Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing.

BACKGROUND: Advances in single-cell sequencing provide unprecedented opportunities for clinical examination of circulating tumor cells, cancer stem cells, and other rare cells responsible for disease progression and drug resistance. On the genomic level, single-cell whole exome sequencing (scWES) started to gain popularity with its unique potentials in characterizing mutational landscapes at a single-cell level. Currently, there is little known about the performance of different exome capture kits in scWES. Nextera rapid capture (NXT; Illumina, Inc.) has been the only exome capture kit recommended for scWES by Fluidigm C1, a widely accessed system in single-cell preparation. RESULTS: In this study, we compared the performance of NXT following Fluidigm's protocol with Agilent SureSelectXT Target Enrichment System (AGL), another exome capture kit widely used for bulk sequencing. We created DNA libraries of 192 single cells isolated from spheres grown from a melanoma specimen using Fluidigm C1. Twelve high-yield cells were selected to perform dual-exome capture and sequencing using AGL and NXT in parallel. After mapping and coverage analysis, AGL outperformed NXT in coverage uniformity, mapping rates of reads, exome capture rates, and low PCR duplicate rates. For germline variant calling, AGL achieved better performance in overlap with known variants in dbSNP and transition-transversion ratios. Using calls from high coverage bulk sequencing from blood DNA as the golden standard, AGL-based scWES demonstrated high positive predictive values, and medium to high sensitivity. Lastly, we evaluated somatic mutation calling by comparing single-cell data with the matched blood sequence as control. On average, 300 mutations were identified in each cell. In 10 of 12 cells, higher numbers of mutations were identified using AGL than NXT, probably caused by coverage depth. When mutations are adequately covered in both AGL and NXT data, the two methods showed very high concordance (93-100% per cell). CONCLUSIONS: Our results suggest that AGL can also be used for scWES when there is sufficient DNA, and it yields better data quality than the current Fluidigm's protocol using NXT.

NY-ESO-1 expression predicts an aggressive phenotype of ovarian cancer.

OBJECTIVES: NY-ESO-1 is a cancer testis antigen and a promising target for immunotherapy. The purpose of this study was to determine the expression frequency, immunogenicity, and clinical impact of NY-ESO-1 in ovarian cancer. METHODS: Immunohistochemistry (IHC), reverse-transcription polymerase chain reaction (RT-PCR), and quantitative-PCR (qRT-PCR) were utilized in an ovarian cancer (including Fallopian tube and primary peritoneal cancers) patient cohort; humoral responses against NY-ESO-1 were determined by ELISA. Clinicopathologic outcomes including progression-free (PFS) and overall (OS) survival were evaluated based on NY-ESO-1 expression. Cohen's kappa (κ) tested agreement between expression tests. RESULTS: NY-ESO-1 expression was detected by any method in 40.7% of 1002 patients' tumors (NY-ESO-1+) and baseline humoral response was identified in 19.0% of 689 tested patients. NY-ESO-1+ patients were older (p<0.001), higher stage (85% stage III/IV vs. 76.4%, p=0.015), less likely to have a complete response to initial therapy (53.9% vs. 68.9%, p=0.002), had more serous histotype (74.5% vs. 66.9%, p=0.011), and had more grade 3 tumors (83.7% vs. 70.8%, p<0.001). There was a trend towards shorter PFS (22.2 vs. 25.0months, p=0.07) and significantly shorter OS (42.9 vs. 50.0months, p=0.003) among NY-ESO-1+ patients. A subset analysis of NY-ESO-1+ patients that received immunotherapy demonstrated improved OS by >2years (52.6 vs. 27.2months, p<0.001). CONCLUSIONS: This study is the first demonstration of an association between NY-ESO-1 expression and an aggressive cancer phenotype. The relatively high expression frequency of NY-ESO-1 in ovarian cancer patients coupled with the poor clinical outcomes in NY-ESO-1+ patients reveals an underappreciated need for targeted therapy against this antigen. In support, our study reveals that NY-ESO-1+ patients enrolled on immunotherapy trials targeting the antigen exhibited an improvement in OS.

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer.

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as "stitchers," to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication-licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer.

