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.     

Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells

The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the non-dividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the -701 to -921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.     

VAMP-Associated Protein B (VAPB) Promotes Breast Tumor Growth by Modulation of Akt Activity

VAPB (VAMP- associated protein B) is an ER protein that regulates multiple biological functions. Although aberrant expression of VAPB is associated with breast cancer, its function in tumor cells is poorly understood. In this report, we provide evidence that VAPB regulates breast tumor cell proliferation and AKT activation. VAPB protein expression is elevated in primary and metastatic tumor specimens, and VAPB mRNA expression levels correlated negatively with patient survival in two large breast tumor datasets. Overexpression of VAPB in mammary epithelial cells increased cell growth, whereas VAPB knockdown in tumor cells inhibited cell proliferation in vitro and suppressed tumor growth in orthotopic mammary gland allografts. The growth regulation of mammary tumor cells controlled by VAPB appears to be mediated, at least in part, by modulation of AKT activity. Overexpression of VAPB in MCF10A-HER2 cells enhances phosphorylation of AKT. In contrast, knockdown of VAPB in MMTV-Neu tumor cells inhibited pAKT levels. Pharmacological inhibition of AKT significantly reduced three-dimensional spheroid growth induced by VAPB. Collectively, the genetic, functional and mechanistic analyses suggest a role of VAPB in tumor promotion in human breast cancer.     

Numb在三阴乳腺癌中抑制HDM2泛素化降解p53

目的:探究Numb蛋白在三阴乳腺癌患者中的表达降低情况,及Numb蛋白在三阴乳腺癌中对抑癌因子p53蛋白水平的影响及调控机制,进一步研究Numb蛋白的降低与三阴乳腺癌发生发展的相关性,从而为缺乏有效治疗方法的三阴乳腺癌提供一个潜在的治疗新靶点。方法:40例三阴乳腺癌患者病理组织切片取自重庆医科大学临床病理诊断中心,采用免疫组化法检测Numb蛋白在三阴乳腺癌患者中的表达情况。MCF-10A细胞株和MDA-MB-231细胞株均为ATCC来源,采用qPCR和Western blot法检测对比Numb、HDM2、p53三者的转录水平和蛋白质水平在以上两个细胞株中差异。采用增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)质粒转染的方法在MDA-MB-231细胞中重表达Numb,采用q PCR和Western blot法验证Numb、HDM2、p53三者表达的变化。结果:转染NUMB-EGFP后MDA-MB-231细胞中Numb的mRNA和蛋白质水平均明显上调,HDM2无显著改变,p53在转录水平无明显变化,但在蛋白质水平显著升高。在231细胞中上调Numb蛋白可以在转录后水平调节p53水平,使p53蛋白随之显著升高。结论:Numb蛋白在三阴乳腺癌患者中表达降低的比列很高,为55%,且Numb蛋白在三阴乳腺癌细胞MDA-MB-231中可以调控抑癌因子p53蛋白水平,Numb蛋白水平与p53蛋白水平呈正相关。     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

Plakoglobin Represses SATB1 Expression and Decreases In Vitro Proliferation,Migration and Invasion

Plakoglobin (γ-catenin) is a homolog of β-catenin with dual adhesive and signaling functions. Plakoglobin participates in cell-cell adhesion as a component of the adherens junction and desmosomes whereas its signaling function is mediated by its interactions with various intracellular protein partners. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in the human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We observed that the mRNA levels of SATB1, the oncogenic chromatin remodeling factor, were decreased approximately 3-fold in SCC9-PG cells compared to parental SCC9 cells. Here, we showed that plakoglobin decreased levels of SATB1 mRNA and protein in SCC9-PG cells and that plakoglobin and p53 associated with the SATB1 promoter. Plakoglobin expression also resulted in decreased SATB1 promoter activity. These results were confirmed following plakoglobin expression in the very low plakoglobin expressing and invasive mammary carcinoma cell line MDA-MB-231 cells (MDA-231-PG). In addition, knockdown of endogenous plakoglobin in the non-invasive mammary carcinoma MCF-7 cells (MCF-7-shPG) resulted in increased SATB1 mRNA and protein. Plakoglobin expression also resulted in increased mRNA and protein levels of the metastasis suppressor Nm23-H1, a SATB1 target gene. Furthermore, the levels of various SATB1 target genes involved in tumorigenesis and metastasis were altered in MCF-7-shPG cells relative to parental MCF-7 cells. Finally, plakoglobin expression resulted in decreased in vitro proliferation, migration and invasion in different carcinoma cell lines. Together with the results of our previous studies, the data suggests that plakoglobin suppresses tumorigenesis and metastasis through the regulation of genes involved in these processes.     

