Knockdown Of Slug Research Articles

Lentivirus-mediated shRNA Interference Targeting SLUG Inhibits Lung Cancer Growth and Metastasis

Lung cancer is a deadly cancer, whose kills more people worldwide than any other malignancy. SLUG (SNAI2, Snail2) is involved in the epithelial mesenchymal transition in physiological and in pathological contexts and is implicated in the development and progression of lung cancer. We constructed a lentivirus vector with SLUG shRNA (LV-shSLUG). LV-shSLUG and a control lentivirus were infected into the non-small cell lung cancer cell A549 and real-time PCR, Western blot and IHC were applied to assess expression of the SLUG gene. Cell proliferation and migration were detected using MTT and clony formation methods. Real-time PCR, Western Blot and IHC results confirmed down-regulation of SLUG expression by its shRNA by about 80%~90% at both the mRNA and protein levels. Knockdown of SLUG significantly suppressed lung cancer cell proliferation. Furthermore, knockdown of SLUG significantly inhibited lung cancer cell invasion and metastasis. Finally, knockdown of SLUG induced the down-regulation of Bcl-2 and up-regulation of E-cadherin. These results indicate that SLUG is a newly identified gene associated with lung cancer growth and metastasis. SLUG may serve as a new therapeutic target for the treatment of lung cancer in the future.

High Motility of Triple-negative Breast Cancer Cells Is Due to Repression of Plakoglobin Gene by Metastasis Modulator Protein SLUG

One of highly pathogenic breast cancer cell types are the triple negative (negative in the expression of estrogen, progesterone, and ERBB2 receptors) breast cancer cells. These cells are highly motile and metastatic and are characterized by high levels of the metastasis regulator protein SLUG. Using isogenic breast cancer cell systems we have shown here that high motility of these cells is directly correlated with the levels of the SLUG in these cells. Because epithelial/mesenchymal cell motility is known to be negatively regulated by the catenin protein plakoglobin, we postulated that the transcriptional repressor protein SLUG increases the motility of the aggressive breast cancer cells through the knockdown of the transcription of the plakoglobin gene. We found that SLUG inhibits the expression of plakoglobin gene directly in these cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Blocking of SLUG function with a double-stranded DNA decoy that competes with the E2-box binding of SLUG also increased the levels of plakoglobin mRNA, protein, and promoter activity in the SLUG-high triple negative breast cancer cells. Overexpression of SLUG in the SLUG-deficient cells elevated the motility of these cells. Knockdown of plakoglobin in these low motility non-invasive breast cancer cells rearranged the actin filaments and increased the motility of these cells. Forced expression of plakoglobin in SLUG-high cells had the reverse effects on cellular motility. This study thus implicates SLUG-induced repression of plakoglobin as a motility determinant in highly disseminating breast cancer.

Prediction of lymph node metastasis and prognosis in colorectal cancer patients using slug and vimentin expression.

e14029 Background: Slug plays a critical role in regulating the epithelial-mesenchymal transition (EMT) by down-regulation of epithelial markers and up-regulation of mesenchymal markers. The purpose of the present study was to evaluate the clinical significance of Slug and Vimentin expression in colorectal cancer (CRC) and to perform in vitro characterization of Slug’s function. Methods: At first, the biological role of Slug in CRC was assessed by RNA interference in CRC cell lines to assess tumor progression, invasion and migration. We next analyzed Slug and Vimentin expression in surgical tissue specimens from 181 CRC patients by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Results: Knockdown of Slug in siRNA knockdown studies resulted in induction of EMT markers, inhibited cancer cell proliferation, invasion and migration abilities. Slug and Vimentin expression in cancer tissues was significantly higher in patients with high T stage, lymph node involvement, liver metastasis and advanced TNM stage. We also observed a significant correlation between Slug and Vimentin expression in CRC (rho = 0.467). Increased Slug and Vimentin expression were significantly associated with poor prognosis in Stage I-IV (p<0.0001, p=0.0005, log-rank test) and Stage II (p= 0.04, p=0.012, log-rank test) Furthermore, increased Slug expression was an independent predictive marker of lymph node metastasis (p=0.012) and prognostic factor (p= 0.025). Conclusions: Our data demonstrate that evaluation of Slug and Vimentin could be valuable in the identification of patients with lymph node metastasis or poor prognosis in CRC.

