Evolutionary fingerprints of EMT in pancreatic cancers.

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.

SMARCB1 regulates the hypoxic stress response in sickle cell trait.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Association of High-Intensity Exercise with Renal Medullary Carcinoma in Individuals with Sickle Cell Trait: Clinical Observations and Experimental Animal Studies.

Renal medullary carcinoma (RMC) is a lethal malignancy affecting individuals with sickle hemoglobinopathies. Currently, no modifiable risk factors are known. We aimed to determine whether high-intensity exercise is a risk factor for RMC in individuals with sickle cell trait (SCT). We used multiple approaches to triangulate our conclusion. First, a case-control study was conducted at a single tertiary-care facility. Consecutive patients with RMC were compared to matched controls with similarly advanced genitourinary malignancies in a 1:2 ratio and compared on rates of physical activity and anthropometric measures, including skeletal muscle surface area. Next, we compared the rate of military service among our RMC patients to a similarly aged population of black individuals with SCT in the U.S. Further, we used genetically engineered mouse models of SCT to study the impact of exercise on renal medullary hypoxia. Compared with matched controls, patients with RMC reported higher physical activity and had higher skeletal muscle surface area. A higher proportion of patients with RMC reported military service than expected compared to the similarly-aged population of black individuals with SCT. When exposed to high-intensity exercise, mice with SCT demonstrated significantly higher renal medulla hypoxia compared to wild-type controls. These data suggest high-intensity exercise is the first modifiable risk factor for RMC in individuals with SCT.

Epithelial memory of inflammation limits tissue damage while promoting pancreatic tumorigenesis.

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma (PDAC). When occurring in the context of pancreatitis, KRAS mutations accelerate tumor development in mouse models. We report that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, pancreatic epithelial cells display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thereby limiting tissue damage through a rapid decrease of zymogen production. We propose that because activating mutations of KRAS maintain an irreversible ADM, they may be beneficial and under strong positive selection in the context of recurrent pancreatitis.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Loss of ARID1A Promotes Epithelial-Mesenchymal Transition and Sensitizes Pancreatic Tumors to Proteotoxic Stress.

Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in ARID1A represent one of the most frequent molecular alterations in human cancers. ARID1A mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial-mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both in vitro and in vivo. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in ARID1A associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment. SIGNIFICANCE: This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.

Accuracy of a Symptom-Based Approach to Identify Hypertensive Emergencies in the Emergency Department.

BACKGROUND: A hierarchical symptoms-based diagnostic strategy relying on the presence of five main symptoms (chest pain, acute dyspnea, neurological symptoms, headache, visual impairment) was recently proposed to diagnose patients with hypertensive emergency. However, poor scientific evidence is available about the role of symptoms in both diagnosis and management of acute hypertensive disorders. METHODS: Data from 718 patients presenting to the emergency department of the "Città della Salute e della Scienza" Hospital of Turin with systolic blood pressure > 180 and/or diastolic blood pressure > 110 mm/Hg were retrospectively analyzed. The accuracy of the typical symptoms for identification of hypertensive emergencies was assessed. RESULTS: A total of 79 (11%) out of 718 patients were diagnosed with hypertensive emergencies (51% had cardiovascular and 49% neurovascular acute organ damage). Patients with hypertensive emergencies were older and with higher prevalence of coronary artery disease and chronic heart failure than patients with uncontrolled hypertension. Typical symptoms could discriminate true hypertensive emergency from uncontrolled hypertension with 64% accuracy, 94% sensitivity, and 60% specificity. CONCLUSION: Typical symptoms might be used as a simple screening test (99% negative predictive value) in the emergency department to select for further evaluations of patients with suspected hypertensive emergencies among those with acute hypertensive disorders.

Comprehensive Molecular Characterization Identifies Distinct Genomic and Immune Hallmarks of Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.

Oncogenic KRAS-Driven Metabolic Reprogramming in Pancreatic Cancer Cells Utilizes Cytokines from the Tumor Microenvironment.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ-IL4rα and IL2rγ-IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ-IL4rα and IL2rγ-IL13rα1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

CD147 Promotes Cell Small Extracellular Vesicles Release during Colon Cancer Stem Cells Differentiation and Triggers Cellular Changes in Recipient Cells.

