Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Role of TGF-β signaling regulatory microRNAs in the pathogenesis of colorectal cancer

Transforming growth factor β (TGF-β) modulates tumor progression by regulating cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. Biological and pharmacological agonists/antagonists, the interplay between intracellular signaling pathways, and microRNAs (miRNAs) control the initiation and activation of the TGF-β signaling pathway. It has been proposed that the expression profiles of tumor suppressor and oncogenic TGF-β miRNAs may be used for the classification, diagnosis, and prognosis of human malignancies. Deregulated miRNAs and aberrant activation of TGF-β signaling are frequently found in human colorectal cancers (CRCs), but a little is known about their mechanisms of action in the development and progression of colorectal carcinoma. This review summarizes the current knowledge of the role of TGF-β signaling regulatory miRNAs in the pathogenesis of CRC for a better understanding and hence better management of this disease.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type II of de-differentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hyp0Xic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological featureof hypoxic pulmonary vascular remodeling.However,the origin of the cells involved in this process is stillnot well understood.The present study was undertaken to test the hypothesis that transforming growthfactor-β1 (TGF-β1) can induce transdifferentiation of fibroblasts into myofibroblasts,which might play akey role in the muscularization of non-muscular pulmonary arterioles.It was found that mean pulmonaryarterial pressure increased significantly after 7 d of hypoxia.Pulmonary artery remodeling index and rightventricular hypertrophy became evident after 14 d of hypoxia.The distribution of nonmuscular,partiallymuscular,and muscular vessels was significantly different after 7 d of hypoxia.Immunocytochemistryresults demonstrated that the expression of co-smooth muscle actin was increased in intra-acinar pulmonaryarteries with increasing hypoxic time.TGF-β1 mRNA expression in pulmonary arterial walls was increasedsignificantly after 14 d of hypoxia,but showed no obvious changes after 3 or 7 d of hypoxia.In pulmonarytunica adventitia and tunica media,TGF-β1 protein staining was poorly positive in control rats,but wasmarkedly enhanced after 3 d of hypoxia,reaching its peak after 7 d Of hypoxia.The myofibroblast phenotypewas confirmed by electron microscopy,which revealed microfilaments and a well-developed rough endo-plasmic reticulum.Taken together,our results suggested that TGF-β1 induces transdifferentiation of fibro-blasts into myofibroblasts,which is important in hypoxic pulmonary vascular remodeling.     

Hypoxia Induces Transforming Growth Factor-β1 Gene Expression in the Pulmonary Artery of Rats via Hypoxia-inducible Factor-1α

The present study was undertaken to investigate the dynamic expression of hypoxia induciblefactor-1 α (HIF-1α) and transforming growth factor-β1 (TGF-β1) in hypoxia-induced pulmonary hypertensionof rats.It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d ofhypoxia.Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d ofhypoxia.HIF-1α mRNA staining was less positive in the control,hypoxia for 3 d and hypoxia for 7 d,butbegan to enhance significantly after 14 d of hypoxia,then remained stable.Expression of HIF-1 α protein inthe control was less positive,but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats.TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, butshowed no obvious changes after 3 or 7 d of hypoxia.In pulmonary tunica adventitia and tunica media,TGF-β1 protein staining was less positive in control rats,but was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d of hypoxia,and then weakening after 14 and 21 d of hypoxia.Western blottingshowed that HIF- 1α protein levels increased significantly after 7 d of hypoxia and then remained at a highlevel. TGF-β1 protein level was markedly enhanced after 3 d of hypoxia,reaching its peak after 7 d ofhypoxia,and then decreasing after 14 and 21 d of hypoxia.Linear correlation analysis showed that HIF-1αmRNA, TGF-β1 mRNA, TGF-β1 protein were positively correlated with mPAP,vessel morphometry andright ventricular hypertrophy index.TGF-β1 protein (tunica adventitia) was negatively correlated withHIF-lα mRNA.Taken together,our results suggest that changes in HIF-lα and TGF-β1 expression afterhypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.     

Crosstalk between TGF-β and hedgehog signaling in cancer

Hedgehog (HH) and TGF-β signals control various aspects of embryonic development and cancer progression. While their canonical signal transduction cascades have been well characterized, there is increasing evidence that these pathways are able to exert overlapping activities that challenge efficient therapeutic targeting. We herein review the current knowledge on HH signaling and summarize the recent findings on the crosstalks between the HH and TGF-β pathways in cancer.     

Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Role of TGF-β signaling in the mechanisms of tamoxifen resistance

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling.     

TRAF4 mediates activation of TGF-β signaling and is a biomarker for oncogenesis in breast cancer

The tumor-promoting arm of transforming growth factor beta(TGF-β)receptor signaling contributes to advanced cancer progression and is considered a master regulator of breast cancer metastasis.In mammals,there are six distinct members in the tumor-necrosis factor receptor(TNFR)-associated factor(TRAF)family(TRAF1–TRAF6),with the function of TRAF4 not being extensively studied in the past decade.Although numerous studies have suggested that there is elevated TRAF4 expression in human cancer,it is still unknown in which oncogenic pathway TRAF4 is mainly implicated.This review highlights TGF-β-induced SMAD-dependent signaling and non-SMAD signaling as the major pathways regulated by TRAF4 involved in breast cancer metastasis.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transition of ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.     

Dynamics of TGF-β induced epithelial-to-mesenchymal transition monitored by electric cell-substrate impedance sensing

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell-cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24-30 h after exposure to TGF-β1.     

Negative regulation of TGF-β signaling in development

The TGF-β superfamily members have important roles in controlling patterning and tissue formation in both invertebrates and vertebrates. Two types of signal transducers, receptors and Smads, mediate the signaling to regulate expression of their target genes. Despite of the relatively simple signal transduction pathway, many modulators have been found to contribute to a tight regulation of this pathway in a variety of mechanisms. This article reviews the negative regulation of TGF-β signaling with focus on its roles in vertebrate development.     

Extracellular matrix as a contextual determinant of transforming growth factor-β signaling in epithelial-mesenchymal transition and in cancer

Extracellular matrix (ECM) provides both structural support and contextual information to cells within tissues and organs. The combination of biochemical and biomechanical signals from the ECM modulates responses to extracellular signals toward differentiation, proliferation, or apoptosis; alterations in the ECM are necessary for development and remodeling processes, but aberrations in the composition and organization of ECM are associated with disease pathology and can predispose to development of cancer. The primary cell surface sensors of the ECM are the integrins, which provide the physical connection between the ECM and the cytoskeleton and also convey biochemical information about the composition of the ECM. Transforming growth factor-β (TGF-β) is an extracellular signaling molecule that is a powerful controller of a variety of cellular functions, and that has been found to induce very different outcomes according to cell type and cellular context. It is becoming clear that ECM-mediated signaling through integrins is reciprocally influenced by TGF-β: integrin expression, activation, and responses are affected by cellular exposure to TGF-β, and TGF-β activation and cellular responses are in turn controlled by signaling from the ECM through integrins. Epithelial-mesenchymal transition (EMT), a physiological process that is activated by TGF-β in normal development and in cancer, is also affected by the composition and structure of the ECM. Here, we will outline how signaling from the ECM controls the contextual response to TGF-β, and how this response is selectively modulated during disease, with an emphasis on recent findings, current challenges, and future opportunities.