PDF - 96K, Legend for Supplemental Figures S1-S9.

PDF - 82K, Description of additional methods and procedures used in the study.

PDF - 1790K, ALCAM expression is upregulated in response to the loss of ECadherin (S1); TGFbeta-induced shedding (S2); ALCAM serum half-life in mice (S3); ALCAM shedding by prostate and breast cancer cells in vitro and in vivo (S4); Compound-32/ADAM17 inhibitor dosing in vivo (S5); TGFbeta-induced migration is abrogated by the loss of ALCAM in breast and lung cancer cells (S6); Representative TRAcP stained bone lesion created by PC3 shControl and shALCAM after intratibial injection into immunodeficient mice (S7); Immune response modulation and wound healing does not effect serum ALCAM (S8); Graph represents tumor cell proliferation over time as assessed by total cell counts over 72 hours (S9).

Supplementary Figures 1-7 from Gr-1+CD11b+ Myeloid Cells Tip the Balance of Immune Protection to Tumor Promotion in the Premetastatic Lung

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a severe fibrotic component that compromises treatment, alters the immune cell profile and contributes to patient mortality. It has been shown that early on in this process, dynamic changes in tissue biomechanics play an integral role in supporting pancreatic cancer development and progression. Despite the acknowledgement of its importance, a granular view of how stromal composition changes during the course of PDAC progression remains largely unknown. To mimic the quasi-mesenchymal phenotype and pronounced desmoplastic response observed clinically, we utilized a genetically engineered mouse model of PDAC that is driven by a KrasG12D mutation and loss of Tgfbr2 expression. Application of compartment resolved proteomics revealed that PDAC progression in this KTC model is associated with dynamic stromal alterations that are indicative of a wound healing program. We identified an early provisional matricellular fibrosis that was accompanied by markers of macrophage activation and infiltration, consistent with the inflammatory phase of wound healing. At 20 weeks a proliferative phenotype was observed with increased fibroblast markers, further collagen deposition and loss of basement membrane and native cell markers.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.

The cellular and noncellular components surrounding the tumor cells influence many aspects of tumor progression. Transforming growth factor β (TGF-β), bone morphogenetic proteins (BMPs), and activins have been shown to regulate the phenotype and functions of the microenvironment and are attractive targets to attenuate protumorigenic microenvironmental changes. Given the pleiotropic nature of the cytokines involved, a full understanding of their effects on numerous cell types in many contexts is necessary for proper clinical intervention. In this review, we will explore the various effects of TGF-β, BMP, and activin signaling on stromal phenotypes known to associate with cancer progression. We will summarize these findings in the context of their tumor suppressive or promoting effects, as well as the molecular changes that these cytokines induce to influence stromal phenotypes.

Abstract The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFβRIIKO), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF–blocking antibody decreased tumor growth. Anti–G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718–29. ©2017 AACR.

Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

Tumors are fibrotic and characterized by abundant, remodeled, and cross-linked collagen that stiffens the extracellular matrix stroma. The stiffened collagenous stroma fosters malignant transformation of the tissue by increasing tumor cell tension to promote focal adhesion formation and potentiate growth factor receptor signaling through kinase. Importantly, collagen cross-linking requires fibronectin (FN). Fibrotic tumors contain abundant FN, and tumor cells frequently up-regulate the FN receptor α5β1 integrin. Using transgenic and xenograft models and tunable two- and three-dimensional substrates, we show that FN-bound α5β1 integrin promotes tension-dependent malignant transformation through engagement of the synergy site that enhances integrin adhesion force. We determined that ligation of the synergy site of FN permits tumor cells to engage a zyxin-stabilized, vinculin-linked scaffold that facilitates nucleation of phosphatidylinositol (3,4,5)-triphosphate at the plasma membrane to enhance phosphoinositide 3-kinase (PI3K)-dependent tumor cell invasion. The data explain why rigid collagen fibrils potentiate PI3K activation to promote malignancy and offer a perspective regarding the consistent up-regulation of α5β1 integrin and FN in many tumors and their correlation with cancer aggression.

