Migration Of Mammary Epithelial Cells Research Articles

Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland

BackgroundAdvanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship.MethodsTo address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal–Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups.ResultsHistological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion.ConclusionsWe observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

Abstract B023: Oncostatin M induces HIF1A-dependent collective cell migration in mammary epithelial cells

Abstract Over 40,000 deaths due to breast cancer occur annually in the United States, and metastatic status is the most important determinant in prognosis. The metastatic potential of cancer cells is strongly influenced by interactions with the tumor microenvironment, which are mediated in large part by growth factors and cytokines that can modulate cellular phenotype. One such ligand is Oncostatin M (OSM), a cytokine produced by macrophages in breast cancer tumors. An emerging body of literature suggests that OSM is capable of shifting cancer cells into a metastatic cell state, however the mechanistic relationship between OSM signaling and cancer progression remains unclear. Gaining a better understanding of the molecular and microenvironmental factors that promote metastasis is critical for design of therapeutic strategies that can mitigate metastasis for patients with breast cancer. In previous studies, we found that MCF10A mammary epithelial cells exposed to OSM form multicellular clusters that migrate collectively, a process termed collective cell migration (CCM). This migratory phenotype has been linked to an increase in metastatic potential in breast cancer and provides an explanation for the association between OSM and cancer progression. Understanding the mechanism that underlies OSM-induced CCM may reveal how this phenotype contributes to metastasis. Toward this goal, we performed RNAseq and proteomic profiling of OSM-treated MCF10A cells, which revealed broad changes in molecular activity. Notably, OSM induced upregulation of transcription factors associated with migration (e.g., CEBPB, HIF1A) as well as a set of transcripts that contribute to cell junction formation (e.g., CDH3, GJA1). Network analysis revealed key nodes that may be associated with the phenotypic response, and functional screening with siRNA knock-down identified HIF1A as an essential mediator of OSM-induced CCM. Cellular activation of HIF1A is typically observed in response to hypoxia, a microenvironmental condition commonly found in human breast tumors, and patients with increased HIF1A expression have been found to be at a greater risk of metastasis. Our findings suggest that OSM released from tumor infiltrating myeloid cells may offer an alternative hypoxia-independent pathway to induce tumor cell upregulation of HIF1A and activation of metastatic cascades. Single-cell RNA-seq will be utilized to identify the transcriptional programs regulated by HIF1A in OSM-treated MCF10A cells. To further explore this hypothesis, we have performed additional experiments investigating the HIF1A signaling axis using organoids derived from MMTV-PyMT mice, cultured ex vivo in collagen gels. Initial results indicate that exposure to OSM increase cancer cell invasion in this mouse model of breast cancer. Future experiments will explore the relationship between HIF1A activation and MMTV-PyMT invasion and dissemination. Collectively, these results demonstrate an important connection between OSM, HIF1A, and CCM and help clarify the signaling consequences of OSM on cancer cell invasion. Citation Format: Ian C. McLean. Oncostatin M induces HIF1A-dependent collective cell migration in mammary epithelial cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B023.

The Role of Early Growth Response Family Members 1-4 in Prognostic Value of Breast Cancer.

Early growth response family members (EGRs), EGR1–4, have increasingly attracted attention in multiple cancers. However, the exact expression patterns and prognostic values of EGRs in the progress of breast cancer (BRCA) remain largely unknown. The mRNA expression and prognostic characteristics of EGRs were examined by the Cancer Genome Atlas (TCGA), Oncomine, and Kaplan-Meier plotter. Enrichment analyses were conducted based on protein-protein interaction (PPI) network. The Tumor Immune Estimation Resource (TIMER) database and MethSurv were further explored. The protein expression of EGR1 in BRCA was measured by western blotting and immunohistochemistry. The migration of mammary epithelial cells was determined by Boyden chamber assay. The transcriptional levels of EGR1/2/3 displayed significantly low expression in BRCA compared with that in normal tissues, while EGR4 was shown adverse expression pattern. Survival analysis revealed upregulated EGR1–4 were remarkably associated with favorable relapse-free survival (RFS). A close correlation with specific tumor-infiltrating immune cells (TIICs) and several CpG sites of EGRs were exhibited. Immunohistochemistry assays showed that the protein expression of EGR1 was remarkably downregulated in BRCA compared with that in paracancerous tissues. The migration of MCF10A mammary epithelial cells was increased after the silence of EGR1 by siRNA transfection. This study provides a novel insight to the role of EGRs in the prognostic value of BRCA.

