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Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors.Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer.Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

Supplementary Data

PDF file, 104KB, Effects of MET and MTD HA-TXL therapy on tumor nodules in ovarian cancer models.

Supplementary Figures S1-S5.

PDF file - 2689K, Common differentially expressed genes in both OVCA432 and SKOV3 cells transfected with the FGFR4-specific siRNAs Hs_FGFR4_5 and Hs_FGFR4_6

Supplementary Data from Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

The supporting information file contains figures with experimental data such as tandem mass spectrometry mass spectra, 2D ion images, representative mass spectra of cancer and normal tissues, and mass spectra of standard compounds. Tables are also included which present identification of molecular markers, and predictive accuracy results for the molecular models.

Supplementary Figures S1-S5; Supplementary Table S1.

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.

This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therapy regimen of metronomic topotecan and pazopanib, a multireceptor tyrosine kinase inhibitor. In vitro (Western blot) and in vivo dose-finding experiments were done following pazopanib therapy in ovarian cancer models. Pazopanib and metronomic (daily) oral topotecan therapy was examined in an orthotopic model of ovarian cancer. Tumor weights, survival, and markers of the tumor microenvironment [angiogenesis (CD31 and pericyte coverage), proliferation (Ki-67), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling)] were analyzed by immunostaining following therapy. Pazopanib therapy reduced vascular endothelial growth factor receptor 2 (VEGFR-2) activity in vitro and vivo in a dose-dependent manner. Compared with control mice, pazopanib reduced tumor weight by 28% to 82% (P < 0.01 in the SKOV3ip1 model) and metronomic topotecan reduced tumor weight by 40% to 59% in the HeyA8 (P = 0.13) and SKOV3ip1 (P = 0.07) models. Combination therapy had the greatest effect with 79% to 84% reduction (P < 0.01 for both models). In the SKOV3ip1 and A2780 models, mouse survival was significantly longer (P < 0.001 versus controls) with pazopanib and metronomic topotecan therapy. Pazopanib therapy reduced murine endothelial cell migration in vitro in a dose-dependent manner following VEGF stimulation and decreased tumor microvessel density and pericyte coverage when given in combination with metronomic topotecan. Tumor cell proliferation decreased in all treatment arms compared with controls (P < 0.01 for combination groups) and increased tumor cell apoptosis by 4-fold with combination therapy. Pazopanib therapy in combination with metronomic topotecan therapy showed significant antitumor and antiangiogenic properties in preclinical ovarian cancer models and warrants further investigation as a novel therapeutic regimen in clinical trials. Mol Cancer Ther; 9(4); 985–95. ©2010 AACR.

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P < 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor–initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%–90% reduction; P < 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. Mol Cancer Ther; 9(12); 3186–99. ©2010 AACR.

Supplementary Data from c-Jun-NH2-kinase-1 Inhibition Leads to Antitumor Activity in Ovarian Cancer

PDF file - 959KB, Supplementary Figure 1. PDGFRalpha expression and activation in patients with uterine cancer. Supplementary Figure 2. In vivo therapeutic and biological effects of PDGFRalpha blockade (3G3) on uterine cancer. Supplementary 3. Network analysis of angiogenesis factors down-regulated by 3G3 treatment in PDGFRalpha-positive cells and PDGFRalpha-negative cells.

Supplementary Data from Therapeutic Targeting of ATP7B in Ovarian Carcinoma

Suppl Methods and Figure Legend

Clinical correlation of miR-155 expression with survival in leukemia. Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in two CLL cohorts, CLL - NEJM (A) and CLL - Italy (B), and in one ALL cohort, ALL - MDACC (C).

