USP7 controls NGN3 stability and pancreatic endocrine lineage development.

Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.

Active mesh and neural network pipeline for cell aggregate segmentation.

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Are There Specific Cancer Stem Cell Markers?

The cancer stem cell (CSC) model states that heterogeneous tumor cell populations are organized in a hierarchical manner, with a small population of CSCs at the apex. These CSCs are capable of self-renewal and giving rise to other cancer cell populations, conceptually analogous to the function of normal adult stem cells present in almost all organs. However, there has been significant controversy regarding the existence and identification of CSCs. We argue that technical differences in experimentation and CSC assays, CSC niche-dependency and plasticity, and CSC heterogeneity itself may explain some of the differences observed.

A small Rho GTPase RAB25 with a potential role in chemotherapy resistance in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the major human health challenges with minimal therapeutic benefits due to its late detection, and de novo - and acquired chemotherapy resistance. OBJECTIVE: In this work we unravel the potential pro-survival role of RAB25 in pancreatic cancer chemotherapy resistance and aim to identify if RAB25 is a prognostic marker of patients' survival in PDA. METHODS: We used RNA sequencing, shRNA mediated gene knockdown, BioGRID open repository of CRISPR screens (ORCS), GEPIA, kmplot.com, and cBioPortal.org databases to identify the role of RAB25 in PDA cell proliferation, chemotherapy response, expression in tumour versus normal tissues, and overall patients' survival. RESULTS: RNA sequencing show Rab25 to be one of the top upregulated genes in gemcitabine resistance mouse PDA cells. Knockdown of Rab25 in these cells enhanced gemcitabine toxicity. In addition, re-analysis of previously published CRISPR/Cas9 data confirm RAB25 to be responsible for chemotherapy resistance in KRASG12D mutant human pancreatic cancer cell line. Finally, we used publicly available TCGA datasets and identify the upregulation of RAB25 in tumour tissues compared to the adjacent normal tissue, co-occurrence of KRASG12 mutations with RAB25 amplifications, and poor patients' survival in cohorts with higher mRNA expression of RAB25. CONCLUSION: RAB25 expression is a prognostic marker for patient's survival and gemcitabine resistance in PDA.

Micro-CT acquisition and image processing to track and characterize pulmonary nodules in mice.

X-ray computed tomography is a reliable technique for the detection and longitudinal monitoring of pulmonary nodules. In preclinical stages of diagnostic or therapeutic development, the miniaturized versions of the clinical computed tomography scanners are ideally suited for carrying out translationally-relevant research in conditions that closely mimic those found in the clinic. In this Protocol, we provide image acquisition parameters optimized for low radiation dose, high-resolution and high-throughput computed tomography imaging using three commercially available micro-computed tomography scanners, together with a detailed description of the image analysis tools required to identify a variety of lung tumor types, characterized by specific radiological features. For each animal, image acquisition takes 4-8 min, and data analysis typically requires 10-30 min. Researchers with basic training in animal handling, medical imaging and software analysis should be able to implement this protocol across a wide range of lung cancer models in mice for investigating the molecular mechanisms driving lung cancer development and the assessment of diagnostic and therapeutic agents.

An intrinsic temporal order of c-JUN N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment.

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-JUN proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-JUN multisite phosphorylation has remained elusive. Here we show that c-JUN phosphorylation by JNK exhibits defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identify the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis reveals three c-JUN phosphorylation states: unphosphorylated c-JUN recruits the MBD3 repressor, serine63/73 doubly-phosphorylated c-JUN binds to the TCF4 co-activator, whereas the fully phosphorylated form disfavours TCF4 binding attenuating JNK signalling. Thus, c-JUN phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.

The roles of intratumour heterogeneity in the biology and treatment of pancreatic ductal adenocarcinoma.

Intratumour heterogeneity (ITH) has become an important focus of cancer research in recent years. ITH describes the cellular variation that enables tumour evolution, including tumour progression, metastasis and resistance to treatment. The selection and expansion of genetically distinct treatment-resistant cancer cell clones provides one explanation for treatment failure. However, tumour cell variation need not be genetically encoded. In pancreatic ductal adenocarcinoma (PDAC) in particular, the complex tumour microenvironment as well as crosstalk between tumour and stromal cells result in exceptionally variable tumour cell phenotypes that are also highly adaptable. In this review we discuss four different types of phenotypic heterogeneity within PDAC, from morphological to metabolic heterogeneity. We suggest that these different types of ITH are not independent, but, rather, can inform one another. Lastly, we highlight recent findings that suggest how therapeutic efforts may halt PDAC progression by constraining cellular heterogeneity.

