New insights into the Immune TME of adult-type diffuse gliomas.

PURPOSE OF REVIEW: Adult-type diffuse gliomas are highly heterogeneous tumors. Bulk transcriptome analyses suggested that the composition of the tumor microenvironment (TME) corresponds to genetic and clinical features. In this review, we highlight novel findings on the intratumoral heterogeneity of IDH-wildtype and IDH-mutant gliomas characterized at single-cell resolution, and emphasize the mechanisms shaping the immune TME and therapeutic implications. RECENT FINDINGS: Emergent evidence indicates that in addition to genetic drivers, epigenetic mechanisms and microenvironmental factors influence the glioma subtypes. Interactions between glioma and immune cells contribute to immune evasion, particularly in aggressive tumors. Spatial and temporal heterogeneity of malignant and immune cell subpopulations is high in recurrent gliomas. IDH-wildtype and IDH-mutant tumors display distinctive changes in their myeloid and lymphoid compartments, and D-2HG produced by IDH-mutant cells impacts the immune TME. SUMMARY: The comprehensive dissection of the intratumoral ecosystem of human gliomas using single-cell and spatial transcriptomic approaches advances our understanding of the mechanisms underlying the immunosuppressed state of the TME, supports the prognostic value of tumor-associated macrophages and microglial cells, and sheds light on novel therapeutic options.

Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma.

BACKGROUND: Knowing the genetic status of patients affected by paragangliomas and pheochromocytomas (PPGL) is important for the guidance of their management and their relatives. Our objective was to improve the diagnostic performances of PPGL genetic testing by next-generation sequencing (NGS). METHODS: We developed a custom multigene panel, which includes 17 PPGL genes and is compatible with both germline and tumour DNA screening. The NGS assay was first validated in a retrospective cohort of 201 frozen tumour DNAs and then applied prospectively to 623 DNAs extracted from leucocytes, frozen or paraffin-embedded PPGL tumours. RESULTS: In the retrospective cohort, the sensitivity of the NGS assay was evaluated at 100% for point and indels mutations and 86% for large rearrangements. The mutation rate was re-evaluated from 65% (132/202) to 78% (156/201) after NGS analysis. In the prospective cohort, NGS detected not only germline and somatic mutations but also co-occurring variants and mosaicism. A mutation was identified in 74% of patients for whom both germline and tumour DNA were available. CONCLUSION: The analysis of 824 DNAs from patients with PPGL demonstrated that NGS assay significantly improves the performances of PPGL genetic testing compared with conventional methods, increasing the rate of identified mutations and identifying rare genetic mechanisms.

PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance.

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas.

Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.

The senescent microenvironment promotes the emergence of heterogeneous cancer stem-like cells.

There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state.

Telomere crisis in kidney epithelial cells promotes the acquisition of a microRNA signature retrieved in aggressive renal cell carcinomas.

Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We discovered that human epithelial kidney cells undergoing T-CIN display massive microRNA (miR) expression changes that are not related to local losses or gains. This widespread miR deregulation encompasses a miR-200-dependent epithelial-to-mesenchymal transition (EMT) that confers to immortalized pre-tumoral cells phenotypic traits of metastatic potential. Remarkably, a miR signature of these cells, comprising a downregulation of miRs with conserved expression in kidney, was retrieved in poorly differentiated aggressive renal cell carcinomas. Our results reveal an unanticipated connection between telomere crisis and the activation of the EMT program that occurs at pre-invasive stages of epithelial cancers, through mechanisms that involve miR deregulation. Thus, this study provides a new rational into how telomere instability contributes to the acquisition of the malignant phenotype.

Pro-tumorigenic role of lnc-ZNF30-3 as a sponge counteracting miR-145-5p in prostate cancer.

