Regulatory role of the transforming growth factor-β signaling pathway in the drug resistance of gastrointestinal cancers

Gastrointestinal (GI) cancer, including esophageal, gastric, and colorectal cancer, is one of the most prevalent types of malignant carcinoma and the leading cause of cancer-related deaths. Despite significant advances in therapeutic strategies for GI cancers in recent decades, drug resistance with various mechanisms remains the prevailing cause of therapy failure in GI cancers. Accumulating evidence has demonstrated that the transforming growth factor (TGF)-β signaling pathway has crucial, complex roles in many cellular functions related to drug resistance. This review summarizes current knowledge regarding the role of the TGF-β signaling pathway in the resistance of GI cancers to conventional chemotherapy, targeted therapy, immunotherapy, and traditional medicine. Various processes, including epithelial-mesenchymal transition, cancer stem cell development, tumor microenvironment alteration, and microRNA biogenesis, are proposed as the main mechanisms of TGF-β-mediated drug resistance in GI cancers. Several studies have already indicated the benefit of combining antitumor drugs with agents that suppress the TGF-β signaling pathway, but this approach needs to be verified in additional clinical studies. Moreover, the identification of potential biological markers that can be used to predict the response to TGF-β signaling pathway inhibitors during anticancer treatments will have important clinical implications in the future.     

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-β signaling pathway

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.     

Targeting the transforming growth factor-β signalling pathway in metastatic cancer

Transforming growth factor (TGF)-β signalling plays a dichotomous role in tumour progression, acting as a tumour suppressor early and as a pro-metastatic pathway in late-stages. There is accumulating evidence that advanced-stage tumours produce excessive levels of TGF-β, which acts to promote tumour growth, invasion and colonisation of secondary organs. In light of the pro-metastasis function, many strategies are currently being explored to antagonise the TGF-β pathway as a treatment for metastatic cancers. Strategies such as using large molecule ligand traps, reducing the translational efficiency of TGF-β ligands using antisense technology, and antagonising TGF-β receptor I/II kinase function using small molecule inhibitors are the most prominent methods being explored today. Administration of anti-TGF-β therapies alone, or in combination with immunosuppressive or cytotoxic therapies, has yielded promising results in the preclinical and clinical settings. Despite these successes, the temporal- and context-dependent roles of TGF-β signalling in cancer has made it challenging to define patient subgroups that are most likely to respond, and the therapeutic regimens that will be most effective in the clinic. Novel mouse models and diagnostic tools are being developed today to circumvent these issues, which may potentially expedite anti-TGF-β drug development and clinical application.     

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

ABSTRACT: INTRODUCTION: Transforming 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. METHODS: Fluorescently 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. RESULTS: Intravital 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. CONCLUSION: Fibroblast-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.     

Targeting transforming growth factor-β signaling in liver metastasis of colon cancer

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-β can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-β receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-β type I and type II receptors, LY2109761, inhibits TGF-β-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-β on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.     

Tumor-associated macrophage-derived transforming growth factor-β promotes colorectal cancer progression through HIF1-TRIB3 signaling

Tumor-associated macrophages (TAMs), one of the most common cell components in the tumor microenvironment, have been reported as key contributors to cancer-related inflammation and enhanced metastatic progression of tumors. To explore the underlying mechanism of TAM-induced tumor progression, TAMs were isolated from colorectal cancer patients, and the functional interaction with colorectal cancer cells was analyzed. Our study found that coculture of TAMs contributed to a glycolytic state in colorectal cancer, which promoted the stem-like phenotypes and invasion of tumor cells. TAMs produced the cytokine transforming growth factor-β to support hypoxia-inducible factor 1α (HIF1α) expression, thereby upregulating Tribbles pseudokinase 3 (TRIB3) in tumor cells. Elevated expression of TRIB3 resulted in activation of the β-catenin/Wnt signaling pathway, which eventually enhanced the stem-like phenotypes and cell invasion in colorectal cancer. Our findings provided evidence that TAMs promoted colorectal cancer progression in a HIF1α/TRIB3-dependent manner, and blockade of HIF1α signals efficiently improved the outcome of chemotherapy, describing an innovative approach for colorectal cancer treatment.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-β signaling pathway

Interstitial lung disease (ILD) is a serious side-effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) treatment. Therefore, it is necessary to study underlying mechanisms for the development of pulmonary fibrosis induced by EGFR-TKI and potential approaches to attenuate it. Metformin is a well-established and widely prescribed oral hypoglycemic drug, and has gained attention for its potential anticancer effects. Recent reports have also demonstrated its role in inhibiting epithelial-mesenchymal transition and fibrosis. However, it is unknown whether metformin attenuates EGFR-TKI-induced pulmonary fibrosis. The effect of metformin on EGFR-TKI-induced exacerbation of pulmonary fibrosis was examined in vitro and in vivo using MTT, Ki67 incorporation assay, flow cytometry, immunostaining, Western blot analysis, and a bleomycin-induced pulmonary fibrosis rat model. We found that in lung HFL-1 fibroblast cells, TGF-β or conditioned medium from TKI-treated lung cancer PC-9 cells or conditioned medium from TKI-resistant PC-9GR cells, induced significant fibrosis, as shown by increased expression of Collegen1a1 and α-actin, while metformin inhibited expression of fibrosis markers. Moreover, metformin decreased activation of TGF-β signaling as shown by decreased expression of pSMAD2 and pSMAD3. In vivo, oral administration of gefitinib exacerbated bleomycin-induced pulmonary fibrosis in rats, as demonstrated by HE staining and Masson staining. Significantly, oral co-administration of metformin suppressed exacerbation of bleomycin-induced pulmonary fibrosis by gefitinib. We have shown that metformin attenuates gefitinib-induced exacerbation of TGF-β or bleomycin-induced pulmonary fibrosis. These observations indicate metformin may be combined with EGFR-TKI to treat NSCLC patients.     

