Suppressed Breast Cancer Cell Migration Research Articles

Identification of TG100-115 as a new and potent TRPM7 kinase inhibitor, which suppresses breast cancer cell migration and invasion

BackgroundTransient receptor potential melastatin 7 (TRPM7) regulates breast cancer cell proliferation, migration, invasion and metastasis in its ion channel- and kinase domain-dependent manner. The pharmacological effects of TRPM7 ion channel inhibitors on breast cancer cells have been studied, but little is known about the effects of TRPM7 kinase domain inhibitors due to lack of potent TRPM7 kinase inhibitors. MethodsScreening was performed by using TRPM7 kinase assay. Effects of TG100-115 on breast cancer cell proliferation, migration, invasion, myosin IIA phosphorylation, and TRPM7 ion channel activity were assessed by using MTT, wound healing, transwell assay, Western blotting, and patch clamping, respectively. ResultsWe found that CREB peptide is a potent substrate for the TR-FRET based TRPM7 kinase assay. Using this method, we discovered a new and potent TRPM7 kinase inhibitor, TG100-115. TG100-115 inhibited TRPM7 kinase activity in an ATP competitive fashion with over 70-fold stronger activity than that of rottlerin, known as a TRPM7 kinase inhibitor. TG100-115 has little effect on proliferation of MDA-MB-231 cells, but significantly decreases cell migration and invasion. Moreover, TG100-115 inhibits TRPM7 kinase regulated phosphorylation of the myosin IIA heavy chain and phosphorylation of focal adhesion kinase. TG100-115 also suppressed TRPM7 ion channel activity. ConclusionsTG100-115 can be used as a potent TRPM7 kinase inhibitor and a potent inhibitor of breast cancer cell migration. General significanceTG100-115 could be a useful tool for studying the pharmacological effects of TRPM7 kinase activity aimed at providing insight into new therapeutic approaches to the treatment of breast cancer.

Pax-5 is a potent regulator of E-cadherin and breast cancer malignant processes.

Pax-5, an essential transcription factor for B lymphocyte development, has been linked with the development and progression of lymphoid cancers and carcinoma. In contrast to B-cell cancer lesions, the specific expression signatures and roles of Pax-5 in breast cancer progression are relatively unknown. In the present study, we set out to profile Pax-5 expression in mammary tissues and elucidate the cellular and molecular roles of Pax-5 in breast cancer processes. Using immunohistology on mammary tissue arrays, Pax-5 was detected in a total of 298/306 (97.6%) samples tested. Interestingly, our studies reveal that Pax-5 inhibits aggressive features and confers anti-proliferative effects in breast carcinoma cells in contrast to its oncogenic properties in B cell cancers. More precisely, Pax-5 suppressed breast cancer cell migration, invasion and tumor spheroid formation while concomitantly promoting cell adhesion properties. We also observed that Pax-5 inhibited and reversed breast cancer epithelial to mesenchymal phenotypic transitioning. Mechanistically, we found that the Pax-5 transcription factor binds and induces gene expression of E-cadherin, a pivotal regulator of epithelialisation. Globally, we demonstrate that Pax-5 is predominant expressed factor in mammary epithelial cells. We also present an important role for Pax-5 in the phenotypic transitioning processes and aggressive features associated with breast cancer malignancy and disease progression.

New Ligand Binding Function of Human Cerberus and Role of Proteolytic Processing in Regulating Ligand–Receptor Interactions and Antagonist Activity

Cerberus is a key regulator of vertebrate embryogenesis. Its biological function has been studied extensively in frog and mouse embryos. Its ability to bind and antagonize the transforming growth factor-β (TGF-β) family ligand Nodal is well established. Strikingly, the molecular function of Cerberus remains poorly understood. The underlying reason is that Cerberus is a complex, multifunctional protein: It binds and inhibits multiple TGF-β family ligands, it may bind and inhibit some Wnt family members, and two different forms with distinct activities have been described. In addition, sequence homology between frog and mammalian Cerberus is low, suggesting that previous studies, which analyzed frog Cerberus function, may not accurately describe the function of mammalian Cerberus. We therefore undertook to determine the molecular activities of human Cerberus in TGF-β family signaling. Using purified proteins, surface plasmon resonance, and reporter gene assays, we discovered that human Cerberus bound and inhibited the TGF-β family ligands Activin B, BMP-6, and BMP-7, but not the frog Cerberus ligand BMP-2. Notably, full-length Cerberus successfully blocked ligand binding to type II receptors, but the short form was less effective. In addition, full-length Cerberus suppressed breast cancer cell migration but the short form did not. Thus, our findings expand the roles of Cerberus as TGF-β family signaling inhibitor, provide a molecular rationale for the function of the N-terminal region, and support the idea that Cerberus could have regulatory activities beyond direct inhibition of TGF-β family signaling.