Adverse Effects of Coumestrol and Genistein on Mammary Morphogenesis and Future Milk Production Ability of Mammary Epithelial Cells.

Isoflavones are a class of flavonoids present in legumes and are called phytoestrogens because of their estrogen-like activity. Endogenous estrogen is well known to regulate mammary gland morphogenesis during pregnancy. Each isoflavone also has different physiological activities. However, it is difficult to investigate the direct effect of each isoflavone in mammary morphogenesis in vivo because isoflavones are metabolized into different isoflavones by enteric bacteria. In this study, investigated are the direct influences of coumestrol, daidzein, and genistein on mammary structure development and future milk production ability of mammary epithelial cells (MECs) using in vitro culture models. Mouse MECs are cultured in Matrigel with basic fibroblast growth factor and epidermal growth factor to induce ductal branching and alveolar formation, respectively. Coumestrol and genistein inhibit ductal branching and alveolar formation by affecting the proliferation and migration of MECs with the induction of apoptosis. Daidzein hardly influences mammary structure development. Furthermore, pretreatment with coumestrol adversely affects the induction of milk production ability of MECs. These results suggest that each isoflavone differentially influences mammary morphogenesis and future milk production by affecting MEC behaviors. These results also suggest that the culture models are effective to study mammary epithelial morphogenesis in vitro.

Polarized Secretion of Extracellular Vesicles by Mammary Epithelia.

Extracellular vesicles (EVs) are secreted by many cell types and are increasingly investigated for their role in human diseases including cancer. Here we focus on the secretion and potential physiological function of non-pathological EVs secreted by polarized normal mammary epithelial cells. Using a transwell system to allow formation of epithelial polarity and EV collection from the apical versus basolateral compartments, we found that impaired secretion of EVs by knockdown of RAB27A or RAB27B suppressed the establishment of mammary epithelial polarity, and that addition of apical but not basolateral EVs suppressed epithelial polarity in a dose-dependent manner. This suggests that apical EV secretion contributes to epithelial polarity, and a possible mechanism is through removal of certain intracellular molecules. In contrast, basolateral but not apical EVs promoted migration of mammary epithelial cells in a motility assay. The protein contents of apical and basolateral EVs from MCF10A and primary human mammary epithelial cells were determined by mass spectrometry proteomic analysis, identifying apical-EV-enriched and basolateral-EV-enriched proteins that may contribute to different physiological functions. Most of these proteins differentially secreted by normal mammary epithelial cells through polarized EV release no longer showed polarized secretion in MCF10A-derived transformed epithelial cells. Our results suggest an essential role of EV secretion in normal mammary epithelial polarization and distinct protein contents and functions in apical versus basolateral EVs secreted by polarized mammary epithelia.

Abstract 4141: Role of interferon regulatory factor 5 in anti-tumor immunity: Orchestration of a functional tertiary lymphoid structure

Abstract The ability of breast cancer cells to metastasize is due to intrinsic intratumoral and extrinsic microenvironment changes. Many breast cancers are heavily infiltrated by immune cells and these infiltrating leukocytes are commonly observed around tumors in patients with invasive ductal carcinoma. Several recent studies show that the presence of tumor infiltrating leukocytes in human breast cancer tissue is associated with improved outcome, suggesting that the immune system is participating in the control and elimination of tumor cells. One way in which epithelial-derived cancers evade immunity is through dysregulation of cytokines and chemokines that “call in” specific immune cells to form a functional ectopic lymphoid-like structure termed a tertiary lymphoid structure (TLS) near the tumor. TLS are important for cell-mediated and humoral immunity and are extremely efficient immunological tools as the presentation of antigen occurs to both B and T cells. Although very little is known about the processes that drive TLS formation, data indicate that the presence of a TLS next to a solid cancer, including breast cancer, is a strong positive prognostic indicator. Recent data from our lab and others show that expression of the transcription factor interferon regulatory factor 5 (IRF5) in a human breast cancer molecular signature predicts increased survival and lower incidence of metastasis. We recently identified IRF5 as an important intrinsic regulator of mammary epithelial cell migration and metastasis. We also found that IRF5 within a breast tumor regulates the expression of cytokines/chemokines, such as CXCL13, that mediate immune cell trafficking. We previously reported that tumor-conditioned media (TCM) from IRF5-positive breast tumor cells contains CXCL13 and induces trafficking of CD19+CXCR5+ B cells and CD3+CD4+CXCR5+ T follicular helper-like (Tfh) cells to the TCM. CXCL13 is a key player in the formation of a TLS as it recruits in the specific cells and activates the B cells to produce immunoglobulins. Further analysis of immune cell trafficking to TCM from IRF5+ and IRF5- breast tumor cells reveals specificity for the trafficking of anti-tumorigenic B and T cell subsets to IRF5-positive TCM, such as transitional, memory, and plasmablast/plasma B cells, along with Th1 and Tfh cells, rather than pro-tumorigenic, immunosuppressive Bregs, Th2, and Tregs that were found to specifically migrate to IRF5-negative TCM. Analysis of formalin-fixed paraffin-embedded breast tumors by immunofluorescence microscopy revealed a significant correlation between IRF5 intratumoral expression and the presence of a TLS. Furthermore, preliminary data from a murine model of invasive mammary tumorigenesis supports a critical role for IRF5 in anti-tumor immunity. Together, these data implicate IRF5 in the generation of a functional TLS and as a critical mediator of anti-tumor immunity. Citation Format: Erica Pimenta, Ryan Weiss, Dan Li, Betsy Barnes. Role of interferon regulatory factor 5 in anti-tumor immunity: Orchestration of a functional tertiary lymphoid structure. [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 4141.