Livin promotes progression of breast cancer through induction of epithelial–mesenchymal transition and activation of AKT signaling

The inhibitor of apoptosis proteins (IAP) are closely correlated with proliferation, apoptosis, motility, and metastasis. Livin is the most recently identified IAP, and its role in breast progression remains unknown. In our study, analyses of 50 patients with breast cancer revealed that the positive expression rate of Livin was higher in breast cancer tissues (62%) relative to that in adjacent (35%) and normal tissues (25%). Livin expression in breast cancer correlated with the clinical stage and axillary lymph node metastasis and could be used as a prognostic marker. Our in vitro experiment revealed that Livin was highly expressed in high-invasive MDA-MB-231 cells as compared to low-invasive cells (MCF-7). Suppression of Livin by short-hairpin RNA reduced the Livin expression of MDA-MB-231 cells and subsequently inhibited tumor cell growth, proliferation, and colony formation and induced tumor cell apoptosis, motility, migration, and invasion. Overexpression of Livin in MCF7 cells resulted in increased migration and invasion capabilities of the cells without affecting proliferation and apoptosis. In addition, epithelial–mesenchymal transition (EMT) was induced by Livin expression in breast cancer cell lines. The high level of phosphorylated AKT in MDA-MB-231 cells was suppressed by Livin knockdown. Further, Livin-induced migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or knockdown of AKT expression using small-interfering RNA. In conclusion, Livin serves as an independent prognostic indicator for breast cancer. Livin expression promotes breast cancer metastasis through the activation of AKT signaling and induction of EMT in breast cancer cells both in vitro and in vivo.     

MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer

A search for regulators of estrogen receptor alpha (ERalpha) expression has yielded a set of microRNAs (miRNAs) for which expression is specifically elevated in ERalpha-negative breast cancer. Here we show distinct expression of a panel of miRNAs between ERalpha-positive and ERalpha-negative breast cancer cell lines and primary tumors. Of the elevated miRNAs in ERalpha-negative cells, miR-221 and miR-222 directly interact with the 3'-untranslated region of ERalpha. Ectopic expression of miR-221 and miR-222 in MCF-7 and T47D cells resulted in a decrease in expression of ERalpha protein but not mRNA, whereas knockdown of miR-221 and miR-222 partially restored ERalpha in ERalpha protein-negative/mRNA-positive cells. Notably, miR-221- and/or miR-222-transfected MCF-7 and T47D cells became resistant to tamoxifen compared with vector-treated cells. Furthermore, knockdown of miR-221 and/or miR-222 sensitized MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis. These findings indicate that miR-221 and miR-222 play a significant role in the regulation of ERalpha expression at the protein level and could be potential targets for restoring ERalpha expression and responding to antiestrogen therapy in a subset of breast cancers.     

Acetyl-CoA synthetases ACSS1 and ACSS2 are 4-hydroxytamoxifen responsive factors that promote survival in tamoxifen treated and estrogen deprived cells

