Enhanced Breast Cancer Cell Migration Research Articles

Exosomal miRNA-92a derived from cancer-associated fibroblasts promote invasion and metastasis in breast cancer by regulating G3BP2

Cancer-associated Fibroblasts (CAFs) exert a tumor-promoting effect in various cancers, including breast cancer. CAFs secrete exosomes containing miRNA and proteins, influencing the tumor microenvironment. In this study, we identified CAF-derived exosomes that transport functional miR-92a from CAFs to tumor cells, thereby intensifying the aggressiveness of breast cancer. CAFs downregulate the expression of G3BP2 in breast cancer cells, and a significant elevation in miR-92a levels in CAF-derived exosomes was observed. Both in vitro and in vivo experiments demonstrate that miR-92a enhances breast cancer cell migration and invasion by directly targeting G3BP2, functioning as a tumor-promoting miRNA. We validated that the RNA-binding proteins SNRPA facilitate the transfer of CAF-derived exosomal miR-92a to breast cancer cells. The reduction of G3BP2 protein by CAF-derived exosomes releases TWIST1 into the nucleus, promoting epithelial-mesenchymal transition (EMT) and further exacerbating breast cancer progression. Moreover, CAF-derived exosomal miR-92a induces tumor invasion and metastasis in mice. Overall, our study reveals that CAF-derived exosomal miR-92a serves as a promoter in the migration and invasion of breast cancer cells by reducing G3BP2 and may represent a potential novel tumor marker for breast cancer.

Human Mesenchymal Stem Cells Derived from the Placenta and Chorion Suppress the Proliferation while Enhancing the Migration of Human Breast Cancer Cells.

Breast cancer is the most frequently diagnosed malignancy among women, resulting from abnormal proliferation of mammary epithelial cells. The highly vascularized nature of breast tissue leads to a high incidence of breast cancer metastases, resulting in a poor survival rate. Previous studies suggest that human mesenchymal stem cells (hMSCs) play essential roles in the growth, metastasis, and drug responses of many cancers, including breast cancer. However, hMSCs from different sources may release different combinations of cytokines that affect breast cancer differently. In this study, we have isolated hMSCs from the placenta (PL-hMSCs) and the chorion (CH-hMSCs) and determined how these hMSCs affect the proliferation, migration, invasion, and gene expression of two human breast cancer cells, MCF-7 and MDA-MB-231, as well as the possible mechanisms underlying those effects. The results showed that the soluble factors derived from PL-hMSCs and CH-hMSCs inhibited the proliferation of MCF-7 and MDA-MB-231 cells but increased the migration of MDA-MB-231 cells. The study of gene expression showed that PL-hMSCs and CH-hMSCs downregulated the expression levels of the protooncogene CyclinD1 while upregulating the expression levels of tumor suppressor genes, P16 and P21 in MCF-7 and MDA-MB-231 cells. Furthermore, hMSCs from both sources also increased the expression levels of MYC, SNAI1, and TWIST, which promote the epithelial-mesenchymal transition and migration of breast cancer cells in both cell lines. The functional study suggests that the suppressive effect of CH-hMSCs and PL-hMSCs on MCF-7 and MDA-MB231 cell proliferation was mediated, at least in part, through IFN-γ. Our study suggests that CH-hMSCs and PL-hMSCs inhibited breast cancer cell proliferation by negatively regulating CYCLIND1 expression and upregulating the expression of the P16 and P21 genes. In contrast, hMSCs from both sources enhanced breast cancer cell migration, possibly by increasing the expression of MYC, SNAI1, and TWIST genes in those cells.

The effect of marrow secretome and culture environment on the rate of metastatic breast cancer cell migration in two and three dimensions.

