Breast Cancer Motility Research Articles

The Actin Motor Protein Myosin 6 Contributes to Cell Migration and Expression of GIPC1 and Septins in Breast Cancer Cells.

Breast cancer is highly metastatic. One protein that may participate in breast cancer cell migration is the actin motor protein myosin 6 (MYO6), which is likely regulated by the GIPC1 protein. Additionally, septins (SEPTs) appear to participate in breast cancer motility. Here, we investigated the effects of loss of MYO6 on cell morphology, migration, and expression of GIPC1, SEPT2, and SEPT7 in two breast cancer cell lines. The research material consisted of two breast cancer cell lines, MCF-7 and MDA-MB-231, in which the level of MYO6 was reduced and the effect of knockdown on the migration potential and the expression of GIPC1, SEPT2 and SEPT7 was determined. The levels of these proteins were also analyzed in silico. siRNA-mediated knock down of MYO6 altered the morphology of MCF-7 cells and reduced the expression of GIPC1 and SEPT7 in both MCF-7 and MDA-MB-231 cells. In in silico data, GIPC1, SEPT2, and SEPT7 were all overexpressed in breast cancer tissue samples from patients. Finally, MYO6 knock down impaired migration and adhesion in both MCF-7 and MDA-MB-231 cells. Our study substantiates that downregulation of MYO6 diminishes the migratory abilities of breast cancer cell lines with varying invasiveness. Furthermore, we have demonstrated that decreased MYO6 protein leads to reduced expression of GIPC1, SEPT2, and SEPT7 in breast cancer cells. These findings contribute to a more comprehensive understanding of the pathways influencing breast cancer cell migration, a critical aspect of metastasis.

The adhesion-GPCR ADGRF5 fuels breast cancer progression by suppressing the MMP8-mediated antitumorigenic effects

ADGRF5 (GPR116) has been identified as a facilitator of breast cancer cell migration and metastasis, yet the underlying mechanisms remain largely elusive. Our current study reveals that the absence of ADGRF5 in breast cancer cells impairs extracellular matrix (ECM)-associated cell motility and impedes in vivo tumor growth. This correlates with heightened expression of matrix metalloproteinase 8 (MMP8), a well-characterized antitumorigenic MMP, and a shift in the polarization of tumor-associated neutrophils (TANs) towards the antitumor N1 phenotype in the tumor microenvironment (TME). Mechanistically, ADGRF5 inhibits ERK1/2 activity by enhancing RhoA activation, leading to decreased phosphorylation of C/EBPβ at Thr235, hindering its nuclear translocation and subsequent activation. Crucially, two C/EBPβ binding motifs essential for MMP8 transcription are identified within its promoter region. Consequently, ADGRF5 silencing fosters MMP8 expression and CXCL8 secretion, attracting increased infiltration of TANs; simultaneously, MMP8 plays a role in decorin cleavage, which leads to trapped-inactivation of TGF-β in the TME, thereby polarizing TANs towards the antitumor N1 neutrophil phenotype and mitigating TGF-β-enhanced cell motility in breast cancer. Our findings reveal a novel connection between ADGRF5, an adhesion G protein-coupled receptor, and the orchestration of the TME, which dictates malignancy progression. Overall, the data underscore ADGRF5 as a promising therapeutic target for breast cancer intervention.

Retraction Note: TMED3 promotes cell proliferation and motility in breast cancer and is negatively modulated by miR-188-3p

Retraction Note: TMED3 promotes cell proliferation and motility in breast cancer and is negatively modulated by miR-188-3p

Caveolin-1 signaling-driven mitochondrial fission and cytoskeleton remodeling promotes breast cancer migration.

