Myrthala Moreno-Smith

Tumour angiogenesis regulation by the miR-200 family

The miR-200 family is well known to inhibit the epithelial-mesenchymal transition, suggesting it may therapeutically inhibit metastatic biology. However, conflicting reports regarding the role of miR-200 in suppressing or promoting metastasis in different cancer types have left unanswered questions. Here we demonstrate a difference in clinical outcome based on miR-200s role in blocking tumour angiogenesis. We demonstrate that miR-200 inhibits angiogenesis through direct and indirect mechanisms by targeting interleukin-8 and CXCL1 secreted by the tumour endothelial and cancer cells. Using several experimental models, we demonstrate the therapeutic potential of miR-200 delivery in ovarian, lung, renal and basal-like breast cancers by inhibiting angiogenesis. Delivery of miR-200 members into the tumour endothelium resulted in marked reductions in metastasis and angiogenesis, and induced vascular normalization. The role of miR-200 in blocking cancer angiogenesis in a cancer-dependent context defines its utility as a potential therapeutic agent.

Impact of stress on cancer metastasis.

The influence of psychosocial factors on the development and progression of cancer has been a longstanding hypothesis since ancient times. In fact, epidemiological and clinical studies over the past 30 years have provided strong evidence for links between chronic stress, depression and social isolation and cancer progression. By contrast, there is only limited evidence for the role of these behavioral factors in cancer initiation. Recent cellular and molecular studies have identified specific signaling pathways that impact cancer growth and metastasis. This article provides an overview of the relationship between psychosocial factors, specifically chronic stress, and cancer progression.

Stress effects on FosB- and interleukin-8 (IL8)-driven ovarian cancer growth and metastasis.

A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250–300% increase in IL8 protein and 240–320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5–4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5- and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.

Dopamine Blocks Stress-Mediated Ovarian Carcinoma Growth

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.

Targeting pericytes with a PDGF-B aptamer in human ovarian carcinoma models

Purpose: On the basis of the known role of platelet-derived growth factor (PDGF)-BB/PDGF receptor (PDGFR) β in pericyte regulation, highly specific inhibitors of this target are needed. We tested the efficacy of a highly selective aptamer against PDGF-B with or without anti-VEGF therapy in ovarian cancer models. Experimental Design: The therapeutic efficacy of targeting endothelial cells (bevacizumab) and/or pericytes (PDGF-aptamer, AX102) was examined using HeyA8 and SKOV3ip1 orthotopic models of ovarian cancer metastasis. Following therapy, tumors were examined for microvessel density (MVD), proliferating cell nuclear antigen (PCNA), and vascular maturation (pericyte coverage). Results: Bevacizumab inhibited tumor growth by 45% and 48% in the HeyA8 and SKOV3ip1 models, respectively. AX102 had minimal effect on the HeyA8 model, but increased tumor growth in the SKOV3ip1 model. However, bevacizumab plus AX102 was more effective than bevacizumab alone, and resulted in 76 - 88% inhibition of tumor growth in both models. A longitudinal study in the HeyA8 model using bioluminescence imaging showed that combination of bevacizumab, AX102 and paclitaxel caused tumor reduction by 65% (based on bioluminescence imaging). In the HeyA8 model, MVD and PCNA counts were significantly reduced in the bevacizumab treatment groups, and pericyte coverage was significantly decreased in the AX102 treatment groups. In the SKOV3ip1 model, MVD and PCNA was significantly reduced in the bevacizumab treatment group, and even lower in the bevacizumab and AX102 combination treatment group. Conclusions: Dual targeting of endothelial cells and pericytes holds potential as an anti-vascular therapeutic approach in ovarian carcinoma.

ATP11B mediates platinum resistance in ovarian cancer

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

Converging Evidence for Efficacy from Parallel EphB4-Targeted Approaches in Ovarian Carcinoma

EphB4 is a transmembrane receptor tyrosine kinase that plays an important role in neural plasticity and angiogenesis. EphB4 is overexpressed in ovarian cancer and is predictive of poor clinical outcome. However, the biological significance of EphB4 in ovarian cancer is not known and is the focus of the current study. Here, we examined the biological effects of two different methods of EphB4 targeting (a novel monoclonal antibody, EphB4-131 or siRNA) using several ovarian cancer models. EphB4 gene silencing significantly increased tumor cell apoptosis and decreased migration (P < 0.001) and invasion (P < 0.001). Compared with controls, EphB4 siRNA–1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone significantly reduced tumor growth in the A2780-cp20 (48%, P < 0.05) and IGROV-af1 (61%, P < 0.05) models. Combination therapy with EphB4 siRNA–1,2-dioleoyl-sn-glycero-3-phosphatidylcholine and docetaxel resulted in the greatest reduction in tumor weight in both A2780-cp20 and IGROV-af1 models (89–95% reduction versus controls; P < 0.05 for both groups). The EphB4-131 antibody, which reduced EphB4 protein levels, decreased tumor growth by 80% to 83% (P < 0.01 for both models) in A2780-cp20 and IGROV-af1 models. The combination of EphB4-131 and docetaxel resulted in the greatest tumor reduction in both A2780-cp20 and IGROV-af1 models (94–98% reduction versus controls; P < 0.05 for both groups). Compared with controls, EphB4 targeting resulted in reduced tumor angiogenesis (P < 0.001), proliferation (P < 0.001), and increased tumor cell apoptosis (P < 0.001), which likely occur through modulation of phosphoinositide 3-kinase signaling. Collectively, these data identify EphB4 as a valuable therapeutic target in ovarian cancer and offer two new strategies for further development. Mol Cancer Ther; 9(8); 2377–88. ©2010 AACR.

