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Kamarajan P.,University of California at San Francisco | Rajendiran T.M.,Michigan Center for Translational Pathology | Kinchen J.,Metabolon | Bermudez M.,National Autonomous University of Mexico | And 2 more authors.
Journal of Proteome Research | Year: 2017

Cancer cells use alternate energetic pathways; however, cancer stem cell (CSC) metabolic energetic pathways are unknown. The purpose of this study was to define the metabolic characteristics of head and neck cancer at different points of its pathogenesis with a focus on its CSC compartment. UPLC-MS/MS-profiling and GC-MS-validation studies of human head and neck cancer tissue, saliva, and plasma were used in conjunction with in vitro and in vivo models to carry out this investigation. We identified metabolite biomarker panels that distinguish head and neck cancer from healthy controls, and confirmed involvement of glutamate and glutaminolysis. Glutaminase, which catalyzes glutamate formation from glutamine, and aldehyde dehydrogenase (ALDH), a stemness marker, were highly expressed in primary and metastatic head and neck cancer tissues, tumorspheres, and CSC versus controls. Exogenous glutamine induced stemness via glutaminase, whereas inhibiting glutaminase suppressed stemness in vitro and tumorigenesis in vivo. Head and neck CSC (CD44hi/ALDHhi) exhibited higher glutaminase, glutamate, and sphere levels than CD44lo/ALDHlo cells. Glutaminase drove transcriptional and translational ALDH expression, and glutamine directed even CD44lo/ALDHlo cells toward stemness. Glutaminolysis regulates tumorigenesis and CSC metabolism via ALDH. These findings indicate that glutamate is an important marker of cancer metabolism whose regulation via glutaminase works in concert with ALDH to mediate cancer stemness. Future analyses of glutaminolytic-ALDH driven mechanisms underlying tumorigenic transitions may help in the development of targeted therapies for head and neck cancer and its CSC compartment. © 2017 American Chemical Society.


Shukla S.,University of Michigan | Shukla S.,Michigan Center for Translational Pathology | Malik R.,University of Michigan | Malik R.,Michigan Center for Translational Pathology | And 11 more authors.
Journal of the National Cancer Institute | Year: 2017

Background: Precision therapy for lung cancer will require comprehensive genomic testing to identify actionable targets as well as ascertain disease prognosis. RNA-seq is a robust platform that meets these requirements, but microarray-derived prognostic signatures are not optimal for RNA-seq data. Thus, we undertook the first prognostic analysis of lung adenocarcinoma RNA-seq data and generated a prognostic signature. Methods: Lung adenocarcinoma RNA-seq and clinical data from The Cancer Genome Atlas (TCGA) were divided chronologically into training (n=255) and validation (n=157) cohorts. In the training cohort, prognostic association was assessed by univariate Cox analysis. A prognostic signature was built with stepwise multivariable Cox analysis. Outcomes by risk group, stage, and mutation status were analyzed with Kaplan-Meier and multivariable Cox analyses. All the statistical tests were two-sided. Results: In the training cohort, 96 genes had prognostic association with P values of less than or equal to 1.00x10-4, including five long noncoding RNAs (lncRNAs). Stepwise regression generated a four-gene signature, including one lncRNA. Signature high-risk cases had worse overall survival (OS) in the TCGA validation cohort (hazard ratio [HR] = 3.07, 95% confidence interval [CI] = 2.00 to 14.62) and a University of Michigan institutional cohort (n=67; HR=2.05, 95% CI=1.18 to 4.55), and worse metastasis-free survival in the TCGA validation cohort (HR=3.05, 95% CI=2.31 to 13.37). The four-gene prognostic signature also statistically significantly stratified overall survival in important clinical subsets, including stage I (HR=2.78, 95% CI=1.91 to 11.13), EGFR wild-type (HR=3.01, 95% CI=1.73 to 14.98), and EGFR mutant (HR=8.99, 95% CI=62.23 to 141.44). The fourgene prognostic signature also stood out on top when compared with other prognostic signatures. Conclusions: Here, we present the first RNA-seq prognostic signature for lung adenocarcinoma that can provide a powerful prognostic tool for precision oncology as part of an integrated RNA-seq clinical sequencing program. © The Author 2016. Published by Oxford University Press. All rights reserved.