Metastasis is responsible for over 90% of cancer-related deaths, and bone is the most common site for breast cancer metastasis. Metastatic breast cancer cells home to trabecular bone, which contains hematopoietic and stromal lineage cells in the marrow. As such, it is crucial to understand whether bone or marrow cells enhance breast cancer cell migration toward the tissue. To this end, we quantified the migration of MDA-MB-231 cells toward human bone in two- and three-dimensional (3D) environments. First, we found that the cancer cells cultured on tissue culture plastic migrated toward intact trabecular bone explants at a higher rate than toward marrow-deficient bone or devitalized bone. Leptin was more abundant in conditioned media from the cocultures with intact explants, while higher levels of IL-1β, IL-6, and TNFα were detected in cultures with both intact bone and cancer cells. We further verified that the cancer cells migrated into bone marrow using a bioreactor culture system. Finally, we studied migration toward bone in 3D gelatin. Migration speed did not depend on stiffness of this homogeneous gel, but many more dendritic-shaped cancer cells oriented and migrated toward bone in stiffer gels than softer gels, suggesting a coupling between matrix mechanics and chemotactic signals.

Mechanically stimulated osteocytes promote the proliferation and migration of breast cancer cells via a potential CXCL1/2 mechanism

Bone represents the most common site for breast cancer metastasis. Bone is a highly dynamic organ that is constantly adapting to its biophysical environment, orchestrated largely by the resident osteocyte network. Osteocytes subjected to physiologically relevant biophysical conditions may therefore represent a source of key factors mediating breast cancer cell metastasis to bone. Therefore, we investigated the potential proliferative and migratory capacity of soluble factors released by mechanically stimulated osteocytes on breast cancer cell behaviour. Interestingly the secretome of mechanically stimulated osteocytes enhanced both the proliferation and migration of cancer cells when compared to the secretome of statically cultured osteocytes, demonstrating that mechanical stimuli is an important physiological stimulus that should be considered when identifying potential targets. Using a cytokine array, we further identified a group of mechanically activated cytokines in the osteocyte secretome, which potentially drive breast cancer metastasis. In particular, CXCL1 and CXCL2 cytokines are highly expressed, mechanically regulated, and are known to interact with one another. Lastly, we demonstrate that these specific factors enhance breast cancer cell migration independently and in a synergistic manner, identifying potential osteocyte derived factors mediating breast cancer metastasis to bone.

RNF20 Is Critical for Snail-Mediated E-Cadherin Repression in Human Breast Cancer.

BackgroundE-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often repressed due to Snail-mediated epigenetic modification; however, the exact mechanism remains unclear. There is an urgent need to understand the determinants of tumor aggressiveness and identify potential therapeutic targets in breast cancer.Experimental designWe studied the association of RNF20 with Snail and G9a by co-immunoprecipitation. We employed quantitative real-time PCR, ChIP, transwell assay, colony formation assay, and mammosphere assay to dissect the molecular events associated with the repression of E-cadherin in human breast cancer. We used a proteogenomic dataset that contains 105 breast tumor samples to determine the clinical relevance of RNF20 by Kaplan-Meier analyses.ResultsIn this study, we identified that Snail interacted with RNF20, an E3 ubiquitin-protein ligase responsible for monoubiquitination of H2BK120, and G9a, a methyltransferase for H3K9me2. RNF20 expression led to the inhibition of E-cadherin expression in the human breast cancer cells. Mechanically, we showed that RNF20 and H3K9m2 were enriched on the promoter of E-cadherin and knockdown of Snail reduced the enrichment of RNF20, showing a Snail-dependent manner. RNF20 expression enhanced breast cancer cell migration, invasion, tumorsphere and colony formation. Clinically, patients with high RNF20 expression had shorter overall survival.ConclusionRNF20 expression contributes to EMT induction and breast cancer progression through Snail-mediated epigenetic suppression of E-cadherin expression, suggesting the importance of RNF20 in breast cancer.

Lysyl oxidase expression is regulated by the H3K27 demethylase Jmjd3 in tumor-associated M2-like macrophages.