BACKGROUND: Prostate cancer remains one of the deadliest neoplasms in developed countries. Identification of new molecular markers that predict the onset and progression of the disease could improve its clinical management. Low miR-145-5p expression is consistently found in primary tumors and metastases, but the regulatory mechanisms governing its functions remain largely unknown. METHODS: Bioinformatics analysis was conducted to identify [1] a set of novel potential competing endogenous lncRNAs for sponging of miRNA-145-5p in prostate cancer and [2] miR-145-5p and other EMT-related miRNAs response elements in lnc-ZNF30-3. Quantification of miR-145-5p, lnc-ZNF30-3, and TWIST1 expression levels in tumor tissues in RNA sequencing datasets of our and TCGA PRAD cohorts revealed a correlation with clinical outcome of prostate cancer patients. Biochemical and cell biology approaches, such as RNA pull-down, western blot, immunostaining, and wound healing assays were used for evaluation of the impact of TWIST1/miR-145/ lnc-ZNF30-3 interactions in prostate cancer cells altered in miRNA and lncRNA expression. RESULTS: We identified a few potential lncRNA sponges of miR-145-5p, including lnc-ZNF30-3. It contains five response elements for miR-145-5p, but also other miRNAs targeting EMT transcription factors. Lnc-ZNF30-3 is significantly upregulated in prostate cancer cell lines and tumor tissues, and its high expression is correlated with poor patient prognosis. We demonstrated that lnc-ZNF30-3 is associated with AGO2 and specifically interacts with the miR-145-5p seed region. Knockdown of lnc-ZNF30-3 results in decreased migration of prostate cancer cells and downregulation of EMT drivers such as TWIST1 and ZEB1 at both the RNA and protein levels. These phenotypic and molecular features of lnc-ZNF30-3-depleted cells are partially rescued by miR-145-5p inhibition. CONCLUSIONS: Collectively, our results point to lnc-ZNF30-3 as a novel competing endogenous lncRNA for miR-145-5p and other miRNAs that target TWIST1 as well as other EMT transcription factors. Prostate cancer patients with high lncRNA expression in primary tumors show lower survival rate suggesting that lnc-ZNF30-3 may contribute to prostate cancer progression and metastasis.

TET-Mediated Hypermethylation Primes SDH-Deficient Cells for HIF2α-Driven Mesenchymal Transition.

Loss-of-function mutations in the SDHB subunit of succinate dehydrogenase predispose patients to aggressive tumors characterized by pseudohypoxic and hypermethylator phenotypes. The mechanisms leading to DNA hypermethylation and its contribution to SDH-deficient cancers remain undemonstrated. We examine the genome-wide distribution of 5-methylcytosine and 5-hydroxymethylcytosine and their correlation with RNA expression in SDHB-deficient tumors and murine Sdhb-/- cells. We report that DNA hypermethylation results from TET inhibition. Although it preferentially affects PRC2 targets and known developmental genes, PRC2 activity does not contribute to the DNA hypermethylator phenotype. We also prove, in vitro and in vivo, that TET silencing, although recapitulating the methylation profile of Sdhb-/- cells, is not sufficient to drive their EMT-like phenotype, which requires additional HIF2α activation. Altogether, our findings reveal synergistic roles of TET repression and pseudohypoxia in the acquisition of metastatic traits, providing a rationale for targeting HIF2α and DNA methylation in SDH-associated malignancies.

