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The integrin LFA-1 mediates activation-dependent leukocyte adhesion. The beta subunit cytoplasmic domain has been demonstrated previously to modulate the adhesiveness of LFA-1. To investigate whether the alpha subunit cytoplasmic domain is also involved in the regulation of LFA-1-adhesive function, we stably expressed cytoplasmic domain truncated forms of the alpha subunit in a Jurkat mutant (Jurkat-beta2.7) deficient in the endogenous LFA-1 alpha subunit and in K562 cells. Clones expressing similar levels of cell surface LFA-1 were tested for their ability to bind to immobilized ICAM-1. Truncation of the alpha subunit cytoplasmic domain before, but not after, the conserved GFFKR sequence motif resulted in constitutive ICAM-1 binding of both Jurkat-beta2.7 and K562 transfectants. However, truncation after the GFFKR motif reduced sensitivity to stimulation by PMA or stimulatory Abs. Internal deletion of the GFFKR motif, or point mutations of the Gly (G), the two Phe (F), or the Arg (R) in the GFFKR motif to Ala (A) rendered LFA-1 constitutively active. Mutation of the Lys (K) did not affect LFA-1 adhesion to ICAM-1. These findings indicate that the GFFKR motif maintains the low adhesive state of LFA-1, possibly by restraining the receptor conformation. We further demonstrate that the alpha subunit cytoplasmic domain and the conserved GFFKR motif are also required for efficient formation of LFA-1 alphabeta heterodimers.

Introduction Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Objectives Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. Methods The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Results Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Discussion Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression.

Metabotropic glutamate receptors (mGluRs), members of family 3 G protein-coupled receptors, play pivotal roles in the modulation of synaptic transmission and are important drug targets for various neurological diseases. The structures of the extracellular ligand-binding domain (ECD) of mGluRs and its changes upon ligand binding have been well studied by various techniques, including X-ray crystallography. In contrast, little is known about the structure and structural changes of the seven-transmembrane domain (TMD). Here we searched for constitutively active mutation (CAM) sites in the TMD of mGluR8 to get insight into the epicenter of the structural changes in TMD, a potential target for allosteric ligands. Mutational analyses based on the knowledge of activating mutations of calcium-sensing receptor showed the presence of several CAM sites in the TMD of mGluR8. Among them, the site at position 764 in helix V, where threonine is present, is unique in that any substitution resulted in elevation of the basal activity, and some substitutions caused a loss of responsiveness to agonist. Further comprehensive mutational analyses indicated that the additional mutation of the CAM site at position 663 in helix III, where a tyrosine residue is present, generated a revertant phenotype. Mutations at these sites also increased the agonist binding affinity, although these sites are far from the ECD. These results indicated that the specific pair of amino acids at these CAM sites forms an activation switch that stabilizes the inactive state of mGluR8 and mediates the signal flow from the ECD to the cytoplasmic G protein-interacting site.

Up to date, three thyroid-stimulating hormone receptor (TSHR) germline variants have been reported for which no functional consequences have been detected by in vitro characterizations. However, familial nonautoimmune hyperthyroidism and hot nodules are clearly associated with constitutively activating TSHR germline mutations. We describe a family with a new TSHR germline mutation that is associated with euthyroidism in 13 family members and hyperthyroidism in 1 family member.Mutation analysis of the TSHR gene was performed by denaturing gradient gel electrophoresis. TSHR constructs were characterized by determination of cell surface expression, 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation, and constitutive cAMP activity.A novel TSHR germline mutation (N372T) was found in a man who presented with thyrotoxicosis. The mutation was also detected in 13 family members, all of whom were euthyroid. Interestingly, an additional constitutively active somatic mutation (S281N) was identified on the second parental TSHR allele of the hyperthyroid index patient. Linear regression analysis showed a lack of constitutive activity for N372T. Moreover, coexpression studies of N372T with S281N did not reveal any evidence for a functional influence of N372T on the constitutively active mutation (CAM).N372T is unlikely to cause altered thyroid function. This is consistent with the finding that only the index patient with the additional somatic mutation S281N was hyperthyroid.

