Search

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Hepatocellular carcinoma (HCC) has a high rate of recurrence and poor prognosis, even after curative surgery. Multikinase inhibitors have been applied for HCC patients, but their effect has been restricted. This study aims to clarify the clinical impact of SUV420H1/KMT5B, one of the methyltransferases for histone H4 at lysine 20, and elucidate the novel mechanisms of HCC progression. We retrospectively investigated SUV420H1 expression using HCC clinical tissue samples employing immunohistochemical analysis (n = 350). We then performed loss-of-function analysis of SUV420H1 with cell cycle analysis, migration assay, invasion assay and RNA sequence for Gene Ontology (GO) pathway analysis in vitro, and animal experiments with xenograft mice in vivo. The SUV420H1-high-score group (n = 154) had significantly poorer prognosis for both 5-year overall and 2-year/5-year disease-free survival than the SUV420H1-low-score group (n = 196) (p < 0.001 and p < 0.05, respectively). The SUV420H1-high-score group had pathologically larger tumor size, more tumors, poorer differentiation, and more positive vascular invasion than the SUV420H1-low-score group. Multivariate analysis demonstrated that SUV420H1 high score was the poorest independent factor for overall survival. SUV420H1 knockdown could suppress cell cycle from G1 to S phase and cell invasion. GO pathway analysis showed that SUV420H1 contributed to cell proliferation, cell invasion, and/or metastasis. Overexpression of SUV420H1 clinically contributed to poor prognosis in HCC, and the inhibition of SUV420H1 could repress tumor progression and invasion both in vitro and in vivo; thus, further analyses of SUV420H1 are necessary for the discovery of future molecularly targeted drugs., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro ; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24 , but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020, Spandidos Publications.)

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks., (© 2022. The Author(s).)

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β (TGF-β) family, are multifunctional cytokines. BMPs have a broad range of functions, and abnormalities in BMP signaling pathways are involved in cancer progression. BMPs activate the proliferation of certain cancer cells. Malignant phenotypes of cancer cells, such as increased motility, invasiveness, and stemness, are enhanced by BMPs. Simultaneously, BMPs act on various cellular components and regulate angiogenesis in the tumor microenvironment. Thus, BMPs function as pro-tumorigenic factors in various types of cancer. However, similar to TGF-β, which shows both positive and negative effects on tumorigenesis, BMPs also act as tumor suppressors in other types of cancers. In this article, we review important findings published in the recent decade and summarize the pro-oncogenic functions of BMPs and their underlying mechanisms. The current status of BMP-targeted therapies for cancers is also discussed., Competing Interests: SE and KM were partly supported by Eisai, Co., Ltd., (Copyright © 2022 Ehata and Miyazono.)

We developed an in vivo serial passaging model for renal cancer with orthotopic renal subcapsular inoculation. We detail the procedures for renal subcapsular inoculation of cancer cells in mice, followed by in vivo and ex vivo bioluminescence imaging, tumor-bearing kidney dissociation, and in vivo passaging. This protocol is capable of reproducing the coevolution between cancer cells and the primary tumor microenvironment. It enables us to unveil the systemic dynamics of metastasis and develop a therapeutic strategy for metastatic renal cancer. For complete details on the use and execution of this protocol, please refer to Nishida et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Tissue-clearing technology is an emerging imaging technique currently utilized not only in neuroscience research but also in cancer research. In our previous reports, tissue-clearing methods were used for the detection of metastatic tumors. Here, we showed that the cell cycles of primary and metastatic tumors were visualized by tissue-clearing methods using a reporter system. First, we established cancer cell lines stably expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) reporter with widely used cancer cell lines A549 and 4T1. Fluorescence patterns of the Fucci reporter were investigated in various tumor inoculation models in mice. Interestingly, fluorescence patterns of the Fucci reporter of tumor colonies were different between various organs, and even among colonies in the same organs. The effects of antitumor drugs were also evaluated using these Fucci reporter cells. Of the three antitumor drugs studied, 5-fluorouracil treatment on 4T1-Fucci cells resulted in characteristic fluorescent patterns by the induction of G 2 /M arrest both in vitro and in vivo. Thus, the combination of a tissue-clearing method with the Fucci reporter is useful for analyzing the mechanisms of cancer metastasis and drug resistance., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG + ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor β and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG + population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG - population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG + and ENG - cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG + cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG + cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

