Significant Decrease In Tumor Growth Research Articles

Antiproliferative effect of sweet orange peel and its bioactive compounds against human hepatoma cells, in vitro and in vivo

The ethyl acetate extracts from sweet orange peel (EESP) and its bioactive compounds against human hepatoma cells, in vitro and in vivo, is investigated. EESP (50–500µg/ml) exhibits significant cytotoxicity on HepG2 cells. This antiproliferative action is partly attributable to cell cycle arrest in the G0/G1 phase and apoptosis induction. EESP induces apoptosis through an increase in the Bax/Bcl-2 ratio, the down-regulation of mitochondrial membrane potential and the up-regulation of caspase-3. Excessive ROS generated by EESP contributes to apoptosis of HepG2 cells. According to the HPLC-DAD and HPLC-MS/MS analysis, nobiletin, tangeretin and another twelve compounds were present in EESP, which may account for its antiproliferative effect. When HepG2 cells are grown as xenografts in nude mice, the oral administration of EESP induces a significant decrease in tumor growth. Collectively, these results suggest that EESP could be a potential useful source for the future application of EESP in liver cancer therapy.

Dasatinib Changes Immune Cell Profiles Concomitant with Reduced Tumor Growth in Several Murine Solid Tumor Models

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157-69. ©2017 AACR.

Gene delivery of apoptin-derived peptide using an adeno-associated virus vector inhibits glioma and prolongs animal survival

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of the secreted HSP70-targeted peptide APOPTIN derived from Apoptin to GBM tumors. We applied this therapy to GBM models using human U87MG glioma cells and GBM xenograft models in mice. In U87MG and U251MG cells, conditioned medium from AAV2-apoptin-derived peptide (ADP)-expressing cells induced 83% and 78% cell death. In mice bearing intracranial U87MG tumors treated with AAV2-ADP, treatment resulted in a significant decrease in tumor growth and longer survival in mice bearing orthotopic invasive GBM brain tumors. These data indicate that ssAAV2-ADP injection in the left hemisphere effectively prevented ipsilateral tumor growth but was insufficient to prevent distal tumor growth in the contralateral hemisphere. However, the systemic route is the most effective approach for treating widely dispersed tumors. In summary, systemic delivery of AAV2-ADP is an attractive approach for invasive GBM treatment.

TTI-281, an Orally Bioavailable Bromodomain and Extraterminal Domain (BET) Inhibitor, Is a Promising Candidate for the Treatment of Multiple Myeloma

Although recent advances in the development of multiple myeloma (MM) therapies such as proteasome inhibitors and immunomodulatory agents have improved patient outcomes, MM remains incurable. Additional therapeutic agents with high efficacy, low toxicity and the convenience of oral administration are in high demand. BET inhibitors, such as JQ-1, have been considered as potential therapeutic agents for MM. In the present study, we report that TTI-281, an orally bioavailable BET inhibitor, displays anti-MM activity with a low toxicity profile in preclinical studies.First, TTI-281 was tested for binding and anti-tumor activity in vitro. BROMOscan and AlphaScreen assays demonstrated that TTI-281 bound to bromodomains of BRD2/BRD3/BRD4 with Kd values less than 10 nM. In MTS assays, TTI-281 inhibited the growth of MM cell lines (MM.1s, NCIH929, and RPMI-8826) with cell growth-inhibition (IC50) values less than 300 nM.Next, in vitro ADME screening and in vivo PK studies were conducted. Permeability assays using murine gastrointestinal epithelial cells indicated that TTI-281 had good permeability with little efflux liability (efflux ratio <1), suggesting favorable properties for oral absorption. Indeed, TTI-281 displayed excellent oral bioavailability in both mice and rats (93.1% and 91.8%, respectively). In addition, TTI-281 did not interfere with the metabolism of representative CYP isozyme substrates at concentrations up to 50 μM in pooled human liver microsomes. Data also suggested minimal potential for drug-drug interactions, allowing for the possible combination with first-line therapy to improve therapeutic and survival outcomes.Finally, TTI-281 was tested for anti-myeloma efficacy and tolerability in vivo. NOD-SCID mice (n=10/group) subcutaneously engrafted with the human myeloma cell line MM.1S were treated orally once daily for 21 days with different doses of TTI-281, vehicle control or the benchmark drug carfilzomib. TTI-281 reduced tumor growth in a dose-dependent manner in this MM xenograft model. At 30 mg/kg/day, TTI-281 led to a statistically significant decrease in tumor growth compared with the vehicle control and carfilzomib (reduced tumor volume: 67% after TTI-281 treatment vs 33% after carfilzomib treatment, p<0.0003). Furthermore, TTI-281 treatment was well tolerated, with no effect on body weight or other obvious toxicity.In summary, our preclinical data suggest that the orally available BET inhibitor TTI-281 has an excellent efficacy and safety profile, highlighting its potential as a promising drug candidate for myeloma therapy. DisclosuresWang:Trillium Therapeutics: Employment, Patents & Royalties. Choi:Trillium Therapeutics: Employment. Dove:Trillium Therapeutics: Employment, Patents & Royalties. Wang:Trillium Therapeutics: Employment. Schimmer:Novartis: Honoraria. Petrova:Trillium Therapeutics Inc: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Uger:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Slassi:Trillium Therapeutics: Employment, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

