Chintda Santiskulvong

Dual Targeting of Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Using NVP-BEZ235 as a Novel Therapeutic Approach in Human Ovarian Carcinoma

Purpose: This study evaluates the effect of dual PI3K and mTOR inhibition using NVP-BEZ235 in preclinical models of ovarian cancer as a potential novel therapeutic strategy. Experimental Design: Inhibition of PI3K/Akt/mTOR signaling by NVP-BEZ235 was demonstrated by immunoblotting. The effect on cell proliferation was assessed in 18 ovarian cancer cell lines, including four pairs of syngeneic cisplatin-sensitive and cisplatin-resistant cell lines. The in vivo effects of NVP-BEZ235 on established tumor growth were evaluated using an immunocompetent, transgenic murine ovarian cancer model (LSL-K-rasG12D/+PtenloxP/loxP). Results: NVP-BEZ235 decreased cell proliferation in all ovarian cancer cell lines assayed and sensitized cisplatin-resistant cells to the cytotoxic effects of cisplatin. Cell lines with PI3K-activating mutations or Pten deletions were significantly more sensitive to the effect of NVP-BEZ235 than cell lines without these mutations (P < 0.05). A statistically significant correlation was found between relative levels of p4E-BP1 and the IC50 for NVP-BEZ235. In LSL-K-rasG12D/+PtenloxP/loxP mice with established intraperitoneal tumor disease, oral administration of NVP-BEZ235 decreased pAkt, p4E-BP1 and Ki67 in tumor tissue, and resulted in significantly longer survival compared to control animals (P < 0.05). NVP-BEZ235 also induced cell cycle arrest, caspase 3 activity, and reduced cell migration. Conclusions: Targeting PI3K and mTOR simultaneously using NVP-BEZ235 effectively inhibits ovarian cancer cell growth even in the presence of platinum resistance and prolongs survival of mice with intra-abdominal ovarian tumor disease. We propose that dual PI3K and mTOR inhibition using NVP-BEZ235 may be an effective novel therapeutic approach in patients with ovarian cancer. Clin Cancer Res; 17(8); 2373–84. ©2011 AACR.

Galardin (GM 6001), a broad-spectrum matrix metalloproteinase inhibitor, blocks bombesin- and LPA-induced egf receptor transactivation and DNA synthesis in Rat-1 cells

Matrix metalloproteinases (MMPs) have been implicated in the transactivation of the epidermal growth factor receptor (EGFR) induced by G-protein coupled receptor (GPCR) agonists. Although EGFR phosphorylation and downstream signaling have been shown to be dependent on MMP activity in many systems, a role for MMPs in GPCR-induced DNA synthesis has not been studied in any detail. In this study we utilized the broad-spectrum matrix metalloproteinase inhibitor, galardin (Ilomastat, GM 6001), to study the mechanism of bombesin- or LPA-induced EGFR transactivation and the role of MMPs in early and late response mitogenic signaling in Rat-1 cells stably transfected with the bombesin/GRP receptor (BoR-15 cells). Addition of galardin to cells stimulated with bombesin or LPA specifically inhibited total EGFR phosphorylation, as well as site-specific phosphorylation of tyrosine 845, a putative Src phosphorylation site, and tyrosine 1068, a typical autophosphorylation site. Galardin treatment also inhibited extracellular signal-regulated kinase (ERK) activation induced by bombesin or LPA, but not by EGF. In addition, galardin inhibited bombesin- or LPA-induced DNA synthesis in a dose dependent manner, when stimulated by increasing concentrations of bombesin, and when added after bombesin stimulation. Furthermore, addition of galardin post-bombesin stimulation indicated that by 3 h sufficient accumulation of EGFR ligands had occurred to continue to induce transactivation despite an inhibition of MMP activity. Taken together, our results suggest that MMPs act as early as 5 min, and up to around 3 h, to mediate GPCR-induced EGFR transactivation, ERK activation, and stimulation of DNA synthesis.

Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells

UNLABELLED The exact molecular mechanisms of ovarian cancer platinum resistance are not well understood, and biomarkers to reliably predict ovarian cancer resistance to platinum and other chemotherapeutic agents are lacking. Biomechanics of cisplatin-treated ovarian cancer cells were measured quantitatively at nanoscale level using atomic force microscopy. We demonstrate that cisplatin modulates the cellular nanomechanics of ovarian cancer cells; sensitive cells show dose-dependent increase in cell stiffness, which is effected by disrupting the F-actin polymerization. In contrast, resistant cells show no significant changes in cell stiffness upon cisplatin treatment. Further, stimulated emission depletion, an emerging super-resolution microscopy, shows that at the molecular level, F-actin is indeed remodeled considerably in cisplatin-sensitive and cisplatin-resistant cells. These findings reveal a direct role of the actin remodeling mechanism in cisplatin resistance of ovarian cancer cells, suggesting potential future applications of nanomechanical profiling as a marker for cancer drug sensitivity. FROM THE CLINICAL EDITOR In this paper, nanomechanical profiling and an emerging super-resolution microscopy method was utilized to decipher the mechanisms of cisplatin resistance in ovarian cancer cells, paving the way to future studies of this and similar other problems with drug resistance in cancer biology.

p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet‐derived growth factor‐BB (PDGF‐BB) through p38 mitogen‐activated protein (MAP) kinase and extracellular signal‐regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF‐BB stimulated membrane ruffling, migration, and proliferation. PDGF‐BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF‐BB‐stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF‐BB‐induced proliferation. Selective antagonism of ERK kinase inhibited PDGF‐BB‐induced ERK phosphorylation and proliferation, but did not affect PDGF‐BB‐stimulated migration. Concentrations of PDGF‐BB that stimulated migration and proliferation did not influence myosin‐dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF‐BB‐induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF‐BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF‐BB‐stimulated migration, but not proliferation, ERKs mediate PDGF‐induced proliferation, but not migration, and (3) increases in myosin‐dependent contractility are not required for PDGF‐BB‐stimulated migration.

Cisplatin and PI3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix‐metalloproteinase expression

Targeting of the PI3K (phosphoinositide3‐kinase)/Akt/mTOR pathway in human ovarian cancer cells is a promising novel therapeutic strategy. We investigated the effects of cisplatin and the PI3K inhibitor LY294002 on invasion, migration and the expression of essential matrix metalloproteinases (MMPs) in ovarian cancer cells. SKOV3, OVCAR5 and IGROV1 human ovarian cancer cell lines were treated with cisplatin, LY294002 and a combination of both drugs. Invasion and migration of treated cells was assessed using Matrigel and uncoated PET membrane assays. Expression levels of pro‐MMP2, MMP2, TIMP1, TIMP2 and MT1‐MMP were determined using Western Blotting. Gel zymography was used to quantitate the functional levels of active MMP2. All three cell lines showed significantly reduced invasion and migration after treatment with cisplatin, LY294002, and the combination of both drugs compared to untreated controls. In SKOV3 cells, cisplatin alone and in combination with LY294002 resulted in a 6.3 and 7.1‐fold reduction in the total amount of activated MMP2. TIMP1 expression decreased by 5.0, 6.6 and 28.4‐fold with cisplatin, LY294002 and the combination respectively (P < 0.05). In contrast, only cisplatin and the combination of both drugs resulted in a significant, 3.7 and 5.1‐fold reduction in the level of TIMP2. Expression levels of MT1‐MMP remained unchanged. These observations were corroborated in IGROV1 cell lines that showed similar changes of activated MMP2 and TIMP2 expression, but no significant decrease in TIMP1 levels. Our data suggests that inhibition of ovarian cancer cell motility is mediated via down‐regulation of activated MMP2, TIMP1 and TIMP2 expression under these treatment conditions.

