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NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. Cancer Res; 74(4); 1091–104. ©2013 AACR.

PDF file - 82K, Growth effects of NOTCH1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. NOTCH1 was downregulated in UPCI-SCC090 (090, top, NOTCH1 wt) and SCC61 (bottom, NOTCH1 wt) by siRNA as described in methods. The effect of NOTCH1 downregulation on cell proliferation for these two cell lines is shown in the middle. The effect of siNOTCH1 on NOTCH1 expression (left) or expression of its targets (HES1 and HEY1, right) is also shown. The mRNA expression levels were calculated relative to GAPDH and were normalized to 1 in the control experiments with scrambled siRNA sequences. Experiments were performed in pentaplicates, mean plus-minus SEM were shown. *, p-value < 0.05.

PDF file - 73K, Significant overexpression of JAG1 (A) and PSEN1 (B) in the tumors with increased copy number gains in comparison to that in the normal tissues in the cohort of 44 HNSCC tumors and 25 normal mucosa. N, normal mucosa; T, HNSCC tumors; -, without JAG1(A) or PSEN1 (B) copy number gain; +, with JAG1 (A) or PSEN1 (B) copy number gain. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

PDF file - 169K, NOTCH pathway activation in HNSCC. A, Immunohistochemical analysis of NOTCH1, HES1 and HEY1 protein expression in HNSCC. Tissue microarray arrays with quadruplicates of samples from each of 56 HNSCC tumor and 11 non-tumor patients were stained with anti-NOTCH1, anti-HES1 or anti-HEY1 antibody, as described in methods. Top, the heat-map of immunohistochemical analysis for tissue microarray slides. Rows, genes; column, each individual sample. The average staining for four tissues was categorized to Strong (brown), Moderate (grey) or Weak/Negative (blue) staining. Samples are ranked by the pathology ID numbers in tumor (left) and normal (right) groups. Bottom: quantification of immunohistochemical data. The number and percentage of tumor (Tu) and normal (No) patient in strong, moderate and weak/negative category was quantified. P-values were calculated by comparing numeric scores in tumor vs normal sample by independent t test. The samples 57964, 58602, 22146, 5931, 26514 represent tumor samples X6, X7, X11, X28 and X34 from the original discovery cohort of 44 HNSCC tumors. B, Representative immunohistochemical results. The representative slides of positive or moderate staining are shown for tumors and compared to normal tissue. Bar, 100 um equivalent. Tumor (T) and normal (N) tissues on each sample are labeled. ID 22146 represented X11 samples, which were used both in array and in immunohistochemical analysis, where protein and RNA expression strongly correlate.

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PDF file - 54K, Effect of gamma-secretase inhibitor on the NOTCH1 pathway genes and cell growth in HNSCC cells with NOTCH1 wildtype status. UPCI-SCC090 cells were growing on the media supplemented with 0.2 uM gamma-secretase inhibitor XXI (GSI-XXI) for up to 72 hours. The effect of GSI-XXI on the NOTCH1 targets HES1 and HEY1 is shown on the left for UPSI-SCC090 cell line.

PDF file - 68K, Growth effects of HEY1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. HEY1 was downregulated in UPCI-SCC090 and SCC61 by siRNA as described in methods. The effect of siHEY1 on the expression of HEY1 is shown on the left. The effect of HEY1 downregulation on cell proliferation for these two cell lines is shown on the right. Note, there is over 90% transfection efficiency of HEY1 downregulation for each cell line, however the strong growth inhibition effect was shown only for NOTCH1 wildtype 090 cell line.

PDF file - 108K, Validation of JAG1, JAG2, NOTCH1 and PSEN1 overexpression in TCGA HNSCC cohort. A, Overexpression of JAG1, JAG2 and NOTCH1 in tumors in comparison to that in normal tissues in the TCGA HNSCC cohort. B, Overexpression of JAG1 and PSEN1 in the tumors with increased copy number gains in comparison to that in the normal tissues as well as to that in the tumors without copy number gains in the TCGA HNSCC cohort. N, normal mucosa; T, HNSCC tumors; -, without JAG1 or PSEN1 copy number gain; +, with JAG1 or PSEN1 copy number gain. Dots, relative expression levels in different tissue samples. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

PDF file - 88K, Supplementary Table I. Baseline characteristics of the HNSCC patients for microarray analysis. Supplementary Table II. Short Tandem Repeat Profile of the 2 HNSCC Cell Lines. Supplementary Table III. NOTCH Signaling Pathway Genes assigned in KEGG pathway database and shown on expression microarrays. Supplementary Table IV. Summary of the mRNA levels of NOTCH signaling pathway genes in the cohort of 44 HNSCC tumors and 25 normal mucosas measured by Affymetrix expression array.

Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.

Recently, the KIF1B beta gene on 1p36, a region commonly deleted in neural crest cancers, was found to be a proapoptotic factor for sympathetic precursors. KIF1B beta mutations were detected in pheochromocytomas and neuroblastomas, two sympathetic lineage tumors, suggesting a role for this gene in cancer. Here, we studied five individuals from a three-generation cancer-prone family with a KIF1B beta germline variant and seven of their tumors, both of neural crest and nonneural origin. Genetic studies including sequencing, copy number analysis and fluorescence in situ-hybridization (FISH) showed retention of both KIF1B beta alleles in all neural crest-derived tumors in this family, consistent with haploinsufficiency or methylation of the wild-type allele. In contrast, the lung adenocarcinoma from one mutation carrier had somatic loss of the wild-type allele in agreement with a classical two-hit inactivation. Global transcription analysis of KIF1B beta mutant pheochromocytomas revealed that these tumors are transcriptionally related to pheochromocytomas with RET and NF1 mutations but independent from SDH- and VHL-associated tumors. Furthermore, KIF1B beta-mutant tumors are uniquely enriched for pathways related to glutamate metabolism and the oxidative stress response. Our data start to delineate the signals that are disrupted by KIF1B beta dysfunction in pheochromocytomas and suggest that loss of this gene may also be permissive to the development of nonneural crest malignancies. This may imply the existence of a tissue-specific gene dosage requirement for its tumorigenesis.

NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. Cancer Res; 74(4); 1091–104. ©2013 AACR.

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.

Pheochromocytomas, which are catecholamine-secreting tumors of neural crest origin, are frequently hereditary. However, the molecular basis of the majority of these tumors is unknown. We identified the transmembrane-encoding gene TMEM127 on chromosome 2q11 as a new pheochromocytoma susceptibility gene. In a cohort of 103 samples, we detected truncating germline TMEM127 mutations in approximately 30% of familial tumors and about 3% of sporadic-appearing pheochromocytomas without a known genetic cause. The wild-type allele was consistently deleted in tumor DNA, suggesting a classic mechanism of tumor suppressor gene inactivation. Pheochromocytomas with mutations in TMEM127 are transcriptionally related to tumors bearing NF1 mutations and, similarly, show hyperphosphorylation of mammalian target of rapamycin (mTOR) effector proteins. Accordingly, in vitro gain-of-function and loss-of-function analyses indicate that TMEM127 is a negative regulator of mTOR. TMEM127 dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR, suggesting a subcompartmental-specific effect. Our studies identify TMEM127 as a tumor suppressor gene and validate the power of hereditary tumors to elucidate cancer pathogenesis.

In the classic retinoid cycle, 11-cis retinol is synthesized in the retinal pigment epithelium (RPE) by two enzymes: Isomerase I (RPE65) and lecithin:retinol acyltransferase (LRAT). The purpose of this study is to provide experimental evidence for two active isomerases in the cone-dominated chicken eye: an LRAT-dependent Isomerase I in the RPE and an ARAT (acyl CoA:retinol acyltransferase)-dependent isomerase (Isomerase II) in the retina. First, we show that whole chicken retina in vitro, removed from the RPE/choroid and sclera, produces 11-cis retinoids upon light exposure, indicating the existence of RPE-independent isomerase (Isomerase II) activity in the retina. Reverse transcriptase polymerase chain reaction studies show high levels of RPE65 expression in the RPE, low levels in the retina, and none in primary Muller cell cultures, indicating the presence of Isomerase I in the RPE and a minimal amount in the retina. Activities of the RPE and retina isomerases were then measured by enzyme assays with specific enzyme inhibitors. 2,2'-Bipyridine, a known Isomerase I inhibitor, and N-ethylmaleimide (NEM), a known LRAT inhibitor, significantly reduced Isomerase I activity but not Isomerase II activity. Progesterone, a known ARAT inhibitor, completely blocked Isomerase II activity but not Isomerase I activity. Thus, this study reports novel results for distinguishing the biochemical properties of Isomerase I from those of Isomerase II, as well a difference in their locations in the chicken eye. On the basis of these differences, the cone-dominated chicken eye must contain two retinoid cycles: a classic visual cycle for retinoid exchange between the RPE and the retina supported by Isomerase I in the RPE and an additional visual cycle for retinoid processing in the retina supported by Isomerase II.

