Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.

Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death.

Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.

The checkpoint kinase ATM (ataxia telangiectasia mutated) transduces genomic stress signals to halt cell cycle progression and promote DNA repair in response to DNA damage. Here, we report the characterisation of an essential cofactor for ATM, ATMIN (ATM INteracting protein). ATMIN interacts with ATM through a C-terminal motif, which is also present in Nijmegen breakage syndrome (NBS)1. ATMIN and ATM co-localised in response to ATM activation by chloroquine and hypotonic stress, but not after induction of double-strand breaks by ionising radiation (IR). ATM/ATMIN complex disruption by IR was attenuated in cells with impaired NBS1 function, suggesting competition of NBS1 and ATMIN for ATM binding. ATMIN protein levels were reduced in ataxia telangiectasia cells and ATM protein levels were low in primary murine fibroblasts lacking ATMIN, indicating reciprocal stabilisation. Whereas phosphorylation of Smc1, Chk2 and p53 was normal after IR in ATMIN-deficient cells, basal ATM activity and ATM activation by hypotonic stress and inhibition of DNA replication was impaired. Thus, ATMIN defines a novel NBS1-independent pathway of ATM signalling.

Meiosis is a germ cell-specific division that is indispensable for the generation of haploid gametes. However, the regulatory mechanisms of meiotic initiation remain elusive. Here, we report that the Wdr62 (WD40-repeat protein 62) is involved in meiotic initiation as a permissive factor rather than an instructive factor. Knock-out of this gene in a mouse model resulted in female meiotic initiation defects. Further studies demonstrated that Wdr62 is required for RA-induced Stra8 expression via the activation of JNK signaling, and the defects in meiotic initiation from Wdr62-deficient female mice could be partially rescued by JNK1 overexpression in germ cells. More importantly, two novel mutations of the WDR62 gene were detected in patients with premature ovarian insufficiency (POI), and these mutations played dominant-negative roles in regulating Stra8 expression. Hence, this study revealed that Wdr62 is involved in female meiotic initiation via activating JNK signaling, which displays a novel mechanism for regulating meiotic initiation, and mutation of WDR62 is one of the potential etiologies of POI in humans.

The tumor suppressor FBW7 targets oncoproteins such as c-MYC for ubiquitylation and is mutated in several human cancers. We noted that in a substantial percentage of colon cancers, FBW7 protein is undetectable despite the presence of FBW7 mRNA. To understand the molecular mechanism of FBW7 regulation in these cancers, we employed proteomics and identified the deubiquitinase (DUB) USP9X as an FBW7 interactor. USP9X antagonized FBW7 ubiquitylation, and Usp9x deletion caused Fbw7 destabilization. Mice lacking Usp9x in the gut showed reduced secretory cell differentiation and increased progenitor proliferation, phenocopying Fbw7 loss. In addition, Usp9x inactivation impaired intestinal regeneration and increased tumor burden in colitis-associated intestinal cancer. c-Myc heterozygosity abrogated increased progenitor proliferation and tumor burden in Usp9x-deficient mice, suggesting that Usp9x suppresses tumor formation by regulating Fbw7 protein stability and thereby reducing c-Myc. Thus, we identify a tumor suppressor mechanism in the mammalian intestine that arises from the posttranslational regulation of FBW7 by USP9X independent of somatic FBW7 mutations.

Ductal cells have been proposed as a source of adult β cell neogenesis, but this has remained controversial. By combining lineage tracing, 3D imaging, and single-cell RNA sequencing (scRNA-seq) approaches, we show that ductal cells contribute to the β cell population over time. Lineage tracing using the Neurogenin3 (Ngn3)-CreERT line identified ductal cells expressing the endocrine master transcription factor Ngn3 that were positive for the δ cell marker somatostatin and occasionally co-expressed insulin. The number of hormone-expressing ductal cells was increased in Akita+/- diabetic mice, and ngn3 heterozygosity accelerated diabetes onset. scRNA-seq of Ngn3 lineage-traced islet cells indicated that duct-derived somatostatin-expressing cells, some of which retained expression of ductal markers, gave rise to β cells. This study identified Ngn3-expressing ductal cells as a source of adult β cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to β cell proliferation, maintains the adult islet β cell population.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. In vivo this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.

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 β cells. Loss of Fbw7 stabilized the transcription factor Ngn3, a key regulator of endocrine cell differentiation. The induced β cells resemble islet β cells in morphology and histology, express genes essential for β 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.

The ERK MAPK signalling pathway is a highly conserved kinase cascade linking transmembrane receptors to downstream effector mechanisms. To investigate the function of ERK in neurons, a constitutively active form of MEK1 (caMEK1) was conditionally expressed in the murine brain, which resulted in ERK activation and caused spontaneous epileptic seizures. ERK activation stimulated phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and augmented NMDA receptor 2B (NR2B) protein levels. Pharmacological inhibition of NR2B function impaired synaptic facilitation in area cornus ammonicus region 3 (CA3) in acute hippocampal slices derived from caMEK1-expressing mice and abrogated epilepsy in vivo. In addition, expression of caMEK1 caused phosphorylation of the transcription factor, cAMP response element-binding protein (CREB) and increased transcription of ephrinB2. EphrinB2 overexpression resulted in increased NR2B tyrosine phosphorylation, which was essential for caMEK1-induced epilepsy in vivo, since conditional inactivation of ephrinB2 greatly reduced seizure frequency in caMEK1 transgenic mice. Therefore, our study identifies a mechanism of epileptogenesis that links MAP kinase to Eph/Ephrin and NMDA receptor signalling.

