Model Of Ovarian Cancer Metastasis Research Articles

Abstract 2922: Sensory innervation as a novel driver of ovarian cancer progression

Abstract One of the main challenges currently faced in ovarian cancer treatment is that the primary platinum/taxane therapy used to treat patients does not meaningfully extend survival. The overall survival for ovarian cancer patients with advanced disease is less than 30% with most patients recurring within 5 years of initial therapy. Once resistant disease develops there are few second line therapy options, which inevitably leads to disease progression. Thus, there is a critical need to identify new therapies that can overcome ovarian cancer chemoresistance and produce meaningful increases in patient survival. In recent years, new efforts have focused on targeting the tumor microenvironment (TME), the complex mixture of non-cancer cells that surround and support the cancer cells within the tumor. Different strategies have evolved to target the TME, such as immune checkpoint inhibitors or angiogenesis inhibitors. However, these TME targeting therapies have limited effect when used to treat ovarian cancer patients. Most ovarian tumors have a microenvironment that is highly immunosuppressive and do not respond or are resistant to angiogenesis inhibitors. Thus, new TME targeting therapies must be developed that can induce a therapeutic effect in ovarian tumors and can be used to treat most ovarian cancer patients. A novel TME component ripe for therapeutic targeting are the nerve fibers that infiltrate tumors in a process termed tumor innervation. Recent studies in several cancers have shown that tumor innervation can promote tumor growth/metastasis. However, in ovarian cancer the role of innervation in promoting cancer progression remains undefined. Here we show that Transient Receptor Potential cation channel subfamily V member 1 (TRPV1)+ sensory innervation plays a significant role in driving ovarian cancer growth and metastasis. Analysis of patient samples shows that sensory nerve fiber innervation is much higher in ovarian tumors vs benign reproductive tissue. In addition, ablation of TRPV1 sensory nerve fibers in vivo causes reduced tumor burden and prolongs survival in a syngeneic mouse model of ovarian cancer metastasis. Taken together, our results establish TRPV1+ sensory innervation as a novel driver of ovarian cancer growth/metastasis and a potential therapeutic target for ovarian cancer treatment. Citation Format: Matthew Knarr, Katherine Cummins, Dusan Racordon, Hunter Reavis, Timothy Lippert, Ryan Hausler, Priyanka Rawat, Suyeon Ryu, Jamie Moon, Dave Hoon, Roger Greenberg, Paola Vermeer, Ronny Drapkin. Sensory innervation as a novel driver of ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2922.

Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice

BackgroundIn a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a activating receptor...

Complementary anti-cancer pathways triggered by inhibition of sideroflexin 4 in ovarian cancer

DNA damaging agents are a mainstay of standard chemotherapy for ovarian cancer. Unfortunately, resistance to such DNA damaging agents frequently develops, often due to increased activity of DNA repair pathways. Sideroflexin 4 (SFXN4) is a little-studied inner mitochondrial membrane protein. Here we demonstrate that SFXN4 plays a role in synthesis of iron sulfur clusters (Fe-S) in ovarian cancer cells and ovarian cancer tumor-initiating cells, and that knockdown of SFXN4 inhibits Fe-S biogenesis in ovarian cancer cells. We demonstrate that this has two important consequences that may be useful in anti-cancer therapy. First, inhibition of Fe-S biogenesis triggers the accumulation of excess iron, leading to oxidative stress. Second, because enzymes critical to multiple DNA repair pathways require Fe-S clusters for their function, DNA repair enzymes and DNA repair itself are inhibited by reduction of SFXN4. Through this dual mechanism, SFXN4 inhibition heightens ovarian cancer cell sensitivity to DNA-damaging drugs and DNA repair inhibitors used in ovarian cancer therapy, such as cisplatin and PARP inhibitors. Sensitization is achieved even in drug resistant ovarian cancer cells. Further, knockout of SFXN4 decreases DNA repair and profoundly inhibits tumor growth in a mouse model of ovarian cancer metastasis. Collectively, these results suggest that SFXN4 may represent a new target in ovarian cancer therapy.

Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer.

Near-infrared dye-labeled antibody COC183B2 enables detection of tiny metastatic ovarian cancer and optimizes fluorescence-guided surgery.

