Charvann K. Bailey

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells.

The regulation of vitamin D receptor (VDR), a key mediator in the vitamin D pathway, in breast cancer etiology has long been of interest. We have shown here that the transcriptional repressor protein SLUG inhibits the expression of VDR in human breast cancer cells. To explore the possibility that SLUG regulates the VDR gene promoter, we cloned a 628bp fragment (-613 to +15) of the human VDR gene promoter. This region contains three E2-box sequences (CAGGTG/CACCTG), the classical binding site of SLUG. SLUG specifically inhibited VDR gene promoter activity. Chromatin-immunoprecipitation (ChIP) assays revealed that SLUG is recruited on the native VDR gene promoter along with the co-repressor protein CtBP1 and the effector protein HDAC1. These data suggests that SLUG binds to the E2-box sequences of the VDR gene promoter and recruits CtBP1 and HDAC1, which results in the inhibition of VDR gene expression by chromatin remodeling.

High motility of triple-negative breast cancer cells is due to repression of plakoglobin gene by metastasis modulator protein SLUG.

Background: High motility of aggressive breast cancer cells is associated with high SLUG and low plakoglobin levels. Results: SLUG binds to plakoglobin gene promoter and represses its expression. Conclusion: SLUG-induced increase in breast cancer cell motility is due to repression of plakoglobin by SLUG. Significance: Management of SLUG level should diminish the motility and thus aggressiveness in breast cancer cells. 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.

Mode of action of the retrogene product SNAI1P, a SNAIL homolog, in human breast cancer cells

SNAI1P, a protein coded by a retrogene, is a member of the SNAI family of E2-box binding transcriptional repressors. To evaluate whether the mode of action of SNAI1P is similar to those of the other predominant members of the SNAI family, we studied its action on human claudin 7 (CLDN7) gene promoter which has seven E2-boxes. We over-expressed FLAG-tagged SNAI1P in MCF7 and MDA-MB-468 cells. SNAI1P inhibited the expression of CLDN7 in these recombinant cells. SNAI1P also inhibited cloned CLDN7 gene promoter activity in human breast cancer cells. ChIP assays revealed that SNAI1P is recruited on the CLDN7 gene promoter along with the co-repressor CtBP1 and the effector HDAC1. Treatment of the cells with trichostatin A, an inhibitor of HDAC1, abrogated the repressor activity of SNAI1P. These data suggest that SNAI1P inhibits CLDN7 gene promoter epigenetically in breast cancer cells through chromatin remodeling.

BRCA1 accumulates in the nucleus in response to hypoxia and TRAIL and enhances TRAIL‐induced apoptosis in breast cancer cells

A major contributing factor to the development of breast cancer is decreased functional expression of breast cancer susceptibility gene 1, BRCA1. Another key contributor to tumorigenesis is hypoxia. Here we show that hypoxia increased the nuclear localization of BRCA1 in MCF‐7 and MDA‐MB‐468 human breast cancer cell lines without changing its steady‐state expression level. Nuclear accumulation of BRCA1 was not evident in MCF‐12A or HMEC (human mammary epithelial cell) nonmalignant mammary epithelial cells under the same conditions. Hypoxia also increased the cell surface expression of TRAIL on MDA‐MB‐468 cells. Neutralization of TRAIL precluded the hypoxia‐induced accumulation of BRCA1 in the nucleus, whereas exogenously administered TRAIL mimicked the effect. Treatment of MDA‐MB‐468 cells with TRAIL resulted in a dose‐ and time‐dependent increase in apoptosis. Furthermore, TRAIL‐induced apoptosis in HCC1937 cells, which harbor a BRCA1 mutation, increased synergistically when wild‐type BRCA1 was reconstituted in the cells, and downregulation of BRCA1 expression in MDA‐MB‐468 cells reduced the apoptotic response to TRAIL. These data provide a novel link between hypoxia, TRAIL and BRCA1, and suggest that this relationship may be especially relevant to the potential use of TRAIL as a chemotherapeutic agent.

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 B36: Development of calcitrol-resistance in triple-negative breast cancer cells through SLUG-mediated coordinate repression of CYP2R1, CYP27B1, and VDR gene promoters

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

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.