ETS homologous factor (EHF) belongs to the epithelium-specific subfamily of the E26 transformation-specific (ETS) transcription factor family. Currently, little is known about EHF's function in cancer. We previously reported that ETS1 induces expression of the ZEB family proteins ZEB1/δEF1 and ZEB2/SIP1, which are key regulators of the epithelial-mesenchymal transition (EMT), by activating the ZEB1 promoters. We have found that EHF gene produces two transcript variants, namely a long form variant that includes exon 1 (EHF-LF) and a short form variant that excludes exon 1 (EHF-SF). Only EHF-SF abrogates ETS1-mediated activation of the ZEB1 promoter by promoting degradation of ETS1 proteins, thereby inhibiting the EMT phenotypes of cancer cells. Most importantly, we identified a novel point mutation within the conserved ETS domain of EHF, and found that EHF mutations abolish its original function while causing the EHF protein to act as a potential dominant negative, thereby enhancing metastasis in vivo. Therefore, we suggest that EHF acts as an anti-EMT factor by inhibiting the expression of ZEBs, and that EHF mutations exacerbate cancer progression.

Tissue clearing is one of the most powerful strategies for a comprehensive analysis of disease progression. Here, we established an integrated pipeline that combines tissue clearing, 3D imaging, and machine learning and applied to a mouse tumour model of experimental lung metastasis using human lung adenocarcinoma A549 cells. This pipeline provided the spatial information of the tumour microenvironment. We further explored the role of transforming growth factor-β (TGF-β) in cancer metastasis. TGF-β-stimulated cancer cells enhanced metastatic colonization of unstimulated-cancer cells in vivo when both cells were mixed. RNA-sequencing analysis showed that expression of the genes related to coagulation and inflammation were up-regulated in TGF-β-stimulated cancer cells. Further, whole-organ analysis revealed accumulation of platelets or macrophages with TGF-β-stimulated cancer cells, suggesting that TGF-β might promote remodelling of the tumour microenvironment, enhancing the colonization of cancer cells. Hence, our integrated pipeline for 3D profiling will help the understanding of the tumour microenvironment.

Pancreatic cancer is one of the cancers with the poorest prognosis, with a 5-year survival rate of approximately 5-10%. Thus, it is urgent to identify molecular targets for the treatment of pancreatic cancer. Using serial transplantations in a mouse pancreatic orthotopic inoculation model, we previously produced highly malignant pancreatic cancer sublines with increased tumor-forming abilities in vivo. Here, we used these sublines to screen molecular targets for the treatment of pancreatic cancer. Among the genes with increased expression levels in the sublines, we focused on those encoding cell surface receptors that may be involved in the interactions between cancer cells and the tumor microenvironment. Based on our previous RNA-sequence analysis, we found increased expression levels of neurotensin (NTS) receptor 1 (NTSR1) in highly malignant pancreatic cancer sublines. Furthermore, re-analysis of clinical databases revealed that the expression level of NTSR1 was increased in advanced pancreatic cancer and that high NTSR1 levels were correlated with a poor prognosis. Overexpression of NTSR1 in human pancreatic cancer cells Panc-1 and SUIT-2 accelerated their tumorigenic and metastatic abilities in vivo. In addition, RNA-sequence analysis showed that MAPK and NF-κB signaling pathways were activated upon NTS stimulation in highly malignant cancer sublines and also revealed many new target genes for NTS in pancreatic cancer cells. NTS stimulation increased the expression of MMP-9 and other pro-inflammatory cytokines and chemokines in pancreatic cancer cells. Moreover, the treatment with SR48692, a selective NTSR1 antagonist, suppressed the activation of the MAPK and NF-κB signaling pathways and induction of target genes in pancreatic cancer cells in vitro, while the administration of SR48692 attenuated the tumorigenicity of pancreatic cancer cells in vivo. These findings suggest that NTSR1 may be a prognostic marker and a molecular target for pancreatic cancer treatment., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Tissue-clearing technologies have developed rapidly in the past decade, especially for use in neuroscience research. We previously reported that CUBIC, which is one tissue-clearing method, is useful for applications in cancer research. CUBIC cancer analysis can be used to detect cancer metastasis with single-cell resolution at whole mouse body/organ level. This approach can also analyze the tumor characteristics with high-quality 3D images. Here, we describe a detailed CUBIC cancer protocol from tissue clearing, capturing 3D images and post-immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Kubota et al. (2017)., Competing Interests: K.M. and S.E. were partly supported by Eisai, Co., Ltd. H.R.U. is a co-inventor on patent applications covering the CUBIC reagents (PCT/JP2014/070618 [pending], patent applicant is RIKEN; PCT/JP2017/016410 [pending], patent applicant is RIKEN) and CUBIC-HV reagents (PCT/JP2020/ 31840 [pending], patent applicant is CUBICStars) and is a co-founder of CUBICStars. This work was partly done in collaboration with Olympus Corporation., (© 2020 The Author(s).)