We aimed to evaluate the ability of the fluorescent monoclonal antibody probe COC183B2-Cy7 (Cy7-conjugated COC183B2 antibody) to detect tiny metastatic lesions of ovarian cancer and thus guide precise tumor resection. The expression of the tumor-associated antigen OC183B2 in lymph nodes and SKOV3-Luc cells was detected using immunohistochemistry and immunofluorescence. A subcutaneous mouse tumor model and an intraperitoneal ovarian cancer metastasis model were constructed using SKOV3-Luc cells. Near-infrared fluorescence (NIRF) imaging was performed to determine the imaging parameters and evaluate the ability of COC183B2-Cy7 to detect tiny metastatic lesions. OC183B2 was expressed in metastatic lymph nodes and SKOV3-Luc cells. NIRF imaging of the subcutaneous mouse tumor model showed that the tumor background ratio was significantly higher in the COC183B2-Cy7 group than in the control group at different time points postinjection. Biodistribution study showed that COC183B2-Cy7 did not accumulate in other organs. COC183B2-Cy7 can detect tiny metastatic lesions of ovarian cancer. The smallest intraperitoneal metastatic tumor detected by COC183B2-Cy7 was approximately 1 mm. COC183B2-Cy7 probe has relatively high specificity and sensitivity. Our study suggests that COC183B2-Cy7 probe is a promising diagnostic tool for the complete and accurate resection of malignant lesions in fluorescence-guided surgery.

Abstract A41: ROR1 is associated with ovarian cancer progression and chemoresistance

Abstract Background: New targets for ovarian cancer treatment are critically needed. The Wnt receptors ROR1 and ROR2 are overexpressed in all subtypes of ovarian cancer and appear to play a role in both the tumor and surrounding microenvironment (1). In vitro studies support the approach of targeting these receptors together to inhibit ovarian cancer migration and invasion (2, 3). Aim: To investigate the role of ROR1 and ROR2 in ovarian cancer progression, survival and chemoresistance, to determine their feasibility as therapeutic targets. Methods: Analysis of RNA-Seq data from publicly available ovarian cancer datasets was performed to determine associations with overall survival and platinum resistance. ROR1 IHC using a recently released monoclonal antibody (4A5, BD Bioscience) was performed on a cohort of ovarian cancer patients. ROR1 and/or ROR2 were silenced in a unique preclinical organotypic model of platinum-resistant ovarian cancer metastasis. Results: There is a significant increase (p=0.01) in ROR1 expression in both primary resistant and acquired resistant (p=0.03) high-grade serous ovarian cancer (HGSOC). ROR2 expression is not significantly associated with chemoresistance. High ROR1 (P=0.002, HR 1.93) or high ROR2 (P=0.004, HR 1.9) expression is associated with a significantly shorter overall survival (OS) in the Tothill cohort (4). Patients expressing both high ROR1 and high ROR2 have the shortest OS (P=0.0003, HR 2.38). ROR1 protein expression was detected in 97% of HGSOC patients via IHC, with 59% of HGSOC expressing “high ROR1” (IHC score 2 or 3). Overall, patients with higher-grade tumors expressed significantly higher ROR1 levels (P=0.001) than patients with low-grade tumors. Silencing ROR1 and ROR2 in combination inhibits adhesion and invasion of platinum resistant ovarian cancer in a 3D organotypic coculture model. Conclusion: Our data support targeting both ROR1 and ROR2 as a new approach to treating ovarian cancer. The development of a number of monoclonal antibodies targeting ROR1 and ROR2 that are currently in phase 1 trials for other tumor types makes this clinically feasible in the future. These data also suggest that this may be most effective in the context of HGSOC with platinum resistance.

A new mouse model of ovarian cancer metastasis

A new mouse model of ovarian cancer metastasis

Abstract A12: The metabolic stress mediator LKB1 is required for ovarian cancer metastasis

