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An emerging model in cancer biology supports a dual role for TGFβ signaling in tumorigenesis, acting as a tumor suppressor in early stages and a strong promoter of cell proliferation, migration and invasion in advanced tumor stages.1,2 TGFβ blocks cell proliferation in untransformed cells through the induction of a cell cycle arrest at late G1 phase.3 Two critical molecular events underlie TGFβ anti-proliferative response: the initial transcriptional repression of the basic helix-loop-helix leucine zipper transcription factor c-Myc and the subsequent induction of the cyclin dependent kinase inhibitors p15Ink4b and p21Cip1.3,4 The down-regulation of c-Myc serves two major purposes - deprive the cell of growth-promoting functions by downstream targets (e.g. cyclin D) and to facilitate the induction of the cell cycle regulators, the Cdk inhibitors p15Ink4b and p21Cip1. c-Myc repression by TGFβ requires the receptor mediated activation of a Smad3-4 complex to transduce its stimulus into the nucleus. Here, the Smads complex with the transcription factors E2F4/5 and DP1, and corepressor p107 to represses c-Myc promoter through the binding to the TIE element (TGFβ-inhibitory element) located upstream of the P2 transcription initiation site of the c-Myc gene.7 An alternative Smad-dependent c-Myc/TIE repression mechanism is mediated by Smad3-KLF11 complex; KLF11 is a Sp/KLF-like repressor that silences target gene promoters through recruitment of Sin3A/HDAC corepressor complexes.8 Ligand activation of the TGFβ pathway promotes KLF11 interaction with Smad3 and binding to the TIE element. This alternative silencing pathway is interesting as KLF11 itself is an early TGFβ-response gene, thus implicating a self-enabling mechanism whereby Smads induces expression of its partner protein. Together these findings define the TGFβ growth inhibitory response as a Smad-dependent function requiring the formation of transcriptional repressor complexes at the promoter of the c-Myc oncogene.
During carcinogenesis, tumor cells change their transcriptional responsiveness to TGFβ and virtually all epithelial-derived cancer cells become resistant to the growth inhibitory effects due to either mutational or functional inactivation of the TGFβ-Smad pathway. Interestingly, impaired Smad signaling only causes an incomplete loss of TGFβ growth inhibitory function while other tumor-related functions remain unaffected. Depending on the cell type and the activation status of the cell, TGFβ then signals through Smad-independent pathways (e.g. PI3K and MAPK pathways) to promote the acquisition of a mesenchymal phenotype and stimulate tumor cell migration. In addition, TGFβ can switch to a growth promoter pathway in epithelial-derived cancer cells.1,9 Although this cellular event is clearly established, the molecular mechanisms underlying the TGFβ switch to a growth-promoter in cancer cells has not been characterized until recently. Singh and colleagues reported the existence of a novel TGFβ downstream pathway operating on the c-Myc gene to stimulate its expression, cell cycle transition and tumor growth.9 TGFβ induces expression and nuclear accumulation of Nuclear Factor of Activated T (NFAT) c1 and c2, two members of the NFAT transcription factor family, which comprises four members of Ca2/calcineurin regulated proteins particularly recognized for their central roles in gene regulation during T-lymphocyte activation.10 However, a multitude of studies established that NFAT proteins are also expressed in cells outside the immune system, where they participate in the regulation of the expression of genes influencing cell growth and differentiation. Emerging evidence supports a key role for NFATc1 and NFATc2 during carcinogenesis by regulating crucial aspects of neoplastic transformation and tumor progression.10 Both isoforms are frequently overexpressed and active in epithelial malignancies, and are associated with a highly malignant and aggressive phenotype.11
In resting cells, NFAT proteins are located in the cytoplasm in a hyperphosphorylated, inactive form. Under these conditions, NFAT phosphorylation is maintained bythe combined action of several kinases, including CK1 and DYRK2, which phosphorylate specific serine residues in NFAT regulatory domains. Signaling through calcium/calcineurin results in NFAT proteins dephosphorylation, causing a conformational switch that unmasks their nuclear localization sequence and allows their translocation to the nucleus, where they bind to their target genes either as homodimers, heterodimers or through interaction with other transcription factors.10,11 Based on the work by Singh et al., it is apparent that NFAT factors are also key players in the TGFβ switch from a growth suppressor to a promoter of cell proliferation.9 Induction and activation of NFATc1 and NFATc2 occurs in a Smad independent manner but requires activation of the calcineurin phosphatase. Upon activation and nuclear translocation, NFAT factors accumulate in the nucleus and displace pre-existing Smad3 repressor complexes from the TIE element. Upon promoter binding, NFAT initiates p300-dependent histone acetylation, and creates a local chromatin structure permissive for the inducible recruitment of Ets-like gene (ELK)-1, a protein required for maximal activation of the c-Myc promoter. NFAT genetic silencing not only prevents c-Myc induction and proliferation, but also restores TGFβ growth suppressor functions in cancer cells as indicated by downregulation of D-type cyclins and a halt of cancer cells in the G1 cell cycle phase. In conclusion, these results positioned NFAT transcription factors as a novel class of TGFβ downstream effectors with a key function in cell cycle control and growth in cancer; and identify these transcription factors as novel therapeutic target for tumors with an active oncogenic TGFβ signaling.
V.E. is supported by the Deutsche Forschungsgemeinschaft (DFG, SFB-TR17), the LOEWE-Schwerpunkt “Tumor and Inflammation” and the Max Eder programme of the German Cancer Research Foundation (Deutsche Krebshilfe, 70-3022-El I). M.E.F-.Z. is supported by the Division of Oncology Research, Mayo Clinic Cancer Center, Miles and Shirley Fiterman Center for Digestive Disease, CA136526, Mayo Clinic Pancreatic SPORE P50 CA102701, and Mayo Clinic Center for Cell Signaling in Gastroenterology NIDDK P30 DK084567.