Prensner J.R.,Michigan Center for Translational Pathology | Chen W.,University of Michigan | Iyer M.K.,Michigan Center for Translational Pathology | Cao Q.,Michigan Center for Translational Pathology | And 21 more authors.
Cancer Research | Year: 2014

Impairment of double-stranded DNA break (DSB) repair is essential to many cancers. However, although mutations in DSB repair proteins are common in hereditary cancers, mechanisms of impaired DSB repair in sporadic cancers remain incompletely understood. Here, we describe the first role for a long noncoding RNA (lncRNA) in DSB repair in prostate cancer. We identify PCAT-1, a prostate cancer outlier lncRNA, which regulates cell response to genotoxic stress. PCAT-1 expression produces a functional deficiency in homologous recombination through its repression of the BRCA2 tumor suppressor, which, in turn, imparts a high sensitivity to smallmolecule inhibitors of PARP1. These effects reflected a posttranscriptional repression of the BRCA2 30UTR by PCAT-1. Our observations thus offer a novel mechanism of "BRCAness" in sporadic cancers.©2014 American Association for Cancer Research.


Mehra R.,University of Michigan | Udager A.M.,University of Michigan | Ahearn T.U.,Harvard University | Cao X.,Michigan Center for Translational Pathology | And 6 more authors.
European Urology | Year: 2015

The long noncoding RNA SChLAP1 is overexpressed in a subset of prostate cancers (PCa), and high SChLAP1 expression by in situ hybridization (ISH) independently predicts biochemical recurrence after radical prostatectomy. Importantly, although biochemical recurrence is a significant clinical outcome, it is not a validated surrogate for PCa-related mortality. Thus, we evaluated the association between SChLAP1 expression and development of lethal PCa in a large cohort of American men with PCa and long-term follow-up. SChLAP1 ISH was performed on tissue microarrays containing representative formalin-fixed, paraffin-embedded PCa tissue from all patients and scored using a semiquantitative method (ISH score range 0-400). Hazard ratios (HRs) for the association between SChLAP1 expression and time to development of lethal PCa were estimated using multivariable Cox regression analysis. Of the 937 patients evaluated, 89 (9.5%) had high SChLAP1 expression (ISH score ≥100), which in patients treated with radical prostatectomy was strongly associated with development of lethal PCa independent of age, Gleason score, pathologic stage, and PTEN status (HR 2.2, 95% confidence interval 1.1-4.1). These results suggest that SChLAP1 may be a useful tissue-based biomarker for identifying PCa patients at higher risk of lethal progression. Patient summary: We examined expression of the RNA molecule SChLAP1 in a large group of prostate cancer patients with long-term follow-up and found that patients with high SChLAP1 expression had a significantly higher chance of developing lethal disease. High SChLAP1 expression by ISH independently predicts lethal disease in a large cohort of American men with prostate cancer and long-term follow-up, suggesting that SChLAP1 ISH may be a promising biomarker for identifying patients at higher risk of lethal progression. © 2015 European Association of Urology.


Hua Y.,Northwestern University | Larsen N.,University of Aarhus | Kalyana-Sundaram S.,Michigan Center for Translational Pathology | Kjems J.,University of Aarhus | And 2 more authors.
BMC Genomics | Year: 2013

Background: Based on their function in cancer micro(mi)RNAs are often grouped as either tumor suppressors or oncogenes. However, miRNAs regulate multiple tumor relevant signaling pathways raising the question whether two oncogenic miRNAs could be functional antagonists by promoting different steps in tumor progression. We recently developed a method to connect miRNAs to biological function by comparing miRNA and gene array expression data from the NCI60 cell lines without using miRNA target predictions (miRConnect).Results: We have now extended this analysis to three primary human cancers (ovarian cancer, glioblastoma multiforme, and kidney renal clear cell carcinoma) available at the Cancer Genome Atlas (TCGA), and have correlated the expression of the clustered miRNAs with 158 oncogenic signatures (miRConnect 2.0). We have identified functionally antagonistic groups of miRNAs. One group (the agonists), which contains many of the members of the miR-17 family, correlated with c-Myc induced genes and E2F gene signatures. A group that was directly antagonistic to the agonists in all three primary cancers contains miR-221 and miR-222. Since both miR-17 ~ 92 and miR-221/222 are considered to be oncogenic this points to a functional antagonism of different oncogenic miRNAs. Analysis of patient data revealed that in certain patients agonistic miRNAs predominated, whereas in other patients antagonists predominated. In glioblastoma a high ratio of miR-17 to miR-221/222 was predictive of better overall survival suggesting that high miR-221/222 expression is more adverse for patients than high miR-17 expression.Conclusion: miRConnect 2.0 is useful for identifying activities of miRNAs that are relevant to primary cancers. The new correlation data on miRNAs and mRNAs deregulated in three primary cancers are available at miRConnect.org. © 2013 Hua et al.; licensee BioMed Central Ltd.


