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BACKGROUND: Angiogenin is a multifunctional secreted ribonuclease that is upregulated in human cancers and downregulated or mutationally inactivated in neurodegenerative diseases. A role for angiogenin in glioblastoma was inferred from the inverse correlation of angiogenin expression with patient survival but had not been experimentally investigated. METHODS: Angiogenin knockout mice were generated and the effect of angiogenin deficiency on glioblastoma progression was examined. Angiogenin and plexin-B2 genes were knocked down in glioblastoma cells and the changes in cell proliferation, invasion and vascular association were examined. Monoclonal antibodies of angiogenin and small molecules were used to assess the therapeutic activity of the angiogenin-plexin-B2 pathway in both genetic and xenograft animal models. RESULTS: Deletion of Ang1 gene prolonged survival of PDGF-induced glioblastoma in mice in the Ink4a/Arf(−/−):Pten(−/−) background, accompanied by decreased invasion, vascular association and proliferation. Angiogenin upregulated MMP9 and CD24 leading to enhanced invasion and vascular association. Inhibition of angiogenin or plexin-B2, either by shRNA, monoclonal antibody or small molecule inhibitor, decreases sphere formation of patient-derived glioma stem cells, reduces glioblastoma proliferation and invasion and inhibits glioblastoma growth in both genetic and xenograft animal models. CONCLUSIONS: Angiogenin and its receptor, plexin-B2, are a pair of novel regulators that mediate invasion, vascular association and proliferation of glioblastoma cells. Inhibitors of the angiogenin-plexin-B2 axis have therapeutic potential against glioblastoma.

Autophagy is a highly conserved cellular process that maintains homeostasis by degrading and recycling long-lived or misfolded proteins and damaged organelles. In addition, autophagy can protect cells from diverse types of cellular stress produced by amino acid starvation and hypoxia, providing energy to retain the function of organelles and cellular signaling pathways. In cancer, autophagy can play dual roles, inhibiting tumor cell growth and viability or sustaining cancer cells in hypoxic, nutrient poor microenvironments. DIRAS3 (ARHI), a maternally imprinted tumor suppressor gene, encodes a 26-kDa GTPase that has 60% homology to Ras and is downregulated in the majority of ovarian cancers. Re-expression of DIRAS3 inhibits growth of ovarian cancer cells, slows their motility and induces autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway. Previously, we have shown that DIRAS3-induced autophagy inhibits ovarian cancer cell growth and promotes autophagic cell death in culture while inducing tumor dormancy in vivo. We have demonstrated that growth factors and extracellular membrane components including fibronectin (FN) in the tumor microenvironment can rescue ovarian cancer cells from DIRAS3-induced autophagic death in cell culture and the use of antibodies against those growth factors or their receptors can eliminate dormant autophagic ovarian cancer cells in ovarian cancer xenografts. In the current study, we explored whether FN, a ubiquitous extracellular matrix (ECM) glycoprotein also plays an important role in DIRAS3-induced tumor dormancy. We hypothesized that FN blocks DIRAS3-induced growth inhibition in ovarian cancer. We investigated the effect of FN on cultured ovarian cancer cells in the presence and absence of DIRAS3. We found that DIRAS3-induced autophagy was inhibited by FN in ovarian cancer cells, judged by decreased conversion of LC3I to LC3II on Western blots and reduced LC3 puncta detected by fluorescence staining. We also found that FN partially rescues ovarian cancer cells from DIRAS3-induced cell death in culture. Mechanistically, FN weakened the inhibitory effect of DIRAS3 on p-FAK and p-AKT, whereas addition of neutralizing antibody against integrin β1 decreased p-FAK and p-AKT. In addition, FN and FN-mediated FAK/AKT signaling is elevated in tumor xenograft tissue when compared to cancer cells in culture. Pharmacologic inhibition of FAK using defactinib, enhanced DIRAS3-induced autophagy, inducing cell death in ovarian cancer cells in the presence of FN. Together, our data demonstrate that factors such as FN, block DIRAS3-induced autophagic cell death, contributing to the survival of ovarian cancer cells. Citation Format: Janice M. Santiago-OFarrill, Jing Guo, Hailing Yang, Maggie Mao, Zhen Lu, Robert Bast. DIRAS3 suppresses ovarian cancer cell growth through the inhibition of fibronectin-mediated FAK/AKT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2515.

Background Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. Methods By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. Results Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. Conclusions Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

The present study aimed to identify microRNAs (miRNAs) that may be crucial for the mechanism of mesenchymal stem cell (MSC) treatment in cisplatin‑induced acute kidney injury (AKI) and to investigate other potential drugs that may have a similar function. Transcriptomics (GSE85957) and miRNA expression (GSE66761) datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) were identified using the linear models for microarray data method and mRNA targets of DEMs were predicted using the miRWalk2.0 database. The crucial DEGs were screened by constructing a protein‑protein interaction (PPI) network and module analysis. Functions of target genes were analyzed using the database for annotation, visualization and integrated discovery. Small molecule drugs were predicted using the connectivity map database. As a result, 5 DEMs were identified to be shared and oppositely expressed in comparisons between AKI model and control groups, and between MSC treatment and AKI model groups. The 103 DEGs were overlapped with the target genes of 5 common DEMs, and the resulting list was used for constructing the miRNA‑mRNA regulatory network, including rno‑miR‑210/Serpine1 and rno‑miR‑378/Fos. Serpine1 (degree=17) and Fos (degree=42) were predicted to be hub genes according to the topological characteristic of degree in the PPI network. Function analysis indicated Serpine1 and Fos may be inflammation‑related. Furthermore, gliclazide was suggested to be a potential drug for the treatment of AKI because the enrichment score was the closest to ‑1 (‑0.9). In conclusion, it can be speculated that gliclazide may have a similar mechanism to MSC as a potential therapeutic agent for cisplatin‑induced AKI, by regulating miR‑210/Serpine1 and miR‑378‑/Fos‑mediated inflammation and cell apoptosis.