Mitochondria are highly dynamic organelles that constantly divide and fuse to maintain their proper structure and function. Cancer cells are often accompanied by an imbalance of mitochondrial fusion and fission, cancer progression is greatly affected by this imbalance. Here, we found that high-metastatic breast cancer MDA-MB-231 cells possess higher caveolin-1 (Cav-1) expression compared with low-metastatic breast cancer MCF-7 cells or normal breast epithelial MCF-10A cells. Downregulation of Cav-1 decreases the migratory and invasive abilities of MDA-MB-231 cells. Our results further demonstrated that downregulation of Cav-1 facilitated DRP1 and MFN2 to translocate to mitochondria, increasing the inhibitory phosphorylation level of DRP1 at Ser637 by protein kinase A (PKA), resulting in mitochondria elongation. We also showed that downregulation of Cav-1 significantly reduced the Rac1 activity by affecting intracellular reactive oxygen species (ROS) generation, which then inhibited F-actin formation. Based on these findings, we proposed that Cav-1 mediated mitochondrial fission-affected intracellular ROS generation and activated Rho GTPases, leading to F-actin-dependent formation of lamellipodia and promotion of breast cancer motility.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes‐associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain‐ and loss‐of‐function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro‐taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho‐associated kinase (ROCK), blocked shear stress–induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow‐sensing mechanotransduction pathways.

ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1

Breast cancer progression features ECM stiffening due to excess deposition and crosslinking of collagen, which dramatically influence tumor behaviour and fate. The mechanisms by which extracellular matrix (ECM) stiffening drives breast cancer invasion is an area of active research. Here we demonstrate the role of exosomes in ECM stiffness triggered breast cancer invasiveness. Using stiffness tuneable hydrogel ECM scaffolds, we show that stiff ECMs promote exosome secretion in a YAP/TAZ pathway-dependent manner. Interestingly, blocking exosome synthesis and secretion by GW4869 abrogated stiffness regulated motility and contractility in breast cancer cells. Reciprocally, exogenous addition of ECM stiffness-tuned exosomes orchestrated a series of changes in cell morphology, adhesion, protrusion dynamics resulting in fostered cell motility and invasion. Proteomic analysis of exosomal lysates followed by overrepresentation analysis and interactome studies revealed enrichment of cell adhesion and cell migration proteins in exosomes from stiff ECM cultures compared to that of soft ones. Quantitative proteomics of exosomes combined with genomic analysis of human breast tumor tissues (TCGA database) identified thrombospondin-1 (THBS1) as a prospective regulator of stiffness-dependent cancer invasion. Knockdown studies confirmed that the pro-invasive effects of stiffness-tuned exosomes are fuelled by exosomal THBS1. We further demonstrated that exosomal THBS1 mediates these stiffness-induced effects by engaging matrix metalloproteinase and focal adhesion kinase. Our studies establish the pivotal role of exosomal communication in ECM stiffness dependent cell migration with exosomal THBS1 as a master regulator of cancer invasion, which can be further exploited as a potential theranostic for improved breast cancer management.

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

Abstract PO-089: Morphokinetic single-cell analysis and machine learning as a tool to characterize breast cancer cell motility and response to therapy

