Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function

In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.     

Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines

The role of transforming growth factor beta in breast cancer is controversial with tumor suppressor and pro-oncogenic activities having been demonstrated. To address whether the same or different signal transduction pathways mediate these opposing activities, we manipulated the Smad2/3 signaling pathway in cells of common origin but differing degrees of malignancy derived from MCF10A human breast cells. We show that interference with endogenous Smad2/3 signaling enhances the malignancy of xenografted tumors of premalignant and well-differentiated tumor cells but strongly suppresses lung metastases of more aggressive carcinoma cells after tail vein injection. Overexpression of Smad3 in the same cells has opposite effects. The data demonstrate that the Smad2/3 signaling pathway mediates tumor suppressor and prometastatic signals, depending on the cellular context.     

TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells

Transforming growth factor (TGF)-beta has been reported to exert growth inhibitory activity in normal epithelial cells whereas it induces cell proliferation and invasive phenotypes in advanced carcinomas. Our previous study showed that MCF10A, a spontaneously immortalized "normal" breast epithelial cell line, is resistant to TGF-beta-induced growth inhibition, suggesting that conversion of TGF-beta growth inhibitory signaling into an oncogenic pathway may occur at the early stage of tumor development/progression. To address this issue, we investigated the TGF-beta signaling pathway and its role in phenotypic transformation of MCF10A cells. TGF-beta treatment induced changes in the MCF10A cell morphology from cuboidal to an elongated spindle-like shape, accompanied with down-regulation of epithelial cell marker E-cadherin. TGF-beta treatment was sufficient to induce migrative and invasive phenotypes in these cells, an important phenotypic conversion during tumor progression. We also showed that TGF-beta treatment rapidly activated ERK-1/2 and p38 MAPK leading to upregulation of matrix metalloproteinase (MMP)-2 and MMP-9. Using chemical inhibitors and dominant negative mutants of MAPKs, we provide evidence that while both p38 MAPK and ERKs are required for TGF-beta-induced MCF10A cell migration and invasion, TGF-beta-induced MMP-2 and MMP-9 expression depends on p38 MAPK signaling, but is independent of ERK activity. This study demonstrates the roles of TGF-beta signaling pathways for induction of oncogenic signaling in preneoplastic human breast epithelial cells and will deepen our understanding of TGF-beta signaling in the progress of breast cancer.     

Restoration of transforming growth factor Beta signaling by functional expression of smad4 induces anoikis

Smad proteins transduce signals carried by the transforming growth factor beta (TGF-beta) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-beta signaling was restored as determined by TGF-beta-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-beta1 also resulted in an altered cell morphology that coincided with enhanced beta1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-beta-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-beta signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.     

Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells

Transforming growth factor-beta (TGF-beta) signals through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, TGF-beta activates intracellular Smad proteins and regulates proliferation and apoptosis in various cell types. To demonstrate the effects of TGF-beta/Smad signal on growth and apoptosis of human embryonal rhabdomyosarcoma (RMS) cells, a strategy of RNAi-mediated 'gene silencing' of Smad4 was used to interrupt endogenous TGF-beta/Smad signaling in an RMS cell line, RD, and the regulation of exogenous TGF-beta1 to growth and apoptosis of the cells was also determined. Physiologically, TGF-beta/Smad signaling was essential for the normal growth of RD. The interruption of endogenous TGF-beta/Smad signaling by RNAi significantly suppressed the growth of RD cells and dramatically induced apoptosis of RD cells. Exogenous TGF-beta1 also inhibited the growth of RD cells, but had no effect on apoptosis. It also partially counteracted the growth inhibition and apoptosis induced by Smad4 silencing in RD cells. These findings provide a new insight into how TGF-beta/Smad signaling regulates the growth and apoptosis of cancer cells. Moreover, as a powerful tool, shRNA interference suppresses endogenous Smad4 gene expression and subsequently modulates cell growth and apoptosis, which may provide a novel basis for the development of rational intervention strategies in RMS therapy.     