SCNVSim: somatic copy number variation and structure variation simulator.

BACKGROUND: Somatically acquired structure variations (SVs) and copy number variations (CNVs) can induce genetic changes that are directly related to tumor genesis. Somatic SV/CNV detection using next-generation sequencing (NGS) data still faces major challenges introduced by tumor sample characteristics, such as ploidy, heterogeneity, and purity. A simulated cancer genome with known SVs and CNVs can serve as a benchmark for evaluating the performance of existing somatic SV/CNV detection tools and developing new methods. RESULTS: SCNVSim is a tool for simulating somatic CNVs and structure variations SVs. Other than multiple types of SV and CNV events, the tool is capable of simulating important features related to tumor samples including aneuploidy, heterogeneity and purity. CONCLUSIONS: SCNVSim generates the genomes of a cancer cell population with detailed information of copy number status, loss of heterozygosity (LOH), and event break points, which is essential for developing and evaluating somatic CNV and SV detection methods in cancer genomics studies.

MAC: identifying and correcting annotation for multi-nucleotide variations.

BACKGROUND: Next-Generation Sequencing (NGS) technologies have rapidly advanced our understanding of human variation in cancer. To accurately translate the raw sequencing data into practical knowledge, annotation tools, algorithms and pipelines must be developed that keep pace with the rapidly evolving technology. Currently, a challenge exists in accurately annotating multi-nucleotide variants (MNVs). These tandem substitutions, when affecting multiple nucleotides within a single protein codon of a gene, result in a translated amino acid involving all nucleotides in that codon. Most existing variant callers report a MNV as individual single-nucleotide variants (SNVs), often resulting in multiple triplet codon sequences and incorrect amino acid predictions. To correct potentially misannotated MNVs among reported SNVs, a primary challenge resides in haplotype phasing which is to determine whether the neighboring SNVs are co-located on the same chromosome. RESULTS: Here we describe MAC (Multi-Nucleotide Variant Annotation Corrector), an integrative pipeline developed to correct potentially mis-annotated MNVs. MAC was designed as an application that only requires a SNV file and the matching BAM file as data inputs. Using an example data set containing 3024 SNVs and the corresponding whole-genome sequencing BAM files, we show that MAC identified eight potentially mis-annotated SNVs, and accurately updated the amino acid predictions for seven of the variant calls. CONCLUSIONS: MAC can identify and correct amino acid predictions that result from MNVs affecting multiple nucleotides within a single protein codon, which cannot be handled by most existing SNV-based variant pipelines. The MAC software is freely available and represents a useful tool for the accurate translation of genomic sequence to protein function.

Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

A pilot study comparing the effect of flaxseed, aromatase inhibitor, and the combination on breast tumor biomarkers.

Use of complementary approaches is common among breast cancer survivors. Potential interactions between aromatase inhibitors (AI) and high phytoestrogen foods, such as flaxseed (FS), are not often described. We conducted a pilot 2 × 2 factorial, randomized intervention study between tumor biopsy and resection, in 24 postmenopausal women with estrogen receptor positive (ER+) breast cancer, to assess the effects of FS and anastrozole, and possible interactions between them, on serum steroid hormone and tumor-related characteristics associated with long-term survival (Roswell Park Cancer Institute, 2007-2010). The effect of each treatment vs. placebo on outcomes was determined by linear regression adjusting for pretreatment measure, stage, and grade. Although not statistically significant, mean ERß expression was approximately 40% lower from pre- to postintervention in the FS + AI group only. We observed a statistically significant negative association (ß ± SE -0.3 ± 0.1; P = 0.03) for androstenedione in the FS + AI group vs. placebo and for DHEA with AI treatment (ß ± SE -1.6 ± 0.6; P = 0.009). Enterolactone excretion was much lower in the FS + AI group compared to the FS group. Our results do not support strong effects of FS on AI activity for selected breast tumor characteristics or serum steroid hormone levels but suggest AI therapy might reduce the production of circulating mammalian lignans from FS.

ESR1 and p53 interactome alteration defines mechanisms of tamoxifen response in luminal breast cancer.

