Defective TGF-β Signaling Research Articles

TGFβ signaling in head and neck squamous cell carcinoma

Transforming growth factor beta (TGFβ) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFβ signaling maintains epithelial homeostasis, dysregulated TGFβ signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFβ signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFβ production by tumor epithelial cells with TGFβ signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFβ signaling in HNSCC.

Abstract 5357: TLR4-dependent Nanog+ cancer stem cells exhibit defective TGF-β signaling through IGF-Akt-Yap1 pathway

Abstract [Background] We have recently identified stem cell marker Nanog as a novel target gene of Toll-like receptor-4 (TLR4) in the genesis of CD133+/CD49f+ cancer stem cells (CSC) in HCV Ns5a Tg mice fed alcohol (PNAS, 1548, 2009). Defective TGF-β pathway as seen in mice haploinsufficient for β2 spectrin (β2SP), a key adaptor protein for nuclear translocation of Smads, leads to spontaneous development of HCC (PNAS, 2445, 2008). However, the relationship between pro-oncogenic TLR4 signaling and defective TGF-β tumor suppressor pathway in liver oncogenesis is unknown. [Aim] We aimed to: 1) validate the oncogenic activity of the putative CSCs isolated from the Ns5a Tg model; and 2) determine the role of defective TGF-β signaling in the TLR4-dependent CSCs. [Methods] The CSCs and CD133-/CD49f+ (control) cells from alcohol-fed Ns5a Tg mice were examined for growth in soft agar. Their lentiviral cDNA libraries were created and tested for transformation of the oval cell line and screening for oncogenic genes. GFP-labeled CSCs were injected in nude mice to monitor tumor growth upon repetitive LPS injection by GFP imaging. Interactions of TGF-β pathway and TLR4-dependent oncogenic activity of the CSCs were also examined. [Results] Anchorage-independent growth of the CSCs but not the control cells is accelerated by LPS. The CSCs but not the control cells formed growing tumors in nude mice injected repetitively with LPS, and knockdown of Nanog abrogates this growth. Transduction of lentiviral cDNA library from the CSCs but not the control cells, transforms the oval cell line as shown by colony formation in soft agar, and known (Yap1 and Stat3) and novel oncogenic genes (IGFBP) are isolated from these colonies. The CSCs are defective in phosphorylation and nuclear translocation of Smad3, and TGF-β response element promoter activity under TLR4 activation with LPS. Further inactivation of TGF-β pathway by β2SP knockdown accelerates both spontaneous and TLR4-induced growth and Yap1 and Stat3 expression by the CSCs. IGFBP overexpression induces phosphorylation of Akt and Yap1, leading to inhibition of Smad3 nuclear translocation and Sorafenib (FDA-approved liver cancer drug)-induced cell death by cytoplasmic retension of Smad3 through interaction between Smad3 and Yap1. [Conclusions] TLR4-dependent CSCs isolated from Ns5a Tg mice are tumorigenic and have heightened IGF-Akt-Yap1 signaling and defective TGF-β signaling associated with impaired Smad3 nuclear trasnlocation under TLR4 activation. Our results suggest heightened TLR4 activation in the Nanog+ CSCs is causally associated and interactive with defective TGF-β tumor suppressor pathway for their oncogenic activity of the Nanog+ CSCs through coupling between growth signal and inhibition of tumor-killing mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5357.

Abstract 3848: Abrogation of TGF-β signaling promotes mammary carcinoma progression by enhanced Th17 response

Abstract We have shown that TGF-β signaling in carcinoma cells can suppress the expression of chemokines that are responsible for the expansion and recruitment of myeloid derived suppressor cells (MDSCs) in tumor bearing mice. Using mice with conditional deletion of the type II TGF-β receptor gene (Tgfbr2) in mammary epithelial cells we have shown that derivative MMTV-PyMT induced carcinomas were better able to recruit tumor-promoting MDSCs than carcinomas with intact TGF-β signaling. This recruitment correlated with higher levels of Cxcl1/5 expression in tumors lacking intact TGF-β signaling. Cxcl1/5 signal through the CXCR2 receptor and it has been shown that CXCR2 signaling in vitro was able to promote migration and invasion of MDSC. IL-17 can up-regulate secretion of Cxc chemokines by epithelial cells. Differentiation of Th17 cells in the tumor microenvironment depends on the balance of cytokines such as TGF-β, IL-6 and IL-23, which are primarily produced by tumor infiltrating myeloid cells. Based on this information, we hypothesize that loss of TGF-β signaling in carcinoma cells results in increased tumor volume through an enhanced Th17 response. In vivo studies were performed using a spontaneous tumor model MMTV-PyMT and MMTV-PyMT/Tgfbr2 KO. On 7-9 days after tumor formation we isolated tumor tissue and prepared single cell suspensions which were used for flow cytometry analysis and for FACS sorting. CD4+ cells were isolated by CD4 Microbeads from Miltenyi Biotec. For in vitro studies we used immortalized epithelial cell lines which were prepared from PyMT tumors of MMTV-WT and MMTV-KO mice. Levels of cytokines were measured by ELISA. We found that MDSCs don't express CXCR2 in the lung and tumor tissue compared with MDSCs from the spleen and bone marrow. Carcinoma cells without Tgbr2 produce more CXCL1/5 and ELISA data showed that release of these two chemokines is upregulated by IL-17. Also, we have found increased expression of IL-17r on knockout epithelial cells and these cells are 4 times more sensitive to low concentrations of IL-17. In parallel, we found that TGF-β decreases secretion of CXCL1/5 from normal epithelial cells but not from cells without Tgbr2. Levels of TGF-β and IL-6, which are important for differentiation of Th17 cells, are increased in tumor tissue without Tgbr2. These data are paralleled with an increased number of Th-17 cells and level of IL-17. In addition, we discovered a secondary effect of IL-17 in increasing the suppressive function of MDSC's on T cells through the upregulation of Arg, IDO and COX2. We conclude that the tumor microenvironment of mammary carcinomas with defective TGF-β signaling in epithelial cells have increased levels of IL-17 which enhance tumor progression thru increased MDSCs migration and enhanced suppressive function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3848.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Defective TGF-β signaling sensitizes human cancer cells to rapamycin

mTOR, the mammalian target of rapamycin, is a critical target of survival signals in many human cancers. In the absence of serum, rapamycin induces apoptosis in MDA-MB-231 human breast cancer cells. However, in the presence of serum, rapamycin induces G(1) cell cycle arrest-indicating that a factor(s) in serum suppresses rapamycin-induced apoptosis. We report here that transforming growth factor-beta (TGF-beta) suppresses rapamycin-induced apoptosis in serum-deprived MDA-MB-231 cells in a protein kinase Cdelta (PKCdelta)-dependent manner. Importantly, if TGF-beta signaling or PKCdelta was suppressed, rapamycin induced apoptosis rather than G(1) arrest in the presence of serum. And, if cells were allowed to progress into S phase, rapamycin induced apoptosis in the presence of serum. BT-549 and MDA-MB-468 breast, and SW-480 colon cancer cells have defects in TGF-beta signaling and rapamycin induced apoptosis in these cells in the presence of either serum or TGF-beta. Thus, in the absence of TGF-beta signaling, rapamycin becomes cytotoxic rather than cytostatic. Importantly, this study provides evidence indicating that tumors with defective TGF-beta signaling--common in colon and pancreatic cancers--will be selectively sensitive to rapamycin or other strategies that target mTOR.

Transforming Growth Factor-βs and Vascular Disorders

Transforming growth factor-β (TGF-β) superfamily members, TGF-β and bone morphogenetic proteins (BMPs), are potent regulatory cytokines with diverse functions on vascular cells. They signal through heteromeric type I and II receptor complexes activating Smad-dependent and Smad-independent signals, which regulate proliferation, differentiation, and survival. They are potent regulators of vascular development and vessel remodeling and play key roles in atherosclerosis and restenosis, regulating endothelial, smooth muscle cell, macrophage, T cell, and probably vascular calcifying cell responses. In atherosclerosis, TGF-β regulates lesion phenotype by controlling T-cell responses and stimulating smooth muscle cells to produce collagen. It contributes to restenosis by augmenting neointimal cell proliferation and collagen accumulation. Defective TGF-β signaling in endothelial cells attributable to mutations in endoglin or the type I receptor ALK-1 leads to hereditary hemorrhagic telangiectasia, whereas defective BMP signaling attributable to mutations in the BMP receptor II has been associated with development of primary pulmonary hypertension. The development of mouse models with either cell type–specific or general inactivation of TGF-β/BMP signaling has started to reveal the importance of the regulatory network of TGF-β/BMP pathways in vivo and their significance for atherosclerosis, hereditary hemorrhagic telangiectasia, and primary pulmonary hypertension. This review highlights recent findings that have advanced our understanding of the roles of TGF-β superfamily members in regulating vascular cell responses and provides likely avenues for future research that may lead to novel pharmacological therapies for the treatment or prevention of vascular disorders.