UM-164: A Potent c-Src/p38 Kinase Inhibitor with In Vivo Activity against Triple-Negative Breast Cancer.

c-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis. In the clinic, however, the limited efficacy and high toxicity of existing c-Src inhibitors have tempered the enthusiasm for targeting c-Src. We developed a novel c-Src inhibitor (UM-164) that specifically binds the DFG-out inactive conformation of its target kinases. We hypothesized that binding the inactive kinase conformation would lead to improved pharmacologic outcomes by altering the noncatalytic functions of the targeted kinases. We have analyzed the anti-triple-negative breast cancer (TNBC) activity of UM-164 in a comprehensive manner that includes in vitro cell proliferation, migration, and invasion assays (including a novel patient-derived xenograft cell line, VARI-068), along with in vivo TNBC xenografts. We demonstrate that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified that Src and p38 kinase families were potently inhibited by UM-164. We further demonstrate that dual c-Src/p38 inhibition is superior to mono-inhibition of c-Src or p38 alone. We demonstrate that UM-164 alters the cell localization of c-Src in TNBC cells. In xenograft models of TNBC, UM-164 resulted in a significant decrease of tumor growth compared with controls, with limited in vivo toxicity. In contrast with c-Src kinase inhibitors used in the clinic (1, 2), we demonstrate in vivo efficacy in xenograft models of TNBC. Our results suggest that the dual activity drug UM-164 is a promising lead compound for developing the first targeted therapeutic strategy against TNBC. Clin Cancer Res; 22(20); 5087-96. ©2016 AACR.

Cationic microRNA-delivering nanocarriers for efficient treatment of colon carcinoma in xenograft model.

Manipulation of tumor microRNAs (miRNAs) may offer novel avenues for treatment of cancer. However, development of safe, robust, non-viral delivery methods remains a main challenge to obtain the promise of gene therapy. The miR-145 is dysregulated in many cancers, including colon carcer, and further in vitro investigation established antiproliferative and proapoptotic roles of miR-145. Herein, we study a PLGA/PEI (poly (d, l-lactide-co-glycolide)/polyethylenimine)-mediated miRNA vector delivery system; the validation of the method was carried out using a colon cancer xenograft model with miR-145 vector encoding for the expression of miR-145 (pDNA). First, high-molecular-weight PEI (25000 Da) was conjugated with cetyl to formulate reducible cetylated PEI (PEI-cet), and then PEI-cet was introduced to PLGA suspension. Next, PLGA/PEI-cet was crosslinked with hyaluronic acid (HA) to facilitate cellular uptake of miRNA plasmid vector via HA receptor-mediated endocytosis. After local administration of PLGA/PEI/HA complexes, intact miRNA plasmid vectors were delivered into HCT-116 colon cancer cells and xenograft tumor-bearing mice, and significant antitumor effects were achieved. The results show that the HA-based miR-145 nanocarrier could efficiently facilitate cellular uptake and significantly enhance miR-145 expression in HCT-116 cells. Consequently, the increased miR-145 induced G1 cell cycle arrest, reduced tumor proliferation and increased apoptosis, inhibited HCT-116 cell migration and suppressed c-MYC expressions, a regulatory target of miR-145. Of particular importance is the significant decrease in tumor growth in the mice model of colon cancer with the targeting miR-145 delivery system. The results in this work show that miR-145 has been effectively delivered to colon carcinomas through a PLGA/PEI/HA vehicle, indicating a promising miRNA replacement therapy strategy.

668. Platelets Engineered to Express Interleukin-24 Inhibited Melanoma Tumor Growth in Mice

Background: Activated platelets secrete agents (e.g., cytokines) that can promote solid tumor growth & cancer metastasis. Previous studies showed that hematopoietic stem cells (HSC) can be genetically modified to induce platelets to express & secrete proteins to establish hemostasis in animal models of bleeding disorders. Interleukin-24 (IL-24) is a cytokine (normally produced by activated monocytes, macrophages & Thelper cells) with cytotoxic & anti-angiogenic activity preferentially towards cancer cells. Thus, we hypothesized it may be feasible use HSC gene transfer to target platelet synthesis, storage & secretion of IL-24 to inhibit tumor growth. Aims: To investigate if HSC gene transfer targeting IL-24 synthesis, storage & secretion from platelets can inhibit melanoma in mice. Methods: C57BlL/6 mice were transplanted with bone marrow transduced with a lentiviral construct encoding a megakaryocytespecific ITGA2B gene promoter driving synthesis of IL-24 gene. Four weeks after transplant, IL-24 protein was characterized in platelets by flow cytometry. Murine melanoma cells (1×106) were implanted in mice at 5 weeks after transplant. Tumor size was measured in IL24 & control groups using a digital caliper periodically for 4 weeks. Then mice were sacrificed to record tumor mass. Results: Flow cytometry showed that HSC gene transfer of murine bone marrow led to synthesis & storage of IL-24 in platelets. An elisa assay showed that activated platelets could secrete IL-24 in vitro. At 30 days after implant of melanoma cells, platelet IL24 mice displayed significantly smaller (≈50%) tumor size (550 ± 101mm3) & weight (599 ± 120 mg) compared to control mice with larger tumor size (1120 ± 114 mm3) & weight (1391 ± 134 mg) n≈5 mice/group (p<0.05) indicating that platelet IL24 inhibited tumor growth in vivo. Conclusion: HSC gene transfer can be utilized to induce synthesis, storage and secretion of anti-oncogenic agent IL24 in platelets. Melanoma tumor challenge in mice with platelet IL24 showed a significant decrease in tumor growth suggesting that platelets may serve as an ideal therapeutic vehicle to treat cancer.

