not only promotes cancer progression and metastasis, it may also increase the risk of certain cancers like squamous epithelial cancer of esophagus, head and neck cancer and also breast and colon cancer (Seitz and Stickel, 2007). Furthermore, several experimental studies suggested that Snail and EMT are not only involved in cancer progression but also in cancer initiation (Hugo et al., 2007; Thiery, 2002; Thiery and Sleeman, 2006; Turley et al., 2008; Weinberg, 2008). For example, overexpression of Snail in transgenic mice results in multiple tumor types and EMT itself is associated with increased genomic instability which in turn could contribute to cancer initiation as well as cancer progression (Radisky et al., 2005). Our findings in normal, nontransformed intestinal cells that alcohol promotes EMT in noncancer cells support the notion that alcohol-induced stimulation of EMT pathway in normal epithelial cells may be a mechanism for increased risk of cancers in alcoholics. Further studies are needed to confirm our findings in different noncancer cell lines and primary epithelial cells.
In summary, collectively our data present evidence for a novel pathway for alcohol promotion of cancer progression through induction of the EMT gene program in cancer cells. To our knowledge these data are the first to show that alcohol activates and increases mRNA expression of the key EMT transcription factor Snail or to show EGFR transactivation and induction of MMPs by alcohol in epithelial cells. Together with the vimentin, E-cadherin reporter, and cell migration (a surrogate marker for metastasis) data our study strongly supports the hypothesis that alcohol induces the EMT program in cancer cells and promotes metastasis. The mechanisms for exactly how EMT promotes cancer progression are currently being unraveled. Future studies will be needed to identify the individual roles of EGFR, MMP, and Snail signaling in alcohol induction of EMT as well as the possible roles for other EMT transcription factors. Identification of this novel mechanism for alcohol promotion of cancer progression could open new avenues for prevention and treatment of alcohol-related cancers.