Maturity onset diabetes of the young type 3 (MODY3) is a consequence of heterozygous germline mutation in HNF1A. A subtype of hepatocellular adenoma (HCA) is also caused by biallelic somatic HNF1A mutations (H-HCA), and rare HCA may be related to MODY3. To better understand a relationship between the development of MODY3 and HCA, we compared both germline and somatic spectra of HNF1A mutations.

Genetic studies have shown that the tuberous sclerosis complex (TSC) 1-TSC2-mammalian target of Rapamycin (mTOR) and the Hippo-Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. Whether and how mTOR regulates YAP remains elusive. Here we describe a novel mouse model of TSC which develops renal mesenchymal lesions recapitulating human perivascular epithelioid cell tumors (PEComas) from patients with TSC. We identify that YAP is up-regulated by mTOR in mouse and human PEComas. YAP inhibition blunts abnormal proliferation and induces apoptosis of TSC1-TSC2-deficient cells, both in culture and in mosaic Tsc1 mutant mice. We further delineate that YAP accumulation in TSC1/TSC2-deficient cells is due to impaired degradation of the protein by the autophagosome/lysosome system. Thus, the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth-permissive conditions. YAP may serve as a potential therapeutic target for TSC and other diseases with dysregulated mTOR activity.

Liver metastases are a major adverse event during the evolution of digestive endocrine tumors. However, little is known about their natural history and the determinants of their growth. In particular, whereas liver endocrine metastases, like their primary counterparts, are hypervascular, the role of tumor-associated angiogenesis has been little explored. We therefore designed an experimental model to study the intrahepatic growth of tumor endocrine cells; murine enteroendocrine STC-1 cells were injected into the spleen of nude mice to obtain their hepatic dissemination through the portal vein. Three stages of intrahepatic tumor growth were identified. Engraftment stage, until day 4 after intrasplenic injection of STC-1 cells, was avascular. Early growth, until day 17, resulted in small, infralobular nodules. Late growth, after day 17, was characterized by the development of large nodules associated with peritumoral vessels and containing abnormal intratumoral vessels. To test the effects of potentially anti-angiogenic agents on tumor growth, we then used STC-1 cells stably transfected with the endostatin-coding sequence. Intrahepatic tumor volume showed no significant change at days 4 and 8, but a dramatic decrease at day 28 (9.7 +/- 1.7% of liver tissue versus 25.2 +/- 2.4% in controls), because of a markedly lower number of large nodules (11 +/- 1.8% versus 42 +/- 5.8%) likely to result from an increased apoptotic index (39.4 +/- 5.6% versus 18.3 +/- 3.4). Our results suggest that active angiogenesis is not necessary for the engraftment and early growth of endocrine cells metastatic to the liver but is required at a later stage of progression.

The aim of this study was to investigate (a) in vitro the relationship between [(18)F]fluorocholine ([(18)F]FCH) uptake and cell growth in endocrine cell lines and (b) in vivo the uptake of [(18)F]FCH by tumoral sites in an animal model of metastasized endocrine tumor.

Ileal epithelial cell apoptosis and the local microbiota modulate the effects of oxaliplatin against proximal colon cancer by modulating tumor immunosurveillance. Here, we identified an ileal immune profile associated with the prognosis of colon cancer and responses to chemotherapy. The whole immune ileal transcriptome was upregulated in poor-prognosis patients with proximal colon cancer, while the colonic immunity of healthy and neoplastic areas was downregulated (except for the Th17 fingerprint) in such patients. Similar observations were made across experimental models of implanted and spontaneous murine colon cancer, showing a relationship between carcinogenesis and ileal inflammation. Conversely, oxaliplatin-based chemotherapy could restore a favorable, attenuated ileal immune fingerprint in responders. These results suggest that chemotherapy inversely shapes the immune profile of the ileum-tumor axis, influencing clinical outcome.