The fibrotic phenotype of pancreatic ductal carcinoma contributes to patient mortality. Nevertheless, anti-stromal therapies have had mixed results, suggesting there are multifaceted, anti and pro-tumorigenic roles for fibrosis. We found that the genotype of pancreatic ductal carcinomas tunes the tension of the malignant epithelium to mechanically prime the stroma and promote tumor progression through epithelial pSTAT3 and YAP. Kras pancreatic tumors in mice lacking epithelial TGF signaling developed a highly stiff, matricellular-enriched fibrosis and exhibited increased epithelial myosin activity with elevated cytokine, Jak, Rock, Fak and Yap signaling, and Stat3-dependent inflammation. Increasing pancreatic epithelial mechanosignaling accelerated Kras-dependent transformation that was accompanied by stromal stiffening and a matricellular-enriched fibrosis with high epithelial Yap and pStat3 activity. Elevating pStat3 increased tissue tension and matricellular-enriched fibrosis and activated Yap to accelerate Krasdependent pancreatic transformation. By contrast, epithelial Stat3 ablation attenuated pancreatic malignancy and reduced the matricellular-enriched fibrosis, stromal stiffening, epithelial contractility and Yap activation induced by Kras/TGF knockout. Tissue arrays revealed that the least differentiated human pancreatic tumors from patients with the shortest survival had matricellular-enriched fibrosis and a highly contractile, mesenchymal-like epithelium that lacked pSMAD and had elevated pSTAT3, YAP and SOX2. Our data underscore the importance of tumor genotype in tuning stromal-epithelial interactions and illustrate how tissue tension can force malignancy, drive tumor aggression and compromise patient survival. Introduction Pancreatic ductal adenocarcinomas (PDACs) are profoundly fibrotic and PDAC patients have a high mortality rate. PDAC fibrosis induces interstitial fluid pressure to disrupt blood vessel integrity and induce hypoxia that compromise drug delivery and promote disease aggression and therapy resistance . Consequently, considerable resources have been expended to develop strategies to reduce PDAC fibrosis. To this end, inhibition of stromal sonic hedgehog (SHH) signaling in a mouse model of PDAC significantly reduced fibrosis and increased intratumoral vascular density to increase drug update that, at least transiently, stabilized the disease. Similarly, reducing mouse pancreatic tumor hyaluronan, using hyaluronidase, or treating xenografted human pancreatic tumors with an angiotensin inhibitor to reduce tissue tension, decreased interstitial fluid pressure and normalized the vasculature to facilitate chemotherapy response. Yet, phase II clinical trials in PDAC patients treated with the SHH inhibitors IPI-926 or GDC-0449, or with a monoclonal antibody against the collagen cross-linking enzyme LOXL2, failed (NCT01472198). Experiments in mouse models of PDAC revealed that, while depletion of proliferating -smooth muscle actin ( SMA) positive stromal cells reduced fibrosis, the vasculature remained abnormal and the tumor, while smaller, was hypoxic and less differentiated, with accelerated mortality. Despite a frank reduction in fibrosis and enhancement of tissue vascularity, genetic ablation of SHH or treatment with a smoothened inhibitor induced mouse PDACs that were less differentiated and more, not less, aggressive. These data imply that the stroma can both promote and restrain tumor progression, and suggest stromal dependency may be context dependent. Whether such complexity could be explained by distinct tumor genotype stromal interactions or by the natural evolution of PDACs remains unclear. Malignant transformation of an epithelial tissue is universally accompanied by extracellular matrix (ECM) deposition and remodeling. Nevertheless, the extent and nature of the fibrosis and the responsiveness of the transformed epithelium to the desmoplastic ECM can vary widely across cancers, amongst tumor subtypes and even within one tumor . Indeed, the fibrotic response in patients with aggressive, treatment-resistant, quasi-mesenchymal PDACs (QM-PDA) is less prominent and tumor cells isolated from QM-PDA patients are only marginally anchorage-dependent for their growth and survival. By contrast, patients with classical PDACs have a better prognosis and classical PDACs are more differentiated, and tumor cells isolated from these cancers retain Ras dependence and express higher levels of cell adhesion molecules. Although the origins of the QM and classical histophenotypes have yet to be determined, PDAC development has been irrevocably linked to a handful of genetic modifications. Thus, pre-malignant pancreatic lesions (PanINs) frequently possess activating point mutations in the Kras proto-oncogene and PDAC progression correlates with either the genetic and/or epigenetic inactivation of the tumor suppressor genes p16INK4a ( 90%), p53 ( 75%) and SMAD4 (DPC4, 55%). Consistently, genetically-engineered