G protein-coupled receptors are classified into several families on the basis of their amino acid sequences and the members of the same family exhibit sequence similarity but those of different families do not. In family 1 GPCRs such as rhodopsin and adrenergic receptor, extensive studies have revealed the stimulus-dependent conformational change of the receptor: the rearrangement of transmembrane helices III and VI is essential for G protein activation. In contrast, in family 3 GPCRs such as metabotropic glutamate receptor (mGluR), the inter-protomer relocation upon ligand binding has been observed but there is much less information about the structural changes of the transmsmbrane helices and the cytoplasmic domains. Here we identified constitutively active mutation sites at the cytoplasmic borders of helices II and IV of mGluR8 and successfully inhibited the G protein activation ability by engineering disulfide cross-linking between these cytoplasmic regions. The analysis of all possible single substitution mutants of these residues revealed that some steric interactions around these sites would be important to keep the receptor protein inactive. These results provided the model that the conformational changes at the cytoplasmic ends of helices II and IV of mGluR are involved in the efficient G protein coupling.

Purpose: Factors affecting the efficacy of therapeutic monoclonal antibodies (mAb) directed to the epidermal growth factor receptor (EGFR) remain relatively unknown, especially in glioma. Experimental Design: We examined the efficacy of two EGFR-specific mAbs (mAbs 806 and 528) against U87MG-derived glioma xenografts expressing EGFR variants. Using this approach allowed us to change the form of the EGFR while keeping the genetic background constant. These variants included the de2-7 EGFR (or EGFRvIII), a constitutively active mutation of the EGFR expressed in glioma. Results: The efficacy of the mAbs correlated with EGFR number; however, the most important factor was receptor activation. Whereas U87MG xenografts expressing the de2-7 EGFR responded to therapy, those exhibiting a dead kinase de2-7 EGFR were refractory. A modified de2-7 EGFR that was kinase active but autophosphorylation deficient also responded, suggesting that these mAbs function in de2-7 EGFR–expressing xenografts by blocking transphosphorylation. Because de2-7 EGFR–expressing U87MG xenografts coexpress the wild-type EGFR, efficacy of the mAbs was also tested against NR6 xenografts that expressed the de2-7 EGFR in isolation. Whereas mAb 806 displayed antitumor activity against NR6 xenografts, mAb 528 therapy was ineffective, suggesting that mAb 528 mediates its antitumor activity by disrupting interactions between the de2-7 and wild-type EGFR. Finally, genetic disruption of Src in U87MG xenografts expressing the de2-7 EGFR dramatically enhanced mAb 806 efficacy. Conclusions: The effective use of EGFR-specific antibodies in glioma will depend on identifying tumors with activated EGFR. The combination of EGFR and Src inhibitors may be an effective strategy for the treatment of glioma.

Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-κB kinase-ε specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-κB kinase-ε did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-xL but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect.

Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.

The trp gene of Drosophila encodes a subunit of a class of Ca 2+ -selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate “store-operated Ca 2+ entry,” which is important in Ca 2+ homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.

The protein kinase BRaf plays an important role in the mitogen-activated protein kinase (MAPK) pathway initiated by Ras. Raf, after activated by Ras activates Erk and its downstream effectors to carry out normal cellular functions, which include differentiation, proliferation, survival and senescence. BRafV600E mutation has been implicated in 50-60% of all melanoma cases. It is a constitutively active mutation that has been linked to the progression of melanoma. Many microRNAs have been implicated in cancer proliferation and metastasis. Of special interest is the miR-10b, which is known to play a crucial role in tumor invasion and metastasis as previously observed in a mouse mammary tumor model. Our preliminary data show that miR-10b was upregulated in melanoma cell-lines that carried either N-Ras gain-of-function mutation or BRafV600E. BRafV600E knockdown in melanoma cell lines reduces the expression of TWIST, a transcriptional activator of miR-10b, as well as the expression of miR-10b. Further, BrafV600E is known to mediate senescence through several factors including the transcriptional regulator DMTF1. Concordantly, we detected an increase in the expression of DMTF1 on BRafV600E knockdown. Thus, we hypothesize that in melanoma, BRafV600E activates the expression of TWIST which then activates miR-10b, leading to inhibition of DMTF1 and ultimately a failure to bring about cell-cycle arrest. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5045. doi:1538-7445.AM2012-5045