Copper is one of the essential micronutrients, and copper-containing enzymes contribute to crucial functions in the body. Lysyl oxidase is a copper-containing enzyme that remodels the extracellular matrix by cross-linking collagen and elastin. The overexpression of lysyl oxidase was recently shown to promote tumor metastasis. M2-like macrophages were also found to significantly accumulate in the tumor microenvironment, and correlated with a poor patient's outcome. We speculate that M2-like macrophages promote tumor progression via lysyl oxidase expression. Epigenetics, a mitotically heritable change in gene expression without any change in DNA sequencing, is also associated with tumor progression. However, the relationship between lysyl oxidase expression in M2-like macrophages and epigenetics remains unclear. Lysyl oxidase expression was significantly induced in human leukemic THP-1 cell-derived M2-like macrophages. Furthermore, the level of histone H3 tri-methylation at lysine 27 was decreased, and a pre-treatment with a H3K27 demethylase inhibitor notably suppressed lysyl oxidase expression in M2-like macrophages. Lysyl oxidase derived from M2-like macrophages also enhanced breast cancer cell migration, and this was suppressed by a H3K27 demethylase inhibitor. The present results suggest the mechanism of lysyl oxidase expression in M2-like macrophages as an aspect of epigenetics, particularly histone methylation.

Long non-coding RNA TTN-AS1 promotes the metastasis in breast cancer by epigenetically activating DGCR8.

Breast cancer (BC) is one of the most common fatal cancers. Recent studies have identified the vital roles of long non-coding RNAs (lncRNAs) in the development and progression of BC. This research aimed to investigate the underlying mechanisms of lncRNA TTN-AS1 in the metastasis of BC. TTN-AS1 expression of tissues was detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) in 50 BC patients. Wound healing assay and transwell assay were used to observe the phenotypic alteration of BC cells after knockdown or overexpression of TTN-AS1. Moreover, RT-qPCR and Western blot assay were performed to discover the potential targets of TTN-AS1 in BC. TTN-AS1 expression in BC samples was significantly higher than that of the adjacent tissues. Besides, the migration and invasion of BC cells were markedly inhibited after TTN-AS1 was silenced, while promoted after TTN-AS1 overexpression. In addition, a remarkable decrease of DGCR8 was observed after TTN-AS1 was inhibited in BC cells, while DGCR8 was upregulated after overexpression of TTN-AS1. Furthermore, DGCR8 expression showed significant enhancement in BC tissues and was positively associated with TTN-AS1 level. Our study uncovered a new oncogene in BC and suggested that TTN-AS1 could enhance BC cell migration and invasion via sponging DGCR8, which provided a novel therapeutic target for the treatment of breast cancer.

ZnR/GPR39 upregulation of K+/Cl−-cotransporter 3 in tamoxifen resistant breast cancer cells

Expression of the zinc receptor, ZnR/GPR39, is increased in higher grade breast cancer tumors and cells. Zinc, its ligand, is accumulated at larger concentrations in the tumor tissue and can therefore activate ZnR/GPR39-dependent Ca2+ signaling leading to tumor progression. The K+/Cl− co-transporters (KCC), activated by intracellular signaling, enhance breast cancer cell migration and invasion. We asked if ZnR/GPR39 enhances breast cancer cell malignancy by activating KCC. Activation of ZnR/GPR39 by Zn2+ upregulated K+/Cl− co-transport activity, measured using NH4+ as a surrogate to K+ while monitoring intracellular pH. Upregulation of NH4+ transport was monitored in tamoxifen resistant cells with functional ZnR/GPR39-dependent Ca2+ signaling but not in MCF-7 cells lacking this response. The NH4+ transport was Na+-independent, and we therefore focused on KCC family members. Silencing of KCC3, but not KCC4, expression abolished Zn2+-dependent K+/Cl− co-transport, suggesting that KCC3 is mediating upregulated NH4+ transport. The ZnR/GPR39-dependent KCC3 activation accelerated scratch closure rate, which was abolished by inhibiting KCC transport with [(DihydroIndenyl) Oxy] Alkanoic acid (DIOA). Importantly, silencing of either ZnR/GPR39 or KCC3 attenuated Zn2+-dependent scratch closure. Thus, a novel link between KCC3 and Zn2+, via ZnR/GPR39, promotes breast cancer cell migration and proliferation.

Doxorubicin Promotes Migration and Invasion of Breast Cancer Cells through the Upregulation of the RhoA/MLC Pathway.

PurposeCancer cells develop acquired resistance induced by chemotherapeutic drugs. In this study, we investigated the effects of brief treatment with cytotoxic drugs on the phenotype of breast cancer cells.MethodsBreast cancer cells MCF7 and BT-474 were briefly treated with paclitaxel or doxorubicin. Clonogenic, migration, and invasion assays were performed on the treated cells. Western blot analysis and RhoA activity assay were also performed.ResultsBreast cancer cells when briefly treated with paclitaxel or doxorubicin showed reduced clonogenic ability. Doxorubicin, but not paclitaxel, augmented cell migration and invasion. The invasion-promoting effects of doxorubicin were lost when the two drugs were sequentially used in combination. Myosin light chain (MLC) 2 phosphorylation and RhoA activity were upregulated by doxorubicin and downregulated by paclitaxel. Pretreatment with RhoA inhibitors abolished the migration- and invasion-promoting effects of doxorubicin.ConclusionDoxorubicin activates the RhoA/MLC pathway and enhances breast cancer cell migration and invasion. Therefore, this pathway might be explored as a therapeutic target to suppress anthracycline-enhanced tumor progression.

Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression

BackgroundAdipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis.MethodsBreast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro.ResultsGene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways.ConclusionsOur study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.

3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition.

Solid tumors are 3D assemblies of cancer cells, together with multiple stromal cell types within an extracellular matrix. Yet, the vast majority of cell-based studies to characterize oncogenesis and discovery of new anti-cancer drugs is conducted using conventional 2D monolayer culture systems, where cells are grown on plastic substratum under normoxic environments. In current study, we generated 3D breast cancer cell culture platform consists of photocrosslinkable hydrogels and encapsulated isogenic primary (21PT) and a metastatic (21MT-2) breast cancer cell lines derived from the primary tumor and pleural effusion from the same patient. We demonstrated that hypoxia decreased cellular assembly size and density, and promoted epithelial to mesenchymal transition (EMT) process, without affecting cell viability. Next, we showed hypoxia enhanced breast cancer cell migration, and expression and secretion of lysyl oxidase (LOX), which is copper-dependent amine oxidase and has the primary function to drive the crosslinking of collagen and elastin and is regulated by hypoxia. Furthermore, to recapitulate in vivo situation, we generated breast cancer and lung cells (derived from the same patient) contact model by stacking 3D hydrogel constructs with breast cancer cells onto lung mesenchymal cells (LMC) laden-hydrogel and then showed breast cancer cells migrated towards LMC during hypoxia. Lastly, as a validation of this model for future screen of therapeutic agents, we demonstrated that LOX inhibitor exhibited a significant decrease in breast cancer cell viability, migration, and EMT. Taken together, these results validate the use of hydrogels based models to examine hypoxia related EMT in breast cancer cells.

An Engineered Human Adipose/Collagen Model forIn VitroBreast Cancer Cell Migration Studies

Adipocytes are one of the major stromal cell components of the human breast. These cells play a key role in the development of the gland and are implicated in breast tumorigenesis. Frequently, directional stromal collagen I fibers are found surrounding aggressive breast tumors. These fibers enhance breast cancer cell migration and are associated with poor patient prognosis. We sought to recapitulate these stromal components in vitro to provide a three-dimensional (3D) model comprising human adipose tissue and anisotropic collagen fibers. We developed a human mesenchymal stem cell (hMSC) cell line capable of undergoing differentiation into mature adipocytes by immortalizing hMSCs, isolated from breast reduction mammoplasties, through retroviral transduction. These immortalized hMSCs were seeded in engineered collagen I scaffolds with directional internal architecture, and adipogenesis was chemically induced, resulting in human adipose tissue being synthesized in vitro in an architectural structure associated with breast tumorigenesis. Subsequently, fluorescently labeled cells from an established breast cancer cell line were seeded into this model, cocultured for 7 days and imaged using multiphoton microscopy. Enhanced breast cancer cell migration was observed in the adipose-containing model over empty scaffold controls, demonstrating an adipocyte-mediated influence on breast cancer cell migration. Thus, this 3D in vitro model recapitulates the migratory effects of adipocytes observed on breast cancer cells and suggests that it could have utility with fresh breast tumor biopsies as an assay for cancer therapeutic efficacy in personalized medicine strategies.