Transcriptome Analysis of lncRNAs in Pheochromocytomas and Paragangliomas.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors explained by germline or somatic mutations in about 70% of cases. Patients with SDHB mutations are at high risk of developing metastatic disease, yet no reliable tumor biomarkers are available to predict tumor aggressiveness. OBJECTIVE: We aimed at identifying long noncoding RNAs (lncRNAs) specific for PPGL molecular groups and metastatic progression. DESIGN AND METHODS: To analyze the expression of lncRNAs, we used a mining approach of transcriptome data from a well-characterized series of 187 tumor tissues. Clustering consensus analysis was performed to determine a lncRNA-based classification, and informative transcripts were validated in an independent series of 51 PPGLs. The expression of metastasis-related lncRNAs was confirmed by RT-qPCR. Receiver operating characteristic (ROC) curve analysis was used to estimate the predictive accuracy of potential markers. MAIN OUTCOME MEASURE: Univariate/multivariate and metastasis-free survival (MFS) analyses were carried out for the assessment of risk factors and clinical outcomes. RESULTS: Four lncRNA-based subtypes strongly correlated with mRNA expression clusters (chi-square P-values from 1.38 × 10-32 to 1.07 × 10-67). We identified one putative lncRNA (GenBank: BC063866) that accurately discriminates metastatic from benign tumors in patients with SDHx mutations (area under the curve 0.95; P = 4.59 × 10-05). Moreover, this transcript appeared as an independent risk factor associated with poor clinical outcome of SDHx carriers (log-rank test P = 2.29 × 10-05). CONCLUSION: Our findings extend the spectrum of transcriptional dysregulations in PPGL to lncRNAs and provide a novel biomarker that could be useful to identify potentially metastatic tumors in patients carrying SDHx mutations.

Emerging molecular markers of metastatic pheochromocytomas and paragangliomas.

Metastatic pheochromocytoma/paraganglioma (PPGL) represents a major clinical challenge due to limitations in accurate diagnostic tools and effective treatments. Currently, patients classified at high-risk by means of clinical, biochemical and genetic criteria, require a lifelong monitoring, while it remains difficult to determine the metastatic potential of PPGL only on the basis of histopathological features. Thus, tumor molecular markers that improve the risk stratification of these patients are needed. In the past few years, we have witnessed an unprecedented molecular characterization of PPGL, which led to the emergence of promising candidate biomarkers predictive of metastatic behavior. Here, we briefly discuss these breakthroughs and provide some insights for the prospective implementation of molecular markers of metastatic PPGL in the clinical setting in years to come.

Telomerase Activation and ATRX Mutations Are Independent Risk Factors for Metastatic Pheochromocytoma and Paraganglioma.

PURPOSE: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. Whereas most PPGLs are benign, up to 20% may become metastatic with SDHB- and FH-mutated tumors showing the higher risk. We aimed at determining the contribution of immortalization mechanisms to metastatic progression.Experimental Design: Immortalization mechanisms were investigated in 200 tumors. To identify telomerase (+) tumors, we analyzed genomic alterations leading to transcriptional activation of TERT comprising promoter mutations, hypermethylation and gain copy number. To identify tumors that activated the alternative lengthening of telomere (ALT) mechanism, we combined analyses of telomere length by slot blot, telomere heterogeneity by telomere FISH, and ATRX mutations by next-generation sequencing. Univariate/multivariate and metastasis-free survival (MFS) and overall survival (OS) analyses were carried out for assessment of risk factors and clinical outcomes. RESULTS: Only 37 of 200 (18.5%) tumors achieved immortalization. Telomerase activation occurred in 12 metastatic tumors and was prevalent in SDHB-mutated paragangliomas (P = 2.42e-09). ALT features were present in 25 tumors, mostly pheochromocytomas, regardless of metastatic status or molecular group (P = 0.169), yet ATRX mutations were found preferentially in SDHB/FH-mutated metastatic tumors (P = 0.0014). Telomerase activation and ATRX mutations were independent factors of poor prognosis: MFS (hazard ratio, 48.2 and 33.1; P = 6.50E-07 and 1.90E-07, respectively); OS (hazard ratio, 97.4 and 44.1; P = 4.30E-03 and 2.00E-03, respectively) and were associated with worse MFS and OS (log-rank tests P < 0.0001). CONCLUSIONS: Assessment of telomerase activation and ATRX mutations could be used to identify metastatic PPGLs, particularly in tumors at high risk of progression.

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

Germline Mutations in the Mitochondrial 2-Oxoglutarate/Malate Carrier SLC25A11 Gene Confer a Predisposition to Metastatic Paragangliomas.

Comprehensive genetic analyses have identified germline SDHB and FH gene mutations as predominant causes of metastatic paraganglioma and pheochromocytoma. However, some suspicious cases remain unexplained. In this study, we performed whole-exome sequencing of a paraganglioma exhibiting an SDHx-like molecular profile in the absence of SDHx or FH mutations and identified a germline mutation in the SLC25A11 gene, which encodes the mitochondrial 2-oxoglutarate/malate carrier. Germline SLC25A11 mutations were identified in six other patients, five of whom had metastatic disease. These mutations were associated with loss of heterozygosity, suggesting that SLC25A11 acts as a tumor-suppressor gene. Pseudohypoxic and hypermethylator phenotypes comparable with those described in SDHx- and FH-related tumors were observed both in tumors with mutated SLC25A11 and in Slc25a11Δ/Δ immortalized mouse chromaffin knockout cells generated by CRISPR-Cas9 technology. These data show that SLC25A11 is a novel paraganglioma susceptibility gene for which loss of function correlates with metastatic presentation.Significance: A gene encoding a mitochondrial carrier is implicated in a hereditary cancer predisposition syndrome, expanding the role of mitochondrial dysfunction in paraganglioma. Cancer Res; 78(8); 1914-22. ©2018 AACR.

Primary mediastinal large B-cell lymphoma: transcriptional regulation by miR-92a through FOXP1 targeting.

BACKGROUND: Primary mediastinal large B-cell lymphoma (PMBL) shares pathological features with diffuse large B-cell lymphoma (DLBCL), and molecular features with classical Hodgkin lymphoma (cHL). The miR-17~92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. METHODS: Here we compared the expression of each member of the miR-17~92 oncogenic cluster in samples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. RESULTS: We found a higher level of miR-92a in PMBL than in DLBCL, but not in cHL. A combination of in silico prediction and transcriptomic analyses enabled us to identify FOXP1 as a main miR-92a target gene in PMBL, a result so far not established. This was confirmed by 3'UTR, and RNA and protein expressions in transduced cell lines. In vivo studies using the transduced cell lines in mice enabled us to demonstrate a tumor suppressor effect of miR-92a and an oncogenic effect of FOXP1.A higher expression of miR-92a and the down-regulation of FOXP1 mRNA and protein expression were also found in human samples of PMBL, while miR-92a expression was low and FOXP1 was high in DLBCL. CONCLUSIONS: We concluded to a post-transcriptional regulation by miR-92a through FOXP1 targeting in PMBL, with a clinico-pathological relevance for better characterisation of PMBL.

The MITF, p.E318K Variant, as a Risk Factor for Pheochromocytoma and Paraganglioma.

CONTEXT: The microphthalmia-associated transcription factor (MITF) regulates the survival, proliferation, and differentiation of neural crest-derived lineages. Recent studies reported an increased risk of melanoma in individuals carrying the rare variant MITF, p.E318K (rs149617956). Whether this variant plays a role in other neural crest-derived tumors is unknown. OBJECTIVE: In the present study, we aimed at determining the prevalence of the MITF, p.E318K variant, in a well-characterized French cohort of pheochromocytomas/paragangliomas (PCC/PGL). DESIGN AND METHODS: Genomic DNA from 555 unrelated patients with PCC/PGL was genotyped for the p.E318K variant in MITF using Sanger sequencing. MAIN OUTCOME MEASURE: The prevalence of the mutation in the PCC/PGL cohort was compared with a population-based sample of 2348 ethnically matched controls. RESULTS: We identified seven carriers (five patients with sporadic PCCs, two with PGLs). The prevalence of the MITF, p.E318K variant, was higher in the PCC/PGL cohort than in controls, and appears to be a significant risk factor (odds ratio, 3.19; 95% confidence interval, 1.34-7.59; P = .005). Noteworthy, two patients were homozygous for the p.E318K risk allele, a patient with metastatic PCC and an SDHB-mutated patient with PGL. CONCLUSION: Our results indicate that the germline variant MITF, p.E318K is associated with an increased risk of other neural crest-derived tumors such as PCC/PGL.