BACKGROUND Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. METHODS Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. RESULTS Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P

Recently, severe acute respiratory syndrome (SARS) coronavirus (CoV) 2 (SARS­CoV­2)­causing CoV disease 2019 (COVID­19) emerged in China and has become a global pandemic. SARS­CoV­2 is a novel CoV originating from s­CoVs. Major distinctions in the gene sequences between SARS­CoV and SARS­CoV­2 include the spike gene, open reading frame (ORF) 3b and ORF 8. SARS­CoV­2 infection is initiated when the virus interacts with angiotensin­converting enzyme 2 (ACE2) receptors on host cells. Through this mechanism, the virus infects the alveolar, esophageal epithelial, ileum, colon and other cells on which ACE2 is highly expressed, causing damage to target organs. To date, host innate immunity may be the only identified direct factor associated with viral replication. However, increased ACE2 expression may upregulate the viral load indirectly by increasing the baseline level of infectious virus particles. The peak viral load of SARS­CoV­2 is estimated to occur ~10 days following fever onset, causing patients in the acute stage to be the primary infection source. However, patients in the recovery stage or with occult infections can also be contagious. The host immune response in patients with COVID­19 remains to be elucidated. By studying other SARS and Middle East respiratory syndrome coronaviruses, it is hypothesized that patients with COVID­19 may lack sufficient antiviral T­cell responses, which consequently present with innate immune response disorders. This may to a certain degree explain why this type of CoV triggers severe inflammatory responses and immune damage and its associated complications.

The present study aimed to screen potential target genes for the early diagnosis and treatment of early metastatic clear cell renal cell carcinoma (ccRCC) using the microarray data of early metastatic and non-metastatic ccRCC samples. The DNA microarray dataset GSE47352 was downloaded from Gene Expression Omnibus and included 4 early metastatic and 5 non-metastatic ccRCC samples. Differentially expressed genes (DEGs) were screened using the limma package. Then, pheatmap package was used to conduct two-way clustering for the DEGs. Subsequently, MAPPFinder and GenMAPP were employed separately to perform functional and pathway enrichment analysis for the DEGs. Additionally, a protein-protein interaction (PPI) network was constructed using Cytoscape, and small drug molecules were searched using Connectivity map (cmap). In total, 196 upregulated and 163 downregulated genes were identified. DEGs, including JUN, tumor necrosis factor (TNF), Ras homolog family member B (RHOB) and transforming growth factor β2 (TGFβ2) were significantly enriched in the signaling pathway of renal cell carcinoma. Furthermore, nuclear receptor subfamily 4 group A member 1 (NR4A1) was significantly enriched in the mitogen-activated protein kinase signaling pathway; in addition, laminin subunit α (LAMA) 1, LAMA2 and LAMA4 were significantly enriched in extracellular matrix-receptor interaction. JUN (degree=6) had the highest degree in the PPI network. Thapsigargin (score=−0.913) possessed the highest performance in terms of the treatment of early metastatic ccRCC. In the present study, it was discovered that certain DEGs, including JUN, TNF, RHOB, NR4A1, TGFβ2, LAMA1, LAMA2 and LAMA4 were potential target genes associated with early metastatic ccRCC. In addition, thapsigargin could be used as an efficient small drug molecule for the treatment of early metastatic ccRCC.

Purpose: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. Results: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose–phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. Conclusions: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.

Salt-induced kinase 2 (SIK2) is a serine-threonine kinase that regulates centrosome splitting, activation of PI3 kinase and phosphorylation of class IIa HDACs, affecting gene expression. Previously, we found that inhibition of SIK2 enhanced sensitivity of ovarian cancer cells to paclitaxel. Carboplatin and paclitaxel constitute first-line therapy for most patients with ovarian carcinoma, producing a 70% clinical response rate, but curing &lt, 20% of patients with advanced disease. We have asked whether inhibition of SIK2 with ARN-3261 enhances sensitivity to carboplatin in ovarian cancer cell lines and xenograft models. ARN-3261-induced DNA damage and apoptosis were measured with &gamma, H2AX accumulation, comet assays, and annexin V. ARN-3261 inhibited growth of eight ovarian cancer cell lines at an IC50 of 0.8 to 3.5 &micro, M. ARN-3261 significantly enhanced sensitivity to carboplatin in seven of eight ovarian cancer cell lines and a carboplatin-resistant cell line tested. Furthermore, ARN-3261 in combination with carboplatin produced greater inhibition of tumor growth than carboplatin alone in SKOv3 and OVCAR8 ovarian cancer xenograft models. ARN-3261 enhanced DNA damage and apoptosis by downregulating expression of survivin. Thus, a SIK2 kinase inhibitor enhanced carboplatin-induced therapy in preclinical models of ovarian cancer and deserves further evaluation in clinical trials.