PO-34 - Optimal doses of tinzaparin to reduce both cancer-associated thrombosis and tumor growth in a mouse model of ectopic pancreatic syngeneic tumor

In clinical studies, thromboprophylaxis with low-molecular-weight heparins (LMWHs) has been demonstrated to reduce the risk of venous thromboembolism and to improve outcomes in cancer patients. Moreover, preclinical models have previously suggested that LMWHs may also offer additional benefits through direct antitumor properties. However, the optimal doses of LMWHs that may prevent both cancer-related thrombosis and tumor development are yet unknown. The goal of this study was to determine the optimal doses of tinzaparin that may prevent both cancer-related thrombosis and tumor development in a syngeneic ectopic model of pancreatic cancer. The optimal doses of tinzaparin to generate a plasma anti-Xa activity >0.2IU/mL were determined in vivo following injection into wild type mice.The syngeneic ectopic model of cancer was induced in wild-type mice using the mouse pancreatic cancer cell line Panc02. Mice were injected daily with 200, 300IU/kg or 400IU/kg, or placebo from day 8 to 25 following tumor induction. Kinetics of thrombus formation and fibrin generation were determined in real time by digital real time intravital microscopy in mice bearing a tumor treated with tinzaparin or placebo. The growth of the tumor and the bleeding times were measured and compared in the different groups of mice. Plasma anti-Xa levels <0.2IU/mL were observed with tinzaparin doses ranging from 0 to 150IU/kg, whereas plasma anti-Xa activities >0.2IU/mL were obtained with >200IU/kg tinzaparin doses. At day 25 following tumor induction, the kinetics of thrombosis were not affected in mice treated with daily 200IU/kg tinzaparin compared to controls whereas it was strongly affected in mice treated with daily 300 and 400IU/kg tinzaparin. Interestingly, a significant decrease in tumor growth was observed in mice treated with 200, 300 and 400IU/kg tinzaparin in comparison to controls, with no significant difference between these groups. Bleeding times were similar to control mice in mice treated with 200IU/kg tinzaparin, but significantly increased in mice treated with 300IU/kg and 400IU/kg tinzaparin. At the dose of 200IU/kg, tinzaparin treatment significantly inhibits tumor growth but did not affect the thrombotic phenotype in mice developing a cancer. When 300 and 400IU/kg dose are used, tinzaparin treatment decreases both cancer-related thrombotic phenotype and tumor growth, but at the price of a significant increase in the bleeding time.