Abstract 1287: High mobility of triple-negative breast cancer cells is due to repression of plakoglobin gene by SLUG

Abstract One of highly pathogenic breast cancer cell types are the SLUG-high claudin-low triple negative (negative in the expression of estrogen, progesterone and ERBB2 receptors) breast cancer (TNBC) cells. These cells are highly metastatic and have low levels of the motility regulatory catenin plakoglobin. In order to link high levels of the transcriptional repressor SLUG in the TNBC cells with low levels of plakoglobin we found that SLUG inhibits the expression of plakoglobin gene directly in these cells and thus, among other downstream effects, help disseminating these tumor cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high cancer cells elevated the levels of plakoglobin. Overexpression of SLUG in the SLUG-deficient cells elevated the invasiveness and motility of these cells. On the other hand, knockdown of plakoglobin in these low motility non-invasive breast cancer cells did not affect the ability of the cancer cells to penetrate Matrigel matrix but increase the growth and migration rates of these cells. This study thus implicates high levels of SLUG and low levels of plakoglobin as determinants in the progression of highly disseminating breast cancer. Supported in parts by the DOD-CDMRP grants BC050641, BC086542 and BC103645 to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1287. doi:1538-7445.AM2012-1287

Abstract 4677: Slug is a potential target to restore transforming growth factor-β mediated apoptosis in lung cancer cells

Abstract Transforming growth factor-β (TGFβ) inhibits carcinogenesis at early stages by inhibiting cell growth and inducing apoptosis. Cancer cells acquire resistance to TGFβ induced apoptosis and divert the TGF-β signaling to stimulate tumor promoting processes such as Epithelial-Mesenchymal-Transition (EMT), immunosuppression and angiogenesis. Hence, the identification of molecular targets/ pathways enabling cancer cells to overcome TGFβ-mediated apoptosis is critical for exploiting tumor suppressive functions of TGFβ for therapy. We have identified slug (snai2), a zinc-finger transcription factor, as a critical regulator of TGF-β mediated apoptosis in lung cancer cells. In EMT models of lung cancer slug is up-regulated by several fold upon TGFβ treatment. Knock down of Smad 3 or Smad 4, but not Smad 2, completely abrogated the TGF-β induced up-regulation of Slug. Chromatin immunoprecipitation analysis revealed increased occupancy of Smad 3 to the slug promoter on TGFβ treatment indicating slug might be a direct target gene of Smad 3. Slug is often described as a potent E-cadherin suppressor and an important regulator of EMT along with other E-box proteins. In contrast siRNA knock-down of slug (slug KD) in lung cancer cells did not prevent TGFβ induced E-cadherin suppression or acquisition of mesenchymal markers such as N-cadherin, vimentin and fibronectin; instead lead to increased cell death. Slug KD cells on TGFβ treatment underwent apoptosis through the typical apoptosis cascade characterized by increased annexin-v staining and caspase-3 activation. This apoptotic cell death is abrogated by inhibitors of caspase-3 and caspase-9. Inhibition of caspase-8, an initiator of extrinsic apoptotic pathway or siRNA knockdown of RIP1 that promotes necroptosis did not inhibit the death of TGFβ treated slug KD cells. Together these finding suggests slug KD results in cell death via intrinsic apoptosis pathway in response to TGFβ. This study indicates slug induction during EMT allows cancer cells to escape from apoptotic functions of TGFβ and might function as a critical regulatory switch to shift TGFβ role from tumor suppressor to tumor promoter. Thus slug inhibition might serve as a potential mechanism to reinstate the apoptotic functions of TGFβ in lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4677. doi:1538-7445.AM2012-4677