Advanced clear cell renal cell carcinoma (ccRCC) frequently causes systemic inflammation. Recent studies have shown that cancer cells reshape the immune landscape by secreting cytokines or chemokines. This phenotype, called cancer-cell-intrinsic inflammation, triggers a metastatic cascade. Here, we identified the functional role and regulatory mechanism of inflammation driven by advanced ccRCC cells. The inflammatory nature of advanced ccRCC was recapitulated in a preclinical model of ccRCC. Amplification of cancer-cell-intrinsic inflammation during ccRCC progression triggered neutrophil-dependent lung metastasis. Massive expression of inflammation-related genes was transcriptionally activated by epigenetic remodelling through mechanisms such as DNA demethylation and super-enhancer formation. A bromodomain and extra-terminal motif inhibitor synchronously suppressed C-X-C-type chemokines in ccRCC cells and decreased neutrophil-dependent lung metastasis. Overall, our findings provide insight into the nature of inflammatory ccRCC, which triggers metastatic cascades, and suggest a potential therapeutic strategy.

Thyroid transcription factor-1 (TTF-1, encoded by the NKX2-1 gene) is highly expressed in small-cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome-wide distributions of TTF-1 binding regions and the transcriptional programs regulated by TTF-1 between NCI-H209 (H209), a human SCLC cell line, and NCI-H441 (H441), a human LADC cell line, using chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq). TTF-1 binding regions in H209 and H441 cells differed by 75.0% and E-box motifs were highly enriched exclusively in the TTF-1 binding regions of H209 cells. Transcriptome profiling revealed that TTF-1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF-1 and achaete-scute homolog 1 (ASCL1, also known as ASH1, an E-box binding basic helix-loop-helix transcription factor, and a lineage-survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF-1 regulated expression of the Bcl-2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF-1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Receptor activator of NF-κB ligand (RANKL) is a multifunctional cytokine known to affect immune and skeletal systems, as well as oncogenesis and metastasis 1-4 . RANKL is synthesized as a membrane-bound molecule, and cleaved into its soluble form by proteases 5-7 . As the soluble form of RANKL does not contribute greatly to bone remodelling or ovariectomy-induced bone loss 8 , whether soluble RANKL has a role in pathological settings remains unclear. Here we show that soluble RANKL promotes the formation of tumour metastases in bone. Mice that selectively lack soluble RANKL (Tnfsf11 ΔS/ΔS ) 5-7,9 have normal bone homoeostasis and develop a normal immune system but display markedly reduced numbers of bone metastases after intracardiac injection of RANK-expressing melanoma and breast cancer cells. Deletion of soluble RANKL does not affect osteoclast numbers in metastatic lesions or tumour metastasis to non-skeletal tissues. Therefore, soluble RANKL is dispensable for physiological regulation of bone and immune systems, but has a distinct and pivotal role in the promotion of bone metastases.