Purpose: EphA2 overexpression is frequently observed in endometrial cancers and is predictive of poor clinical outcome. Here, we use an antibody drug conjugate (MEDI-547) composed of a fully human monoclonal antibody against both human and murine EphA2 (1C1) and the tubulin polymerization inhibitor monomethylauristatin F.Experimental Design: EphA2 expression was examined in endometrial cancer cell lines by Western blot. Specificity of MEDI-547 was examined by antibody degradation and internalization assays. Viability and apoptosis were investigated in endometrial cancer cell lines and orthotopic tumor models.Results: EphA2 was expressed in the Hec-1A and Ishikawa cells but was absent in the SPEC-2 cells. Antibody degradation and internalization assays showed that the antibody drug conjugate decreased EphA2 protein levels and was internalized in EphA2-positive cells (Hec-1A and Ishikawa). Moreover, in vitro cytotoxicity and apoptosis assays showed that the antibody drug conjugate decreased viability and increased apoptosis of Hec-1A and Ishikawa cells. In vivo therapy experiments in mouse orthotopic models with this antibody drug conjugate resulted in 86% to 88% growth inhibition (P < 0.001) in the orthotopic Hec-1A and Ishikawa models compared with controls. Moreover, the mice treated with this antibody drug conjugate had a lower incidence of distant metastasis compared with controls. The antitumor effects of the therapy were related to decreased proliferation and increased apoptosis of tumor and associated endothelial cells.Conclusions: The preclinical data for endometrial cancer treatment using MEDI-547 show substantial antitumor activity. Clin Cancer Res; 16(9); 2562–70. ©2010 AACR.

Supplementary Figures 1-6. Supplementary Figure S1: In vivo and in vitro effects of ZA on apoptosis. Supplementary Figure S2: Nab-paclitaxel inhibits tube formation.Supplementary Figure S3: RPPA analysis in HeyA8 MDR treated with ZA. Supplementary Figure S4: Functional changes induced by ZA in HeyA8 MDR. Supplementary Figure S5: ZA decreases angiogenic factors. Supplementary Figure S6: ZA inhibits cell invasion

Supplementary Figure 1 from Targeting Aldehyde Dehydrogenase Cancer Stem Cells in Ovarian Cancer

Purpose: Endoglin (CD105) is a membranous protein overexpressed in tumor-associated endothelial cells, chemoresistant populations of ovarian cancer cells, and potentially stem cells. Our objective was to evaluate the effects and mechanisms of targeting endoglin in ovarian cancer.Experimental Design: Global and membranous endoglin expression was evaluated in multiple ovarian cancer lines. In vitro, the effects of siRNA-mediated endoglin knockdown with and without chemotherapy were evaluated by MTT assay, cell-cycle analysis, alkaline comet assay, γ-H2AX foci formation, and quantitative PCR. In an orthotopic mouse model, endoglin was targeted with chitosan-encapsulated siRNA with and without carboplatin.Results: Endoglin expression was surprisingly predominantly cytoplasmic, with a small population of surface-positive cells. Endoglin inhibition decreased cell viability, increased apoptosis, induced double-stranded DNA damage, and increased cisplatin sensitivity. Targeting endoglin downregulates expression of numerous DNA repair genes, including BARD1, H2AFX, NBN, NTHL1, and SIRT1. BARD1 was also associated with platinum resistance, and was induced by platinum exposure. In vivo, antiendoglin treatment decreased tumor weight in both ES2 and HeyA8MDR models when compared with control (35%–41% reduction, P < 0.05). Endoglin inhibition with carboplatin was associated with even greater inhibitory effect when compared with control (58%–62% reduction, P < 0.001).Conclusions: Endoglin downregulation promotes apoptosis, induces significant DNA damage through modulation of numerous DNA repair genes, and improves platinum sensitivity both in vivo and in vitro. Antiendoglin therapy would allow dual treatment of both tumor angiogenesis and a subset of aggressive tumor cells expressing endoglin and is being actively pursued as therapy in ovarian cancer. Clin Cancer Res; 19(1); 170–82. ©2012 AACR.

Supplementary Methods includes experimental details on western blotting, invasion, wound healing, tube formation, EdU cell proliferation assays, quantitativce RT-PCR, luciferase reporter assay, gene expression analysis and Ingenuity Pathway Analysis.

In vivo orthotopic lung cancer model for the role of miR-155 in chemoresistance. (A) Injection and treatment schedule for CDDP (green arrows) and anti-miR negative control (NC) or anti-miR-155 liposomal nanoparticles (red stars) for four different treatment groups: mice that were injected with A549-LVEV cells and untreated (group 1), injected with A549-LVEV cells and treated with anti-miR-NC and CDDP (group 2), injected with A549-155LV cells and treated with anti-miR-NC and CDDP (group 3), and injected with A549-155LV cells and treated with anti-miR-155 and CDDP (group 4). (B) Representative pictures of dissected mice belonging to each of the treatment groups described in panel A of this Figure. Tumor nodules are marked by dotted white circles. (C-D) Graphs of the primary tumor size (C) and aggregate mass of nodules in mediastinum (D) of the four treatment groups mentioned above. (E) In situ hybridization for miR-155 for each of the four treatment groups mentioned above. (F) Immunohistochemical analysis for Ki-67 (proliferation) and CD31 (angiogenesis), as well as the TUNEL assay (apoptosis) and TP53 immunostaining for each of the four treatment groups mentioned above.

Purpose: The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells.Experimental Designs: miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion.Results: miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression.Conclusions: Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC. Clin Cancer Res; 16(4); 1119–28

PDF file 106K, Table S1. Molecules whose expression significantly differed between cells treated with KX-01 and control cells in RMUG-S cells Table S2. Molecules whose expression significantly differed between cells treated with KX-01 and control cells in RMUG-L cells

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling–tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan–Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial–mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1–RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway–based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697–709. ©2018 AACR.

Although hypoxia has been shown to reprogram cancer cells toward glycolytic shift, the identity of extrinsic stimuli that induce metabolic reprogramming independent of hypoxia, especially in ovarian cancer, is largely unknown. In this study, we use patient-derived ovarian cancer cells and high-grade serous ovarian cancer cell lines to demonstrate that lysophosphatidic acid (LPA), a lipid growth factor and GPCR ligand whose levels are substantially increased in ovarian cancer patients, triggers glycolytic shift in ovarian cancer cells. Inhibition of the G protein α-subunit Gαi2 disrupted LPA-stimulated aerobic glycolysis. LPA stimulated a pseudohypoxic response via Rac-mediated activation of NADPH oxidase and generation of reactive oxygen species, resulting in activation of HIF1α. HIF1α in turn induced expression of glucose transporter-1 and the glycolytic enzyme hexokinase-2 (HKII). Treatment of mice bearing ovarian cancer xenografts with an HKII inhibitor, 3-bromopyruvate, attenuated tumor growth and conferred a concomitant survival advantage. These studies reveal a critical role for LPA in metabolic reprogramming of ovarian cancer cells and identify this node as a promising therapeutic target in ovarian cancer.Significance: These findings establish LPA as a potential therapeutic target in ovarian cancer, revealing its role in the activation of HIF1α-mediated metabolic reprogramming in this disease. Cancer Res; 78(8); 1923–34. ©2018 AACR.

CARS imaging of ex vivo ovarian cancer samples

Purpose: To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma.Experimental Design: The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models.Results:In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2–M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01–sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules.Conclusion: Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. Clin Cancer Res; 19(23); 6532–43. ©2013 AACR.

PDF file, 67KB, Effects of HA-TXL MTD and HA-TXL MET on white blood count (WBC)and hemoglobin levels.

Purpose: Nuclear factor κB (NFκB) activity may increase survival and protect cancer cells from chemotherapy. Therefore, NFκB activity may be prognostic, and inhibition of NFκB may be useful for pancreatic cancer therapy. To test these hypotheses, we examined NFκB activity and the effects of inhibiting NFκB in several pancreatic cancer cell lines with differing sensitivities to gemcitabine.Experimental Design: The gemcitabine sensitivity of pancreatic cancer cell lines BxPC-3, L3.6pl, CFPAC-1, MPanc-96, PANC-1, and MIA PaCa-2 were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and fluorescence-activated cell sorting assays. NFκB levels were determined by electrophoretic mobility shift assay and reporter assays. The effects of gemcitabine on NFκB activity were determined in vitro and in vivo. NFκB was inhibited by silencing of the p65/relA subunit using small interfering RNA in vitro and by neutral liposomal delivery of small interfering RNA in vivo, and the effects were evaluated on gemcitabine sensitivity.Results: The cell lines L3.6pl, BxPC-3, and CFPAC-1 were sensitive, whereas MPanc-96, PANC-1, and MIA PaCa-2 were resistant to gemcitabine. No significant correlation was observed between basal NFκB activity and gemcitabine sensitivity. Gemcitabine treatment did not activate NFκB either in vitro or in vivo. Silencing of p65/relA induced apoptosis and increased gemcitabine killing of all gemcitabine-sensitive pancreatic cancer cells. No significant effects, however, were observed on gemcitabine-resistant pancreatic cancer cell lines either in vitro or in vivo.Conclusions: NFκB activity did not correlate with sensitivity to gemcitabine. Silencing of p65/relA was effective alone and in combination with gemcitabine in gemcitabine-sensitive but not gemcitabine-resistant pancreatic cancer cells. Thus, NFκB may be a useful therapeutic target for a subset of pancreatic cancers.

Fig. S1. Sustained adrenergic signaling increases nerve counts in tumors. Fig. S2. Characterization of tumoral innervation. Fig. S3. NE induces BDNF expression. Fig. S4. NE-induced BDNF expression is mediated by ADRB3/Epac/Jnk. Fig. S5. BDNF increases nerve counts. Fig. S6. Adrenergic-mediated mTrkB activation leads to increased in vivo tumor nodule counts. Table S1. Alteration in pathways associated with neuronal growth and function after NE treatment (HeyA8 and SKOV3ip1 cells). Table S2. Association of Clinicopathologic variables with BDNF protein expression. Table S3. Association of Clinicopathologic variables with Nerve Counts.

Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Normalized expression of DANCR (KIAA0114) in MYC-OFF or MYC-ON P493-6 cells measured by exon microarrays (PLoS One 6, e26057). Three independent array experiments were performed. Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Control and DANCR siRNAs were transduced into PC3 cells. After 48 and 72 hours, the mRNA expression levels of the DANCR adjacent protein-coding genes (ERVMER34-1 and UPS46) were analyzed by qRT-PCR. Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression. A. RT-PCR analysis of DANCR expression in P493-6 after being treated by various concentrations of the two DANCR antisense morpholinos. B. Effects of two different morpholinos targeting DANCR on proliferation of P493-6 cells as compared with control morpholino. C. Induction of P21 by loss of DANCR expression in P493-6 cells treated by two concentrations of the two DANCR antisense morpholinos.

Purpose: VEGF-targeted therapies have modest efficacy in cancer patients, but acquired resistance is common. The mechanisms underlying such resistance are poorly understood.Experimental Design: To evaluate the potential role of immune cells in the development of resistance to VEGF blockade, we first established a preclinical model of adaptive resistance to anti-VEGF therapy. Additional in vitro and in vivo studies were carried out to characterize the role of macrophages in such resistance.Results: Using murine cancer models of adaptive resistance to anti-VEGF antibody (AVA), we found a previously unrecognized role of macrophages in such resistance. Macrophages were actively recruited to the tumor microenvironment and were responsible for the emergence of AVA resistance. Depletion of macrophages following emergence of resistance halted tumor growth and prolonged survival of tumor-bearing mice. In a macrophage-deficient mouse model, resistance to AVA failed to develop, but could be induced by injection of macrophages. Downregulation of macrophage VEGFR-1 and VEGFR-3 expression accompanied upregulation of alternative angiogenic pathways, facilitating escape from anti-VEGF therapy.Conclusions: These findings provide a new understanding of the mechanisms underlying the modest efficacy of current antiangiogenesis therapies and identify new opportunities for combination approaches for ovarian and other cancers. Clin Cancer Res; 23(22); 7034–46. ©2017 AACR.

Table S1. MYC-regulated genes in P493-6 cell line Table S2. MYC-regulated lncRNAs in P493-6 cell line Table S3. MYC-regulated lncRNAs in CCLE Table S4. GO analysis of genes that affected by DANCR knockdown in PC3 cells Table S5. Top 500 up-regulated and down-regulated genes by knockdown of DANCR in PC3 cells. Figure S1. The expression of DANCR is upregulated by MYC activation in P493-6 cells Figure S2. Human-mouse sequence alignment analysis of DANCR and mDancr Figure S3. The mRNA expression of the DANCR adjacent protein-coding genes in cells transduced with DANCR-specific siRNA Figure S4. Effects of two DANCR targeting antisense morpholinos on P493-6 cell proliferation and p21 expression.

Supplemental Methods. The supplementary methods section describes in greater detail how platelets were isolated from whole mouse blood, how the in vitro apoptosis assay was performed, how cleaved caspace 3 immunohistochemistry was performed, how platelet transfusion was performed, and how aspirinization of platelets prior to transfusion was performed. The supplementary data includes a table detailing the demographic characteristics of the patients included in the clinical component of the work as well as the details of platelet change over the course of primary therapy.

PDF file - 3119K, Effect of FGFR4 overexpression and exogenous FGF1 and on ovarian cancer cell proliferation

Supplementary Video 1. 3-D SKOV3ip1 spheroids expressing pGFP-RFP-LC3 were treated with control IgG

Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth.Experimental Design: Expression of dopamine receptors (DR1–DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP).Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely blocked with daily dopamine treatment. Dopamine treatment also blocked the stress-induced increase in angiogenesis. Endothelial and ovarian cancer cells expressed all dopamine receptors except for the lack of DR3 expression in ovarian cancer cells. DR2 was responsible for the inhibitory effects of dopamine on tumor growth and microvessel density as well as the stimulatory effect on apoptosis, as the DR2 antagonist eticlopride reversed these effects. Dopamine significantly inhibited cell viability and stimulated apoptosis in vitro. Moreover, dopamine reduced cyclic AMP levels and inhibited norepinephrine and vascular permeability factor/VEGF-induced Src kinase activation.Conclusions: Dopamine depletion under chronic stress conditions creates a permissive microenvironment for tumor growth that can be reversed by dopamine replacement. Clin Cancer Res; 17(11); 3649–59. ©2011 AACR.

Supplementary Data from Bridging the Gap between Cytotoxic and Biologic Therapy with Metronomic Topotecan and Pazopanib in Ovarian Cancer

Supplementary Figures S1-5, Tables S1-5. Figure S1 The Cytotoxic Effect of KPT-185 is Tumor Suppressor-independent. Figure S2 Combination Index of KPT-185 with Chemotherapeutic Agents. Figure S3 1D Gel Analysis of Cytoplasmic Protein. Figure S4 Effects of Selinexor (KPT-330) in an Ovarian Cancer Mouse Model. Figure S5 The Efficacy of Selinexor (KPT-330) on Tumor Regression and Metastasis Table S1 p53 status and IC50 concentration of KPT-185 after 72 hours of incubation in human cancer cell lines Table S2 Combination index (CI) of KPT-185 with cytotoxic agents Table S3 Proteins showing altered mitochondrial localization after treatment with KPT-185 Table S4 1D Proteomic analysis of cytoplasmic proteins immunoprecipitated with eIF5A Table S5 siRNA Sequences

Purpose: Chronic adrenergic activation has been shown to associate with adverse clinical outcomes in cancer patients, but the underlying mechanisms are not well understood. The focus of the current study was to determine the functional and biologic effects of adrenergic pathways on response to chemotherapy in the context of ovarian cancer.Experimental Design: Increased DUSP1 production by sympathetic nervous system mediators (e.g., norepinephrine) was analyzed by real-time quantitative RT-PCR and by Western blotting. In vitro chemotherapy-induced cell apoptosis was examined by flow cytometry. For in vivo therapy, a well-characterized model of chronic stress was used.Results: Catecholamines significantly inhibited paclitaxel- and cisplatin-induced apoptosis in ovarian cancer cells. Genomic analyses of cells treated with norepinephrine identified DUSP1 as a potential mediator. DUSP1 overexpression resulted in reduced paclitaxel-induced apoptosis in ovarian cancer cells compared with control; conversely, DUSP1 gene silencing resulted in increased apoptosis compared with control cells. DUSP1 gene silencing in vivo significantly enhanced response to paclitaxel and increased apoptosis. In vitro analyses indicated that norepinephrine-induced DUSP1 gene expression was mediated through ADRB2 activation of cAMP–PLC–PKC–CREB signaling, which inhibits JNK-mediated phosphorylation of c-Jun and protects ovarian cancer cells from apoptosis. Moreover, analysis of The Cancer Genome Atlas data showed that increased DUSP1 expression was associated with decreased overall (P = 0.049) and progression-free (P = 0.0005) survival.Conclusions: These findings provide a new understanding of the mechanisms by which adrenergic pathways can impair response to chemotherapy and have implications for cancer management. Clin Cancer Res; 22(7); 1713–24. ©2015 AACR.

S Figure 1 (A) C57BL/6 mice that received intra-peritoneal injections of IG10 cells were randomly assigned to one of two groups (control or B20 treatment). S Figure 2 (A) Bar graph represents the CD11b+/F4/80+ macrophages in tumor tissue by FACS analysis. S Figure 3 Anti-VEGF therapyââ,¬â€œresistant macrophages have heightened viability, migration, and invasion. S Figure 4 (A) Bar graph represents the percentage of viability of B20 treated macrophages. S Figure 5. VEGFR1-positive macrophages increase the effectiveness of anti-VEGF therapy.

CARS imaging of ex vivo ovarian cancer samples

Supplemental Table 1: Patient Demographics.

Table S1

Purpose: We hypothesized that platelet levels during therapy could serve as a biomarker for response to therapy and that manipulation of platelet levels could impact responsiveness to chemotherapy.Experimental Design: The medical records of patients with recurrent or progressive ovarian cancer were retrospectively queried for changes in platelet and CA-125 levels during primary therapy. In vitro coculture experiments and in vivo orthotopic models of human ovarian cancer in mice were used to test the effect of modulating platelet levels on tumor growth and responsiveness to docetaxel.Results: Thrombocytosis at the diagnosis of ovarian cancer was correlated with decreased interval to progression (P = 0.05) and median overall survival (P = 0.007). Mean platelet levels corrected during primary therapy and rose at recurrence. Contrary to treatment-responsive patients, in a cohort of patients refractory to primary therapy, platelet levels did not normalize during therapy. In A2780, HeyA8, and SKOV3-ip1 ovarian cancer cell lines, platelet coculture protected against apoptosis (P < 0.05). In orthotopic models of human ovarian cancer, platelet depletion resulted in 70% reduced mean tumor weight (P < 0.05). Compared with mice treated with docetaxel, mice treated with both docetaxel and platelet-depleting antibody had a 62% decrease in mean tumor weight (P = 0.04). Platelet transfusion increased mean aggregate tumor weight 2.4-fold (P < 0.05), blocked the effect of docetaxel on tumor growth (P = 0.55) and decreased tumor cell apoptosis. Pretransfusion aspirinization of the platelets blocked the growth-promoting effects of transfusion.Conclusions: Platelet-driven effects of chemotherapy response may explain clinical observations. Clin Cancer Res; 21(3); 602–10. ©2014 AACR.

Purpose: To study the role of survivin and its splice variants in taxane-resistant ovarian cancer.Experimental Design: We assessed the mRNA levels of survivin splice variants in ovarian cancer cell lines and ovarian tumor samples. siRNAs targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA) in vitro and orthotopic murine models of ovarian cancer. The mechanism of cell death was studied in taxane-resistant ovarian cancer cells and in tumor sections obtained from different mouse tumors.Results: Taxane-resistant ovarian cancer cells express higher survivin mRNA levels than their taxane-sensitive counterparts. Survivin 2B expression was significantly higher in taxane-resistant compared with -sensitive cells. Silencing survivin 2B induced growth inhibitory effects similar to silencing total survivin in vitro. In addition, survivin 2B-siRNA incorporated into DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) nanoliposomes resulted in significant reduction in tumor growth (P < 0.05) in orthotopic murine models of ovarian cancer, and these effects were similar to T-siRNA-DOPC. The antitumor effects were further enhanced in combination with docetaxel chemotherapy (P < 0.01). Finally, we found a significant association between survivin 2B expression and progression-free survival in 117 epithelial ovarian cancers obtained at primary debulking surgery.Conclusions: These data identify survivin 2B as an important target in ovarian cancer and provide a translational path forward for developing new therapies against this target. Clin Cancer Res; 17(11); 3716–26. ©2011 AACR.

PDF file - 38K

PDF file - 96K, Notch Pathway Alterations in Patients with HGS-OvCa.

Tissue type transglutaminase (TG2) is a unique multifunctional protein that plays a role in many steps in the cancer metastatic cascade. Here, we examined the clinical (n = 93 epithelial ovarian cancers) and biological (in vitro adhesion, invasion, and survival and in vivo therapeutic targeting) significance of TG2 in ovarian cancer. The overexpression of TG2 was associated with significantly worse overall patient survival in both univariate and multivariate analyses. Transfection of TG2 into SKOV3ip1 cells promoted attachment and spreading on fibronectin-coated surfaces and increased the in vitro invasive potential of these cells. Conversely, TG2 silencing with small interfering RNA (siRNA) of HeyA8 cells significantly decreased the invasive potential of the cells and also increased docetaxel-induced cell death. In vivo therapy experiments using chemotherapy-sensitive (HeyA8) and chemotherapy-resistant (HeyA8-MDR and RMG2) models showed significant antitumor activity both with TG2 siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone and in combination with docetaxel chemotherapy. This antitumor activity was related to decreased proliferation and angiogenesis and increased tumor cell apoptosis in vivo. Taken together, these findings indicate that TG2 overexpression is an adverse prognostic factor in ovarian carcinoma and TG2 targeting may be an attractive therapeutic approach. [Cancer Res 2008;68(14):5849–58]