A p53 Super-tumor Suppressor Reveals a Tumor Suppressive p53-Ptpn14-Yap Axis in Pancreatic Cancer.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells.

Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection.

Efficient generation of pancreatic ß-like cells from the mouse gallbladder.

Direct reprogramming is a promising approach for the replacement of ß cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to ß cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional ß cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of ß cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with ß cells and had a metabolic gene expression profile resembling neonatal ß cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.

Dicer regulates differentiation and viability during mouse pancreatic cancer initiation.

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

Pancreatic islet ß-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional ß-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native ß-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark ß-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation.

Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet ß-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.

Genetic interplays between Msx2 and Foxn1 are required for Notch1 expression and hair shaft differentiation.

Hair shafts are produced from stem cells located in the bulge. Our knowledge of the genetic pathways regulating cell fate acquisition in the immediate descendents of these stem cells, and fate maintenance in their committed progeny, is still incomplete. One pathway involved in fate maintenance within the hair matrix is the Notch pathway. Here we use compound genetic mutants to demonstrate that two transcription factors, Msx2 and Foxn1, are both required to maintain Notch1 expression in the hair follicle matrix. In their absence, Notch1 is markedly reduced in hair matrix; as a consequence, medulla and inner root sheath (IRS) differentiation is impaired. Our studies also suggest that Foxn1 is a direct activator of the Notch1 promoter activity through one or more putative Foxn1 consensus binding sites located within the 4.7 kb of mouse Notch1 promoter. Since recombinant human BMP4 can induce Foxn1 expression in Msx2-deficient hair follicles, and that their effect on cortical keratin expression appears synergistic, we suggest that these two genes function in parallel pathways downstream of BMP signaling and upstream of Notch1. Independent from their role in Notch activation, Msx2 and Foxn1 also contribute to the expression of several cortical and cuticle keratins. The impact of these additional defects is the complete loss of all visible external hairs, not seen in Notch1 mutants. Our results position Msx2 and Foxn1 upstream of Notch1 within the hair matrix and demonstrate that together these factors play a pivotal role in IRS, cortex and medulla differentiation.

Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity.

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j-dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes.

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfbeta and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfbeta ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfbeta receptors, consistent with paracrine Tgfbeta signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.

Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA.

Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. We report here the genomic structure of the human PTK7 gene by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon-intron structure of the PTK7 gene in the region that encodes the catalytic domain was distinct from those of other RPTKs with strong homology. The 5'-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, it was demonstrated that the 883-bp 5'-flanking sequence is functional as a promoter of the PTK7 gene. We identified four new splicing variants in testis that could be derived from alternative splicing of exons 8-10, 10, a part of 12-13, and 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. Our findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling.

Genetic mosaic analysis indicates that the bulb region of coat hair follicles contains a resident population of several active multipotent epithelial lineage progenitors.

The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia.