Julie K. Allen

Adrenergic modulation of focal adhesion kinase protects human ovarian cancer cells from anoikis

Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.

Src activation by adrenoreceptors is a key switch for tumour metastasis

Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment.

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.

EphA2 overexpression promotes ovarian cancer growth

Background: Silencing EphA2 has been shown to result in anti-tumor efficacy. However, it is not known whether increasing EphA2 expression specifically results in increased tumor growth and progression. We examined the effects of stable EphA2 transfection into poorly invasive ovarian cancer cells with regard to in vitro invasive and in vivo metastatic potential. Methods: EphA2 gene was introduced into A2780 cells by retroviral infection. The effects of increased EphA2 expression were examined on cellular morphology, and anchorage-dependent and independent cell growth. Furthermore, the effect of EphA2 overexpression on metastatic ability was determined using an orthotopic nude mouse model of ovarian carcinoma. Results: In low cell density, EphA2-overexpressing A2780 cells (A2780-EphA2) displayed less cell-cell contact, increased cell-extracellular matrix (ECM) attachment and anchorage-independent cell growth compared to empty vector controls. There was no significant effect on anchorage-dependent cell proliferation, migration, or invasion. Increased expression of EphA2 promoted tumor growth and enhanced the metastatic potential in A2780-EphA2 human ovarian cancer xenografts. The overexpression of EphA2 resulted in enhanced microvessel density (MVD), but had no effect on tumor cell proliferation. Conclusions: EphA2 promotes tumor growth by enhancing cell-ECM adhesion, increasing anchorage-independent growth, and promoting angiogenesis.

Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis

Adrenergic stimulation adversely affects tumor growth and metastasis, but the underlying mechanisms are not well understood. Here, we uncovered a novel mechanism by which catecholamines induce inflammation by increasing prostaglandin E2 (PGE2) levels in ovarian cancer cells. Metabolic changes in tumors isolated from patients with depression and mice subjected to restraint stress showed elevated PGE2 levels. Increased metabolites, PTGS2 and PTGES protein levels were found in Skov3-ip1 and HeyA8 cells treated with norepinephrine (NE), and these changes were shown to be mediated by ADRB2 receptor signaling. Silencing PTGS2 resulted in significantly decreased migration and invasion in ovarian cancer cells in the presence of NE and decreased tumor burden and metastasis in restraint stress orthotopic models. In human ovarian cancer samples, concurrent increased ADRB2, PTGS2 and PTGES expression was associated with reduced overall and progression-free patient survival. In conclusion, increased adrenergic stimulation results in increased PGE2 synthesis via ADRB2–Nf-kB–PTGS2 axis, which drives tumor growth and metastasis.

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.

Adrenergic-mediated increases in INHBA drive CAF phenotype and collagens

Adrenergic signaling is known to promote tumor growth and metastasis, but the effects on tumor stroma are not well understood. An unbiased bioinformatics approach analyzing tumor samples from patients with known biobehavioral profiles identified a prominent stromal signature associated with cancer-associated fibroblasts (CAFs) in those with a high biobehavioral risk profile (high Center for Epidemiologic Studies Depression Scale [CES-D] score and low social support). In several models of epithelial ovarian cancer, daily restraint stress resulted in significantly increased CAF activation and was abrogated by a nonspecific β-blocker. Adrenergic signaling-induced CAFs had significantly higher levels of collagen and extracellular matrix components than control tumors. Using a systems-based approach, we found INHBA production by cancer cells to induce CAFs. Ablating inhibin β A decreased CAF phenotype both in vitro and in vivo. In preclinical models of breast and colon cancers, there were increased CAFs and collagens following daily restraint stress. In an independent data set of renal cell carcinoma patients, there was an association between high depression (CES-D) scores and elevated expression of ACTA2, collagens, and inhibin β A. Collectively, our findings implicate adrenergic influences on tumor stroma as important drivers of CAFs and establish inhibin β A as an important regulator of the CAF phenotype in ovarian cancer.

