Mian M.K. Shahzad

Paraneoplastic Thrombocytosis in Ovarian Cancer

BACKGROUND The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Sustained Small Interfering RNA Delivery by Mesoporous Silicon Particles

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA). Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma. Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the ανβ3 integrin–positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls. Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease. Clin Cancer Res; 16(15); 3910–22. ©2010 AACR.

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.

Surgical stress promotes tumor growth in ovarian carcinoma.

Purpose: Surgical stress has been suggested to facilitate the growth of preexisting micrometastases as well as small residual tumor postoperatively. The purpose of this study was to examine the effects of surgical stress on ovarian cancer growth and to determine underlying mechanisms responsible for increased growth. Experimental Design: To mimic the effects of surgery, we did a laparotomy or mastectomy under isoflurane inhalation on athymic nude mice 4 days after i.p. tumor cell injection. Propranolol infusion via Alzet pumps was used to block the influence of sympathetic nervous system activation by surgical stress. Results: In both HeyA8 and SKOV3ip1 models, the mice in the laparotomy and mastectomy groups had significantly greater tumor weight (P < 0.05) and nodules (P < 0.05) compared with anesthesia only controls. There was no increase in tumor weight following surgery in the β-adrenergic receptor–negative RMG-II model. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for β-adrenergic receptor signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and vascular endothelial growth factor expression, which were blocked by propranolol treatment. Conclusion: These results indicate that surgical stress could enhance tumor growth and angiogenesis, and β-blockade might be effective in preventing such effects.

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.

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.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma

Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results:ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH2-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. Conclusion: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

EphA2 Immunoconjugate as Molecularly Targeted Chemotherapy for Ovarian Carcinoma

BACKGROUND EphA2 is overexpressed in many types of human cancer but is absent or expressed at low levels in normal epithelial tissues. We investigated whether a novel immunoconjugate containing an anti-EphA2 monoclonal antibody (1C1) linked to a chemotherapeutic agent (monomethyl auristatin phenylalanine [MMAF]) through a noncleavable linker maleimidocaproyl (mc) had antitumor activity against ovarian cancer cell lines and tumor models. METHODS Specificity of 1C1-mcMMAF was examined in EphA2-positive HeyA8 and EphA2-negative SKMel28 ovarian cancer cells by antibody binding and internalization assays. Controls were phosphate-buffered saline (PBS), 1C1, or control IgG-mcMMAF. Viability and apoptosis were investigated in ovarian cancer cell lines and tumor models (10 mice per group). Antitumor activities were tested in the HeyA8-luc and SKOV3ip1 orthotopic mouse models of ovarian cancer. Endothelial cells were identified by use of immunohistochemistry and anti-CD31 antibodies. All statistical tests were two-sided. RESULTS The 1C1-mcMMAF immunoconjugate specifically bound to EphA2-positive HeyA8 cells but not to EphA2-negative cells and was internalized by HeyA8 cells. Treatment with 1C1-mcMMAF decreased the viability of HeyA8-luc cells in an EphA2-specific manner. In orthotopic mouse models, treatment with 1C1-mcMMAF inhibited tumor growth by 85%-98% compared with that in control mice (eg, for weight of HeyA8 tumors, 1C1-mcMMAF = 0.05 g and control = 1.03 g; difference = 0.98 g, 95% confidence interval [CI] = 0.40 to 1.58 g; P = .001). Even in bulkier disease models with HeyA8-luc cells, 1C1-mcMMAF treatment, compared with control treatment, caused regression of established tumors and increased survival of the mice (eg, 1C1-mcMMAF vs control, mean = 60.6 days vs 29.4 days; difference = 31.2 days, 95% CI = 27.6 to 31.2 days; P = .001). The antitumor effects of 1C1-mcMMAF therapy, in SKOV3ip1 tumors, for example, were statistically significantly related to decreased proliferation (eg, 1C1-mcMMAF vs control, mean = 44.1% vs 55.8% proliferating cells; difference = 11.7%, 95% CI = 2.45% to 20.9%; P = .01) and increased apoptosis of tumor cells (eg, 1C1-mcMMAF vs control, mean = 8.6% vs 0.9% apoptotic cells; difference = 7.7%, 95% CI = 3.8% to 11.7%; P < .001) and of mouse endothelial cells (eg, 1C1-mcMMAF vs control, mean 2.8% vs 0.4% apoptotic endothelial cells; difference = 2.4%, 95% CI = 1.4% to 4.6%; P = .034). CONCLUSION The 1C1-mcMMAF immunoconjugate had antitumor activity in preclinical models of ovarian carcinoma.