Abstract Most epithelial ovarian cancer (EOC) patients with metastatic disease initially respond to cytotoxic chemotherapy, yet almost all will relapse with resistant disease. Thus, improving patient outcomes will require novel approaches to limit metastasis and overcome chemo-resistance. Liver Kinase B1 (LKB1), encoded by the STK11 gene, is a key intracellular regulator of metabolic stress and is considered a putative tumor suppressor in some cancers. However, we have demonstrated that LKB1 is intact and required for EOC cell viability and growth in an in vitro spheroid model of ovarian cancer metastasis. We propose that LKB1 signaling enables malignant EOC cells to maintain viability and survive in metabolically challenging environments like that encountered during intraperitoneal metastasis. To further investigate the therapeutic potential of targeting LKB1 activity in metastatic EOC, we generated STK11-knockout cell lines—normal FT190 cells, and EOC cell lines OVCAR8, HeyA8, and iOvCa147—using CRISPR technology. STK11KO resulted in decreased malignant properties of EOC cells in vitro, including clonogenicity and anchorage-independent growth; however, loss of LKB1 in FT190 cells had no effect on cell proliferation, clonogenicity, or anchorage-independent growth, indicating LKB1 does not likely act as a tumor suppressor in EOC. Loss of LKB1 sensitized EOC cells to the growth-inhibiting effects of specific metabolic stresses. OVCAR8-STK11KO cells were more sensitive to nutrient deprivation in adherent culture, and to carboplatin and paclitaxel treatment in spheroid culture, as compared with OVCAR8 cells. Among the three EOC cell lines, STK11KO yielded variable sensitivity to inhibition of mitochondrial ATP production via oligomycin treatment. Interestingly, STK11KO did not affect induction of AMP-activated protein kinase (AMPK) phosphorylation in EOC spheroids, indicating that metabolic stress signaling to support EOC cell survival in spheroids during metastasis may occur via alternative pathways. In support of our previous knockdown results, EOC spheroids completely lacking LKB1 had markedly impaired growth in suspension culture compared to parental cell controls. In contrast, FT190 spheroids exhibited rapid cell attrition in spheroid culture regardless of LKB1 status. These results indicate that LKB1 may be specifically required in EOC cells to evade anoikis during metastatic spread. Finally, to test directly whether loss of LKB1 activity affects the metastatic potential of EOC cells, we performed intraperitoneal injections of OVCAR8-STK11KO and HeyA8-STK11KO cells with their respective parental cell controls. Loss of LKB1 in both aggressive EOC cell lines exhibited a dramatic reduction on tumor burden. STK11KO significantly decreased the establishment of large, solid tumor masses, reduced adhesion of OVCAR8 tumor nodules, and even changed the metastatic trajectory of HeyA8 cells with evidence of tumor cell growth only as a thin layer on peritoneal walls. Histologic analysis revealed evidence of extensive necrosis in STK11KO tumors, which was likely the major contributor to reduced tumor burden. These results strongly indicate that loss of LKB1 activity abrogates the metabolic stress response necessary during EOC metastasis both in spheroids and establishment of intraperitoneal tumors. Overall, LKB1 or its AMPK-independent signaling mediators represent unique yet very potent therapeutic vulnerabilities in metastatic EOC. Citation Format: Adrian Buensuceso, Yudith R. Valdes, Rene Figueredo, Gabriel E. DiMattia, Trevor G. Shepherd. The metabolic stress mediator LKB1 is required for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A12.

Silencing ROR1 and ROR2 inhibits invasion and adhesion in an organotypic model of ovarian cancer metastasis.

OBJECTIVEElevated expression of the ROR1 and ROR2 Wnt receptors has been noted in both the tumour and stromal compartments of ovarian cancer patient tissue samples. In vitro studies have suggested these receptors play a role in ovarian cancer metastasis. However, these previous studies have utilised simple 2D in vitro models to investigate cancer cell growth and migration, which does not allow investigation of stromal involvement in Wnt driven metastasis.AIMTo investigate targeting ROR1 and ROR2 using a primary co-culture 3D model of epithelial ovarian cancer dissemination to the omentum.METHODSPrimary fibroblasts (NOF) and mesothelial (HPMC) cells were isolated from fresh samples of omentum collected from women with benign or non-metastatic conditions and cultured with collagen to produce a organotypic 3D model. Stable shRNA knockdown of ROR1, ROR2 and double ROR1/ROR2 in OVCAR4 cells were plated onto the 3D model to measure adhesion, or using a transwell to measure invasion. Gene expression changes in primary cells upon OVCAR4 interaction was evaluated using indirect transwell co-culture.RESULTSDouble knockdown of ROR1 and ROR2 strongly inhibited cell adhesion (p<0.05) and invasion (P<0.05) to the omentum model. ROR2 was up regulated in primary fibroblasts when cultured with OVCAR4 (P=0.05) and ectopic overexpression of ROR2 in NOFs inhibited cell proliferation (P<0.01) but increased cell migration.CONCLUSIONThe combination of ROR1 and ROR2 signalling influences ovarian cancer dissemination to the omentum, however ROR2 may also play a role in stromal activation during metastasis. Therefore, targeting both ROR1 and ROR2 may be a powerful approach to treating ovarian cancer.