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

BackgroundThe epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.MethodsAgilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.ResultsMorphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.ConclusionsThis work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Abstract B36: Development of calcitrol-resistance in triple-negative breast cancer cells through SLUG-mediated coordinate repression of CYP2R1, CYP27B1, and VDR gene promoters

Abstract Metastatic breast and other cancers are often found to be refractory to vitamin D therapy. Vitamin D is synthesized from 7-dehydrocholesterol, an intermediate metaboltie in cholesterol synthesis. Ultraviolet irradiation in sunlight-exposed skin induces a photochemical reaction of 7–25-position by vitamin D 25-hydroxylase (CYP2R1), to yield 25-hydroxyvitamin D3 (cholecalciferol). Vitamin D3 is hydroxylated at the 25-position by vitamin D 25-hydroxylase (CYP2R1), to yield 25-hydroxyvitamin D3 (25(OH)D3; 25-hydroxycholecalciferol), the major form of vitamin D in the circulation. 25(OH)D3 is further hydroxylated at the 1 -position by the 25-hydroxyvitamin D 1 -hydroxylase (CYP27B1) to produce calcitrol (VD3). VD3 exhibits physiological and pharmacological effects by binding to the vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Although photoactivated cholecalciferol is mainly hydroxylated in the liver and kidney, many other cells including breast cells have significant expressions of CYP2R1, CYP27B1 and VDR. Breast cells thus should be able to activate and utilize cholecalciferol if these proteins are not suppressed. We report here that the metastasis modulator protein SLUG, which is often overexpressed in metastatic triple negative breast cancer cells, coordinately repressse the levels of CYP2R1, CYP27B1 and VDR proteins in the breast cancer cells to induce vitamin D-resistance in these cells. SLUG inhibited CYP2R1, CYP27B1 and VDR gene promoter activities in these cells. ChIP assays revealed that SLUG is recruited at the CYP2R1, CYP27B1 and VDR gene promoters. Knockdown of SLUG in highly invasive MDA-MB-231 and BT549 cells increased their CYP2R1, CYP27B1 and VDR gene expression and decreased their resistance their resistance to VD3 in vitro. Our data established that SLUG regulates vitamin D metabolism and contributes to the induction of VD3-resistance in human breast cancer cells through the inhibition of CYP2R1, CYP27B1 and VDR gene promoters epigenetically through chromatin remodeling. Supported in parts by the DOD-CDMRP grants BC050641 and BC103645. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B36.

Abstract B35: SLUG-induced plakoglobin gene repression in triplenegative breast cancer cells

Abstract Breast cancer with the triple-negative phenotype (TNBC) are ER-negative, PR-negative and ERBB2 (HER2)-negative and represent one of the most aggressive and difficult to treat subtypes of human breast cancer. TNBC is highly over-represented in African American breast cancer patients and determining the genetic and molecular basis for this incidence and the biological basis for the aggressiveness of TNBCs are largely unknown and of high priority. The transcription factor SLUG, which controls epithelial to mesenchymal transition, stem cell phenotypes and therapeutic responsiveness, is highly expressed in the basal-type TNBC cells, making it a candidate master regulator of the TNBC phenotype. Plakoglobin, also known as γ-catenin or JUP, is a member of the armadillo motifcontaining proteins. This protein is a critical component of the desmosomal structure conferring structural integrity and resistance from mechanical stress to epithelial cells in tissues. We report here that SLUG inhibits the expression of several desmosomal proteins including the plakoglobin gene directly and thus, among other downstream effects, help disseminating the TNBC cells. Overexpression of SLUG in the SLUG-deficient cancer cells significantly decreased the levels of mRNA and protein of plakoglobin. On the contrary, knockdown of SLUG in SLUG-high TNBC cells elevated the levels of plakoglobin. Inhibition of SLUG activity with a molecular decoy in the TNBC cells abrogated the inhibitory effect of SLUG on plakoglobin gene expression. Although overexpression of SLUG in the SLUG-deficient cells elevated the invasiveness and motility of these cells, knockdown of plakoglobin only affected the growth and migration rates of these cells. This study thus implicates high levels of SLUG and low levels of plakoglobin as determinants in the progression of highly disseminating breast cancer of the TNBC type. Supported by the DOD-CDMRP BCRP Grants W81XWH-06-1-0466, W81XWH-08-1-0446, BC086542, and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B35.

