Transforming growth factor-β (TGF-β) is known as a multifunctional cytokine, and it exhibits four major biological activities; i.e. growth inhibition, extracellular matrix deposition, induction of epithelial-mesenchymal transition (EMT), and immunosuppression. Through these bioactivities, TGF-β elicits both pro-tumorigenic and anti-tumorigenic effects during progression of cancer. We present here our recent findings on TGF-β signaling in progression of cancer, focusing on induction of EMT by TGF-β.
　　TGF-β induces EMT through activation of Smad and non-Smad signaling pathways. Inhibition of TGF-β signaling by an inhibitory Smad, Smad7, results in prevention of cancer metastasis in mouse models. EMT-transcription factors (EMT-TFs), including Snail, Slug, ZEB1, and ZEB2, play critical roles in TGF-β-induced EMT, and activation of K-Ras is involved in the induction of EMT in cancer cells. Mesenchymal-epithelial transition (MET) is the inverse process of EMT; TGF-β-mediated EMT is a crucial event in the invasion of cancer, while MET occurs when cancer cells form metastatic foci at distant organs.
　　Snail is a key regulator of EMT, which is induced by TGF-β in cancer cells. Induction of Snail by TGF-β is dependent on active Ras signals, and knockdown of KRAS suppresses the Snail induction by TGF-β in pancreatic cancer PANC1 cells. Overexpression of active K-Ras into HeLa cells results in strong induction of Snail expression by TGF-β, while many other direct targets of TGF-β, including SMAD7 and SERPINE1, are not increased by Ras signaling. These observations indicate that Ras and TGF-β signaling cooperate in the induction of Snail and progression of the EMT process. Although mutations in the SMAD4/DPC4 gene can be found in ~50% of pancreatic cancer patients, KRAS mutations occur in earlier phase of ~90% of pancreatic cancer patients; therefore, TGF-β may mainly play pro-tumorigenic functions during progression of pancreatic cancer.
　　Thyroid transcription factor-1 (TTF-1/Nkx2-1) is a tissue-specific transcription factor in lung epithelial cells, and is expressed in lung cancer. TTF-1-positive lung adenocarcinoma patients show better prognosis than TTF-1-negative patients. TTF-1 inhibits the TGF-β-induced EMT, induces the expression of E-cadherin, and restores epithelial phenotype in lung adenocarcinoma cells. TTF-1 disrupts the nuclear Smad3/Smad4 complex without affecting the nuclear localization of phospho-Smad3. This effect of TTF-1 is accompanied by down-regulation of TGF-β target genes, including SNAI1 and SNAI2 (encoding Snail and Slug, respectively). Knockdown of TTF-1 expression enhances TGF-β-mediated EMT. Thus, TTF-1 exhibits a tumor-suppressive effect through attenuation of the pro-tumorigenic activity of TGF-β. TTF-1 represses the synthesis of TGF-β2 in A549 cells, while TGF-β in turn suppresses the expression of TTF-1, suggesting that modulation of TTF-1 expression is a potentially interesting strategy for treatment of lung adenocarcinoma.
　　EMT is known to be a reversible biological process. Chronic TGF-β treatment induces stabilized EMT in breast carcinoma cells. In the stabilized EMT, breast carcinoma cells show stem cell-like properties and chemoresistance. Chronic TGF-β treatment activates mTOR signaling, and inhibition of mTOR signaling results in repression of stem cell-like properties and chemoresistance.
　　We have recently developed a tissue-clearing technology, termed CUBIC-based cancer (CUBIC-Cancer) analysis, which allows whole-body/organ imaging of cancer. By CUBIC-Cancer analyses, spatiotemporal quantification of metastatic cancer progression at single-cell resolution is possible. Importance of EMT in the invasion and intravasation of cancer cells is well-known; however, involvement of EMT in the extravasation of cancer cells has not been clearly demonstrated. Immunodeficient mice were intravenously injected with A549 lung adenocarcinoma cells pre-treated with or without TGF-β. CUBIC-Cancer analyses clearly showed that TGF-β treatment significantly increased the number of metastatic foci from 1 to 14 days, demonstrating that EMT promotes not only intravasation but also cell survival and extravasation of cells at metastatic sites.
Recent Publications
1. Miyazono K, Katsuno Y, Koinuma D, Ehata S, Morikawa M. Intracellular and extracellular TGF-β signaling in cancer: some recent topics. Front Med, 2018, 12, 387-411.
2. Katsuno Y, Meyer DS, Zhang Z, Shokat KM, Akhurst RJ, Miyazono K, Derynck R. Chronic TGF-β exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition. Sci Signal, 2019, 12, pii: eaau8544.
3. Kubota SI, Takahashi K, Nishida J, Morishita Y, Ehata S, Tainaka K, Miyazono K, Ueda HR. Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep, 2017, 20, 236-250.