Abstract AS19: The role of the receptor tyrosine kinase AXL in an immunocompetent mouse model of invasive endometrioid ovarian cancer

Abstract Objective: To evaluate the role of AXL in an immunocompetent model of ovarian cancer metastasis. Methods: Immunohistochemical staining of ovarian cancer tissue microarrays of advanced stage, high-grade epithelial ovarian cancer specimens for AXL expression was performed and correlated with overall survival. Metastatic endometrioid ovarian cancer was generated through intrabursal AdCre-infected LSL-K-rasG12D/+Ptenfl/fl mouse model. LSL-K-rasG12D/+Ptenfl/fl Axl+/+ and LSL-K-rasG12D/+Ptenfl/flAxl−/−were used to evaluate the role of AXL. Necropsy was performed to evaluate burden of the primary and metastatic tumors. Hematoxylin and eosin (H&amp;E) staining was used to confirm tumor formation or lack of tumor. Western blotting was performed to evaluate protein expression. Results: Of the 80 patient-derived primary ovarian tumor specimens, 76% (61) of advanced stage, high-grade epithelial ovarian cancer specimens had 3+ transmembrane AXL staining whereas 24% (19) of advanced stage, high-grade epithelial ovarian cancer specimens had no AXL staining. When correlated with overall survival, patients with tumors that had no AXL expression had a median survival of 53 months vs. 45 months for those with 3+AXL expression (p=0.12). The majority of intrabursal AdCre-infected LSL-K-rasG12D/+Ptenfl/fl Axl+/+ mice had primary (78%) and metastatic tumor formation (86%) whereas the majority of LSL-K-rasG12D/+Ptenfl/flAxl−/− had primary tumor formation (64%) but few had metastatic tumors (28%) (p&amp;lt;0.05). There was no difference in primary tumor weight between the two groups: 120mg for Axl+/+ vs. 110mg for Axl−/−. Metastatic tumors were microscopic and therefore unable to be resected and weighed prior to identification by H&amp;E staining. Western blotting of primary and metastatic tumors from LSL-KrasG12D/+Ptenfl/fl Axl+/+ showed high AXL expression compared to no AXL expression in the ovaries of normal mice. Conclusions: AXL expression in advanced stage epithelial ovarian cancer correlated with lower overall survival. Primary ovarian and metastatic tumors in this intrabursal AdCre-infected LSL-K-rasG12D/+Ptenfl/fl mouse model express AXL. Deletion of the receptor tyrosine kinase, AXL, in this conditional genetically engineered mouse model of endometrioid ovarian cancer resulted in inhibition of metastatic tumor formation. AXL is a critical factor driving metastasis in this immunocompetent murine model of metastatic ovarian cancer. Citation Format: Katherine Fuh, Erinn Rankin, Colleen Wu, Anh Diep, Amato J. Giaccia. The role of the receptor tyrosine kinase AXL in an immunocompetent mouse model of invasive endometrioid ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS19.

Abstract AS23: Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis

Abstract The cross-talk between ovarian cancer cells with the microenvironment of the site of metastasis is an essential determinant of successful metastatic colonization. microRNAs have been well established to play critical roles in various stages of cancer progression, including metastasis. However, the role of the signals from the microenvironment in regulating key microRNAs in metastasizing cancer cells has not been studied. Using a 3D culture model mimicking the human omentum, one of the principal sites of ovarian cancer metastasis, we have identified the microenvironment-induced downregulation of a tumor suppressor microRNA miR-193b in the metastasizing ovarian cancer cells. The direct interaction of the ovarian cancer cells with the mesothelial cells covering the surface of the omentum caused DNA methyltransferase 1 (DNMT1) mediated methylation and decreased expression of miR-193b. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate in the omentum both in vitro and in a mouse xenograft model of ovarian cancer metastasis. The functional effects of miR-193b were mediated at least partly through the concomitant increased expression of its target urokinase-type plasminogen activator (uPA). Our findings link paracrine signals from the microenvironment with the regulation of a key microRNA in cancer cells that is essential for the initial steps of ovarian cancer metastatic colonization. Targeting miR-193b would be a promising approach to treat ovarian cancer metastasis. Citation Format: AK Mitra, CY Chiang, P Tiwari, ME Peter, E Lengyel. Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS23.

Abstract POSTER-TECH-1124: Modeling antibody penetration into metastatic ovarian tumors after intravenous or intraperitoneal delivery

Abstract Although targeted therapy with monoclonal antibodies has been successful against certain types of cancer, these treatments have had little success in metastatic ovarian cancer. Limited response may in part be due to suboptimal delivery of antibodies to tumors within the peritoneal cavity, as well as heterogeneous expression of surface receptors. Recent studies have shown that intraperitonal (i.p.) delivery of cisplatin has a survival advantage over intravenous (i.v.) delivery, yet few studies have examined how route of delivery influences the efficacy of antibody treatment. We have combined experimental and mathematical models to explore whether between-patient variation in tumor vascular density can predict which route of delivery is optimal for targeted treatment of metastatic ovarian tumors. The vascular density of paired bowel and omental metastatic ovarian tumors from ten patients was calculated and found to be uniform for tumors from the same patient, but vascular density among patients ranged from 1% to 10% indicating that vascular density may influence a patient’s response to treatment. Antibody penetration was measured in 3-D spheroid cultures and in vivo in an orthotopic mouse model of ovarian cancer metastasis. For our spheroid model, SKOV3.ip-GFP human ovarian cancer cells that overexpress erbB2 were allowed to form spheroids for 48 hours and then incubated with a fluorescently tagged anti-erbB2 therapeutic antibody, pertuzumab. Pertuzumab bound to the surface of the spheroids within one hour of treatment, but penetration into the center of the spheroid was slow, only reaching the center of the spheroid by 24 hours. In contrast, the chemotherapeutic agent doxorubicin completely penetrated the spheroids within 90 minutes. In our mouse model, SKOV3.ip cells were injected directly into the peritoneal cavity. Fluorescently-tagged pertuzumab was delivered via intravenous or intraperitoneal injection two weeks post-injection by which time tumors were seeded throughout the peritoneal cavity. Excised tumors were imaged on a two-photon microscope to observe antibody penetration. By 24 hours post-injection, antibody had fully penetrated into peritoneal tumors regardless of delivery route, suggesting that any advantage for i.p. delivery will likely occur at early time points. Using parameters derived from our experimental data, we have built a 3-D mathematical model, the OvTM (Ovarian Tumor Model), which simulates cell-cell interactions within an ovarian tumor. The simulated tumor can be avascular to simulate penetration of drugs into spheroids within the ascites fluid, or it can be populated with vessels that are matched to the vascular density of each patient’s tumors. We can use this individualized model to investigate the effect of different modes of drug delivery on drug distribution. Ultimately, we hope that our model will provide improved methods for stratifying patients by treatment route in order to optimize antibody penetration into metastatic ovarian tumors. Citation Format: Mara P. Steinkamp, Kimberly Kanigel Winner, Melanie Moses, Yi Jiang, Bridget S. Wilso. Modeling antibody penetration into metastatic ovarian tumors after intravenous or intraperitoneal delivery [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1124.

Abstract 4111: The salt inducible kinase 2 (SIK2) links lipid metabolism to survival of ovarian cancer metastasis

