SRC2 controls CD4+ T cell activation via stimulating c-Myc-mediated upregulation of amino acid transporter Slc7a5.

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.

Tumor and Peritoneum-Associated Macrophage Gene Signature as a Novel Molecular Biomarker in Gastric Cancer.

A spectrum of immune states resulting from tumor resident macrophages and T-lymphocytes in the solid tumor microenvironment correlates with patient outcomes. We hypothesized that in gastric cancer (GC), macrophages in a polarized immunosuppressive transcriptional state would be prognostic of poor survival. We derived transcriptomic signatures for M2 (M2TS, MRC1; MS4A4A; CD36; CCL13; CCL18; CCL23; SLC38A6; FGL2; FN1; MAF) and M1 (M1TS, CCR7; IL2RA; CXCL11; CCL19; CXCL10; PLA1A; PTX3) macrophages, and cytolytic T-lymphocytes (CTLTS, GZMA; GZMB; GZMH; GZMM; PRF1). Primary GC in a TCGA stomach cancer dataset was evaluated for signature expressions, and a log-rank test determined overall survival (OS) and the disease-free interval (DFI). In 341 TCGA GC entries, high M2TS expression was associated with histological types and later stages. Low M2TS expression was associated with significantly better 5-year OS and DFI. We validated M2TS in prospectively collected peritoneal fluid of a GC patient cohort (n = 28). Single-cell RNA sequencing was used for signature expression in CD68+CD163+ cells and the log-rank test compared OS. GC patients with high M2TS in CD68+CD163+ cells in their peritoneal fluid had significantly worse OS than those with low expression. Multivariate analyses confirmed M2TS was significantly and independently associated with survival. As an independent predictor of poor survival, M2TS may be prognostic in primary tumors and peritoneal fluid of GC patients.

Kinesin Family Member C1 (KIFC1/HSET) Underlies Aggressive Disease in Androgen Receptor-Low and Basal-Like Triple-Negative Breast Cancers.

Quadruple-negative breast cancer (QNBC) lacks traditional actionable targets, including androgen receptor (AR). QNBC disproportionately afflicts and impacts patients of African genetic ancestry. Kinesin family member C1 (KIFC1/HSET), a centrosome clustering protein that prevents cancer cells from undergoing centrosome-amplification-induced apoptosis, has been reported to be upregulated in TNBCs and African-American (AA) TNBCs. Herein, we analyzed KIFC1 RNA levels and their associations with clinical features and outcomes among AR-low and AR-high TNBC tumors in three distinct publicly available gene expression datasets and in the breast cancer gene expression database (bc-GenExMiner). KIFC1 levels were significantly higher in AR-low and basal-like TNBCs than in AR-high and non-basal-like TNBCs, irrespective of the stage, grade, tumor size, and lymph node status. KIFC1 levels were also upregulated in AR-low tumors relative to AR-high tumors among Black and premenopausal women with TNBC. High KIFC1 levels conferred significantly shorter overall survival, disease-free survival, and distant metastasis-free survival among AR-low and basal-like TNBC patients in Kaplan-Meier analyses. In conclusion, KIFC1 levels may be upregulated in AR-low tumors and, specifically, in those of African descent, wherein it may promote poor outcomes. KIFC1 may be an actionable cancer-cell-specific target for the AR-low TNBC subpopulation and could aid in alleviating racial disparities in TNBC outcomes.

Molecular Profiling of Innate Immune Response Mechanisms in Ventilator-associated Pneumonia.

Ventilator-associated pneumonia (VAP) is a common hospital-acquired infection, leading to high morbidity and mortality. Currently, bronchoalveolar lavage (BAL) is used in hospitals for VAP diagnosis and guiding treatment options. Although BAL collection procedures are invasive, alternatives such as endotracheal aspirates (ETA) may be of diagnostic value, however, their use has not been thoroughly explored. Longitudinal ETA and BAL were collected from 16 intubated patients up to 15 days, of which 11 developed VAP. We conducted a comprehensive LC-MS/MS based proteome and metabolome characterization of longitudinal ETA and BAL to detect host and pathogen responses to VAP infection. We discovered a diverse ETA proteome of the upper airways reflective of a rich and dynamic host-microbe interface. Prior to VAP diagnosis by microbial cultures from BAL, patient ETA presented characteristic signatures of reactive oxygen species and neutrophil degranulation, indicative of neutrophil mediated pathogen processing as a key host response to the VAP infection. Along with an increase in amino acids, this is suggestive of extracellular membrane degradation resulting from proteolytic activity of neutrophil proteases. The metaproteome approach successfully allowed simultaneous detection of pathogen peptides in patients' ETA, which may have potential use in diagnosis. Our findings suggest that ETA may facilitate early mechanistic insights into host-pathogen interactions associated with VAP infection and therefore provide its diagnosis and treatment.