To assess the metabolomic fingerprint of small intestine neuroendocrine tumors (SI-NETs) and related hepatic metastases, and to investigate the influence of the hepatic environment on SI-NETs metabolome. Ninety-four tissue samples, including 46 SI-NETs, 18 hepatic NET metastases and 30 normal SI and liver samples, were analyzed using 1H-magic angle spinning (HRMAS) NMR nuclear magnetic resonance (NMR) spectroscopy. Twenty-seven metabolites were identified and quantified. Differences between primary NETs vs. normal SI and primary NETs vs. hepatic metastases, were assessed. Network analysis was performed according to several clinical and pathological features. Succinate, glutathion, taurine, myoinositol and glycerophosphocholine characterized NETs. Normal SI specimens showed higher levels of alanine, creatine, ethanolamine and aspartate. PLS-DA revealed a continuum-like distribution among normal SI, G1-SI-NETs and G2-SI-NETs. The G2-SI-NET distribution was closer and clearly separated from normal SI tissue. Lower concentration of glucose, serine and glycine, and increased levels of choline-containing compounds, taurine, lactate and alanine, were found in SI-NETs with more aggressive tumors. Higher abundance of acetate, succinate, choline, phosphocholine, taurine, lactate and aspartate discriminated liver metastases from normal hepatic parenchyma. Higher levels of alanine, ethanolamine, glycerophosphocholine and glucose was found in hepatic metastases than in primary SI-NETs. The present work gives for the first time a snapshot of the metabolomic characteristics of SI-NETs, suggesting the existence of complex metabolic reality, maybe characteristic of different tumor evolution.

The classification of neuroendocrine neoplasms (NENs) differs between organ systems and currently causes considerable confusion. A uniform classification framework for NENs at any anatomical location may reduce inconsistencies and contradictions among the various systems currently in use. The classification suggested here is intended to allow pathologists and clinicians to manage their patients with NENs consistently, while acknowledging organ-specific differences in classification criteria, tumor biology, and prognostic factors. The classification suggested is based on a consensus conference held at the International Agency for Research on Cancer (IARC) in November 2017 and subsequent discussion with additional experts. The key feature of the new classification is a distinction between differentiated neuroendocrine tumors (NETs), also designated carcinoid tumors in some systems, and poorly differentiated NECs, as they both share common expression of neuroendocrine markers. This dichotomous morphological subdivision into NETs and NECs is supported by genetic evidence at specific anatomic sites as well as clinical, epidemiologic, histologic, and prognostic differences. In many organ systems, NETs are graded as G1, G2, or G3 based on mitotic count and/or Ki-67 labeling index, and/or the presence of necrosis; NECs are considered high grade by definition. We believe this conceptual approach can form the basis for the next generation of NEN classifications and will allow more consistent taxonomy to understand how neoplasms from different organ systems inter-relate clinically and genetically.

Despite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression.

Tumor-associated macrophages (TAMs) having immunosuppressive properties are one of the most abundant immune cells in the tumor microenvironment (TME). Preclinical studies have highlighted the potential role of TAMs in resistance to immune checkpoint blockers (ICBs). Here, we investigated the predictive value of TAM infiltration in patients with non-small cell lung cancer (NSCLC) treated with ICBs and characterized their transcriptomic profiles.

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ß-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2⁻/⁻γ(c)⁻/⁻ mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2⁻/⁻γc⁻/⁻ mice, mature single-positive (SP) CD4⁺ and CD8⁺ cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2⁻/⁻γ(c)⁻/⁻ animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.

The Hedgehog signaling is a determinant pathway for tumor progression. However, while inhibition of the Hedgehog canonical pathway-Patched-Smoothened-Gli-has proved efficient in human tumors with activating mutations in this pathway, recent clinical data have failed to show any benefit in other cancers, even though Sonic Hedgehog (SHH) expression is detected in these cancers. Cell-adhesion molecule-related/down-regulated by Oncogenes (CDON), a positive regulator of skeletal muscle development, was recently identified as a receptor for SHH. We show here that CDON behaves as a SHH dependence receptor: it actively triggers apoptosis in the absence of SHH. The pro-apoptotic activity of unbound CDON requires a proteolytic cleavage in its intracellular domain, allowing the recruitment and activation of caspase-9. We show that by inducing apoptosis in settings of SHH limitation, CDON expression constrains tumor progression, and as such, decreased CDON expression observed in a large fraction of human colorectal cancer is associated in mice with intestinal tumor progression. Reciprocally, we propose that the SHH expression, detected in human cancers and previously considered as a mechanism for activation of the canonical pathway in an autocrine or paracrine manner, actually provides a selective tumor growth advantage by blocking CDON-induced apoptosis. In support of this notion, we present the preclinical demonstration that interference with the SHH-CDON interaction triggers a CDON-dependent apoptosis in vitro and tumor growth inhibition in vivo. The latter observation qualifies CDON as a relevant alternative target for anticancer therapy in SHH-expressing tumors.