Abstract Background: High Grade Serous Ovarian Cancer (HGSOC) metastatic disease is strongly dependent on the adipocyte-rich microenvironment of the omentum. Altough previous work has shown that adipocytes are required for driving cancer metastasis to fat-rich environments, the mechanisms involved in the process of linking metabolism to cancer cell survival at this niche have remained poorly understood and this has, therefore, hindered therapeutic exploitation. We have previously shown that the expression of the Salt Inducible Kinase 2 (SIK2) is important for ovarian cancer cell survival and that it correlates with poor patient survival (Ahmed et al., Cancer Cell, 2010). In this work we present a previously unrecognised role for SIK2 in driving cancer cell metabolism and proliferation at the omental metastatic niche. Methods: We established a system for the co-culture of cancer cells with adipocytes obtained from freshly excised normal omentum from women undergoing surgical staging. This system was used in combination with a chemical biology approach utilizing cells expressing gate-keeper mutants of SIK2 and a type I kinase inhibitor to test the specificity of observed phenotypic effects. Our results were validated using immunohistochemistry of a panel of ovarian cancers and a xenograft model of ovarian cancer metastasis. Results: SIK2 was significantly overexpressed in omental metastases of ovarian tumours compared to paired ovarian cancer primary lesions from the same patients. In a xenograft model of ovarian cancer metastasis SIK2-overexpressing cells implanted orthotopically at the ovarian bursa formed significantly larger omental metastases compared to cells with endogenous levels of SIK2. We first observed that co-culture of ovarian cancer cells with adipocytes resulted in an increased SIK2 autophosphorylation and activation which, in turn stimulated cancer cell proliferation. Next, we showed that PLC-dependent activation of calcium release was required for adipocyte-induced SIK2 autophosphorylation in ovarian cancer cells. Surprisingly, we identified a role for adipocyte-activated SIK2 in stimulating cancer cell metabolism throught augmenting AMPK-induced phosphorylation of ACC and activation of carnitine palmitoyltransferase 1 (CPT1) transcription. Concurrently, SIK2 was required to activate the PI3K complex through direct p85α-S154 phosphorylation. This was confirmed using rapamycin as a known robust paradoxical PI3K activator showing that SIK2 siRNA-mediated depletion or its chemical inhibition abolished rapamycin-induced AKT phosphorylation and sensitized ovarian cancer cells to its cytotoxic effect. Conclusion: Our results suggested that SIK2 phosphorylation and activation were required to establish ovarian cancer lesions at the adipocyte-rich omental environment. Therefore we suggest a therapeutic role for targeting SIK2 in preventing ovarian cancer metastases. Citation Format: Fabrizio Miranda, Shujuan Liu, Sandra Herrero-Gonzalez, David Mannion, Stefan Knapp, Ahmed A. Ahmed. The salt inducible kinase 2 (SIK2) links lipid metabolism to survival of ovarian cancer metastasis. [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 4111. doi:10.1158/1538-7445.AM2015-4111

Abstract 1406: A new ovarian cancer metastasis model using multicellular spheroids generated from human ovarian cancers tissues

Abstract Objective Most research aimed at dissecting the mechanism by which epithelial ovarian cancer (EOC) metastasizes has focused on the ability of individual cells to attach and create tumor implants. However, multicellular aggregates known as spheroids are frequently found in ovarian cancer ascites and these aggregates are increasingly thought to play an important role in metastasis not only for ovarian cancer but also other solid tumors where similar phenomena have been observed. We investigated characteristics of spheroids compared to cells grown in monolayer to further characterize these spheroids. Methods IRB approval was obtained for collection of human tissue. Fresh specimens of ovarian cancer were minced, enzymatically digested, rinsed and incubated either in DMEM to generate monolayer cultures or Mammosphere media to generate spheroids. Tumorigenicity was assessed by inoculating individual spheroids or an equal number of dissociated cells into the peritoneal cavity of SCID mice. Gene expression was examined using Western blot and quantitative real-time PCR. Results We found that cells from most ovarian cancers do generate spheroids and most of the spheroids generated are able to reliably induce peritoneal implants in SCID mice. At a molecular level, the spheroids were enriched for expression of specific markers including CD133, CD44, NANOG and OCT4, associated with populations of cancer initiating cells (CICs) as well as other stem cell markers. MiR-26b, characteristic of CICs, was significantly up regulated in spheroids compared to monolayer cells. Spheroids from CD133 positive cells induced ovarian and abdominal tumors in SCID mice, however, CD44 spheroids only generated localized tumor, suggesting that CD133 is important in metastasis of ovarian cancer. Conclusion Our results indicate that populations of cells from human EOC are capable of spontaneously generating multicellular aggregates that resemble the spheroids found in ovarian cancer ascites de novo. These spheroids are enriched for expression of markers including CD133, CD44, NANOG and OCT4, suggesting that spheroid formation enhances stem cell-like markers. CD133 might also serve as a necessary marker for metastasis of ovarian cancer. The increased expression of miR-26b in spheroids compared to monolayer culture is a novel finding that needs further investigation. 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 1406. doi:1538-7445.AM2012-1406

Activatable Optical Imaging with a Silica-Rhodamine Based Near Infrared (SiR700) Fluorophore: A comparison with cyanine based dyes

Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields; however, if deeper imaging is needed then near-infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to nonspecific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching, enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5 and better biostability compared to Av-Alexa Fluor 680.