Disparate and Alarming Impact of Gastrointestinal Cancers in Young Adult Patients.

BACKGROUND: The rise in the incidence of gastric cancer (GC) and colorectal cancer (CRC) in young adults (YA) remains unexplained. We aim to identify differences in these malignancies between YA and older patients. PATIENTS AND METHODS: We retrospectively analyzed the California Cancer Registry for all GC and CRC cases from 2000 to 2012. Pearson's Chi square analysis and stepwise regression model with backward elimination were used to analyze differences in demographic, clinical, and histopathologic features, and log-rank test to compare survival between young (≤ 40 years) and older adults (41-90 years) with GC or CRC, separately. RESULTS: We analyzed 19,368 cases of GC and 117,415 cases of CRC. YA accounted for 4.6% of GC (n = 883) and 2.8% of CRC (n = 3273) patients. Compared with older patients, YA were more likely to be Hispanic (P < 0.0001) and have poorly differentiated (P < 0.0001), higher histologic grade (P < 0.0001), and signet ring features (P < 0.0001). Synchronous peritoneal metastases were more common in YA patients (32.1% vs. 14.1% GC, 8.8% vs. 5.4% CRC, P < 0.0001). The 5-year overall survival (OS) of YA with CRC or GC was longer than that of older patients with the same stage of malignancy; except YA with stage I GC, who demonstrated poor OS and disease-specific survival (DSS) (65.1% and 67.9%, respectively) which were significantly worse than those of adults aged 41-49 years (70.7% and 76.2%, respectively) and 50-64 years (69.1% and 78.1%, respectively). CONCLUSIONS: YA with GC or CRC have distinctly worse clinical and histopathologic features compared with older patients and are disproportionately of Hispanic ethnicity. These results contribute to improving understanding of younger versus older GI cancer patients.

Rlip depletion prevents spontaneous neoplasia in TP53 null mice.

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

Overexpression of Flap Endonuclease 1 Correlates with Enhanced Proliferation and Poor Prognosis of Non-Small-Cell Lung Cancer.

Flap endonuclease 1 (FEN1) plays a crucial role in both DNA replication and damage repair. In this study, FEN1 expression and its clinical-pathologic significance in non-small-cell lung cancer (NSCLC) was investigated. Quantitative RT-PCR and immunohistochemistry analysis identified that both FEN1 mRNA and protein were highly overexpressed in about 36% of 136 cancer tissues compared to adjacent tissues, in which FEN1 was generally undetectable. Notably, patients with FEN1-overexpressed cancers were prone to have poor differentiation and poor prognosis. A strong positive correlation between the levels of FEN1 and Ki-67 staining was identified in these NSCLC tissues (r = 0.485), suggesting overexpressed FEN1 conferred a proliferative advantage to NSCLC. Furthermore, knockdown of FEN1 resulted in G1/S or G2/M phase cell cycle arrest and suppressed in vitro cellular proliferation in NSCLC cancer cells. Consistently, a selective FEN1 inhibitor was shown to effectively inhibit cellular proliferation of NSCLC cells in a dose-dependent manner. Additionally, knockdown of FEN1 significantly attenuated homologous DNA repair efficiency and enhanced cytotoxic effects of cisplatin in NSCLC cells. Taken together, these findings have indicated that overexpressed FEN1 represents a prognostic biomarker and potential therapeutic target for NSCLC treatment, which warrants further study.

CCNE1 amplification is associated with poor prognosis in patients with triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is aggressive with limited treatment options upon recurrence. Molecular discordance between primary and metastatic TNBC has been observed, but the degree of biological heterogeneity has not been fully explored. Furthermore, genomic evolution through treatment is poorly understood. In this study, we aim to characterize the genomic changes between paired primary and metastatic TNBCs through transcriptomic and genomic profiling, and to identify genomic alterations which may contribute to chemotherapy resistance. METHODS: Genomic alterations and mRNA expression of 10 paired primary and metastatic TNBCs were determined through targeted sequencing, microarray analysis, and RNA sequencing. Commonly mutated genes, as well as differentially expressed and co-expressed genes were identified. We further explored the clinical relevance of differentially expressed genes between primary and metastatic tumors to patient survival using large public datasets. RESULTS: Through gene expression profiling, we observed a shift in TNBC subtype classifications between primary and metastatic TNBCs. A panel of eight cancer driver genes (CCNE1, TPX2, ELF3, FANCL, JAK2, GSK3B, CEP76, and SYK) were differentially expressed in recurrent TNBCs, and were also overexpressed in TCGA and METABRIC. CCNE1 and TPX2 were co-overexpressed in TNBCs. DNA mutation profiling showed that multiple mutations occurred in genes comprising a number of potentially targetable pathways including PI3K/AKT/mTOR, RAS/MAPK, cell cycle, and growth factor receptor signaling, reaffirming the wide heterogeneity of mechanisms driving TNBC. CCNE1 amplification was associated with poor overall survival in patients with metastatic TNBC. CONCLUSIONS: CCNE1 amplification may confer resistance to chemotherapy and is associated with poor overall survival in TNBC.