Fe65 Suppresses Breast Cancer Cell Migration and Invasion through Tip60 Mediated Cortactin Acetylation.

Fe65 is a brain-enriched adaptor protein known for its role in the action of the Aβ amyloid precursor protein in neuronal cells and Alzheimer’s disease, but little is known about its functions in cancer cells. The present study documents for the first time a role of Fe65 in suppressing breast cancer cell migration and invasion. Mechanistic studies suggest that the suppression is mediated through its phosphotyrosine binding domain 1 that mediates the recruitment of Tip60 to cortactin to stimulate its acetylation. The studies identify the Tip60 acetyltransferase as a cytoplasmic drug target for the therapeutic intervention of metastatic breast cancers.

Hit-to-lead optimization of sipholanes for the control of invasive breast cancer through suppression of BRK and FAK signaling

Sipholane triterpenes are natural products isolated from the Red Sea sponge Callyspongia siphonella. Semisynthetic modifications afforded sipholenol A 4β-O-3',4'-dichlorobenzoate (SPA) as a potent breast cancer migration inhibitor, with an IC50 of 1.3µM in the wound-healing assay, without cytotoxicity to the non-tumorigenic breast cells MCF10A. The effects of SPA on the growth, migration, and invasion of diverse human breast cancer cells were studied. Results showed that SPA inhibited the growth of the human breast cancer cells MDA-MB-231, MCF-7, BT-474, and T-47D in a dose-dependent manner. SPA suppressed breast cancer cell migration, invasion, and decreased Brk and FAK activation in a dose-dependent manner. Molecular docking study suggested a perfect fitting at the FAK's FERM domain, inhibiting the main autophosphorylation site Y397, which was further confirmed by Western blot analysis. In vivo studies showed that SPA treatment suppressed breast tumor growth, CD31, p-Brk and p-FAK expression in orthotopic breast cancer in nude mice. Pharmacophore modeling and 3D-QSAR studies highlighted the important pharmacophoric features responsible for the antimigratory activity and Brk phosphorylation inhibition, including rings A and B (perhydrobenzoxepine) together with substituted aromatic ester moiety, creating a simpler structure and eliminating rings C and D ([5,3,0]bicyclodecane system). This opens new horizons for future design of novel sipholane-inspired active leads with perhydrobenzoxepine-aromatic cores, feasibly and cost-effectively.

Stanniocalicin 2 suppresses breast cancer cell migration and invasion via the PKC/claudin-1-mediated signaling.

Stanniocalcin (STC), a glycoprotein hormone, is expressed in a wide variety of tissues to regulate Ca2+ and PO4- homeostasis. STC2, a member of STC family, has been reported to be associated with tumor development. In this study, we investigated whether the expression of STC2 is associated with migration and invasion of breast cancer cells. We found that breast cancer cell line 231 HM transfected with STC2 shRNA displayed high motility, fibroblast morphology, and enhanced cell migration and invasion. Introduction of STC2 in 231 cells reduced cell migration and invasion. In response to irradiation, silencing of STC2 in 231 HM cells reduced apoptosis, whereas overexpression of STC2 in 231 cells promoted apoptosis, compared with in control cells. Mechanistic study showed that STC2 negatively regulated PKC to control the expression of Claudin-1, which subsequently induced the expressions of EMT-related factors including ZEB1, ZO-1, Slug, Twist, and MMP9. Suppression of PKC activity by using a PKC inhibitor (Go 6983) restored the normal motility of STC2-silenced cells. Furthermore, in vivo animal assay showed that STC2 inhibited tumorigenesis and metastasis of breast cancer cells. Collectively, these results indicate that STC2 may inhibit EMT at least partially through the PKC/Claudin-1-mediated signaling in human breast cancer cells. Thus, STC2 may be exploited as a biomarker for metastasis and targeted therapy in human breast cancer.

