Amy C. Ladd

Preservation of Fine-Needle Aspiration Specimens for Future Use in RNA-Based Molecular Testing

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.

Genes involved in radiation therapy response in head and neck cancers.

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.

Mitochondrial DNA Copy Numbers in Pyramidal Neurons are Decreased and Mitochondrial Biogenesis Transcriptome Signaling is Disrupted in Alzheimer's Disease Hippocampi

Alzheimers disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

Postmortem Alzheimer's Disease Hippocampi Show Oxidative Phosphorylation Gene Expression Opposite that of Isolated Pyramidal Neurons

Causes of initiation and progression of sporadic Alzheimers disease (sAD) are likely multiple and include impairment 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-captured hippocampal pyramidal neurons (PyNs, ~1000 neurons from each case) from 8 sAD and 7 CTL cases. Nuclear-encoded OXPHOS genes in WH were significantly increased in sAD, whereas in isolated sAD PyNs, these same genes were significantly decreased. Mitochondrial DNA-encoded genes were increased in sAD PyNs but showed a non-significant downward trend in sAD WH. Relationships among WH and PyN gene expression levels in sAD distributed in a different population compared to CTL. 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 samples clustered together, whereas sAD samples varied widely. From the perspective of OXPHOS bioenergetics, sAD is a heterogeneous syndrome and not likely due to a single abnormality. Increased stimulation of nuclear-encoded OXPHOS gene expression in PyNs is a rational therapeutic approach for most but not all cases of sAD.

RNA-seq analyses reveal that cervical spinal cords and anterior motor neurons from amyotrophic lateral sclerosis subjects show reduced expression of mitochondrial DNA-encoded respiratory genes, and rhTFAM may correct this respiratory deficiency

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 hNSCs rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis

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.

Abstract LB-023: Caspase-9b directly interacts with cIAP1 to drive agonist-independent NF-κB activation and tumorigenesis in non-small cell lung cancer

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