To determine the function of the Dictyostelium Gα1 subunit during aggregation and multicellular development, we analyzed the phenotypes of gα1 null cells and strains overexpressing either wild-type Gα1 or two putative constitutively active mutations of Gα1. Strains overexpressing the wild-type or mutant Gα1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between Gα1 overexpression and expression of a putative constitutively active Gα1 subunit suggests that these phenotypes are due to increased Gα1 activity rather than resulting from a non-specific interference of other pathways. In contrast, gα1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing Gα1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a Gα1/lacZ reporter construct, we show that Gα1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of Gα1 overexpression and the cell-type-specific expression of Gα1 suggest that Gα1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation. During the aggregation phase of development, gα1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while Gα1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that Gα1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaf fected in gα1 null cells and cell lines overexpressing wild-type Gα1 or the Gα1(Q206L) putative dominant activating mutation. However, the Gα1(G45V) putative constitutively active mutation showed significantly reduced adenylyl cyclase activity in response to cAMP. All Gα1 mutant cell lines aggregated normally; however, aggregates of cells expressing Gα1(G45V) developed ring-like structures that then developed a polarity and a small mound-like structure before forming a slug. Immunoprecipitation results suggest that the G45V phenotypes may be due to altered properties of this subunit and its association with the βg subunit.

Abstract 577 Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like JAK family kinases and Src family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials. Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases and Src family kinases. In HEL92.1.7 cells with constitutively active mutation of JAK2, OPB-31121 treatment inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). Src-dependent constitutive phosphorylation of STAT3 was also inhibited by OPB-31121 without inhibition of Src in H1650 cells that had active mutantation of EGF receptor. In addition, STAT3 immunoprecipitated from OPB-31121-treated cells was neither phosphorylated by JAK2 nor Lyn, a Src family kinase, in vitro without decrease in auto phosphorylation of upstream kinases, OPB-31121 demonstrated strong growth inhibitory effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including myeloma, AML with JAK2 mutation and CML. It is revealed that STAT3 is constitutively activated by oncogenic autocrine of IL-6 pathway or tyrosine kinase signal from oncoprotein in these cell lines. We also demonstrated growth inhibition or reduction of cell lines including HEL92.1.7 (AML with JAK2 mutation, T/C: 16%), KU812 (CML, T/C: 2%), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation, T/C: 5.9%) in NOD/SCID mice. For further analyses, we used human leukemia model mice where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth reduction of leukemia cells of BCR-ABL-positive ALL (T/C: 4%, Figure B), CML-BC with T315I mutation in BCR-ABL (T/C: 15.3%), and AML (T/C: 15.9%). Notably, OPB-31121-induced growth reduction was extremely selective for leukemia cells. Normal hematopoietic cells of mice were hardly affected by OPB-31121, whereas, cytarabine showed non-specific growth suppression of both leukemia cells and normal hematopoietic cells (Figure C). The safety of OPB-31121 on normal hematopoietic cells was also confirmed by colony formation assay, where OPB-31121 hardly affected colony formation of human cord blood cells at 100 nM. For further analyses, we transplanted human cord blood cells into NOG mice and investigated the growth inhibitory effect of OPB-31121 on normal hematopoietic cells in vivo. No significant growth suppression of human normal hematopoietic cells was observed in OPB-31121 treated mice (T/C: 99.9%, Figure D). Taken together, we conclude that OPB-31121 holds promise as a non-myelosuppressive therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase I/II trial in Japan. Disclosures: Hayakawa: Otsuka Pharmaceutical Co. Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co. Ltd.: Employment. Sumida:otsuka Pharmaceutical Co. Ltd.: Employment. Naoe:Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