Abstract B19: MET and IL6 signaling in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers and is associated with advanced stage at diagnosis and poorer outcome compared to other breast cancer subtypes. There is an unmet need for targeted therapeutic strategies for TNBC patients since current treatment options are restricted to standard chemotherapy. Both receptor tyrosine kinase (RTK) and inflammatory signaling have been shown to promote cancer progression and are promising therapeutic targets. Our laboratory was the first to demonstrate that the MET receptor tyrosine kinase is highly expressed in TNBC. Hepatocyte growth factor (HGF), the MET ligand, is highly expressed in breast carcinoma and breast carcinoma-associated fibroblasts (CAFs) and is able to induce paracrine or autocrine MET signaling. MET/HGF signaling is also connected with the pro-inflammatory cytokine interleukin 6 (IL6). HGF and IL6 have been shown to interact to enhance invasion of lung cancer cells and progression of multiple myeloma. In breast cancer patients, high serum expression of HGF and IL6 distinguishes metastatic breast cancers. Nonetheless, there is a gap in knowledge as to whether MET and IL6 signaling pathways directly or indirectly interact and how MET/IL6 activation promotes TNBC progression. We are examining the novel concept that MET and IL6 signaling pathways act through a positive signaling feedback loop to drive TNBC progression. By understanding the interactions between these signaling networks, we will be able to design therapeutic strategies that target critical signaling nodes in TNBC. Analysis of gene expression profiles in the four molecular TNBC subtypes defined by Burstein et al. revealed that MET, HGF, and IL6 are expressed in each of the TNBC subtypes. Immunohistochemical analysis of HGF and IL6 expression in breast cancer tissues revealed significantly higher HGF and IL6 expression in TNBC compared to ER+ breast cancers. To determine the effect of MET and IL6 inhibition, we established Mammary Architecture and Microenvironment Engineering (MAME) 3D co-culture models of TNBC cells ± fibroblasts. In these models, TNBC cells have high MET expression, moderate to high IL6 expression, and minimal IL6 receptor (IL6R) expression; whereas the CAF cells have high HGF expression and moderate IL6R expression. Our preliminary studies revealed that an IL6 neutralizing antibody (siltuximab) reduced TNBC structure volumes relative to IL6 expression in the TNBC cells, whereas an IL6 receptor (IL6R) neutralizing antibody (tocilizumab) had no effect. These results correlate with IL6 and IL6R expression levels in TNBC cell lines. We evaluated the efficacy of MET inhibition using XL184 (cabozantinib) and observed that XL184 significantly inhibited TNBC growth, proliferation, and invasion of diverse TNBC cell lines, yet was ineffective against MET-negative breast cancer cells. We are currently evaluating the effect of HGF-mediated MET activation on IL6 signaling and inhibition in our 3D TNBC models. To evaluate the effect of MET and/or IL6 inhibition in vivo we utilized a novel xenograft mouse model that expresses human HGF (hHGFtg SCID). In TNBC cell lines MDA-MB-231 and HCC70, IL6R inhibition slowed tumor progression marginally, whereas MET inhibition with XL184 and the combination of XL184 + anti-IL6R drastically inhibited tumor growth. In a model of established tumor growth, we started treatment when tumor reached 500 mm3. Again we observed a significant decrease in tumor growth with XL184 treatment (p&amp;lt;0.005), but also observed a decrease in tumor growth with anti-IL6R treatment (p&amp;lt;0.03). In our current studies we are evaluating the effects of XL184 and siltuximab treatment in TNBC + CAF tumors. These studies will identify the RTK and inflammatory signaling networks that are critical for TNBC progression and are potential targets for combination therapy. Citation Format: Elizabeth A. Tovar, Mansoureh Sameni, Curt J. Essenburg, Anita Chalasani, Erik S. Linklater, David M. Cherba, Aruselvi Anbalagan, Mary E. Winn, Bonnie F. Sloane, Carrie R. Graveel. MET and IL6 signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B19.

Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival.

Abstract 2842: Personalized pre-clinical trials in BRAF inhibitor resistant patient derived xenograft models of melanoma identify c-Met as an effective second line combination therapy target

Abstract Melanoma has seen a remarkable improvement in the treatment of advanced disease. However, the majority of patients still relapse with the emergence of resistant disease. This is especially true for BRAF inhibitors, were multiple mechanisms of resistance have been uncovered. However, the lack of dependable models to develop approaches to overcome resistance, poses a real challenge for clinical decisions on second line therapies. In addition, combination therapies necessary to achieve long term responses increase the number of possibilities to be assessed. In this study, we developed patient derived xenograft (PDX) models from BRAF inhibitor resistant patients and, after characterization, tested rational second line combination therapies in co-clinical trials.Fresh tumor tissue from 12 patients after progression on vemurafenib or dabrafenib was implanted into immune deficient NSG mice. Once the tumor grafts were established, mice were continually dosed with PLX4720 to approximate clinical plasma levels to preserve the resistant phenotype. Xenografts were then characterized for genomic alterations and pathway activation status using a targeted sequencing array and a reverse phase protein array, respectively. We found at least 2 and up to 9 known deleterious alterations in all samples, many of which have been described as conferring resistance. The protein array clustered into two major groups: MAPK pathway activated and PI3K pathway activated, again consistent with previous reports. Integrating these data allowed us to design rational combination therapies for in vivo testing. We treated a PDX model with MET amplification and high phospho c-Met with the BRAF inhibitor LGX818 20mg/kg QD, the MEK inhibitor MEK162 3mg/kg QD, and the c-Met inhibitor INC280 25mg/kg QD (all p.o.). Although INC280 alone led to significant decrease in tumor growth, only the LGX818/ MEK162/ INC280 triple combination led to complete and sustained tumor regression in all mice. Further, we tested a second PDX model with a MET amplification (albeit broader and lower) but baseline pMET on the protein level. In this model, INC280 did not show any antitumor effect confirming the specificity for c-Met and the importance of validating genomic data.In summary, using our BRAF inhibitor resistant PDX model pipeline we were not only able to identify actionable alterations in all models, but also to test rational second line combination therapies in pre-clinical in vivo trials. Genomic data alone might not be sufficient to accurately predict therapy responses and protein arrays were a valuable tool to validate deleterious alteration calls. This study represents an important step towards improving personalized medicine in melanoma, and highlights the potential use of c-Met inhibitors in melanoma combination therapy in a defined subset of patients. Citation Format: Clemens Krepler, Min Xiao, Katrin Sproesser, Patricia Brafford, Batool Shannan, Marilda Beqiri, Wei Xu, Bradley Garman, Katherine L. Nathanson, Xiaowei Xu, Giorgos Karakousis, Gordon B. Mills, Yiling Lu, Giordano Caponigro, Markus Boehm, Malte Peters, Lynn Schuchter, Meenhard Herlyn. Personalized pre-clinical trials in BRAF inhibitor resistant patient derived xenograft models of melanoma identify c-Met as an effective second line combination therapy target. [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 2842. doi:10.1158/1538-7445.AM2015-2842

Arsenic trioxide sensitizes glioblastoma to a myc inhibitor.

