Angela Sanguino

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)–protein kinase A (PKA) signaling pathway by the β2 adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify β-adrenergic activation of the cAMP–PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.

Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth

BACKGROUND Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. METHODS Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. RESULTS Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively. CONCLUSIONS Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p

An anticancer C-Kit kinase inhibitor is reengineered to make it more active and less cardiotoxic

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modification to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We established the molecular blueprint for target discrimination in vitro using spectrophotometric and colorimetric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibitory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up approach to control drug specificity.

Stress Hormones Regulate Interleukin-6 Expression by Human Ovarian Carcinoma Cells through a Src-dependent Mechanism

Recent studies have demonstrated that chronic stress promotes tumor growth, angiogenesis, and metastasis. In ovarian cancer, levels of the pro-angiogenic cytokine, interleukin 6 (IL-6), are known to be elevated in individuals experiencing chronic stress, but the mechanism(s) by which this cytokine is regulated and its role in tumor growth remain under investigation. Here we show that stress hormones such as norepinephrine lead to increased expression of IL-6 mRNA and protein levels in ovarian carcinoma cells. Furthermore, we demonstrate that norepinephrine stimulation activates Src tyrosine kinase and this activation is required for increased IL-6 expression. These results demonstrate that stress hormones activate signaling pathways known to be critical in ovarian tumor progression.

Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer

There is growing evidence that chronic stress and other behavioral conditions are associated with cancer pathogenesis and progression, but the mechanisms involved in this association are poorly understood. We examined the effects of two mediators of stress, norepinephrine and epinephrine, on the activation of signal transducer and activator of transcription-3 (STAT3), a transcription factor that contributes to many promalignant pathways. Exposure of ovarian cancer cell lines to increasing concentrations of norepinephrine or epinephrine showed that both independently increased levels of phosphorylated STAT3 in a dose-dependent fashion. Immunolocalization and ELISA of nuclear extracts confirmed increased nuclear STAT3 in response to norepinephrine. Activation of STAT3 was inhibited by blockade of the beta1- and beta2-adrenergic receptors with propranolol, and by blocking protein kinase A with KT5720, but not with the alpha receptor blockers prazosin (alpha1) and/or yohimbine (alpha2). Catecholamine-mediated STAT3 activation was not inhibited by pretreatment with an anti-interleukin 6 (IL-6) antibody or with small interfering RNA (siRNA)-mediated decrease in IL-6 or gp130. Regarding the effects of STAT3 activation, exposure to norepinephrine resulted in an increase in invasion and matrix metalloproteinase (MMP-2 and MMP-9) production. These effects were completely blocked by STAT3-targeting siRNA. In mice, treatment with liposome-incorporated siRNA directed against STAT3 significantly reduced isoproterenol-stimulated tumor growth. These studies show IL-6-independent activation of STAT3 by norepinephrine and epinephrine, proceeding through the beta1/beta2-adrenergic receptors and protein kinase A, resulting in increased matrix metalloproteinase production, invasion, and in vivo tumor growth, which can be ameliorated by the down-regulation of STAT3.

Intraperitoneal delivery of liposomal siRNA for therapy of advanced ovarian cancer

PURPOSE. Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows efficient intravenous delivery to tumor tissue, and has therapeutic efficacy in pre-clinical proof-of-concept studies using EphA2-targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as intravenous delivery. EXPERIMENTAL DESIGN. SiRNA was incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Alexa555-siRNA-DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared. RESULTS. Alexa555-siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2-targeting siRNA-DOPC reduced tumor growth by 48-81% compared to paclitaxel/control siRNA-DOPC IP (HeyA8: 0.34g v 0.66g; SKOV3ip1: 0.04 v 0.21, p

Therapeutic significance of elevated tissue transglutaminase expression in pancreatic cancer

Purpose: Tissue transglutaminase (TG2) is a multifunctional protein that is implicated in development of drug resistance and metastasis. Therefore, we examined therapeutic targeting of TG2 for inhibiting growth and metastasis of in vivo growing pancreatic ductal adenocarcinoma (PDAC) in nude mice. Experimental Design: We implanted Panc-28 pancreatic cancer cells to induce orthotopic PDAC tumors in nude mice and determined the efficacy of liposomal TG2 small interfering RNA (siRNA) either alone or in combination with gemcitabine. Results: We show that down-regulation of endogenous TG2 by siRNA could effectively block the growth of PDAC. Moreover, down-regulation of TG2 significantly enhanced the therapeutic efficacy of gemcitabine against PDAC and inhibited metastatic spread of the disease. The antitumor activity was related to inhibition of proliferation, angiogenesis, and Akt phosphorylation. Conclusion: siRNA-mediated down-regulation of TG2 represents a promising therapeutic approach for improved treatment of PDAC.

Biochemotherapy in Patients with Metastatic Anorectal Mucosal Melanoma

Patients with metastatic anorectal melanoma generally have an unfavorable prognosis, but no effective systemic therapy has been reported.

Rational Drug Redesign to Overcome Drug Resistance in Cancer Therapy: Imatinib Moving Target

Protein kinases are central targets for drug-based cancer treatment. To avoid functional impairment, the cell develops mechanisms of drug resistance, primarily based on adaptive mutations. Redesigning a drug to target a drug-resistant mutant kinase constitutes a therapeutic challenge. We approach the problem by redesigning the anticancer drug imatinib guided by local changes in interfacial de-wetting propensities of the C-Kit kinase target introduced by an imatinib-resistant mutation. The ligand is redesigned by sculpting the shifting hydration patterns of the target. The association with the modified ligand overcomes the mutation-driven destabilization of the induced fit. Consequently, the redesigned drug inhibits both mutant and wild-type kinase. The modeling effort is validated through molecular dynamics, test tube kinetic assays of downstream phosphorylation activity, high-throughput bacteriophage-display kinase screening, cellular proliferation assays, and cellular immunoblots. The inhibitor redesign reported delineates a molecular engineering paradigm to impair routes for drug resistance.