A conserved region within interferon regulatory factor 5 controls breast cancer cell migration through a cytoplasmic and transcription-independent mechanism.

BackgroundMigration of breast cancer cells out of a duct or lobule is a prerequisite for invasion and metastasis. However, the factors controlling breast cancer cell migration are not fully elucidated. We previously found that expression of the transcription factor interferon regulatory factor 5 (IRF5) is significantly decreased as a breast lesion progresses from a non-malignant stage to ductal carcinoma in situ and is eventually lost in ~80% of invasive ductal carcinomas examined. Human in vitro and murine in vivo models of invasive breast cancer confirmed an important role for IRF5 in regulating cell motility, invasion and/or metastasis; yet, the mechanism(s) by which this occurs is not known. Since IRF5 is primarily expressed in the cytoplasm of human mammary epithelial cells, we hypothesized that IRF5 may function in a transcription-independent manner to control intrinsic cell migration.ResultsA series of IRF5 deletion mutants were tested in cell motility, invasion and migration assays. A novel, conserved 10 amino acid domain was identified that regulates mammary epithelial cell migration. This region (∆115-125) is downstream of IRF5′s DNA binding domain and therefore when absent, retains IRF5 transcription activity but loses cell migration control. An IRF5 construct with a mutated nuclear localization signal further confirmed that IRF5 controls migration in a cytoplasmic and transcription-independent manner. Candidate cytoskeletal molecules were identified in MDA-MB-231 cells to interact with IRF5 by immunoprecipitation and mass spectrometry analysis. α6-tubulin was independently confirmed to interact with endogenous IRF5 in MCF-10A cells. Alterations in F-actin bundling after staining EV- and IRF5-231 cells with phalloidin suggests that IRF5 may control cell migration/motility through its interaction with cytoskeletal molecules that contribute to the formation of F-actin networks. Last and most notably, we found that IRF5′s control of cell migration is not restricted to mammary epithelial cells but functions in other epithelial cell types suggesting a more global role for this newly identified cell migratory function of IRF5.ConclusionsThese findings are significant as they identify a new regulator of epithelial cell migration and provide specific insight into the mechanism(s) by which loss of IRF5 expression in mammary epithelial cells contributes to breast cancer metastasis.

Signal transducer and activator of transcription 5a inhibited by pimozide may regulate survival of goat mammary gland epithelial cells by regulating parathyroid hormone-related protein