Glioblastoma multiforme (GBM) is associated with high mortality due to infiltrative growth and recurrence. Median survival of the patients is less than 15 months, increasing requirements for new therapies. We found that both arsenic trioxide and 10058F4, an inhibitor of Myc, induced differentiation of cancer stem-like cells (CSC) of GBM and that arsenic trioxide drastically enhanced the anti-proliferative effect of 10058F4 but not apoptotic effects. EGFR-driven genetically engineered GBM mouse model showed that this cooperative effect is higher in EGFRvIII-expressing INK4a/Arf-/- neural stem cells (NSCs) than in control wild type NSCs. In addition, treatment of GBM CSC xenografts with arsenic trioxide and 10058F4 resulted in significant decrease in tumor growth and increased differentiation with concomitant decrease of proneural and mesenchymal GBM CSCs in vivo. Our study was the first to evaluate arsenic trioxide and 10058F4 interaction in GBM CSC differentiation and to assess new opportunities for arsenic trioxide and 10058F4 combination as a promising approach for future differentiation therapy of GBM.

Effect of baicalin-copper on the induction of apoptosis in human hepatoblastoma cancer HepG2 cells.

The medical properties of baicalin have been well known for many years. However, the discovery that baicalin in the presence of metal ions is more effective than baicalin alone changed the course of drug research. The present study was designed to investigate the effect and possible mechanism of apoptosis induced by baicalin-copper in a human hepatoblastoma cancer cell line (HepG2) and in vivo. This study demonstrated that baicalin-copper suppresses the proliferation of HepG2 cells in a dose-dependent manner. Intraperitoneal injection of baicalin-copper resulted in a significant decrease in tumor growth in xenografts in nude mice. Acridine orange staining and flow cytometry analysis demonstrated that baicalin-copper induced apoptosis in HepG2 cells and caused cells to arrest in G2-M phase of the cell cycle. Furthermore, baicalin-copper treatment significantly increased the Bax/Bcl-2 ratio and p38 levels, as well as decreased the expression of caspase-3, p-PI3K, p-Akt and p-mTOR (P < 0.01). All of the evidences above indicate that baicalin-copper induces apoptosis in HepG2 cells by down-regulating the PI3K/Akt/mTOR signaling pathway.

Evaluation of thymosin α 1 in nonclinical models of the immune-suppressing indications melanoma and sepsis

Objectives: Recent understanding of the complex pathophysiology of melanoma and severe sepsis suggests that immune-modulating compounds such as thymosin alpha 1 (INN: thymalfasin; abbreviated Ta1) could be useful in the treatment of these two unrelated immune-suppressing indications.Research design and methods: Three nonclinical murine models were utilized, including: i) a lung metastasis B16 model; ii) a B16-based tumor growth model; and iii) a cecal-ligation and puncture (CLP) sepsis model.Results: In the lung metastasis model, Ta1 treatment alone led to a 32% decrease in metastases (p < 0.05). Additionally, combinations of Ta1 and an anti-PD-1 antibody led to significantly fewer metastases than vehicle. In the tumor growth model, significant decreases in tumor growth were seen: 34% (p = 0.015) to 46% (p = 0.001) depending on the Ta1 dose. In the CLP sepsis model, Ta1 treatment showed a positive trend towards increased survival and decreased bacterial load. In this CLP model, Ta1 also appeared to have an effect on the levels of some biomarkers.Conclusions: All three models demonstrated a benefit after treatment with Ta1, with no evidence of toxicity. These initial pilot studies support the hypothesis that immune-suppressive indications, including sepsis and melanoma, may be treated with Ta1 alone or by Ta1 in combination with other immunotherapies.

Engineering Oncogenic Kinases to Activate Apoptotic Machinery: A Novel Rewiring of Bcr-Abl and PKCβII

