Suppresses Breast Cancer Cell Invasion Research Articles

KLF14 alleviated breast cancer invasion and M2 macrophages polarization through modulating SOCS3/RhoA/Rock/STAT3 signaling

PurposeTo study the functions and underlying network of KLF14 in breast cancer invasion and tumor-associated macrophages (TAMs). MethodsThe expressions of gene or protein were assessed by qRT-PCR and western blot assays, respectively. Cell proliferation and invasion were investigated by colony formation, CCK-8 and transwell assays, respectively. Macrophage M2 polarization was identified by flow cytometry assay. The methylation level was tested by methylation Specific PCR (MSP). The molecular relationship between KLF14 and SOCS3 was validated by dual luciferase and ChIP assays. In vivo model was established to confirm effect of KLF14 on tumor growth and metastasis. ResultsKLF14 was downregulated in breast cancer, and its level was modified by CpG-mediated methylation. Overexpression of KLF14 significantly inhibited the proliferation and invasion of breast cancer in vitro and in vivo. Moreover, KLF14-overexpressing breast cancer cells notably reduced M2 macrophages polarization and it related promoting factor of tumor microenvironment (EGF, TGFβ, MMP9 and VEGF). Mechanistically, KLF14 could positively activate SOCS3 transcription, then blocking the activation of RhoA/Rock/STAT3 signaling. Further rescue experiments identified that either SOCS3 silencing and activation of RhoA/Rock/STAT3 signaling dramatically restrained the regulatory roles of KLF14 overexpression in breast cancer invasion and M2 macrophages polarization. ConclusionCollectively, KLF14 suppressed breast cancer cell invasion and M2 macrophage polarization through modulating SOCS3/RhoA/Rock/STAT3 signaling, and these findings would provide a new potential target against breast cancer.

Interactome Profile of Visceral Adipose Tissue in Obesity Links Key Genes to Cancer Pathogenesis

Obesity increases the risk of the development of several malignancies. The visceral adipose tissue (VAT) depot is one of the pivotal contributors behind the obesity-related pathogenetic mechanisms. In this study, we analyzed the differential gene expression profile in the VAT of obese children using two Gene Expression Omnibus datasets. GSE29718 and GSE9624 were sorted and 68 common differentially expressed genes (DEG) with fold change 1.5 upregulation or downregulation (cutoff |logFC|≥0.58496) were obtained. Gene ontology and functional enrichment and protein-protein interaction (PPI) network for the DEG were analyzed in Search Tool for the Retrieval of Interacting Genes (STRING), which revealed 37 biological processes, 3 cellular components, and 1 molecular function to be significantly associated. Reactome pathway analysis showed the DEG to be involved in- one carbon pool by folate, glycine degradation, transcriptional regulation by TP53, ERK inactivation, G1/S-specific transcription, Fanconi anemia pathway, beta-catenin phosphorylation cascade, RAF activation, and negative regulation of the MAPK pathway. The PPI network was set with a minimum interaction score of 0.400 and a maximum of 10 interactions, and it was significantly enriched (p-value 0.047) with 66 nodes and 46 edges. Target prediction was performed using miRNet. Several miRNA, including hsa-miR-1-3p, hsa-let-7b-5p, hsa-miR-16-5p, hsa-miR-27a-3p and hsa-miR-34a-5p were part of the mRNA-miRNA interaction network. Using the CytoHubba plugin in Cytoscape, the top 10 hub genes from the PPI network were discovered. Thymidine phosphorylase (TYMP) and dihydrofolate reductase (DHFR), essential components of nucleic acid metabolism, have been shown to be involved in angiogenesis and endothelial cell growth, and correlated to p53 mutations, respectively. Protein phosphatase 2, regulatory subunit A & regulatory subunit B (PPP2R1A and PPP2R1B) mutations are involved in ovarian, endometrial, lung and colorectal cancers. HLA-DQA1 mutation is involved cervical cancer, and it is involved in increased immune sensitivity and liver damage in breast cancer patients. The RAB7Ab and RAB7-interacting lysosomal protein (RILP) are regulators of endo-lysosomal trafficking and suppresses breast cancer cell invasion. To conclude, this study identifies several genes and their regulatory pathways in VAT which may contribute to the increased risk of cancer pathogenesis in obese individuals.