SLUG promotes prostate cancer cell migration and invasion via CXCR4/CXCL12 axis

BackgroundSLUG is a zinc-finger transcription factor of the Snail/Slug zinc-finger family that plays a role in migration and invasion of tumor cells. Mechanisms by which SLUG promotes migration and invasion in prostate cancers remain elusive.MethodsExpression level of CXCR4 and CXCL12 was examined by Western blot, RT-PCR, and qPCR analyses. Forced expression of SLUG was mediated by retroviruses, and SLUG and CXCL12 was downregulated by shRNAs-expressing lentiviruses. Migration and invasion of prostate cancer were measured by scratch-wound assay and invasion assay, respectively.ResearchWe demonstrated that forced expression of SLUG elevated CXCR4 and CXCL12 expression in human prostate cancer cell lines PC3, DU145, 22RV1, and LNCaP; conversely, reduced expression of SLUG by shRNA downregulated CXCR4 and CXCL12 expression at RNA and protein levels in prostate cancer cells. Furthermore, ectopic expression of SLUG increased MMP9 expression and activity in PC3, 22RV1, and DU-145 cells, and SLUG knockdown by shRNA downregulated MMP9 expression. We showed that CXCL12 is required for SLUG-mediated MMP9 expression in prostate cancer cells. Moreover, we found that migration and invasion of prostate cancer cells was increased by ectopic expression of SLUG and decreased by SLUG knockdown. Notably, knockdown of CXCL12 by shRNA impaired SLUG-mediated migration and invasion in prostate cancer cells. Lastly, our data suggest that CXCL12 and SLUG regulate migration and invasion of prostate cancer cells independent of cell growth.ConclusionWe provide the first compelling evidence that upregulation of autocrine CXCL12 is a major mechanism underlying SLUG-mediated migration and invasion of prostate cancer cells. Our findings suggest that CXCL12 is a therapeutic target for prostate cancer metastasis.

SLUG-induced Elevation of D1 Cyclin in Breast Cancer Cells through the Inhibition of Its Ubiquitination

UbcH5c, a member of the UbcH5 family of protein ubiquitin conjugase E2 enzymes, is a critical component of biological processes in human cells, being the initial ubiquitinating enzyme of substrates like IκB, TP53, and cyclin D1. We report here that the metastasis regulator protein SLUG inhibits the expression of UbcH5c directly through chromatin remodeling and thus, among other downstream effects, elevates the level of cyclin D1, thus enhancing the growth rates of breast cancer cells. Overexpression of SLUG in the SLUG-deficient breast cancer cells significantly decreased the levels of mRNA and protein of UbcH5c but only elevated the protein levels of cyclin D1. On the contrary, knockdown of SLUG in SLUG-high breast cancer cells elevated the levels of UbcH5c while decreasing the level of cyclin D1 protein. SLUG is recruited at the E2-box sequence at the UbcH5c gene promoter along with the corepressor CtBP1 and the effector HDAC1 to silence the expression of this gene. Knockdown of UbcH5c in the SLUG-deficient human breast cells elevated the level of cyclin D1 as well as the rates of proliferation and invasiveness of these cells. Whereas the growth rates of the cells are enhanced due to overexpression of SLUG or knockdown of UbcH5c in the breast cancer cells tested, ER(+) cells also acquire resistance to the anti-estrogen 4-hydroxytamoxifen due to the rise of cyclin D1 levels in these cells. This study thus implicates high levels of SLUG and low levels of UbcH5c as a determinant in the progression of metastatic breast cancer.

