Evan Gomes

Increased heating efficiency and selective thermal ablation of malignant tissue with DNA-encased multiwalled carbon nanotubes.

Nanoparticles, including multiwalled carbon nanotubes (MWNTs), strongly absorb near-infrared (nIR) radiation and efficiently convert absorbed energy to released heat which can be used for localized hyperthermia applications. We demonstrate for the first time that DNA-encasement increases heat emission following nIR irradiation of MWNTs, and DNA-encased MWNTs can be used to safely eradicate a tumor mass in vivo. Upon irradiation of DNA-encased MWNTs, heat is generated with a linear dependence on irradiation time and laser power. DNA-encasement resulted in a 3-fold reduction in the concentration of MWNTs required to impart a 10 degrees C temperature increase in bulk solution temperature. A single treatment consisting of intratumoral injection of MWNTs (100 microL of a 500 microg/mL solution) followed by laser irradiation at 1064 nm, 2.5 W/cm(2) completely eradicated PC3 xenograft tumors in 8/8 (100%) of nude mice. Tumors that received only MWNT injection or laser irradiation showed growth rates indistinguishable from nontreated control tumors. Nonmalignant tissues displayed no long-term damage from treatment. The results demonstrate that DNA-encased MWNTs are more efficient at converting nIR irradiation into heat compared to nonencased MWNTs and that DNA-encased MWNTs can be used safely and effectively for the selective thermal ablation of malignant tissue in vivo.

Unique dual targeting of thymidylate synthase and topoisomerase1 by FdUMP[10] results in high efficacy against AML and low toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy that leads to marrow failure and death. There is a desperate need for new therapies. The novel fluoropyrimidine, FdUMP[10], was highly active against both human AML cell lines, (IC(50) values, 3.4nM-21.5nM) and murine lines (IC(50) values, 123.8pM-131.4pM). In all cases, the IC(50) of FdUMP[10] was lower than for cytarabine and ∼ 1000 times lower than 5-fluorouracil (5-FU). FdUMP[10] remained effective against cells expressing the Flt3 internal tandem duplication, BCR-ABL, MN1, and an shRNA against p53. It had activity against patient samples at concentrations that did not affect normal hematopoietic cells. FdUMP[10] inhibited thymidylate synthase (TS) and trapped topoisomerase I cleavage complexes (Top1CCs), leading to DNA damage and apoptosis. All cell lines and nearly all primary AML samples examined expressed both TS and Top1. In vivo, FdUMP[10] was active against a syngeneic AML model with a survival advantage equivalent to doxorubicin plus cytarabine. 5-FU treatment was toxic and did not improve survival. FdUMP[10] was better tolerated than 5-FU or cytarabine plus doxorubicin and did not affect normal HSCs, while 5-FU dramatically impaired their ability to engraft. In summary, FdUMP[10] was highly efficacious and better tolerated than standard therapies.

Targeting the yin and the yang: combined inhibition of the tyrosine kinase c-Src and the tyrosine phosphatase SHP-2 disrupts pancreatic cancer signaling and biology in vitro and tumor formation in vivo.

Objectives Although c-Src (Src) has emerged as a potential pancreatic cancer target in preclinical studies, Src inhibitors have not demonstrated a significant therapeutic benefit in clinical trials. The objective of these studies was to examine the effects of combining Src inhibition with inhibition of the protein tyrosine phosphatase SHP-2 in pancreatic cancer cells in vitro and in vivo. Methods SHP-2 and Src functions were inhibited by siRNA or small molecule inhibitors. The effects of dual Src/SHP-2 functional inhibition were evaluated by Western blot analysis of downstream signaling pathways; cell biology assays to examine caspase activity, viability, adhesion, migration, and invasion in vitro; and an orthotopic nude mouse model to observe pancreatic tumor formation in vivo. Results Dual targeting of Src and SHP-2 induces an additive or supra-additive loss of phosphorylation of Akt and ERK-1/2 and corresponding increases in expression of apoptotic markers, relative to targeting either protein individually. Combinatorial inhibition of Src and SHP-2 significantly reduces viability, adhesion, migration, and invasion of pancreatic cancer cells in vitro and tumor formation in vivo, relative to individual Src/SHP-2 inhibition. Conclusions These data suggest that the antitumor effects of Src inhibition in pancreatic cancer may be enhanced through simultaneous inhibition of SHP-2.

Discovery of molecular biomarker signatures for the detection of bladder cancer.