Oh H.-K.,National University of Singapore | Tan A.L.-K.,National University of Singapore | Das K.,National University of Singapore | Ooi C.-H.,National University of Singapore | And 10 more authors.
Clinical Cancer Research | Year: 2011

Purpose: MicroRNAs (miRNA) play pivotal oncogenic and tumor-suppressor roles in several human cancers. We sought to discover novel tumor-suppressor miRNAs in gastric cancer (GC). Experimental Design: Using Agilent miRNA microarrays, we compared miRNA expression profiles of 40 primary gastric tumors and 40 gastric normal tissues, identifying miRNAs significantly downregulated in gastric tumors. Results: Among the top 80 miRNAs differentially expressed between gastric tumors and normals (false discovery rate < 0.01), we identified hsa-miR-486 (miR-486) as a significantly downregulated miRNA in primary GCs and GC cell lines. Restoration of miR-486 expression in GC cell lines (YCC3, SCH and AGS) caused suppression of several pro-oncogenic traits, whereas conversely inhibiting miR-486 expression in YCC6 GC cells enhanced cellular proliferation. Array-CGH analysis of 106 primary GCs revealed genomic loss of the miR-486 locus in approximately 25% to 30% of GCs, including two tumors with focal genomic losses specifically deleting miR-486, consistent with miR-486 playing a tumor-suppressive role. Bioinformatic analysis identified the secreted antiapoptotic glycoprotein OLFM4 as a potential miR-486 target. Restoring miR-486 expression in GC cells decreased endogenous OLFM4 transcript and protein levels, and also inhibited expression of luciferase reporters containing an OLFM4 3′ untranslated region with predicted miR-486 binding sites. Supporting the biological relevance of OLFM4 as a miR-486 target, proliferation in GC cells was also significantly reduced by OLFM4 silencing. Conclusions: miR-486 may function as a novel tumor-suppressor miRNA in GC. Its antioncogenic activity may involve the direct targeting and inhibition of OLFM4. ©2011 AACR.


Trerotola M.,Thomas Jefferson University | Jernigan D.L.,Drexel University | Liu Q.,Wistar Institute | Siddiqui J.,Michigan Center for Translational Pathology | And 4 more authors.
Cancer Research | Year: 2013

The molecular mechanisms underlying metastatic dissemination are still not completely understood. We have recently shown that β1 integrin-dependent cell adhesion to fibronectin and signaling is affected by a transmembrane molecule, Trop-2, which is frequently upregulated in human carcinomas. Here, we report that Trop-2 promotes metastatic dissemination of prostate cancer cells in vivo and is abundantly expressed in metastasis from human prostate cancer. We also show here that Trop-2 promotes prostate cancer cell migration on fibronectin, a phenomenon dependent on β1 integrins. Mechanistically, we demonstrate that Trop-2 and the α5β1 integrin associate through their extracellular domains, causing relocalization of α5β1 and the β1-associated molecule talin from focal adhesions to the leading edges. Trop-2 effect is specific as this molecule does not modulate migration on vitronectin, does not associate with the major vitronectin receptor, αvβ3 integrin, and does not affect localization of αvβ3 integrin as well as vinculin in focal adhesions. We show that Trop-2 enhances directional prostate cancer cell migration, through modulation of Rac1 GTPase activity. Finally, we show that Trop-2 induces activation of PAK4, a kinase that has been reported to mediate cancer cell migration. In conclusion, we provide the first evidence that β1 integrin-dependent migratory and metastatic competence of prostate cancer cells is enhanced by Trop-2. Cancer Res; 73(10); 3155-67. © 2013 AACR.


Udager A.M.,University of Michigan | Alva A.,University of Michigan | Chen Y.-B.,Sloan Kettering Cancer Center | Siddiqui J.,Michigan Center for Translational Pathology | And 8 more authors.
American Journal of Surgical Pathology | Year: 2014