MiR-335 inhibits migration of breast cancer cells through targeting oncoprotein c-Met

Metastasis is the leading cause of death in patients with breast cancer and aberrantly expressed microRNAs (miRNAs) are highly associated with this process. A previous study has shown that miR-335 is downregulated in breast cancer and can suppress tumor invasion and metastasis. Emerging evidences indicate that c-Met is implicated in cell scattering, migration, and invasion. However, little is known about the relationship between miR-335 expression and c-Met alteration in breast cancer. In the present study, we found that miR-335 expression was downregulated and c-Met protein expression was upregulated in two human breast cell lines. MiR-335 was found to negatively regulate c-Met protein level by directly targeting its 3' untranslated region (UTR). Forced expression of miR-335 decreased c-Met expression at protein levels and consequently diminished hepatocyte growth factor (HGF)-induced phosphorylation of c-Met and subsequently inhibited HGF promotion of breast cancer cell migration in a c-Met-dependent manner. MiR-335 expression was increased after 5-aza-2'-deoxycytidine (5-AZA-CdR) treatment, and 5-AZA-CdR treatment resulted in the same phenotype as the effect of miR-335 overexpression. Taken together, these results demonstrate that miR-335 suppresses breast cancer cell migration by negatively regulating the HGF/c-Met pathway.

Rhapontigenin suppresses cell migration and invasion by inhibiting the PI3K-dependent Rac1 signaling pathway in MDA-MB-231 human breast cancer cells.

The invasive behavior of cancer cells resulting in metastasis is the major cause of cancer-related deaths. Rhapontigenin (1) has various biological activities including anticancer activities. However, whether and how 1 affects cancer invasion has never been explored. Here, we examined the anti-invasive effects of 1 and its underlying molecular mechanisms in the highly invasive human breast cancer cell line designated MDA-MB-231. At noncytotoxic concentrations, 1 strongly suppressed serum-induced cell migration and invasion as judged by Boyden chamber analysis and wound-healing assays, respectively. Compound 1 strikingly reduced Rac1 activity as judged by both absorbance-based and pull-down assays. In addition, its downstream effectors such as WASP-family verprolin homologous proteins 2 (WAVE-2) and p21-activated kinase 1 (PAK1) signaling cascades were attenuated after treatment with 1. Immunofluorescence staining showed that 1 diminished lamellipodia formation at the leading edge of cells. Finally, 1 decreased the phosphorylation of phosphoinisitide-3-kinase (PI3K) and AKT. Rac1 activity was inhibited by the PI3K inhibitor wortmannin. Taken together, these results suggest that 1 suppresses breast cancer cell migration and invasion, which is involved in inhibiting the PI3K-dependent Rac1 signaling pathway.

The marine-derived sipholenol A-4-O-3',4'-dichlorobenzoate inhibits breast cancer growth and motility in vitro and in vivo through the suppression of Brk and FAK signaling.

Sipholenol A is a natural sipholane triterpenoid isolated from the Red Sea sponge, Callyspongia siphonella. Previous studies showed the antimigratory and antiproliferative activities of the semisynthetic sipholenol A esters against breast cancer cell lines. This study investigated the effects of sipholenol A-4-O-3′,4′-dichlorobenzoate (SPA) on the growth, migration and invasion of diverse human breast cancer cells. Results showed that SPA inhibited the growth of the human breast cancer cells, MDA-MB-231, MCF-7, BT-474 and T-47D, in a dose-dependent manner. Immunofluorescent analysis showed that SPA significantly reduced Ki-67-positive cells in MDA-MB-231 cells. Flow cytometry and Western blot analyses revealed that SPA treatment suppressed MDA-MB-231 cell growth by inducing cell cycle arrest at the G1 phase. In addition, SPA suppressed breast cancer cell migration, invasion and decreased Brk and FAK activation in a dose-dependent manner. Molecular docking study suggested a perfect fitting at the FAK’s FERM domain, inhibiting the main autophosphorylation site, Y397, which was further confirmed by Western blot analysis. Most known small molecule FAK inhibitors target the kinase domain, creating several off-target side effects. The in vivo studies showed that SPA treatment suppressed breast tumor growth and Ki-67, CD31, p-Brk and p-FAK expression in orthotopic breast cancer in nude mice. In conclusion, SPA inhibited the growth, invasion and migration of breast cancer cells possibly via deactivating Brk and FAK signaling, suggesting good potential for therapeutic use to control invasive breast cancer.

12-O-Tetradecanoylphorbol-13-acetate Promotes Breast Cancer Cell Motility by Increasing S100A14 Level in a Kruppel-like Transcription Factor 4 (KLF4)-dependent Manner

The S100 protein family represents the largest subgroup of calcium binding EF-hand type proteins. These proteins have been reported to be involved in a wide range of biological functions that are related to normal cell development and tumorigenesis. S100A14 is a recently identified member of the S100 protein family and differentially expressed in a number of different human malignancies. However, the transcriptional regulation of S100A14 and its role in breast cancer needs to be further investigated. Here, we determined that 12-O-tetradecanoylphorbol-13-acetate (TPA) up-regulated the expression of KLF4 and facilitated its binding directly to two conserved GC-rich DNA segments within the S100A14 promoter, which is essential for the transactivation of KLF4 induced S100A14 expression. Furthermore, stable silencing of KLF4 significantly suppressed breast cancer cell migration induced by TPA. Collectively, these results offer insights into the fact that TPA provokes cell motility through regulating the expression and function of S100A14 in a KLF4-dependent manner.

Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion

Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis.

Bif-1 suppresses breast cancer cell migration by promoting EGFR endocytic degradation

Dysregulation of EGFR expression and signaling is well documented to contribute to disease progression and metastasis in many types of cancer including breast cancer. EGF-stimulated EGFR activation leads to receptor internalization and endocytic degradation to control EGFR-mediated signaling. This process is frequently deregulated in cancer cells, leading to increased EGFR expression and mitogenic signaling. Here, we demonstrate that Bif-1, a tumor suppressor, plays a role in EGFR endocytic degradation and chemotactic migration in MDA-MB-231 breast cancer cells. Our data reveal that suppression of Bif-1 expression delays EGFR degradation and sustains Erk1/2 activation in response to EGF stimulation. Mechanistically, loss of Bif-1 sequesters internalized EGF in Rab5-positive endosomes and delays EGFR trafficking to lysosomes. Recruitment of Rab7 to EGF-positive vesicles and the activation of Rab7 are impaired in Bif-1 knockdown cells. Additionally, intracellular pH and the localization of acidic vesicles are altered by suppression of Bif-1. Furthermore, inhibition of Bif-1 increases chemotactic cell migration in response to EGF or serum, which correlates with prolonged cytoskeletal reorganization. Importantly, the effect of Bif-1 on EGF-induced cell migration is abolished by gefitinib, an EGFR-specific inhibitor. Taken together, these data suggest a novel function for Bif-1 as a suppressor of breast cancer cell migration by promoting EGFR degradation through the regulation of endosome maturation.

Opposing roles of the oncogene Akt isoforms in tumour progression: is there a dark side to Akt pathway inhibition?

Opposing roles of the oncogene Akt isoforms in tumour progression: is there a dark side to Akt pathway inhibition?

MicroRNA let-7a suppresses breast cancer cell migration and invasion through downregulation of C-C chemokine receptor type 7

IntroductionC-C chemokine receptor type 7 (CCR7) plays an important role in chemotactic and metastatic responses in various cancers, including breast cancer. In the present study, the authors demonstrated that microRNA (miRNA) let-7a downregulates CCR7 expression and directly influences the migration and invasion of breast cancer cells.MethodsThe expression of CCR7, its ligand CCL21, and let-7a was detected in breast cancer cell lines and in breast cancer patient tissues. Synthetic let-7a and an inhibitor of let-7a were transfected into MDA-MB-231 and MCF-7 breast cancer cells, respectively, and cell proliferation, cell migration, and invasion assays were performed. To confirm the fact that 3'UTR of CCR7 is a direct target of let-7a, a luciferase assay for the reporter gene expressing the let-7a binding sites of CCR7 3'UTR was used. An in vivo invasion animal model system using transparent zebrafish embryos was also established to determine the let-7a effect on breast cancer cell invasion.ResultsFirst, a higher expression of both CCR7 and CCL21 in malignant tissues than in their normal counterparts from breast cancer patients was observed. In addition, a reverse correlation in the expression of CCR7 and let-7a in breast cancer cell lines and breast cancer patient tissues was detected. Synthetic let-7a decreased breast cancer cell proliferation, migration, and invasion, as well as CCR7 protein expression in MDA-MB-231 cells. The let-7a inhibitor reversed the let-7a effects on the MCF-7 cells. The 3'UTR of CCR7 was confirmed as a direct target of let-7a by using the luciferase assay for the reporter gene expressing let-7a CCR7 3'UTR binding sites. Notably, when analyzing in vivo invasion, MDA-MB 231 cells after synthetic let-7a transfection were unable to invade the vessels in zebrafish embryos.ConclusionsThe results from the present study suggest that targeting of CCL21-CCR7 signaling is a valid approach for breast cancer therapy and that let-7a directly binds to the 3'UTR of CCR7 and blocks its protein expression, thereby suppressing migration and invasion of human breast cancer cells. Furthermore, the present study underscores the therapeutic potential of let-7a as an antitumor and antimetastatic manager in breast cancer patients.