Although transforming growth factor beta (TGF-β) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-β in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-β signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-β and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-β type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-β signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-β was assessed. Although TGF-β signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-β. Taken together, these findings suggest that c-Ski suppresses TGF-β signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-β., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Bone is one of the preferred sites for the metastasis of malignant tumours, such as breast cancer, lung cancer and malignant melanoma. Tumour cells colonizing bone have the capacity to induce the expression of receptor activator of nuclear factor-κB ligand (RANKL), which promotes osteoclast differentiation and activation. Tumour-induced osteoclastic bone resorption leads to a vicious cycle between tumours and bone cells that fuels osteolytic tumour growth, causing bone pain and hypercalcaemia. Furthermore, RANKL contributes to bone metastasis by acting as a chemoattractant to bone for tumour cells that express its receptor, RANK. Thus inhibition of the RANKL-RANK pathway is a promising treatment for bone metastasis, and a human monoclonal anti-RANKL antibody, denosumab, has been used in the clinic. However, orally available drugs targeting RANKL must be developed to increase the therapeutic benefits to patients. Here we report the efficacy of the small-molecule RANKL inhibitor AS2676293 in treating bone metastasis using mouse models. Oral administration of AS2676293 markedly inhibited bone metastasis of human breast cancer cells MDA-MB-231-5a-D-Luc2 as well as tumour-induced osteolysis. AS2676293 suppressed RANKL-mediated tumour migration in the transwell assay and inhibited bone metastasis of the murine cell line B16F10, which is known not to trigger osteoclast activation. Based on the results from this study, RANKL inhibition with a small-molecule compound constitutes a promising therapeutic strategy for treating bone metastasis by inhibiting both osteoclastic bone resorption and tumour migration to bone., Competing Interests: K.Okamoto and A.Terashima declare that they belong to an endowment department, Department of Osteoimmunology, supported with an unrestricted grant from Chugai Pharmaceutical Co., Ltd., AYUMI Pharmaceutical Corporation and Noevir Co., Ltd. The remaining authors declare no conflict of interest.

Transforming growth factor (TGF)-β regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-β have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-β as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-β, in relation to crosstalk with some other signaling pathways, and the roles of TGF-β in lung and pancreatic cancers, in which TGF-β has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-β signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-β plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-β is produced as latent high molecular weight complexes, and the latent TGF-β complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-β activities through regulation of the latent TGF-β complex activation will be discussed.

Tumor microenvironment plays a pivotal role in cancer progression; however, little is known regarding how differences in the microenvironment affect characteristics of cancer cells. Here, we investigated the effects of tumor microenvironment on cancer cells by using mouse tumor models. After three cycles of inoculation and extraction of human pancreatic cancer cells, including SUIT-2 and Panc-1 cells, from tumors, distinct cancer cell lines were established: 3P cells from the pancreas obtained using the orthotopic tumor model and 3sc cells from subcutaneous tissue obtained using the subcutaneous tumor model. On re-inoculation of these cells, the 3sc cells and, more prominently, the 3P cells, exhibited higher tumorigenic activity than the parental cells. The 3P cells specifically exhibited low E-cadherin expression and high invasiveness, suggesting that they were endowed with the highest malignant characteristics. RNA-sequence analysis demonstrated that distinct signaling pathways were activated in each cell line and that the 3P cells acquired a cancer stem cell-like phenotype. Among cancer stem cell-related genes, those specifically expressed in the 3P cells, including NES, may be potential new targets for cancer therapy. The mechanisms underlying the development of highly malignant cancer cell lines were investigated. Individual cell clones within the parental cells varied in tumor-forming ability, indicating the presence of cellular heterogeneity. Moreover, the tumor-forming ability and the gene expression profile of each cell clone were altered after serial orthotopic inoculations. The present study thus suggests that both selection and education processes by tumor microenvironment are involved in the development of highly malignant cancer cells.