Pheochromocytomas and paragangliomas are catecholamine-secreting tumors of neural crest origin caused by germline mutations in at least six distinct genes. This genetic heterogeneity has provided a rich source for both the discovery and functional characterization of new tumor-related genes. However, the genetic repertoire of these tumors is still not fully known, and current evidence points to the existence of additional pheochromocytoma susceptibility genes. Here, the unique contributions of three hereditary models of pheochromocytoma that can advance our knowledge of the disease pathogenesis are presented. The first model, loss of succinate dehydrogenase (SDH) function, illustrates how SDHB, C, or D mutations, components of the energy metabolism pathway, serve as a unique system to explore the pervasive metabolic shift of cancer cells towards glycolysis as a source of energy (also known as the Warburg effect) in contrast to the characteristic oxidative phosphorylation of normal cells. In the second model, mechanisms of tumorigenesis distinct from classical pheochromocytoma susceptibility genes are discussed in the context of a novel putative suppressor of neural crest-derived tumors, the KIF1B beta gene. Finally, NF1 loss is highlighted as a valuable study model to investigate the cell lineage selectivity of the Egln3-mediated developmental apoptotic defect of chromaffin precursor cells. Results from these studies may offer clues to understand the tissue specificity of hereditary pheochromocytoma syndromes. These distinct hereditary disease models illustrate how genetic-driven progress has the potential to narrow current gaps in our knowledge of pheochromocytoma and paraganglioma pathogenesis.

Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

3630 Background: The variable prognoses of stage II colon cancer subjects have provoked a therapeutic dilemma of who should receive adjuvant therapy. Presently, prognosis is evaluated on often subjective histopathological features. Reliable, objective markers that can accurately predict recurrence risk are needed. Using retrospectively collected tumor samples with known clinical outcome, we evaluated the prognostic utility of miRNA together with clinicopathological features and molecular markers with suggestive prognostic value. Methods: We evaluated the expression of miRNA in 118 stage IIA FFPE tumors (73 non-recurrent, NR; 45 recurrent, R) using the Affymetrix Gene Chip V1.0 microarray, and performed RT-qPCR to verify select miRNAs. We also sequenced KRAS codons 12/13, and BRAF codon 600, and assessed MSI status using a fluorescent PCR-based assay. MMR protein expression was determined by IHC. An empirical approach was applied to screen miRNA using combinatorial criteria of microarray 2-way ANOVA analysis, Kaplan-Meier curve logrank, and single and multivariate Cox-regression. miRNA was the most significant predictor among all histopathological and molecular features (FDR 0.6), and associated with non-recurrent disease. Conclusions: These data provide strong support for miRNAs as independent prognostic biomarkers for stage IIA colon cancer.Validation work is ongoing to demonstrate the potential utility of these miRNAs in patient stratification.

MicroRNAs (miRNAs) are naturally occurring, short non-coding RNAs believed to be involved in many biological processes including normal development and adult cancer. It seems logical that miRNAs would be particularly important in pediatric cancers as these malignancies generally involve perturbations of normal developmental and differentiation processes, yet relatively little is known about the role of miRNAs in pediatric cancers. Here, we show that oncogenesis in Wilms’ tumor - the most common pediatric renal cancer - is mediated by miRNAs. MiRNA microarray analysis identified a cohort of miRNAs that show altered expression in Wilms’ tumors when compared with normal matched control kidney. Our results reveal that several of these miRNAs show similar expression patterns both in Wilms’ tumors and fetal kidney, suggesting that they may play an important regulatory role in kidney development and Wilms’ tumorigenesis. Supporting this notion are our findings that show a number of differentially expressed miRNAs to target genes known to play a role in kidney development and Wilms’ tumorigenesis, including the Six1 homeobox gene, a potent oncogene reported to be overexpressed in several solid tumors. Analyses of primary Wilms’ tumors and ovarian cancer tissues as well as multiple tumor cell lines from breast cancer, ovarian cancer and rhabdomyosarcoma showed decreased expression of miRNAs targeting the Six1 3′UTR, paralleling an increase in Six1 levels. Further investigation revealed a potent tumor suppressor role for Six1 targeting miRNAs, as ectopic expression suppressed the ability of cancer cell lines to form colonies and tumor growth in nude mice. Interestingly, the reduced expression of these miRNAs in Wilms’ tumors was due in part to genomic loss of loci that contain these miRNAs, as revealed by high resolution comparative genomic hybridization analysis. In summary, our findings suggest that miRNAs are integral to the molecular circuitry that plays an important role in Wilms’ tumorigenesis by regulating events during normal kidney development. In addition, our discovery of novel tumor suppressor miRNAs targeting the Six1 oncogene promises to unravel a unique regulatory mechanism for Six1 deregulation in pediatric as well adult tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2107.