Metabolic subtypes of clear cell renal cell carcinoma defined by tobacco smoking.

Tobacco smoking (TS) results in reprogramming of major metabolic pathways, including glycolysis, the citric acid (TCA) cycle, oxidative phosphorylation, and metabolism of aspartate, glutamate and glutamine in clear cell renal cell carcinoma (ccRCC). TS alters the distribution and activities of cadmium, arsenic and copper in a manner mechanistically supporting metabolic remodeling. Alterations in metabolism and metal distribution identify new actionable targets for treatment of ccRCC.

Molecular and Metabolic Subtypes in Sporadic and Inherited Clear Cell Renal Cell Carcinoma.

The promise of personalized medicine is a therapeutic advance where tumor signatures obtained from different omics platforms, such as genomics, transcriptomics, proteomics, and metabolomics, in addition to environmental factors including metals and metalloids, are used to guide the treatments. Clear cell renal carcinoma (ccRCC), the most common type of kidney cancer, can be sporadic (frequently) or genetic (rare), both characterized by loss of the von Hippel-Lindau (VHL) gene that controls hypoxia inducible factors. Recently, several genomic subtypes were identified with different prognoses. Transcriptomics, proteomics, metabolomics and metallomic data converge on altered metabolism as the principal feature of the disease. However, in view of multiple biochemical alterations and high level of tumor heterogeneity, identification of clearly defined subtypes is necessary for further improvement of treatments. In the future, single-cell combined multi-omics approaches will be the next generation of analyses gaining deeper insights into ccRCC progression and allowing for design of specific signatures, with better prognostic/predictive clinical applications.

Tobacco smoking induces metabolic reprogramming of renal cell carcinoma.

BACKGROUNDClear cell renal cell carcinoma (ccRCC) is the most common histologically defined renal cancer. However, it is not a uniform disease and includes several genetic subtypes with different prognoses. ccRCC is also characterized by distinctive metabolic reprogramming. Tobacco smoking (TS) is an established risk factor for ccRCC, with unknown effects on tumor pathobiology.METHODSWe investigated the landscape of ccRCCs and paired normal kidney tissues using integrated transcriptomic, metabolomic, and metallomic approaches in a cohort of white males who were long-term current smokers (LTS) or were never smokers (NS).RESULTSAll 3 Omics domains consistently identified a distinct metabolic subtype of ccRCCs in LTS, characterized by activation of oxidative phosphorylation (OXPHOS) coupled with reprogramming of the malate-aspartate shuttle and metabolism of aspartate, glutamate, glutamine, and histidine. Cadmium, copper, and inorganic arsenic accumulated in LTS tumors, showing redistribution among intracellular pools, including relocation of copper into the cytochrome c oxidase complex. A gene expression signature based on the LTS metabolic subtype provided prognostic stratification of The Cancer Genome Atlas ccRCC tumors that was independent of genomic alterations.CONCLUSIONThe work identified the TS-related metabolic subtype of ccRCC with vulnerabilities that can be exploited for precision medicine approaches targeting metabolic pathways. The results provided rationale for the development of metabolic biomarkers with diagnostic and prognostic applications using evaluation of OXPHOS status. The metallomic analysis revealed the role of disrupted metal homeostasis in ccRCC, highlighting the importance of studying effects of metals from e-cigarettes and environmental exposures.FUNDINGDepartment of Defense, Veteran Administration, NIH, ACS, and University of Cincinnati Cancer Institute.

The Art of Selective Cannulation at ERCP.

PURPOSE OF REVIEW: ERCP is undergoing its 50th anniversary with a steady conversion from a diagnostic procedure to a therapeutic procedure. This paper shows a reflection of my 42 years of experience in the accession of biliary and pancreatic ducts and how the techniques have evolved to achieve a high rate of primary duct cannulation. RECENT FINDINGS: There has been an increase in the primary approach to cannulation involving different cannulation catheters and guidewires, and we will review some of those available, realizing that the selection may vary depending on operator preference and team approach. Different views and experiences with these techniques will be presented. ERCP has evolved from a diagnostic to primarily a therapeutic procedure. This has resulted in the development of a variety of devices and techniques to access the ducts of interest. I have presented several of the techniques and my team approach that has evolved over a number of years. A more prospective evaluation of devices and wires is needed to see if one is superior to the other.

Translation control of the immune checkpoint in cancer and its therapeutic targeting.

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

Development of a stress response therapy targeting aggressive prostate cancer.

Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2-α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.

Noninvasive Measurement of mTORC1 Signaling with 89Zr-Transferrin.

Purpose: mTOR regulates many normal physiological processes and when hyperactive can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling noninvasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need.Experimental Design: Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity.Results: After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that 89Zr-labeled transferrin (Tf) can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with PET. Finally, we show that 89Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard-of-care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer.Conclusions: In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues, and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man. Clin Cancer Res; 23(12); 3045-52. ©2016 AACR.

Protein and nucleotide biosynthesis are coupled by a single rate-limiting enzyme, PRPS2, to drive cancer.

Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis-regulatory element within the Prps2 5' UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PAPERFLICK:

Decreased genetic dosage of hepatic Yin Yang 1 causes diabetic-like symptoms.

Insulin sensitivity in liver is characterized by the ability of insulin to efficiently inhibit glucose production and fatty acid oxidation as well as promote de novo lipid biosynthesis. Specific dysregulation of glucose and lipid metabolism in liver is sufficient to cause insulin resistance and type 2 diabetes; this is seen by a selective inability of insulin to suppress glucose production while remaining insulin-sensitive to de novo lipid biosynthesis. We have previously shown that the transcription factor Yin Yang 1 (YY1) controls diabetic-linked glucose and lipid metabolism gene sets in skeletal muscle, but whether liver YY1-targeted metabolic genes impact a diabetic phenotype is unknown. Here we show that decreased genetic dosage of YY1 in liver causes insulin resistance, hepatic lipid accumulation, and dyslipidemia. Indeed, YY1 liver-specific heterozygous mice exhibit blunted activation of hepatic insulin signaling in response to insulin. Mechanistically, YY1, through direct recruitment to promoters, functions as a suppressor of genes encoding for metabolic enzymes of the gluconeogenic and lipogenic pathways and as an activator of genes linked to fatty acid oxidation. These counterregulatory transcriptional activities make targeting hepatic YY1 an attractive approach for treating insulin-resistant diabetes.

New connections between old pathways: PDK1 signaling promotes cellular transformation through PLK1-dependent MYC stabilization.

Limited understanding of the functional link between multiple oncogenic pathways is a major barrier in the ongoing effort of cancer biologists to design an effective therapeutic approach to treat malignancies characterized by driver oncogenic network signals. In this issue of Cancer Discovery, Tan and colleagues elucidate a novel PDK1-PLK1-MYC signaling pathway connecting two fundamental oncogenic programs, phosphoinositide 3-kinase and MYC. They define the functional role for PDK1-PLK1-MYC signaling in cancer cell survival and tumor formation and show the therapeutic benefit of inhibiting PDK1 and PLK1 pharmacologically in cancer, tackling the most undruggable tumors defined by elevated levels of the MYC oncoprotein.

Investigating Myc-dependent translational regulation in normal and cancer cells.

There is an increasing realization that a primary role for Myc in driving cellular growth and cell cycle progression relies on Myc's ability to increase the rate of protein synthesis. Myc induces myriad changes in both global and specific mRNA translation. Herein, we present three assays that allow researchers to measure changes in protein synthesis at the global level as well as alterations in the translation of specific mRNAs. Metabolic labeling of cells with (35)S-containing methionine and cysteine is presented as a method to measure the overall rate of global protein synthesis. The bicistronic reporter assay is employed to determine levels of cap-dependent and cap-independent translation initiation in the cell. Finally, isolation of polysome-associated mRNAs followed by next-generation sequencing, microarray or quantitative real-time PCR (qRT-PCR) analysis is utilized to detect changes in the abundance of specific mRNAs that are regulated upon Myc hyperactivation. The protocols described in this chapter can be used to understand how and to what extent Myc-dependent regulation of translation influences normal cellular functions as well as tumorigenesis.

ER stress-mediated autophagy promotes Myc-dependent transformation and tumor growth.

The proto-oncogene c-Myc paradoxically activates both proliferation and apoptosis. In the pathogenic state, c-Myc-induced apoptosis is bypassed via a critical, yet poorly understood escape mechanism that promotes cellular transformation and tumorigenesis. The accumulation of unfolded proteins in the ER initiates a cellular stress program termed the unfolded protein response (UPR) to support cell survival. Analysis of spontaneous mouse and human lymphomas demonstrated significantly higher levels of UPR activation compared with normal tissues. Using multiple genetic models, we demonstrated that c-Myc and N-Myc activated the PERK/eIF2α/ATF4 arm of the UPR, leading to increased cell survival via the induction of cytoprotective autophagy. Inhibition of PERK significantly reduced Myc-induced autophagy, colony formation, and tumor formation. Moreover, pharmacologic or genetic inhibition of autophagy resulted in increased Myc-dependent apoptosis. Mechanistically, we demonstrated an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute (L24+/-) mutant, which resulted in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we showed that Myc-induced UPR activation was reversed. Our findings establish a role for UPR as an enhancer of c-Myc-induced transformation and suggest that UPR inhibition may be particularly effective against malignancies characterized by c-Myc overexpression.