TGF-β regulates both the tumor-forming and migratory abilities of various types of cancer cells. However, it is unclear how the loss of TGF-β signaling components affects these abilities in clear-cell renal cell carcinoma (ccRCC). In this study, we investigated the role of TGFBR3 (TGF-β type III receptor, also known as betaglycan) in ccRCC. Database analysis revealed decreased expression of TGFBR3 in ccRCC tissues, which correlated with poor prognosis in patients. Orthotopic inoculation experiments using immunocompromised mice indicated that low TGFBR3 expression in ccRCC cells enhanced primary tumor formation and lung metastasis. In the presence of TGFBR3, TGF-β2 decreased the aldehyde dehydrogenase (ALDH)-positive ccRCC cell population, in which renal cancer-initiating cells are enriched. Loss of TGFBR3 also enhanced cell migration in cell culture and induced expression of several mesenchymal markers in a TGF-β-independent manner. Increased lamellipodium formation by FAK-PI3K signaling was observed with TGFBR3 downregulation, and this contributed to TGF-β-independent cell migration in ccRCC cells. Taken together, our findings reveal that loss of TGFBR3 endows ccRCC cells with multiple metastatic abilities through TGF-β-dependent and independent pathways.

The mammalian target of rapamycin (mTOR) pathway is commonly activated in human cancers. The activity of mTOR complex 1 (mTORC1) signaling is supported by the intracellular positioning of cellular compartments and vesicle trafficking, regulated by Rab GTPases. Here we showed that tuftelin 1 (TUFT1) was involved in the activation of mTORC1 through modulating the Rab GTPase-regulated process. TUFT1 promoted tumor growth and metastasis. Consistently, the expression of TUFT1 correlated with poor prognosis in lung, breast and gastric cancers. Mechanistically, TUFT1 physically interacted with RABGAP1, thereby modulating intracellular lysosomal positioning and vesicular trafficking, and promoted mTORC1 signaling. In addition, expression of TUFT1 predicted sensitivity to perifosine, an alkylphospholipid that alters the composition of lipid rafts. Perifosine treatment altered the positioning and trafficking of cellular compartments to inhibit mTORC1. Our observations indicate that TUFT1 is a key regulator of the mTORC1 pathway and suggest that it is a promising therapeutic target or a biomarker for tumor progression., Competing Interests: N.K., K.I., D.K., and K.M. have submitted a patent related to this work to the Japan Patent Office under application no. 2015-089220. The other authors declare that they have no competing interests.

Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y 12 at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y 12 phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.

Zinc finger E-box binding protein 1 (ZEB1) and ZEB2 induce epithelial-mesenchymal transition (EMT) and enhance cancer progression. However, the global view of transcriptional regulation by ZEB1 and ZEB2 is yet to be elucidated. Here, we identified a ZEB1-regulated inflammatory phenotype in breast cancer cells using chromatin immunoprecipitation sequencing and RNA sequencing, followed by gene set enrichment analysis (GSEA) of ZEB1-bound genes. Knockdown of ZEB1 and/or ZEB2 resulted in the downregulation of genes encoding inflammatory cytokines related to poor prognosis in patients with cancer, including IL6 and IL8, therefore suggesting that ZEB1 and ZEB2 have similar functions in terms of the regulation of production of inflammatory cytokines. Antibody array and ELISA experiments confirmed that ZEB1 controlled the production of the IL-6 and IL-8 proteins. The secretory proteins regulated by ZEB1 enhanced breast cancer cell proliferation and tumor growth. ZEB1 expression in breast cancer cells also affected the growth of fibroblasts in cell culture, and the accumulation of myeloid-derived suppressor cells in tumors in vivo. These findings provide insight into the role of ZEB1 in the progression of cancer, mediated by inflammatory cytokines, along with the initiation of EMT., (© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Poor prognoses for colorectal cancer patients with metastatic lesions have driven demand for the development of novel targeted therapies. Here, we demonstrate that expression of bone morphogenetic protein 4 (BMP-4) is universally upregulated in human colorectal cancer cells and tissues, resulting in activated BMP signaling. Inhibition of endogenous BMP signaling by the BMP type I receptor inhibitor LDN-193189 elevated expression of the phosphatase DUSP5 in colorectal cancer cells, inducing apoptosis via dephosphorylation of Erk MAPK. Administering LDN-193189 to mice diminished tumor formation of colorectal cancer cells. Our findings suggest inhibition of autocrine BMP-4 as a candidate treatment strategy for colorectal cancer. Cancer Res; 77(15); 4026-38. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Stochastic and proliferative events initiated from a single cell can disrupt homeostatic balance and lead to fatal disease processes such as cancer metastasis. To overcome metastasis, it is necessary to detect and quantify sparsely distributed metastatic cells throughout the body at early stages. Here, we demonstrate that clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC)-based cancer (CUBIC-cancer) analysis with a refractive index (RI)-optimized protocol enables comprehensive cancer cell profiling of the whole body and organs. We applied CUBIC-cancer analysis to 13 mouse models using nine cancer cell lines and spatiotemporal quantification of metastatic cancer progression at single-cell resolution. CUBIC-cancer analysis suggests that the epithelial-mesenchymal transition promotes not only extravasation but also cell survival at metastatic sites. CUBIC-cancer analysis is also applicable to pharmacotherapeutic profiling of anti-tumor drugs. CUBIC-cancer analysis is compatible with in vivo bioluminescence imaging and 2D histology. We suggest that a scalable analytical pipeline with these three modalities may contribute to addressing currently incurable metastatic diseases., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The p53 family of transcription factors includes p63, which is a master regulator of gene expression in epithelial cells. Determining whether p63 is tumor-suppressive or tumorigenic is complicated by isoform-specific and cellular context-dependent protein associations, as well as antagonism from mutant p53. ΔNp63 is an amino-terminal-truncated isoform, that is, the predominant isoform expressed in cancer cells of epithelial origin. In HaCaT keratinocytes, which have mutant p53 and ΔNp63, we found that mutant p53 antagonized ΔNp63 transcriptional activity but that activation of Ras or transforming growth factor-β (TGF-β) signaling pathways reduced the abundance of mutant p53 and strengthened target gene binding and activity of ΔNp63. Among the products of ΔNp63-induced genes was dual-specificity phosphatase 6 (DUSP6), which promoted the degradation of mutant p53, likely by dephosphorylating p53. Knocking down all forms of p63 or DUSP6 and DUSP7 (DUSP6/7) inhibited the basal or TGF-β-induced or epidermal growth factor (which activates Ras)-induced migration and invasion in cultures of p53-mutant breast cancer and squamous skin cancer cells. Alternatively, overexpressing ΔNp63 in the breast cancer cells increased their capacity to colonize various tissues upon intracardiac injection in mice, and this was inhibited by knocking down DUSP6/7 in these ΔNp63-overexpressing cells. High abundance of ΔNp63 in various tumors correlated with poor prognosis in patients, and this correlation was stronger in patients whose tumors also had a mutation in the gene encoding p53. Thus, oncogenic Ras and TGF-β signaling stimulate cancer progression through activation of the ΔNp63 transcriptional program., (Copyright © 2016, American Association for the Advancement of Science.)

Background: A recent study reported that long non-coding RNA activated by TGF-β (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-β (TGF-β)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients., Materials and Methods: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-β, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-β1 or TGF-β receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype., Results: The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-β in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression., Conclusion: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

Transforming growth factor-β (TGF-β) induces apoptosis in many types of cancer cells and acts as a tumor suppressor. We performed a functional analysis of TGF-β signaling to identify a molecular mechanism that regulated survival in small cell lung cancer cells. Here, we found low expression of TGF-β type II receptor (TβRII) in most small cell lung cancer cells and tissues compared to normal lung epithelial cells and normal lung tissues, respectively. When wild-type TβRII was overexpressed in small cell lung cancer cells, TGF-β suppressed cell growth in vitro and tumor formation in vivo through induction of apoptosis. Components of polycomb repressive complex 2, including enhancer of zeste 2 (EZH2), were highly expressed in small cell lung cancer cells; this led to epigenetic silencing of TβRII expression and suppression of TGF-β-mediated apoptosis. Achaete-scute family bHLH transcription factor 1 (ASCL1; also known as ASH1), a Smad-dependent target of TGF-β, was found to induce survival in small cell lung cancer cells. Thus, EZH2 promoted small cell lung cancer progression by suppressing the TGF-β-Smad-ASCL1 pathway.

Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell-like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer-initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer-initiating cells. Pancreatic cancer cells contained ALDH1(hi) cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1(hi) cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer-initiating cells., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Dysregulation of signalling pathways by changes of gene expression contributes to hallmarks of cancer. The ubiquitously expressed chaperone protein AAG8 (aging-associated gene 8 protein, encoded by the SIGMAR1 gene) is often found to be overexpressed in various cancers. AAG8 is involved in ER (endoplasmic reticulum)-associated degradation and has been intensively elaborated in neuroscience. However, its rationale in carcinogenesis has rarely been noticed. In this study, we explored the intrinsic oncogenetic roles of AAG8 in cancer cells and found that AAG8 promoted carcinogenesis both in vitro and in vivo. We further characterized AAG8, for the first time to our knowledge, as a STAT3 activator and elucidated that it alternatively activated STAT3 in addition to IL6/JAK pathway. Based on these findings and a drug screening study, we demonstrated that combined inhibition of AAG8 and IL6/JAK signalling synergistically limits cancer cell growth. Taken together, our findings shed light on the fundamental evidences for identification of AAG8 as an oncoprotein and potential target for cancer prevention, as well as highlight the importance of ER proteins in contributing to JAK/STAT signaling and carcinogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-β is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-β. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-β in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-β. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-β type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-β of anti-apoptotic DEC1 and pro-apoptotic Bim proteins. Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-β-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-β functions may be restricted to apoptosis. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-β signaling., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Bone morphogenetic proteins (BMPs) are multi-functional cytokines, which belong to the transforming growth factor-β (TGF-β) family. In some cancer tissues, aberrant expression of various BMP signal components has been detected. Here, we describe the divergent roles of BMPs during the progression of cancer. BMPs exhibit various effects on both cancer cells and on tumor microenvironments. BMPs inhibit the proliferation of cancer cells, with some exceptions. BMPs also induce the differentiation of certain cancer stem cells, and attenuate their aggressiveness. In parallel, BMPs play a critical role in the regulation of tumor angiogenesis and the metastasis of cancer cells. Some mouse xenograft models have revealed that cancer metastases are prevented by the inhibition of BMP signaling. Together, these findings imply that BMPs function as both suppressors and promoters of tumors in a context dependent manner. The bi-directional characteristics of BMPs in cancer are similar to those of TGF-β, which was previously described as a molecular 'Jekyll and Hyde.', (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Aldehyde dehydrogenase 1 (ALDH1) has been shown to serve as a marker for cancer-initiating cells (CICs), but little is known about the regulation of the CIC functions of ALDH1+ cancer cells. We isolated ALDH1+ cells from human diffuse-type gastric carcinoma cells and characterized these cells using an Aldefluor assay. ALDH1+ cells constituted 5-8% of the human diffuse-type gastric carcinoma cells, OCUM-2MLN and HSC-39; were more tumourigenic than ALDH1- cells; and were able to self-renew and generate heterogeneous cell populations. Using gene expression microarray analyses, we identified REG4 (regenerating islet-derived family, member 4) as one of the genes up-regulated in ALDH1+ cells, and thus as a novel marker for ALDH1+ tumour cells. Induced expression of REG4 enhanced the colony-forming ability of OCUM-2MLN cells, while knockdown of REG4 inhibited the tumourigenic potential of ALDH1+ cells. We further found that TGF-β signalling reduces the expression of ALDH1 and REG4, and the size of the ALDH1+ cell population. In human diffuse-type gastric carcinoma tissues, the expression of ALDH1 and REG4 correlated with each other, as assessed by immunohistochemistry, and ALDH1 expression correlated inversely with Smad3 phosphorylation as a measure of TGF-β signalling. These findings illustrate that, in diffuse-type gastric carcinoma, REG4 is up-regulated in ALDH1+ CICs, and that the increased tumourigenic ability of ALDH1+ cells depends on REG4. Moreover, TGF-β down-regulates ALDH1 and REG4 expression, which correlates with a reduction in CIC population size and tumourigenicity. Targeting REG4 in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse-type gastric carcinoma., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.