Baicalin inhibits breast cancer development via inhibiting IĸB kinase activation in vitro and in vivo.

The aim of the present study was to investigate the effect and therapeutic potential of baicalin in breast cancer. Baicalin is used to treat inflammatory diseases. The effects of baicalin were assessed in breast cancer MCF-7 and MDA-MB‑231 cells, and human breast cancer xenograft mice. Cells were treated with 0, 20 or 30µM baicalin for 48h, while xenograft mice were treated with intraperitoneal injection of 0, 100 or 200mg/kg baicalin for 30days. The results demonstrated that treatment with baicalin dose-dependently suppressed breast cancer cell invasion, migration and proliferation, and also induced G1/S-phase cell cycle arrest invitro and invivo. Baicalin alleviated inflammation injury and inhibited the secretion of tumor necrosis factor(TNF)-α and interleukin(IL)-1β, thus suppressing nuclear factor(NF)-ĸB-p65 activation via inhibition of IĸB kinase. Investigation of the mechanism underlying baicalin activity indicated that it inhibited protein expression of NF-ĸB-p65, leading to NF-ĸB‑induced increased expression of CCND1, BCL2, BIRC2 and BIRC3, thus inhibiting cell proliferation, invasion and migration and suppressing anti-apoptotic factors invitro and invivo. In addition, baicalin did not affect non-tumorigenic normal breast epithelial cells. These results indicate that baicalin may exert therapeutic effects in breast cancer.

MicroRNA-34a Inhibition of the TLR Signaling Pathway Via CXCL10 Suppresses Breast Cancer Cell Invasion and Migration

Breast cancer (BC) starts as a local disease, but it can metastasize to the lymph nodes and distant organs. However, the metastatic process is still poorly understood. The mRNA microarray datasets GSE26910 and GSE33447 show that CXCL10 is up-regulated in BC, and the microRNA microarray dataset GSE38167 and a network meta-analysis of microRNA expression profile studies in human BC suggest that microRNA-34a (miR-34a) is down-regulated in BC. CXCL10 was predicted as a target of miR-34a by microRNA.org. In this study, we uncovered a CXCL10-independent mechanism by which miR-34a exerts its antimetastatic activity in BC. To investigate the clinical significance of miR-34a in BC, we collected cancer tissues and paracancerous tissues from 258 patients with BC. In addition, a series of inhibitors, mimics, and siRNAs was introduced into MCF-7 and T47D cells to validate the regulatory mechanisms by which miR-34a regulates CXCL10. Next, to better understand the pivotal role of TLR signaling pathway inhibition in MCF-7 and T47D cells, we blocked the TLR signaling pathway using OxPAPC, an antagonist of TLR signaling. Among BC patients, miR-34a was down-regulated, CXCL10 was up-regulated, and the TLR signaling pathway was activated. Determination of luciferase activity revealed that CXCL10 was a target of miR-34a. Through gain- and loss-of-function studies, miR-34a was demonstrated to negatively regulate CXCL10; inhibit activation of the TLR signaling pathway; significantly suppress in vitro cell proliferation, migration, and invasion; and induce apoptosis. Our findings suggest that functional loss or suppression of the tumor suppressor CXCL10 due to induction of miR-34a leads to inhibition of the TLR signaling pathway during breast tumorigenesis, providing a novel target for the molecular treatment of breast malignancies.

Guanylate-binding protein 2 regulates Drp1-mediated mitochondrial fission to suppress breast cancer cell invasion

Guanylate-binding protein 2 (GBP2) is a member of the large GTPase superfamily that is strongly induced by interferon-γ (IFN-γ). Although the biochemical characteristics of GBP2 have been reported in detail, its biological function has not been thoroughly elucidated to date. To the best of our knowledge, this study presents the first demonstration that GBP2 inhibits mitochondrial fission and cell metastasis in breast cancer cells both in vitro and in vivo. Our previous work demonstrated that dynamin-related protein 1 (Drp1)-dependent mitochondrial fission has a key role in breast cancer cell invasion. In this study, we demonstrate that GBP2 binds directly to Drp1. Elimination of Drp1 by shRNA or Mdivi-1 (a Drp1-specific inhibitor) suppressed GBP2’s regulatory function. Furthermore, GBP2 blocks Drp1 translocation from the cytosol to mitochondria, thereby attenuating Drp1-dependent mitochondrial fission and breast cancer cell invasion. In summary, our data provide new insights into the function and molecular mechanisms underlying GBP2’s regulation of breast cancer cell invasion.

Inhibition of cell invasion and migration by CEACAM1-4S in breast cancer.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cell-cell adhesion molecule, has been revealed to perform an important role in tumor progression. Although there are a number of studies on CEACAM1 in patients with breast cancer, there is limited information on the roles of CEACAM1 in breast cancer metastasis. The present study aimed to identify whether CEACAM1 is involved in breast cancer development and to investigate the underlying mechanisms. First, the expression of CEACAM1 was observed in patients with breast cancer, and the association between CEACAM1 expression levels and migration and invasion of breast cancer cells was analyzed. As there are 12 isoforms of CEACAM1, of which CEACAM1-4S dominates in the human breast epithelium, subsequent study focused on CEACAM1-4S as a representative of all the isoforms. Results of the present study demonstrated that CEACAM1-4S suppresses breast cancer cell invasion and migration in a manner that is dependent on the balance between matrix metalloproteinase 2/tissue inhibitor of metalloproteinase 2 and E-/N-cadherin expression. In addition, CEACAM1-4S was likely to cause reversal of epithelial-mesenchymal transition of breast cancer cells through repressing Smad2 and signal transducer and phosphorylation of activator of transcription 3. In conclusion, the present study demonstrated that CEACAM1-4S performs an inhibitory role in breast cancer metastasis, and restoring CEACAM1-4S expression may provide a novel strategy for therapy of patients with metastatic breast cancer.

Resveratrol suppresses breast cancer cell invasion by inactivating a RhoA/YAP signaling axis

Hippo/YAP signaling is implicated in tumorigenesis and progression of various cancers. By inhibiting a plethora signaling cascades, resveratrol has strong anti-tumorigenic and anti-metastatic activity. In the present study, we demonstrate that resveratrol decreases the expression of YAP target genes. In addition, our data showed that resveratrol attenuates breast cancer cell invasion through the activation of Lats1 and consequent inactivation of YAP. Strikingly, we also demonstrate that resveratrol inactivates RhoA, leading to the activation of Lats1 and induction of YAP phosphorylation. Further, resveratrol in combination with other agents that inactivate RhoA or YAP showed more marked suppression of breast cancer cell invasion compared with single treatment. Collectively, these findings indicate the beneficial effects of resveratrol on breast cancer patients by suppressing the RhoA/Lats1/YAP signaling axis and subsequently inhibiting breast cancer cell invasion.

MiR-34a modulates ErbB2 in breast cancer.

Breast cancer is the second highest cause of carcinoma-related death caused by distant metastasis in women. Estrogen receptor (ER), human epidermal growth factor receptor 2, (HER2) and progesterone receptor (PR) are three classified makers of breast cancer, which are defined as ER+, HER2+, and the most serious ER-PR-HER2- (triple-negative). It is well known that ErbB2 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2) plays an important part in breast cancer. However, the molecular mechanisms underlying ErbB2 action needs to be well studied. In this report, we discovered that the decreased expression levels of miR-34a were inversely correlated with the increased ErbB2 levels in breast cancer. A luciferase reporter assay was done to understand the potential correlation between ErbB2 and miR-34a. Over-expression of miR-34a reduces ErbB2 expression and suppresses breast cancer cell invasion and growth in vitro. What's more, reduced expression of ErbB2 inhibits breast Cancer cell proliferation and re-expression of ErbB2 reversed miR-34a-dependent tumor suppression. Meanwhile, miR-34a levels were correlated inversely with breast cancer malignancy. Our study demonstrates that miR-34a, like ErbB2, might be a diagnostic target in breast cancer.

The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis.

Metastasis is a critical event affecting breast cancer patient survival. To identify molecules contributing to the metastatic process, we analysed The Cancer Genome Atlas (TCGA) breast cancer data and identified 41 genes whose expression is inversely correlated with survival. Here we show that GABAA receptor alpha3 (Gabra3), normally exclusively expressed in adult brain, is also expressed in breast cancer, with high expression of Gabra3 being inversely correlated with breast cancer survival. We demonstrate that Gabra3 activates the AKT pathway to promote breast cancer cell migration, invasion and metastasis. Importantly, we find an A-to-I RNA-edited form of Gabra3 only in non-invasive breast cancers and show that edited Gabra3 suppresses breast cancer cell invasion and metastasis. A-to-I-edited Gabra3 has reduced cell surface expression and suppresses the activation of AKT required for cell migration and invasion. Our study demonstrates a significant role for mRNA-edited Gabra3 in breast cancer metastasis.

Shikonin as an inhibitor of the LPS-induced epithelial-to-mesenchymal transition in human breast cancer cells.

Shikonin (SK), a natural naphthoquinone isolated from the Chinese medicinal herb, has been known to suppress the proliferation of several cancer cells. However, its role in the epithelial mesenchymal transition (EMT) has yet to be demonstrated. The aim of the present study was to examine the effects of SK on EMT. Lipopolysaccharide (LPS) induced EMT-like phenotypic changes, enhancing cell migration and invasion. SK markedly reduced the expression of the LPS-induced EMT markers, including N-cadherin in MDA-MB‑231 cells, and increased the expression of E-cadherin in MCF-7 cells. SK also inhibited cell migration and invasion in vitro. The effects of SK on the LPS-induced EMT were mediated by the inactivation of the NF-κB-Snail signaling pathway. The results provided new evidence that SK suppresses breast cancer cell invasion and migration by inhibiting the EMT. Therefore, SK is a potentially effective anticancer agent for breast tumors, by inhibiting metastasis.

Abstract 4125: SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have been reported for striking chemopreventive activity in various types of human cancer, including breast cancer. However, few studies have reported that sulindac is able to prevent tumor metastasis. In this study, we aim to study anti-metastatic activity of sulindac in breast cancer and explore the mechanistic basis responsible for this activity, which we hypothesize may involve a cyclooxygenase (COX) independent mechanism. Numerous studies have reported that the anticancer properties of sulindac might involve, at least partially, a non-COX mediated mechanism, which provides an insight into development of safer and more efficacious new derivatives. We have previously described a chemical derivative of sulindac, referred to as sulindac sulfide amide (SSA), which lacks inhibitory effect on COX-1 or -2, yet shows greater potency to inhibit tumor cell growth compared with sulindac sulfide (SS). Our results showed that SSA treatment at a sub-cytotoxic concentration (4μM) for 36 h significantly inhibits both migration and invasion of highly aggressive MDA-MB-231, BT-20 and SK-BR-3 breast tumor cells. When treating non-invasive breast MCF-7 cells with TGF-β1 to induce their motility, we found that SSA could significantly counteract the induced motility of these cells. These results indicate that SSA can suppress breast tumor cell invasion by modulation of TGF-β1 signaling. We further studied Smad-2/3 that are important downstream components in the TGF-β1 signaling pathway and found that SSA could inhibit Smad-2/3 phosphorylation and reduce their translocation to the nucleus. In addition, Smad-2/3 knockdown could mimic the anti-invasive effect of SSA in MDA-MB-231 cells, regardless of the presence of TGF-β1. In summary, we demonstrate that the non-COX inhibitory derivative, SSA can suppress breast cancer cell invasion and migration. These observations suggest that inhibition of TGF-β1/Smad signaling is responsible for the inhibitory effect of SSA on breast tumor cell invasion and migration. Citation Format: Bin Yi, Xingling Feng, Ruixia Ma, Xiaoguo Zhang, Hong Chang, Hongyou Zhao, Ziping Liang, Xi Chen, Xiuhua Hu, Gary Piazza, Yaguang Xi. SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2015-4125

Sinomenine inhibits breast cancer cell invasion and migration by suppressing NF-κB activation mediated by IL-4/miR-324-5p/CUEDC2 axis

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a vital transcription factor that regulates multiple important biological processes, including the epithelial–mesenchymal transition (EMT) and metastasis of breast cancer. Sinomenine is an isoquinoline well known for its remarkable curative effect on rheumatic and arthritic diseases and can induce apoptosis of several cancer cell types. Recently, sinomenine was reported as a tumor suppressor via inhibiting cell proliferation and inducing apoptosis. However, the role and mechanism of sinomenine in invasion and metastasis of breast cancer are largely unknown. Here, we report that sinomenine suppressed the invasion and migration of MDA-MB-231 and 4T1 breast cancer cells in a dose-dependent manner. We detected binding of NF-κB to the inhibitor of NF-κB (IκB) after the MDA-MB-231 cells were treated with 0.25, 0.5, and 1 mM sinomenine. Co-IP analysis revealed that sinomenine enhanced the binding of NF-κB and IκB in a dose-dependent manner, suggesting that sinomenine had an effect on inactivation of NF-κB. Western blotting and ELISA approaches indicated that the suppression effect was closely associated with the phosphorylation of IκB kinase (IKK) and its negative regulator CUEDC2. Sinomenine treatment decreased miR-324-5p expression, thus increased the level of its target gene CUEDC2, and then blocked the phosphorylation of IKK through altering the upstream axis. Finally, transfection of a miR-324-5p mimic inhibited the suppression of invasion and metastasis of MDA-MB-231 and 4T1 cell by sinomenine, providing evidence that sinomenine treatment suppressed breast cancer cell invasion and metastasis via regulation of the IL4/miR-324-5p/CUEDC2 axis. Our findings reveal a novel mechanism by which sinomenine suppresses cancer cell invasion and metastasis, i.e., blocking NF-κB activation.

SUV420H2 suppresses breast cancer cell invasion through down regulation of the SH2 domain-containing focal adhesion protein tensin-3

The genome-wide loss of histone H4 lysine 20 tri-methylation (H4K20me3) is observed in multiple types of cancer, including breast tumors. Since H4K20me3 is preferentially targeted to repetitive elements in the pericentromeric and telomeric heterochromatin and plays a role in chromatin integrity, the pathological effects of disrupted H4K20me3 in tumors have been attributed to genomic instability. However, in this report, we show that loss of H4K20me3 modulates gene expression profiles, leading to increased cell invasion. Reduced H4K20me3 levels in tumor cells are often accompanied by a decrease in the expression of the H4K20-specific methyltransferase, SUV420H2. Exogenous delivery of SUV420H2 into MDA-MB-231 human breast cancer cells induced selective and specific changes in the expression of cancer-related genes. One of the most downregulated genes in response to SUV420H2 expression was the Src substrate, tensin-3, a focal adhesion protein that contributes to cancer cell migration. Depletion of tensin-3 suppressed breast cancer cell invasiveness. Furthermore, silencing of tensin-3 was associated with enrichment of H4K20me3 immediately upstream of the tensin-3 transcription start site, suggesting that the loss of H4K20me3 in tumor cells induced the expression of cancer-promoting genes. These findings connect the loss of H4K20me3 with tumor progression, through the transcriptional activation of cancer-promoting genes.

GPER in CAFs regulates hypoxia-driven breast cancer invasion in a CTGF-dependent manner.

Recent advances indicate that cancer‑associated fibroblasts (CAFs) play a key role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors often experience low oxygen tension environments, which induce gene expression changes and biological features leading to poor outcomes. The G-protein estrogen receptor (GPER) exhibits a stimulatory role in diverse types of cancer cells and in CAFs under hypoxic conditions. We investigated the role of CAFs and hypoxia in breast cancer aggressiveness, and examined the effect of GPER in CAFs on hypoxia-driven breast cancer progression. The results showed that hypoxia upregulated HIF-1α, GPER and α-SMA expression in CAFs, and induced the secretion of Interleukin-6 (IL-6), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in CAFs. However, GPER silencing abrogated the above hypoxia-driven cytokine expression in CAFs. Moreover, knockdown of GPER in CAFs suppressed breast cancer cell invasion induced by CAF conditioned media (CM). Furthermore, GPER silencing in CAFs inhibited hypoxia-increased CTGF expression in CAFs and breast cancer cells cultured with CM from CAFs under hypoxic conditions. In addition, CTGF is responsible for the observed effects of GPER on CAFs activation and breast cancer invasion. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.

A regulatory loop involving miR-22, Sp1, and c-Myc modulates CD147 expression in breast cancer invasion and metastasis.