FOXP1 enhances tumor cell migration by repression of NFAT1 transcriptional activity in MDA-MB-231 cells.

Until now, forkhead box P1 (FOXP1) has been identified as a tumor suppressor in several correlation studies in breast cancer. Although FOXP1 is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. This study demonstrated the repressor function of FOXP1 on nuclear factor of activated T cells (NFAT1) and the migratory effect of this repression in MDA-MB-231 breast cancer cells. Co-immunoprecipitation experiments were performed for the investigation of protein-protein interaction between two transcription factors. Protein-protein interaction on DNA was investigated with EMSA and transcriptional effects of FOXP1 on NFAT1, luciferase reporter assay was performed. Wound healing assay was used to analyze the effects of overexpression of FOXP1 on tumor cell migration. This study showed that FOXP1 has protein-protein interaction with NFAT1 on DNA and enhances breast cancer cell migration by repressing NFAT1 transcriptional activity and FOXP1 shows oncogenic function by regulating breast cancer cell motility.

Identification of NEK3 Kinase Threonine 165 as a Novel Regulatory Phosphorylation Site That Modulates Focal Adhesion Remodeling Necessary for Breast Cancer Cell Migration

Accumulating evidence supports a role for prolactin (PRL) in the development and progression of human breast cancer. Although PRL is an established chemoattractant for breast cancer cells, the precise molecular mechanisms of how PRL regulates breast cancer cell motility and invasion are not fully understood. PRL activates the serine/threonine kinase NEK3, which was reported to enhance breast cancer cell migration, invasion, and the actin cytoskeletal reorganization necessary for these processes. However, the specific mechanisms of NEK3 activation in response to PRL signaling have not been defined. In this report, a novel PRL-inducible regulatory phosphorylation site within the activation segment of NEK3, threonine 165 (Thr-165), was identified. Phosphorylation at NEK3 Thr-165 was found to be dependent on activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway using both pharmacological inhibition and siRNA-mediated knockdown approaches. Strikingly, inhibition of phosphorylation at NEK3 Thr-165 by expression of a phospho-deficient mutant (NEK3-T165V) resulted in increased focal adhesion size, formation of zyxin-positive focal adhesions, and reorganization of the actin cytoskeleton into stress fibers. Concordantly, NEK3-T165V cells exhibited migratory defects. Together, these data support a modulatory role for phosphorylation at NEK3 Thr-165 in focal adhesion maturation and/or turnover to promote breast cancer cell migration.

Abstract 629: The impact of BMP4 on breast cancer metastasis in an in vivo xenograft mouse model