Rho and p38 MAP Kinase Signaling Pathways Mediate LPA-Stimulated Hepatic Myofibroblast Migration

Although hepatic myofibroblast migration plays a key role in the livers injury response, the signal transduction pathways mediating the migration of this cell type are uncertain. Recently, we reported that lysophosphatidic acid (LPA) stimulates the migration of hepatic myofibroblasts. The goal of this study was to test the hypothesis that rho and p38 MAP kinase signaling pathways mediate LPA-stimulated hepatic myofibroblast migration. We measured migration, myosin regulatory light chain and p38 MAP kinase phosphorylation, and contractile force generation by human hepatic myofibroblasts. LPA stimulated migration in a dose-dependent and saturable manner that was partially blocked by Y-27632, a rho-associated kinase inhibitor, as well as by SB-202190, a p38 MAP kinase inhibitor. LPA also induced myosin regulatory light chain phosphorylation and contractile force generation in a Y-27632 dependent, and SB-202190 independent fashion. Moreover, LPA stimulated a dose-dependent and saturable phosphorylation of p38 MAP kinase, which was not altered by Y-27632 or C3 transferase, a rho inactivator. These novel results suggest that LPA stimulates hepatic myofibroblast migration via distinct pathways that signal through rho and p38 MAP kinase.

[d-Arg1,d-Trp5,7,9,Leu11]Substance P Inhibits Bombesin-induced Mitogenic Signal Transduction Mediated by Both Gq and G12 in Swiss 3T3 Cells

Substance P (SP) analogues including [d-Arg1,d-Trp5,7,9,Leu11]SP are broad spectrum neuropeptide antagonists and potential anticancer agents, but their mechanism of action is not fully understood. Here, we examined the mechanism of action of [d-Arg1,d-Trp5,7,9,Leu11]SP as an inhibitor of G protein-coupled receptor (GPCR)-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells. Addition of [d-Arg1,d-Trp5,7,9,Leu11]SP, at 10 μm, caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, or vasopressin and markedly inhibited the activation of p42mapk (ERK-2) and p44mapk (ERK-1) induced by these GPCR agonists. In addition, this SP analogue also prevented the protein kinase C-dependent activation of protein kinase D induced by these agonists. [d-Arg1,d-Trp5,7,9,Leu11]SP, at a concentration (10 μm) that inhibited these Gq-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G12subfamily. These include bombesin-induced assembly of focal adhesions, formation of parallel arrays of actin stress fibers, increase in the tyrosine phosphorylation of focal adhesion kinase (FAK), p130Cas, and paxillin, and formation of a complex between FAK and Src. We conclude that [d-Arg1,d-Trp5,7,9,Leu11]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both Gq and G12.

Screening cell mechanotype by parallel microfiltration

Cell mechanical phenotype or ‘mechanotype’ is emerging as a valuable label-free biomarker. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and cancer progression. Here we describe a simple and scalable technique to measure cell mechanotype: this parallel microfiltration assay enables multiple samples to be simultaneously measured by driving cell suspensions through porous membranes. To validate the method, we compare the filtration of untransformed and HRasV12-transformed murine ovary cells and find significantly increased deformability of the transformed cells. Inducing epithelial-to-mesenchymal transition (EMT) in human ovarian cancer cells by overexpression of key transcription factors (Snail, Slug, Zeb1) or by acquiring drug resistance produces a similar increase in deformability. Mechanistically, we show that EMT-mediated changes in epithelial (loss of E-Cadherin) and mesenchymal markers (vimentin induction) correlate with altered mechanotype. Our results demonstrate a method to screen cell mechanotype that has potential for broader clinical application.

Abstract 226: Parallel microfiltration (PMF): A novel method to screen cell mechanotype