Transforming growth factor-β (TGF-β) elicits both tumor-suppressive and tumor-promoting functions during cancer progression. Here, we describe the tumor-promoting functions of TGF-β and how these functions play a role in cancer progression. Normal epithelial cells undergo epithelial-mesenchymal transition (EMT) through the action of TGF-β, while treatment with TGF-β and fibroblast growth factor (FGF)-2 results in transdifferentiation into activated fibroblastic cells that are highly migratory, thereby facilitating cancer invasion and metastasis. TGF-β also induces EMT in tumor cells, which can be regulated by oncogenic and anti-oncogenic signals. In addition to EMT promotion, invasion and metastasis of cancer are facilitated by TGF-β through other mechanisms, such as regulation of cell survival, angiogenesis, and vascular integrity, and interaction with the tumor microenvironment. TGF-β also plays a critical role in regulating the cancer-initiating properties of certain types of cells, including glioma-initiating cells. These findings thus may be useful for establishing treatment strategies for advanced cancer by inhibiting TGF-β signaling.

A relationship exists between defects in bone morphogenetic protein (BMP) signaling and formation of hamartoma and adenoma in the gastric epithelium; however, the role of BMP signaling in the progression of diffuse-type gastric carcinoma remains unknown. We investigated whether BMP functions as a tumor suppressor in human diffuse-type gastric carcinoma using three different human diffuse-type gastric carcinoma cell lines (OCUM-12, HSC-39, and OCUM-2MLN). Overexpression of the dominant-negative form of BMP-2/4-specific type I receptor (ALK-3) in OCUM-12 and HSC-39 cells accelerated their growth in vivo. BMP-4 induced cell cycle arrest in these cells via p21 induction through the SMAD pathway. Moreover, overexpression of the constitutively active form of ALK-3 in HSC-39 and OCUM-2MLN cells suppressed the proliferation of these cells in vitro and in vivo. Our findings suggest that BMP-2 and BMP-4 function as potent tumor suppressors in diffuse-type gastric carcinoma., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells.

The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-β induced EMT and that long-term exposure to TGF-β elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-β induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-β-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-β and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-β and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.

Cancer cells undergo multi-step processes in obtaining the ability to metastasize, and are constantly exposed to signals that induce apoptosis. Acquisition of anti-apoptotic properties by cancer cells is important for metastasis, and recent studies suggest that transforming growth factor (TGF)-β promotes the survival of certain types of cancer cells. Here, we found that in highly metastatic breast cancer cells, JygMC(A), JygMC(B) and 4T1, TGF-β ligands were produced in autocrine fashion. Pharmacological inhibition of endogenous TGF-β signalling by a TGF-β type I receptor kinase inhibitor in serum-free conditions increased the expression of BH3-only protein, Bim (also known as Bcl2-like 11) in JygMC(A) and JygMC(B) cells, and caused apoptotic cell death. We also found that induction of Bim by TGF-β was not observed in Foxc1 knocked-down cancer cells. These findings suggest that TGF-β plays a crucial role in the regulation of survival of certain types of cancer cells through the TGF-β-Foxc1-Bim pathway, and that specific inhibitors of TGF-β signalling might be useful as apoptosis inducers in breast cancer cells.

Transforming growth factor-beta (TGF-beta) signaling facilitates tumor growth and metastasis in advanced cancer. In the present study, we identified differentially expressed in chondrocytes 1 (DEC1, also known as SHARP2 and Stra13) as a downstream target of TGF-beta signaling, which promotes the survival of breast cancer cells. In the mouse mammary carcinoma cell lines JygMC(A) and 4T1, the TGF-beta type I receptor kinase inhibitors A-44-03 and SB431542 induced apoptosis of cells under serum-free conditions. Oligonucleotide microarray and real-time reverse transcription-PCR analyses revealed that TGF-beta induced DEC1 in these cells, and the increase of DEC1 was suppressed by the TGF-beta type I receptor kinase inhibitors as well as by expression of dominant-negative TGF-beta type II receptor. Overexpression of DEC1 prevented the apoptosis of JygMC(A) cells induced by A-44-03, and knockdown of endogenous DEC1 abrogated TGF-beta-promoted cell survival. Moreover, a dominant-negative mutant of DEC1 prevented lung and liver metastasis of JygMC(A) cells in vivo. Our observations thus provide new insights into the molecular mechanisms governing TGF-beta-mediated cell survival and metastasis of cancer.