Abstract Background. Breast cancer is the most common cancer in women worldwide. Bone morphogenetic proteins (BMPs), members of the transforming growth factor β superfamily, are known to regulate cell proliferation, differentiation and motility, and have also been shown to be involved in cancer pathogenesis, also in breast cancer. We have previously demonstrated that BMP4 is able to consistently reduce breast cancer cell proliferation through G1 cell cycle arrest and to simultaneously induce migration and invasion in a subset of breast cancer cell lines. Similarly, our clinical data revealed a correlation between elevated BMP4 expression in primary breast tumors and reduced proliferation as well as increased risk of recurrence. The growth inhibitory effects of BMP4 have also been demonstrated in vivo but its possible metastasis promoting functions are less well characterized. Here we set out to investigate this topic using a xenograft mouse model. Methods. MDA-MB-231 breast cancer cells were transduced with a luciferase-expressing vector to allow monitoring of the metastasis formation using bioluminescence imaging. Cells (2 × 105) were injected into the mice intracardially and BMP4 (100 ng/g, 10 animals) or vehicle control (11 animals) was administered through tail vein three times a week. After seven weeks, the mice were sacrificed and metastases collected for histological analyses. Results. The overall amount of metastases was similar in both groups (13 in BMP4-treatment group vs. 12 in control group). There was a slight but non-significant trend of metastases developing earlier in the BMP4 group compared to controls. Most of the metastases occurred in bone and adrenal glands. There were somewhat more metastases in bone in the BMP4-treated mice (10 vs. 7) and more adrenal gland metastases in vehicle-treated animals (5 vs. 1). To assess the contribution of BMP4 to the characteristics of the metastases, the tumors were stained for pSMAD1/5/9 (BMP signaling activation), Ki67 (proliferation), MECA32 (blood vessels), mesenchymal marker vimentin, α-SMA (cancer-associated fibroblasts) and basal markers K5 and K14. No major dissimilarities were observed between the BMP4 and vehicle tumor groups in the staining patterns. Interestingly, the osteoclast marker Tartrate-resistant acid phosphatase (TRAP) was expressed in both groups in the cancer cells whereas Toluidine Blue staining revealed that the bone morphology was not detrimentally affected by BMP4 treatment. Conclusions. Despite its ability to enhance breast cancer cell migration and invasion in vitro, BMP4 does not seem to have a dramatic impact on in vivo metastasis formation, although a small acceleration in appearance of the metastases was observed. However, the limitations of the xenograft model do not allow us to exclude the possible long-term effects of BMP4 that might be more applicable to human situation. Citation Format: Minna Ampuja, Emma L. Alarmo, Philip Owens, Riikka Havunen, Agnes E. Gorska, Harold L. Moses, Anne Kallioniemi. The impact of BMP4 on breast cancer metastasis in an in vivo xenograft mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 629.

Abstract 4150: TNFα and TGFβ1 secreted by macrophages enhance breast cancer cell migration dynamics via the induction of NF-κB dependent MMP-1 expression

Abstract Metastasis, which is a major cause of cancer death, depends on cancer cell's ability to migrate through the dense extracellular matrix (ECM) within the solid tumor. Recent studies have revealed that macrophages within the breast tumor microenvironment could promote tumor metastasis. However, it is still unclear how macrophages affect the migration dynamics (e.g. total speed and directedness) of cancer cells in 3D ECM that mimics the in vivo environment. To address this question, we co-cultured MDA-MB-231 breast cancer cells with Raw 264.7 macrophages inside a collagen I ECM embedded in a microfluidic device. Time-lapse microscopy was used to quantify the effects of macrophages on the total speed and directedness of cancer cell 3D migration. Various biochemical assays were also performed to investigate the mechanisms underlying the observed effects. We found that macrophages enhanced both cancer cell migration total speed and directedness, allowing cancer cells to migrate faster and more persistently. Since macrophages are the major producers of TNFα and TGFβ1, we used blocking antibodies against these two factors to investigate their contribution to macrophage-assisted cancer cell migration. We found that inhibiting macrophage-secreted TNFα or TGFβ1 alone only slightly diminished cancer cell migration directedness. In contrast, simultaneous inhibition of both TNFα and TGFβ1 completely abolished macrophage-enhanced cancer cell migration directedness. To investigate this result further, we quantified the migration directedness of cancer cells treated with TNFα and/or TGFβ1. We found that the treatment of cancer cells with TNFα or TGFβ1 alone did not significantly increase their migration directedness. However, treatment of cancer cells with both TNFα and TGFβ1 synergistically enhanced the migration directedness of cancer cells to the level when cancer cells were co-cultured with macrophages. This result suggests that macrophage-released TNFα and TGFβ1 are the main contributors to the increase in cancer cell migration directedness. Furthermore, using a MMP inhibitor, we found that the inhibition of cancer cells’ ability to breakdown ECM led to reduced migration directedness. Therefore, we showed, using western blotting, that macrophage-released TNFα and TGFβ1 synergistically induced the nuclear localization of NF-κB in cancer cells, leading to a synergistically enhanced expression of MMP-1. Taken together, our results indicate that macrophage-secreted TNFα and TGFβ1 could enhance cancer cell migration directedness via the induction of NF-κB nuclear localization and MMP-1 expression within cancer cells. Our results further suggest that both macrophage-released TNFα and TGFβ1 need to be simultaneously inhibited in order to effectively neutralize macrophage-enhanced cancer cell migration and metastasis. Citation Format: Ran Li, Tara Lee, Roger D. Kamm. TNFα and TGFβ1 secreted by macrophages enhance breast cancer cell migration dynamics via the induction of NF-κB dependent MMP-1 expression. [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 4150. doi:10.1158/1538-7445.AM2015-4150