302 Background: Bladder cancer (BCa) is among the five most common malignancies world-wide, and due to high rates of recurrence, one of the most prevalent. Improvements in non-invasive urine-based assays to detect BCa would benefit both patients and healthcare systems. In this study, the goal was to identify urothelial cell transcriptomic signatures associated with BCa. METHODS Gene expression profiling (Affymetrix U133 Plus 2.0 arrays) was applied to exfoliated urothelia obtained from a cohort of 92 subjects with known bladder disease status. Computational analyses identified candidate biomarkers of BCa and an optimal predictive model was derived. Selected targets from the profiling analyses were monitored in an independent cohort of 81 subjects using quantitative real-time PCR (RT-PCR). RESULTS Data analysis identified 52 genes associated with BCa (p≤0.001), and gene models that optimally predicted class label were derived. RT-PCR analysis of 48 selected targets in an independent cohort identified a 14-gene diagnostic signature that predicted the presence of BCa with a specificity of 100% at 90% sensitivity. CONCLUSIONS Exfoliated urothelia sampling provides a robust analyte for the evaluation of patients with suspected BCa. The refinement and validation of the multi-gene urothelial cell signatures identified in this preliminary study may lead to accurate, non-invasive assays for the detection of BCa. The development of an accurate, non-invasive BCa detection assay would benefit both the patient and healthcare systems through better detection, monitoring and control of disease.

Tumor burden reduction in an orthotopic non-muscle-invasive bladder cancer model using IL-15 analogue (ALT-803) targeting T regulatory cells.

298 Background: A recent NCI review listed IL-15 as the most promising product candidate among twelve immunotherapy drugs that could potentially cure cancer. Preclinical studies using ALT-803 (mutated IL-15 analogue combined with IL-15Rα-Fc fusion) have shown promising results for obtaining prolonged drug half-life and stimulating CD8+ T cells and NK cells. Based on these results, we hypothesized that the administration of ALT-803 will generate an immunologic response which will reduce tumor burden in a rodent carcinogen induced orthotopic non muscle invasive bladder cancer model (NMIBC). METHODS We tested intravesical ALT-803 alone and ALT-803 in combination with Bacillus Calmette-Guérin (BCG) in a rodent carcinogen induced orthotopic NMIBC model. Rats were anesthetized then a 22-gauge Teflon transurethral catheter was placed in the bladder and urine completely drained from the bladder. Next, the saline (negative control), ALT-803 (experimental agent) or BCG (positive control) therapy was delivered by transurethral instillation and allowed to dwell in the bladder for 1 hr by occlusion of the urethra with a purse string suture. The intravesical therapy was administered weekly for a total of six weeks to mimic intravesical BCG therapy in humans. RESULTS Herein we demonstrate that ALT-803 was safe and well tolerated alone or in combination with BCG. Furthermore, ALT-803 alone reduced tumor burden by 23% whereas BCG alone reduced tumor burden by 11% compared to control. The combination of ALT-803 and BCG reduced tumor burden by 30% compared to control. Tumoral responses of the combinational treatment were associated with 76% and 80% reduction in angiogenesis and proliferation, respectively, whereas combinational therapy was associated with a 7.7-fold increase in apoptotic index compared to control. Immune monitoring suggested that the antitumor response was linked to the activation of CD8+ cells and NK cells. CONCLUSIONS The enhanced therapeutic index provided by ALT-803 plus BCG therefore provides a powerful justification for the development of this agent for future clinical trials in subjects with non-muscle invasive bladder cancer.

Induction of endothelial proliferation and angiogenesis through activating the ERK1/2/EGF pathway mediate by CXC chemokine receptor 2 by chemokine (C-X-C motif) ligand 1.

138 Background: Endothelial cell growth and proliferation are critical for tumoral angiogenesis. We report here that blockade of Chemokine (C-X-C motif) ligand 1 (CXCL1) results in reduction of human endothelial cell proliferation and its ability to induce angiogenesis. METHODS Two human endothelial cell lines, HUVEC and HDMEC, were used in the in vitro assays. Proliferation assay and matrigel tube formation assay were performed to test the inhibitory effect of anti-CXCL antibody on the activity of endothelial cells in vitro. Matrigel plug assay in nude mice was performed to test the in vivo angiogenic activity of CXCL1. RESULTS CXCL1 interacts with its receptor CXC chemokine Receptor 2 and induces endothelial cell proliferation, whereas blockade of CXCL1 is associated with reduction in cellular proliferation through a decrease in levels of cyclin D and cdk4 and inhibition of angiogenesis through EGF and ERK 1/2. Targeting CXCL1 inhibits neoangiogenesis but has no effect on disrupting established vasculature. Furthermore targeting CXCL1 is associated with reduction in migration of human endothelial cells in an in vitro model. Additionally, neutralizing antibody against CXCL1 in a xenograft angiogenesis model resulted in inhibition of angiogenesis. CONCLUSIONS CXCL1-induced regulation of angiogenesis has not been studied extensively in human cancers, thus these findings illustrate a novel contribution of CXCL1 interactions in pathological angiogenesis. Therefore, the ability to selectively modulate CXCL1, specifically in tumoral angiogenesis, may promote the development of novel oncologic therapeutic strategies.

