A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

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.

A Biomimetic Tumor Model of Heterogeneous Invasion in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a complex, heterogeneous, and genetically unstable disease. Its tumor microenvironment (TME) is complicated by heterogeneous cancer cell populations and strong desmoplastic stroma. This complex and heterogeneous environment makes it challenging to discover and validate unique therapeutic targets. Reliable and relevant in vitro PDAC tumor models can significantly advance the understanding of the PDAC TME and may enable the discovery and validation of novel drug targets. In this study, an engineered tumor model is developed to mimic the PDAC TME. This biomimetic model, named ductal tumor-microenvironment-on-chip (dT-MOC), permits analysis and experimentation on the epithelial-mesenchymal transition (EMT) and local invasion with intratumoral heterogeneity. This dT-MOC is a microfluidic platform where a duct of murine genetically engineered pancreatic cancer cells is embedded within a collagen matrix. The cancer cells used carry two of the three mutations of KRAS, CDKN2A, and TP53, which are key driver mutations of human PDAC. The intratumoral heterogeneity is mimicked by co-culturing these cancer cells. Using the dT-MOC model, heterogeneous invasion characteristics, and response to transforming growth factor-beta1 are studied. A mechanism of EMT and local invasion caused by the interaction between heterogeneous cancer cell populations is proposed.

BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice.

BATF functions in T cells and B cells to control the host response to antigen and promote the production of class switched immunoglobulins. In this study, we demonstrate that BATF expression increases rapidly, and transiently, following B cell stimulation and use an inducible murine model of BATF deletion to show that this induction is necessary, and sufficient, for immunoglobulin (Ig) class switch recombination (CSR). We examine two genes (Nfil3 and miR155gh) that are positively regulated, and one gene (Wnt10a) that is negatively regulated by BATF during CSR. These genes play essential roles in CSR and each impacts the expression and/or function of the others. Our observations allow these targets of BATF regulation to be positioned in a network upstream of the activation of germline transcripts (GLT) from the IgH locus and of transcriptional activation of Aicda - the gene encoding the enzyme directing Ig gene rearrangements. This work extends the knowledge of the molecular control of CSR and, importantly, positions the induction and function of BATF as an early event in this process.

Induced Mist1 expression promotes remodeling of mouse pancreatic acinar cells.

BACKGROUND & AIMS: Early embryogenesis involves cell fate decisions that define the body axes and establish pools of progenitor cells. Development does not stop once lineages are specified; cells continue to undergo specific maturation events, and changes in gene expression patterns lead to their unique physiological functions. Secretory pancreatic acinar cells mature postnatally to synthesize large amounts of protein, polarize, and communicate with other cells. The transcription factor MIST1 is expressed by only secretory cells and regulates maturation events. MIST1-deficient acinar cells in mice do not establish apical-basal polarity, properly position zymogen granules, or communicate with adjacent cells, disrupting pancreatic function. We investigated whether MIST1 directly induces and maintains the mature phenotype of acinar cells. METHODS: We analyzed the effects of Cre-mediated expression of Mist1 in adult Mist1-deficient (Mist1(KO)) mice. Pancreatic tissues were collected and analyzed by light and electron microscopy, immunohistochemistry, real-time polymerase chain reaction analysis, and chromatin immunoprecipitation. Primary acini were isolated from mice and analyzed in amylase secretion assays. RESULTS: Induced expression of Mist1 in adult Mist1(KO) mice restored wild-type gene expression patterns in acinar cells. The acinar cells changed phenotypes, establishing apical-basal polarity, increasing the size of zymogen granules, reorganizing the cytoskeletal network, communicating intercellularly (by synthesizing gap junctions), and undergoing exocytosis. CONCLUSIONS: The exocrine pancreas of adult mice can be remodeled by re-expression of the transcription factor MIST1. MIST1 regulates acinar cell maturation and might be used to repair damaged pancreata in patients with pancreatic disorders.

Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.

OBJECTIVE: Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated. DESIGN: Expression of HNF6 and Sox9 was measured by immunostaining in normal and diseased human pancreas. The function of the factors was tested in cultured cells and in mouse models of ADM by a combination of gain and loss of function experiments. RESULTS: Expression of HNF6 and Sox9 was ectopically induced in acinar cells in human ADM as well as in mouse models of ADM. HNF6 and, to a lesser extent, Sox9 were required for repression of acinar genes, for modulation of ADM-associated changes in cell polarity and for activation of ductal genes in metaplastic acinar cells. CONCLUSIONS: HNF6 and Sox9 are new biomarkers of ADM and constitute candidate targets for preventive treatment in cases when ADM may lead to cancer. This work also shows that ectopic activation of transcription factors may underlie metaplastic processes occurring in other organs.

Polo-like kinase 1 facilitates loss of Pten tumor suppressor-induced prostate cancer formation.

Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the molecular mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress and that nuclear function of Pten, but not its phosphatase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a critical regulator of the cell cycle. We show that overexpression of Plk1 correlates with genetic inactivation of Pten during prostate neoplasia formation. Significantly, we find that elevated Plk1 is critical for Pten-depleted cells to adapt to mitotic stress for survival and that reintroduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.

Nonproteolytic properties of murine alternatively spliced tissue factor: implications for integrin-mediated signaling in murine models.

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a ß3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via ß3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

Extensive pancreas regeneration following acinar-specific disruption of Xbp1 in mice.

BACKGROUND & AIMS: Progression of diseases of the exocrine pancreas, which include pancreatitis and cancer, is associated with increased levels of cell stress. Pancreatic acinar cells are involved in development of these diseases and, because of their high level of protein output, they require an efficient, unfolded protein response (UPR) that mediates recovery from endoplasmic reticulum (ER) stress following the accumulation of misfolded proteins. METHODS: To study recovery from ER stress in the exocrine organ, we generated mice with conditional disruption of Xbp1 (a principal component of the UPR) in most adult pancreatic acinar cells (Xbp1fl/fl). We monitored the effects of constitutive ER stress in the exocrine pancreas of these mice. RESULTS: Xbp1-null acinar cells underwent extensive apoptosis, followed by a rapid phase of recovery in the pancreas that included expansion of the centroacinar cell compartment, formation of tubular complexes that contained Hes1- and Sox9-expressing cells, and regeneration of acinar cells that expressed Mist1 from the residual, surviving Xbp1+ cell population. CONCLUSIONS: XBP1 is required for homeostasis of acinar cells in mice; ER stress induces a regenerative response in the pancreas that involves acinar and centroacinar cells, providing the needed capacity for organ recovery from exocrine pancreas disease.

The Xbp1-regulated transcription factor Mist1 restricts antibody secretion by restraining Blimp1 expression in plasma cells.

Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23-Cre Bhlha15 fl/fl mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared with Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 (Prdm1) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1 Gfp/+ Cd23-Cre Bhlha15 fl/fl mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.