Abstract Triple-negative breast cancer (TNBC) is a type of aggressive breast cancer lacking the expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2). TNBC patients have a high propensity for distant metastasis and limited treatment options. Cancer cell motility and invasion are fundamental steps in metastasis. MET, a tyrosine kinase receptor, and its ligand, Hepatocyte Growth Factor/Scatter Factor (HGF/SF), induce specific signal transduction in tumor cells leading to cell motility and metastasis. MET's induced tumorigenesis and metastatic processes make it an ideal target for anti-cancer therapy. We characterized the motility patterns of m-Cherry labeled TNBC cells (BT-549, MDA-MB-231) and ER-positive cells (MCF7) subjected to time-lapse fluorescence wound healing assay. We also studied the effect of MET inhibition, chemotherapy, and their combined impact on breast cancer motility. Time-lapse images were subjected to commercial packages for segmentation and tracking - allowing us to extract morphokinetic (MK) information at a single-cell resolution. To better understand cell motility patterns, we developed the Tool for Analysis of Single-Cell (TASC) infrastructure, which utilizes unsupervised machine learning methods to demonstrate a high-dimensional feature set at a single-cell resolution. We compared our experimental MK results to simulations of three modified classical physical models: Lévy flight (LF), fractal Brownian motion, and random walk (RW) with or without a back-propagating wave (BPW). TASC analysis demonstrated a fundamental difference between ER-positive and TNBC cells: both cell types contained two subpopulations harboring similar MK characteristics. TNBC cells presented an additional subpopulation characterized by 1) dominantly increasing cumulative kinetics values (mean square displacement - MSD), 2) highest temporal wave values (velocity starting-time), and 3) a decrease in non-cumulative kinetics values. The combined treatment of TNBC cells with MET inhibition and chemotherapy eliminates this high-motility group. Cell motility models and motion simulations were compared using Wasserstein's analysis. Untreated TNBC cells showed high similarities to RW with BPW simulations, while MET-activated TNBC cells transformed into LF without BPW simulations, indicating more aggressive behavior. TASC analysis revealed a previously undefined high motility subpopulation of TNBC that was eradicated by combining MET inhibition and chemotherapy. The primary benefit of TASC is the ability to cluster and characterize similar behaviors across different motility models while still detecting subtle changes within each one. MK analysis combined with TASC can lead to discovering novel targets for precise therapy. Moreover, this infrastructure may determine patient tumor susceptibility to chemotherapy and biological treatments and operate as an analytical tool for personalized, targeted therapy. Citation Format: Ilan Tsarfaty, Or Megides, Ohad Doron, Hagar Alaloof, Ori Moskowitz, Judith Horev. Morphokinetic single-cell analysis and machine learning as a tool to characterize breast cancer cell motility and response to therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-089.

MTA1 coregulator regulates LDHA expression and function in breast cancer

Metastasis Associated Protein1 (MTA1) is a chromatin modifier and its expression is significantly associated with prognosis of many cancers. However, its role in glucose metabolism remains unexplored. Here, we report that MTA1 has a significant role in glucose metabolism where MTA1 regulates the LDHA expression and activity and subsequently its function in breast cancer motility. The results showed that MTA1 expression is positively correlated with the LDHA expression levels in breast cancer patients. Further, it was found that MTA1 is necessary for the optimal expression of LDHA. The underlying molecular mechanism involves the interaction of MTA1 with c-Myc and recruitment of MTA1-c-Myc complex on to the LDHA promoter to regulate its transcription. Consequently, the LDHA knock down using LDHA specific siRNA in MCF7 cells stably expressing MTA1 reduced the migration of MCF7 cells. Altogether these findings revealed the regulatory role for MTA1 in LDHA expression and its resulting biological function.

Ganoderma lucidum Extract Reduces the Motility of Breast Cancer Cells Mediated by the RAC⁻Lamellipodin Axis.

Breast cancer (BC) is the second leading cause of cancer death among women worldwide. The main cause of BC morbidity and mortality is the invasiveness capacity of cancer cells that may lead to metastasis. Here, we aimed to investigate the therapeutic efficacy of Ganoderma lucidum extract (GLE)—a medicinal mushroom with anticancer properties—on BC motility via the Rac/Lamellipodin pathway. GLE treatment effects were tested on MDA-MB-231 breast cancer cells. The effects were tested on cell viability, migration and invasion. Pulldowns, immunoblotting, and immunofluorescence were used to measure Rac activity and the expression of proteins involved in cell migration and in lamellipodia formation, respectively. As a result, GLE suppressed BC cell viability, migration, and invasion capacity. GLE impaired Rac activity, as well as downregulated Lamellipodin, ENA/VASP, p-FAK (Tyr925), Cdc42, and c-Myc expression. Lamellipodia formation was significantly reduced by GLE. In conclusion, we demonstrate that GLE reduces Rac activity and downregulates signaling molecules involved in lamellipodia formation. These novel findings serve as basis for further studies to elucidate the potential of GLE as a therapeutic agent regulating the Rac/Lamellipodin pathway in BC metastasis.