Gene Expression Differences in Prostate Cancers between Young and Old Men.

Prostate cancer incidence is increasing in younger men. We investigated whether men diagnosed with Gleason 7 (3+4) T2 prostate cancer at younger ages (≤ 45 years, young cohort) had different mRNA and miRNA expression profiles than men diagnosed at older ages (71-74 years, older cohort). We identified differentially expressed genes (DEGs) related to tumor-normal differences between the cohorts. Subsequent pathway analysis of DEGs revealed that the young cohort had significantly more pronounced inflammatory and immune responses to tumor development compared to the older cohort. Further supporting a role of inflammation-induced immune-suppression in the development of early-onset prostate cancer, we observed significant up-regulation of CTLA4 and IDO1/TDO2 pathways in tumors of the young cohort. Moreover, over-expression of CTLA4 and IDO1 was significantly associated with biochemical recurrence. Our results provide clues on the mechanisms of tumor development and point to potential biomarkers for early detection and treatment for prostate cancer in young men.

Targeting histone methyltransferase G9a inhibits growth and Wnt signaling pathway by epigenetically regulating HP1α and APC2 gene expression in non-small cell lung cancer.

BACKGROUND: Dysregulated histone methyltransferase G9a may represent a potential cancer therapeutic target. The roles of G9a in tumorigenesis and therapeutics are not well understood in non-small cell lung cancer (NSCLC). Here we investigated the impact of G9a on tumor growth and signaling pathways in NSCLC. METHODS: Immunohistochemistry analyzed G9a expression in NSCLC tissues. Both siRNA and selective inhibitor were used to target G9a. The impact of targeting G9a on key genes, signaling pathways and growth were investigated in NSCLC cells by RNA sequencing analysis, rescue experiments, and xenograft models. RESULTS: Overexpression of G9a (≥ 5% of cancer cells showing positive staining) was found in 43.2% of 213 NSCLC tissues. Multiple tumor-associated genes including HP1α, APC2 are differentially expressed; and signaling pathways involved in cellular growth, adhesion, angiogenesis, hypoxia, apoptosis, and canonical Wnt signaling pathways are significantly altered in A549, H1299, and H1975 cells upon G9a knockdown. Additionally, targeting G9a by siRNA-mediated knockdown or by a selective G9a inhibitor UNC0638 significantly inhibited tumor growth, and dramatically suppressed Wnt signaling pathway in vitro and in vivo. Furthermore, we showed that treatment with UNC0638 restores the expression of APC2 expression in these cells through promoter demethylation. Restoring HP1α and silencing APC2 respectively attenuated the inhibitory effects on cell proliferation and Wnt signaling pathway in cancer cells in which G9a was silenced or suppressed. CONCLUSIONS: These findings demonstrate that overexpressed G9a represents a promising therapeutic target, and targeting G9a potentially suppresses growth and Wnt signaling pathway partially through down-regulating HP1α and epigenetically restoring these tumor suppressors such as APC2 that are silenced in NSCLC.

Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma.

Deregulated monoubiquitination of histone H2B (H2Bub1), mainly catalyzed by E3 ubiquitin-protein ligase RNF20/RNF40 complex, may play an important role in cancer. Here we investigate potential roles of H2Bub1 and the underlying mechanisms through which it contributes to cancer development and progression in lung adenocarcinoma. We show that downregulation of H2Bub1 through RNF20 knockdown dramatically decreases H3K79 and H3K4 trimethylation in both normal and malignant lung epithelial cell lines. Concurrently, global transcriptional profiling analysis reveals that multiple tumor-associated genes such as CCND3, E2F1/2, HOXA1, Bcl2 modifying factor (BMF), Met, and Myc; and signaling pathways of cellular dedifferentiation, proliferation, adhesion, survival including p53, cadherin, Myc, and anti-apoptotic pathways are differentially expressed or significantly altered in these lung epithelial cells upon downregulation of H2Bub1. Moreover, RNF20 knockdown dramatically suppresses terminal squamous differentiation of cultured bronchial epithelial cells, and significantly enhances proliferation, migration, invasion, and cisplatin resistance of lung cancer cells. Furthermore, immunohistochemistry analysis shows that H2Bub1 is extremely low or undetectable in >70% of 170 lung adenocarcinoma samples. Notably, statistical analysis demonstrates that loss of H2Bub1 is significantly correlated with poor differentiation in lung adenocarcinoma (p = 0.0134). In addition, patients with H2Bub1-negative cancers had a trend towards shorter survival compared with patients with H2Bub1-positive cancers. Taken together, our findings suggest that loss of H2Bub1 may enhance malignancy and promote disease progression in lung adenocarcinoma probably through modulating multiple cancer signaling pathways.

