Xiao, Yinan

Abstract Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine–rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody–drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases. Significance: This study identifies that LRRC15 activates β1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody–drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.

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 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.

We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that 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. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (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 induced overexpression 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 promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results 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. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited oncogenesis. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

Abstract Objectives: Endometrial cancer (EC) is the most common gynecological cancer in the developed countries with rising incidence rates. However, the knowledge about targeted therapies that can improve response rates to chemotherapy in EC is limited. Of the cancer afflicting women, women with metabolic disorders such as diabetes and obesity have an increased risk of developing EC. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a glycolytic enzyme, is found to be highly expressed in many solid tumors. This study aimed to investigate the role and molecular mechanism of PFKFB3 in endometrial cancer. Methods: HEC-1B and ARK-2 endometrial cancer cell lines were used as models to evaluate the potential antitumor effects of pharmacological (treatment with PFK158, a selective PFKFB3 inhibitor) and genetic (PFKFB3 knockdown using CRISPR/Cas9 technology) inhibition of PFKFB3 on EC. In vitro, we evaluated the effects of PFKFB3 inhibition, and its synergistic effects when combined with carboplatin (CBPt) or cisplatin on cell viability, glucose metabolism, autophagy, apoptosis, and changes of cell signaling using MTT and colony formation assays, Chou-Talalay methodology, immunofluorescence assay, flow cytometry and western blotting. In vivo, two EC subcutaneous xenograft models derived from HEC-1B and ARK-2 cell lines were used to investigate the effect of single-agent PFK158 and in combination with CBPt on tumor growth, followed by western blotting, TUNEL and immunohistochemistry staining of excised tumor tissue. Results: Targeting PFKFB3 via PFK158 considerably reduced glucose uptake, ATP production, lactate release in EC cell lines. In vitro, inhibition of PFKFB3 induced cell death and synergistically enhanced CBPt/cisplatin sensitivity in EC cell lines by suppressing cell viability, inducing autophagy and apoptosis in a time- and dose-dependent manner. In vivo xenograft studies revealed that treatment with PFK158, either alone or in combination with CBPt, led to a marked reduction in tumor growth. In addition, PFK158 caused significant upregulation of cleaved PARP, cleaved caspase 3, and reduction of mTOR, Akt, ERK phosphorylation levels, both in vitro and in vivo. Conclusions: Our results indicate that PFKFB3 plays a prominent role in the growth and chemoresistance in EC cells. Therefore, a selective PFKFB3 inhibitor or in combination with chemotherapy, may be used as a novel therapeutic strategy for patients with chemoresistant EC. Citation Format: Yinan Xiao, Ling Jin, Sayantani Sarkar Bhattacharya, Julie Staub, Chaolin Deng, Xiaoling Fang, Viji Shridhar. Inhibition of 6-Phosphofructo-2-kinase (PFKFB3) induces cell death and synergistically enhances chemosensitivity in endometrial cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6222.