Citations

Abstract There is growing evidence showing that sex disparity in lung cancer risk cannot be fully explained by sex differences in smoking behavior, implying existence of genetic and molecular basis for sex disparity in lung cancer development. However, the information about sex dimorphism in lung cancer risk is quite limited despite the great success in lung cancer association studies. By adopting a stringent two-stage analysis strategy, we performed a genome-wide gene-sex interaction analysis using genotype from a lung cancer cohort including ~ 47,000 individuals with European ancestry. Three low-frequency variants (minor allele frequency < 0.05), rs17662871 (OR=0.71, p=4.29x10-8); rs79942605 (OR=2.17, p=2.81x10-8); and rs208908 (OR=0.70, p=4.54x10-8), were identified with different risk effect of lung cancer between men and women. Further eQTL and functional annotation analysis suggested rs208908 affect lung cancer risk through differential regulation of CXADR (Coxsackie Virus And Adenovirus Receptor) gene expression in lung tissues between men and women. Our study is one of the first studies to provide novel insights about the genetic and molecular basis for sex disparity in lung cancer development.

Introduction Central sensitization syndromes (CSS) comprise an overlapping group of clinical conditions with the core feature of “pain arising as a direct consequence of a lesion or disease affecting the somatosensory system.” Patients with CSS are known to have challenging interactions with healthcare providers contributing to psychological distress and increased healthcare utilization. CSS symptom severity has been associated with psychologic comorbidities, but little is known about how symptom severity relates to provider interactions. Methods We performed a cross-sectional survey among patients with CSS in our primary care practices to examine the relationship between CSS symptom severity and experiences with doctors. Results A total of 775 respondents completed the survey (775/5000; 15.5%) with 72% reporting high CSS symptom severity. About 44% of respondents had a prior diagnosis of fibromyalgia, 72% had migraines, and 28% had IBS. Patients with high CSS symptom severity were more likely to report that doctor(s) had often/always told them that they don’t need treatment when they feel like they do (OR = 3.6, 95% CI 1.9-7.5), that doctor(s) often/always don’t understand them (OR = 3.1, 95% CI 1.9-5.4), and that doctor(s) often/always seem annoyed with them when compared with respondents with low-moderate CSS symptom severity (OR = 4.8, 95% CI 2.2-12.5). Patients with high CSS symptom severity were at greater than 5 times odds of reporting being told that their symptoms were “all in their head” when compared to patients with low-moderate symptom severity (OR = 5.4, 95% CI 3.3-9.0). Conclusion Patients with CSS spectrum disorders experience frequent pain and decreased quality of life. A high degree of CSS symptom severity is associated with negative experiences with healthcare providers, which deters the establishment of a positive provider-patient relationship. Further research is needed to help understand symptom severity in CSS and harness the power of the therapeutic alliance as a treatment modality.

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3 (bag3) gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the bag3-/- fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed ex vivo assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on bag3 -based cardiomyopathy model. Different from the other three GBT mutants, GBT0411+/- , which tags the long isoform of dnajb6 b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the bag3-/- fish, suggesting dnajb6b as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the GBT0411+/-;bag3-/- double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.

The zebrafish has emerged as an excellent model for cardiovascular research thanks to its ex-utero and rapid embryonic development, its embryonic transparent nature, and its capacity to survive in the absence of a functional cardiovascular system during the first week of development, which enables functional characterization of mutations that would otherwise induce lethality in traditional murine models. The aim of this project is to identify and characterize novel genes involved in zebrafish cardiovascular development using mutagenic gene trapping, a technique that generates random insertional mutations across the genome. We use the well-defined RP2 gene-breaking transposon system, which not only mutates, but also fluorescently tags the trapped gene product(s) (Clark, Nat Methods, 2011). RP2 also introduces loxP sites into the mutant locus, which can be used for Cre-mediated phenotype rescue by microinjection of Cre recombinase, or by crossing to tissue-specific Cre lines. From over 3000 RP2-injected embryos, 141 fish showed germline transmission. Among them, 51 expressed strong fluorescence in different tissues, including the heart, vessels, notochord, central nervous system (CNS), and eyes. Three cardiovascular lines were selected for phenotypic characterization and functional studies. RP2#C2 strain expressed fluorescence in the heart valves, CNS, eyes and pectoral fin buds. Inverse PCR in RP2#C2 demonstrated a trapped gene at meis4.1a, which encodes a homeobox transcription factor that has not been previously studied in zebrafish. RP2#121 strain expressed strong fluorescence in cardiac and skeletal muscles. RP2#91 strain showed expression in the vasculature and demonstrated a trapped gene at pdgfra. Homozygous RP2#91 mutants showed severe defects in the heart, blood flow, and other body parts including the head and musculature. We are currently creating a panel of tissue-specific Cre lines targeting tissues such as cardiomyocytes, endothelial cells and smooth muscle cells for spatiotemporal rescuing of the mutant phenotype. The generated zebrafish protein-trap lines are invaluable tools to annotate gene function, dissect the molecular mechanisms of cardiovascular development, and potentially serve as disease models.