Mechanistic basis and clinical relevance of the role of transforming growth factor-β in cancer

Transforming growth factor-β (TGF-β) is a key factor in cancer development and progression. TGF-β can suppress tumorigenesis by inhibiting cell cycle progression and stimulating apoptosis in early stages of cancer progression. However, TGF-β can modulate cancer-related processes, such as cell invasion, distant metastasis, and microenvironment modification that may be used by cancer cells to their advantage in late stages. Corresponding mechanisms include angiogenesis promotion, anti-tumor immunity suppression, and epithelial-to-mesenchymal transition (EMT) induction. The correlation between TGF-β expression and cancer prognosis has also been extensively investigated. Results suggest that TGF-β pathway can be targeted to treat cancer; as such, the feasibility of this treatment is investigated in clinical trials.KEYWORDS : Transforming growth factor-β (TGF-β), neoplasms, prognosis, therapeutics     

Expression of adhesion molecules and transforming growth factor-β in pleomorphic carcinomas of the lung

Pleomorphic carcinoma (PC) of the lung consists of an epithelial component showing the histology of poorly differentiated non-small cell carcinoma of the lung and a sarcomatous component, that is more aggressive compared to non-small cell carcinoma of the lung. To determine the differences between an epithelial component of PC and poorly differentiated non-small cell carcinoma, the expression of adhesion molecules (E-cadherin, β-catenin and N-cadherin) and transforming growth factor-β (TGF-β) was compared immunohistochemically among 14 poorly differentiated adenocarcinomas of the lung (PDAs) and 14 PCs of the lung, with an epithelial component, showing the histology of PDA. Expression levels of E-cadherin and β-catenin were significantly lower in epithelial or sarcomatous components of PCs than in PDAs while that of TGF-β was significantly higher in epithelial components of PCs than in PDAs. No significant difference was found in incidences of the expression of these molecules between epithelial and sarcomatous components of PCs. No significant difference was noted in the expression level of N-cadherin among PDAs and epithelial and sarcomatous components of PCs. The present results showed that E-cadherin and β-catenin expression is reduced and TGF-β expression is increased in epithelial components of PCs with the same histology as PDA when compared to PDAs, suggesting that an epithelial component of PC is distinct from non-small cell carcinoma with the same histology.     

Inhibition of transforming growth factor-β signals suppresses tumor formation by regulation of tumor microenvironment networks

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial–mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks.     

Improving function of cytotoxic T-lymphocytes by transforming growth factor-β inhibitor in oral squamous cell carcinoma

Immunotherapy with immune-checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor-β (TGF-β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T-cells (Tregs) and cancer-associated fibroblasts and inhibiting the function of cytotoxic T-lymphocytes (CTLs) and natural killer cells. TGF-β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF-β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T-cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF-β suppressed the function of antigen-specific CTLs in the priming and effector phases in vitro. Additionally, TGF-β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8⁺ T-cell/Treg ratio and between TGFB1 mRNA expression and the Ki-67 expression in CD8⁺ T-cells, indicating that TGF-β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF-β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune-checkpoint inhibitors and TGF-β inhibitors, for OSCCs.     

On the role of transforming growth factor-β in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells

Background  

Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-β2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-β in retinoic acid-mediated growth inhibition in pancreatic cancer cells.     

Aberrant expression in multiple components of the transforming growth factor-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene-induced hamster buccal-pouch squamous-cell carcinogenesis

Transforming growth factor (TGF)-β1 signaling controls a plethora of cellular processes including tumorigenesis. The TGF-β1 ligand initiates signaling by binding to TGF-βreceptor II (TβRII) and allowing heterodimerization with TGF-βreceptor I (TβRI); thus, TβRI is phosphorylated by TβRII. After phosphorylation, Smad2 and Smad3 heterodimerize with Smad4, and this complex migrates to the nucleus to regulate the expression of specific target genes. However, Smad7 interrupts above signal transduction by preventing phosphorylation of Smad2 or Smad3. The objective of this study was to examine the TGF-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch squamous-cell carcinogenesis. Fifty 6-week-old male Syrian golden hamsters were divided into three experimental and two control groups (10 animals in each). Both pouches of each animal in the experimental groups were painted with 0.5% DMBA solution, and both pouches of each animal of one of the control groups were similarly treated with mineral oil; the other control group remained untreated throughout the experiment. Animals from three experimental groups were sacrificed at the end of 3rd, 9th, and 14th-weeks after DMBA treatment, respectively, and animals from two control groups were all sacrificed at 14th-weeks after the treatment. Immunohistochemical staining for TGF-β1, TβRI, TβRII, Smad2-4 and Smad7 were performed. RESULTS: A significant increase in the expression of Smad7 and significant decreases in the expression of TβRII, Smad 2, Smad3 and Smad4 were noted during hamster buccal-pouch carcinogenesis induced by DMBA. Our findings indicate that a disruption in TGF-β1-induced Smad signaling occurs as a result of aberrant expression of multiple components in the TGF-β1 signaling pathway during DMBA-induced hamster buccal-pouch carcinogenesis, leading to loss of TGF-β1 growth-suppressive effects on transformed pouch keratinocytes.