Slug Confers Resistance to the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

Non-small cell lung cancers carrying epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), but patients ultimately develop drug resistance and relapse. Although epithelial-mesenchymal transition (EMT) can predict resistance to EGFR TKIs, the molecular mechanisms are still unknown. To examine the role of EMT regulators in resistance to gefitinib. The expression level of EMT regulators in gefitinib-sensitive cells (PC9) and gefitinib-resistant cells (PC9/gef) was determined using quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Molecular manipulations (silencing or overexpression) were performed to investigate the effects of EMT regulators on gefitinib resistance in vitro, and a xenograft mouse model was used for in vivo confirmation. In addition, cancer cells from 44 patients with malignant pleural effusions of lung adenocarcinoma were collected for analysis of EMT regulator mRNA by quantitative real-time reverse transcription-polymerase chain reaction. Slug expression, but not that of snail, twist, or zeb-1, was significantly increased in PC9/gef compared with PC9 cells. Slug knockdown in PC9/gef cells reversed resistance to gefitinib, and overexpression of Slug in PC9 cells protected cells from gefitinib-induced apoptosis. Silencing of Slug in gefitinib-resistant cells restored gefitinib-induced apoptosis primarily through Bim up-regulation and activation of caspase-9. Slug enhanced tumor growth in a xenograft mouse model, even with gefitinib treatment. In clinical samples, Slug expression was significantly higher in cancer cells with resistance to EGFR TKIs than in treatment-naive cancer cells. Slug contributes to the resistance to gefitinib and may be a potential therapeutic target for treating resistance to EGFR TKIs.

Slug regulates proliferation and invasiveness of esophageal adenocarcinoma cells in vitro and in vivo

Slug is a transcription factor and E-cadherin repressor, which has recently been demonstrated to be important for cancer cells to down-regulate epithelial markers and up-regulate mesenchymal markers in order to become motile and invasive. In the present study, we assessed the relevance of Slug for invasion and growth potential of esophageal adenocarcinoma (EA) cells in vitro and in vivo. Slug expression was detected in nine human esophageal cancer cell lines. OE33 cell line was infected with Slug siRNA to knockdown of Slug; TE7 cell line was infected with full Slug cDNA to increase Slug expression. Then, Bcl-2 and E-cadherin expression and Caspase-3 activity were analyzed. MTT assay was applied to detect growth curve. The flow cytometric and Hoechst33258 staining was performed to detect apoptosis. The cells invasion in vitro was detected with a Boyden chamber. A pseudometastatic model of OE33 and TE7 in immunodeficient mice was used to assess the effects of knockdown of Slug and Slug overexpression on metastasis development. A subcutaneously nude mice xenograft model of OE33 and TE7 was used to assess the effects of knockdown of Slug and Slug overexpression on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug, bcl-2 and E-cadherin, and TUNEL was used to detected apoptosis in vivo. Western blotting and RT-PCR showed that Slug expression was detectable in 7 of 9 human esophageal cancer cell lines. Bcl-2 was down-regulated and E-cadherin was up-regulated significantly in Slug siRNA-infected OE33 cell line (P<0.01). Bcl-2 was upregulated and E-cadherin was downregulated significantly in Slug cDNA-infected TE7 cells (P<0.05). OE33 cells with Slug knockdown were shown to possess markedly decreased invasiveness (P<0.05) and markedly increased apoptosis (P<0.05). Slug cDNA-infected TE7 cells were shown only to possess markedly increased invasiveness (P<0.05). There was significant relationship between Slug knockdown or Slug overexpression and cells proliferation (respectively, P<0.05). Animals injected with Slug-silenced OE33 cells had fewer seeded tumor (P<0.01), more apoptosis cells (P<0.05) and significantly xenograft tumor growth regression (P<0.05). But in Slug cDNA-infected TE7 cells, more seeded tumor number and significantly xenograft tumor growth were found in xenograft tumor (respectively, P<0.05). It was showed in the subcutaneously nude mice xenograft model tumor tissue, bcl-2 expression was reduced followed by the decrease of Slug expression in Slug-silenced tumor, and bcl-2 expression was increased followed by the increase of Slug expression. In pseudometastatic model, E-cadherin overexpression was found in Slug siRNA tumor tissue, but less E-cadherin expression was found in Slug cDNA tissue. Slug is an important modulator of apoptosis, growth and invasion in EA in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of EA.