ATRA-induced cellular differentiation and CD38 expression inhibits acquisition of BCR-ABL mutations for CML acquired resistance.

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD+) levels and blocked acquired resistance by inhibiting the activity of the NAD+-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.

Mutations in DNA2 link progressive myopathy to mitochondrial DNA instability.

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance.

The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro.

COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1-defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5- and DR-green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent.

Down-regulation of programmed cell death 4 (PDCD4) is associated with aromatase inhibitor resistance and a poor prognosis in estrogen receptor-positive breast cancer.

Progression or recurrence due to resistance to aromatase inhibitors (AIs) is a significant clinical problem for a considerable number of patients with breast cancer. Programmed cell death 4 (PDCD4), a tumor suppressor protein, is targeted for degradation during tumor progression. In the current study, we aimed to examine PDCD4 expression and regulation in AI-resistant breast cancer cells, and its association with survival in patients with estrogen receptor (ER)-positive breast cancer. We determined PDCD4 expression levels in AI-resistant breast cancer cell lines and ER-positive breast cancer tumors, investigated the regulation of PDCD4 in AI-resistant breast cancer cell lines, and carried out a Kaplan-Meier survival analysis in two independent cohorts that included a total of 420 patients with ER-positive breast cancer. We found that PDCD4 expression was down-regulated in AI-resistant breast cancer cells, and this down-regulation was inversely correlated with activation of HER2 signaling. Moreover, lower expression of PDCD4 was significantly associated with HER2 positive status in ER-positive breast tumors. Down-regulation of PDCD4 was mediated through up-regulation of HER2 via the mitogen-activated protein kinase (MAPK), protein kinase B (PKB/AKT), and miR-21 in AI-resistant breast cancer cells. MiR-21 inhibitor and the ER down-regulator fulvestrant induced PDCD4 expression and decreased cell proliferation in AI-resistant breast cancer cells. Furthermore, forced overexpression of PDCD4 resensitized AI-resistant cells to AI or hormone deprivation. Finally, we identified that down-regulation of PDCD4 was associated with a lower rate of disease-free survival in patients with ER-positive breast cancer and high histologic grade of breast tumors. In summary, our study shows that expression of PDCD4 is down-regulated by HER2 signaling in AI-resistant breast cancer. Down-regulation of PDCD4 is associated with AI resistance and a poor prognosis in patients with ER-positive breast cancer.

YY1 suppresses FEN1 over-expression and drug resistance in breast cancer.

BACKGROUND: Drug resistance is a major challenge in cancer therapeutics. Abundant evidence indicates that DNA repair systems are enhanced after repetitive chemotherapeutic treatments, rendering cancers cells drug-resistant. Flap endonuclease 1 (FEN1) plays critical roles in DNA replication and repair and in counteracting replication stress, which is a key mechanism for many chemotherapeutic drugs to kill cancer cells. FEN1 was previously shown to be upregulated in response to DNA damaging agents. However, it is unclear about the transcription factors that regulate FEN1 expression in human cancer. More importantly, it is unknown whether up-regulation of FEN1 has an adverse impact on the prognosis of chemotherapeutic treatments of human cancers. METHODS: To reveal regulation mechanism of FEN1 expression, we search and identify FEN1 transcription factors or repressors and investigate their function on FEN1 expression by using a combination of biochemical, molecular, and cellular approaches. Furthermore, to gain insights into the impact of FEN1 levels on the response of human cancer to therapeutic treatments, we determine FEN1 levels in human breast cancer specimens and correlate them to the response to treatments and the survivorship of corresponding breast cancer patients. RESULTS: We observe that FEN1 is significantly up-regulated upon treatment of chemotherapeutic drugs such as mitomycin C (MMC) and Taxol in breast cancer cells. We identify that the transcription factor/repressor YY1 binds to the FEN1 promoter and suppresses the expression of FEN1 gene. In response to the drug treatments, YY1 is dissociated from the FEN1 promoter region leading over-expression of FEN1. Overexpression of YY1 in the cells results in down-regulation of FEN1 and sensitization of the cancer cells to MMC or taxol. Furthermore, we observe that the level of FEN1 is inversely correlated with cancer drug and radiation resistance and with survivorship in breast cancer patients. CONCLUSION: Altogether, our current data indicate that YY1 is a transcription repressor of FEN1 regulating FEN1 levels in response to DNA damaging agents. FEN1 is up-regulated in human breast cancer and its levels inversely correlated with cancer drug and radiation resistance and with survivorship in breast cancer patients.