Transformation of castration-resistant prostate cancer (CRPC) into an aggressive neuroendocrine disease (CRPC-NE) represents a major clinical challenge and experimental models are lacking. A CTC-derived eXplant (CDX) and a CDX-derived cell line are established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis from a CRPC patient resistant to enzalutamide. The CDX and the derived-cell line conserve 16% of primary tumor (PT) and 56% of CTC mutations, as well as 83% of PT copy-number aberrations including clonal TMPRSS2-ERG fusion and NKX3.1 loss. Both harbor an androgen receptor-null neuroendocrine phenotype, TP53, PTEN and RB1 loss. While PTEN and RB1 loss are acquired in CTCs, evolutionary analysis suggest that a PT subclone harboring TP53 loss is the driver of the metastatic event leading to the CDX. This CDX model provides insights on the sequential acquisition of key drivers of neuroendocrine transdifferentiation and offers a unique tool for effective drug screening in CRPC-NE management.

The embryonic transcription factors TWIST1/2 are frequently overexpressed in cancer, acting as multifunctional oncogenes. Here we investigate their role in neuroblastoma (NB), a heterogeneous childhood malignancy ranging from spontaneous regression to dismal outcomes despite multimodal therapy. We first reveal the association of TWIST1 expression with poor survival and metastasis in primary NB, while TWIST2 correlates with good prognosis. Secondly, suppression of TWIST1 by CRISPR/Cas9 results in a reduction of tumor growth and metastasis colonization in immunocompromised mice. Moreover, TWIST1 knockout tumors display a less aggressive cellular morphology and a reduced disruption of the extracellular matrix (ECM) reticulin network. Additionally, we identify a TWIST1-mediated transcriptional program associated with dismal outcome in NB and involved in the control of pathways mainly linked to the signaling, migration, adhesion, the organization of the ECM, and the tumor cells versus tumor stroma crosstalk. Taken together, our findings confirm TWIST1 as promising therapeutic target in NB.

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient's tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies.

Several fibroblast growth factor receptor (FGFR) inhibitors are approved or in clinical development for the treatment of FGFR-driven urothelial cancer, and molecular mechanisms of resistance leading to patient relapses have not been fully explored. We identified 21 patients with FGFR-driven urothelial cancer treated with selective FGFR inhibitors and analyzed postprogression tissue and/or circulating tumor DNA (ctDNA). We detected single mutations in the FGFR tyrosine kinase domain in seven (33%) patients (FGFR3 N540K, V553L/M, V555L/M, E587Q; FGFR2 L551F) and multiple mutations in one (5%) case (FGFR3 N540K, V555L, and L608V). Using Ba/F3 cells, we defined their spectrum of resistance/sensitivity to multiple selective FGFR inhibitors. Eleven (52%) patients harbored alterations in the PI3K-mTOR pathway (n = 4 TSC1/2, n = 4 PIK3CA, n = 1 TSC1 and PIK3CA, n = 1 NF2, n = 1 PTEN). In patient-derived models, erdafitinib was synergistic with pictilisib in the presence of PIK3CA E545K, whereas erdafitinib-gefitinib combination was able to overcome bypass resistance mediated by EGFR activation.