Slug inhibits proliferation of human prostate cancer cells via downregulation of cyclin D1 expression

Slug is a transcription factor of the Snail/Slug zinc-finger family and is implicated in metastasis of tumors, but its role in cell proliferation of prostate cancers is unclear. Expression level of Slug and other genes was examined by Western blot, RT-PCR, and QPCR analyses. The forced expression of Slug was mediated by retroviruses and adenoviruses. Slug was downregulated by shRNA. Cell growth was measured by the MTT assay and the quick cell proliferation assay. Here, we demonstrated that Slug expression is elevated in mouse prostate tumors, and human prostate cancer cell lines LNCaP, PC-3, and 22RV1. Forced expression of Slug-inhibited proliferation of prostate cancer cells PC-3 and DU-145. Conversely, reduced expression of Slug by shRNA promoted growth of PC-3 cancer cells. Consistent with these data, we found that forced expression of Slug in prostate cancer cells led to G1 cell-cycle arrest. Furthermore, ectopic expression of Slug decreased cyclin D1 expression in both PC-3 and DU-145 cells, and knockdown of Slug by shRNA upregulated cyclin D1 expression in these cancer cells. In addition, we demonstrated that ectopic expression of cyclin D1 relieved Slug-mediated inhibition of proliferation of prostate cancer cells. We provide the first compelling evidence that Slug is a negative regulator of proliferation of prostate cancer cells. Our findings in this study are distinct from the previously reported role of Slug as a promoter for tumor metastasis, and suggest that Slug is a prognostic marker and potential therapeutic target.

Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells

Mutations in the p53 tumour suppressor gene are associated clinically with tumour progression and metastasis. Downregulation of the E-cadherin cell-cell adhesion molecule is a key event for epithelial to mesenchymal transition (EMT) in tumour progression. Here, we show that wild-type p53 induced to adopt a mutant conformation, and hot-spot p53 mutants, which are both transcriptionally inactive, downregulate E-cadherin expression in the colon carcinoma cell line HCT116. Downregulation of E-cadherin occurred concomitantly with the upregulation of Slug and Zeb-1, transcriptional factors known to repress E-cadherin gene expression. In addition, knockdown of Slug and Zeb-1 expression diminished p53-mediated E-cadherin repression. Knocking down endogenous mutant p53 in MDA-MB-231 and SW620 cancer cell lines lacking E-cadherin protein restored the expression of E-cadherin. Complete loss of E-cadherin expression in HCT116 cells induced morphological alterations along with upregulation of vimentin, a mesenchymal marker. These changes characteristic of the EMT phenotype were, however, not sufficient to confer invasiveness in a three-dimensional matrix. Downregulation of E-cadherin by mutant p53 was not required to promote the invasive phenotype induced by inactivation of p53. These findings indicate that independent control of E-cadherin expression and cell motility could be essential molecular events in p53 mutant-induced invasive phenotypes.

Mechanisms of SLUG-Induced Drug Resistance Development in Breast Cancer Cells.