From Nf1 to Sdhb knockout: Successes and failures in the quest for animal models of pheochromocytoma.

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors characterized by a high frequency of hereditary forms. Based on transcriptome classification, PPGL can be classified in two different clusters. Cluster 1 tumors are caused by mutations in SDHx, VHL and FH genes and are characterized by a pseudohypoxic signature. Cluster 2 PPGL carry mutations in RET, NF1, MAX or TMEM127 genes and display an activation of the MAPK and mTOR signaling pathways. Many genetically engineered and allografted mouse models have been generated these past 30 years to investigate the mechanisms of PPGL tumorigenesis and test new therapeutic strategies. Among them, only Cluster 2-related models have been successful while no Cluster 1-related knockout mouse was so far reported to develop a PPGL. In this review, we present an overview of existing, successful or not, PPGL models, and a description of our own experience on the quest of Sdhb knockout mouse models of PPGL.

The High Expression of the microRNA 17-92 Cluster and its Paralogs, and the Downregulation of the Target Gene PTEN, Is Associated with Primary Cutaneous B-Cell Lymphoma Progression.

The oncogenic microRNA (miR) 17-92 cluster has a causative role in the lymphomagenesis of nodal B-cell lymphomas, by activating proliferation and inhibiting apoptosis. Here we analyzed primary cutaneous B-cell lymphomas for the miR-17-92 cluster and its paralogs miR-106a-363 and miR-106b-25. In 22 primary cutaneous diffuse large B-cell lymphomas, leg type (PCLBCL-LT) compared with 22 primary cutaneous follicle center lymphomas (PCFCLs), we found that miR-20a and miR-106a were overexpressed. Multivariate Cox analysis showed that higher miR-20a and miR-20b expression levels were associated with shorter disease-free and overall survival, independently from histological type. Gene expression profiling also showed a downregulation of 8 candidate target genes of miR-20a, miR-20b, and miR-106a in PCLBCL-LT compared with PCFCL. Among the candidate target genes, PTEN, NCOA3, and CAPRIN2 were confirmed to be underexpressed in PCLBCL-LT using quantitative reverse transcriptase-PCR on CD20-positive laser-microdissected tumor cells. In multivariate Cox analysis, lower PTEN mRNA expression level was associated with shorter disease-free survival (DFS), independently from the histological type. Altogether, this molecular and bioinformatic study of 44 patient skin biopsy samples showed that the oncogenic miR-17-92 cluster and its paralogs were involved in cutaneous B-cell lymphoma progression, and that the downregulation of the target gene PTEN was associated with shorter DFS.

Deciphering the molecular basis of invasiveness in Sdhb-deficient cells.

Metastatic pheochromocytomas and paragangliomas (PPGL) are malignant neuroendocrine tumors frequently associated with germline mutations in the SDHB gene. SDHB-mutated PPGL display a hypermethylator phenotype associated with hallmarks of epithelial-to-mesenchymal transition (EMT). In the present study, we report the characterization of a unique model of Sdhb knockout in mouse chromaffin cells. Sdhb deficient cells exhibit a metastatic phenotype as highlighted by increased individual cell migration (characterized by faster motility and increased persistence) as well as high invasive and adhesion abilities. This phenotype is associated with the modulation of Twist1, Twist2, Tcf3, Snai1, N-cadherin or Krt19 expression, reflecting an EMT-like reprogramming of cells. Krt19 is epigenetically silenced in Sdhb-deficient cells and re-expressed after treatment by the demethylating agent decitabine. Krt19 rescue by lentiviral transduction in Sdhb-deficient cells and Krt19 inhibition by RNA interference in wild-type cells were performed. Both studies revealed the involvement of KRT19 in the invasive phenotype by modulating collective and individual migration and cell/extra-cellular matrix adhesion properties. These findings underline the role of hypermethylation and EMT in the in vitro acquisition of metastatic properties, following SDHB loss of function.

Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism.

The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.