Melisi, Davide (2009) Smad- dependent and Smad- independent pathways in TGF-β signaling as therapeutic targets in pancreatic cancer. [Tesi di dottorato] (Unpublished)

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Pancreatic cancer (PC) remains one of the most lethal and poorly understood human malignancies. Most PC patients present with inoperable disease, or develop metastases after surgery, and conventional therapies are usually ineffective in treating this disease. It is evident that novel therapies remain to be developed. Transforming Growth Factor β (TGF-β) is a family of dimeric olypeptide growth factors that initiate cell signaling by dimerizing the TGF-β type I (TβRI) and type II (TβRII) serine/threonine kinase receptors, which, in turn, propagate the signal by activating downstream Smad-dependent and Smad-independent pathways. TGF-β signaling plays a key role in the tumor progression and metastasis of many different types of tumor cells. The overexpression of TGF-β has been reported in most tumor types. In human PC, increased levels of TGF-β have been significantly associated with venous invasion, advanced tumor stages, progressive disease, shorter patient survival duration, and liver metastases. The effect of TGF-β is not limited to the tumor cells themselves in vivo. It has been recently highlighted its paracrine mechanism in the interactions between various cell populations in the tumor microenvironment. Hence its pleiotropic role in PC biology, the TGF-β Smad- dependent and independent pathways represent a critical area for the development of novel strategies for the treatment of PC patients. We initially hypothesized that targeting the Smad- dependent pathways by inhibiting TRI/II kinase activity with the novel inhibitor LY2109761 would suppress PC growth and metastatic processes. The effect of LY2109761 was evaluated on soft-agar growth, migration, invasion using a fibroblast coculture model, and anoikis by annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival and reduction of spontaneous metastasis was evaluated in an orthotopic murine model of metastatic PC expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether PC cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft-agar growth; suppressed both basal and TGF-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761 in combination with gemcitabine (GEM) significantly reduced the tumor burden, prolonged the survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Resistance to chemotherapeutic drugs poses one of the greatest challenges in PC research. The Smad-independent effector TGF-- Activated Kinase- 1 (TAK-1) is a MAP3K essential for the activation of NFB and AP1 transcription factors. cIAP2 is a NFB and AP1 target gene that regulates apoptosis by direct inhibition of caspases. We hypothesized that TAK1 is responsible for PC chemoresistance by regulating NFB- and AP-1- mediated transcription of cIAP2. The expression of TAK-1 in PC cell lines was studied by Western blot. TAK-1 expression was silenced by shRNA in AsPC-1, Panc-1, and Panc-28 cell lines. NFB and AP-1 activation was analyzed by EMSA. Apoptosis was quantified using cleavage of caspase-3 and PARP1 and DNA fragmentation. MTT assays were used to assess the in vitro chemopotentiation of GEM, oxaliplatin (OX), and SN-38. TAK-1 kinase activity was targeted using an orally available small molecule selective inhibitor (Ki in enzymatic assay: TAK1 = 13 nM; p38 > 20 μM; IKK > 20 μM) provided by Eli Lilly Research Labs. In vivo activity of the TAK-1 inhibitor alone and in combination with GEM was evaluated in an orthotopic nude mouse model with luciferase-expressing AsPC-1 PC cells. TAK-1 protein was overexpressed in all PC cell lines studied but not in normal pancreatic ductal epithelial cells. shRNA knockdown of TAK-1 completely suppressed both NFB and AP1 DNA binding activities. As a result, cIAP2 expression was completely suppressed, inducing a proapoptotic phenotype as demonstrated by higher levels of cleaved caspase-3 and PARP-1 and by DNA fragmentation. shRNA silencing of TAK-1 in AsPC-1, Panc-1, and Panc-28 cell lines resulted in significantly higher in vitro sensitivity to GEM, OX, and SN-38, compared to the respective control cell lines. In vitro, the TAK1 inhibitor alone demonstrated potent cytotoxic activity (IC50 5-39 nM) and suppressed NFB DNA binding activity in all three PC cell lines studied. In combination, the TAK1 inhibitor strongly potentiated the cytotoxic activities of GEM, OX, or SN-38 in all three PC cell lines. In nude mice, oral administration of the TAK-1 inhibitor plus GEM significantly reduced tumor burden and prolonged mice survival. In our study we demonstrated that targeting the Smad- dependent pathways by inhibiting TRI/II kinase activity on PC cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent PC metastasis. Our study is the first to demonstrate that the genetic silencing or inhibition of the Smad-independent effector TAK1 is a valid approach to revert in vivo the intrinsic chemoresistance of PC. Specifically designed clinical trials need to be conducted to evaluate the therapeutic effect of LY2109761 as a neoadjuvant treatment in patients with resectable PC. The TAK-1 inhibitor used in this study is an exciting drug that warrants further development for the treatment of advanced PC.

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