Genomic instability is a recognized hallmark of cancer. Germline mutations in critical DNA-repair and DNA-damage response genes predispose to cancer development, but also create vulnerabilities that can be exploited for cancer therapy. Poly (ADP-ribose) polymerase (PARP) inhibitors are selectively active in cells with homologous recombination deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other DNA repair genes. PARP inhibitors elicit significant responses in ovarian and breast cancers from BRCA1 or BRCA2 mutation carriers. However, many cancers that initially respond to PARP inhibitors eventually develop drug resistance. Thus, it is important to develop new strategies to enhance PARP inhibitor sensitivity and to increase the duration of response. We have identified Salt Induced Kinase 2 (SIK2) inhibitors (ARN3236 and ARN3261) that induce double strand breaks (DSBs) in DNA of HR-competent cells and produce synthetic lethality with multiple PARP inhibitors. SIK2 is an AMP-activated protein kinase (AMPK)-related protein kinase that is required for ovarian cancer cell proliferation and metastasis. SIK2 is overexpressed and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma and triple negative breast cancer. SIK2 inhibition enhances paclitaxel sensitivity in both cancer types. We have demonstrated that olaparib-induced-growth inhibition was significantly enhanced by concurrent treatment with either ARN3236 or ARN3261 in each of 12 ovarian and breast cancer cell lines tested, but not in 3 non-tumorigenic cell lines. Co-administration of olaparib with SIK2 inhibitors suppressed tumor growth and increased the survival of mice with human ovarian (OC316) and breast (MDA-MB-231) cancer xenografts without affecting animal weight. ARN3261 produced little toxicity in preclinical toxicology studies. SIK2 inhibitors decrease the phosphorylation of class-IIa HDAC4/5/7 and abolish class-IIa HDAC 4/5/7-associated transcriptional activity of Myocyte Enhancer Factor 2D (MEF2D). Genome-wide chromatin immunoprecipitation (CHIP) sequencing revealed that SIK2 inhibitors reduce MEF2D binding to regulatory regions with high-chromatin accessibility in DNA repair genes, including FANCD2, EXO1 and XRCC4, resulting in repression of critical genes in DNA DSB repair pathway and induction of apoptosis. In addition, SIK2 inhibitors significantly decreased olaparib-mediated PARP activity and enhanced olaparib-induced cytotoxicity. Together, our data argue that the combination of a SIK2 inhibitor and a PARP inhibitor has the potential to increase the magnitude and duration of PARP inhibitor activity with tolerable toxicity. Use of a SIK2 inhibitor in combination with a PARP inhibitor provides a novel therapeutic strategy for ovarian and triple negative breast cancers with or without BRCA gene mutation. Citation Format: Zhen Lu, Weiqun Mso, Janice M. Santiago-O'Farrill, Hailing Yang, Lan Pang, Ahmed A. Ahmed, Hariprasad Vankayalapati, Robert C. Bast Jr. Novel SIK2 inhibitors sensitize ovarian and breast cancer to PARP inhibitors [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 5210.

Purpose: Most patients with ovarian cancer receive paclitaxel chemotherapy, but less than half respond. Pre-treatment microtubule stability correlates with paclitaxel response in ovarian cancer cell lines. Microtubule stability can be increased by depletion of individual kinases. As microtubule stability can be regulated by phosphorylation of microtubule-associated proteins (MAPs), we reasoned that depletion of pairs of kinases that regulate phosphorylation of MAPs could induce microtubule stabilization and paclitaxel sensitization. Experimental Design: Fourteen kinases known to regulate paclitaxel sensitivity were depleted individually in 12 well-characterized ovarian cancer cell lines before measuring proliferation in the presence or absence of paclitaxel. Similar studies were performed by depleting all possible pairs of kinases in six ovarian cancer cell lines. Pairs that enhanced paclitaxel sensitivity across multiple cell lines were studied in depth in cell culture and in two xenograft models. Results: Transfection of siRNA against 10 of the 14 kinases enhanced paclitaxel sensitivity in at least six of 12 cell lines. Dual knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity more than silencing single kinases. Sequential knockdown was superior to concurrent knockdown. Dual silencing of IKBKB/STK39 or EDN2/TBK1 stabilized microtubules by inhibiting phosphorylation of p38 and MAP4, inducing apoptosis and blocking cell cycle more effectively than silencing individual kinases. Knockdown of IKBKB/STK39 or EDN2/TBK1 enhanced paclitaxel sensitivity in two ovarian xenograft models. Conclusions: Sequential knockdown of dual kinases increased microtubule stability by decreasing p38-mediated phosphorylation of MAP4 and enhanced response to paclitaxel in ovarian cancer cell lines and xenografts, suggesting a strategy to improve primary therapy. Clin Cancer Res; 24(20); 5072–84. ©2018 AACR.

Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200(th) of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13(th) of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety.

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear–centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling. Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945–54. ©2016 AACR.

Numerous studies have implicated mutations in tubulin or the overexpression of specific tubulin genes in resistance to microtubule-targeted drugs. Much less is known about the role of accessory proteins that modulate microtubule behavior in the genesis of drug resistance. Here, we examine mitotic centromere–associated kinesin (MCAK), a member of the kinesin family of microtubule motor proteins that has the ability to stimulate microtubule depolymerization, and show that overexpressing the protein confers resistance to paclitaxel and epothilone A, but increases sensitivity to colcemid. Cells transfected with FLAG-tagged MCAK cDNA using a tet-off–regulated expression system had a disrupted microtubule cytoskeleton and were able to survive a toxic concentration of paclitaxel in the absence, but not in the presence of tetracycline, showing that drug resistance was caused by ectopic MCAK production. Moreover, a population that was heterogeneous with respect to FLAG-MCAK expression became enriched with cells that produced the ectopic protein when it was placed under paclitaxel selection. Similar to previously isolated mutants with altered tubulin, paclitaxel resistant cells resulting from MCAK overexpression were found to have decreased microtubule polymer and a seven-fold increase in the frequency of microtubule detachment from centrosomes. These data are consistent with a model for paclitaxel resistance that is based on stability of the attachment of microtubules to their nucleating centers, and they implicate MCAK in the mechanism of microtubule detachment. Mol Cancer Ther; 10(6); 929–37. ©2011 AACR.

We previously described the isolation of Tax 18 and Tax 11-6, two paclitaxel-dependent cell lines that assemble low amounts of microtubule polymer and require the drug for cell division. In the present studies, fluorescence time-lapse microscopy was used to measure microtubule dynamic instability behavior in these cells. The mutations were found to cause small decreases in microtubule growth and shortening, but the changes seemed unable to explain the defects in microtubule polymer levels or cell division. Moreover, paclitaxel further suppressed microtubule dynamics at low drug concentrations that were insufficient to rescue the mutant phenotype. Wild-type (WT) cells treated with similar low drug concentrations also had highly suppressed microtubules, yet experienced no problems with cell division. Thus, the effects of paclitaxel on microtubule dynamics seemed to be unrelated to cell division in both WT and mutant cell lines. The higher drug concentrations needed to rescue the mutant phenotype instead inhibited the formation of unstable microtubule fragments that appeared at high frequency in the drug-dependent, but not WT, cell lines. Live cell imaging revealed that the fragments were generated by microtubule detachment from centrosomes, a process that was reversed by paclitaxel. We conclude that paclitaxel rescues mutant cell division by inhibiting the detachment of microtubule minus ends from centrosomes rather than by altering plus-end microtubule dynamics. Mol Cancer Ther; 9(11); 2914–23. ©2010 AACR.

