Shridhar, Vijayalakshmi

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.

11119 Background: Evaluation of prognostic factors in carcinoid tumors of the lung is limited due to the rarity of disease. This study assessed Ki-67 expression and other clinical variables as prognostic factors in 262 patients. Methods: A systematic search of Mayo Clinic lung cancer epidemiology and tumor registry databases from 1997 to 2009 identified 449 consecutive patients, with 262 having available tissue blocks [40 atypical carcinoids (AC) and 222 typical carcinoids (TC)]. Clinical data were collected by chart review. Tissue blocks were reviewed by 1/3 pathologists using WHO criteria. Tumors were tested for the Ki-67 index using digital image analysis (tumor tracing) by two operators. The association and predictive value of the factors with recurrence-free and overall survival (RFS and OS) were explored using univariable Cox proportional Hazards model and concordance (c) index. Results: Age, stage, smoking history, lymph node (LN) involvement and Ki-67 index were significant prognostic factors for RFS and OS. Median follow-up on alive-patients is 5 years (range: 0.006-5). Median percentage of Ki-67 index of AC and TC were 1.61% and 0.56% (P<0.0001), respectively. Patients with Ki-67 ≥ 1% had significantly worse RFS (HR=3.69, P<0.0001) and OS (HR=3.69, P=0.0007) compared with Ki-67 < 1% group. The c-index of Ki-67 (0.65) was comparable to the pathologic distinction between AC and TC (0.62 for original diagnosis and 0.63 for central-reviewed diagnosis). Conclusions: Ki-67 index cutoff value of 1% is a valuable prognostic biomarker for pulmonary carcinoids based on this large cohort. Our data also provide strong evidence for clinical variables such as age, stage, smoking history, and LN involvement as clinical prognostic factors in pulmonary carcinoids. A prognostic calculator incorporating Ki-67 and clinical variables is under development. [Table: see text]

Abstract UNC-45A, a highly conserved member of the UCS (UNC45A/CRO1/SHE4P) protein family of cochaperones, plays an important role in regulating cytoskeletal-associated functions in invertebrates and mammalian cells, including cytokinesis, exocytosis, cell motility, and neuronal development. Here, for the first time, UNC-45A is demonstrated to function as a mitotic spindle-associated protein that destabilizes microtubules (MT) activity. Using in vitro biophysical reconstitution and total internal reflection fluorescence microscopy analysis, we reveal that UNC-45A directly binds to taxol-stabilized MTs in the absence of any additional cellular cofactors or other MT-associated proteins and acts as an ATP-independent MT destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles, and its depletion causes severe defects in chromosome congression and segregation. UNC-45A is overexpressed in human clinical specimens from chemoresistant ovarian cancer and that UNC-45A–overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and restores sensitivity to paclitaxel. Implications: These findings reveal novel and significant roles for UNC-45A in regulation of cytoskeletal dynamics, broadening our understanding of the basic mechanisms regulating MT stability and human cancer susceptibility to paclitaxel, one of the most widely used chemotherapy agents for the treatment of human cancers.

6087 Background: NTRK1-3 fusions, identified in pediatric radiation-associated thyroid cancers, are low frequency oncogenic drivers that can trigger constitutive activation of TrK1-3 tyrosine kinase domains with responsively heightened downstream signaling through RAS-MAPK, PI3K-AKT, and/or PLCγ-PKC. NTRK point mutations are rare and of largely uninvestigated oncogenic potential, but some appear oncogenic. Methods: Advanced thyroid cancer (TC) patients (pts) requiring systemic therapy at Mayo Clinic sites underwent tumor genetic interrogation using Foundation One analyses with the goal of informing salvage therapeutic strategies. In this IRB-approved study, data from pts with TCs bearing NTRK point mutations were retrospectively analyzed, as this occurrence was unexpected but potentially of prognostic and/or therapeutic relevance. Results: Five of 55 pts (9%) with advanced TCs subject to Foundation One tumor interrogation had NTRK1-3 point mutations (allele frequency, AF, 15-49%); none had fusions. All 5 tumors had microsatellite stability; 4/5 had low tumor mutation burden (2-5 mutations/megabase). NTRK mutations in 3 anaplastic TC (ATC) pts (15% prevalence) were: NTRK3 I749M (AF 15%; overall survival, OS, 15 mos), NTRK1 R744H (AF not available; OS 9.5 mos), and NTRK1 R583H (AF 20.1%; OS > 4.9 mos); NTRK2 S167Y (AF 49%; OS > 51 mos) or NTRK1 R6W (AF 42%; OS > 60 mos) were found in one papillary (PTC) and one Hürthle cell (HCC) TC pt respectively (summary table below). Conclusions: NTRK point mutations, but not fusions, were unexpectedly identified in 9% of assessed advanced TCs - but remarkably in 15% of ATCs. Mutated NTRKs, along with oncogenic p53 and TERT, may contribute to TC progression, especially in ATC, consequent to downstream activation of MAPK and PI3K-AKT pathways. We posit that NTRK point mutations may have therapeutic implications for treatment of refractory disease. [Table: see text]