Acetyl-CoA synthetases ACSS1 and ACSS2 promote conversion of acetate to acetyl-CoA for use in lipid synthesis, protein acetylation, and energy production. These enzymes are elevated in some cancers and important for cell survival under hypoxia and nutrient stress. 4-hydroxytamoxifen (4-OHT) can induce metabolic changes that increase cancer cell survival. An effect of 4-OHT on expression of ACSS1 or ACSS2 has not been reported. We found ACSS1 and ACSS2 are increased by 4-OHT in estrogen receptor-α positive (ER+) breast cancer cells and 4-OHT resistant derivative cells. ERα knockdown blocked ACSS1 induction by 4-OHT but not ACSS2. 4-OHT also induced ACSS2 but not ACSS1 expression in triple negative breast cancer cells. Long-term estrogen deprivation (LTED) is a model for acquired resistance to aromatase inhibitors. We found LTED cells and tumors express elevated levels of ACSS1 and/or ACSS2 and are especially sensitive to viability loss caused by depletion of ACSS1 and ACSS2 or treatment with an ACSS2-specific inhibitor. ACSS2 inhibitor also increased toxicity in cells treated with 4-OHT. We conclude ACSS1 and ACSS2 are 4-OHT regulated factors important for breast cancer cell survival in 4-OHT-treated and long-term estrogen deprived cells.     

A fine balance between CCNL1 and TIMP1 contributes to the development of breast cancer cells

Cyclin L1 (CCNL1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) are candidate genes involved in several types of cancer. However, the expression of CCNL1 and the relationship between CCNL1 and TIMP1 in breast cancer cells is unknown. Using patients’ breast cancer tissues, the expression of CCNL1 and TIMP1 was measured by cDNA microarray and further confirmed by real-time RT-PCR and western blotting. Overexpression or repression of CCNL1 and TIMP1, individually or together, was performed in breast cancer MDA-MB-231 cells by transient transformation methods to investigate their role in breast cancer cell growth. Simultaneously, mRNA and protein expression levels of CCNL1 and TIMP1 were also measured. CCNL1 and TIMP1 expression was significantly elevated in breast cancer tissues compared with that in peri-breast cancer tissues of patients by cDNA microarray and these results were further confirmed by real-time RT-PCR and western blotting. Interestingly, in vitro experiments showed a stimulatory effect of TIMP1 and an inhibitory effect of CCNL1 on growth of MDA-MB-231 cells. Co-expression or co-repression of these two genes did not affect cell growth. Overexpression of CCNL1 and TIMP1 individually induced overexpression of each other. These data demonstrate that there is a fine balance between CCNL1 and TIMP1, which may contribute to breast cancer development.     

Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene

Ron, the tyrosine kinase receptor for the Macrophage-stimulating protein, is involved in cell dissociation, motility, and matrix invasion. DeltaRon, a constitutively active isoform that confers increased motility to expressing cells, is generated through the skipping of exon 11. We show that abnormal accumulation of DeltaRon mRNA occurs in breast and colon tumors. Skipping of exon 11 is controlled by a silencer and an enhancer of splicing located in the constitutive exon 12. The strength of the enhancer parallels the relative abundance of DeltaRon mRNA and depends on a sequence directly bound by splicing factor SF2/ASF. Overexpression and RNAi experiments demonstrate that SF2/ASF, by controlling the production of DeltaRon, activates epithelial to mesenchymal transition leading to cell locomotion. The effect of SF2/ASF overexpression is reverted by specific knockdown of DeltaRon mRNA. This demonstrates a direct link between SF2/ASF-regulated splicing and cell motility, an activity important for embryogenesis, tissue formation, and tumor metastasis.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.     

Regulation of the P2X7R by microRNA-216b in human breast cancer

Breast cancer is the most common cancer in women around the world. However, the molecular mechanisms underlying breast cancer pathogenesis are only partially understood. Here, in this study, we found that P2X7R was up-regulated and miR-216b was down-regulated in breast cancer cell lines and tissues. Using bioinformatic analysis and 3′UTR luciferase reporter assay, we determined P2X7R can be directly targeted by miR-216b, which can down-regulate endogenous P2X7R mRNA and protein levels. Ectopic expression of miR-216b mimics leads to inhibited cell growth and apoptosis, while blocking expression of the miR-216b results in increased cell proliferation. Furthermore, our findings demonstrate that knockdown of P2X7R promotes apoptosis in breast cancer cells through down-regulating Bcl-2 and increasing the cleavage caspase-3 protein level. Finally, we confirmed that down-regulation of miR-216b in breast cancer is inversely associated with P2X7R expression level. Together, these findings establish miR-216b as a novel regulator of P2X7R and a potential therapeutic target for breast cancer.