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Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non–small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977–89. ©2016 AACR.

Methods used for Supplemental Data only: WST-1 cell proliferation assay, Ki-67 mmunohistochemical analysis

Supplemental Figure 1: Larger legend table for symbols in fig 1A. Supplemental Figure 2: The expression of BIRC5, CDKN1A, CXCL1 and IER3 correlates with expression of C9b, but not C9a, in human NSCLC tissues. Supplemental Figure 3: Down-regulation of RIP1 abolishes the effect of C9b on IκBα level.. Supplemental Figure 4: The role of C9b in the NF-κB pathway is translational to other cell lines, H838 and HBEpC. Supplemental Figure 5: C9b activates the canonical NF-κB pathway in H838 cells and HBEpCs. Supplemental Figure 6: C9b level inversely correlates with IκBα level in NSCLC cells. Supplemental Figure 7: Down-regulation of cIAP2 has no effect on NF-κB activation and does not inhibit the effect of C9b on the NF-κB pathway. Supplemental Figure 8: Cleavage of C9b is not important for interaction with cIAP1. Supplemental Figure 9: Tumors formed from NSCLC cells stably expressing WT C9b have higher Ki-67 expression (cell proliferation) than tumors formed from control cells or cells expressing IBM-mutated (Mut) C9b. Supplemental Figure 10: The interaction with cIAP1 mediates the enhancing effect of C9b on the cell viability and survival/AIG of NSCLC cells and HBEpCs. Supplemental Figure 11: Suppression of the canonical NF-κB pathway by ectopically expressing the suppressor, IκBα, increases NIK level. Supplemental Figure 12: Down-regulation of NIK does not affect the survival/AIG of NSCLC cells. Supplemental Table 1: qPCR primers for NF-κB target genes utilized in the study Supplemental Table 2: List of NF-κB-related genes that expression was affected by down-regulation of C9b in A549 cells or up-regulation of C9b in HBEpC cells (identified by deep RNA sequencing). Supplemental Table 3: List of NF-κB target genes that expression is contrastingly affected by down-regulation of C9b in A549 cells versus by up-regulation of C9b in HBEpCs. Supplemental Table 4: NSCLC cell lines used in Figure 2D or Supplemental Figure 6. Supplemental Table 5: Sequences of negative control si-RNA and caspase-9a or -9b si-RNA utilized in the study.

Supplemental Figure Legends for Supplemental Figures 1 through 12.

Background Approximately 20% of cancer survivors treated with chemotherapy experience worsening heart failure (HF) symptoms post-cancer treatment. While research has predominantly investigated the role of cardiotoxic treatments, much less attention has focused on other risk factors, such as adiposity. However, emerging data in cancer survivors indicates that adiposity may also impact a variety of cardiovascular outcomes. Methods: In a prospective study of 62 patients diagnosed with cancer followed for 24 months from cancer diagnosis through to survivorship (post-cancer treatment), we ascertained baseline fat depots including intermuscular fat (IMF) of the erector spinae muscles; and pre- and post-cancer treatment left ventricular ejection fraction (LVEF) and HF symptoms at baseline and 24-months, respectively. Linear regression was used to model independent variables in relation to HF symptoms at 24-months. Results Baseline IMF and LVEF change over 24-months significantly interacted to predict HF score at 24-months. The highest HF symptom score was observed for participants who experienced high IMF at baseline and a high decline in LVEF over 24-months (HF score = 11.0) versus all other categories of baseline IMF and LVEF change. Conclusions Together IMF and LVEF decline may play an important role in the worsening of HF symptoms in cancer survivors. The finding that IMF at cancer diagnosis led to elevated HF scores post-treatment suggests that IMF may be a potential target for intervention studies.

Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSC's rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.