Abstract We have previously shown that loss of HSulf-1, an endosulfatase that removes 6-O sulfation of HSPGs augmented heparin binding growth factor (HB-GF) signaling resulting in enhanced tumorigenesis, angiogenesis and metastasis in ovarian cancer. However, the role of HSulf-1 in energy metabolism and how the increased growth factor signaling in cells with loss of HSulf-1 altered cellular metabolism in cancer yet to be investigated. Here, we show for the first time that batch clonal lines of HSulf-1 knockdown cells [OV202 (Sh1/2)] and HSulf-1 knock out (KO) MEFs showed a significantly higher glycolytic flux (glucose uptake and lactate production) and reduced oxygen consumption rate (OCR) compared to a non-transduced control (OV202NTC) and wild-type (WT) MEF cells respectively. Conversely, enhanced expression of HSulf-1 in TOV21G cells resulted in lower glycolytic flux and higher OCR. Microarray analysis revealed that several of the genes in the glycolytic pathway such as Glut1, HK2, PFKFB3, ALDOC, PGAM, PKM2 and LDHA were upregulated in Sh1/2, cells compared to NTC cells. Expression of these genes and their respective products were validated both at the mRNA and protein levels in OV202Sh1/2, TOV21G Vec and MEFs KO cells compared to OV202NTC, TOV21G Cl13 and MEF WT respectively. Importantly, we found that loss of HSulf-1 decouples the energy flow from glycolysis to mitochondria by lowering the PDH and up-regulating PDK1 expression. We further demonstrated that loss of HSulf-1 reduced the total cellular ATP levels in Sh1/Sh2, TOV21G VEC, and MEFs KO cells, all consistent with lower mitochondrial activity as evidenced by decreased total number of mitochondria, its cross sectional area and perimeter in Sh1/Sh2, TOV21G Vec and MEF KO cells. This augmented aerobic glycolysis with loss of HSulf-1 was further corroborated by metabolomics study which showed significantly higher glycolytic metabolites such as G-6P, F1,6BP, F2,6BP, 3PG. While most of the TCA intermediates were downregulated in Sh1 and Sh2 cells; glutamate, glutamine, α-ketoglutarate and 2-hydroxyglutarate were higher upon loss of HSulf-1. These results suggests that loss of HSulf-1 plays a pivotal role in reprogramming the energy metabolism towards the Warburg effect which are critical to sustain cancer cells proliferation, transformation and angiogenesis. Citation Format: Susmita Mondal, Debarshi Roy, Juliana Camacho Pereira, Eduardo N. Chini, Vijayalakshmi Shridhar. Loss of sulf-1 reprograms energy metabolism in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2014-3360

Abstract PG545 is a synthetic heparan sulfate (HS) mimetic purported to inhibit angiogenesis via the blockade of VEGF and FGF-2 signaling, and block the enzymatic activity of heparanase - a molecule closely associated with metastatic dissemination. The attenuation of VEGF and FGF-2 signaling is supported by recent clinical data from the first four advanced cancer patients with multiple tumor types treated with PG545 which exhibited increases in plasma VEGF and FGF-2 levels, often in close association with the drug treatments/exposure profile during the first treatment cycle over 4 weeks. Unfortunately, due to local injection site reactions associated with subcutaneous (SC) administration, the trial was recently terminated. However, to support a re-entry to the clinic using the intravenous route, we tested PG545 in a preclinical model of ovarian cancer.The rationale to investigate PG545 in ovarian cancer is derived from recently generated data which found PG545 (5 µM) almost completely inhibited migration of SKOV3 ovarian carcinoma cells stimulated by each of the following HS binding growth factors: VEGF, HGF, FGF-2, SDF-1 and HB-EGF. Because HB-EGF is thought to play a major role in the spread of this cancer, PG545 was also tested against HB-EGF dependant SKOV3 cell invasion and was found to be a potent inhibitor of this process. The effect of PG545 on HB-EGF signaling pathways in these experiments were investigated and the results indicated that PG545 reduces activation of the HB-EGF receptor (EGFR) and, consequently, both AKTand MAPK signaling are also reduced. Given the strong mechanism of action data for ovarian carcinoma, PG545 was next tested in the A2780 ovarian xenograft model. The signficant antitumor activity using PG545 at 7.5 mg/kg twice weekly intravenous (IV) and 15 mg/kg once weekly IV was similar to a 20 mg/kg once weekly SC dose. Pharmacokinetic analysis confirmed that drug exposure (AUC) over the course of a week was consistent between groups. PG545 significantly reduced tumor burden in a cisplatin resistant ovarian tumor model (C200) when given intraperitoneally at the dose of 20mg/kg, once a week until the end of the study. Most importantly, PG545 completely reduced tumor mass and reduced ascites accumulation in a syngeneic immunocompetent mouse model of ovarian cancer (ID8). Given the known effects of PG545 on heparanase and in metastatic models, these emerging data on growth factors in the clinic and HB-EGF in preclinical models provide a strong rationale to re-enter the clinic using the IV route and to focus on ovarian cancer as the clinical indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-309. doi:1538-7445.AM2012-LB-309

