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.     

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n−6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.     

SOX7基因启动子甲基化水平对乳腺癌MDA-MB-231细胞株体外迁移和侵袭的影响

目的:探讨乳腺癌MDA-MB-231细胞中,Y性别决定区基因7(SOX7)基因启动子甲基化水平对细胞的体外迁移和侵袭的影响。方法:脂质体转染pcDNA3.0-DNA甲基转移酶3a(DNMT3a)质粒至MDA-MB-231细胞中,并于24h、48h及72h后,采用蛋白质免疫印迹实验(WB)检测细胞内DNMT3a蛋白表达水平;甲基化特异性定量PCR(Q-MSP)检测DNMT3a处理组、5-aza-C处理组及对照(Control)组MDA-MB-231细胞中的SOX7基因启动子DNA甲基化水平;实时荧光定量PCR(qRT-PCR)及WB实验检测各组MDA-MB-231细胞中的SOX7 m RNA和蛋白表达水平;细胞划痕实验及细胞侵袭实验检测各组MDA-MB-231细胞的迁移和侵袭能力。结果:pcDNA3.0-DNMT3a质粒转染MDA-MB-231细胞24h时,细胞内的DNMT3a蛋白表达水平最高。DNMT3a能够显著提高SOX7基因启动子DNA甲基化水平,而5-aza-C则抑制了SOX7基因启动子DNA甲基化水平(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞中,SOX7的m RNA及蛋白表达水平均明显下降,而5-aza-C处理组SOX7的m RNA及蛋白表达水平均明显增加(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞的迁移和侵袭能力均显著增强(P0.05),而5-aza-C处理组的MDA-MB-231细胞的迁移和侵袭能力变化不大(P0.05)。结论:在恶性肿瘤中,SOX7低表达表受其基因启动子高甲基化调节,且乳腺癌MDA-MB-231细胞中低表达的SOX7能够影响细胞的外迁移和侵袭能力。     

SMURF1 Plays a role in EGF-induced breast cancer cell migration and invasion

Epidermal growth factor (EGF) is a well-known growth factor that induces cancer cell migration and invasion. Previous studies have shown that SMAD ubiquitination regulatory factor 1 (SMURF1), an E3 ubiquitin ligase, regulates cell motility by inducing RhoA degradation. Therefore, we examined the role of SMURF1 in EGF-induced cell migration and invasion using MDA-MB-231 cells, a human breast cancer cell line. EGF increased SMURF1 expression at both the mRNA and protein levels. All ErbB family members were expressed in MDA-MB-231 cells and receptor tyrosine kinase inhibitors specific for the EGF receptor (EGFR) or ErbB2 blocked the EGF-mediated induction of SMURF1 expression. Within the signaling pathways examined, ERK1/2 and protein kinase C activity were required for EGF-induced SMURF1 expression. The overexpression of constitutively active MEK1 increased the SMURF1 to levels similar to those induced by EGF. SMURF1 induction by EGF treatment or by the overexpression of MEK1 or SMURF1 resulted in enhanced cell migration and invasion, whereas SMURF1 knockdown suppressed EGF- or MEK1-induced cell migration and invasion. EGF treatment or SMURF1 overexpression decreased the endogenous RhoA protein levels. The overexpression of constitutively active RhoA prevented EGF- or SMURF1-induced cell migration and invasion. These results suggest that EGFinduced SMURF1 plays a role in breast cancer cell migration and invasion through the downregulation of RhoA.     

Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells

The small GTPase Cdc42 has been implicated as an important regulator of cell migration. However, whether Cdc42 plays similar role in all cancer cells irrespective of metastatic potential remains poorly defined. Here, we show by using three different breast cancer cell lines with different metastatic potential, the role of Cdc42 in cell migration/invasion and its relationship with a number of downstream signaling pathways controlling cell migration. Small interfering RNA (siRNA)-mediated knockdown of Cdc42 in two highly metastatic breast cancer cell lines (MDA-MB-231 and C3L5) resulted in enhancement, whereas the same in moderately metastatic (Hs578T) cell line resulted in inhibition of intrinsic cellular migration/invasion. Furthermore, Cdc42 silencing in MDA-MB-231 and C3L5 but not Hs578T cells was shown to be accompanied by increased RhoA activity and phosphorylation of protein kinase C (PKC)-δ, extracellular signal regulated kinase1/2 (Erk1/2), and protein kinase A (PKA). Pharmacological inhibition of PKCδ, MEK-Erk1/2, or PKA was shown to inhibit migration of both control and Cdc42-silenced MDA-MB-231 cells. Furthermore, introduction of constitutively active Cdc42 was shown to decrease migration/invasion of MDA-MB-231 and C3L5 but increase migration/invasion of Hs578T cells. This decreased migration/invasion of MDA-MB-231 and C3L5 cells was also shown to be accompanied by the decrease in the phosphorylations of PKCδ, Erk1/2, and PKA. These results suggested that endogenous Cdc42 could exert a negative regulatory influence on intrinsic migration/invasion and some potentially relevant changes in phosphorylation of PKCδ, Erk1/2, and PKA of some aggressive breast cancer cells.     

Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. A synthetic library was screened to identify inhibitors of tumor cell migration. From this, we discovered that CAC-1098 (aurintricarboxylic acid) and CBI-0997 (5-(2,4-dimethoxy-5-ethylphenyl)-4-(4-bromophenyl) isoxazole) inhibited migration of MDA-MB-231 cells with IC50 = 5 and 50 nM, respectively. We synthesized KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) by replacing the bromide group of CBI-0997 with a methoxyl group. Like CBI-0997, KRIBB3 has anti-migratory and anti-invasive activities in MDA-MB-231 cells. Because KRIBB3 has a better drug-like structure, we focused our effort on further understanding its anti-migratory mechanism. Biotinyl-KRIBB3 was synthesized as an affinity probe for identification of KRIBB3-binding proteins. Using affinity chromatography, we identified Hsp27 as a target protein of KRIBB3 in vitro. Treatment of MDA-MB-231 cells with phorbol 12-myristate 13-acetate induced protein kinase C-dependent phosphorylation of Hsp27 and tumor cell migration. In contrast, treatment of MDA-MB-231 cells with KRIBB3 blocked phorbol 12-myristate 13-acetate-induced phosphorylation of Hsp27 and tumor cell migration. Furthermore, overexpression of Hsp27 antagonized the inhibitory effect of KRIBB3 on tumor cell invasion, and knockdown of Hsp27 using small interfering RNA inhibited tumor cell migration. Overall, our results demonstrate that KRIBB3 inhibits tumor cell migration and invasion by blocking protein kinase C-dependent phosphorylation of Hsp27 through its direct binding to Hsp27.     

Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells

In the current study, we examined the function of N-myc downstream-regulated gene 2 (NDRG2) expression in breast cancer cells, especially focusing on the role of bone morphogenetic protein-4 (BMP-4) induced by NDRG2. NDRG2 expression in MDA-MB-231 cells inhibited the mRNA expression of several matrix metalloproteinases (MMPs) and the gelatinolytic activity of MMP-9. Interestingly, a specific induction of active BMP-4 was exclusively observed in MDA-MB-231-NDRG2 cells but not in MDA-MB-231-mock cells. Neutralization of BMP-4 in MDA-MB-231-NDRG2 cells resulted in the rescue of MMP-9 mRNA expression and migration capacity. In addition, treatment with recombinant BMP-4 dramatically suppressed MMP-9 mRNA expression, gelatinolytic MMP-9 activity, migration, and invasion capacity both in MDA-MB-231 and PMA-treated MCF-7 cells. Collectively, our data show that BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity.     

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.