Breast cancer is the most common cancer in women for which the metastatic process is still poorly understood. CD147 is upregulated in breast cancer and has been associated with tumor progression, but little is known about its regulatory mechanisms. In this study, we demonstrated that CD147 was overexpressed in breast cancer tissues and cell lines, and the high expression correlated with tumor invasion and metastasis. We also found that the transcription factors Sp1 and c-Myc could bind to the CD147 promoter and enhance its expression. The CD147 mRNA has a 748-bp 3'-untranslated region (UTR) with many miRNA target sites, suggesting possible regulation by miRNAs. We discovered that miR-22 repressed CD147 expression by directly targeting the CD147 3'UTR. We also determined that miR-22 could indirectly participate in CD147 modulation by downregulating Sp1 expression. miR-22 could form an autoregulatory loop with Sp1, which repressed miR-22 transcription by binding to the miR-22 promoter. Together with the c-Myc-mediated inhibition of miR-22 expression, our investigation identified a miR-22/Sp1/c-Myc network that regulates CD147 gene transcription. In addition, miR-22 overexpression suppressed breast cancer cell invasion, metastasis, and proliferation by targeting CD147 in vitro and in vivo. Furthermore, we found that miR-22 was significantly downregulated in breast cancer tissues and that its expression was inversely correlated with the tumor-node-metastasis stage and lymphatic metastasis in patients. Our study provides the first evidence that an miR-22/Sp1/c-Myc network regulates CD147 upregulation in breast cancer and that miR-22 represses breast cancer invasive and metastatic capacities.

Suppression of endoplasmic reticulum stress-induced invasion and migration of breast cancer cells through the downregulation of heparanase

Tumor metastasis is the ultimate stage of cancer, and the primary cause of mortality in patients. Tumor cells breaking through the natural barrier consisting of the basement membrane (BM) and extracellular matrix (ECM) is the a crucial step in tumor invasion and metastasis. Thus, protecting this barrier is the key to reducing mortality. Heparanase is a mammalian endo-β-glucuronidase which has been found to promote the cleavage of heparan sulfate (HS), and plays a significant role in tumor cell invasion and metastasis. Although chemotherapeutic reagents have a strong antitumor activity, they may promote the invasion and migration of cancer cells, as has been observed during clinical treatment. Chemotherapeutic reagents can induce endoplasmic reticulum (ER) stress; in this study, we used adriamycin (ADM) and a classical ER stress inducer, tunicamycin (TM). We report that the activation of ER stress is involved in the enhanced invasion and migration ability of breast cancer cells and we hypothesized that this effect is associated with the activation of heparanase. In support of this, we used the heparanase inhibitor, OGT2115, and low molecular weight heparin (LMWH) to inhibit the expression and activity of heparanase, and we found that the invasion and migration ability of the cells was suppressed. Our findings demonstrate that heparanase inhibitors suppress breast cancer cell invasion and migration induced by ER stress, and provide a strong rationale for the development of heparanase-based therapeutics for the prevention of metastasis induced by chemotherapeutic reagents.

Differential Remodeling of Actin Cytoskeleton Architecture by Profilin Isoforms Leads to Distinct Effects on Cell Migration and Invasion

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

Piceatannol Suppresses Breast Cancer Cell Invasion through the Inhibition of MMP-9: Involvement of PI3K/AKT and NF-κB Pathways

Cancer invasion and metastasis are the main causes of treatment failure and death in cancer patients. Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene) is a natural analogue of resveratrol. This study investigated the anti-invasive mechanisms of piceatannol in MDA-MB-231 cells. Piceatannol significantly reduced serum-induced cell invasion and migration as well as adhesion without affecting the viability of cells. Furthermore, piceatannol markedly inhibited matrix metalloproteinase-9 (MMP-9) activity and expression at both protein and mRNA levels. Piceatannol attenuated phosphoinisitide-3-kinase (PI3K) and phosphorylation of AKT and mammalian target of rapamycin (mTOR), whereas phosphatase and tensin homologue (PTEN) was increased. Moreover, piceatannol inhibited nuclear factor kappa B (NF-κB) transcriptional activity and DNA binding of NF-κB on MMP-9 promoter. In addition, piceatannol diminished NF-κB nuclear translocation through blocking the inhibitor of NF-κB alpha (IκBα) phosphorylation in the cytoplasm. These results proposed piceatannol as a potential anti-invasive agent by inhibiting MMP-9 involved in PI3K/AKT and NF-κB pathways.