The signal transducer and activator of transcription 5a (Stat5a) modulates genes involved in proliferation and survival and plays pivotal roles in regulating the function of the mammary gland during pregnancy, lactation, and involution. However, there is little information about the effects of Stat5a on apoptosis of goat mammary gland epithelial cells (GMECs). In addition, parathyroid hormone-related protein (PTHrP) is a key regulator in cellular calcium transport, mammary gland development and breast tumor biology. This study aimed to explore the interaction of Stat5a and PTHrP in GMEC apoptosis. Quantitative real time PCR (qRT-PCR) suggested that Stat5a was predominantly expressed in the mammary gland, lung, liver and spleen of goats. Treating the GMECs with pimozide, an inhibitor of Stat5a that decreases Stat5a tyrosine phosphorylation, increased PTHrP levels in GMECs in a dose-dependent manner and simultaneously promoted apoptosis of the GMECs. We also demonstrated that PTHrP inhibition induced GMEC apoptosis and restrained cell proliferation. In contrast, PTHrP overexpression protected GMECs from pimozide- and calcium-induced apoptosis, and promoted cell proliferation. Furthermore, pimozide and CaCl2 downregulated the antiapoptotic protein Bcl-2 mRNA expression, respectively, and these effects were protected by PTHrP overexpression. Interestingly, we also found that Stat5a suppressed the expression of matrix metalloproteinase 9 (MMP-9) which can induce goat mammary epithelial cell migration, but PTHrP increased MMP-9 mRNA level. Thus, Stat5a may regulate GMEC survival by regulating the expression of PTHrP.

Bone metastasis and RANKL

The mice with a disruption of Rank or Rankl exhibit normal mammary development during puberty, but their mammary epithelium fails to proliferate and form lobuloalveolar structures during pregnancy, resulting in the death of newborns. Hormone replacement therapy is associated with an increased risk of breast cancer. Importantly, specific deletion of RANK in mammary epithelium cells prevents both the onset and progression of medroxyprogesterone acetate (MPA) -driven mammary cancer and impairs self-renewal of breast cancer stem cells. Furthermore, RANK is highly expressed in several cancer cells. Functionally, it has been shown that RANKL can stimulate the directed migration of mammary epithelial cells as well as prostate cancer and melanoma cells toward a source of RANKL. In an in vivo metastasis model, OPG reduced the tumor burden in bones and ameliorated clinical paralysis, but did not affect the frequency of the spread of metastases into other tissues. These findings show that the RANK/RANKL system is crucial for mammary development, breast tumorigenesis and bone metastasis.

Abstract 5191: Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility.

Abstract The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasive-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative feedback loop that restricts Gab2-dependent epithelial cell motility. Based on the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems. Citation Format: Xiaocui Zhang, Geneviève Lavoie, Loic Fort, Edward L. Huttlin, Joseph Tcherkezian, Jacob A. Galan, Haihua Gu, Steven P. Gygi, Sebastien Carreno, Philippe P. Roux. Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2013-5191

Abstract 2616: Rsu1 regulates MCF10A mammary epithelial cell migration and acini formation by binding to the IPP complex but it promotes spreading and Map kinase activation independent of IPP.

Abstract Tumor cell motility is dependent upon integrin activation, focal adhesion (FA) formation and dissolution, and cytoskeleton reorganization. The IPP complex [integrin linked kinase (ILK), PINCH1, Parvin] regulates FA formation via binding of ILK to B-integrin. ILK serves as an adaptor by binding PINCH1 and Parvin thus linking FAs and actin. Rsu1 binds to PINCH1 and also regulates stress kinase signaling. The IPP complex is elevated in fibrotic disease and many common tumors contributing to changes in adhesion and migration. However, the Rsu1 locus is deleted in subsets of gliomas and liver carcinomas and Rsu1 expression is altered by alternative splicing in other advanced tumors. To identify the functional contribution of Rsu1 to the IPP complex regulation of adhesion, migration, and survival, Rsu1 and PINCH1 were independently depleted from MCF10A mammary epithelial cells. siRNA-mediated depletion of either Rsu1 or PINCH1 altered the localization but the not the expression of FA proteins, inhibited adhesion, and blocked migration. Both Rsu1- and PINCH1-depleted cells exhibited disorganized actin stress fibers and constitutive phosphorylation of actin regulatory proteins. Reduction of either Rsu1 or PINCH1 blocked acini formation in 3D culture. However, only Rsu1 depletion blocked the phosphorylation of p38 Map kinase. Depletion of Rsu1 caused significant reduction in PINCH1, but PINCH1 depletion resulted in only a modest reduction in Rsu1 implying a function for Rsu1 in regulating the IPP complex through PINCH1 stabilization. Expression of wt-Rsu1 in Rsu1-depleted cells restored all Rsu1 functions. In contrast, reconstitution of Rsu1-depleted cells with an Rsu1 mutant that is non-binding for PINCH1 failed to induce FA formation or cell migration, suggesting that binding of Rsu1 to PINCH1 is required for Rsu1 regulation of adhesion and migration. Unexpectedly, this non-binding mutant restored both the level of PINCH1 and cell spreading as well as p38 MAP kinase phosphorylation. These data indicate that Rsu1 regulates the stability of PINCH1 by a mechanism other than direct binding and that Rsu1-PINCH1 association is not required to promote cell spreading. Rather it is required to promote formation of FAs and the turnover of FAs necessary for migration. These findings differentiate the critical functions that Rsu1, PINCH1 and IPP complex contribute to cell adhesion and migration and explain how, even when disassociated, members of this complex contribute to tumor cell migration. Supported by W81XWH-10-1-0224, Predoctoral fellowship from the CDMRP Breast Cancer Research Program. Citation Format: Reyda Gonzalez-Nieves, Mary Lou Cutler. Rsu1 regulates MCF10A mammary epithelial cell migration and acini formation by binding to the IPP complex but it promotes spreading and Map kinase activation independent of IPP. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2616. doi:10.1158/1538-7445.AM2013-2616

Gab2 Phosphorylation by RSK Inhibits Shp2 Recruitment and Cell Motility

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems.

Vaccaria segetalis extract can inhibit angiogenesis

Background: Study of antiangiogenic drugs is one of the cutting-edge fields in new antitumor drug development. However, system studies on Vaccaria segetalis extract as an inhibitor of tumor-induced neovascularization have not been published yet. In the current study, we performed a preliminary investigation of the antiangiogenic effect of Vaccaria segetalis extract on angiogenesis. Objective: We studied the inhibitory effect of Vaccaria segetalis extract on neovascularization. Methods: The effects of Vaccaria segetalis extract on in vitro proliferation, migration, and tube formation of human mammary endothelial cells (HMECs) were determined by 3 - (4,5) - dimethylthiahiazo (-z-y1) - 3, 5 -diphenytetrazoliumromide (MTT) assay and Matrigel plug assay. The antiangiogenic effect of Vaccaria segetalis extract was observed on the chick chorioallantoic-membrane (CAM) angiogenesis model. The CD31 expression on HMECs was detected by immunohistochemical staining. The therapeutic effect of Vaccaria segetalis extract on the metastasis of Lewis lung cancer was also observed in the mouse model. Results: The proliferation and migration of human mammary epithelial cells (HMECs) were inhibited significantly by Vaccaria segetalis extract in a dose-dependent manner (IC50 = 50 ?g/mL). Angiogenesis was also inhibited in the Matrigel-plug mouse model and the chick chorioallantoic-membrane model. The Vaccaria segetalis extract treatment reduced the expression of CD31 in HMECs and inhibited the growth and metastasis of Lewis lung carcinoma cells in the mouse model. Conclusion: The Vaccaria segetalis extract, which can obviously inhibit angiogenesis, could be developed as a promising new antiangiogenic drug. Keywords: Angiogenensis, CD31, endothelial cell, human mammary endothelial cells, Vaccaria segetalis

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

Specific β-containing Integrins Exert Differential Control on Proliferation and Two-dimensional Collective Cell Migration in Mammary Epithelial Cells

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration.

Abstract 324: The integrin-linked kinase (ILK) / Rictor complex is required for TGFβ-1-induced epithelial to mesenchymal transition (EMT)

Abstract Epithelial to mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase is a focal adhesion adaptor and serine/threonine protein kinase that regulates cell proliferation, survival, and EMT. Elucidating the molecular mechanisms necessary for the development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1 mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mTORC2 component Rictor as well as its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK-Rictor complex using siRNA-mediated knockdown. Furthermore, we identified formation of the ILK/Rictor complex formation in cancer but not normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells following TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 324. doi:1538-7445.AM2012-324

Role of the integrin-linked kinase (ILK)/Rictor complex in TGFβ-1-induced epithelial–mesenchymal transition (EMT)

Epithelial-to-mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase (ILK) is a focal adhesion adaptor and a serine/threonine protein kinase that regulates cell proliferation, survival and EMT. Elucidating the molecular mechanisms necessary for development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here, we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1-mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mammalian target of rapamycin complex 2 component Rictor and its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK/Rictor complex using small-interfering RNA-mediated knockdown. Furthermore, we identified ILK/Rictor complex formation in cancer but not in normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells after TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT.