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

Abstract 2859: Dual targeting of protein tyrosine kinase c-Src and protein tyrosine phosphatase SHP-2 is a novel therapeutic strategy that induces potent inhibition of pancreatic cancer cell viability in vitro and tumor progression in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pancreatic adenocarcinoma is a deadly human malignancy, with over 95% of patients succumbing within five years. The majority of pancreatic cancer deaths are caused by metastatic dissemination of the primary tumor. The only curative treatment is surgical resection, which is limited by tumor stage, and the standard chemotherapeutic option, gemcitabine, offers only modest survival and quality of life benefits. There is a need for a greater understanding of pancreatic cancer biology and development of novel therapies to inhibit tumor progression and metastasis. The non-receptor protein tyrosine kinase, c-Src (Src) has emerged as a potential target for the treatment of pancreatic cancer, having been demonstrated to promote a number of tumorigenic processes. Src is overexpressed and/or aberrantly activated in greater than 70% of human pancreatic cancers. In addition, SHP-2, a non-receptor protein tyrosine phosphatase, regulates Src family kinase activity. Evidence has established clinical relevance for SHP-2 in human diseases. A recent study demonstrated that inhibition of SHP-2 abrogates in vitro and in vivo angiogenesis and inhibits activity of the MAPK/Erk1/2 and PI3K/Akt survival pathways. SHP-2 and Src function as key signaling intermediates downstream of growth factor receptors, cytokine receptors, and integrins, and are crucial for activation of downstream cascades of tumor progression and metastasis. The purpose of this study was to determine the importance of SHP-2 and Src, for pancreatic cancer signaling, cell biology, tumorigenicity, and metastasis. We have demonstrated elevated SHP-2 expression and phosphorylation in pancreatic cancer cells, relative to normal pancreatic cells. Phosphorylation of SHP-2 in pancreatic cancer cells occurs in a Src kinase-dependent fashion. Functional inhibition of SHP-2 and Src, individually or in combination, resulted in reduced ERK-1/2 phosphorylation, increased cleavage of caspase 3 and PARP, increased expression of the pro-apoptotic protein Bax, and decreased levels of the anti-apoptotic protein Bcl-xL. These results were mirrored by decreased viability and increased caspase activity in the presence of Src and SHP-2 inhibition. Finally, treatment of mice with small molecule inhibitors of Src or SHP-2, produced markedly smaller pancreatic tumors relative to controls in an orthotopic nude mouse model and dual treatment exacerbated this effect. The potential use for the Src and SHP-2 signaling axes as therapeutic targets remains largely untapped. These data suggest the importance of SHP-2 in pancreatic cancer cell biology and its potential value as a therapeutic target. Our studies suggest that the anti-tumor/anti-metastatic effects of Src inhibition may be improved through simultaneous inhibition of SHP-2. 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 2859. doi:1538-7445.AM2012-2859

Abstract 1237: Role of Src in resistance to anoikis in detachedpancreatic cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic adenocarcinoma is an aggressive malignancy currently ranked as the fourth leading cause of cancer related death in the United States, with over 95% of patients succumbing to the disease within 5 years of diagnosis. The vast majority of pancreatic cancer deaths are due to metastatic dissemination of the primary tumor. The non-receptor protein tyrosine kinase, c-Src (Src) has emerged in recent years as a potential target for pancreatic cancer treatment. In vitro studies have demonstrated the importance of Src for pancreatic tumor cell adhesion, migration, invasiveness, and resistance to apoptotic death. Mouse models of pancreatic cancer have indicated an important role for Src in formation of metastases. In addition, SHP-2, a non-receptor protein tyrosine phosphatase that regulates Src family kinase activity, has been recently shown to be a key mediator of various signaling pathways and evidence has established clinical relevance for SHP-2 in human diseases. Our goal is to determine the importance of Src for discrete steps in the progression to pancreatic cancer metastasis and resistance to detachment-induced cell death and the role of SHP-2 in modulating Src activity and downstream biology during this process. We have demonstrated a rapid and robust activation of Src in L3.6 human pancreatic cancer cells that have become detached from the extracellular environment, a crucial step in the development of metastases. Also, we have observed co-immunoprecipitation between Src and SHP-2 in detached cells, suggesting a potential role for SHP-2 in regulating Src activity in detached pancreatic cancer cells. In addition, our results reveal a Src-dependent activation of the Akt cell survival cascade and the stress kinase, Jun N-terminal kinase (JNK) in detached cells. Furthermore, this study confirmed that inhibition of both Src and SHP-2 in combination sensitizes L3.6 pancreatic cancer cells to anoikis in detached cells. We hypothesize that Src activity in detached pancreatic cancer cells promotes metastasis through suppression of anoikis. We will confirm these in vitro studies by testing the effects of inhibition of Src alone or in combination with SHP-2 inhibition on metastatic tumor formation using a nude mouse experimental metastasis model. The potential use for the Src and SHP-2 signaling axes as therapeutic targets for many cancers remains largely untapped. Our studies suggest that the anti-tumor and anti-metastatic effects of Src inhibition may be improved through simultaneous inhibition of SHP-2. 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 1237. doi:10.1158/1538-7445.AM2011-1237