The canonical mechanism behind tamoxifen's therapeutic effect on estrogen receptor α/ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to tamoxifen (Tam) therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remains unclear. In a window-of-opportunity trial on patients with newly diagnosed stage I-III ESR1+/HER2/wild-type p53 breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in functional reactivation of p53 leading to transcriptional reprogramming that favors tumor-suppressive signaling, as well as downregulation of oncogenic pathways. These findings illustrating the convergence of ESR1 and p53 signaling during Tam therapy enrich mechanistic understanding of the impact of p53 on the response to Tam therapy.

Inflammatory myofibroblastic tumor of the mesentery: a clinical dilemma.

Although rare, extra-pulmonary inflammatory myofibroblastic tumors (IMTs) are becoming increasingly recognized. While surgical resection is currently an effective and accepted treatment for IMTs, the optimal management of unresectable or residual IMTs remains a clinical dilemma. We present the case of an incompletely resected IMT treated successfully with anti-inflammatory therapy alone, and describe the rationale for this approach.

Metabolic adaptation of ovarian tumors in patients treated with an IDO1 inhibitor constrains antitumor immune responses.

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD+), which reduced T cell proliferation and function. Because NAD+ metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD+ on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD+-mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma.

Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.

Mismatch repair status and outcomes after adjuvant therapy in patients with surgically staged endometrial cancer.

OBJECTIVES: To determine whether DNA mismatch repair (MMR) modifies the response to chemotherapy or radiotherapy in patients with endometrial cancer. METHODS: Immunohistochemistry (IHC) for the DNA MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed on a tissue microarray of specimens of primary endometrial cancer. MMR deficiency was defined as lack of expression of one or more proteins. Expression of all proteins classified a tumor as having an intact MMR system. Recurrence rates were calculated for women treated with platinum-based chemotherapy or pelvic external beam radiation. Comparisons were made using the log-rank test. Multiple comparisons were controlled for by utilizing the Bonferroni correction method. RESULTS: Four hundred seventy-seven cases of endometrial cancer were evaluated on a tissue microarray (TMA). One hundred fifty-eight patients (41%) received chemotherapy. Sixty-six patients (17%) received pelvic teletherapy. Overall and progression-free survival were not different between patients whose tumors had intact MMR and those with defective MMR when stratified by adjuvant treatment with radiation or chemotherapy. Subgroup analyses stratified by histology (non-endometrioid versus endometrioid) and stage did show significant survival differences. There was a significant increase in overall (p=0.003) and progression-free (p=0.004) survival in those with MMR-deficient, non-endometrioid tumors treated with teletherapy compared to those with an intact MMR system. Improved progression-free survival was noted in patients with intact MMR with stage III/IV disease treated with adjuvant chemotherapy (p=0.031). CONCLUSIONS: Subgroups of patients with non-endometrioid endometrial cancer and defective MMR may have improved survival after adjuvant radiotherapy. Patients with advanced stage endometrial cancer and defects in mismatch repair may receive less benefit from adjuvant chemotherapy.

Early-onset lichenoid graft-vs.-host disease: a unique variant of acute graft-vs.-host disease occurring in peripheral blood stem cell transplant recipients.

BACKGROUND: A complication of stem cell transplantation is chronic graft-vs.-host disease (GvHD), developing months to years after transplant; the two commonest manifestations are lichenoid GvHD and scleroderma. The purpose of this study was to characterize early-onset lichenoid GvHD. METHODS: A retrospective study identified patients diagnosed with early-onset lichenoid GvHD. This diagnosis was correlated with type of transplant and concurrent or prior episodes of acute GvHD. RESULTS: Patients in whom a sex mismatch was present between donor and recipient were included, representing a study population of 17. All received an allogeneic peripheral blood stem cell transplant (PBSCT). All patients had biopsy proven lichenoid GvHD within 60 days or less following transplantation. All had concurrent gastrointestinal symptoms which was biopsy proven GvHD in thirteen of the cases. FISH XY studies revealed that the infiltrating lymphocytes were of donor origin in 12 of the cases, mixed in three and of host origin in two cases. CONCLUSIONS: Early-onset lichenoid GvHD is exclusive to the PBSCT setting and appears to be mediated by donor lymphocytes, reflecting the higher numbers of donor T cells encountered in PBSCT. We consider this reaction pattern a distinctive subtype of acute GvHD.