89. Src activation by beta adrenoreceptors is a key switch for microenvironmental regulation of tumor metastasis

Polyinosinic:polycytidylic acid (poly I:C), an agonist for TLR3, induces type-I interferons and other inflammatory mediators such as TNF-alpha, IL-6 and to a lesser extent IL-1beta. Poly I:C induces sickness behaviour in mice and it is known that type-I interferons impact on behaviour and CNS function. Here we assess the role of type-I interferons in pI:C-induced sickness behaviour. Type-I interferon receptor 1 knockout (IFNAR1 / ) and WT C57BL/6 mice were challenged with pI:C (12 mg/kg i.p). IFNAR1 / animals displayed reduced sickness behaviour compared to WT mice and only WT animals displayed a significant hypothermic response. All mice showed a reduction in weight 28 h post-challenge but full recovery was more rapid in IFNAR1 / mice. In addition to this, 3 h post-challenge, WT mice showed decreased activity in the open field with respect to IFNAR1 / . As expected, the interferon responsive genes PKR and IRF7 were not induced by pI:C in IFNAR1 / mice. While IL-1beta and TNF-alpha responses to pI:C were equivalent in KO and WT in blood, hippocampus and hypothalamus, there was no synthesis of IL-6 in IFNAR1 / in either blood or brain. These results demonstrate a role for type-I interferons in the sickness behaviour response to pI:C and in the induction of IL-6 responses. Further investigations will examine whether the IL-6 deficiency seen in IFNAR1 / mice is responsible for the reduced pI:C-induced sickness behaviour.

Abstract 3368: Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Purpose: Catecholamine mediated stress effects are known to induce production of various pro-inflammatory cytokines. However, the mechanism and functional effect of adrenergic signaling in driving inflammation via pro-inflammatory metabolites is currently unknown. Here we address the functional and biological consequences of adrenergic-induced Cox2/PGE2 axis activation in ovarian cancer metastasis. Methods: We first analyzed global metabolic changes in tumors isolated from patients with known Center for Epidemiologic Studies Depression Scale (CES-D; depressive) scores and tumoral norepinephrine (NE) levels. Beta-adrenergic receptor (ADRB) positive cells (Skov3 and HeyA8) were used to study gene and protein levels of PTGS2 (cyclooxygenase2), PTGES (prostaglandin E synthase) and metabolite PGE2 in vitro and in vivo. To study tumor-specific effects on catecholamine-derived expression of PTGS2, we used a novel DOPC delivery system of PTGS2 siRNA. Results: Our results revealed that levels of PGs were significantly increased in patients with high depressive scores (>16). PGE2 was upregulated by 2.38 fold when compared to the low CES-D scores. A similar trend was also observed with other pro-inflammatory eicosanoids, such as 6-keto prostaglandin F1 Alpha (2.03), prostaglandin A2 (1.39) and prostaglandin E1 (1.39). Exposure to NE resulted in increased PTGS2 and PTGES (prostaglandin E2 synthase) gene expression and protein levels in Skov3 and HeyA8. PGE2 ELISA confirmed that upon treatment with NE, PGE2 levels were increased in conditioned medium from Skov3 and HeyA8 cells. Treatment with a broad ADRB agonist (isoproterenol) or ADRB2 specific agonist (terbutaline) led to increases in expression of PTGS2 and PTGES as well as PGE2 levels in supernatant. Conversely, treatment with a broad antagonist (propranolol) or an ADRB2 specific antagonist (butoxamine) in the presence of NE abrogated gene expression changes of PTGS2 and PTGES. ChIP analysis showed enrichment of Nf-kB binding to the promoter region of PTGS2 and PTGES by 2.4 and 4.0 fold respectively when Skov3ip1 cells were treated with NE. Silencing PTGS2 resulted in significantly decreased migration (40%) and invasion (25%) of Skov3 cells in the presence of NE. Importantly, in the Skov3-ip1 restraint stress orthotopic model, silencing PTGS2 abrogated stress mediated effects and decreased tumor burden by 70% compared to control siRNA with restraint stress. Conclusion Increased adrenergic stimulation results in a pro-inflammatory milieu mediated by prostaglandins that drives tumor progression and metastasis in ovarian cancer. Citation Format: Archana S. Nagaraja, Piotr Dorniak, Nouara Sadaoui, Guillermo Armaiz-Pena, Behrouz Zand, Sherry Y. Wu, Julie K. Allen, Rajesha Rupaimoole, Cristian Rodriguez-Aguayo, Sunila Pradeep, Lin Tan, Rebecca A. Previs, Jean M. Hansen, Peiying Yang, Garbiel Lopez-Berestein, Susan K. Lutgendorf, Steve Cole, Anil K. Sood. Sustained adrenergic signaling activates pro-inflammatory prostaglandin network in ovarian carcinoma. [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 3368. doi:10.1158/1538-7445.AM2015-3368