Oncogenic kinases, such as Bcr-Abl, EGFR, and Src, are crucial for the initiation, maintenance and progression several cancers. Accordingly, these kinases have become a popular target for small molecule based inhibition. However, despite the development of 2nd and 3rd generation inhibitors, resistance to this therapy remains a significant clinical obstacle. Instead of attempting to develop a new inhibitor to block kinase signaling, we hypothesized that we could hijack the oncogenic kinase to activate a rewired 'pro-drug’, leading to the death or differentiation of the cancerous cell. As a model for this rewiring, we selected Bcr-Abl, an oncogenic kinase which is constitutively expressed and critical for chronic myeloid leukemia (CML) cell survival. For our pro-drug, we chose protein kinase C, isoform βII (PKCβII) whose activation has been shown to increase apoptosis and dendritic cell (DC) differentiation in CML cells. PKCβII is a member of the PKC family of serine/threonine kinases and is in an inactive state in the cytosol due to interactions between its pseudosubstrate and kinase domains. Upon activation, the pseudosubstrate domain releases the kinase domain, and PKCβII translocates from the cytoplasm to the plasma membrane. It has been previously reported that substitution of the alanine at position 25 in the pseudosubstrate domain for a phosphomimetic glutamic acid leads to the constitutive activation of PKCβII. To create a pro-drug from PKCβII, we substituted the alanine at position 25 for a phosphorylatable tyrosine (A25Y) along with the corresponding Bcr-Abl kinase target motif into the pseudosubstrate domain of PKCβII. This allows the Bcr-Abl tyrosine kinase to phosphorylate the introduced tyrosine on PKCβII, thereby activating it through an incorporation of a negative charge on the pseudosubstrate domain. We hypothesize that this PKCβII activation induces apoptosis, inhibits growth, and spurs the differentiation in transformed Bcr-Abl+ leukemic cells, while leaving Bcr-Abl-cells unaffected.Using molecular modeling techniques, we designed four mutated PKCβII constructs that contain elements of both the native PKCβII pseudosubstrate sequence as well as the Bcr-Abl kinase target motif yet do not affect the native folding of the protein. To determine whether these mutated constructs were activated by Bcr-Abl, we transfected them into both Bcr-Abl+ and Bcr-Abl- cells and used both confocal microscopy and ImageStream technology to assess their location. Transfection of both WT-PKCβII and mutant A25Y-PKCβII into Bcr-Abl- cells resulted in no significant difference in activation and membrane translocation, however when these constructs were transfected into Bcr-Abl+ cells, we observed a significant increase in activation and membrane translocation of mutant A25Y-PKCβII when compared to WT-PKCβII constructs (p<0.05). When Bcr-Abl was expressed in cells that did not previously have it, we saw that there was a significant increase in activation and membrane translocation for mutant A25Y-PKCβII when compared to WT-PKCβII transfected cells (p<0.05). Cells treated with the Bcr-Abl inhibitor imatinib resulted in a significant decrease in activation of A25Y-PKCβII mutant constructs, suggesting that this activation is dependent upon Bcr-Abl activity (p<0.05). We found that this rewiring produced no effect on the constitutive signaling of Bcr-Abl, as there were no significant differences in the levels of phospho-Stat5, phospho-Bcr, and phospho-CRKL between mutant A25Y-PKCβII and WT-PKCβII transfected cells. This rewiring lead to a significant decrease five days post transfection in the number of Bcr-Abl+ cells, with no differences in Bcr-Abl- cells when transfected with A25Y-PKCβII compared to WT-PKCβII (p<0.05). This decrease was found to be due to both a significant increase in apoptosis (p<0.05) and a decrease in proliferation (p<0.05) in A25Y-PKCβII transfected Bcr-Abl+ cells. Lastly, inducible expression of A25Y-PKCβII mutant constructs resulted in a significant decrease in tumor growth in a murine model of CML (p<0.05). These findings demonstrate that Bcr-Abl mediated activation of PKCβII is feasible and induces death in leukemic cells, and provides a novel mechanism by which oncogenic kinases can be re-wired to activate pro-death agents and can have broad therapeutic applications. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-45: VEGFR3 signaling facilitates mammary tumor cells to resist chemotherapy

Abstract Breast cancer is a deadly disease which accounts for more than 40,000 deaths each year in United States. With the availability of various treatment options, side effects and resistance associated with them is also becoming a major concern for the treatment of breast cancer. How breast cancer cells evade chemotherapy still remains a vast area for research. In this study, we proposed that mammary tumor cells resist cell death via VEGFR3 signaling pathway. To test our hypothesis, we used doxorubicin and paclitaxel resistant murine mammary tumor cells (Cl66-Dox and Cl66-Pac) which have been developed in our laboratory. When implanted in female Balb/c mice they form more aggressive tumor than the parent cells (Cl66) from which they have been derived. We observed that these resistant cells have higher expression of VEGFR3 receptor than parent cells at mRNA level. Treatment with VEGFR3 kinase inhibitor, MAZ51, Cl-66Dox and Cl66-Pac cells showed 50% growth inhibition at 10uM dose, suggesting VEGFR3 receptor activation as one of the mechanism for resistance. Moreover, when implanted in mice, tumor formed by resistant cells responded well to MAZ51 treatment with slower tumor growth in comparison to control group consistent with our in vitro data. However, when we treated mice bearing resistant tumors first with chemotherapeutic drug, docetaxel followed by MAZ51, we observed a significant decrease in tumor growth and tumor weight, examined upon endpoint analysis. These results suggest that combination treatment is more effective in inhibiting drug-resistant tumors growth in comparison to drug and inhibitor alone as determined by reduced tumor volume and weight in combination treatment group. Overall, our in vivo data showed that blocking VEGFR3 signaling pathway makes drug-resistant mammary tumor cells susceptible to chemotherapy suggesting the use of inhibitors for this signaling pathway to be beneficial for drug-resistant mammary tumors. Citation Format: Bhawna Sharma, Michelle L. Varney, Rakesh K. Singh. VEGFR3 signaling facilitates mammary tumor cells to resist chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2014-LB-45

