YAP1 and TAZ Control Pancreatic Cancer Initiation in Mice by Direct Up-regulation of JAK-STAT3 Signaling.

BACKGROUND & AIMS: Pancreatitis is the most important risk factor for pancreatic ductal adenocarcinoma (PDAC). Pancreatitis predisposes to PDAC because it induces a process of acinar cell reprogramming known as acinar-to-ductal metaplasia (ADM)-a precursor of pancreatic intraepithelial neoplasia lesions that can progress to PDAC. Mutations in KRAS are found at the earliest stages of pancreatic tumorigenesis, and it appears to be a gatekeeper to cancer progression. We investigated how mutations in KRAS cooperate with pancreatitis to promote pancreatic cancer progression in mice. METHODS: We generated mice carrying conditional alleles of Yap1 and Taz and disrupted Yap1 and Taz using a Cre-lox recombination strategy in adult mouse pancreatic acinar cells (Yap1fl/fl;Tazfl/fl;Ela1-CreERT2). We crossed these mice with LSL-KrasG12D mice, which express a constitutively active form of KRAS after Cre recombination. Pancreatic tumor initiation and progression were analyzed after chemically induced pancreatitis. We analyzed pancreatic tissues from patients with pancreatitis or PDAC by immunohistochemistry. RESULTS: Oncogenic activation of KRAS in normal, untransformed acinar cells in the pancreatic tissues of mice resulted in increased levels of pancreatitis-induced ADM. Expression of the constitutive active form of KRAS in this system led to activation of the transcriptional regulators YAP1 and TAZ; their function was required for pancreatitis-induced ADM in mice. The JAK-STAT3 pathway was a downstream effector of KRAS signaling via YAP1 and TAZ. YAP1 and TAZ directly mediated transcriptional activation of several genes in the JAK-STAT3 signaling pathway; this could be a mechanism by which acinar cells that express activated KRAS become susceptible to inflammation. CONCLUSIONS: We identified a mechanism by which oncogenic KRAS facilitates ADM and thereby generates the cells that initiate neoplastic progression. This process involves activation of YAP1 and TAZ in acinar cells, which up-regulate JAK-STAT3 signaling to promote development of PDAC in mice.

Requirement for DNA ligase IV during embryonic neuronal development.

The embryonic ventricular and subventricular zones (VZ/SVZ) contain the neuronal stem and progenitor cells and undergo rapid proliferation. The intermediate zone (IZ) contains nonreplicating, differentiated cells. The VZ/SVZ is hypersensitive to radiation-induced apoptosis. Ablation of DNA non-homologous end-joining (NHEJ) proteins, XRCC4 or DNA ligase IV (LigIV), confers ataxia telangiectasia mutated (ATM)-dependent apoptosis predominantly in the IZ. We examine the mechanistic basis underlying these distinct sensitivities using a viable LigIV (Lig4(Y288C)) mouse, which permits an examination of the DNA damage responses in the embryonic and adult brain. Via combined analysis of DNA breakage, apoptosis, and cell-cycle checkpoint control in tissues, we show that apoptosis in the VZ/SVZ and IZ is activated by low numbers of DNA double-strand breaks (DSBs). Unexpectedly, high sensitivity in the VZ/SVZ arises from sensitive activation of ATM-dependent apoptosis plus an ATM-independent process. In contrast, the IZ appears to be hypersensitive to persistent DSBs. NHEJ functions efficiently in both compartments. The VZ/SVZ and IZ regions incur high endogenous DNA breakage, which correlates with VZ proliferation. We demonstrate a functional G(2)/M checkpoint in VZ/SVZ cells and show that it is not activated by low numbers of DSBs, allowing damaged VZ/SVZ cells to transit into the IZ. We propose a novel model in which microcephaly in LIG4 syndrome arises from sensitive apoptotic induction from persisting DSBs in the IZ, which arise from high endogenous breakage in the VZ/SVZ and transit of damaged cells to the IZ. The VZ/SVZ, in contrast, is highly sensitive to acute radiation-induced DSB formation.

The Essential DNA Damage Response Complex MRN Is Dispensable for the Survival and Function of Purkinje Neurons.