Abstract A substantial body of evidence suggests an association between vascular endothelial growth factor (VEGF) and the neovascularization that occurs with ovarian carcinogenesis. VEGF expression has been directly related to ascites accumulation. Phase II clinical trials in ovarian cancer using single-agent bevacizumab (a monoclonal antibody directed against VEGF) showed promising results, clearly highlighting the importance of anti-angiogenesis based-targeted therapies in ovarian cancer. One of the ways that VEGF mediates angiogenesis is by activating NADPH oxidase-ROS pathway. We have developed and engineered nanoparticles (NP) of cerium oxide (Nanoceria, NCe), a rare earth oxides, which have been tested for their ability to serve as free-radical scavengers to provide protection against chemical, biological, and radiological insults that promote the production of free radicals. Here we show for the first time that treatment of human umbilical vascular endothelial cells (HUVECs) with NCe resulted in inhibition of proliferation, capillary tube formation and VEGF165 induced activation of VEGFR2 and MMP2. Additionally, NCe treatment inhibited growth factor induced migration and invasion of ovarian cancer cells. Collectively, these results indicate that cerium oxide based NP could potentially be used as an antiangiogenic therapeutic agent in ovarian cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C48.

Abstract Background: Invasive ovarian cancer is a significant cause of gynecologic cancer mortality. Methods: We examined whether this mortality was associated with inherited variation in approximately 170 candidate genes/regions [993 single-nucleotide polymorphisms (SNPs)] in a multistage analysis based initially on 312 Mayo Clinic cases (172 deaths). Additional analyses used The Cancer Genome Atlas (TCGA; 127 cases, 62 deaths). For the most compelling gene, we immunostained Mayo Clinic tissue microarrays (TMA, 326 cases) and conducted consortium-based SNP replication analysis (2,560 cases, 1,046 deaths). Results: The strongest initial mortality association was in HGF (hepatocyte growth factor) at rs1800793 (HR = 1.7, 95% CI = 1.3–2.2, P = 2.0 × 10−5) and with overall variation in HGF (gene-level test, P = 3.7 × 10−4). Analysis of TCGA data revealed consistent associations [e.g., rs5745709 (r2 = 0.96 with rs1800793): TCGA HR = 2.4, CI = 1.4–4.1, P = 2.2 × 10−3; Mayo Clinic + TCGA HR = 1.6, CI = 1.3–1.9, P = 7.0 × 10−5] and suggested genotype correlation with reduced HGF mRNA levels (P = 0.01). In Mayo Clinic TMAs, protein levels of HGF, its receptor MET (C-MET), and phospho-MET were not associated with genotype and did not serve as an intermediate phenotype; however, phospho-MET was associated with reduced mortality (P = 0.01) likely due to higher expression in early-stage disease. In eight additional ovarian cancer case series, HGF rs5745709 was not associated with mortality (HR = 1.0, CI = 0.9–1.1, P = 0.87). Conclusions: We conclude that although HGF signaling is critical to migration, invasion, and apoptosis, it is unlikely that HGF genetic variation plays a major role in ovarian cancer mortality. Furthermore, any minor role is not related to genetically-determined expression. Impact: Our study shows the utility of multiple data types and multiple data sets in observational studies. Cancer Epidemiol Biomarkers Prev; 20(8); 1638–48. ©2011 AACR.