mouse models (GEMMs) in which an activated Kras is expressed in the pancreatic ductal epithelium develop PanINS and when combined with deletion of a single allele of p53, p16INK4a, Smad4 or Tgfbr2 develop PDACs . Of these genetic modifications, mice with combined Kras mutations and Tgfbr2 deletion are very aggressive and exhibit a mesenchymal-like phenotype following stromal ablation. Moreover, the human mesenchymal-like PDAC phenotype most frequently associates with aberrant TGF signaling in the epithelium. These findings imply that distinct genotypes may dictate unique stromal-epithelial phenotypes. Importantly, as mouse PDACs develop they also increase expression of mesenchymal-like features, as do patients with recurrent PDACs, and ablation of proliferating alpha smooth muscle actin ( SMA) positive cells in Kras/p53 mouse PDAC permits the expansion of mesenchymal-like, aggressive tumors . These observations suggest that the epithelium likely evolves over time towards a less stromallydependent phenotype and imply that this evolution may be linked to the engagement of pathways that promote a mesenchymal-like transition. High grade PDACs express more Sex-determining region Y (SRY)-Box2 (SOX2), a transcription factor that drives an epithelial-to-mesenchymal transition (EMT), with elevated Sox2 levels in PDACs linked to poor PDAC patient prognosis. Poorly differentiated, mesenchymal-like PDACs (QM-PDACs) also express higher levels and activity of the Hippo transcription factor Yes-associated protein 1 (YAP), and YAP directly induces SOX2 and an EMT . These findings suggest QM-PDACs may arise through elevated YAP and SOX2 activity. YAP is exquisitely sensitive to mechanical stimuli such that cells interacting with a stiff ECM activate more ROCK to increase nuclear YAP and induce YAP-dependent gene expression. Importantly, PDACs are mechanically-activated tumors composed of a progressively stiffened ECM and high interstitial pressure. Thus, the elevated tissue mechanics mediated by the stiffened tissue stroma and high interstitial pressure could eventually activate YAP to drive tumor aggression and induce an EMT. Yet, many oncogenes also induce tissue tension by increasing Rock-dependent contractility. Indeed, the majority of PDACs have activated Kras, and Kras activity, per se, increases ROCK to drive cell contractility which, in turn, induces ECM remodeling and stiffening to drive integrin-dependent mechanosignaling and malignant transformation. It is therefore also equally plausible that the genotype of the pancreatic tumor epithelium additionally elevates tissue tension to drive tumor progression. Here, we examined human pancreatic tumor tissue of differing grades and survival, and exploited a series of PDAC GEMMs to explore the relationship between tumor genotype, stromal-epithelial interactions and tissue tension in PDAC progression and aggression. Results Human PDAC aggression is characterized by low epithelial TGF signaling and high myosin activity PDACs are fibrotic, and contain abundant fibrillar collagen.Yet, recent findings suggest collagen abundance may associate with better, not worse, patient prognosis and that high collagen content correlates with a more differentiated PDAC phenotype . Nevertheless, high fibrillar collagen has repeatedly been implicated in PDAC aggression and treatment resistance. To address this discrepancy, we constructed a gene list of major fibrillar collagens (Supplementary Table 1) and used this signature to interrogate a publically-available NCBI GEO agilent gene expression data set (GSE21501) consisting of 67 PDAC patients between the ages of 40 and 65. We could find no statistical differences in the combined expression levels of the major fibrillar collagens and patient survival. However, when we created a median centroid value representing an average high versus low Col1 2, Col2 1 and Col4 1 combined expression score for each patient and graphed each of the groups over time, a Cox-Mantel Logrank analysis revealed that patients with high levels of these fibrillar collagens had a reduced overall survival, as illustrated by Kaplan-Meier survival curves (log rank p 0.04; Fig. 1a). These data suggest elevated gene expression of specific fibrillar collagens can predict poorer patient outcome. Collagens are subjected to a plethora of posttranslational modifications that can modify their organization and mechanical properties. Therefore, to more directly investigate the relationship between fibrillar collagens and PDAC tumor phenotype, we secured PDAC tissue arrays from US Biomax representing a total of 68 patients between the ages of 23 and 78 with well (n=19), moderately (n=23) and poorly differentiated (n=26) tumors. A UCSF pathologist confirmed tumor grade using H&E stained sections. Thereafter, serial sections were stained for total collagen using Massons Trichrome and assessed for fibrillar collagen levels, organization and diameter using polarized imaging

Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status.

The extracellular matrix (ECM) is a guiding force that regulates various developmental stages of the breast. In addition to providing structural support for the cells, it mediates epithelial-stromal communication and provides cues for cell survival, proliferation, and differentiation. Perturbations in ECM architecture profoundly influence breast tumor progression and metastasis. Understanding how a dysregulated ECM can facilitate malignant transformation is crucial to designing treatments to effectively target the tumor microenvironment. Here, we address the contribution of ECM mechanics to breast cancer progression, metastasis, and treatment resistance and discuss potential therapeutic strategies targeting the ECM.

The TGF-β pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-β pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-β type III receptor (TGFBR3) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TβRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TβRIII (sTβRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTβRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTβRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TβRIII has distinct roles not only in cancer-associated fibroblasts but that sTβRIII has distinct paracrine functions in the tumor microenvironment.

Abstract Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality. Nevertheless, anti-stromal therapies for pancreatic cancer patients have had mixed results, suggesting there are multifaceted, anti and pro-tumorigenic roles of fibrosis in tumor pathogenesis. We found that human PDACs lacking epithelial TGFβ activity have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis that associates with high epithelial tension and shorter patient survival. Using several Kras-driven mouse models, we found that both the loss of TGFβ signaling and elevated β1 integrin mechanosignaling engage a positive feedback loop whereby Stat3 signaling modulates pancreatic cancer malignancy by increasing matricellular fibrosis and tissue tension. By contrast, epithelial Stat3 ablation attenuated pancreatic malignancy by reducing the stromal stiffening and epithelial contractility induced by loss of TGFβ signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial actomyosin tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 as a key driver of the phenotype. These data illustrate how tumor genotype can directly tune tissue mechanics and support the development of genotype-driven, anti-stromal therapies targeting PDAC. Citation Format: Hanane Laklai, Yekaterina Miroshnikova, Michael Pickup, Eric Collisson, Kim Grace, Alex Barrett, Ryan Hill, Johnathon Lakins, David Schlaepfer, Janna Mouw, Valerie LeBleu, Sergey Novitskiy, Julie Johansen, Valeria Poli, Rahgu Kalluri, Laura Wood, Matthias Hebrok, Kirk Hansen, Harold Moses, Valerie Weaver.{Authors}. Genotype tunes PDAC tension to induce matricellular-fibrosis and tumor aggression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A50.

Impaired TGF-β signaling due to SMAD4 mutation in PDAC tumors initiates a STAT3-dependent signaling cascade that leads to increased stromal stiffening and disease progression. Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

Abstract Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor β (TGFβ) superfamily. BMP ligands have recently been shown to be overexpressed in human breast cancers. Normal and cancerous breast display active BMP signaling as indicated by phosphorylated Smads 1, 5 and 9. We combined mice expressing the MMTV.PyVmT oncogene with mice lacking BMPR1a in mammary epithelial cells and found this deletion resulted in delayed tumor onset and extended survival significantly (p-value = <0.001). We examined the histopathology of BMPR1a knockout (cKO) tumors and found a striking loss of epithelial characteristics combined with stromal desmoplasia. Immunofluorescence staining revealed that cKO tumors co-expressed Keratin 5 and mesenchymal cell markers such as Vimentin. This indicated that epithelial-to-mesenchymal (EMT)-like transitions were occurring in cKO tumors. We performed microarray analysis on these tumors and found changes that supported EMT-like changes with increased Snail mRNA expression. We established primary tumor cell lines and found that BMPR1a cKO had slower growth in vitro and upon in vivo implantation. Additionally, cKO tumor cells had reduced migration yet not invasion in vitro. We next analyzed human databases from TCGA and survival data from microarrays to confirm BMPR1a tumor promoting functions and found high BMPR1a gene expression correlated with worse survival regardless of molecular breast cancer subtype. In conclusion, we found that loss of the BMPR1a impairs tumor formation and progression and does not exhibit tumor suppressive functions in mouse and human breast cancer. Citation Format: Laura D. Hover, Michael W. Pickup, Agnieszka E. Gorska, Anna Chytil, Yan Guo, Sergey V. Novitskiy, Harold L. Moses, Philip Owens. Deletion of the BMP receptor BMPR1a results in EMT and impairs mammary gland tumor formation and metastasis. [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 4083. doi:10.1158/1538-7445.AM2015-4083

Introduction: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a Mesenchymal-Stem Like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-β) pathway-associated genes relative to other subtypes, including the TGF-β receptor type III (TβRIII). We hypothesize that TβRIII is tumor promoter in mesenchymal-stem like TNBC cells. Methods: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TβRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TβRIII (TβRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TβRIII expression patterns across all TNBC subtypes. Results: TβRIII was the most differentially expressed TGF-β signaling gene in the MSL subtype. Silencing TβRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TβRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TβRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TβRIII-KD. Stable knockdown of integrin-α2 in TβRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. Conclusions: We have found that TβRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TβRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TβRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TβRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity. Citation Format: Bojana Jovanovic, J Scott Beeler, Michael W Pickup, Anna Chytil, Agnieszka E Gorska, William J Ashby, Brian D Lehmann, Andries Zijlstra, Jennifer A Pietenpol, Harold L Moses. TGF-β receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-04.