Causes of initiation and progression of sporadic Alzheimer’s disease (sAD) are likely multiple and include impair-ment of mitochondrial bioenergetics. We analyzed RNA expression levels of multiple mitochondrial oxidative phosphorylation(OXPHOS) and biogenesis (mitobiogenesis) genes in unfixed hippocampal (WH) frozen sections (10 sAD; 9 CTL) and laser-capturedhippocampalpyramidalneurons(PyNs,∼1000neuronsfromeachcase)from8sADand7CTLcases.Nuclear-encodedOXPHOS genes in WH were significantly increased in sAD, whereas in isolated sAD PyNs, these same genes were significantlydecreased. Mitochondrial DNA-encoded genes were increased in sAD PyNs but showed a non-significant downward trend insAD WH. Relationships among WH and PyN gene expression levels in sAD distributed in a different population compared toCTL. Principal component analysis (PCA) revealed clustering of CTL but widespread heterogeneity of sAD samples. In sAD,mitochondrial bioenergetics at the gene expression level are depressed in vulnerable PyNs. PCA revealed that CTL samplesclusteredtogether,whereassADsamplesvariedwidely.FromtheperspectiveofOXPHOSbioenergetics,sADisaheterogeneoussyndrome and not likely due to a single abnormality. Increased stimulation of nuclear-encoded OXPHOS gene expression inPyNs is a rational therapeutic approach for most but not all cases of sAD.Keywords: Laser capture microdissection, mitochondria, mitochondrial biogenesis, relative gene expression, quantitative PCR

Origins of onset and progression of motor neurodegeneration in amyotrophic lateral sclerosis (ALS) are not clearly known, but may include impairment of mitochondrial bioenergetics. We used quantitative PCR approaches to analyze the mitochondrial oxidative phosphorylation (OXPHOS) transcriptomes of spinal cord tissue and peripheral blood mononuclear cells (PBMC) from persons with sporadic ALS compared with those without neurological disease. Expression measurements of 88 different nuclear (n) and mitochondrial (mt) DNA-encoded OXPHOS genes showed mtDNA-encoded respiratory gene expression was significantly decreased in ALS spinal cord by 78-84% (ANOVA p < 0.002). We observed the same phenomenon in freshly isolated PBMC from ALS patients (reduced 24-35%, ANOVA p < 0.001) and reproduced it in a human neural stem cell model treated with 2',3'-dideoxycytidine (ddC) (reduced 52-78%, ANOVA p < 0.001). nDNA-encoded OXPHOS genes showed heterogeneously and mostly decreased expression in ALS spinal cord tissue. In contrast, ALS PBMC and ddC-treated stem cells showed no significant change in expression of nDNA OXPHOS genes compared with controls. Genes related to mitochondrial biogenesis (PGC-1α, TFAM, ERRα, NRF1, NRF2 and POLG) were queried with inconclusive results. Here, we demonstrate there is a systemic decrease in mtDNA gene expression in ALS central and peripheral tissues that support pursuit of bioenergetic-enhancing therapies. We also identified a combined nDNA and mtDNA gene set (n = 26), downregulated in spinal cord tissue that may be useful as a biomarker in the development of cell-based ALS models.

BACKGROUND: The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. METHODS: FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). RESULTS: Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. CONCLUSIONS: FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies. Cancer (Cancer Cytopathol) 2011;000:000–000. V C 2011 American Cancer Society.

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non–small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977–89. ©2016 AACR.

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with

Objectives: This is a pilot study designed to identify gene expression profiles able to stratify head and neck squamous cell carcinoma (HNSCC) tumors that may or may not respond to chemoradiation or radiation therapy. Study Design: We prospectively evaluated 14 HNSCC specimens, arising from patients undergoing chemoradiotherapy or radiotherapy alone with curative intent. A complete response was assessed by clinical evaluation with no evidence of gross tumor after a 2-year follow-up period. Methods: Residual biopsy samples from eight complete responders (CR) and six nonresponders (NR) were evaluated by genome-wide gene expression profiling using HG-U133A 2.0 arrays. Univariate parametric t-tests with proportion of false discoveries controlled by multivariate permutation tests were used to identify genes with significantly different gene expression levels between CR and NR cases. Six different prediction algorithms were used to build gene predictor lists. Three representative genes showing 100% crossvalidation support after leave-one-out crossvalidation (LOOCV) were further validated using real-time QRT-PCR. Results: We identified 167 significant probe sets that discriminate between the two classes, which were used to build gene predictor lists. Thus, 142 probe sets showed an accuracy of prediction ranging from 93% to 100% across all six prediction algorithms. The genes represented by these 142 probe sets were further classified into different functional networks that included cellular development, cellular movement, and cancer. Conclusions: The results presented herein offer encouraging preliminary data that may provide a basis for a more precise prognosis of HNSCC, as well as a molecular-based therapy decision for the management of these cancers. Laryngoscope, 119:91–101, 2009