Abstract 435: Reduction of the invasive phenotype of SNAI overexpressing human breast cancer cells by peptide aptamer-mediated inhibition of SNAI protein functions

Two members of the SNAI superfamily of zinc-finger transcriptional repressors: SNAI1 (also known as SNAIL) and SNAI2 (also known as SLUG) are implicated in the induction of aggressiveness and metastatic phenotypes in human breast cancer cells. SNAI1 and SNAI2 are very similar in their amino acid sequences at the C-terminal zinc-finger domains, but the N-terminal repressor domain is somewhat different. SNAI2 has two essential sub-domains, SNAG and SLUG domains, required for its repressor activity. The SNAG domain is also important in SNAI1. We have developed a recombinant protein containing thioredoxin, a tri-repeat of SNAG and SLUG domains of SNAI2 and a membrane translocation motif (MTM; AAVLLPVLLAAP) for the protein to be targeted to the nucleus. We expressed and purified this protein from E. coli using the His-patch thiofusion expression system (Invitrogen). When delivered to MDA-MB-231 and BT549 cells, this aptamer inhibited the functions of the SNAI proteins. The effect of SNAI protein knockdown by the peptide aptamer on the transformed phenotypes of these cells was evaluated by investigating the following parameters: (i) Measurement of doubling time and saturation density by plating cells and performing daily counts. (ii) Evaluation of cellular proliferation by measuring incorporation of 5′-bromodeoxyuridine into logarithmically growing cells. (iii) Determination of anchorage-independent growth in soft agar assays and analysis of the number and size of colonies formed. (iv) Evaluation of the cellular migration in scratch assays and in modified Boyden Chamber/transwell migration assays (directional migration). (v) Measurement of cellular invasion in vitro by using Matrigel-coated chambers. (vi) Assessment of the sensitivity to anoikis by plating cells on poly-HEMA coated plates and determination of cells undergoing apoptosis with propidium iodine staining or annexin labeling and flow cytometric analysis. (vii) Performing 3D culture assays to analyze invasiveness, proliferation or matrix degradation. All these data indicated reduction of the invasive phenotypes of the SNAI protein over expressing highly aggressive and metastatic human breast cancer cells by the peptide aptamer. Wrapsome-mediated targeted delivery of this aptamer alone or with other drugs may be used to reduce the progression of breast cancer. Supported by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 435.

Abstract LB-22: Repression of alpha-, beta- and gamma-catenin genes by SNAI2 in human breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC SNAI2 is a transcriptional repressor protein implicated in the proliferation and metastasis of several human cancer cells. It binds to the E2-box sequence of its target gene promoters and down regulates their expressions by chromatin remodeling. While studying SNAI2-binding gene promoters in the human breast cells by ChIP-DSL technique, we found that SNAI2 binds to the promoters of the cytoskeleton proteins alpha-, beta- and gamma-catenins. These proteins help cells to maintain their epithelial phenotype and apico-basal polarity. Reduced expression of these proteins has been shown by IHC in high grade metastatic breast carcinoma. Here we report the molecular characterization of this repression. Highly invasive breast cancer cells (MDA-MB-231 and BT-549) express high levels of SNAI2 while mildly invasive (MCF 7 and MDA-MB-468) cells do not express SNAI2. Over-expression of SNAI2 in the SNAI2-negative MCF7 and MDA-MB-468 cells down regulated the mRNA and protein levels of the catenins in these cells. siRNA-mediated knock down of SNAI2 in the SNAI2-positive MDA-MB-231 and BT549 cells elevated the mRNA and protein levels of these proteins in these cells. SNAI2 over-expression induces the invasion and migration in non invasive cells, while down-regulation of the SNAI2 decreases the invasion and migration potential of highly invasive breast cancer cells. Chromatin immunoprecipitation data revealed co-recruitment of CtBP1 and HDAC1 at the catenin gene promoters in the SNAI2-expressing cells further indicating that SNAI2 represses these genes through chromatin remodeling. Our study thus adds to the mode of action of SNAI2 in mediating metastatic conversion of human breast cancer cells. Supported by the DOD-CDMRP IDEA Grant# W81XWH-06-1-0466 and the Susan G. Komen Breast Cancer Foundation grant# BCTR0707627 to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-22.