Abstract Background: Lysosomes are acidic organelles that contain numerous cathepsins and acid hydrolases. Lysosomal membrane permeabilization (LMP) and the presence of cathepsins in the cytosol triggers intrinsic apoptotic pathway through cathepsin L (CTSL) mediated degradation of critical proteins. This process is mediated by the proteolytic activation of the pro-apoptotic molecule Bid, resulting in mitochondrial outer membrane permeability (MOMP), cyt c release and then, cell death. Based on our previous study that quinacrine (QC) induces autophagy in ovarian cancer, the current study is aimed to investigate the potential role of cathepsins in QC induced LMP and MOMP in ovarian cancer. Methods: MagicRed and Mitotracker Red staining was used to assess the QC induced CTSL activation and LMP. To identify the role of specific cathepsin in QC mediated cytotoxicity, different specific cathepsin inhibitors pepstatin A, CA-074Me, Z-FY(tBU)-DMK were used and cell death was assessed by clonogenic assay, PARP cleavage and annexin v-PI staining. Using ATG5 and/or CTSL knockdown cells and pharmacological inhibition with 3-MA/chloroquine and/or CTSL overexpression clones, QC-induced flux in CTSL activation was determined using several different approaches including evaluating autophagy flux using tandem dual labeled RFP/GFP LC3BII by immunoflourescence analysis. Recombinant CTSL was used to validate identify p62 as novel substrate of CTSL. QC-induced autophagy and CTSL activation were validated by western blot analysis in HeyA8MDR xenograft tumors. Results: QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, MOMP, and mitochondrial cytochrome c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas ectopic expression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL suggesting that CTSL plays a role in QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockdown cells or with autophagy inhibitor chloroquine. Importantly we show upregulation of CTSL in QC treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome c, tBid, cleaved PARP, and caspase3. Conclusion: Taken together, the data suggests that QC-induced autophagy and CTSL upregulation promotes a positive feedback loop leading to excessive autophagic flux, LMP, MOMP to promote QC-induced cell death in ovarian cancer cells. Citation Format: Prabhu Thirusangu, Christopher L. Pathoulas, Upasana Ray, Yinan xiao, Julie Staub, Ashwani Khurana, Vijayalakshmi Shridhar. Quinacrine-induced autophagy in ovarian cancer triggers cathepsin-L mediated lysosomal/mitochondrial membrane permeabilization and cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1937.

Abstract Objective: Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Increasing evidence suggests that Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, a recently recognized critical regulator of lipid metabolism is associated with cancer metastasis. Though recent studies reveal that high expression of PLA2G3 significantly correlates with poor prognosis, however, there is lack of a single paradigm that determines the contribution of secreted PLAs in ovarian cancer (OC) progression that may open new therapeutic opportunities. This study aimed to investigate the role and molecular regulatory mechanism of PLA2G3 in OC. Methods: CRISPR and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal microscopy, cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Metabolic inhibitor PFK-158 induced autophagy in combination with platinum drugs was assessed by immunofluorescence assay, immunoblot and MTT cell viability assay. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and in combination with chemotherapy was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results: CRISPR and shRNA mediated downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. Accumulating evidence also shows that increased lipogenic activity in cancer cells is associated with loss of primary cilia (PC), which coordinates cellular signaling homeostasis, and is thus associated with tumorigenesis. We found that both PFK158-mediated downregulation of PLA2G3 and genetic KD of PLA2G3 resulted in increased number of percent ciliated cells and inhibited oncogenesis. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore PC in OC cells. Of clinical relevance, PFK158 induced an increase in percent ciliated cells in patient-derived primary ascites culture through autophagic degradation of PLA2G3 to reduce cell viability and sensitized cells to chemotherapy. Conclusion: Taken together, this will be the first comprehensive study that emphasizes the in depth crucial role of PLA2G3 in regulating the OC metastasis and further suggests the therapeutic potential of targeting phospholipases and/or restoration of primary cilia in cancer cells for future treatment. Thus, the elucidation of pathways and molecular inhibitors towards ciliogenesis in cancer cells will be of interest to forge forward to be tested in a novel clinical setting. Citation Format: Upasana Ray, Debarshi Roy, Ling Jin, Prabhu Thirusangu, Julie Staub, Yinan Xiao, Viji Shridhar. Autophagy-dependent therapeutic targeting of group III phospholipase A2 attenuates ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1936.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity but its mechanism of action was poorly understood. We report the high‐resolution crystal structures of the Did2‐ and Vps60‐binding N‐terminal domain and the Vps4‐binding C‐terminal domain of S. cerevisiae Vta1. The C‐terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C‐terminal domain stabilizes the ATP‐dependent double ring assembly of Vps4. In addition, the MIT motif containing N‐terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT‐III proteins. This provides an additional level of regulation and coordination for ESCRT‐III assembly and disassembly.

MHC Class II molecules are essential in antigen presentation and selection of the CD4+ T cell repertoire. They are mainly expressed on professional Antigen Presenting Cells (APC), i.e. B lymphocytes, dendritic cells and macrophages. Activated human T lymphocytes also express MHC Class II but MHC Class II expression is not detected on activated mouse T cells. HLA‐DR3 can be expressed as a transgene on CD4+ T cells in complete MHC Class II knock‐out (AEo) mice, but not in I‐Ab knock‐out (Abo) mice, suggesting that a gene, which could modify the expression of HLA‐DR3, might exist between I‐A and I‐E. Expression sequence tag (EST) databases were searched using the flank regions between I‐A and I‐E and a new potential gene was identified. No similar sequence was found in the human genome database. Expression in multiple mouse tissues, but not in human cell lines, was verified by RT‐PCR. However, this gene was found to be expressed in B6 mouse B cells and two mouse B cell lines, but not in T cells, indicating that it might not be related to the expression of HLA‐DR3. Transduction of this gene into the LG2 cell line had no effect on MHC II expression. In summary, we report the discovery of a new gene between the I‐Aa and I‐Eb1 region in C57BL/6 mice and its function remains unknown. (Supported by NIH‐AI30554)