Pancreatic ductal adenocarcinoma (PDAC) shows great cellular heterogeneity, with pronounced epithelial and mesenchymal cancer cell populations. However, the cellular hierarchy underlying PDAC cell diversity is unknown. Here we identify the tetraspanin CD9 as a marker of PDAC tumour-initiating cells. CD9high cells had increased organoid formation capability, and generated tumour grafts in vivo at limiting dilutions. Tumours initiated from CD9high cells recapitulated the cellular heterogeneity of primary PDAC, whereas CD9low cells produced only duct-like epithelial progeny. CD9 knockdown decreased the growth of PDAC organoids, and heterozygous CD9 deletion in Pdx1-Cre; LSL-KRasG12D; p53F/F mice prolonged overall survival. Mechanistically, CD9 promoted the plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in PDAC cells. Thus, our study identifies a PDAC subpopulation capable of initiating PDAC and giving rise to PDAC heterogeneity, suggesting that the cellular diversity of PDAC is generated by PDAC stem cell differentiation.

Background and Aims: ALPPS (associating liver partition and portal vein ligation for staged hepatectomy), a novel 2-staged hepatectomy, dramatically accelerates liver regeneration and thus enables extensive liver tumor resection. The signaling networks underlying the ALPPS-induced accelerated regeneration process are largely unknown. Methods: We performed transcriptome profiling (TP) of liver tissue obtained from a mouse model of ALPPS, standard hepatectomy (68% model), and additional control surgeries (sham, PVL and Tx). We also performed TP using human liver biopsies (n = 5) taken from the occluded lobe and the future liver remnant (FLR) during the first step of ALPPS surgery (4-5 h apart). We used Oncofinder computational tools, which covers 378 ISPs, for unsupervised, unbiased quantification of ISP activity. Results: Gene expression cluster analysis revealed an ALPPS specific signature: the IGF1R Signaling Pathway (Cell survival), the ILK Pathway (Induced cell proliferation), and the IL-10 Pathway (Stability determination) were significantly enriched, whereas the activity of the Interferon Pathway (Transcription) was reduced (p < 0.05). Further, the PAK- and ILK-associated ISPs were activated at an earlier time point, reflecting significant acceleration of liver regeneration (p < 0.001). These pathways, which were also recovered in human liver biopsies, control cell growth and proliferation, inflammatory response, and hypoxia-related processes. Conclusions: ALPPS is not a straightforward addition of portal vein ligation (PVL) plus transection-it is more. The early stages of normal and accelerated liver regeneration are clearly discernible by a significantly increased and earlier activation of a small number of signaling pathways. Compounds mimicking these responses may help to improve the ALPPS method and further reduce the hospitalization time of the patient.

The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs), duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development.

Brown adipose tissue (BAT) is a specialized form of adipose tissue, able to increase energy expenditure by heat generation in response to various stimuli. Recently, its pathological activation has been implicated in the pathogenesis of cancer cachexia. To establish a causal relationship, we retrospectively investigated the longitudinal changes in BAT and cancer in a large FDG-PET/CT cohort.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterized, the mechanism of activation by Ras was elusive. Here we identify the uncharacterized ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras-Raf-MEK-ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 co-activator RACO-1, leading to RACO-1 protein stabilization. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation.

The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9-10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of β-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug screening.

The skin is a squamous epithelium that is continuously renewed by a population of basal layer stem/progenitor cells and can heal wounds. Here, we show that the transcription regulators YAP and TAZ localise to the nucleus in the basal layer of skin and are elevated upon wound healing. Skin-specific deletion of both YAP and TAZ in adult mice slows proliferation of basal layer cells, leads to hair loss and impairs regeneration after wounding. Contact with the basal extracellular matrix and consequent integrin-Src signalling is a key determinant of the nuclear localisation of YAP/TAZ in basal layer cells and in skin tumours. Contact with the basement membrane is lost in differentiating daughter cells, where YAP and TAZ become mostly cytoplasmic. In other types of squamous epithelia and squamous cell carcinomas, a similar control mechanism is present. By contrast, columnar epithelia differentiate an apical domain that recruits CRB3, Merlin (also known as NF2), KIBRA (also known as WWC1) and SAV1 to induce Hippo signalling and retain YAP/TAZ in the cytoplasm despite contact with the basal layer extracellular matrix. When columnar epithelial tumours lose their apical domain and become invasive, YAP/TAZ becomes nuclear and tumour growth becomes sensitive to the Src inhibitor Dasatinib.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.