Jung, Deok‐Beom

A considerable subset of gynecologic cancer patients experience disease recurrence or acquired resistance, which contributes to high mortality rates in ovarian cancer (OC). Our prior studies showed that quinacrine (QC), an antimalarial drug, enhanced chemotherapy sensitivity in treatment-refractory OC cells, including artificially generated chemoresistant and high-grade serous OC cells. In this study, we investigated QC-induced transcriptomic changes to uncover its cytotoxic mechanisms of action. Isogenic pairs of OC cells generated to be chemoresistant and their chemosensitive counterparts were treated with QC followed by RNA-seq analysis. Validation of selected expression results and database comparison analyses indicated the ribosomal biogenesis (RBG) pathway is inhibited by QC. RBG is commonly upregulated in cancer cells and is emerging as a drug target. We found that QC attenuates the in vitro and in vivo expression of nucleostemin (NS/GNL3), a nucleolar RBG and DNA repair protein, and the RPA194 catalytic subunit of Pol I that results in RBG inhibition and nucleolar stress. QC promotes the redistribution of fibrillarin in the form of extranuclear foci and nucleolar caps, an indicator of nucleolar stress conditions. In addition, we found that QC-induced downregulation of NS disrupted homologous recombination repair both by reducing NS protein levels and PARylation resulting in reduced RAD51 recruitment to DNA damage. Our data suggest that QC inhibits RBG and this inhibition promotes DNA damage by directly downregulating the NS–RAD51 interaction. Additionally, QC showed strong synergy with PARP inhibitors in OC cells. Overall, we found that QC downregulates the RBG pathway, induces nucleolar stress, supports the increase of DNA damage, and sensitizes cells to PARP inhibition, which supports new therapeutic stratagems for treatment-refractory OC. Our work offers support for targeting RBG in OC and determines NS to be a novel target for QC.

Abstract The metabolic signatures of cancer cells are often associated with elevated glycolysis which promotes alteration in cellular bioenergetics. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway has emerged as a potential anti-cancer target associated with many aspects of neoplastic progression. Pharmacological (PFK158 treatment) and genetic inhibition of PFKFB3 activity, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and inhibits cell proliferation in vitro by arresting the cells in the G0/G1 phase induced cell death in malignant pleural mesothelioma (MPM). It induces an early response of increased macropinocytosis and drastic endosomal vacuolization to overcome the nutrient stress both in sarcomatoid (H28) and epithelioid (EMMeso) MPM cells. Inhibition of PFKFB3 leads to the activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called “methuosis”. Transmission Electron Microscopy analysis showed that pharmacological and genetic inhibition of PFKFB3 activity leads to the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Confocal microscopy revealed F-actin and α-tubulin disassembly leading to the disruption of cell structure integrity. In parallel, PFKFB3 expression inversely modulates endoplasmic reticulum (ER) stress in MPM. Pharmacological and genetic inhibition of PFKFB3 led to an escalation in ER activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression levels. However, EHT1864, a Rac1 inhibitor, could not rescue MPM cells from ER stress, suggesting that the activation of macropinocytosis and ER stress are a result of two independent PFKFB3 modulated adaptive responses. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that the two crucial biomolecules of each pathway, Rac1 and Calnexin, interact with each other only at the later stages of PFKFB3 inhibition. Finally, PFK158, in a combination of with CBPt, demonstrated a significant reduction in tumor weight in mesothelin overexpressed EMMeso xenograft model. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM. Citation Format: Sayantani Sarkar Bhattacharya, Ling Jin, Debarshi Roy, Deokbeom Jung, Prabhu Thirusangu, Yinan Xiao, Julie Staub, Julian Molina, Viji Shridhar. PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2658.

Metabolic alterations are increasingly recognized as important novel anti‐cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158‐induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co‐immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158‐mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX‐resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

Abstract We have previously shown that a frequent loss of endosulfatase HSulf-1 in ovarian cancer enhances the heparin binding growth factor signaling which subsequently induces and potentiates tumorigenesis. Our recent data indicate that knockdown of HSulf-1 in ovarian cancer cells significantly alter levels of several metabolites which is comparable to the deranged metabolite profiles in primary ovarian tumors. Here we report that the metabolites associated with lipid metabolism are altered in such a way that the cells start manifesting a “lipogenic phenotype” characterized by an increased fatty acid synthesis, storage and mitochondrial beta oxidation. The lipogenic phenotype is associated with an accumulation of cytoplasmic lipid droplets (LDs) in a stably HSulf-1 knockdown cells [OV202 (Sh1/2) and OV2223 cells]. Presence of LDs is evidenced by BODIPY staining and transmission electron microscopy (TEM) analysis in Sh1/2 and a non-transduced control (NTC) cells. Conversely, enhanced expression of HSulf-1 in TOV21G, SKOV3, and C13 cells also resulted in reducing the number of LDs. More importantly, MEFs (mouse embryonic fibroblasts) from HSulf-1 knockout mice also showed an increased LD biogenesis compared to the wild type MEFs. Microarray analysis identified cytoplasmic phospholipase A2 (cPLA2; an enzyme involved in LD biogenesis) as one of the genes that was significantly upregulated in OV202 Sh1 and Sh2 cells. We found that Sh1 and Sh2 cells show an increased phosphorylation (ser505) of cytoplasmic phospholipase A2 (p-cPLA2) compared to NTC cells. Phosphorylation at ser505 is vital for the enzyme activity of cPLA2 and also necessary for LD biogenesis. We inhibited the LD biogenesis in Sh1 cells by a pharmacological inhibitor of cPLA2 (AACOCF3 and MAFP) as well by shRNA mediated knockdown of endogenous cPLA2. Further, we stimulated NTC and Sh1 cells with heparin binding growth factor (50ng/ml) for different time points and found an elevated phosphorylation of cPLA2 and extracellular signal related kinase (ERK), which is also inhibited by a pre-treatment of cells with cPLA2 inhibitor. Interestingly, inhibition of phospho-ERK by U0126 (MEK inhibitor) also inhibits the phosphorylation of cPLA2. These findings strongly suggest that loss of HSulf-1 promotes lipid metabolism in the ovarian cancer cells via activation of cPLA2 via an ERK dependent pathway. The present work highlights a novel role HSulf-1 in regulating lipid metabolism which could lead to a better understanding of tumorigenesis and identification of new targets for tumor therapy. Citation Format: Debarshi Roy, Xiaoping He, Ashwani Khurana, Susmita Mondal, Deok-Beom Jung, Sung Kim, Thomas Dierks, Clifford Folmes, Andre Terzic, Viji Shridhar. HSulf-1 loss in ovarian cancer cells induces lipid droplet biogenesis. [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 2439. doi:10.1158/1538-7445.AM2014-2439