RETRACTED ARTICLE: TMED3 promotes cell proliferation and motility in breast cancer and is negatively modulated by miR-188-3p

BackgroundThe role of TMED3 involved in cancers has been seldom described, let alone in breast cancer. To explore the clinicopathological significance of TMED3 expression and the biological roles involved in breast cancer cells, we undertook the study.MethodsImmunohistochemistry was performed to observe the pattern of TMED3 expression in breast cancer tissues, totaling 224 cases; followed by detailed statistical analysis between TMED3 expression versus clinicopathological information available. To explore the role of TMED3 involved in the malignant behaviors of breast cancer cells, wound-healing and Transwell assays were conducted to evaluate the variation of migration and invasion of MCF-7 and MDA-MB-231 cells whose TMED3 has been stably silenced using lenti-viral based short hairpin RNA (shRNA) vectors. MTT, clonogenic assay and xenograft nude mice model were undertaken to observe the variation of proliferation both in vitro and in vivo.ResultsIt was shown that elevated TMED3 markedly correlated with ER, PR, Her-2 status, and lymph nodes metastases in addition to significant association with poor overall prognosis. In vitro, TMED3 was shown to promote proliferation, migration and invasion of breast cancer cells. Moreover, miR-188-3p was identified as a novel negative regulator of TMED3 in breast cancer, which can slow down the proliferation, migration and invasion of MCF-7 cells. Results from in vivo xenograft nude mice models showed that lenti-viral based miR-188-3p re-expression can markedly impair the tumor growth.ConclusionsOur data define and bolster the oncogenic role of TMED3 in breast cancer.

A DAAM1 3\u2032-UTR SNP mutation regulates breast cancer metastasis through affecting miR-208a-5p-DAAM1-RhoA axis

BackgroundDishevelled-associated activator of morphogenesis 1 (DAAM1) is a member of microfilament-related formins and mediates cell motility in breast cancer (BrCa). However, the genetic mutation status of DAAM1 mRNA and its correlation with pathological characteristics are still unclearly.MethodsA patient cohort and BrCa cells were recruited to demonstrate the role of functional SNP in microRNA-208a-5p binding site of DAAM1 3′-UTR and underlying mechanism in BrCa metastasis.ResultsThe expression and activation of DAAM1 increased markedly in lymphnode metastatic tissues. A genetic variant (rs79036859 A/G) was validated in the miR-208a-5p binding site of DAAM1 3′-UTR. The G genotype (AG/GG) was a risk genotype for the metastasis of BrCa by reducing binding affinity of miR-208a-5p for the DAAM1 3′-UTR. Furthermore, the miR-208a-5p expression level was significantly suppressed in lymphnode metastatic tissues compared with that in non-lymphnode metastatic tissues. Overexpression of miR-208a-5p inhibited DAAM1/RhoA signaling pathway, thereby leading to the decrease of the migratory ability.ConclusionOverall, the rs79036859 G variant of DAAM1 3′-UTR was identified as a relevant role in BrCa metastasis via the diversity of miR-208a-5p binding affinity.

TRIM59 promotes breast cancer motility by suppressing p62-selective autophagic degradation of PDCD10.

Cancer cells adopt various modes of migration during metastasis. How the ubiquitination machinery contributes to cancer cell motility remains underexplored. Here, we report that tripartite motif (TRIM) 59 is frequently up-regulated in metastatic breast cancer, which is correlated with advanced clinical stages and reduced survival among breast cancer patients. TRIM59 knockdown (KD) promoted apoptosis and inhibited tumor growth, while TRIM59 overexpression led to the opposite effects. Importantly, we uncovered TRIM59 as a key regulator of cell contractility and adhesion to control the plasticity of metastatic tumor cells. At the molecular level, we identified programmed cell death protein 10 (PDCD10) as a target of TRIM59. TRIM59 stabilized PDCD10 by suppressing RING finger and transmembrane domain-containing protein 1 (RNFT1)-induced lysine 63 (K63) ubiquitination and subsequent phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa (p62)-selective autophagic degradation. TRIM59 promoted PDCD10-mediated suppression of Ras homolog family member A (RhoA)-Rho-associated coiled-coil kinase (ROCK) 1 signaling to control the transition between amoeboid and mesenchymal invasiveness. PDCD10 overexpression or administration of a ROCK inhibitor reversed TRIM59 loss-induced contractile phenotypes, thereby accelerating cell migration, invasion, and tumor formation. These findings establish the rationale for targeting deregulated TRIM59/PDCD10 to treat breast cancer.