The utilization of genome-wide gene expression microarray technology in tumor stratification has proven a powerful tool to identify gene expression signatures associated with cancer prognosis and is currently under evaluation in clinical laboratories. Standardized protocols, including tumor tissue postoperatively handling guidelines are yet to be defined. We aimed at assessing a systematic effect of devitalization in ovarian tumors' gene expression profiling, using high-density oligonucleotide microarrays, under a standardized protocol following strict quality control criteria. Residual tissue from the surgical pathology specimen was divided into 5 samples. Half of each was immediately snap frozen in liquid nitrogen. The remaining halves were kept at room temperature for 0, 15, 30, 60, and 120 minutes, at which time the tissue was snap frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. The entire process from RNA extraction through feature intensity distribution was rigorously monitored for quality. Identification of altered gene expression among each pair of snap frozen and devitalized samples per ovarian tumor specimen was assessed by using the Significance score (S-score) method. We identified only 4 probe sets that seemed to correlate with devitalization time in one of the ovarian tumor specimens, suggesting that they are not likely to have an impact on gene expression profiling tumor stratification. Our study suggests that with proper sample handling and rigorous quality control procedures for RNA extraction and microarray analysis, tumor classification based on global gene expression data will not be adversely affected if devitalization times are kept within a 120-minute window.

Caspase-9 has two isoforms with opposing functions, pro-apoptotic caspase-9a (C9a) and anti-apoptotic caspase-9b (C9b). C9b expression has been reported to augment the anchorage-dependent growth (AIG) and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. The mechanism of this biological observation was revealed in this study. Specifically, C9b was demonstrated to have a dual caspase-9a-independent function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway. In particular, C9b was shown to activate the canonical arm and inhibit of the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for C9b contributes to the enhanced survival and AIG of NSCLC cells conferred by C9b expression. The link between C9b expression and NF-κB activation was also validated in human NSCLC tumors. Further mechanistic studies revealed a direct association of C9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif (IBM). Through this interaction, C9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the viability, AIG and tumorigenicity of NSCLC cells. Hence, C9b/cIAP1 interaction is a new attractive molecular target for developing therapeutics to treat NSCLC. Citation Format: Ngoc T. Vu, Margaret A. Park, Michael D. Shultz, Amy C. Ladd, Charles E. Chalfant. Caspase-9b directly interacts with cIAP1 to drive agonist-independent NF-κB activation and tumorigenesis in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-023. doi:10.1158/1538-7445.AM2015-LB-023

Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A260/A280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). Conclusions: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Cryopreservation of patient bone marrow specimens allows for future purification of tumor cells for research or diagnostic purposes. It has not been determined whether this practice causes significant changes in gene expression. In order to evaluate this, we performed microarray analysis (Affymetrix U133A and U133A 2.0 gene chips) on mononuclear cells from 5 acute myelogenous leukemia (AML) patient samples (>70% blast count) that had been either a) cryopreserved, b) snap frozen, or c) resuspended in TRIzol and stored at −80°C. Samples remained frozen for a period of 1–4 weeks. While gene expression changes between groups were minor, there were some differences. Thirteen probe sets between TRIzol and cryopreserved samples, and 60 probe sets between TRIzol and snap frozen samples, varied significantly (>2-fold difference and p