Interleukin-18 enhances breast cancer cell migration via down-regulation of claudin-12 and induction of the p38 MAPK pathway

Interleukin-18 (IL-18) was recently reported to have a pro-tumor effect in various cancers. Increased IL-18 levels in the serum of cancer patients correlated with malignancy, and IL-18 acts a crucial factor for cell migration in gastric cancer and melanoma. Claudins, which are the most important tight junction proteins, are also linked with cancer progression and metastasis. However, the relationship between claudins and IL-18 is not well-understood. Here, we show that the migratory ability of MCF-7 cells was reduced when endogenous IL-18 expression was inhibited with IL-18 siRNA. Moreover, exogenous IL-18 enhanced breast cancer cell migration and suppressed the expression of the tight junction proteins claudin-1, claudin-3, claudin-4, and claudin-12 in MCF-7 cells. Knockdown of claudin-3, claudin-4, and claudin-12, but not claudin-1, increased breast cancer migration with maximal effects observed in claudin-12 siRNA-transfected cells. To investigate whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in IL-18-induced cell migration and claudin-12 expression, cells were pretreated with SB203580 (an inhibitor of p38 MAPK) or PD98059 (an inhibitor of ERK1/2) prior to the addition of IL-18. Although pretreatment of MCF-7 cells with SB203580 blocked both the enhanced cell migration and the decreased claudin-12 expression, PD98059 only blocked cell migration and did not affect claudin-12 expression. In addition, exogenous IL-18 induced rapid phosphorylation of p38 MAPK. These results suggest that IL-18 is an important factor inducing breast cancer cell migration through down-regulation of claudin-12 and activation of the p38 MAPK pathway.

Effects of exposure to benzo[a]pyrene on metastasis of breast cancer are mediated through ROS-ERK-MMP9 axis signaling

Metastasis is the leading cause of deaths in patients with breast cancer. Benzo[a]pyrene is a cumulative carcinogen and ubiquitous environmental pollutant with potent carcinogenic properties. As we report here, we established an accumulative mouse model mimicking the cumulative effects of benzo[a]pyrene exposure in human breast carcinogenesis. Our focus was on elucidating the mechanisms by which benzo[a]pyrene contributes to the process of breast cancer metastasis. Our study indicated that benzo[a]pyrene increased the migration of breast cancer cells both in vitro and in vivo. Specifically, we demonstrated that benzo[a]pyrene enhances breast cancer cell migration and invasion by up-regulating ROS-induced ERK signaling, leading to the activation of matrix metalloproteinases 9. Our results suggest that benzo[a]pyrene-induced mammary gland cancer metastasis is an important and intricate process facilitated by cumulative benzo[a]pyrene exposure leading to activation of the ROS-ERK-MMP9 signaling pathway.

Preferential, enhanced breast cancer cell migration on biomimetic electrospun nanofiber 'cell highways'.