P-cadherin cooperates with insulin-like growth factor-1 receptor to promote metastatic signaling of gonadotropin-releasing hormone in ovarian cancer via p120 catenin

Gonadotropin-releasing hormone (GnRH) is a potent prometastatic factor in ovarian cancer, but the intracellular signaling events are not well understood. The classical Gα(q)-phospholipase C signal transduction pathway known to operate in the pituitary is not involved in GnRH actions at non-pituitary targets. Here we showed that GnRH treatment of ovarian cancer cells led to a rapid and remarkable tyrosine phosphorylation of p120 catenin (p120(ctn)), which was mediated by P-cadherin. The use of P-cadherin small interfering RNA or neutralizing antibodies to inhibit P-cadherin expression and function resulted in diminished p120(ctn) activation, confirming that the effect was P-cadherin specific. On exploring how P-cadherin, which lacks intrinsic kinase activity, might regulate the activation of p120(ctn), we found that P-cadherin could induce the ligand-independent activation of insulin-like growth factor-1 receptor (IGF-1R). Inhibition of IGF-1R expression or its activity significantly inhibited GnRH-induced p120(ctn) activation, and the subsequent cell migration and invasion. In addition, we showed that IGF-1R regulation by P-cadherin was associated with complex formation between IGF-1R and P-cadherin, and this regulation was also observed to be in vivo correlated with metastasis. Furthermore, using a mouse model of ovarian cancer metastasis, GnRH receptor knockdown was shown to diminish peritoneal dissemination of tumors and ascites formation. These findings suggest for the first time that GnRH can initiate an outside-in p120(ctn) signal transduction through the cross-talk between P-cadherin and IGF-1R, thus providing a novel molecular mechanism by which GnRH may control the high level of aggressiveness and invasion and metastasis potential that are characteristic of ovarian cancer.

Abstract 2363: Positive inter-regulation between β-catenin and endothelin signaling in ovarian cancer cells: Epigenetic regulation of gene transcription

Abstract Although endothelin-1 (ET-1) and the endothelin A receptor (ETAR) regulate different steps of ovarian tumor progression, the molecular mechanism controlling the expression of ET-1 in this tumor is unknown. We previously demonstrated that ET-1 activates β-catenin/TCF4 transcriptional activity to promote cell invasion and metastasis. Here, we show that ET-1 gene is directly regulated by β-catenin in ovarian cancer cells. Thus, inhibition of β-catenin signalling results in lowered ET-1 promoter activity and expression, while enhanced β-catenin signalling leads to further activation of this gene. Chromatin immunoprecipitation (ChIP) demonstrated that, after ET-1 stimulus, β-catenin and its cognate DNA binding partner, TCF4, are recruited on the specific DNA element within the ET-1 promoter. Unravelling the role of the scaffold protein β-arrestin-1 as nucleus chaperone controlling β-catenin transcription activity, we demonstrated that β-arrestin-1 is required for the activation of ET-1 promoter, and for the recruitment of β-catenin/TCF4 on this promoter. In agreement with these findings, immunoprecipitation assay demonstrated that ET-1 promotes the interaction between endogenous β-catenin and β-arrestin-1 both in the cytoplasm and in the nucleus. Moreover, β-arrestin-1 siRNA leads to the loss of ET-1 mRNA expression and ET-1 secretion, as well as a significant inhibition of ET-1 promoter activity, thus suggesting the critical role of β-arrestin-1 in the β-catenin-dependent ET-1 gene expression. Further experiments showed that ET-1 promotes the nuclear association between histone acetyltransferase p300 and β-arrestin and the recruitment of p300 on the ET-1 promoter, resulting in H3 and H4 histone acetylation and enhanced ET-1 transcription. Besides ET-1, β-arrestin-1 represents a platform for achieving signal specificity that converges on β-catenin-mediated transcription of defined genes, such as cyclin D1 and matrix metalloprotease (MMP)-2, but not MMP-9. Moreover, ETAR blockade with the specific ETAR antagonist, zibontentan (ZD4054), abrogates both the engagement of β-arrestin and the interplay between ET-1 and the β-catenin in controlling gene transcription. In an intraperitoneal model of ovarian cancer metastasis, HEY cells expressing the mutant β-arrestin-1, or treatment with zibontentan, significantly reduced peritoneal tumor-cell dissemination and β-catenin expression, highlighting the importance of β-arrestin-mediated signaling in metastasis formation. Altogether these results reveal a positive inter-regulation between β-catenin and ET-1 that amplify the ET-1/ETAR autocrine loop in ovarian cancer cells, in which β-arrestin-1 acts as a nuclear messenger mediating epigenetic mechanism in β-catenin-mediated ET-1 transcription. Supported by AIRC, AstraZeneca Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2363.