De novo expression of αvβ6 integrin by myoepithelial cells in ductal carcinoma in situ may be an important marker of disease progression

Myoepithelial cells (MEC) are essential to the maintenance of normal breast function, and loss of normal MEC function is commonly associated with breast cancer. Most established invasive breast carcinomas develop through an in situ phase known as ductal carcinoma in situ (DCIS). We have identified up-regulation of β6 integrin on MEC in a subset of DCIS. Normal MEC exhibit potent tumour suppressor function, but it is not clear whether this is compromised in DCIS. The aim of the present study is to investigate the effect of β6 expression on myoepithelial tumour suppressor function. Immunohistochemical analysis of DCIS of different grades with and without an invasive breast cancer was carried out to determine the β6 status. For in vitro studies magnetic bead sorting was used to isolate a pure normal-like myoepithelial cell line from the immortalised 1089 cell line (N-1089 MEC). These were used to generate overexpressing myoepithelial cells (DCIS-modified MEC) by β6 retroviral transduction. The lines were characterised by immuno-fluorescence and flow cytometry, and the tumour suppressor function of both lines was compared with primary normal and DCIS MEC in coculture with breast cancer cell lines. Analysis of a series of primary DCIS tissues (n > 400) demonstrated induction of β6 in MEC in a subset of cases, predominantly high grade. Upregulation was almost universal in DCIS associated with invasive disease. Initial characterisation of the 1089 line revealed a mixed phenotype from which pure MEC were selected on the basis of β4 integrin. This population exhibited all characteristic myoepithelial markers. Coculture assays demonstrated that N-1089 MEC could produce the same tumour suppressor effect as primary MEC, leading to significant reduction in tumour invasion (P < 0.001) and proliferation (P < 0.001). N-1089 MEC transduced with αvβ6 (DCIS-1089 MEC) demonstrated enhanced binding and migration to the β6 ligand LAP, and activation of a transforming growth factor beta reporter, indicating that the αvβ6 was functional. Whilst primary DCIS MEC showed loss of suppressor function (P < 0.05), DCIS-1089 MEC exhibit altered behaviour with a more migratory phenotype then the normal counterpart, but at least some of the tumour-suppressor function was maintained. We have shown that MEC exhibit an altered phenotype in DCIS with de novo expression of αvβ6. We have generated normal-like cell lines that exhibit all the characteristics of primary MEC and recapitulate primary MEC tumour suppressor function. Primary DCIS MEC show loss of suppressor function whereas β6-overexpressing MEC (which resemble DCIS-like MEC) promote or suppress breast cancer cell invasion in a cell-type-specific manner. These findings suggest that changes in MEC during DCIS may influence disease progression, and these cell lines provide a powerful model to study further the mechanisms involved.

Reactivation of SYK expression by inhibition of DNA methylation suppresses breast cancer cell invasiveness.

The gene product of spleen tyrosine kinase (SYK) has been implicated in the suppression of breast cancer invasion. We previously reported that SYK expression is lost in a subset of breast cancer; primarily by methylation-mediated gene silencing. In our study, we explored the possibility of using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), to suppress breast cancer cell invasion by restoring SYK expression. We found that AZA treatment reestablished the expression of SYK(L) that was accompanied by suppression of the invasion capacity of SYK-negative cells. This invasion inhibition was not due to global cellular toxicity since this treatment did not affect overall cell proliferation. This decreased invasiveness by AZA treatment was diminished by piceatannol, a SYK inhibitor, suggesting that SYK play a significant role in AZA-inducible invasion suppression. SYK promoter hypermethylation was found infrequent in pathologically normal mammary tissues or benign lesions (<5%). In contrast, SYK methylation was frequently identified in ductal carcinoma in situ ( approximately 45%) and invasive ductal carcinoma (47% in node-negative and 40% in node-positive cases), indicating that the hypermethylation of SYK occurs at a stage prior to the development of invasion phenotypes. All these results suggested a potential use of SYK methylation as a valuable biomarker to detect early cancerous lesions and support the use of AZA as a new reagent to the management of advanced breast cancer.