Abstract 2989: High-dose, intermittent sunitinib as an alternative treatment strategy

Abstract Background: Increased exposure to sunitinib treatment is associated with improved outcome indicative of the importance of dose intensity in the efficacy/toxicity balance. The currently approved schedule (50 mg daily, 4 weeks on, 2 weeks off) precludes further dose intensification. We hypothesized that high doses of sunitinib would improve tumor response with limited toxicity, when applied intermittently. Methods: In vitro proliferation was studied with MTT assays, one week after 3-9 hrs exposure of a panel of cancer cell lines (786-0, HT29, MDA-MB231, HCC827, A431) to high (5 and 20uM) concentrations of tyrosine kinase inhibitors (TKIs), namely sunitinib, sorafenib, erlotinib, pazopanib and axitinib. Apo-ONETM Homogenous Caspase 3/7 Assay (Promega) was applied to detect activation of apoptosis. The chick embryo chorioallantoic membrane (CAM) in vivo model was employed to further test the efficacy of the new scheduling, where sunitinib was topically administered on HT29 tumors growing on the CAM either daily at the commonly used dose of 40 mg/kg or once weekly at 200 mg/kg. Microvessel density (MVD) was determined by CD31 immunohistochemical staining. Plasma sunitinib concentrations were determined by LC-MS-MS from refractory patients with solid tumors participating in a phase I clinical trial (classical 3+3 design, EudraCT No: 2012-005756-41) in which high-dose, intermittent sunitinib treatment is being studied. Results: Sunitinib was identified as one of the most potent inhibitors of cell growth from the panel of TKIs; 6 hrs exposure to 20 uM of sunitinib resulted in tumor cell death. This effect was tumor cell line-independent and mediated via activation of caspase 3/7 pathway. High dose treatment of CAM tumors resulted in significant decrease in tumor growth compared to the control (mean 0.17 mg vs. 0.23 mg respectively, p=0.04), irrespective of angiogenesis inhibition as denoted by lack of inhibitory effect on MVD. It also led to higher intratumoral sunitinib concentrations compared to the daily schedule (14.3 vs. 8 ng/mg tissue, respectively), while blood concentrations were identical between the two schedules, averaging at 170 ng/ml. Patients enrolled in the first cohort of the clinical trial received 200 mg sunitinib orally weekly. This schedule was well tolerated and no dose limiting toxicities occurred. Overall, sunitinib exhibited moderate interpatient variability. Maximum plasma concentrations on Day 1 averaged on 161 ng/ml (range 118-239.5 ng/ml) and were reached approximately 6 hours after administration. Plasma drug concentrations decreased to undetectable from one cycle to the other. We have proceeded to escalate the dose for the next cohort of patients. Conclusion: Optimization of therapy with targeted agents remains an unmet challenge. Thus far, high-dose, intermittent sunitinib is well tolerated. These findings open new avenues that might contribute to improved treatment with sunitinib. Citation Format: Maria Rovithi, Richard R. de Haas, Richard J. Honeywell, Johannes Voortman, Arjan W. Griffioen, Mariette Labots, Anne Marije Luik, Godefridus J. Peters, Henk J. Broxterman, Henk M.w. Verheul. High-dose, intermittent sunitinib as an alternative treatment strategy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2014-2989

Abstract 774: EGFR-regulated RGS expression contributes to paclitaxel resistance in human lung cancer cells

Abstract Lung cancer is one of the main causes of cancer-related death worldwide, and the 5-year survival of lung cancer is less than 20%. Paclitaxel is a widely used chemotherapeutic agent to treat a variety of cancers, but its therapeutic efficacy tends to be limited due to the development of drug resistance, raising the need to understand the molecular mechanisms underlying paclitaxel resistance. In the current study, we investigated the mechanisms of paclitaxel resistance in non-small cell lung cancers (NSCLCs). First, we established paclitaxel-resistant NSCLC cell lines, including H460 TxR and SK-MES TxR, by pulse- or prolonged treatment of paclitaxel. The IC50 value of paclitaxel-resistant cell lines was 50 fold less than that of parental cells. Upon paclitaxel treatment, H460 TxR and SK-MES TxR sublines experienced markedly greater anchorage-dependent and -independent colony forming abilities with reduced apoptosis than did the parental cell lines. Next, we investigated the underlying molecular mechanism of acquired resistance against paclitaxel. Through microarray analysis, we found 19 genes commonly-increased both in H460 TxR and SK-MES TxR. Among them, the expression of regulator of G-protein signaling (RGS) gene increased the most. Knockdown of RGS gene expression by siRNA sensitized the paclitaxel-resistant cells to the drug treatment in vitro. Furthermore, H460 TxR xenograft mice showed a significant decrease in tumor growth after combined treatment with siRGS and paclitaxel, implying the association of RGS with paclitaxel resistance in NSCLC. To identify the mechanisms involved in increase in RGS expression, we assessed the phosphorylation level of several kinases involved in cell proliferation and survival. Among them, epidermal growth factor receptor (EGFR) showed the most significant phosphorylation. EGFR inhibition by erlotinib down-regulated RGS expression in a transcriptional level, leading to the enhanced sensitivity to paclitaxel in H460 TxR. Taken together, our results indicate that RGS overexpression is a mediator of paclitaxel resistance in NSCLCs and represent the first link between RGS and chemoresistance. We suggest that RGS might be a potential target of therapeutic strategies for lung cancer patients with paclitaxel resistance. Citation Format: Shin-Hyung Park, Myung-A Sung, Ho-Young Lee. EGFR-regulated RGS expression contributes to paclitaxel resistance in human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 774. doi:10.1158/1538-7445.AM2014-774