Rapid ("warm") autopsies of patients with advanced metastatic cancer provide invaluable insight into the natural history, pathobiology, and morphology of advanced and treatment-resistant tumors. Here, we report a rapid autopsy case of a hereditary leiomyomatosis and renal cell carcinoma (HLRCC) patient with advanced metastatic renal cell carcinoma (RCC) - the first such case described for either a primary renal tumor or HLRCC-related cancer. Mutations in the fumarate hydratase (FH) gene underlie HLRCC, a rare syndrome involving cutaneous and uterine leiomyomata and aggressive kidney tumors. Loss of heterozygosity at the wild-type FH gene locus results in profound cellular metabolic derangement, "pseudohypoxic" upregulation of hypoxia-inducible factor 1α (HIF-1α)-dependent transcription, and aberrant protein succination; these molecular changes drive oncogenesis of kidney tumors in HLRCC patients. The current index patient had a high-grade RCC with classic morphologic features of HLRCC, including large nuclei with prominent eosinophilic nucleoli and perinucleolar clearing. In addition, this patient's RCC demonstrated extensive sarcomatoid and rhabdoid features - morphologies not previously well described in HLRCC-associated kidney tumors. Here, we report the extent of metastatic dissemination and supplement this unique tumor morphology with mitochondrial enzyme histochemistry and extended immunohistochemical analysis. Tumor cells strongly expressed PAX8, vimentin, CD10, and the HIF target GLUT1 and showed increased nuclear p53 accumulation; the expression of other RCC markers was negative. We also detail microscopic tubular epithelial changes in the grossly uninvolved ipsilateral renal parenchyma and demonstrate sporadic, aberrant upregulation of the HIF targets GLUT1 and CAIX in dysplastic peritumoral tubules. © 2014 by Lippincott Williams & Wilkins.


Somashekar B.S.,University of Michigan | Kamarajan P.,University of Michigan | Danciu T.,University of Michigan | Kapila Y.L.,University of Michigan | And 5 more authors.
Journal of Proteome Research | Year: 2011

High-resolution magic-angle spinning (HR-MAS) proton NMR spectroscopy is used to explore the metabolic signatures of head and neck squamous cell carcinoma (HNSCC) which included matched normal adjacent tissue (NAT) and tumor originating from tongue, lip, larynx and oral cavity, and associated lymph-node metastatic (LN-Met) tissues. A total of 43 tissues (18 NAT, 18 Tumor and 7 LN-Met) from 22 HNSCC patients were analyzed. Principal Component Analysis of NMR data showed a clear classification between NAT and tumor tissues, however, LN-Met tissues were classified among tumor. A partial least-squares discriminant analysis model generated from NMR metabolic profiles was used to differentiate normal from tumor samples (Q 2 > 0.80, Receiver Operator Characteristic area under the curve >0.86, using 7-fold cross validation). HNSCC and LN-Met tissues showed elevated levels of lactate, amino acids including leucine, isoleucine, valine, alanine, glutamine, glutamate, aspartate, glycine, phenylalanine and tyrosine, choline containing compounds, creatine, taurine, glutathione, and decreased levels of triglycerides. These elevated metabolites were associated with highly active glycolysis, increased amino acids influx (anaplerosis) into the TCA cycle, altered energy metabolism, membrane choline phospholipid metabolism, and oxidative and osmotic defense mechanisms. Moreover, decreased levels of triglycerides may indicate lipolysis followed by β-oxidation of fatty acids that may exist to deliver bioenergy for rapid tumor cell proliferation and growth. © 2011 American Chemical Society.


Udager A.M.,University of Michigan | Mehra R.,University of Michigan | Mehra R.,Michigan Center for Translational Pathology
Archives of Pathology and Laboratory Medicine | Year: 2016

Molecular and morphologic interrogation has driven a much-needed reexamination of renal cell carcinoma (RCC). Indeed, the recently released 2016 World Health Organization classification now recognizes 12 distinct RCC subtypes, as well as several other emerging/provisional RCC entities. From a clinical perspective, accurate RCC classification may have important implications for patients and their families, including prognostic risk stratification, targeted therapeutics selection, and identification for genetic testing. In this review, we provide a conceptual framework for approaching RCC diagnosis and classification by categorizing RCCs as tumors with clear cytoplasm, papillary architecture, and eosinophilic (oncocytic) cytoplasm. The currently recognized 2016 World Health Organization classification for RCC subtypes is briefly discussed, including new diagnostic entities (clear cell papillary RCC, hereditary leiomyomatosis and RCCassociated RCC, succinate dehydrogenase-deficient RCC, tubulocystic RCC, and acquired cystic disease-associated RCC) and areas of evolving RCC classification, such as transcription elongation factor B subunit 1 (TCEB1)-mutated RCC/RCC with angioleiomyoma-like stroma/RCC with leiomyomatous stroma, RCC associated with anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangement, thyroidlike follicular RCC, and RCC in neuroblastoma survivors. For each RCC subtype, relevant clinical, molecular, gross, and microscopic findings are reviewed, and ancillary studies helpful for its differential diagnosis are presented, providing a practical approach to modern RCC classification.

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