Abstract Down regulation of vitamin D receptor and over-expression of cyclin D1 are often associated with breast cancers. Depending upon the genetic context of the breast cancer cells, these alterations also often lead to development of resistance of the breast cancer cells against drugs like vitamin D3, all-trans retinoic acid and tamoxifen. We report here that the metastasis promoter protein SLUG indirectly induces the elevation of cyclin D1 levels in human breast cancer cells by repressing vitamin D3 receptor (VDR) and ubiquitin conjugating enzyme UbE2D3 genes. SLUG over-expression repressed the mRNA and protein levels of VDR and UbE2D3 in SLUG-negative human breast cells whereas knockdown of SLUG in the SLUG-expressing human breast cells had the reverse effect. SLUG also E2-box-dependently inhibited the activity of VDR and UbE2D3 gene promoters. CtBP1 and HDAC1 are co-recruited at the VDR and UbE2D3 gene promoters with SLUG, further indicating that SLUG represses this gene through chromatin remodeling. Knockdown of UbE2D3 or VDR in the SLUG-negative human breast cells elevated the levels of cyclin D1 as well as the rates of proliferation/invasiveness of these cells. While SLUG over expression has no effect on tamoxifen-resistance of ER-negative breast cancer cells, it significantly decreases tamoxifen-sensitivity of ER-positive breast cancer cells. On the other hand, knockdown of VDR gene expression by SLUG increased the resistance of both ER-positive and ER-negative breast cancer cells to 1, 25(OH)2D3 and all-trans retinoic acid. Our data thus suggest that SLUG not only promotes metastatic development of breast cancer cells, but also conspire to the development of resistance of these cells against several anti-cancer compounds. Supported by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1128.

Slug Is Required for Cell Survival during Partial Epithelial-Mesenchymal Transition of HGF-induced Tubulogenesis

Transcription factors of the Snail family are key regulators of epithelial-mesenchymal transition (EMT). In many processes during development or disease, cells do not acquire all the characteristics associated with EMT, leading to what we refer to as partial EMT (p-EMT). However, little is known of the implications of the Snail transcription factors in processes that only involve a p-EMT. To assess this, we used the hepatocyte growth factor (HGF)-induced Madin-Darby canine kidney tubulogenesis system, which provides a three-dimensional culture model of a morphogenetic process including a p-EMT. We found that although Slug (Snail2) is highly and transitory up-regulated during the p-EMT phase of tubulogenesis, it is not a repressor of E-cadherin during this process. Using inducible knockdown of Slug, we demonstrate that Slug is not an inducer of cell movement and instead is required for survival during p-EMT. We conclude that in epithelial cells, promoting cell survival can be a primary function of Slug, rather than being acquired concomitantly with EMT.

Oncogenic Raf1 induces epithelial cell transformation by activating Slug and suppressing Occludin promoter activity

The apical junctional complex (AJC) is important in regulating epithelial intercellular adhesion. Disruption of the AJC with the loss of concomitant junctional protein expression is a hallmark of cancer cell invasion. Activation of the Ras-Raf signaling pathway has been implicated in some epithelial cancers. Thus, our studies examined mechanisms by which Raf-1 induces epithelial mesenchymal transition (EMT) and oncogenic transformation. Using a rat salivary gland epithelial cell line (Pa4), we have previously demonstrated that constitutive expression of the Raf-1 oncogene in Pa4 cells disrupts functional AJC and induces oncogenic transformation by downregulating occludin expression. To better characterize the mechanism by which Raf-1 influences occludin expression and EMT, we analyzed the influence of Raf-1 on occludin promoter activity. A 1853 bp fragement of the occludin promoter was isolated and a series of deletion constructs were generated. Transfection of these constructs into Raf-1 transformed cells revealed that the minimal Occludin promoter segment (−50/+240) was repressed by Raf-1. Oncogenic Raf-1 induced upregulation of the transcription repressor Slug with subsequent down regulation of occludin. These events correlated with epithelial cell transformation. Interestingly, in contrast to other studies analyzing EMT, E-cadherin expression remained unchanged. Knockdown of Slug in Raf-1 transformed cells induced occludin expression and transition to an epithelial phenotype. These findings support a role of Slug in mediating Raf-1 induced suppression of occludin and subsequent EMT.