Background Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown. Methods We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging. Results Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [18F]-FDG uptake, and significantly altered choline metabolism. Conclusions ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2850-8) contains supplementary material, which is available to authorized users.

NDN is a maternally imprinted gene consistently expressed in normal ovarian epithelium, is dramatically downregulated in the majority of ovarian cancers. Little or no NDN expression could be detected in 73% of 351 epithelial ovarian cancers. NDN was also downregulated in 10 ovarian cancer cell lines with total loss in 6 of 10. Re-expression of NDN decreased Bcl-2 levels and induced apoptosis, which significantly inhibited ovarian cancer cell growth in cell culture and in xenografts. In addition, re-expression of NDN inhibited cell migration by decreasing actin stress fiber and focal adhesion complex formation through deactivation of Src, FAK and RhoA. Loss of NDN expression in ovarian cancers could be attributed to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 of NDN promoter in 23% and 30% of 43 ovarian cancers, respectively. Promoter hypermethylation was also found in 5 of 10 ovarian cancer cell lines. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored NDN expression in 4 of 7 cell lines with enhanced promoter methylation levels. These observations support the conclusion that NDN is an imprinted tumor suppressor gene which affects cancer cell motility, invasion and growth and that its loss of function in ovarian cancer can be caused by both genetic and epigenetic mechanisms.

Introduction: Most ovarian cancers are diagnosed in a late, incurable stage, which has prompted efforts towards earlier detection and more effective screening strategies. To be considered effective, screening must provide sufficient sensitivity and specificity to impact patient mortality while minimizing false positives. Magnetic relaxometry (MRX), which detects binding of targeted iron oxide nanoparticles (NPs) to cancer cells, offers the promise of improved sensitivity and specificity over conventional early detection modalities. Methods: We investigated the sensitivity and specificity of MRX by scanning ovarian cancer cell samples containing 105, 106, and 107 cells incubated with a fixed amount (57 µg Fe3O4) of anti-HER2 antibody-conjugated, PEG-coated NPs or unconjugated, PEG-coated NPs (Senior Scientific LLC). To further evaluate specificity, we used cell lines with both high and low expression of HER2 (SKOV3 and HEY, respectively). To assess MRX under in vivo conditions, we subcutaneously injected 105, 106, and 107 anti-HER2 NP-labeled SKOV3 cells into nude mice (n = 9) and immediately performed MRX scanning. Prior to performing this study, we verified successful antibody-NP conjugation through an ELISA assay, which confirmed the presence of anti-HER2 antibody in NP pellets. Additionally, we performed flow cytometry to confirm a high level of specific binding between SKOV3 cells and anti-HER2-conjugated NPs. Results: Our in vitro data revealed strong linearity between cell number and MRX signal for both SKOV3 and HEY cells incubated with anti-HER2 NPs (R2 = 0.99 and 1, respectively). Furthermore, there was little to no MRX signal for all cell samples incubated with unconjugated, PEG NPs regardless of cell number. The highest MRX signal was observed when 107 SKOV3 cells were incubated with anti-HER2 NPs, which was 2.1 ± 0.3 and 15.7 ± 1.4 times higher than when 107 Hey cells were incubated with anti-HER2 NPs or when 107 SKOV3 cells were incubated with unconjugated PEG NPs, respectively; significantly higher MRX signals (relative to the controls; p < .01) were also noted for samples containing 106 SKOV3 cells incubated with anti-HER2 NPs. When scanning live mice injected with 107 anti-HER2 NP-labeled SKOV3 cells, the MRX signal was significantly higher than the signal from the mice prior to the injection (p < .001). Additionally, MRX signal versus cell number in the injected mice was highly correlated (r = 0.99) with the MRX data from the corresponding cell sample scans. Conclusion: MRX is sufficiently sensitive to detect 1 million cells in culture or 10 million cells in mice with a high level of specificity. Sensitivity may be improved by using nanoparticles coated with antibodies against antigens that are overexpressed by a larger fraction of ovarian cancers and will be the focus of future work. Citation Format: Kelsey Mathieu, Zhen Lu, Hailing Yang, Lan Pang, Adam Kulp, John Hazle, Robert C. Bast. Feasibility of magnetic relaxometry for early ovarian cancer detection: preliminary evaluation of sensitivity and specificity in cell culture and in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1864. doi:10.1158/1538-7445.AM2017-1864

Breast cancer is the most common cause and epithelial ovarian cancer the fourth most common cause of cancer death among women in the developed world. While breast cancer can be cured in 70% of cases, only 30% of ovarian cancer patients remain free from disease long term. Paclitaxel is an integral component of primary therapy for both forms of cancer, but less than half of breast and ovarian cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases. In recent years several kinases have been identified that regulate the sensitivity of cancer cells to paclitaxel by inhibiting centrosome splitting or enhancing microtubule stability. Much less attention has been given to kinases that affect paclitaxel sensitivity by modulating cancer cell metabolism. We previously performed siRNA kinome-screens to identify molecular targets whose decreased expression overcomes paclitaxel resistance and increases paclitaxel sensitivity in ovarian cancer cells. We showed that 20% of the potential kinase targets whose knockdown modulates paclitaxel sensitivity participate in glucose and energy metabolism. Among these, a leading candidate was 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), an isoform of the glycolytic enzyme phosphofructokinase (PFK2). PFKFB2 is overexpressed in a fraction of ovarian and breast cancers. Knockdown of PFKFB2 inhibited clonogenic growth of ovarian and breast cancer cell lines and enhanced paclitaxel sensitivity in the cell lines with wt TP53. Liposome encapsulated PFKFB2 siRNA significantly inhibited tumor growth and enhanced sensitivity to paclitaxel in xenografts derived from ovarian cancer cell lines. Knockdown of PFKFB2 increased glycolysis, decreasing the flow of glycolytic intermediates to the pentose-phosphate pathway with reduced G6PD activity in wt TP53 cancer cell lines. With decreased NADPH, ROS accumulated after PFKFB2 knockdown, stimulating phosphorylation of JNK, inducing G1 cell cycle arrest, and initiating apoptosis dependent upon upregulation of p21Cip1 and Puma which are downstream targets of TP53. Our studies have shown for the first time that PFKFB2, a glycolytic enzyme, drives tumor cell growth and regulates paclitaxel sensitivity by inducing apoptosis and G1 cell cycle arrest. These findings highlight a remarkable degree of coordination between cancer metabolism with cell proliferation and chemo-sensitivity, which may provide a novel target in patients with ovarian cancers and breast cancers where TP53 function remains intact. Citation Format: Hailing Yang, Shu Zhang, Weiqun Mao, Ahmed A. Ahmed, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Gabriel Lopez-Berestein, Anil K. Sood, Xiao-Feng Le, Zhen Lu, Robert C. Bast. Silencing pfkfb2 enhances paclitaxel sensitivity by modulating metabolism of p53 wt ovarian and breast cancer cells and xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2017-3564

Drugs that target microtubules are potent inhibitors of angiogenesis, but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; however, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish, rather than elongated, cells, rapid but nondirectional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells, whereas more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. Mol Cancer Ther; 12(12); 2837–46. ©2013 AACR.

Drug resistance imposes a major obstacle in ovarian cancer treatment. All patients with newly diagnosed ovarian cancer are treated with a combination of paclitaxel and carboplatin. While 70% of ovarian cancers respond to carboplatin, less than 50% respond to paclitaxel. Inhibition of kinases that modulate primary paclitaxel resistance, could enhance response to therapy. A Kinase siRNA library was screened in ovarian cancer cell lines to determine which kinases regulate paclitaxel sensitivity. Among 45 hits from high throughput screening, 14 target proteins (AATK, ACRBP, BMP2K, CHUK, EDN2, IKBKB, ILK, RAPGEF3, RAPGEF4, RFP, SIK2, STK24, STK39 and TBK1) regulated sensitivity to paclitaxel and were differentially expressed or overexpressed in a fraction of ovarian cancers. siRNAs against each of these kinases were tested for the ability to enhance sensitivity to paclitaxel in each of 12 ovarian cancer cell lines that reflected the heterogeneity observed in ovarian cancers including mutations of TP53, BRCA1/2, KRAS, BRAF, PI3K and PTEN. Knockdown of 10 individual genes enhanced paclitaxel sensitivity by at least two folds in different cell lines. A subscreen using combinations of these 14 kinase siRNAs in the six most responsive cell lines were carried out to select pairs of kinase siRNAs which could further potentiate paclitaxel sensitivity. IKBKB and STK39 kinase siRNAs had the greatest activity. We discovered that knockdown of IKBKB and STK39 stabilized microtubules judged by a microtubule fractionation assay and levels of acetylated and detyrosinated tubulin. IKBKB and STK39 kinase siRNAs also induced apoptosis. Double knockdown of microtubule stabilizing kinases IKBKB and STK39 resulted in greater enhancement in microtubule stability and apoptosis, leading to additive sensitization of paclitaxel. Small molecule inhibitors are available for IKBKB. As inhibition drugs become available for STK39, these observations can be translated to the clinic. Citation Format: Hailing Yang, Xiaoyan Wang, Ahmed Ahmed, Lakesla Iles, Geoffrey Bartholomeusz, Zhen Lu, Robert C. Bast Jr.. Combinations of kinase siRNAs potentiates paclitaxel sensitivity in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4670.

DIRAS3 (also known as ARHI; Aplasia Ras Homolog Member I) is a potent tumor suppressor that has been well characterized for its role in ovarian cancer and autophagy. DIRAS3 is a maternally imprinted tumor suppressor gene that encodes a 26kDa GTPase which shares 60% homology to Ras and Rap. DIRAS3 is downregulated in many tumor types including ovarian cancer. DIRAS3 is downregulated by multiple mechanisms including loss of heterozygosity, transcriptional regulation by E2F1 and E2F4, hypermethylation of the second allele, and regulation by miRNAs 221 and 222. Re-expression of DIRAS3 at physiologic levels inhibits cancer cell growth, reduces motility, induces autophagy and promotes tumor dormancy. The mechanisms by which DIRAS3 induce autophagic cell death in vitro and tumor dormancy in vivo have been well characterized by previous work in our laboratory demonstrating that DIRAS3 is required for the induction of autophagy, and that upon inhibition of autophagy you can prevent outgrowth of dormant ovarian cancer cells in vivo. Interestingly, mice do not have DIRAS3 as it was lost during evolutionary rearrangement of murine chromosomes 3 and 6 nearly 60 million years ago, yet murine cells can still undergo autophagy. The DIRAS family members, DIRAS1 and DIRAS2 share 50-60% homology with DIRAS3 and are found in the murine genome. These 22kDa GTPases differ from DIRAS3 by the truncation of their N-terminal extension. Although DIRAS1 and DIRAS2 have not previously been characterized in ovarian cancer, TCGA analysis suggests that higher mRNA expression of these genes results in a survival advantage for patients with high grade serous ovarian cancer. In this study we demonstrate that re-expression of DIRAS1 and DIRAS2 inhibit ovarian cancer cell growth in vitro and induce autophagy in both human and murine cells. DIRAS1 and DIRAS2 are required for murine autophagy induced by rapamycin or amino acid starvation. Overexpression of DIRAS1 and DIRAS2 inhibits cancer cell growth and motility, and results in the inhibition of both the PI3K and Ras/MAPK signaling pathways. Thus DIRAS1 and DIRAS2 provide many of the functions of DIRAS3 in normal and malignant murine cells. Citation Format: Margie N. Sutton, Zhen Lu, Hailing Yang, Gilbert Huang, Yan Wang, Weiqun Mao, Robert C. Bast. DIRAS1 and DIRAS2 are novel ovarian cancer tumor suppressors that regulate cell growth, motility and autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3659.

Ovarian carcinomas account for 4% of total cancers in women in the United States. Improved outcomes might be attained if sensitivity to primary chemotherapy were enhanced. A recent study discovered that the salt inducible kinase 2 (SIK2) plays a key role in the initiation of mitosis and regulates paclitaxel sensitivity. Here we show that SIK2 is overexpressed in 30% ovarian cancer specimens, associated with a poor prognosis. ARN-3236, a selective, highly potent small molecule SIK2 inhibitor with function similar to SIK2 siRNA, blocks cell proliferation in a panel of ovarian cancer cell lines, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression. ARN-3236 also enhanced response to paclitaxel in ovarian cancer cells (OC316, SKOv3, A2780, HEY, ES2 and UPN251). Similar to the function of SIK2 siRNA, ARN-3236 effectively inhibits AKT phosphorylation at Ser473 and Tyr308, as well as the expression of the downstream effector survivin. ARN-3236 uncouples the centrosome from the nucleus in interphase, and blocks centrosome separation in mitosis, resulting in the accumulation of cells in prometaphase. In addition, ARN-3236 enhances tumor growth inhibition of paclitaxel in ovarian cancer xenograft models. Thus, functional inhibition of SIK2 with ARN-3236 had the same effects on ovarian cancer cell function as knockdown of SIK2 with siRNA. ARN-3236 deserves further study as a SIK2 inhibitor that significantly improves the sensitivity to paclitaxel for treatment of human ovarian cancer cells and xenografts. Citation Format: Jinhua Zhou, Albandri Alfredi, Shu Zhang, Janice M. Santiago-O’Farrill, Ahmed A. Ahmed, Hailing Yang, Weiqun Mao, Yan Wang, Hariprasad Vankayalapati, Zhen Lu, Robert C. Bast Jr. A novel compound ARN-3236 inhibits SIK2 and sensitizes ovarian cancer to paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3032.

Ovarian cancer is the most lethal gynecologic malignancy. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at “second look” operations. Previously, we reported that more than 80% of persistent, dormant drug resistant ovarian cancers express ARHI and are undergoing autophagy. Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Our current experiments were designed to determine whether survival of autophagic cells in vivo requires permissive levels of VEGF, IL-8 and IGF-1 in the tumor microenvironment. In cell culture, co-incubation of VEGF, IGF-1 and IL-8 with SKOv3-ARHI cells, increased AKT activity, reduced ARHI-mediated autophagy formation and decreased autophagic cell death. SKOv3-ARHI cells grown subcutaneously in nude mice exhibited higher levels of AKT activity and lower levels of autophagy than cells grown in cell culture. When cytoplasmic and nuclear fractions were obtained from xenografts, the nuclear fractions showed increased phospho-AKT, and decreased levels of a transcription factor EB (TFEB) and low mRNA expression of LC3, suggesting survival factors in tumor environment may stimulate AKT, downregulate expression of TFEB and inhibit formation of autophagy. Treatment with anti-VEGF, anti-IL-8 and anti-IGF-1 antibodies, individually and in combination, inhibited survival factors-mediated AKT activation and slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability to induce autophagy and to inhibit AKT. These preclinical studies support that the possibility that treatment with a combination of anti-VEGF, anti-IL8 and anti-IGF-1 antibodies could prevent outgrowth of dormant cells in patients with ovarian cancer. Furthermore, these results suggest that survival factors are required to limit autophagic death. Citation Format: Zhen Lu, Aaron Orozco, Weiqun Mao, Yan Wang, Margie Nicole Sutton, Hailing Yang, Douglas A. Levine, Robert C. Bast Jr.. IL-8, VEGF and IGF-1 play the important role in ARHI mediated-tumor dormancy in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1151.

Among gynecological cancers, ovarian cancer has the worst prognosis. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at second look operations. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers and in cancers from several other sites. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin1to form the autophagy initiating complex, inducing ATG4, decorating the autophagosome membrane and co-localizing with LC3II. Re-expression of ARHI in ovarian cancer cells in culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced once again in vivo, xenografts grow promptly to kill the host. Outgrowth of dormant xenografts is markedly delayed, but not prevented by treatment with chloroquine, a functional inhibitor of autophagy. The clinical relevance of this dormancy model is supported by previous studies that found ARHI expression in ovarian cancer cells was significantly increased in 90% of cases where small, dormant nodules of drug resistant ovarian cancer removed from the peritoneal surface at second-look operations, but in only 40% of the primary specimens from the same patients from initial cytoreductive surgery. In this study, we have measured Ki67 and microvascular density (MVD) in the same set of primary human ovarian cancers and in matched positive second look specimens. Second look specimens exhibited significantly lower Ki-67 (p This abstract is also presented as Poster A36. Citation Format: Zhen Lu, Aaron F. Orozco, Hailing Yang, Margie N. Sutton, Yan Wang, Weiqun Mao, Douglas A. Levine, Robert C. Bast, Jr., Robert C. Bast, Jr.. Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR18.

Anutosh Ganguly, Hailing Yang, and Fernando Cabral 1 Department of Integrative Biology and Pharmacology, University of Texas Medical School, 6431 Fannin St., Houston, Texas 77030, USA Keywords: tubulin isotypes; microtubules; dynamic instability; motility; drug resistance Received: March 30, 2011; Accepted: May 3, 2011; Published: May 16, 2011; Correspondence: Fernando Cabral, e-mail: // // Abstract Class III β-tubulin (β3) is associated with tumor aggressiveness, resistance to therapy, and patient relapse. To elucidate its action, we tested β3’s effect on cell migration. Expression of β3 in HeLa and MCF-7 did not alter the intrinsic rate of cell migration, but it prevented the inhibition of migration by low, nontoxic concentrations of paclitaxel. The effects on cell motility were confirmed in CHO cells with tetracycline regulated expression of β3. Cell migration and microtubule dynamics were inhibited by similar concentrations of paclitaxel, but required a 5-10 fold higher drug concentration when β3 was expressed. The directionality of migration was normal in paclitaxel, but cells spent more time in a “paused” state during which there was no net movement. These studies support a model in which paclitaxel inhibits cell migration by suppressing microtubule dynamics and β3-tubulin counteracts paclitaxel action by maintaining microtubule dynamic activity. The results provide a potential explanation for the aggressiveness of β3-expressing tumors.

Ovarian cancers exhibit a distinctive pattern of metastasis over the surface of the peritoneum. The ability of tumor cells to migrate and invade is a prerequisite for metastasis and cancer progression. Imprinted tumor suppressor genes are of particular interest in that only a single allele is expressed and function can be lost in a single event. Here we report that an imprinted tumor suppressor gene, NDN, can suppress ovarian cancer cell migration and inhibit cell motility. NDN is a maternally imprinted gene consistently expressed in normal ovarian epithelium, but dramatically downregulated in the majority of ovarian cancers. Little or no NDN expression could be detected by immunohistochemistry in 73% of 351 epithelial ovarian cancers. NDN was also downregulated in 10 ovarian cancer cell lines with total loss in 6 of 10. Re-expression of NDN in SKOv3ip cells significantly inhibited cell migration and invasion by wound healing and Boyden Chamber assays. NDN re-expression disrupted actin stress fiber formation and prevented focal adhesion kinase (FAK) and paxillin assembly at the focal adhesion site by fluorescence microscopy. NDN re-expression reduced FAKY397 autophosphorylation by 50% and reduced SrcY416 phosphorylation by 25%. NDN re-expression also blocked RhoA signaling pathway by decreasing levels of active GTP-RhoA. Our results indicate that NDN deactivates FAK-RhoA signaling pathway in ovarian cancer cells, leading to disruption in stress fiber and focal adhesion formation, and thus suppresses cancer cell motility. Citation Format: Hailing Yang, Partha Das, Weiqun Mao, Rebecca T. Marquez, Zhen Lu, Robert C. Bast. The imprinted tumor suppressor gene NDN inhibits cell motility in ovarian cancer by reactivating FAK and RhoA. [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 5105. doi:10.1158/1538-7445.AM2015-5105

Glioblastoma (GBM) tumors secrete high level of angiogenic factors and feature vascular abnormality. Targeted therapy, such as Bevacizumab, which neutralizes VEGF and normalizes tumor vasculature, has been approved to treat recurrent GBM. However, it only marginally increases patients' overall survival with inevitable resistance. It suggests that therapy targeting complementary angiogenic pathway in GBM could suppress Bevacizumab resistance and extend patients' survival. Angiogenin (ANG) is involved in tumor angiogenesis where it regulates ribosomal RNA transcription and functions downstream of VEGF and other related angiogenic factors. Like VEGF, ANG is also up-regulated in GBM patients. Inhibition of ANG by neutralizing monoclonal antibody delays U87 human GBM xenografts on nude mice. My preliminary data have shown that ANG is essential for GBM cell line s.c. vessel formation and tumor proliferation. In Rcas/tva orthotropic GBM model, Ang1 knockout mice show lower penetration and better survivals. ANG knockdown increases apoptosis during GBM cell line colony formation in agarose. Notably, ANG's role in tumor proliferation might extend beyond GBM, since oncogenic KRas transformed Ang1 knockout MEFs also fail to grow in agarose. Lastly, I have shown that PlexinB2, ANG cellular receptor, can mediate ANG cellular uptake and also modulate GBM apoptosis in agarose. In conclusion, inhibition of ANG inhibits GBM progression both in vitro and in vivo and thus represents a new molecular target for GBM treatment. Citation Format: Hailing Yang, Guo-fu Hu. Angiogenin and PlexinB2 promote glioblastoma progression by stimulating tumor angiogenesis, cancer cell survival and invasion. [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 3934. doi:10.1158/1538-7445.AM2014-3934

NDN is a maternally imprinted gene on chromosome 15q11.2 that is consistently expressed in normal ovarian epithelium, but dramatically downregulated in the majority of ovarian cancer cell lines and surgical specimens, determined by gene expression array and RT-PCR analysis. On tissue microarrays, little or no NDN protein could be detected by immunohistochemical analysis in73% of 351 epithelial ovarian cancers. Loss of NDN expression could be attributed, in part, to LOH in 28% of 18 informative cases and to hypermethylation of CpG sites 1 and 2 in the NDN promoter region in 21% and 27% of 43 ovarian cancers, respectively. All hypermethylated cases were associated with NDN down-regulation. Promoter hypermethylation and decreased NDN expression were also found in 7 of 10 ovarian cancer cell lines. Treatment with the demethylating agent decitabine increased NDN expression in 4 of 7 hypermethylated cell lines. Treatment with the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased NDN expression in 2 cell lines with normal or reduced methylation. However, when the two drugs were combined, they didn't show additive or synergistic effect. Re-expression of NDN in ovarian cancer cell lines such as SKOv3 and HEY significantly inhibited cell growth, associated with apoptosis, but not autophagy or necrosis. Re-expression of NDN also inhibited cell migration. These observations support the hypothesis that NDN is an imprinted tumor suppressor gene and its loss of function can be caused by multiple mechanisms in ovarian cancer. Citation Format: Hailing Yang, Partha Das, Yinhua Yu, Keith Baggerly, Ying Wang, Weiqun Mao, Rebecca T. Marquez, Zhen Lu, Jinsong Liu, Robert C. Bast. NDN, an imprinted tumor suppressor gene inhibits ovarian cancer cell growth and motility and is downregulated by genetic and epigenetic mechanisms. [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 2311. doi:10.1158/1538-7445.AM2014-2311

Virtually all patients with newly diagnosed ovarian cancer are treated with a combination of paclitaxel and carboplatin. While 70% of ovarian cancers respond to carboplatin, more than half do not respond to the paclitaxel. Inhibition of kinases that modulate primary resistance to paclitaxel, could enhance response to therapy. To identify kinases that regulate sensitivity to paclitaxel, high throughput screening with a kinome wide siRNA library was performed in SKOv3 ovarian cancer cells. Among 45 hits, 14 target proteins (AATK, ACRBP, BMP2K, CHUK, EDN2, IKBKB, ILK, RAPGEF3, RAPGEF4, RFP, SIK2, STK24, STK39 and TBK1) regulated sensitivity to paclitaxel and were expressed or overexpressed in a fraction of ovarian cancers. siRNAs against each of these kinases were tested for the ability to enhance sensitivity to paclitaxel in each of 12 ovarian cancer cell lines that reflected the heterogeneity observed in ovarian cancers including mutations of TP53, BRCA1/2, KRAS, BRAF, PI3K and PTEN. Knockdown of 7 individual genes reduced the IC50 of paclitaxel in more than half of the cell lines by 25 to 80 percent. Knockdown of STK39 and NFKB pathway kinases IKBKB and TBK1 enhanced microtubule stability judged by microtubule fractionation assay. Other kinases are known to affect centrosome function (SIK2, ACRBP). Our studies have identified several proteins that regulate primary sensitivity to paclitaxel in ovarian cancer cells and that could serve as targets to enhance response to primary therapy. Citation Format: Hailing Yang, Xiaoyan Wang, Ahmed A. Ahmed, Zhen Lu, Robert C. Bast. Knockdown of kinases that regulate microtubule stability and centrosome function enhance paclitaxel sensitivity in multiple ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 953. doi:10.1158/1538-7445.AM2013-953

NDN is a monoallelically expressed, maternally imprinted gene located on chromosome 15q11.2. Deletion and uniparental disomy of the NDN gene is causally associated with the Prader-Willi syndrome, a congenital neuro-developmental disorder. Studies of brain development suggest that NDN functions as a suppressor of neuronal growth. Recent studies have shown that NDN gene expression is downregulated in melanoma and urothelial cancers, indicating that it may also act as a tumor suppressor gene. We have now found that NDN gene expression is also downregulated in 50% of 14 ovarian tumors and 9 out of 10 ovarian cancer cell lines. Hypermethylation of 2 CpG sites in NDN promoter was detected in 5 out of 10 cell lines by pyrosequencing, and 5-aza-2′-deoxycytidine treatment revived NDN expression at different levels, suggesting that DNA hypermethylation is one of the mechanisms of NDN downregulation. Re-expression of the NDN gene product Necdin in SKOv3 ovarian cancer cells significantly inhibited growth by clonogenic assays associated with upregulation of caspase-3 activity. Expression of Necdin did not induce autophagy. As SKOv3 cells are p53 null, the mechanism(s) by which Necdin induces cell death is independent of p53 activity. Our observations that Necdin expression is downregulated in majority of ovarian cancers and cancer cell lines and that re-expression of Necdin inhibited cancer cell growth support the notion that NDN is a tumor suppressor gene. 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 2182. doi:1538-7445.AM2012-2182

Paclitaxel has been reported to inhibit cell division by suppressing microtubule dynamics at low concentrations that do not alter microtubule polymer levels. Although our studies confirm that paclitaxel suppresses microtubule dynamics at low concentrations, we show that suppression of dynamics is insufficient to inhibit cell proliferation. Wild-type CHO cells exhibited a dose-dependent decrease in microtubule dynamicity that reached a minimum at concentrations that had no observable effect on the ability of the cells to divide. Moreover, paclitaxel-dependent mutant cells that assemble reduced amounts of microtubule polymer and require paclitaxel for proliferation, exhibited a similar dose response for suppression of dynamics but much higher drug concentrations were needed to restore cell division. Thus, maximal suppression of microtubule dynamics neither inhibited the proliferation of wild-type cells, nor rescued cell division in paclitaxel-dependent cells. Instead, the higher concentrations of drug needed to restore mutant cell division caused the disappearance of microtubule fragments commonly seen in these cell lines. Using live cell imaging, we found that microtubule fragments are generated by detachment from microtubule organizing centers, and that paclitaxel acts to prevent this detachment, thereby raising microtubule polymer levels. We conclude that low paclitaxel concentrations act at microtubule plus ends to inhibit dynamics, but that higher concentrations are needed to inhibit cell division and prevent microtubule detachment from organizing centers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-53.

Vertebrate cells express 6-7 isotypes of both α and β-tubulin, all of which coassemble into microtubules. Our previous studies demonstrated that β3 and β5 (but not β1, β2, and β4b) tubulin overexpression destabilizes microtubules, inhibits cell growth, and confers paclitaxel resistance (Blade, K., et al. (1999). J Cell Sci 112: 2213-21; Bhattacharya, R. and F. Cabral (2004). Mol Biol Cell 15: 3123-31; Hari, M., et al. (2003). Cell Motil Cytoskeleton 56: 45-56.). Overexpression of β3 does not appear to significantly alter microtubule dynamics, but dynamic behavior is relatively resistant to suppression by paclitaxel (Kamath, K., et al. (2005). J Biol Chem 280: 12902-7.). We now report the effects of β5 overexpression on microtubule dynamics. Microtubules were visualized in wild type (WT) or β5 overexpressing CHO cells using eGPF-MAP4. Growth and shrinkage of individual microtubules were measured using ImageJ and dynamic parameters were calculated by analyzing life history plots. We found that microtubules with high β5 content had significantly increased shortening rates and very long depolymerization distances compared to wild type. Addition of paclitaxel at a concentration that rescues cell growth, completely reversed these effects; but the dynamic parameters in such microtubules were significantly different compared to WT microtubules and resembled the inhibited microtubule dynamics of WT cells treated with paclitaxel. We conclude that suppression of microtubule dynamics in presence of paclitaxel is insufficient to explain why β5 overexpressing cells become resistant to the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2489.