COHCAP: an integrative genomic pipeline for single-nucleotide resolution DNA methylation analysis.

COHCAP (City of Hope CpG Island Analysis Pipeline) is an algorithm to analyze single-nucleotide resolution DNA methylation data produced by either an Illumina methylation array or targeted bisulfite sequencing. The goal of the COHCAP algorithm is to identify CpG islands that show a consistent pattern of methylation among CpG sites. COHCAP is currently the only DNA methylation package that provides integration with gene expression data to identify a subset of CpG islands that are most likely to regulate downstream gene expression, and it can generate lists of differentially methylated CpG islands with ∼50% concordance with gene expression from both cell line data and heterogeneous patient data. For example, this article describes known breast cancer biomarkers (such as estrogen receptor) with a negative correlation between DNA methylation and gene expression. COHCAP also provides visualization for quality control metrics, regions of differential methylation and correlation between methylation and gene expression. This software is freely available at https://sourceforge.net/projects/cohcap/.

Mutants TP53 p.R273H and p.R273C but not p.R273G enhance cancer cell malignancy.

Mutation of the tumor suppressor TP53 gene occurs in greater than half of all human cancers. In addition to loss of tumor suppressor function of wild-type TP53, gain-of-function mutations endow cancer cells with more malignant properties. R273 is a mutation hotspot with the p.R273H, p.R273C, and p.R273G variants occurring most commonly in patient samples. To better understand the consequences of these R273 mutations, we constructed cancer cell lines expressing TP53 p.R273H, p.R273C, or p.R273G and explored their characteristics. We found that p.R273H and p.R273C, but not p.R273G, enhanced proliferation, invasion, and drug resistance in vitro. Furthermore, breast cancer susceptibility protein 1 was upregulated by mutant TP53 p.R273H and p.R273C in response to DNA damage and repair. Transcriptional analysis of the TP53-R273 mutants by RNA-seq confirmed that the apoptosis pathway was less active in p.R273H and p.R273C, compared with R273G. Molecular dynamics simulation further revealed that TP53-R273G binds more tightly to DNA than TP53-R273H or TP53-R273C. These findings indicate that mutation of TP53 at a single codon has different effects, and likely clinical implications. p.R273H and p.R273C lead to a more aggressive phenotype than p.R273G. These findings may contribute to future diagnosis and therapy in TP53 mutant cancers.

A germline missense mutation in COQ6 is associated with susceptibility to familial schwannomatosis.

PURPOSE: Schwannomatosis, a subtype of neurofibromatosis, is characterized by multiple benign, nonvestibular, nonintradermal schwannomas. Although the tumor suppressor SMARCB1 gene has been frequently identified as the underlying genetic cause of half of familial and ~10% of sporadic schwannomatosis, for most other cases, further causative genes remain to be discovered. Herein, we characterize the genome of a schwannomatosis family without constitutional inactivation of the SMARCB1 gene to explore novel genomic alterations predisposing individuals to the familial disease. METHODS: We performed whole-genome/exome sequencing on genomic DNA of both schwannomatosis-affected and normal members of the family. RESULTS: We identified a novel missense mutation (p.Asp208His; c.622G>C) in the coenzyme Q10 (CoQ10) biosynthesis monooxygenase 6 gene (COQ6) in schwannomatosis-affected members. The deleterious effects of the COQ6 mutations were validated by their lack of complementation in a coq6-deficient yeast mutant. Our study further indicated that the resultant haploinsufficiency of COQ6 might lead to CoQ10 deficiency and chronic overproduction of reactive oxygen species in Schwann cells. CONCLUSION: Although the exact oncogenetic mechanisms in this schwannomatosis family remain to be elucidated, our data strongly indicate a probable role of COQ6 mutation and CoQ10 deficiency in the development of familial schwannomatosis.Genet Med 16 10, 787-792.