Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.

Elevated FGF21 secretion, PGC-1α and ketogenic enzyme expression are hallmarks of iron-sulfur cluster depletion in human skeletal muscle.

Iron-sulfur (Fe-S) clusters are ancient enzyme cofactors found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF21 (fibroblast growth factor 21). Enhanced muscle FGF21 expression was reflected by elevated circulating FGF21 levels in the patients, and robust FGF21 secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF21 levels.

Exome sequencing identifies novel somatic variants in African American esophageal squamous cell carcinoma.

Esophageal cancer has a strikingly low survival rate mainly due to the lack of diagnostic markers for early detection and effective therapies. In the U.S., 75% of individuals diagnosed with esophageal squamous cell carcinoma (ESCC) are of African descent. African American ESCC (AA ESCC) is particularly aggressive, and its biological underpinnings remain poorly understood. We sought to identify the genomic abnormalities by conducting whole exome sequencing of 10 pairs of matched AA esophageal squamous cell tumor and control tissues. Genomic analysis revealed diverse somatic mutations, copy number alterations (SCNAs), and potential cancer driver genes. Exome variants created two subgroups carrying either a high or low tumor mutation burden. Somatic mutational analysis based on the Catalog of Somatic Mutations in Cancer (COSMIC) detected SBS16 as the prominent signature in the high mutation rate group suggesting increased DNA damage. SBS26 was also detected, suggesting possible defects in mismatch repair and microsatellite instability. We found SCNAs in multiple chromosome segments, encoding MYC on 8q24.21, PIK3CA and SOX2 on 3q26, CCND1, SHANK2, CTTN on 11q13.3, and KRAS on 12p12. Amplifications of EGFRvIII and EGFRvIVa mutants were observed in two patients, representing a novel finding in ESCC that has potential clinical relevance. This present exome sequencing, which to our knowledge, represents the first comprehensive exome analysis exclusively in AA ESCC, and highlights novel mutated loci that might explain the aggressive nature of AA ESCC and lead to the development of diagnostic and prognostic markers as well as therapeutic targets.

Epigenetic regulator MLL2 shows altered expression in cancer cell lines and tumors from human breast and colon.

BACKGROUND: MLL2, an epigenetic regulator in mammalian cells, mediates histone 3 lysine 4 tri-methylation (H3K4me3) through the formation of a multiprotein complex. MLL2 shares a high degree of structural similarity with MLL, which is frequently disrupted in leukemias via chromosomal translocations. However, this structural similarity is not accompanied by functional equivalence. In light of this difference, and previous reports on involvement of epigenetic regulators in malignancies, we investigated MLL2 expression in established cell lines from breast and colon tissues. We then investigated MLL2 in solid tumors of breast and colon by immunohistochemistry, and evaluated potential associations with established clinicopathologic variables. RESULTS: We examined MLL2 at both transcript and protein levels in established cell lines from breast and colon cancers. Examination of these cell lines showed elevated levels of MLL2. Furthermore, we also identified incomplete proteolytic cleavage of MLL2 in the highly invasive tumor cell lines. To corroborate these results, we studied tumor tissues from patients by immunohistochemistry. Patient samples also revealed increased levels of MLL2 protein in invasive carcinomas of the breast and colon. In breast, cytoplasmic MLL2 was significantly increased in tumor tissues compared to adjacent benign epithelium (p < 0.05), and in colon, both nuclear and cytoplasmic immunostaining was significantly increased in tumor tissues compared to adjacent benign mucosa (p < 0.05). CONCLUSION: Our study indicates that elevated levels of MLL2 in the breast and colon cells are associated with malignancy in these tissues, in contrast to MLL involvement in haematopoietic cancer. In addition, both abnormal cellular localization of MLL2 and incomplete proteolytic processing may be associated with tumor growth/progression in breast and colonic tissues. This involvement of MLL2 in malignancy may be another example of the role of epigenetic regulators in cancer.

gamma-Radiation-induced chromosomal mutagen sensitivity is associated with breast cancer risk in African-American women: caffeine modulates the outcome of mutagen sensitivity assay.