MRE11, RAD50, and NBS1 form the MRN complex in response to DNA damage to activate ATM, a gene responsible for Ataxia-Telangiectasia (A-T). Loss of any components of the MRN complex compromises cell life. Mutations in MRE11, RAD50, and NBS1 cause human genomic instability syndromes Ataxia-Telangiectasia-like disorder (A-TLD), NBS-like disorder (NBSLD), and Nijmegen Breakage Syndrome (NBS), respectively. Among other pathologies, neuronal deficits, including microcephaly, intellectual disabilities, and progressive cerebellar degeneration, are common in these disorders. Nbs1 deletion in neural stem cells of mouse models resulted in cerebellar atrophy and ataxia, mimicking the A-T syndrome suggesting an etiological function of MRN-mediated DDR in neuronal homeostasis and neuropathology. Here we show that deletion of Nbs1 or Mre11 specifically in Purkinje neurons of mouse models (Nbs1-PCΔ and Mre11-PCΔ, respectively) is compatible with cerebellar development. Deleting Nbs1 in Purkinje cells disrupts the cellular localization pattern of MRE11 or RAD50 without inducing apparent DNA damage, albeit impaired DNA damage response (judged by 53BP1 focus formation) to ionizing radiation (IR). However, neither survival nor morphology of Purkinje cells and thus locomotor capabilities is affected by Nbs1 deletion under physiological conditions. Similarly, deletion of Mre11 in Purkinje cells does not affect the numbers or morphology of Purkinje cells and causes no accumulation of DNA damage. Mre11-deleted Purkinje cells have regular intrinsic neuronal activity. Taken together, these data indicate that the MRN complex is not essential for the survival and functionality of postmitotic neurons such as Purkinje cells. Thus, cerebellar deficits in MRN defect-related disorders and mouse models are unlikely to be a direct consequence of loss of these factors compromising DDR in postmitotic neurons such as Purkinje cells.

Loss of Fbw7 reprograms adult pancreatic ductal cells into α, δ, and ß cells.

The adult pancreas is capable of limited regeneration after injury but has no defined stem cell population. The cell types and molecular signals that govern the production of new pancreatic tissue are not well understood. Here, we show that inactivation of the SCF-type E3 ubiquitin ligase substrate recognition component Fbw7 induces pancreatic ductal cells to reprogram into α, δ, and ß cells. Loss of Fbw7 stabilized the transcription factor Ngn3, a key regulator of endocrine cell differentiation. The induced ß cells resemble islet ß cells in morphology and histology, express genes essential for ß cell function, and release insulin after glucose challenge. Thus, loss of Fbw7 appears to reawaken an endocrine developmental differentiation program in adult pancreatic ductal cells. Our study highlights the plasticity of seemingly differentiated adult cells, identifies Fbw7 as a master regulator of cell fate decisions in the pancreas, and reveals adult pancreatic duct cells as a latent multipotent cell type.

DNA damage response in microcephaly development of MCPH1 mouse model.

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV-VI), while generate less late-born neurons that form a thinner outer layer (II-III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.

Astrocyte dysfunction associated with cerebellar attrition in a Nijmegen breakage syndrome animal model.

Nijmegen breakage syndrome (NBS) is a genomic instability disorder caused by hypomorphic mutations in the Nbs1 gene. When Nbs1 is conditionally inactivated in the central nervous system of mice (Nbs1-CNS-Δ), they suffer from severe cerebellar atrophy, ataxia, and white matter damage. Here, we show that conditional inactivation of the murine Nbs1 gene has a profound effect on the integrity and the functionality of the glial cells, which suggests their crucial role in the pathogenesis of NBS. Interestingly, in Nbs1-CNS-Δ mice, the dramatic reduction in the numbers of Purkinje and granule cells was also linked to a reduction of microglial cells but not to astrocytes (GFAP+), suggesting an impairment in astrocytic functionality. Nbs1 levels were dramatically reduced in adult astrocyte isolated from Nbs1-CNS-Δ mice, suggesting a major role in cerebellar pathology. In order to investigate the effect of Nbs1 deletion on astrocyte activity, we investigated glutamine synthetase levels in astrocyte and discovered 40% reduction as compared to WT. Furthermore, we found a significant reduction in the secretion of neurotrophic factors, such as brain-derived neurotrophic factor and neurotrophin 3. Understanding the contribution of malfunctioning astrocytes to the etiology of NBS can elucidate a hitherto unknown aspect of this disorder.

MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1-Cdc25 pathway.

Primary microcephaly 1 is a neurodevelopmental disorder caused by mutations in the MCPH1 gene, whose product MCPH1 (also known as microcephalin and BRIT1) regulates DNA-damage response. Here we show that Mcph1 disruption in mice results in primary microcephaly, mimicking human MCPH1 symptoms, owing to a premature switching of neuroprogenitors from symmetric to asymmetric division. MCPH1-deficiency abrogates the localization of Chk1 to centrosomes, causing premature Cdk1 activation and early mitotic entry, which uncouples mitosis and the centrosome cycle. This misorients the mitotic spindle alignment and shifts the division plane of neuroprogenitors, to bias neurogenic cell fate. Silencing Cdc25b, a centrosome substrate of Chk1, corrects MCPH1-deficiency-induced spindle misalignment and rescues the premature neurogenic production in Mcph1-knockout neocortex. Thus, MCPH1, through its function in the Chk1-Cdc25-Cdk1 pathway to couple the centrosome cycle with mitosis, is required for precise mitotic spindle orientation and thereby regulates the progenitor division mode to maintain brain size.