Clonality and antigen-specific responses shape the prognostic effects of tumor-infiltrating T cells in ovarian cancer.

CD8+ tumor-infiltrating lymphocytes (TILs) are not all specific for tumor antigens, but can include bystander TILs that are specific for cancer-irrelevant epitopes, and it is unknown whether the T-cell repertoire affects prognosis. To delineate the complexity of anti-tumor T-cell responses, we utilized a computational method for de novo assembly of sequences from CDR3 regions of 369 high-grade serous ovarian cancers from TCGA, and then applied deep TCR-sequencing for analyses of paired tumor and peripheral blood specimens from an independent cohort of 99 ovarian cancer patients. Strongly monoclonal T-cell repertoires were associated with favorable prognosis (PFS, HR = 0.65, 0.50-0.84, p = 0.003; OS, HR = 0.61, 0.44-0.83, p = 0.006) in TCGA cohort. In the validation cohort, we discovered that patients with low T-cell infiltration but low diversity or focused repertoires had clinical outcomes almost indistinguishable from highly-infiltrated tumors (median 21.0 months versus 15.9 months, log-rank p = 0.945). We also found that the degree of divergence of the peripheral repertoire from the TIL repertoire, and the presence of detectable spontaneous anti-tumor immune responses are important determinants of clinical outcome. We conclude that the prognostic significance of TILs in ovarian cancer is dictated by T-cell clonality, degree of overlap with peripheral repertoire, and the presence of detectable spontaneous anti-tumor immune response in the patients. These immunological phenotypes defined by the TCR repertoire may provide useful insights for identifying "TIL-low" ovarian cancer patients that may respond to immunotherapy.

Copy number gain and oncogenic activity of YWHAZ/14-3-3zeta in head and neck squamous cell carcinoma.

Gene amplification, a common mechanism for oncogene activation in cancers, has been used in the discovery of novel oncogenes. Low-level copy number gains are frequently observed in head and neck squamous cell carcinomas (HNSCCs) where numerous amplification events and potential oncogenes have already been reported. Recently, we applied restriction landmark genome scanning to study gene amplifications in HNSCC and located novel and uncharacterized regions in primary tumor samples. Gain on chromosome 8q22.3, the location of YWHAZ (14-3-3zeta), is found in 30-40% HNSCC cases. Data obtained from fluorescence in situ hybridization and immunohistochemistry on HNSCC tissue microarrays confirmed frequent low-level YWHAZ copy number gain and protein overexpression. YWHAZ mRNA was frequently upregulated in patients' tumor tissues. Furthermore, YWHAZ RNAi significantly suppressed the growth rate of HNSCC cell lines, and overexpression of YWHAZ in HaCaT immortalized human skin keratinocytes promotes overgrowth, as well as morphological changes. Reduced YWHAZ levels increased the G1/G0-phase proportion, decreased the S-phase proportion and the rate of DNA synthesis. Based on this evidence, we suggest that YWHAZ is a candidate proto-oncogene and deserves further investigation into its role in HNSCC carcinogenesis.

Tumor suppressor activity of CCAAT/enhancer binding protein alpha is epigenetically down-regulated in head and neck squamous cell carcinoma.

Tumor suppressor CCAAT enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in cell cycle control and cellular differentiation. In a recent study, microarray expression profiling on head and neck squamous cell carcinoma (HNSCC) samples identified significant C/EBPalpha down-regulation, correlating with poor prognosis. However, the mechanisms of C/EBPalpha down-regulation remained elusive. C/EBPalpha has been previously found to provide an antiproliferative role in lung cancer, and our laboratory showed that its down-regulation involves epigenetic mechanisms. This prompted us to investigate the involvement of epigenetics in down-regulating C/EBPalpha in HNSCC. Here, we show that C/EBPalpha is down-regulated in HNSCC by loss of heterozygosity and DNA methylation, but not by gene mutation. We found a consistently methylated upstream regulatory region (-1,399 bp to -1,253 bp in relation to the transcription start site) in 68% of the HNSCC tumor samples, and DNA demethylation using 5-aza-2'-deoxycytidine treatment was able to significantly restore C/EBPalpha mRNA expression in the HNSCC cell lines we tested. In addition, C/EBPalpha overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In conclusion, we showed for the first time not only that C/EBPalpha has tumor suppressor activity in HNSCC, but also that it is down-regulated by DNA promoter methylation.

