Sunil K. Halder

Antimetastatic Role of Smad4 Signaling in Colorectal Cancer

BACKGROUND & AIMS Transforming growth factor (TGF)-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. However, the function of Smad signaling and the effects of TGF-beta-receptor kinase inhibitors have not been analyzed during CRC metastasis. We investigated the role of TGF-beta/Smad signaling in CRC progression. METHODS We evaluated the role of TGF-beta/Smad signaling on cell proliferation, migration, invasion, tumorigenicity, and metastasis in Smad4-null colon carcinoma cell lines (MC38 and SW620) and in those that transgenically express Smad4. We also determined the effects of a TGF-beta-receptor kinase inhibitor (LY2109761) in CRC tumor progression and metastasis in mice. RESULTS TGF-beta induced migration/invasion, tumorigenicity, and metastasis of Smad4-null MC38 and SW620 cells; incubation with LY2109761 reversed these effects. In mice, LY2109761 blocked metastasis of CRC cells to liver, inducing cancer cell expression of E-cadherin and reducing the expression of the tumorigenic proteins matrix metalloproteinase-9, nm23, urokinase plasminogen activator, and cyclooxygenase-2. Transgenic expression of Smad4 significantly reduced the oncogenic potential of MC38 and SW620 cells; in these transgenic cells, TGF-beta had tumor suppressor, rather than tumorigenic, effects. CONCLUSIONS TGF-beta/Smad signaling suppresses progression and metastasis of CRC cells and tumors in mice. Loss of Smad4 might underlie the functional shift of TGF-beta from a tumor suppressor to a tumor promoter; inhibitors of TGF-beta signaling might be developed as CRC therapeutics.

Targeting Transforming Growth Factor-β signaling in liver metastasis of colon cancer

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

Smad7 induces hepatic metastasis in colorectal cancer

Although Smad signalling is known to play a tumour suppressor role, it has been shown to play a prometastatic function also in breast cancer and melanoma metastasis to bone. In contrast, mutation or reduced level of Smad4 in colorectal cancer is directly correlated to poor survival and increased metastasis. However, the functional role of Smad signalling in metastasis of colorectal cancer has not been elucidated. We previously reported that overexpression of Smad7 in colon adenocarcinoma (FET) cells induces tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis. Here, we have observed that abrogation of Smad signalling by Smad7 induces liver metastasis in a splenic injection model. Polymerase chain reaction with genomic DNA from liver metastases indicates that cells expressing Smad7 migrated to the liver. Increased expression of TGF-β type II receptor in liver metastases is associated with phosphorylation and nuclear accumulation of Smad2. Immunohistochemical analyses have suggested poorly differentiated spindle cell morphology and higher cell proliferation in Smad7-induced liver metastases. Interestingly, we have observed increased expression and junctional staining of Claudin-1, Claudin-4 and E-cadherin in liver metastases. Therefore, this report demonstrates, for the first time, that blockade of TGF-β/Smad pathway in colon cancer cells induces metastasis, thus supporting an important role of Smad signalling in inhibiting colon cancer metastasis.

Oncogenic function of a novel WD-domain protein, STRAP, in human carcinogenesis

The development and progression of malignancies is a complex multistage process that involves the contribution of a number of genes giving growth advantage to cells when transformed. The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex with tumor-suppressor or prooncogenic activities depending on the cell type and the stage of the disease. We have previously reported the identification of a novel WD-domain protein, STRAP, that associates with both TGF-beta receptors and that synergizes with the inhibitory Smad, Smad7, in the negative regulation of TGF-beta-induced transcription. Here, we show that STRAP is ubiquitously expressed and is localized in both cytoplasm and nucleus. STRAP is up-regulated in 60% colon and in 78% lung carcinomas. Stable expression of STRAP results in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and in down-regulation of the cyclin-dependent kinase inhibitor p21(Cip1), which results in retinoblastoma protein hyperphosphorylation. In addition, we have observed that Smad2/3 phosphorylation, TGF-beta-mediated transcription, and growth inhibition are induced in STRAP-knockout mouse embryonic fibroblasts compared with wild-type cells. Ectopic expression of STRAP in A549 lung adenocarcinoma cell line inhibits TGF-beta-induced growth inhibition and enhances anchorage-independent growth of these cells. Moreover, overexpression of STRAP increases tumorigenicity in athymic nude mice. Knockdown of endogenous STRAP by small interfering RNA increases TGF-beta signaling, reduces ERK activity, increases p21(Cip1) expression, and decreases tumorigenicity. Taken together, these results suggest that up-regulation of STRAP in human cancers may provide growth advantage to tumor cells via TGF-beta-dependent and TGF-beta-independent mechanisms, thus demonstrating the oncogenic function of STRAP.

Restoration of TGF-β signalling reduces tumorigenicity in human lung cancer cells

Members of the transforming growth factor-β (TGF-β) family regulate a wide range of biological processes including cell proliferation, migration, differentiation, apoptosis, and extracellular matrix deposition. Resistance to TGF-β-mediated tumour suppressor function in human lung cancer may occur through the loss of type II receptor (TβRII) expression. In this study, we investigated the expression pattern of TβRII in human lung cancer tissues by RT–PCR and Western blot analyses. We observed downregulation of TβRII in 30 out of 46 NSCLC samples (65%) by semiquantitative RT–PCR. Western blot analyses with tumour lysates showed reduced expression of TβRII in 77% cases. We also determined the effect of TβRII expression in lung adenocarcinoma cell line (VMRC-LCD) that is not responsive to TGF-β due to lack of TβRII expression. Stable expression of TβRII in these cells restored TGF-β-mediated effects including Smad2/3 and Smad4 complex formation, TGF-β-responsive reporter gene activation, inhibition of cell proliferation and increased apoptosis. Clones expressing TβRII showed reduced colony formation in soft-agarose assay and significantly reduced tumorigenicity in athymic nude mice. Therefore, these results suggest that reestablishment of TGF-β signalling in TβRII null cells by stable expression of TβRII can reverse malignant behaviour of cells and loss of TβRII expression may be involved in lung tumour progression.

Oncogenic Serine-Threonine Kinase Receptor-Associated Protein Modulates the Function of Ewing Sarcoma Protein through a Novel Mechanism

Although much is known about the oncogenic functions of chimeric Ewing sarcoma (EWS) fusion proteins that result from chromosomal translocations, the cellular role of the normal EWS protein is not well characterized. We have previously identified a WD domain-containing protein, serine-threonine kinase receptor-associated protein (STRAP), which inhibits transforming growth factor beta (TGF-beta) signaling through interaction with receptors and Smad7 and promotes growth and enhances tumorigenicity. Here, we report the interaction between STRAP and EWS using matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry. Although STRAP is localized in both cytoplasm and nucleus, nuclear STRAP colocalizes and associates specifically with EWS in the nucleus through its NH(2) and COOH termini. We have found that normal EWS protein is up-regulated in human cancers, which correlates with the up-regulation of STRAP in 71% of colorectal cancers and 54% of lung cancers, suggesting a cooperative role of these two proteins in human cancers. TGF-beta has no effect on STRAP and EWS interaction. However, EWS, like STRAP, attenuates TGF-beta-dependent transcription. STRAP inhibits EWS-dependent p300-mediated transactivation of EWS target genes, such as ApoCIII and c-fos, in a TGF-beta-independent manner. Interestingly, we have shown that STRAP blocks the interaction between EWS and p300, whereas the complex formation between STRAP and EWS is not affected by p300. These results suggest that STRAP inhibits the transactivation function of EWS by displacing p300 from the functional transcriptional complex. Thus, this study provides a novel TGF-beta-independent function of STRAP and describes a mechanism by which STRAP regulates the function of oncogenic EWS protein.

Role of TGF-β signaling in curcumin-mediated inhibition of tumorigenicity of human lung cancer cells

PurposeCurcumin has been shown to have potent anticancer activities like inhibition of cell proliferation, induction of apoptosis, and suppression of angiogenesis. Transforming growth factor-β (TGF-β) signaling plays a complex role in tumor suppression and promotion depending on the tumor type and stage. However, the effect of curcumin on TGF-β signaling in cancer cells and the role of TGF-β signaling in curcumin-induced anticancer activities have not been determined. Here, we investigate the role of curcumin on TGF-β signaling, and whether TGF-β signaling is involved in the antitumor activities of curcumin.MethodsHuman non-small cell lung cancer (NSCLC) cell lines, ACC-LC-176 (without TGF-β signaling), H358, and A549 (with TGF-β signaling) were treated with curcumin to determine cell growth, apoptosis, and tumorigenicity. Antitumor activities of curcumin were determined using these cell lines and an in vivo mouse model. We also tested the effect of curcumin on TGF-β/Smad signaling by western blotting and by luciferase assays.ResultsCurcumin inhibited cell growth and induced apoptosis of all three NSCLC cell lines in vitro and in vivo. It significantly reduced subcutaneous tumor growth by these three cell lines irrespective of TGF-β signaling status. Curcumin inhibited TGF-β-induced Smad2/3 phosphorylation and transcription in H358 and A549 cells, but not in ACC-LC-176 cells.ConclusionsCurcumin reduces tumorigenicity of human lung cancer cells in vitro and in vivo by inhibiting cell proliferation and promoting apoptosis. These results suggest that TGF-β signaling is not directly involved in curcumin-mediated growth inhibition, induction of apoptosis, and inhibition of tumorigenicity.