Correction: Mechanosensitive caveolin-1 activation-induced PI3K/Akt/mTOR signaling pathway promotes breast cancer motility, invadopodia formation and metastasis in vivo.

[This corrects the article DOI: 10.18632/oncotarget.7583.].

Limited fibrosis accompanies triple-negative breast cancer metastasis in multiple model systems and is not a preventive target.

The lysophosphatidic acid receptor 1 (LPAR1) is mechanistically implicated in both tumor metastasis and tissue fibrosis. Previously, metastasis was increased when fulminant fibrosis was first induced in mice, suggesting a direct connection between these processes. The current report examined the extent of metastasis-induced fibrosis in breast cancer model systems, and tested the metastasis preventive efficacy and fibrosis attenuation of antagonists for LPAR1 and Idiopathic Pulmonary Fibrosis (IPF) in breast and ovarian cancer models. Staining analysis demonstrated only focal, low-moderate levels of fibrosis in lungs from eleven metastasis model systems. Two orally available LPAR1 antagonists, SAR100842 and EPGN9878, significantly inhibited breast cancer motility to LPA in vitro. Both compounds were negative for metastasis prevention and failed to reduce fibrosis in the experimental MDA-MB-231T and spontaneous murine 4T1 in vivo breast cancer metastasis models. SAR100842 demonstrated only occasional reductions in invasive metastases in the SKOV3 and OVCAR5 ovarian cancer experimental metastasis models. Two approved drugs for IPF, nintedanib and pirfenidone, were investigated. Both were ineffective at preventing MDA-MB-231T metastasis, with no attenuation of fibrosis. In summary, metastasis-induced fibrosis is only a minor component of metastasis in untreated progressive breast cancer. LPAR1 antagonists, despite in vitro evidence of specificity and efficacy, were ineffective in vivo as oral agents, as were approved IPF drugs. The data argue against LPAR1 and fibrosis as monotherapy targets for metastasis prevention in triple-negative breast cancer and ovarian cancer.

NON‐CONTACT ELECTRIC FIELDS POTENTLY HINDER EGF PROMOTED BREAST CANCER MOTILITY BY DOWNREGULATING EGFR PHOSPHORYLATION

The objective of the proposed study is to assess the therapeutic potential of non‐contact inductive E‐fields (iEFs) in inhibiting breast cancer motility. Normal breast ducts are lined with monolayer of epithelial cells that have asymmetric distribution of ion channels resulting in a charge separation between the luminal and the basal sides giving rise to trans‐epithelial potentials (TEP). These TEPs are responsible for endogenous E‐fields that are directed radially from luminal to basal side. Consequently, the sites of invasion in breast tumors are depolarized, which suggest that that cell migration in late‐stage tumors occurs in absence of endogenous E‐fields. Furthermore, breast cancer cells in‐vitro have been shown to migrate towards the positive electrodes under the influence of externally applied direct current electric fields (1–10 V/cm). Therefore, we hypothesize that E‐fields applied in the direction of migrating breast cancer cells potently inhibit cell motility. To test our hypothesis, we used a panel of three breast cell lines, 1) normal MCF10A, 2) MCF10CA1a(MIV) (a malignant variant of MCF10A cells with H‐Ras mutation), and 3) triple negative breast cancer (TNBC) cells MDA‐MB‐231 in conjunction with a custom Bi‐directional Microtrack Assay (BMA). The BMA consists of an array of parallel‐aligned tracks that measure 700 μm in length and with a cross‐section of 20 μm × 20 μm. These microtracks mimic the topological cues encountered by migrating tumor cells that have detached from the primary tumor site during invasion. Inductive Electric Fields (iEFs ~ 60 μV/cm), which mimic the endogenous E‐fields in the breast duct, were applied using a custom‐designed air‐cored Helmholtz coils. iEFs applied anti‐parallel to the direction of migration and in absence of a pro‐migratory epidermal growth factor (EGF) gradient on cancer cells significantly increased migration speeds (Fig A) but had no effect on the control epithelial cells which maintained their non‐migratory phenotype. In presence of a stable EGF gradient, iEFs applied parallel in the direction of migration significantly reduce migration speeds for both the cancerous cell lines whereas it has no effect on migration speeds of MCF10A cells. Actin aggregation at the leading and/or trailing edges is a characteristic of migratory cells and for cancer cells that showed significant speed reduction the intracellular actin was uniformly distributed (Fig B) rather aggregating on the tips indicating a possible mechanism controlled through the EGFR/EGF pathway. Furthermore, we verified using Western Blotting techniques that iEFs significantly reduced phosphorylation of EGFR in EGF‐treated MDA‐MB‐231 cells (Fig C) whereas they downregulated the expression of EGFR in EGF‐treated MCF10CA1a(MIV) cells. For each case, the protein levels had a one‐to‐one correlation with the migration speeds for each cell line. Moreover, we also found that a combinatorial treatment of MK2206 (Akt inhibitor) with iEFs significantly reduced migration speeds of these breast cancer cells (Fig A) below the level of their respective controls. Thus, iEFs selectively hindered cancer cells with no adverse effects on normal epithelial cells, making them a potential candidate for cancer control and intervention. From a therapeutic standpoint, iEFs may help augment current therapeutic approaches to hinder the metastasis of breast cancer and confer survival benefit, including in TNBC, which currently lack any targeted therapies.Support or Funding InformationFunding for JWS: American Cancer Society and Institute for Materials Research at The Ohio State University. JWS acknowledge support from Pelotonia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carcinoma associated fibroblasts (CAFs) promote breast cancer motility by suppressing mammalian Diaphanous-related formin-2 (mDia2).

The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.

Serglycin Regulates Cytoskeletal‐related Proteins Associated with Cell Motility in Breast Cancer In Vitro

Despite advances in cancer treatment, triple‐negative (ER−/PR−/HER2−) breast cancer represents a complicated subtype with no specific treatment regime. Serglycin (SRGN) is an intracellular proteoglycan which has been shown to be highly expressed in the triple‐negative MDA‐MB‐231 breast cancer cell line. In this study, we aim to decipher the molecular networks and pathways regulated by SRGN in breast cancer cells using a global gene expression analysis approach. We first analyzed the expression of SRGN in a dataset containing 1,881 breast cancer tissues from the GOBO database (http://co.bmc.lu.se/gobo/gobo.pl). For the in vitro experiments, siRNA‐mediated down‐regulation of the SRGN gene in MDA‐MB‐231 cells was performed. Transcriptomic analysis of si‐SRGN breast cancer cells was then carried out. Subsequently real time PCR for specific gene targets and expression of selected proteins by Western Blot were performed. Cell motility was determined using commercially available cell migration chambers. The Gene Set analysis from the GOBO database verified that the SRGN gene is highly expressed in basal (triple negative) breast tumors. Knockdown of the SRGN gene in MDA‐MB‐231 cells induced differential expression of 225 genes. Gene ontology analysis revealed the presence of a cluster of genes that were highly enriched in cellular processes, including cell movement. Reduced expression of SRGN was accompanied by down‐regulation of several cytoskeletal‐related genes, namely ABLIM1, LIMA1, CFL1, RAC1, RAC2 and RHOA, concomitant with decreased cell motility. Decreased expression of RAC1 and RHOA proteins were also demonstrated. The results suggest that SRGN is important in regulating actin cytoskeletal organization associated with cell migration, a crucial event during cancer metastasis.Support or Funding InformationThis work is supported by the National Medical Research Council Singapore Grant NMRC/CIRG/1370/2013.

MiR520c blocks EMT progression of human breast cancer cells by repressing STAT3.

Breast cancer is one of the most malignant diseases world-wide and it ranks the first among female cancers. Masses of intrinsic and extrinsic factors, especially the inflammatory factors can lead to breast cancer. Aberrant activation and accumulation of key molecules can lead to inflammation associated carcinogenesis. The signal transducers and activators of transcription 3 (STAT3) is one of them. Therefore, to evaluate the novel molecular mechanisms, STAT3 has become our focus for breast cancer targeted therapy. At present, many tumor suppressing microRNAs have been validated, and are the highlights in research on microRNAs. Thus, we predicted microRNAs which could putatively regulate STAT3 through databases and selected six to screen with Dual-luciferase assay. The result hinted that miR520c could bind with STAT3 3'UTR. We mutated the seed sequence of miR520c on STAT3 3'UTR, which illustrated a reverse effect compared with wild-type of STAT3 3'UTR. Subsequently, STAT3, p-STAT3 and miR520c were assessed in three different grades of breast cancer cells, with the degree of malignancy, we found an escalating trend of STAT3 and p-STAT3, on the contrary, a downward trend of miR520c. We observed STAT3 was deactivated by miR520c. Epithelial to mesenchymal transition (EMT) is a fatal transfer of cancer progression. To find out whether the downregulation of STAT3 can repress breast cancer motility and invasion ability, we detected EMT markers. The result implied a suppression effect on EMT. We overexpressed STAT3 to conduct rescue experiments, the result showed a recovery of STAT3 and EMT characteristics. Cell motility and invasion property were regained as well. In the study, we elucidated miR520c could inhibit breast cancer EMT by targeting STAT3. It can enrich the mechanism of breast cancer and may lay the foundation for breast cancer targeted treatment.

Twist induces epithelial-mesenchymal transition and cell motility in breast cancer via ITGB1-FAK/ILK signaling axis and its associated downstream network

Twist, a highly conserved basic Helix-Loop-Helix transcription factor, functions as a major regulator of epithelial-mesenchymal transition (EMT) and tumor metastasis. In different cell models, signaling pathways such as TGF-β, MAPK/ERK, WNT, AKT, JAK/STAT, Notch, and P53 have also been shown to play key roles in the EMT process, yet little is known about the signaling pathways regulated by Twist in tumor cells. Using iTRAQ-labeling combined with 2D LC–MS/MS analysis, we identified 194 proteins with significant changes of expression in MCF10A-Twist cells. These proteins reportedly play roles in EMT, cell junction organization, cell adhesion, and cell migration and invasion. ECM–receptor interaction, MAPK, PI3K/AKT, P53 and WNT signaling were found to be aberrantly activated in MCF10A-Twist cells. Ingenuity Pathways Analysis showed that integrin β1 (ITGB1) acts as a core regulator in linking integrin-linked kinase (ILK), Focal-adhesion kinase (FAK), MAPK/ERK, PI3K/AKT, and WNT signaling. Increased Twist and ITGB1 are associated with breast tumor progression. Twist transcriptionally regulates ITGB1 expression. Over-expression of ITGB1 or Twist in MCF10A led to EMT, activation of FAK/ILK, MAPK/ERK, PI3K/AKT, and WNT signaling. Knockdown of Twist or ITGB1 in BT549 and Hs578T cells decreased activity of FAK, ILK, and their downstream signaling, thus specifically impeding EMT and cell invasion. Knocking down ILK or inhibiting FAK, MAPK/ERK, or PI3K/AKT signaling also suppressed Twist-driven EMT and cell invasion. Thus, the Twist-ITGB1-FAK/ILK pathway and their downstream signaling network dictate the Twist-induced EMT process in human mammary epithelial cells and breast cancer cells.