GREM1 is required to maintain cellular heterogeneity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.

USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a potential therapeutic target in pancreatic cancer.

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we develop a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identifies USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition results in potent growth impairment in PDAC organoids, while normal pancreatic organoids are insensitive, and causes dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinates and stabilizes the HIF-1α transcription factor. PDAC is characterized by a severely hypoxic microenvironment, and USP25 depletion abrogates HIF-1α transcriptional activity and impairs glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1α axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited.

Tissue architecture in tumor initiation and progression.

The 3D architecture of tissues bearing tumors impacts on the mechanical microenvironment of cancer, the accessibility of stromal cells, and the routes of invasion. A myriad of intrinsic and extrinsic forces exerted by the cancer cells, the host tissue, and the molecular and cellular microenvironment modulate the morphology of the tumor and its malignant potential through mechanical, biochemical, genetic, and epigenetic cues. Recent studies have investigated how tissue architecture influences cancer biology from tumor initiation and progression to distant metastatic seeding and response to therapy. With a focus on carcinoma, the most common type of cancer, this review discusses the latest discoveries on how tumor architecture is built and how tissue morphology affects the biology and progression of cancer cells.

METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability.

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3'-untranslated Region (3'-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

The Jun-dependent axon regeneration gene program: Jun promotes regeneration over plasticity.

The regeneration-associated gene (RAG) expression program is activated in injured peripheral neurons after axotomy and enables long-distance axon re-growth. Over 1000 genes are regulated, and many transcription factors are upregulated or activated as part of this response. However, a detailed picture of how RAG expression is regulated is lacking. In particular, the transcriptional targets and specific functions of the various transcription factors are unclear. Jun was the first-regeneration-associated transcription factor identified and the first shown to be functionally important. Here we fully define the role of Jun in the RAG expression program in regenerating facial motor neurons. At 1, 4 and 14 days after axotomy, Jun upregulates 11, 23 and 44% of the RAG program, respectively. Jun functions relevant to regeneration include cytoskeleton production, metabolic functions and cell activation, and the downregulation of neurotransmission machinery. In silico analysis of promoter regions of Jun targets identifies stronger over-representation of AP1-like sites than CRE-like sites, although CRE sites were also over-represented in regions flanking AP1 sites. Strikingly, in motor neurons lacking Jun, an alternative SRF-dependent gene expression program is initiated after axotomy. The promoters of these newly expressed genes exhibit over-representation of CRE sites in regions near to SRF target sites. This alternative gene expression program includes plasticity-associated transcription factors and leads to an aberrant early increase in synapse density on motor neurons. Jun thus has the important function in the early phase after axotomy of pushing the injured neuron away from a plasticity response and towards a regenerative phenotype.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

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.

Ductal Ngn3-expressing progenitors contribute to adult ß cell neogenesis in the pancreas.

Ductal cells have been proposed as a source of adult ß 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 ß 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 ß cells. This study identified Ngn3-expressing ductal cells as a source of adult ß cell neogenesis in homeostasis and diabetes, suggesting that this mechanism, in addition to ß cell proliferation, maintains the adult islet ß cell population.

PARP1 Inhibitor Combined With Oxaliplatin Efficiently Suppresses Oxaliplatin Resistance in Gastric Cancer-Derived Organoids via Homologous Recombination and the Base Excision Repair Pathway.

Oxaliplatin (OXA) resistance in the treatment of different types of cancer is an important and complex problem. The culture of tumor organoids derived from gastric cancer can help us to provide a deeper understanding of the underlying mechanisms that lead to OXA resistance. In this study, our purpose was to understand the mechanisms that lead to OXA resistance, and to provide survival benefits to patients with OXA through targeted combination therapies. Using sequence analysis of OXA-resistant and non-OXA-resistant organoids, we found that PARP1 is an important gene that mediates OXA resistance. Through the patients' follow-up data, it was observed that the expression level of PARP1 was significantly correlated with OXA resistance. This was confirmed by genetic manipulation of PARP1 expression in OXA-resistant organoids used in subcutaneous tumor formation. Results further showed that PARP1 mediated OXA resistance by inhibiting the base excision repair pathway. OXA also inhibited homologous recombination by CDK1 activity and importantly made cancers with normal BRCA1 function sensitive to PARP inhibition. As a result, combination of OXA and Olaparib (PARP-1/2/3 inhibitor), inhibited in vivo and in vitro OXA resistant organoid growth and viability.