Defective mitochondrial morphology and bioenergetic function in mice lacking the transcription factor Yin Yang 1 in skeletal muscle.

The formation, distribution, and maintenance of functional mitochondria are achieved through dynamic processes that depend strictly on the transcription of nuclear genes encoding mitochondrial proteins. A large number of these mitochondrial genes contain binding sites for the transcription factor Yin Yang 1 (YY1) in their proximal promoters, but the physiological relevance is unknown. We report here that skeletal-muscle-specific YY1 knockout (YY1mKO) mice have severely defective mitochondrial morphology and oxidative function associated with exercise intolerance, signs of mitochondrial myopathy, and short stature. Gene set enrichment analysis (GSEA) revealed that the top pathways downregulated in YY1mKO mice were assigned to key metabolic and regulatory mitochondrial genes. This analysis was consistent with a profound decrease in the level of mitochondrial proteins and oxidative phosphorylation (OXPHOS) bioenergetic function in these mice. In contrast to the finding for wild-type mice, inactivation of the mammalian target of rapamycin (mTOR) did not suppress mitochondrial genes in YY1mKO mice. Mechanistically, mTOR-dependent phosphorylation of YY1 resulted in a strong interaction between YY1 and the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), a major regulator of mitochondrial function. These results underscore the important role of YY1 in the maintenance of mitochondrial function and explain how its inactivation might contribute to exercise intolerance and mitochondrial myopathies.

Yin Yang 1 deficiency in skeletal muscle protects against rapamycin-induced diabetic-like symptoms through activation of insulin/IGF signaling.

Rapamycin and its derivatives are mTOR inhibitors used in tissue transplantation and cancer therapy. A percentage of patients treated with these inhibitors develop diabetic-like symptoms, but the molecular mechanisms are unknown. We show here that chronic rapamycin treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as Igf1-2, Irs1-2, and Akt1-3. Importantly, skeletal muscle-specific YY1 knockout mice were protected from rapamycin-induced diabetic-like symptoms. This protection was caused by hyperactivation of insulin/IGF signaling with increased gene expression in this cascade that, in contrast to wild-type mice, was not suppressed by rapamycin. Mechanistically, rapamycin induced YY1 dephosphorylation and recruitment to promoters of insulin/IGF genes, which promoted interaction with the polycomb protein-2 corepressor. This was associated with H3K27 trimethylation leading to decreased gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes such as longevity.

Endoscopic ultrasound with biopsy of omental mass for cholangiocarcinoma diagnosis in cirrhosis.

In this report, a patient had a previous diagnosis of cholangiocarcinoma with an extended cholecystectomy. Three years later, he was evaluated for recurrent ascites. The patient had several large volume paracentesis, without evidence of malignant cells. Subsequently, endoscopic ultrasound (EUS) with fine needle aspiration (FNA) of both lymph and omental nodules was utilized. While the lymph nodes were negative for malignancy, the omental nodule was interrogated with multiple antibodies and was found to be positive for neoplasia. EUS with FNA can safely be used in patients with cirrhosis to spare the patient invasive evaluation such as exploratory laparotomy (ex-lap) for diagnosis and staging of cholangiocarcinoma.

Use of endoscopic ultrasound for diagnosis of cholangiocarcinoma in auto-immune hepatitis.

In this report, a patient was exposed to an herbal remedy for hypercholesterolemia. She became acutely jaundiced while taking the remedy and presented for medical care. Endoscopic ultrasound was utilized, and found a distal common bile duct mass. Endoscopic retrograde cholangiopancreatography guided bile duct biopsies revealed that the mass was cholangiocarcinoma (CCA). This case highlights a unique association between autoimmune hepatitis and CCA. It also highlights that EUS can be safely used in patients with cirrhosis to spare invasive evaluation such as exploratory laporotomy for diagnosis and staging of cholangiocarcinoma.

Large-diameter biliary orifice balloon dilation to aid in endoscopic bile duct stone removal: a multicenter series.