Sustained Adrenergic Signaling Promotes Intratumoral Innervation through BDNF Induction

Mounting clinical and preclinical evidence supports a key role for sustained adrenergic signaling in the tumor microenvironment as a driver of tumor growth and progression. However, the mechanisms by which adrenergic neurotransmitters are delivered to the tumor microenvironment are not well understood. Here we present evidence for a feed-forward loop whereby adrenergic signaling leads to increased tumoral innervation. In response to catecholamines, tumor cells produced brain-derived neurotrophic factor (BDNF) in an ADRB3/cAMP/Epac/JNK-dependent manner. Elevated BDNF levels in the tumor microenvironment increased innervation by signaling through host neurotrophic receptor tyrosine kinase 2 receptors. In patients with cancer, high tumor nerve counts were significantly associated with increased BDNF and norepinephrine levels and decreased overall survival. Collectively, these data describe a novel pathway for tumor innervation, with resultant biological and clinical implications.Significance: Sustained adrenergic signaling promotes tumor growth and metastasis through BDNF-mediated tumoral innervation. Cancer Res; 78(12); 3233-42. ©2018 AACR.

Abstract 406: Mechanisms of stress-buffering effects of dopamine on tumor vasculature

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chronic stress is known to promote tumor growth by activating the sympathetic nervous system (SNS) with resultant increases in norepinephrine (NE) and epinephrine (E). In contrast to NE and E, dopamine (DA) levels are low under chronic stress conditions. We have recently demonstrated that dopamine replacement in mice exposed to daily restraint stress can reverse the stimulatory effects of NE and E on ovarian cancer growth. Ovarian tumor tissues from stressed mice treated with dopamine showed significant increases in pericyte coverage of tumor microvessels. The focus of the current study was to examine the mechanisms by which dopamine treatment affected pericyte coverage of tumor endothelial cells under stress conditions. Female nude mice were subjected to chronic stress using the restraint-stress procedure. Tumor formation was induced by injecting the SKOV3 ip1 or HeyA8 ovarian cancer cells into the peritoneal cavity. Stressed and non-stressed mice were divided into four treatment groups: Control (PBS), DA, DA plus butaclamol (DR1 antagonist) and DA plus eticlopride (DR2 antagonist). Following therapy, harvested tumors were examined for microvessel density (MVD), vascular maturation (pericyte coverage) and proliferating cell nuclear antigen (PCNA). Expression of DA receptors (DR1-DR5) was analyzed by RT-PCR and Western blotting. Mice exposed to daily restraint stress showed 2-fold increased SKOV3ip1-tumor growth compared to non-stressed mice; treatment with DA alone resulted in 78% (p<0.01) decrease in tumor growth and 68% (p<0.01) decrease in MVD compared to control stressed mice. Similarly, DA/butaclamol combined treatment led to 71% (p<0.01) reduction in tumor growth and a 65% (p<0.01) decrease in MVD. Eticlopride in combination with DA reversed the inhibitory effects of DA on tumor growth. Furthermore, in stressed mice, daily dopamine treatment resulted in significant increase in pericyte coverage (51.4%; p<0.001) compared to controls. This effect was abrogated by butaclamol (44%; p<0.01), but not by eticlopride treatment. Similar results were obtained in stressed mice bearing HeyA8-tumors. In T10.5-pericyte like cells, treatment with DA, DA plus NE, DA agonist: [SKF38393][1] or PDGFBB increased significantly cell migration. No significant changes were noted with NE alone or DA plus butaclamol treatments. In MOEC, no significant changes in cell migration were observed under these experimental conditions. Collectively, our data indicate that DA, acting through DR1, could stimulate recruitment of pericytes to tumor endothelial cells, and promote vascular maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2011-406 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=SKF38393&atom=%2Fcanres%2F71%2F8_Supplement%2F406.atom

Abstract 375: Targeting pericytes in ovarian carcinoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Pericytes interact with and closely regulate endothelial cell function. Platelet-derived growth factor (PDGF)-BB/PDGF receptor (PDGFR) β plays a critical role in pericyte regulation, therefore highly specific inhibitors of this target are needed. We hypothesize that dual targeting of endothelial cells and pericytes provide a more efficacious antiangiogenic approach for ovarian cancer therapy. Experimental Design: The therapeutic efficacy of targeting endothelial cells (bevacizumab) and/or pericytes (PDGF-aptamer, AX102) was examined using SKOV3ip1 and HeyA8 orthotopic ovarian cancer models. The biomarkers, such as microvessel density (MVD), vascular maturation (pericyte coverage), and proliferative index (proliferating cell nuclear antigen, PCNA) were examined. Results: Bevacizumab inhibited tumor growth by 48% and 45% in the SKOV3ip1 and HeyA8 models, respectively. AX102 had minimal effect on the HeyA8 model, but increased tumor growth in the SKOV3ip1 model. However, bevacizumab plus AX102 was more effective than bevacizumab alone, and reduced 76 - 88% of tumor growth in both models. A longitudinal study using bioluminescence imaging in the HeyA8 model showed that a combination of bevacizumab, AX102 and paclitaxel caused tumor reduction by 65% (based on bioluminescence imaging). MVD and proliferative index counts were significantly reduced in the bevacizumab treatment groups, and pericyte coverage was significantly decreased in the AX102 treatment groups. Conclusions: Dual targeting of endothelial cells and pericytes demonstrates therapeutic potential as a unique anti-vascular approach in treating ovarian carcinoma. 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 375.