RING finger-dependent ubiquitination by PRAJA is dependent on TGF-beta and potentially defines the functional status of the tumor suppressor ELF

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-beta signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-beta-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P < 0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Delta-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Delta-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t(1/2)) and rate constant for degradation (k(D)) of ELF is 1.91 h and 21.72 min(-1) when coupled with ectopic expression of PRAJA in cells stimulated by TGF-beta, compared to PRAJA-transfected unstimulated cells (t(1/2) = 4.33 h and k(D) = 9.6 min(-1)). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.     

Lentiviral-mediated Smad4 RNAi induced anti-proliferation by p16 up-regulation and apoptosis by caspase 3 down-regulation in hepatoma SMMC-7721 cells

Transforming growth factor-beta (TGF-beta)-Smad signaling pathway participates in the regulation of a variety of cellular activities. Unlike the high incidences of Smad4 mutation or deletion in pancreatic cancer and gastrointestinal cancers, Smad4 gene is seldom mutated or deleted in hepatocellular carcinoma (HCC). The role of TGF-beta-Smad4 signaling pathway in leading to carcinogenesis of liver cells remains unknown. In this study, we succeeded in silencing Smad4 using lentiviral-mediated Smad4 RNA interference (RNAi). We investigated the role of Smad4 in TGF-beta1-induced cell proliferation and apoptosis of HCC cell line SMMC-7721. We determined cell proliferation, apoptosis, and expression of p21, p16, p53 and caspase 3. Results showed that TGF-beta1 not only had a significant anti-proliferation effect but also induced cellular apoptosis in SMMC-7721 cells. These effects induced by TGF-beta1 were almost completely blocked by the knockdown of Smad4. Western blot analysis revealed that p16 was up-regulated and caspase 3 was activated by silencing of Smad4, and the expression of p21 and wild-type p53 were not affected. These results suggest that TGF-beta1-induced cell growth inhibition by up-regulating p16 expression and cellular apoptosis by activating caspase 3 was Smad4-dependent. Additionally, the knock down of a specific gene using lentiviral-mediated RNAi appears to be a promising tool and strategy for analyzing endogenous gene function.     

Stable overexpression of Smad7 in human melanoma cells inhibits their tumorigenicity in vitro and in vivo

We previously identified constitutive Smad signaling in human melanoma cells despite resistance to transforming growth factor-beta (TGF-beta) control of cell proliferation. This led us to investigate the effect of inhibitory Smad7 overexpression on melanoma cell behavior. Using the highly metastatic cell line, 1205-Lu, we thus generated melanoma cell clones constitutively expressing Smad7, and their mock-transfected counterparts. Stable expression of Smad7 resulted in an inhibition of constitutive Smad2/3 phosphorylation, and in a reduced TGF-beta response of Smad3/Smad4-driven gene transactivation, as measured using transfected Smad3/4-specific reporter gene constructs. Smad7 overexpression, however, did not alter their proliferative capacity and resistance to TGF-beta-driven growth inhibition. On the other hand, expression of Smad7 efficiently reduced the capacity of human melanoma cells to invade Matrigel in Boyden migration chambers, while not affecting their motility and adhesion to collagen and laminin. Gelatin zymography identified reduced MMP-2 and MMP-9 secretion by Smad7-expressing melanoma cells as compared with their control counterparts. Smad7-expressing melanoma cells exhibited a dramatically reduced capacity to form colonies under anchorage-independent culture conditions, and, when injected subcutaneously into nude mice, were largely delayed in their ability to form tumors. These results suggest that TGF-beta production by melanoma cells not only affects the tumor environment but also directly contributes to tumor cell aggressiveness through autocrine activation of Smad signaling.     

Transforming growth factor-beta suppresses nonmetastatic colon cancer through Smad4 and adaptor protein ELF at an early stage of tumorigenesis

Although transforming growth factor-beta (TGF-beta) is both a suppressor and promoter of tumorigenesis, its contribution to early tumor suppression and staging remains largely unknown. In search of the mechanism of early tumor suppression, we identified the adaptor protein ELF, a beta-spectrin from stem/progenitor cells committed to foregut lineage. ELF activates and modulates Smad4 activation of TGF-beta to confer cell polarity, to maintain cell architecture, and to inhibit epithelial-to-mesenchymal transition. Analysis of development of colon cancer in (adult) elf+/-/Smad4+/-, elf+/-, Smad4+/-, and gut epithelial cells from elf-/- mutant mouse embryos pinpoints the defect to hyperplasia/adenoma transition. Further analysis of the role of ELF in human colorectal cancer confirms reduced expression of ELF in Dukes' B1 stage tissues (P < 0.05) and of Smad4 in advanced colon cancers (P < 0.05). This study indicates that by modulating Smad 4, ELF has a key role in TGF-beta signaling in the suppression of early colon cancer.     