Disruption of reelin signaling alters mammary gland morphogenesis

Reelin signaling is required for appropriate cell migration and ductal patterning during mammary gland morphogenesis. Dab1, an intracellular adaptor protein activated in response to reelin signaling, is expressed in the developing mammary bud and in luminal epithelial cells in the adult gland. Reelin protein is expressed in a complementary pattern, first in the epithelium overlying the mammary bud during embryogenesis and then in the myoepithelium and periductal stroma in the adult. Deletion in mouse of either reelin or Dab1 induced alterations in the development of the ductal network, including significant retardation in ductal elongation, decreased terminal branching, and thickening and disorganization of the luminal wall. At later stages, some mutant glands overcame these early delays, but went on to exhibit enlarged and chaotic ductal morphologies and decreased terminal branching: these phenotypes are suggestive of a role for reelin in spatial patterning or structural organization of the mammary epithelium. Isolated mammary epithelial cells exhibited decreased migration in response to exogenous reelin in vitro, a response that required Dab1. These observations highlight a role for reelin signaling in the directed migration of mammary epithelial cells driving ductal elongation into the mammary fat pad and provide the first evidence that reelin signaling may be crucial for regulating the migration and organization of non-neural tissues.

Gab2 regulates cytoskeletal organization and migration of mammary epithelial cells by modulating RhoA activation

The docking protein Gab2 is overexpressed in several human malignancies, including breast cancer, and is associated with increased metastatic potential. Here we report that Gab2 overexpression in MCF-10A mammary epithelial cells led to delayed cell spreading, a decrease in stress fibers and mature focal adhesions, and enhanced cell migration. Expression of a Gab2 mutant uncoupled from 14-3-3-mediated negative feedback (Gab2(2xA)) led to a more mesenchymal morphology and acquisition of invasive potential. Expression of either Gab2 or Gab2(2xA) led to decreased activation of RhoA, but only the latter increased levels of Rac-GTP. Expression of constitutively active RhoA in MCF-10A/Gab2 cells restored stress fibers and focal adhesions, indicating that Gab2 signals upstream of RhoA to suppress these structures. Mutation of the two Shp2-binding sites to phenylalanine (Gab2(ΔShp2)) markedly reduced the effects of Gab2 on cellular phenotype and RhoA activation. Expression of Gab2 or Gab2(2xA), but not Gab2(ΔShp2), promoted Vav2 phosphorylation and plasma membrane recruitment of p190A RhoGAP. Knockdown of p190A RhoGAP reversed Gab2-mediated effects on stress fibers and focal adhesions. The identification of a novel pathway downstream of Gab2 involving negative regulation of RhoA by p190A RhoGAP sheds new light on the role of Gab2 in cancer progression.

MLK3 is critical for breast cancer cell migration and promotes a malignant phenotype in mammary epithelial cells

The malignant phenotype in breast cancer is driven by aberrant signal transduction pathways. Mixed-lineage kinase-3 (MLK3) is a mammalian mitogen-activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK pathways. Depending on the cellular context, MLK3 has been implicated in apoptosis, proliferation, migration and differentiation. Here we investigated the effect of MLK3 and its signaling to MAPKs in the acquisition of malignancy in breast cancer. We show that MLK3 is highly expressed in breast cancer cells. We provide evidence that MLK3's catalytic activity and signaling to c-jun N-terminal kinase (JNK) is required for migration of highly invasive breast cancer cells and for MLK3-induced migration of mammary epithelial cells. Expression of active MLK3 is sufficient to induce the invasion of mammary epithelial cells, which requires AP-1 activity and is accompanied by the expression of several proteins corresponding to AP-1-regulated invasion genes. To assess MLK3's contribution to the breast cancer malignant phenotype in a more physiological setting, we implemented a strategy to inducibly express active MLK3 in the preformed acini of MCF10A cells grown in 3D Matrigel. Induction of MLK3 expression dramatically increases acinar size and modestly perturbs apicobasal polarity. Remarkably, MLK3 expression induces luminal repopulation and suppresses the expression of the pro-apoptotic protein BimEL, as has been observed in Her2/Neu-expressing acini. Taken together, our data show that MLK3-JNK-AP-1 signaling is critical for breast cancer cell migration and invasion. Our current study uncovers both a proliferative and novel antiapoptotic role for MLK3 in the acquisition of a malignant phenotype in mammary epithelial cells. Thus, MLK3 may be an important therapeutic target for the treatment of invasive breast cancer.