An Orally Available Small-Molecule Inhibitor of c-Met, PF-2341066, Reduces Tumor Burden and Metastasis in a Preclinical Model of Ovarian Cancer Metastasis

Deregulated expression of the hepatocyte growth factor (HGF) receptor, c-Met, in cancer contributes to tumor progression and metastasis. The objective of this study was to determine whether blocking c-Met with an orally available c-Met inhibitor, PF-2341066, reduces tumor burden and increases survival in a xenograft model of ovarian cancer metastasis. Treatment of mice injected interperitoneally with SKOV3ip1 cells showed reduced overall tumor burden. Tumor weight and the number of metastases were reduced by 55% (P < .0005) and 62% (P < .0001), respectively. Treatment also increased median survival from 45 to 62 days (P = .0003). In vitro, PF-2341066 reduced HGF-stimulated phosphorylation of c-Met in the tyrosine kinase domain as well as phosphorylation of the downstream signaling effectors, Akt and Erk. It was apparent that inhibition of the pathways was functionally important because HGF-induced branching morphogenesis was also inhibited. In addition, proliferation and adhesion to various extracellular matrices were inhibited by treatment with PF-2341066, and the activity of matrix metalloproteinases was decreased in tumor tissue from treated mice compared with those receiving vehicle. Overall, these data indicate that PF-2341066 effectively reduces tumor burden in an in vivo model of ovarian cancer metastasis and may be a good therapeutic candidate in the treatment of patients with ovarian cancer.

Vascular Cell Adhesion Molecule-1 Is a Regulator of Ovarian Cancer Peritoneal Metastasis

Ovarian cancers metastasize by attaching to and invading through the mesothelium, a single layer of mesothelial cells lining the peritoneal cavity. The presence of invasive peritoneal metastases is associated with a poor prognosis for ovarian cancer (5-year survival <25%). Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface receptor that mediates leukocyte attachment and extravasation across endothelial and mesothelial monolayers at sites of inflammation. Membranous VCAM-1 expression was observed on the mesothelium of 13 of 14 women with ovarian cancer compared with 6 of 15 who were cancer-free. Using a cell culture model system of mesothelial invasion, highly tumorigenic SKOV-3 and ES-2 cells were 2.5 to 3 times more efficient in transmigration through the mesothelial monolayer compared with poorly tumorigenic OVCAR-3 cells. Blocking antibodies to, or small interfering RNA knockdown of, VCAM-1 or its ligand alpha(4)beta(1) integrin significantly decreased, but did not completely inhibit, transmigration of SKOV-3 cells through mesothelial monolayers. Furthermore, using a mouse model of ovarian cancer metastasis, treatment with VCAM-1 function-blocking antibodies decreased tumor burden and increased survival. Together, these observations implicate VCAM-1-alpha(4)beta(1) integrin interactions in the regulation of ovarian cancer cell mesothelial invasion and metastatic progression and offer the possibility of novel therapeutic targets.

Inhibition of ovarian cancer cell metastasis by a fusion polypeptide Tat-ELP

Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Cell adhesion plays a critical role in the development of metastatic cancer, and it is mediated by interactions between receptors on the cell surface and ligands of the extracellular matrix or other surfaces. Therefore, inhibition of the cell adhesion process appears to be an effective method of preventing metastasis. This work describes a genetically engineered polypeptide with the potential to prevent cell adhesion and inhibit metastasis. We have found that the cell penetrating peptide Tat, fused with elastin-like polypeptide (ELP) inhibited adhesion, spreading, invasion and migration of SKOV-3 ovarian cancer cells in cell culture. Furthermore, we have also confirmed that Tat-ELP has anti-metastatic potential in an experimental ovarian cancer metastasis model in vivo, causing approximately 80% reduction in the tumor burden. Since cell attachment is an important step in tumor cell invasion and metastasis, these results suggest a novel role of Tat-ELP as a therapeutic intervention in cancer metastasis.