BACKGROUND: The utility and safety of endoscopic biliary orifice balloon dilation (EBD) for bile duct stone removal (with use of large-diameter balloons) after biliary endoscopic sphincterotomy (BES) is currently not well established. OBJECTIVE: Our purpose was to evaluate the efficacy and complications of BES followed by > or = 12 mm diameter EBD for bile duct stone removal. DESIGN: Retrospective, multicenter series. SETTING: Five ERCP referral centers in the United States. PATIENTS AND INTERVENTIONS: Patients who underwent attempted removal of bile duct stones by BES followed by EBD with > or = 12 mm diameter dilating balloons were identified by searching the prospectively recorded endoscopic databases from 1999 to 2007. Clinical parameters, endoscopic data, and outcomes were collected and analyzed. RESULTS: One hundred three patients, mean age 70 +/- 17 years (range 23-98 years), with 56 (54%) women, underwent 107 procedures. Eleven patients (11%) had a prior history of acute pancreatitis. Pancreatogram was performed in 15 (14%) patients. Median stone size and median balloon diameter used was 13 mm. Complete stone removal in the first session of EBD was accomplished in 102 (95%) procedures, and mechanical lithotripsy was required in 29 (27%). Six patients (5.4%) had documented procedure-related complications including one patient with severe bleeding and one with severe cystic duct perforation. No acute pancreatitis occurred. CONCLUSION: EBD with a large-diameter balloon in conjunction with BES for bile duct stone removal is effective and relatively safe. This technique appears to be a reasonable alternative option when standard BES and basket or balloon sweep are inadequate to remove bile duct stones.

Loss of the eukaryotic initiation factor 3f in pancreatic cancer.

Aberrant regulation of the translation initiation is known to contribute to tumorigenesis. eIF3 plays an important role in translation initiation. eIF3f is the p47 subunit of the eIF3 complex whose function in cancer is not clear. Initial studies from our group indicated that eIF3f expression is decreased in pancreatic cancer. Overexpression of eIF3f induces apoptosis in pancreatic cancer cells. The eIF3f gene is located at chromosome band region 11p15.4. Loss of 11p15.4 is a common event in many tumors including pancreatic cancer. In order to investigate the molecular mechanism of the decreased expression of eIF3f in pancreatic cancer, we performed loss of heterozygosity (LOH) analysis in 32 pancreatic cancer specimens using three microsatellite markers encompassing the eIF3f gene. We showed that the prevalence of LOH ranged from 71% to 93%. We also performed eIF3f gene copy number analysis using quantitative real time PCR to further confirm the specific allelic loss of eIF3f gene in pancreatic cancer. We demonstrated a statistically significant decrease of eIF3f gene copy number in pancreatic tumors compared with normal tissues with a tumor/normal ratio of 0.24. Furthermore, RNA in situ hybridization and tissue microarray immunohistochemistry analysis demonstrated that eIF3f expression is significantly decreased in human pancreatic adenocarcinoma tissues compared to normal pancreatic tissues. These data provides new insight into the understanding of the molecular pathogenesis of eIF3f during pancreatic tumorigenesis.

mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex.

Transcriptional complexes that contain peroxisome-proliferator-activated receptor coactivator (PGC)-1alpha control mitochondrial oxidative function to maintain energy homeostasis in response to nutrient and hormonal signals. An important component in the energy and nutrient pathways is mammalian target of rapamycin (mTOR), a kinase that regulates cell growth, size and survival. However, it is unknown whether and how mTOR controls mitochondrial oxidative activities. Here we show that mTOR is necessary for the maintenance of mitochondrial oxidative function. In skeletal muscle tissues and cells, the mTOR inhibitor rapamycin decreased the gene expression of the mitochondrial transcriptional regulators PGC-1alpha, oestrogen-related receptor alpha and nuclear respiratory factors, resulting in a decrease in mitochondrial gene expression and oxygen consumption. Using computational genomics, we identified the transcription factor yin-yang 1 (YY1) as a common target of mTOR and PGC-1alpha. Knockdown of YY1 caused a significant decrease in mitochondrial gene expression and in respiration, and YY1 was required for rapamycin-dependent repression of those genes. Moreover, mTOR and raptor interacted with YY1, and inhibition of mTOR resulted in a failure of YY1 to interact with and be coactivated by PGC-1alpha. We have therefore identified a mechanism by which a nutrient sensor (mTOR) balances energy metabolism by means of the transcriptional control of mitochondrial oxidative function. These results have important implications for our understanding of how these pathways might be altered in metabolic diseases and cancer.

Antegrade bowel intussusception after remote Whipple and Puestow procedures for treatment of pancreas divisum.

To date, antegrade intussusception involving a Roux-en-Y reconstruction has been reported only once. We report a case of acute bowel obstruction due to an intussusception involving two Roux-en-Y limbs in a 40-year-old woman with a history of chronic pancreatitis due to pancreas divisum. Four years preceding this event, the patient had undergone a Whipple procedure, and three years prior to that, a Puestow operation. The patient was successfully treated with bowel resection and a side-to-side anastomosis between the most distal aspect of the bowel and the most distal Roux-en-Y reconstruction, which preserved both Roux-en-Y reconstructions.