The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras G12D mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo. Additionally, Smo-deleted fibroblasts stimulated the growth of Kras G12D /Tp53 R172H pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras G12D -driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knockin mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten FV ), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten FV/FV embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten FV/+ mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organselective cancer development in vivo.

The yeast transcriptional activator Gal4 localizes to UAS GAL sites even in the absence of galactose but cannot activate transcription due to an association with the Gal80 protein. By 4 min after galactose addition, Gal4-activated gene transcription ensues. It is well established that this rapid induction arises through a galactose-triggered association between the Gal80 and Gal3 proteins that decreases the association of Gal80 and Gal4. How this happens mechanistically remains unclear. Strikingly different hypotheses prevail concerning the possible roles of nucleocytoplasmic distribution and trafficking of Gal3 and Gal80 and where in the cell the initial Gal3-Gal80 association occurs. Here we tested two conflicting hypotheses by evaluating the subcellular distribution and dynamics of Gal3 and Gal80 with reference to induction kinetics. We determined that the rates of nucleocytoplasmic trafficking for both Gal80 and Gal3 are slow relative to the rate of induction. We find that depletion of the nuclear pool of Gal3 slows the induction kinetics. Thus, nuclear Gal3 is critical for rapid induction. Fluorescence-recovery-after-photobleaching experiments provided data suggesting that the Gal80-Gal4 complex exhibits kinetic stability in the absence of galactose. Finally, we detect Gal3 at the UAS GAL only if Gal80 is covalently linked to the DNA-binding domain. Taken altogether, these new findings lead us to propose that a transient interaction of Gal3 with Gal4-associated Gal80 could explain the rapid response of this system. This notion could also explain earlier observations.

The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.