The human leukocyte antigen (HLA) system is a major factor controlling cancer immunosurveillance and response to immunotherapy, yet its status in pediatric cancers remains fragmentary. We determined high-confidence HLA genotypes in 576 children, adolescents and young adults with recurrent/refractory solid tumors from the MOSCATO-01 and MAPPYACTS trials, using normal and tumor whole exome and RNA sequencing data and benchmarked algorithms. There was no evidence for narrowed HLA allelic diversity but discordant homozygosity and allele frequencies across tumor types and subtypes, such as in embryonal and alveolar rhabdomyosarcoma, neuroblastoma MYCN and 11q subtypes, and high-grade glioma, and several alleles may represent protective or susceptibility factors to specific pediatric solid cancers. There was a paucity of somatic mutations in HLA and antigen processing and presentation (APP) genes in most tumors, except in cases with mismatch repair deficiency or genetic instability. The prevalence of loss-of-heterozygosity (LOH) ranged from 5.9 to 7.7% in HLA class I and 8.0 to 16.7% in HLA class II genes, but was widely increased in osteosarcoma and glioblastoma (~15-25%), and for DRB1-DQA1-DQB1 in Ewing sarcoma (~23-28%) and low-grade glioma (~33-50%). HLA class I and HLA-DR antigen expression was assessed in 194 tumors and 44 patient-derived xenografts (PDXs) by immunochemistry, and class I and APP transcript levels quantified in PDXs by RT-qPCR. We confirmed that HLA class I antigen expression is heterogeneous in advanced pediatric solid tumors, with class I loss commonly associated with the transcriptional downregulation of HLA-B and transporter associated with antigen processing (TAP) genes, whereas class II antigen expression is scarce on tumor cells and occurs on immune infiltrating cells. Patients with tumors expressing sufficient HLA class I and TAP levels such as some glioma, osteosarcoma, Ewing sarcoma and non-rhabdomyosarcoma soft-tissue sarcoma cases may more likely benefit from T cell-based approaches, whereas strategies to upregulate HLA expression, to expand the immunopeptidome, and to target TAP-independent epitopes or possibly LOH might provide novel therapeutic opportunities in others. The consequences of HLA class II expression by immune cells remain to be established. Immunogenetic profiling should be implemented in routine to inform immunotherapy trials for precision medicine of pediatric cancers.

The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.

Despite evidence that mucin overproduction is critical in the pathogenesis of gallstones, the mechanisms triggering mucin production in gallstone disease are unknown. Here, we tested the potential implication of an inflammation-dependent epidermal growth factor receptor (EGF-R) pathway in the regulation of gallbladder mucin synthesis. In gallbladder tissue sections from subjects with cholesterol gallstones, mucus accumulation was associated with neutrophil infiltration and with increased expressions of EGF-R and of tumor necrosis factor-alpha (TNF-alpha). In primary cultures of human gallbladder epithelial cells, TNF-alpha induced EGF-R overexpression. In the presence of TNF-alpha, EGF-R ligands (either EGF or transforming growth factor-alpha) caused significant increases in MUC5AC mRNA and protein production, whereas expression of the other gallbladder mucins MUC1, MUC3, and MUC5B was unchanged. In addition, on gallbladder tissue sections from subjects with gallstones, increased MUC5AC immunoreactivity was detected in the epithelium and within mucus gel in the lumen. Studies in primary cultures demonstrated that MUC5AC up-regulation induced by the combination of TNF-alpha with EGF-R ligands was completely blunted by inhibitors of EGF-R tyrosine kinase and mitogen-activated protein/extracellular signal-related kinase kinase. In conclusion, an inflammation-dependent EGF-R cascade causes overproduction of the gel-forming mucin MUC5AC, which accumulates in cholesterol gallstone disease. The ability to interrupt this cascade is of potential interest in the prevention of cholesterol gallstones.

In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

mTOR and Unfolded Protein Response (UPR) are two signaling pathways frequently activated in cancer cells. The mTOR pathway has been shown to be up-regulated in most gastroenteropancreatic neuroendocrine tumors. In contrast, little is known about the UPR status in neoplastic neuroendocrine cells. However, these hormone-producing cells are likely to present distinctive adaptations of this pathway, as other secretory cells. We therefore analyzed the status of the three axes of UPR and their relation to mTOR pathway in two gastrointestinal neuroendocrine tumors (GI-NET) cell lines STC-1 and GluTag. At baseline, pharmacological inducers activate the three arms of UPR: PERK, ATF6 and IRE1. Although hypoxia stimulates the PERK, ATF6 and IRE-1 pathways in both cell lines, glucose depletion activates UPR only in STC-1 cell line. Strikingly, P-p70S6K1 increases concomitantly to P-PERK and BiP in response to thapsigargin treatment, glucose depletion or hypoxia. We found that different mTOR inhibitors activate the PERK signaling pathway. To confirm that mTOR inhibition modulates PERK activation, we inhibited PERK and showed that it decreased cell viability when associated to mTOR inhibition, indicating that mTOR drives a PERK-dependent survival pathway. In conclusion, in GI-NET cell lines, UPR signaling is functional and PERK arm is induced by mTOR inhibition. These observations open up new perspectives for therapeutic strategies: the crosstalk between mTOR and UPR might contribute to the resistance to mTOR inhibitors and could be targeted by mTOR and PERK inhibitors in combination therapy.