Activation of bone morphogenetic protein signaling by a Gemini vitamin D3 analogue is mediated by Ras/protein kinase C alpha

Bone morphogenetic proteins (BMP) are members of the transforming growth factor-beta superfamily, and they play an important role for embryonic development, for bone and cartilage formation, and during carcinogenesis. We have previously shown that the novel Gemini vitamin D(3) analogue, Ro-438-3582 [Ro3582; 1 alpha,25-dihydroxy-20S,21(3-hydroxy-3-methylbutyl)-23-yne-26,27-hexafluorocholecalciferol], inhibited cell proliferation and activated the BMP/Smad signaling pathway in MCF10AT1 breast epithelial cells. In this report, we investigated the upstream signaling pathways responsible for the activation of BMP/Smad signaling by Ro3582. Among seven different serine/threonine kinase inhibitors that we tested, protein kinase C (PKC) inhibitors blocked the effects of Ro3582 on the phosphorylation of Smad1/5, mRNA synthesis for BMP-2 and BMP-6, and cell growth in MCF10AT1 cells. Overexpression of PKC alpha, but not PKC epsilon, PKC delta or PKC zeta isoforms, increased Ro3582-induced phosphorylation of Smad1/5, suggesting that PKC alpha mediates the activation of Smad signaling and inhibition of cell proliferation. Interestingly, the activation of Smad signaling by Ro3582 was shown in Ha-ras-transfected MCF10AT1 cells, but not in the parent cell line (MCF10A without Ras). Inhibiting Ras activity blocked the translocation of PKC alpha to the plasma membrane and the phosphorylation of Smad1/5 induced by Ro3582, indicating that Ras is necessary for the activation of PKC alpha and Smad signaling. In conclusion, Ro3582 inhibits cell proliferation and activates BMP/Smad signaling via a Ras and PKC alpha pathway in breast epithelial cells.     

Alterations of Smad signaling in human breast carcinoma are associated with poor outcome: a tissue microarray study.

Based largely on studies of cell lines in vitro and of transgenic mouse models, disruptions of transforming growth factor (TGF) beta signaling are thought to contribute to the development and progression of human breast cancer. However, whether and how TGF-beta signaling becomes disrupted during human breast cancer development in vivo remains largely unknown. To address this question, we have compared the patterns of expression and activation of the postreceptor components of the TGF-beta signaling pathway, the so-called Smads, in human breast cancer cell lines with those in breast carcinoma specimens. None of the breast carcinoma cell lines were growth arrested by TGF-beta in vitro. Each of the tumor cell lines expressed normal levels of Smad2 and -3. Moreover, TGF-beta treatment induced phosphorylation of Smad2 (Smad2P) in each of these lines, except those that lacked TGF-beta type II receptors. Moreover, only one of the cell lines failed to express Smad4. Among 456 cases of human breast carcinoma assembled in tissue microarrays, the majority (92%) expressed Smad2, Smad2P, as well as Smad4, indicating their ability to proliferate within a microenvironment that contains bioactive TGF-beta. Thirty cases (6.6%) failed to express Smad2P, suggesting the loss of TGF-beta receptor signaling. Nine cases (2%) failed to express Smad4, and 3 of these also failed to express Smad2P. Thus, the phenotypes of breast tumors in vivo paralleled that of human breast cancer cell lines in terms of Smad2P and Smad4 expression. Loss of Smad signaling was not associated with any particular histological subtype, histological or nuclear grade, estrogen- or progesterone receptor expression, or HER2/neu expression. Loss of Smad4 was inversely correlated with the presence of axillary lymph node metastases. Most importantly, among patients with stage II breast cancer, lack of Smad2P expression in the tumor was strongly associated with shorter overall survival. Finally, analysis of a small cohort of hereditary breast cancers failed to reveal any association between BRCA1 or BRCA2 genotype and alterations in Smad signaling.