Glioma is the most common and deadly human brain tumor. Epidermal growth factor receptor (EGFR) amplification or constitutively active mutation (EGFRVIII) is one of the most frequent genetic lesions in gliomas. To identify target genes regulated by EGFR signaling, we performed expression array analysis and found that human guanylate binding protein-1 (GBP1), a large GTPase that is induced by inflammatory cytokines, is one of the predominant genes induced by EGFR/EGF or EGFR VIII kinase activity. Our results demonstrate that EGF/EGFR signaling could dramatically induce GBP1 expression at both the mRNA and protein level through the p38 MAPK signaling cascade that is distinct from Stat1 signaling used by interferon γ. Moreover, we identified IRF1 and YY1 as downstream transcriptional regulator for GBP1 enhancement by EGFR/p38 signaling. Interestingly, we show that GBP1 is required for EGFR-mediated matrix metallopeptidase-1 (MMP1) expression, and plays a role in glioma cell invasion in vitro. Overexpression of GBP1 enhances glioma cell invasion through MMP1 induction, which was independent of GBP1 GTPase activity. In contrast, siRNA-mediated knockdown of GBP1 inhibited MMP1 expression and in turn blocked cell invasion as well. Consistently, we found that GBP1 is overexpressed and positively correlated with EGFR/EGFRVIII and MMP1 expression in human glioma clinical samples and glioma cell lines, particularly in neural subtypes of GBM as demonstrated by TCGA database analysis. Therefore, Our results suggest that the dysregulation of EGFR-mediated GBP1 expression may contribute to glioma invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 265.

Glioma is the most common and deadly human brain tumor. Epidermal growth factor receptor (EGFR) amplification or constitutively active mutation (EGFRVIII) is one of the most frequent genetic lesions in gliomas. To identify target genes regulated by EGFR signaling, we performed expression array analysis and found that human guanylate binding protein-1 (GBP-1), a large GTPase that is induced by inflammatory cytokines, is one of the predominant genes induced by EGFR/EGF or EGFR VIII kinase activity. Our results demonstrate that EGF/EGFR signaling could dramatically induce Gbp-1 expression at both the mRNA and protein level through the p38 kinase signaling cascade that is distinct from Stat1 signaling used by interferon gamma. Interestingly, we show that Gbp-1 is required for EGFR-mediated matrix metallopeptidase-1 (MMP1) expression, and plays a role in glioma cell invasion in vitro. Overexpression of Gbp-1 induced MMP1 expression in glioma cells, which was independent of Gbp-1 GTPase activity. In contrast, siRNA-mediated knockdown of Gbp-1 inhibited MMP1 expression and in turn blocked cell invasion as well. Consistently, we found that GBP-1 is overexpressed and positively correlated with EGFR/EGFRVIII and MMP1 expression in human glioma clinical samples and glioma cell lines. Therefore, this study suggests that Gbp-1 may be a novel therapeutic target in glioma. Citation Information: Cancer Res 2009;69(23 Suppl):A4.

Inactivation of the Transforming Growth Factor Beta (TGFβ) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1γ) has recently been proposed to be involved in TGFβ signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1γ in pancreatic carcinogenesis. Using conditional Tif1γ knockout mice (Tif1γlox/lox), we selectively abrogated Tif1γ expression in the pancreas of Pdx1-Cre;Tif1γlox/lox mice. We also generated Pdx1-Cre;LSL-KrasG12D;Tif1γlox/lox mice to address the effect of Tif1γ loss-of-function in precancerous lesions induced by oncogenic KrasG12D. Finally, we analyzed TIF1γ expression in human pancreatic tumors. In our mouse model, we showed that Tif1γ was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-KrasG12D;Smad4lox/lox mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1γ don't have strictly redundant functions. Finally, we report that TIF1γ expression is markedly down-regulated in human pancreatic tumors by quantitative RT–PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1γ is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1γ in the multifaceted functions of TGFβ in carcinogenesis and development., Author Summary Inactivation of the TGFβ tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC), a devastating malignancy. Transcriptional Intermediary Factor 1γ (TIF1γ) has recently been proposed to be involved in TGFβ signaling, a pathway inactivated in virtually all cases of this malignancy. To address the role of TIF1γ in pancreatic carcinogenesis, we used conditional Tif1γ knockout mice. In a genetic background expressing a constitutively active mutation of KRAS oncogene (KrasG12D) recurrently found in patients with PDAC, Tif1γ inactivation induces pancreatic precancerous lesions resembling those observed in the absence of Smad4, a key player involved TGFβ signal transduction. This observation strengthens the notion that TIF1γ plays an active role in TGFβ signaling. Interestingly, we also found that TIF1γ expression was markedly down-regulated in human pancreatic tumors supporting the relevance of our findings to human malignancy. Characterization of new players involved in the outbreak of early pancreatic lesions that will eventually evolve into invasive pancreatic cancer is crucial to detect the disease earlier and eventually develop new therapeutic drugs.