Abstract 2403: Imaging of UVB and UVC-induced DNA damage repair in cancer cells in Gelfoam histoculture and minimal cancer in mice

Abstract We compared the DNA damage repair response of cancer cells after UVB or UVC irradiation. The DNA-damage repair response to UV irradiation was imaged in tumors growing in 3D Gelfoam® histoculture and in superficial tumors grown in mice. UV-induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. 53BP1 forms foci during DNA damage repair. A clonogenic assay initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1 h after UV irradiation, indicating the onset of DNA damage repair response. Induction of UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120 µm in MiaPaCa-2 cells on Gelfoam®, while UVC induced foci only to a depth of 40 µm. The MiaPaCa-2 cells irradiated by both UVB and UVC light in a skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls. Moreover, UVB-treatment had greater inhibition of tumor growth compared to UVC. Our results demonstrate that both UVB and UVC are useful tools for the treatment of residual cancer, and UVB was more effective than UVC in vivo, possibly due to greater tissue penetration of UVB because of its longer wavelength. Citation Format: Fuminari Uehara, Shinji Miwa, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Yukihiko Hiroshima, Shuya Yano, Mako Yamamoto, Yasunori Matsumoto, Elena V. Efimova, Robert M. Hoffman. Imaging of UVB and UVC-induced DNA damage repair in cancer cells in Gelfoam histoculture and minimal cancer in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2014-2403

Abstract 3374: Fasting chemosensitizes tumor cells by affecting their metabolism

Abstract Background/Aim The metabolic properties of cancer cells are significantly different from those of normal cells. Energy production in cancer cells is dependent on aerobic glycolysis, fatty acid synthesis and increased rates of glutamine metabolism. Emerging evidences indicate that these features are linked to therapeutic resistance in cancer treatment. Thus, novel therapeutic strategies able to modify tumor metabolism are warranted. Aim of this study is to investigate the effects and underlying mechanisms of fasting in combination with chemotherapy on tumor metabolism. Methods The in vitro citotoxicity of chemodrugs in combination with short-term-starvation (STS) was tested in various colon cancer cell lines by Trypan Blue staining and Annexin V apoptosis assay. Cancer cell proliferation by BrdUdr and CFSE staining was also evaluated. Reactive oxygen species production were evaluated by DCFDA staining. The effects of STS on the regulation of tumor metabolism were evaluated by 2-[(18)F]-Fluoro-2-deoxyglucose uptake. Expression of different proteins involved in metabolic and signaling pathways were studied by western blot and proteomic analyses. In vivo studies were performed using syngeneic BALB/c mice which were subcutaneously injected with CT26 cells. The in vivo STS protocol allowed mice to consume water but not food for 48 hours before chemotherapy. All mice were monitored daily for weight loss and side effects. Tumor growth was monitored every two days by caliper. The effects of STS in combination with chemotherapy (CT), in terms of glucose uptake/consumption and tumor growth inhibition were also evaluated by dynamic micro-Positron Emission Tomography (micro-PET). Results In vitro experiments showed that STS+CT maximally reduced cell viability and glucose uptake. A metabolic switch from anaerobic glycolysis to oxidative phosphorilation (OXPHOSPH) and beta-lipidic oxidation was induced by STS+CT. Down-regulation of PI3K/AKT pathway by STS+CT was found to be involved in the reduction of different glycolytic enzymes including hexokinases, phosphofructokinase and piruvate kinase. Furthermore, tumor cells treated with STS+CT exhibited a significant induction of respiratory chain complexes I, III, IV associated to a striking uncoupling between oxygen consumption which was induced and ATP synthesis that was reduced. This latter event was related to significant increase of ROS generation by STS+CT, leading to tumor cell apoptosis. Accordingly, in vivo experiments demonstrated that STS in combination with high-dose CT maximally reduced tumor glucose consumption in parallel with tumor cell growth. Conclusions Taken together, these results demonstrated that STS+CT affected tumor metabolism leading to a significant decrease of tumor growth. These findings open a novel scenario in cancer treatment. Citation Format: Giovanna Bianchi, Roberto Martella, Silvia Ravera, Andrea Petretto, Danilo Marimpietri, Laura Emionite, Selene Capitanio, Gianluca Bottone, Annamaria Orengo, Cecilia Marini, Gianmario Sambuceti, Vito Pistoia, Valter D. Longo, Lizzia Raffaghello. Fasting chemosensitizes tumor cells by affecting their metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2014-3374