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

Filamin A regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion

The actin cross-linking protein filamin A (FLNa) functions as a scaffolding protein and couples cell cytoskeleton to extracellular matrix and integrin receptor signaling. In this study, we report that FLNa suppresses invasion of breast cancer cells and regulates focal adhesion (FA) turnover. Two large progression tissue microarrays from breast cancer patients revealed a significant decrease of FLNa levels in tissues from invasive breast cancer compared with benign disease and in lymph node-positive compared with lymph node-negative breast cancer. In breast cancer cells and orthotopic mouse breast cancer models, down-regulation of FLNa stimulated cancer cell migration, invasion, and metastasis formation. Time-lapse microscopy and biochemical assays after FLNa silencing and rescue with wild-type or mutant protein resistant to calpain cleavage revealed that FLNa regulates FA disassembly at the leading edge of motile cells. Moreover, FLNa down-regulation enhanced calpain activity through the mitogen-activated protein kinase-extracellular signal-regulated kinase cascade and stimulated the cleavage of FA proteins. These results document a regulation of FA dynamics by FLNa in breast cancer cells.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G‐protein coupled receptors (GPCRs) promotes breast cancer metastasis. G‐proteins convey GPCR signals by dissociating into Gα and Gβγ subunits. The aim of this study was to determine whether blockade of Gβγ suppresses breast cancer cell migration and invasion, critical steps of metastasis. Transwell chamber assays showed that expression of a Gβγ scavenger peptide derived from β‐adrenergic receptor kinase, attenuated migration and invasion of both breast cancer MDA‐MB‐231 and MDA‐MB‐436 cells by 50%. Migration and invasion of those cells were also blocked by M119K, an inhibitor of Gβγ, with maximum inhibition of 80% and IC50 of 1–2 μM. Activation of Gi‐protein coupled CXC chemokine receptor 4 (CXCR4) promotes breast cancer metastasis. M119K suppressed CXCR4‐dependent MDA‐MB‐231 cell migration by 75% with IC50 of 0.9 μM. Immunostaining analysis indicated that 10 μM M119K attenuated CXCR4‐stimulated, Rac‐dependent formation of lamellipodia, a key structure required for metastasis. In contrast, CXCR4‐dependent inhibition of adenylyl cyclase, a Giα‐mediated response, was not blocked by M119K but was blocked by pertussis toxin, which inactivates Giα. This is the first to directly demonstrate the role of Gβγ in breast cancer metastasis and suggests that targeting Gβγ signaling pathways may provide a novel strategy for suppressing breast cancer metastasis. Supported by Nebraska State LB595.

Gβγ Signaling Promotes Breast Cancer Cell Migration and Invasion

Signaling through G protein-coupled receptors (GPCRs) promotes breast cancer metastasis. G proteins convey GPCR signals by dissociating into Galpha and Gbetagamma subunits. The aim of the present study was to determine whether blockade of Gbetagamma signaling suppresses breast cancer cell migration and invasion, which are critical components of metastasis. Conditioned media (CM) of NIH-3T3 fibroblasts are widely used as chemoattractants in in vitro cancer metastasis studies. Expression of a Gbetagamma scavenger peptide attenuated NIH-3T3 CM-induced migration and invasion of both metastatic breast cancer MDA-MB-231 and MDA-MB-436 cells by 40 to 50% without effects on cell viability. Migration and invasion of cells in response to NIH-3T3 CM were also blocked by 8-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-1-naph-thalene-carboxylic acid) (M119K), a Gbetagamma inhibitor, with maximum inhibition exceeding 80% and half-maximal inhibitory concentration (IC50) values of 1 to 2 microM. M119K also attenuated Rac-dependent formation of lamellipodia, a key structure required for metastasis. Constitutively active Rac1 rescued Gbetagamma blockade-mediated inhibition of breast cancer cell migration, whereas dominant negative Rac1 inhibited cell migration similar to Gbetagamma blockade. Furthermore, M119K suppressed Gi protein-coupled CXC chemokine receptor 4 (CXCR4)-dependent MDA-MB-231 cell migration by 80% with an IC50 value of 1 microM, whereas tyrosine kinase receptor-dependent cell migration was significantly less inhibited. However, CXCR4-dependent inhibition of adenylyl cyclase, a Gialpha-mediated response in MDA-MB-231 cells, was not blocked by M119K but was blocked by pertussis toxin, which selectively inactivates Gialpha. This report is the first to directly demonstrate the role of Gbetagamma in cancer cell migration and invasion and suggests that targeting Gbetagamma signaling pathways may provide a novel strategy for suppressing breast cancer metastasis.