The mechanical phenotype of cells (mechanotype) is emerging as a valuable label-free biomarker. Cells are visco-elastic materials whose mechanotype is altered in diseases from malaria to cancer. For example, marked changes in the viscoelastic characteristics of cells occur during malignant transformation and in response to chemotherapy treatment. Malignant cells across different types of cancers are consistently softer than benign cells, exhibiting a Young9s modulus that is ∼60-70% lower both in vitro and in situ. Cell mechanotype also grades metastatic potential: highly invasive human ovarian carcinoma cells are five-fold more deformable (softer) than less invasive cells. Further, chemotherapeutic drugs can alter mechanotype: human leukemia cells exhibit a nearly two orders of magnitude increase in cell elastic modulus after being treated with dexamethasone and daunorubicin. These published studies suggest the potential of cell mechanotype as a biomarker in cancer prognosis and for identifying effective drug treatments. However, in order to exploit cell mechanical properties for fundamental research and clinical applications a simple and scalable method to measure cell mechanotype is required. Here we describe a parallel microfiltration (PMF) assay, which enables the mechanotype of multiple samples to be simultaneously measured by driving cell suspensions through porous membranes using uniform air pressure. The relative deformability of a cell sample is quantified by the fraction of sample retained above the porous membrane. Based on experimental results and theoretical considerations, we have developed a model that provides a physical explanation of PMF and allows us to relate our experimental data to cell deformability. We employed PMF to study changes in mechanotype in ovarian carcinoma cells. We show that epithelial-to-mesenchymal transition (EMT), a process associated with cancer progression and drug resistance, yields softer cells: OVCA433 cells engineered to overexpress key EMT inducers (Snail, Slug, or Zeb) had much lower retention rates than their epithelial precursors. Similarly, cisplatin-resistant ovarian cancer cell lines (OVCAR5-cisR, SKOV3-cisR) exhibiting features of EMT, such as loss of E-cadherin expression and induction of vimentin, were more deformable than cisplatin-sensitive controls. We also compared normal p53-/- murine ovarian surface epithelial (MOSE) cells with HRasV12-transformed MOSE cells, which lose E-cadherin. PMF confirmed dramatically lower retention rates for the transformed cells relative to normal precursors. We conclude that EMT results in increased cell deformability and that PMF is a robust tool to identify changes in deformability. In summary, we have developed a novel method to assess cell mechanotype that is compatible with high-throughput screening approaches and can complement and enhance existing technology for evaluating cancer cell phenotype and response to drugs. Citation Format: Wolf-Ruprecht Wiedemeyer, Dongping Qi, Navjot Kaur Gill, Chintda Santiskulvong, Oliver Dorigo, JianYu Rao, Barbie Taylor-Harding, Amy C. Rowat. Parallel microfiltration (PMF): A novel method to screen cell mechanotype. [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 226. doi:10.1158/1538-7445.AM2015-226

Abstract 1543: Tumor associated macrophages are associated with poor survival in epithelial ovarian cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background Tumor associated macrophages have been associated with poor outcomes in several solid tumors, but their impact on the outcome of patients with epithelial ovarian cancer has not been well established. Methods We constructed a tissue microarray of 142 epithelial ovarian cancer specimens. Immunohistochemistry was performed for the presence of CD68+ macrophages. The distribution of macrophages was evaluated separately in tumor and stromal tissue. Clinical characteristics were recorded and presence or absence of intratumoral macrophages was correlated with clinical characteristics including survival. Results Median follow-up for the entire cohort was 32.0 months (range, 1.7-170 months), and median progression-free survival (PFS) and overall survival (OS) were 14.1 months and 47.8 months, respectively. Intratumoral macrophages (ITMs) were present in 132/142 patients (91.5%) and absent in 12/142 (8.5%). Patients whose tumor tissue did not contain ITMs had a significantly longer median PFS (not reached, p = 0.009) and median OS (150.1 months, p=0.03), compared with the median PFS and OS of 11.7 and 47.7 months, respectively, among patients with ITMs. Interestingly, we could not demonstrate differences in PFS and OS when stromal macrophages were evaluated separately. The presence of ITMs was an independent prognostic factor for OS in a multivariate analysis (p=0.009). The Cox proportional hazard ratios for presence of ITMs were 3.55 for PFS (95% CI, 1.38-12.13) and 3.62 for OS (95% CI, 1.33-13.08). Conclusions Our study shows that the presence of intratumoral but not stromal macrophages correlates with poor prognosis in epithelial ovarian carcinoma. Citation Format: Wafic El Masri, Caroline Hillerup, Charlotte Topka, Chintda Santiskulvong, Alexander Chiang, Lily Wu, Jianyu Rao, Oliver Dorigo. Tumor associated macrophages are associated with poor survival in epithelial ovarian cancer. [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 1543. doi:10.1158/1538-7445.AM2013-1543