Michael W. Pickup

The roles of TGFβ in the tumour microenvironment

The influence of the microenvironment on tumour progression is becoming clearer. In this Review we address the role of an essential signalling pathway, that of transforming growth factor-β, in the regulation of components of the tumour microenvironment and how this contributes to tumour progression.

The extracellular matrix modulates the hallmarks of cancer

The extracellular matrix regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. The extracellular matrix serves not only as the scaffold upon which tissues are organized but provides critical biochemical and biomechanical cues that direct cell growth, survival, migration and differentiation and modulate vascular development and immune function. Thus, while genetic modifications in tumor cells undoubtedly initiate and drive malignancy, cancer progresses within a dynamically evolving extracellular matrix that modulates virtually every behavioral facet of the tumor cells and cancer‐associated stromal cells. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development, growth and dissemination of all human cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, dysregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. Here, we argue that biophysical and biochemical cues from the tumor‐associated extracellular matrix influence each of these cancer hallmarks and are therefore critical for malignancy. We suggest that the success of cancer prevention and therapy programs requires an intimate understanding of the reciprocal feedback between the evolving extracellular matrix, the tumor cells and its cancer‐associated cellular stroma.

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

The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor 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.

Stromally Derived Lysyl Oxidase Promotes Metastasis of Transforming Growth Factor-β–Deficient Mouse Mammary Carcinomas

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.

TGF-β Receptor II Loss Promotes Mammary Carcinoma Progression by Th17 Dependent Mechanisms

We report that IL-17 significantly increases the secretion of CXCL1 and CXCL5 from mammary carcinoma cells, which is downregulated by TGF-β through the type II TGF-β receptor (TβRII). Carcinoma cells with conditional knockout of TβRII (Tgfbr2(KO)) have enhanced sensitivity to IL-17a in the stimulation of chemokine secretion. During polyoma middle T (PyMT) induced tumor progression, levels of Th17 inducing cytokines TGF-β, IL-6, IL-23 were increased in PyMT/Tgfbr2(KO) tumors, which was associated with an increased number of Th17 cells. IL-17 increased the suppressive function of MDSCs on T cells through the upregulation of Arg, IDO, and COX2. Treatment of PyMT/Tgfbr2(KO) mice with anti-IL-17 Ab decreased carcinoma growth and metastatic burden. Analysis of human breast cancer transcriptome databases showed a strong association between IL-17 gene expression and poor outcome in lymph node positive, estrogen receptor negative or luminal B subtypes suggesting potential therapeutic approaches.

Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN–expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN–expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk

IntroductionTransforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.MethodsFluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.ResultsIntravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.ConclusionFibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

Tissue mechanics promote IDH1-dependent HIF1α-tenascin C feedback to regulate glioblastoma aggression.

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.

Inhibition of BMP signaling suppresses metastasis in mammary cancer.

Bone morphogenetic proteins (BMPs) are secreted cytokines/growth factors that have differing roles in cancer. BMPs are overexpressed in human breast cancers, but loss of BMP signaling in mammary carcinomas can accelerate metastasis. We show that human breast cancers display active BMP signaling, which is rarely downregulated or homozygously deleted. We hypothesized that systemic inhibition of BMP signaling in both the tumor and the surrounding microenvironment could prevent tumor progression and metastasis. To test this hypothesis, we used DMH1, a BMP antagonist, in MMTV.PyVmT expressing mice. Treatment with DMH1 reduced lung metastasis and the tumors were less proliferative and more apoptotic. In the surrounding tumor microenvironment, treatment with DMH1 altered fibroblasts, lymphatic vessels and macrophages to be less tumor promoting. These results indicate that inhibition of BMP signaling may successfully target both the tumor and the surrounding microenvironment to reduce tumor burden and metastasis.