Tumor-associated macrophage expression of interferon regulatory Factor-8 (IRF8) is a predictor of progression and patient survival in renal cell carcinoma.

Tumor-associated macrophages have been well-characterized in solid malignancies, including renal cell carcinoma and generally correlate with poor prognosis. However, the molecular mechanisms which govern intratumoral macrophage behavior and patient outcome are unclear. Here, we investigated whether alterations in macrophage expression of the transcriptional regulator for myeloid commitment and function, interferon regulatory factor-8 (IRF8), could predict survival of clear cell renal cell carcinoma patients. Transcriptional analysis of publicly available data revealed elevated IRF8 expression was associated with prolonged disease-free survival. Evaluation of protein expression within histologic sections of primary clear cell renal cell carcinoma patient samples showed intensity of IRF8 by CD68+ macrophages correlated inversely with stage. Survival outcomes of patients with primary or metastatic disease could be stratified on the basis of IRF8 levels by macrophages. Patients with high levels of IRF8 expression within metastatic sites had prolonged overall survival (log-rank P < 0.01, HR = 0.44, 95% C.I.: 0.23-0.84) compared to patients with low levels of IRF8 expression. When patient cohorts were further separated based on macrophage infiltration within metastatic lesions, patients with a macrophagelo IRF8hi profile had a more than 10 year increase in median overall survival compared to patients with a macrophagelo IRF8lo profile (log-rank, P < 0.001). In summary, we report that macrophage expression of IRF8 is inversely correlated with tumor mass and directly related to survival outcome. These findings support the utilization of IRF8 expression by macrophages to predict patient outcome, which may have important implications for guiding treatment decisions for renal cell carcinoma patients with metastatic disease.

Cytochrome c Deficiency Confers Apoptosome and Mitochondrial Dysfunction in African-American Men with Prostate Cancer.

Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with Caucasian-American (CA) men, the molecular mechanisms of this racial prostate cancer disparity remain undefined. In this study, we provide the first comprehensive evidence that cytochrome c deficiency in AA primary tumors and cancer cells abrogates apoptosome-mediated caspase activation and contributes to mitochondrial dysfunction, thereby promoting therapeutic resistance and prostate cancer aggressiveness in AA men. In AA prostate cancer cells, decreased nuclear accumulation of nuclear respiration factor 1 (Nrf1) and its subsequent loss of binding to the cytochrome c promoter mediated cytochrome c deficiency. The activation of cellular Myc (c-Myc) and NF-κB or inhibition of AKT prevented nuclear translocation of Nrf1. Genetic and pharmacologic inhibition of c-Myc and NF-κB or activation of AKT promoted Nrf1 binding to cytochrome c promoter, cytochrome c expression, caspase activation, and cell death. The lack of p-Drp1S616 in AA prostate cancer cells contributed to defective cytochrome c release and increased resistance to apoptosis, indicating that restoration of cytochrome c alone may be insufficient to induce effective apoptosis. Cytochrome c deficiency promoted the acquisition of glycolytic phenotypes and mitochondrial dysfunction, whereas cytochrome c restoration via inhibition of c-Myc and NF-κB or activation of AKT attenuated glycolysis in AA prostate cancer cells. Inhibition of c-Myc and NF-κB enhanced the efficacy of docetaxel in tumor xenografts. Therefore, restoring cytochrome c may overcome therapeutic resistance and prostate cancer aggressiveness in AA men. Overall, this study provides the first comprehensive experimental, mechanistic, and clinical evidence for apoptosome and mitochondrial dysfunction in prostate cancer racial disparity. SIGNIFICANCE: Mechanistic insights on prostate cancer health disparity among American men provide novel approaches to restore mitochondrial function, which can address therapeutic resistance and aggressiveness in African-American men with prostate cancer.

Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.

Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers.

A role for the WWOX gene in prostate cancer.

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2'-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues.