Susan B. Ingersoll

The role of CD40 in CD40L- and antibody-mediated platelet activation

Our initial finding that CD40- and CD40 ligand (CD40L)-deficient mice displayed prolonged tail bleeding and platelet function analyzer (PFA-100) closure times prompted us to further investigate the role of the CD40-CD40L dyad in primary hemostasis and platelet function. Recombinant human soluble CD40L (rhsCD40L), chemical cross-linking of which suggested a trimeric structure of the protein in solution, activated platelets in a CD40-dependent manner as evidenced by increased CD62P expression. CD40 monoclonal antibody (mAb) M3, which completely blocked rhsCD40L-induced platelet activation, also prolonged PFA-100 closure times of normal human blood. In contrast, CD40 mAb G28-5 showed less potential in blocking rhsCD40L-induced CD62P expression and did not affect PFA-100 closure times. However, when added to the platelets after rhsCD40L, G28-5 significantly enhanced the platelet response by causing clustering of, and signaling through, FcgammaRII. Similarly, higher order multimeric immune complexes formed at a 1/3 molar ratio of M90, a CD40L mAb, to rhsCD40L induced strong Fcgamma RII-mediated platelet activation when translocated to the platelet surface in a CD40-dependent manner, including the induction of morphological shape changes, fibrinogen binding, platelet aggregation, dense granule release, microparticle generation and monocyte-platelet-conjugate formation. The results suggest that CD40 may play a role in primary hemostasis and platelet biology by two independent mechanisms: First, by functioning as a primary signaling receptor for CD40L and, second, by serving as a docking molecule for CD40L immune complexes. The latter would also provide a potential mechanistic explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in recent human and animal studies.

Hyperactive EGF receptor, Jaks and Stat3 signaling promote enhanced colony-forming ability, motility and migration of cisplatin-resistant ovarian cancer cells

We present evidence that the cisplatin-resistant human ovarian cancer lines, A2780S/CP1 (S/CP1), A2780S/CP3 (S/CP3) and A2780S/CP5 (S/CP5), derived by subjecting the sensitive A2780S ovarian cancer line to multiple rounds of cisplatin treatments followed by recovery and are resistant to 1, 3 and 5 μM cisplatin, respectively, have increased colony-forming ability and altered morphology that is consistent with enhanced motility, migration and invasiveness in vitro. The malignant phenotype progresses with increasing resistance and is associated with hyperactive epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (Erk)1/2 and janus kinases (Jaks), aberrant signal transducer and activator of transcription (Stat) 3 activation promoted by EGFR and Jaks, and epithelial-mesenchymal transition (EMT) in vitro. Survivin and FLIP anti-apoptotic factors, vascular endothelial growth factor (VEGF) and matrix metalloproteinase activities are also elevated in the resistant cells. Accordingly, the ectopic expression of constitutively-active Stat3C in the sensitive A2780S cells diminished cisplatin sensitivity. The inhibition of EGFR or Stat3 activity repressed Survivin, VEGF and Vimentin expression and the colony-forming potential, viability, motility and migration of the resistant cells, and sensitized them to cisplatin. Analysis of human ovarian cancer patients’ tumor tissues shows aberrantly-active EGFR and Stat3 that in certain cases correlate with Vimentin over-expression. Intra-peritoneal mouse xenograft studies revealed, compared with the sensitive A2780S line that had low tumor incidence restricted to the ovary, a high tumor incidence for the resistant S/CP3 and S/CP5 lines that formed tumor nodules at several locations on the small intestine and colon, and which responded poorly to cisplatin, but were sensitive to concurrent treatment with cisplatin and EGFR or Stat3 inhibitor. Hyperactive EGFR signaling through Stat3 and the Jak-Stat3 activity together promote ovarian cancer progression to cisplatin resistance and therefore represent targets for preventing the development of cisplatin resistance and the recurrent disease during cisplatin therapy in ovarian cancer.

Experimental metastasis and primary tumor growth in mice with hemophilia A

Summary.  During experimental lung metastasis, tumor cells adhere to the pulmonary microvasculature and activate coagulation via surface‐expressed tissue factor (TF), leading to local fibrin deposition and platelet aggregation. While interventional studies have demonstrated great efficacy of anticoagulants and antiplatelet agents in inhibiting metastasis, no information is available on how tumor biology may be affected by congenital bleeding disorders such as hemophilia A. We therefore used a syngeneic model to study experimental metastasis and primary tumor growth in factor VIII (FVIII)‐deficient mice. By conventional reverse transcription‐polymerase chain reaction, flow cytometry, and one‐stage clotting assays, we demonstrated constitutive expression of TF mRNA, antigen, and procoagulant activity in the murine B16F10 melanoma cell line. In hemophilic mice, B16F10 lung metastasis was significantly (P < 0.001) enhanced by a single dose of human FVIII (100 U kg−1), suggesting that FVIII played a critical role during the early blood‐borne phase of the metastatic cascade. In contrast, lung seeding was significantly (P < 0.05) reduced by lepirudin, a direct thrombin inhibitor, suggesting that thrombin generation contributed to pulmonary metastasis even in the absence of FVIII. Consistent with this finding, intravenous injection of B16F10 cell‐evoked laboratory changes of a hemolytic thrombotic microangiopathy and consumptive coagulopathy in both hemophilic and non‐hemophilic mice. Subcutaneous implantation of B16F10 cells into mice with hemophilia A gave rise to primary tumors in an exponential growth pattern similar to that observed in non‐hemophilic mice. Although TF expression by B16F10 cells may promote thrombin‐dependent metastasis in mice with hemophilia A, amplification of coagulation by host FVIII appears to be necessary for maximum lung seeding.

Tolfenamic acid inhibits ovarian cancer cell growth and decreases the expression of c-Met and survivin through suppressing specificity protein transcription factors

OBJECTIVE The aberrant expression of hepatocyte growth factor and its receptor c-Met are associated with aggressive disease and poor prognosis in a variety of human malignancies including ovarian cancer (OC). Specificity protein (Sp) transcription factors have high relevance in the signaling cascade associated with c-Met activation. Tolfenamic acid (TA), a NSAID, is known to induce the degradation of Sp proteins, which have been negatively associated with survival in some cancer patients. Our aim was to examine the anti-OC activity of TA using in vitro and in vivo models and asses the inhibitory effects of this novel compound. METHODS We developed OC sub-cell lines (AF1-3) derived from the original SKOV3 that are more resistant to IFNα-2b and more tumorigenic in nude mice than the original cells. We tested the anti-cancer activity of TA using ovarian cancer cells, SKOV3-AF2 and ES-2. The cells were treated with DMSO (vehicle) or TA (25/50/100 μM) and cell viability was measured at 24, 48, and 72 h. Cell lysates were prepared following 48 h treatment (50μM) and evaluated the expression of Sp proteins (Sp1/Sp3/Sp4), c-Met, survivin, Bcl2, and cleaved polyADP-ribose polymerase (c-PARP) through Western blot analysis. Caspase-3 activity was assessed with Caspase-Glo kit. Cell cycle distribution and apoptosis were analyzed using BD FACSCalibur flow cytometer. For in vivo studies mice were subcutaneously injected with ES-2 cells and treated with vehicle or TA (50 mg/kg/thrice weekly). RESULTS TA significantly inhibited the growth of SKOV3 (AF1-3) and ES-2 cells. The expression of Sp proteins and c-Met was significantly decreased suggesting that TA could be targeting c-Met through degradation of Sp proteins. TA greatly increased the apoptotic fraction (Annexin V positive), c-PARP expression and caspase-3 activity. TA significantly decreased Bcl2 expression and induced G0/G1 cell cycle arrest. In vivo studies revealed that TA significantly inhibited tumor weight and volume in mice. These results show that TA has a profound inhibitory effect on tumor growth in mice, reduces OC cells proliferation, induces apoptosis, cell cycle arrest and Sp proteins degradation. TA also inhabited the expression of survivin that is associated with radiation resistance and suggests that apart from its tumor suppressant effects, TA can also enhance the tumor response to radiotherapy. CONCLUSIONS These data clearly show that TA effectively inhibits OC cell growth in vitro and in vivo. This study represents potential role(s) of TA that suppresses OC cell growth, and may enhance tumor response to radiotherapy, and have implications in OC treatment.

Cellular Therapy for Ovarian Cancer: Experimental and Clinical Perspectives

Ovarian cancer is the leading cause of death among gynecologic malignancies and the 5th leading cause of cancer deaths for women in the United States. Two-thirds of patients present with advanced-stage disease (Stage III and IV) and the majority will suffer recurrence of disease, require ongoing treatment, and eventually succumb to chemotherapy-resistant disease. To potentially circumvent chemo-resistance in recurrent ovarian cancer, immunotherapy is being explored as a novel treatment option. Our laboratory findings demonstrate that immune effector cells from healthy donors elicit a significant cytotoxic response in the presence of IL-2 and IFN alpha- 2b against ovarian cancer in vitro; however, peripheral blood mononuclear cells (PBMC) isolated from ovarian cancer patients fail to elicit a similar response. A major obstacle to immunotherapy is the immunosuppressive environment supported by tumors, which limits the immune systems ability to fight the tumor. Myeloid-derived suppressor cells are an immature population of myeloid cells, which have recently been implicated to play a major role in immunosuppression and tumor evasion. In addition to novel immunotherapies, new diagnostic and prognostic markers are being identified through applying molecular tools/approaches in clinical and pathological analyses of this malignancy, which will provide additional therapeutic targets. To test these experimental therapeutic options, pre-clinical murine models of ovarian cancer are being developed. Ultimately, treatment of ovarian cancer will benefit from the careful alignment of appropriate target, drug, patient, and trial design. This article provides an objective overview of cellular therapy (the use of immune cells to elicit an anti-tumor response) for ovarian cancer highlighting both experimental and clinical perspectives.

Abstract 2159: Expression of Sp1 and survivin in ovarian cancer specimens: potential novel therapeutic targets in disease treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer (OC) is one of the most common female cancers and is the leading cause of death from gynecologic malignancies. Although, cisplatin is the front-line chemotherapeutic option for OC, its success is compromised due to dose-limiting toxicity and acquired resistance by tumor cells. Specificity protein 1 (Sp1) is a transcription factor that is over-expressed in several cancers and is inversely associated with survival. Survivin, a member of IAP family, is known to cause resistance to chemo- and radiation therapy. Studies from our laboratories have shown that the NSAID, tolfenamic acid (TA), targets Sp1 protein and inhibits expression of survivin in various cancer models. Our primary objective was to evaluate the expression of Sp1 and survivin in OC patients’ specimens and target these candidates using TA for enhancing the cisplatin response. Expression of Sp1 and survivin in clinical specimens was determined by qPCR and Western blots. qPCR showed increased expression of survivin (∼5-fold) and Sp1 (∼2-fold) in tumor samples. Western blot also revealed over-expression of both Sp1 (>2.6-fold) and survivin (>100-fold). OC cell lines (ES2, OVCAR-3) were used to determine the anti-proliferative response to cisplatin and TA. TA and cisplatin showed a dose- and time-dependent inhibition of cell viability in OC cell lines [TA (50 ÂμM) caused 50% (ES2) and 40% (OVCAR-3) growth inhibition and cisplatin (5 ÂμM) caused 60% and 40% inhibition at 48 h post-treatment]. Combination treatment using optimized doses of TA (50 ÂμM) and cisplatin (5 ÂμM) resulted in a synergistic response and caused stronger inhibition (ES2: ∼80%, OVCAR-3: >60) compared to single-agent. Increased inhibition of proliferation by the combination of TA and cisplatin was accompanied by cell-cycle arrest, predominantly in the G2/M phase. A significant increase in apoptosis, as determined by Caspase 3/7 activity, annexin-V staining, and PARP cleavage, was also observed in the combination treatment. Cell invasion and migration was assessed using matrigel coated transwell chambers. Compared to TA or cisplatin treatment alone, their combination significantly inhibited ES2 cell invasion. Analysis of ES2 cells by global proteomic profiling indicated that the combination treatment upregulated proteins associated with oxidative phosphorylation, apoptosis, and electron transport chain; and down-regulated cytoplasmic ribosomal proteins, translational factors, and proteins involved in DNA damage response, and cell cycle. In conclusion, elevated expression of Sp1 and survivin confirmed their association in OC and demonstrated the relevance of targeting these candidates, which may render the OC cells more sensitive to chemotherapy and offer therapeutic potential. Further studies to delineate underlying mechanism(s) of action possibly involving signaling pathways and reactive oxygen species are currently underway. Citation Format: Umesh T. Sankpal, Susan B. Ingersoll, Mohammed I. Shukoor, Chris M. Lee, Nicole M. Stavitzski, Vadiraja B. Bhat, Sarfraz Ahmad, Robert W. Holloway, Liz Abraham, Riyaz Basha. Expression of Sp1 and survivin in ovarian cancer specimens: potential novel therapeutic targets in disease treatment. [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 2159. doi:10.1158/1538-7445.AM2013-2159

Targeting the IL-6 Pathway in Multiple Myeloma and its Implications in Cancer-Associated Gene Hypermethylation

Aberrant methylation of tumor suppressor genes (TSG) is an important epigenetic event in cancer, including multiple myeloma (MM). Interleukin-6 (IL-6), which plays a significant role in the pathogenesis of MM, also regulates DNA methylation. However, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of hypermethylation may be an important pathway leading to rational chemotherapeutic/anti-IL-6 combinations. We first studied the correlation of IL-6 expression and dependence in MM cell lines: U266B1, RPMI8226, and KAS6/1. We confirmed that KAS6/1 is IL-6-dependent whereas U266B1 and RPMI8226 cells are IL-6-independent and that blocking IL-6 inhibited the growth of U266B1 (36% inhibition; p<0.05) and KAS6/1 (68% inhibition; p<0.01), but not the RPMI8226 cells. Using RT-PCR, we showed that U266B1 cells express IL-6, but RPMI8226 and KAS6/1 cells do not. This IL-6 expression pattern correlates with the anti-IL-6 inhibition findings. To correlate IL-6 sensitivity with hypermethylation of TSG, we investigated promoter methylation of CDH1 and DcR1. We found that the promoter of DcR1 and CDH1 is methylated in U266B1 cells and un-methylated in RPMI8226 cells. Furthermore, the DcR1 promoter was un-methylated in KAS6/1 cells. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of TSG in MM. Newer chemotherapeutic agents that affect methylation are being studied in combination with IL-6 blockade.

Deficiencies in the CD40 and CD154 receptor-ligand system reduce experimental lung metastasis

It is established that experimental metastasis requires platelet activity. CD154 expressed on and released from activated platelets induces an inflammatory response in endothelial cells and monocytes, including tissue factor production. CD154 has also been shown to activate platelets in vitro and promote thrombus stability in vivo. These CD154 effects may be mediated, at least in part, by CD40 signaling on platelets and vascular endothelial cells. We have previously demonstrated prolonged bleeding and PFA-100 closure times in mice deficient for Cd154 or its receptor Cd40. In the present study, we hypothesized that Cd40 and Cd154 promote lung tumor formation in experimental metastasis in mice. We created mice doubly deficient in Cd40 and Cd154 (Dbl KO) and found them to be both fertile and viable. Injected tumor cells seeded poorly in mice deficient in Cd40 or Cd154, as well as Dbl KO, compared to wild-type mice. We sought to determine whether blood-borne Cd40 versus endothelial Cd40 contribute differentially to reduced experimental lung metastasis, as observed in Cd40 deficient mice. By bone marrow transplantation, we created mice deficient for Cd40 either in the blood compartment but not in the endothelium, or vice versa. We found that mice deficient in blood compartment Cd40 had fewer lung nodules compared to wild-type mice and mice deficient in endothelial Cd40. Our findings suggest an important contribution of the Cd40-Cd154 pathway to experimental lung metastasis. Furthermore, the data points to a selective role for peripheral blood cell Cd40 in this process.

Abstract 1354: Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Objectives: Specificity protein 1 (Sp1) is a transcription factor that is over-expressed in several cancers and is inversely associated with survival. Survivin is known to cause resistance to chemo- and radiation therapy. Herein, we evaluated the correlation of Sp1 and survivin expression in epithelial ovarian cancer (EOC) patients and tested a pre-clinical strategy to target these candidates using tolfenamic acid (TA) for enhancing the efficacy of a chemotherapeutic agent, Cisplatin (Cis). Methods: Expression of Sp1 and survivin in clinical specimens was determined by qPCR and Western blot analysis. Anti-proliferative response of EOC cell lines (ES2 and OVCAR-3) was assessed in the presence of Cis, TA or Cis+TA using CellTiter-Glo. Apoptosis was determined by flow cytometry using annexin-V staining, measuring the effector caspases activity using caspase3/7 Glo kit. Cell cycle analysis was carried out using flow cytometry. Matrigel coated ranswell chambers were used to assess cell migration/invasion. Proteomics nalysis was performed at Agilent Technologies, Inc. Results: qPCR showed an increase in the expression of urvivin (∼5-fold) and Sp1 (∼2-fold) in clinical specimens, which was corroborated by Western blot analysis [Sp1 (>2.6-fold) and survivin >100-fold)]. TA and Cis showed a dose- and time-dependent inhibition of cell viability in EOC cell lines [50 µM TA caused 50% (ES2) and 40% (OVCAR-3) cell growth inhibition; Cis (5 µM) caused 60% (ES2) and 40% (OVCAR-3) inhibition at 48 h post-treatment]. Combination of TA (50 µM) and Cis (5 µM) resulted in a synergistic response causing higher growth inhibition (ES2: ∼80%, p<0.001; OVCAR-3: 60%, p<0.001) compared to single-agent. This is accompanied by cell-cycle arrest (G2/M phase), an increase in apoptosis, as determined by Caspase 3/7 activity, annexin-V staining, PARP cleavage, and inhibition of ES2 cell invasion and migration. Global proteomic profiling of ES2 cells indicated that the combination treatment up-regulated proteins associated with oxidative phosphorylation, apoptotic execution, and electron transport chain and down-regulated ribosomal proteins, translational actors, DNA damage and cell cycle proteins. Conclusions: Elevated expression of Sp1 and survivin suggests a strong association of these two agents in EOC. Our pre-clinical results confirmed that targeting these candidates with small molecule (TA) potentially sensitizes EOC cells to chemotherapy. Citation Format: Umesh T. Sankpal, Susan B. Ingersoll, Vadiraja B. Bhat, Sarfraz Ahmad, Robert W. Holloway, Riyaz M. Basha. Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets. [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 1354. doi:10.1158/1538-7445.AM2014-1354

Abstract LB-346: EGF receptor, Jaks and Stat3 crosstalk promote enhanced colony-forming, motility, migration, and invasive phenotype of cisplatin-resistant ovarian cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Majority of the ovarian cancer patients who initially respond to cisplatin therapy develop recurrent and resistant disease that is mechanistically not well understood. To define the biological presentations and the molecular phenotype of cisplatin resistant ovarian cancer, we subjected the sensitive A2780S ovarian cancer line to multiple rounds of cisplatin treatments, followed by recovery and derived the lines, A2780S/CP1 (S/CP1), A2780S/CP3 (S/CP3), and A2780S/CP5 (S/CP5), which are resistant to 1, 3, and 5 μM cisplatin, respectively. In vitro studies show the three resistant lines have increased colony-forming ability and altered morphology consistent with enhanced motility, migration, and invasiveness. The malignant phenotype progresses with increasing resistance and is associated with hyperactive epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 and Janus kinases (Jaks), and aberrant Signal Transducer and Activator of Transcription (Stat) 3 activation. Elevated expression of survivin and vascular endothelial growth factor (VEGF), and enhanced matrix metalloproteinase activities are evident in the resistant cells, together with increased vimentin and snail expression, and E-cadherin downregulation, indicative of epithelial-mesenchymal transition (EMT). The inhibition of EGFR or Stat3 activity in the resistant cells repressed the enhanced colony-forming, motility, and migratory phenotype, downregulated survivin, VEGF and vimentin expression, and sensitized the resistant cells to cisplatin. In vivo studies using intra-peritoneal xenograft models in nude mice revealed the resistant lines have a high tumor incidence and formed small, cisplatin-resistant tumor nodules at several locations on the small-intestine and colon, compared to the sensitive A2780S line that exhibited low tumor incidence and formed exclusively ovarian tumors. Treatment with EGFR or Stat3 inhibitor sensitized the resistant tumor nodules to cisplatin. Studies together indicate hyperactive EGFR and Jak signaling predominantly through the induction of aberrantly-active Stat3 promote EMT during cisplatin resistance development. Collectively, these events serve as critical mediators of the invasive phenotype associated with cisplatin resistant ovarian cancer and hence, are targets for preventing the development of resistance and the recurrent disease during cisplatin therapy in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-346. doi:1538-7445.AM2012-LB-346