Cancer cells secrete small extracellular vesicles (sEVs) that are involved in the remodeling of tumor microenvironment (TME) and can promote tumor progression. The role of sEVs and their molecular key players in colon cancer stem cells differentiation are poorly understood. This study aimed to analyze the role and content of sEVs released during the differentiation of colorectal cancer stem cells. Here we show that sEVs secretion during colon cancer stem cells differentiation is partially controlled by CD147, a well-known player involved in colon cancer tumorigenesis. CD147 + sEVs activate a signaling cascade in recipient cells inducing molecular invasive features in colon cancer cells. CD147 knockdown as well as anti-CD147 antibodies impaired sEVs release and downstream effects on recipient cells and blocking multivesicular body maturation prevented sEVs release during the differentiation. Our findings reveal a functional role of CD147 in promoting sEVs release during the differentiation of colon cancer stem cells and in triggering cellular changes in recipient cells.

p53 Is a Master Regulator of Proteostasis in SMARCB1-Deficient Malignant Rhabdoid Tumors.

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRTs) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and ER stress response via a genetically intact MYC-p19ARF-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide a rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRTs.

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells.

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells.

White blood cell and platelet count as adjuncts to standard clinical evaluation for risk assessment in patients at low probability of acute aortic syndrome.

AIMS: Pre-test probability assessment is key in the approach to suspected acute aortic syndromes (AASs). However, most patients with AAS-compatible symptoms are classified at low probability, warranting further evaluation for decision on aortic imaging. White blood cell count, platelet count and fibrinogen explore pathophysiological pathways mobilized in AASs and are routinely assayed in the workup of AASs. However, the diagnostic performance of these variables for AASs, alone and as a bundle, is unknown. We tested the hypothesis that white blood cell count, platelet count and/or fibrinogen at presentation may be applied as additional tools to standard clinical evaluation for pre-test risk assessment in patients at low probability of AAS. METHODS AND RESULTS: This was a retrospective observational study conducted on consecutive patients managed in our Emergency Department from 2009 to 2014 for suspected AAS. White blood cell count, platelet count and fibrinogen were assayed during evaluation in the Emergency Department. The final diagnosis was obtained by computed tomography angiography. The pre-test probability of AAS was defined according to guidelines. Of 1210 patients with suspected AAS, 1006 (83.1%) were classified at low probability, and 271 (22.4%) were diagnosed with AAS. Within patients at low probability, presence of at least one alteration among white blood cell count >9*103/µl, platelet count <200*103/µl and fibrinogen <350 mg/dl was associated with a sensitivity of 95.5% (89.7-98.5%) and a specificity of 18.3% (15.6-21.2%). In patients at low probability, white blood cell count >9*103/µl and platelet count <200*103/µl were found as independent predictors of AAS beyond established clinical risk markers. Within patients at low probability, the estimated risk of AAS based on the number of alterations amongst white blood cell count >9*103/µl and platelet count <200*103/µl was 2.7% (1.2-5.7%) with zero alterations, 11.3% (8.8-14.3%) with one alteration and 31.9% (24.8-40%) with two alterations ( p<0.001). CONCLUSION: In addition to standard clinical evaluation, white blood cell count and platelet count may be used in patients at low pre-test probability to fine-tune risk assessment of AAS.

Combined use of aortic dissection detection risk score and D-dimer in the diagnostic workup of suspected acute aortic dissection.

BACKGROUND: Acute aortic dissection (AD) represents a diagnostic conundrum. Validated algorithms are particularly needed to identify patients where AD could be ruled out without aortic imaging. We evaluated the diagnostic accuracy of a strategy combining the aortic dissection detection (ADD) risk score with D-dimer, a sensitive biomarker of AD. METHODS: Patients from two clinical centers with suspected AD were prospectively enrolled in a registry, from January 2008 to March 2013. The ADD risk score was calculated by retrospective blinded chart review. For D-dimer, a cutoff of 500 ng/ml was applied. RESULTS: AD was diagnosed in 233 of 1035 (22.5%) patients. The ADD risk score was 0 in 322 (31.1%), 1 in 508 (49.1%) and >1 in 205 (19.8%) patients. The sensitivity and the failure rate of D-dimer were 100% and 0% in patients with ADD score 0, versus 97.5% (95% CI 91.4-99.6%) and 4.2% (95% CI 0.7-12.5%) in patients with ADD risk score >1. In patients with ADD risk score ≤ 1, the sensitivity and the failure rate of D-dimer were 98.7% (95% CI 95.3-99.8%) and 0.8% (95% CI 0.1-2.6%). The diagnostic efficiency of D-dimer in patients with ADD risk score 0 and ≤ 1 was 8.9% (95% CI 7.2-10.7%) and 23.6% (95% CI 21.1-26.2%) respectively. CONCLUSIONS: In a large cohort of patients with suspected AD, the presence of ADD risk score 0 or ≤ 1 combined with a negative D-dimer accurately and efficiently ruled out AD.