BackgroundAggressive metastatic breast cancer cells seemingly evade surgical resection and current therapies, leading to colonization in distant organs and tissues and poor patient prognosis. Therefore, high-throughput in vitro tools allowing rapid, accurate, and novel anti-metastatic drug screening are grossly overdue. Conversely, aligned nanofiber constitutes a prominent component of the late-stage breast tumor margin extracellular matrix. This parallel suggests that the use of a synthetic ECM in the form of a nanoscale model could provide a convenient means of testing the migration potentials of cancer cells to achieve a long-term goal of providing clinicians an in vitro platform technology to test the efficacy of novel experimental anti-metastatic compounds.MethodsElectrospinning produces highly aligned, cell-adhesive nanofiber matrices by applying a strong electric field to a polymer-containing solution. The resulting fibrous microstructure and morphology closely resembles in vivo tumor microenvironments suggesting their use in analysis of migratory potentials of metastatic cancer cells. Additionally, a novel interface with a gel-based delivery system creates CXCL12 chemotactic gradients to enhance CXCR4-expressing cell migration.ResultsCellular dispersions of MCF-10A normal mammary epithelial cells or human breast cancer cells (MCF-7 and MDA-MB-231) seeded on randomly-oriented nanofiber exhibited no significant differences in total or net distance traveled as a result of the underlying topography. Cells traveled ~2-5 fold greater distances on aligned fiber. Highly-sensitive MDA-MB-231 cells displayed an 82% increase in net distance traversed in the presence of a CXCL12 gradient. In contrast, MCF-7 cells exhibited only 31% increase and MCF-10A cells showed no statistical difference versus control or vehicle conditions. MCF-10A cells displayed little sensitivity to CXCL12 gradients, while MCF-7 cells displayed early sensitivity when CXCL12 concentrations were higher. MDA-MB-231 cells displayed low relative expression levels of CXCR4, but high sensitivity resulting in 55-fold increase at late time points due to CXCL12 gradient dissipation.ConclusionsThis model could create clinical impact as an in vitro diagnostic tool for rapid assessment of tumor needle biopsies to confirm metastatic tumors, their invasiveness, and allow high-throughput drug screening providing rapid development of personalized therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-825) contains supplementary material, which is available to authorized users.

Abstract 2088: Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis

Abstract The FERM domain containing protein, Kindlin-3, has been recognized as a major regulator of integrin function in hematopoietic cells but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in kindlin-3 mRNA compared to non-neoplastic tissue by quantitative polymerize chain reaction. At the protein level, Kindlin-3 was found to be expressed abundantly in breast cancer tissue, and its expression increases with the advancing stage of tumor development. Mining of the Oncomine database (www.oncomine.com) confirmed this association: Kindlin-3 was in the top 3 % of gene products elevated in breast cancer (p< 10-12). When Kindlin-3 was over-expressed in a breast cancer cell line, primary tumor growth and lung metastasis increased by 2.5- and 3-fold, respectively when implanted into mice compared to cells expressing vector alone. Mechanistically, the Kindlin-cells displayed a 2.2-fold increase in Vascular Endothelial Growth Factor (VEGF) secretion and B1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor which promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production and knockdown of B1 integrins diminished Twist and VEGF production by Kindlin-3 cells while non-targeting si-RNA had no effect on expression of these gene products. The activation of this signaling cascade resulted in the enhancement of breast cancer cell invasion, and tumor angiogenesis and metastasis. Hence, our studies established the pathway in which cross-talk between Kindlin-3 and B1 integrins activates the nuclear translocation of TWIST, which leads to enhanced VEGF-A production and secretion, macrophage infiltration as well as activation of the Epithelial-to-mesenchymal-transition (EMT) program. The activation of this signaling cascade culminates in enhanced breast cancer cell migration, invasion and tumor angiogenesis, all of which are required for tumor progression and metastasis. In summary, by analysis of human tissue samples and in vitro and in vivo studies, we have identified a novel role for Kindlin-3 in the progression of breast cancer. To our knowledge, this is the first demonstration of the presence and implication of Kindlin-3 in breast cancer or any cancer. Citation Format: Khalid Sossey-Alaoui, Elzbieta Pluskota, Gangarao Davuluri, Katarzyna Bialkowska, Mitali Das, Daniel Lindner, Edward F. Plow. Kindlin-3 enhances breast cancer metastasis through upregulation of Twist-mediated tumor angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2088. doi:10.1158/1538-7445.AM2014-2088