Several different cancer studies have indicated that lymphocyte mutagen sensitivity is a marker of DNA repair deficiency and increased cancer risk. We have used a mutagen sensitivity assay (MSA) measuring gamma-radiation-induced chromosomal aberrations in freshly cultured lymphocytes and assessed breast cancer risk in African-American women. Concurrently, we conducted duplicate cultures in the presence of caffeine, which overrides G(2) arrest in cultured cells, decreases time to DNA repair, and hence increases the aberration rate. In comparison with the non-caffeine-treated cells, we are conceptually segregating the contribution of DNA repair and time for DNA repair as individual susceptibility phenotypes. Blood samples were obtained from 61 cases and 86 controls at Howard University Hospital. Two sets of whole-blood cultures were established and gamma-irradiated (1 Gy) at 67 hours, one of which was treated with caffeine (1 mg/mL). Thereafter, cultures were processed for obtaining metaphase spreads. Fifty metaphases were screened for chromatid breaks. The mean breaks per cell (MBPC) for cases (0.34 +/- 0.15) was significantly greater than for controls (0.24 +/- 0.12; P < 0.0001). Using the 75th percentile value of controls as a cutoff to define mutagen sensitivity, the sensitive individuals had an odds ratio of 4.5 (95% confidence intervals, 2.2-9.1) for breast cancer compared with individuals that were not sensitive. The adjusted odds ratio was 3.3 (95% confidence intervals, 0.147-73.917), which was statistically significant but was limited by the small number of subjects. The results for caffeine co-culture were not predictive of breast cancer (MBPC: cases, 1.6 +/- 0.9 versus controls, 1.5 +/- 0.8; P = 0.8663). Comparing the MBPC for caffeine and non-caffeine cultures, there was a correlation in controls (n = 79; Spearman r = 0.4286; P < 0.0001), but not in cases (n = 58; Spearman r = 0.06609; P = 0.6221). This study indicates that the MSA phenotype is a risk factor for breast cancer in African-American women, with a significant effect observable even in small studies. The use of caffeine did not enhance the predictivity of MSA, but the correlation with non-caffeine cultures in controls indicates that the MSA phenotype is due to both DNA repair and G(2) arrest capacity.

Impact of Pre-Analytical Variables on Cancer Targeted Gene Sequencing Efficiency.

Tumor specimens are often preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks, the most common clinical source for DNA sequencing. Herein, we evaluated the effect of pre-sequencing parameters to guide proper sample selection for targeted gene sequencing. Data from 113 FFPE lung tumor specimens were collected, and targeted gene sequencing was performed. Libraries were constructed using custom probes and were paired-end sequenced on a next generation sequencing platform. A PCR-based quality control (QC) assay was utilized to determine DNA quality, and a ratio was generated in comparison to control DNA. We observed that FFPE storage time, PCR/QC ratio, and DNA input in the library preparation were significantly correlated to most parameters of sequencing efficiency including depth of coverage, alignment rate, insert size, and read quality. A combined score using the three parameters was generated and proved highly accurate to predict sequencing metrics. We also showed wide read count variability within the genome, with worse coverage in regions of low GC content like in KRAS. Sample quality and GC content had independent effects on sequencing depth, and the worst results were observed in regions of low GC content in samples with poor quality. Our data confirm that FFPE samples are a reliable source for targeted gene sequencing in cancer, provided adequate sample quality controls are exercised. Tissue quality should be routinely assessed for pre-analytical factors, and sequencing depth may be limited in genomic regions of low GC content if suboptimal samples are utilized.

Somatic Mutation Spectrum of Non-Small-Cell Lung Cancer in African Americans: A Pooled Analysis.

INTRODUCTION: The mutational profile of non-small-cell lung cancer (NSCLC) has become an important tool in tailoring therapy to patients, with clear differences according to the population of origin. African Americans (AAs) have higher lung cancer incidence and mortality than Caucasians, yet discrepant results have been reported regarding the frequency of somatic driver mutations. We hypothesized that NSCLC has a distinct mutational profile in this group. METHODS: We collected NSCLC samples resected from self-reported AAs in five sites from Tennessee, Michigan, and Ohio. Gene mutations were assessed by either SNaPshot or next generation sequencing, and ALK translocations were evaluated by fluorescence in situ hybridization. RESULTS: Two hundred sixty patients were included, mostly males (62.3%) and smokers (86.6%). Eighty-one samples (31.2%) were squamous cell carcinomas. The most frequently mutated genes were KRAS (15.4%), epidermal growth factor receptor (EGFR, 5.0%), PIK3CA (0.8%), BRAF, NRAS, ERBB2, and AKT1 (0.4% each). ALK translocations were detected in two nonsquamous tumors (1.7%), totaling 61 cases (23.5%) with driver oncogenic alterations. Among 179 nonsquamous samples, 54 (30.2%) presented a driver alteration. The frequency of driver alterations altogether was lower than that reported in Caucasians, whereas no difference was detected in either EGFR or KRAS mutations. Overall survival was longer among patients with EGFR mutations. CONCLUSIONS: We demonstrated that NSCLC from AAs has a different pattern of somatic driver mutations than from Caucasians. The majority of driver alterations in this group are yet to be described, which will require more comprehensive panels and assessment of noncanonical alterations.

Genomic Characterization of Non-Small-Cell Lung Cancer in African Americans by Targeted Massively Parallel Sequencing.

PURPOSE: Technologic advances have enabled the comprehensive analysis of genetic perturbations in non-small-cell lung cancer (NSCLC); however, African Americans have often been underrepresented in these studies. This ethnic group has higher lung cancer incidence and mortality rates, and some studies have suggested a lower incidence of epidermal growth factor receptor mutations. Herein, we report the most in-depth molecular profile of NSCLC in African Americans to date. METHODS: A custom panel was designed to cover the coding regions of 81 NSCLC-related genes and 40 ancestry-informative markers. Clinical samples were sequenced on a massively parallel sequencing instrument, and anaplastic lymphoma kinase translocation was evaluated by fluorescent in situ hybridization. RESULTS: The study cohort included 99 patients (61% males, 94% smokers) comprising 31 squamous and 68 nonsquamous cell carcinomas. We detected 227 nonsilent variants in the coding sequence, including 24 samples with nonoverlapping, classic driver alterations. The frequency of driver mutations was not significantly different from that of whites, and no association was found between genetic ancestry and the presence of somatic mutations. Copy number alteration analysis disclosed distinguishable amplifications in the 3q chromosome arm in squamous cell carcinomas and pointed toward a handful of targetable alterations. We also found frequent SMARCA4 mutations and protein loss, mostly in driver-negative tumors. CONCLUSION: Our data suggest that African American ancestry may not be significantly different from European/white background for the presence of somatic driver mutations in NSCLC. Furthermore, we demonstrated that using a comprehensive genotyping approach could identify numerous targetable alterations, with potential impact on therapeutic decisions.

Genome-wide multi-omics profiling of colorectal cancer identifies immune determinants strongly associated with relapse.

The use and benefit of adjuvant chemotherapy to treat stage II colorectal cancer (CRC) patients is not well understood since the majority of these patients are cured by surgery alone. Identification of biological markers of relapse is a critical challenge to effectively target treatments to the ~20% of patients destined to relapse. We have integrated molecular profiling results of several "omics" data types to determine the most reliable prognostic biomarkers for relapse in CRC using data from 40 stage I and II CRC patients. We identified 31 multi-omics features that highly correlate with relapse. The data types were integrated using multi-step analytical approach with consecutive elimination of redundant molecular features. For each data type a systems biology analysis was performed to identify pathways biological processes and disease categories most affected in relapse. The biomarkers detected in tumors urine and blood of patients indicated a strong association with immune processes including aberrant regulation of T-cell and B-cell activation that could lead to overall differences in lymphocyte recruitment for tumor infiltration and markers indicating likelihood of future relapse. The immune response was the biologically most coherent signature that emerged from our analyses among several other biological processes and corroborates other studies showing a strong immune response in patients less likely to relapse.

Cancer stem cells and markers: new model of tumorigenesis with therapeutic implications.

In an effort to improve our understanding and treatment of cancer, a new model of tumorigenesis is being developed - the cancer stem cell model. Building upon traditional concepts of cancer and stem cells, this model is intended to shed new light on the continuing struggle with treatment challenges such as tumor drug-resistance and recurrence. This review describes the cancer stem cell model with an emphasis on delineating markers that represent a "stemness" phenotype within certain tumor cells. The objective of this delineation is to develop targeted therapies for the selective elimination of cancer stem cells with minimal toxicity to normal stem cells and tissues. However, this specific targeting of cancer stem cells has proved to be a significant challenge due to the similarity of markers expressed by both normal and cancer stem cells. Still, research in the area of cancer biomarkers is steadily progressing.

Markers in normal and cancer stem cells.

In an effort to better understand and address the challenges of cancer research and treatment, a new model of tumorigenesis is being developed - the cancer stem cell model. Building upon traditional concepts of cancer and stem cells, this model is intended to shed new light on the continued struggle with treatment issues such as tumor drug-resistance and recurrence. This review describes the cancer stem cell model with an emphasis on markers that represent the "stemness" phenotype. A thorough understanding of normal and cancer stem cells is necessary for a precise delineation of cancer stem cells. The objective of such an improved delineation is to develop targeted therapy for selective elimination of cancer stem cells with minimal toxicity to normal stem cells. Specific targeting of cancer stem cells has proved to be a significant challenge due to the commonality of many markers between normal and cancer stem cells. However, research in the area of cancer biomarkers is slowly, but steadily, progressing.

A novel BRCA1 mutation in an Indian family with hereditary breast/ovarian cancer.

Germ-line mutations in BRCA1 gene contribute to a majority of familial breast and ovarian cancers. A group of 23 Tamil Nadu (south India) patients with positive family history for breast and ovarian cancer were screened for BRCA1 mutations by conformation sensitive gel electrophoresis (CSGE) followed by sequencing. In the present study, we report a novel 1307delT mutation in exon 11 of BRCA1 gene in a 43-year-old woman of Indian origin with breast cancer. This mutation gives rise to a premature stop codon at amino acid residue 409 and also creates a novel DdeI restriction site. The same mutation was also detected in the patient's maternal uncle and his son through extended family analysis. The 1307delT is a novel mutation that has not been documented in any population or published report to the best of our knowledge. Identification of this novel mutation stresses the need for developing a database of BRCA1 mutations, which will aid in breast cancer screening in this population.