Reduced Tumor Cell Proliferation Research Articles

KPT-330, a potent and selective exportin-1 (XPO-1) inhibitor, shows antitumor effects modulating the expression of cyclin D1 and survivin [corrected] in prostate cancer models.

Background and aimsIncreased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.Material and methodsWe evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.Results and conclusionsXPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.

Modulation of transforming growth factor‑β signaling transducers in colon adenocarcinoma cells induced by staphylococcal enterotoxin B.

Colorectal cancer (CRC) is a notable cause of cancer‑associated mortality worldwide, making it a pertinent topic for the study of cancer and its treatment. Staphylococcal enterotoxin B (SEB), an enterotoxin produced by Staphylococcus aureus, has been demonstrated to exert anticancer and antimetastatic effects due to its ability to modify cell immunity and cellular signaling pathways. In the current study, SEB was investigated, including whether it exerts its growth inhibitory effects on colon adenocarcinoma cells. This may occur through the manipulation of a key tumor growth factor, termed transforming growth factor‑β (TGF‑β), and its signaling pathway transducer, Smad2/3. The human colon adenocarcinoma HCT116 cell line was treated with different concentrations of SEB, and cell number was measured using MTT assay at different treatment times. Smad2/3 RNA expression level was analyzed in untreated or SEB‑treated cells using quantitative polymerase chain reaction, which indicated significant differences between cell viability and Smad2/3 expression levels. SEB effectively downregulated Smad2/3 expression in the HCT116 cells at concentrations of 1 and 2 µg/ml (P=0.0021 and P=0.0017, respectively). SEB concentrations that were effective at inhibiting Smad2/3 expression were correlated with those able to inhibit the proliferation of the cancer cells. SEB inhibited Smad2/3 expression at the mRNA level in a concentration‑ and time‑dependent manner. The present study thus proposed SEB as an agent able to significantly reduce Smad2/3 expression in colon cancer cells, provoking moderate TGF‑β growth signaling and the reduction of tumor cell proliferation.

IMPS-38CETUXIMAB THERAPY REQUIRES IMMUNE STIMULATION FOR EFFICACY IN PEDIATRIC GLIOBLASTOMA

Pediatric glioblastoma (GBM) patients have <10% 5-year survival rate, and numerous experimental therapeutic approaches have failed to significantly improve survival. Effective therapies will therefore require a better understanding of the tumor and its environment. Myeloid cells and t-cells are increased in GBM compared to normal brain tissue, and have been shown to be immunosuppressive. This immunosuppressive tumor microenvironment will negatively impact immunotherapeutic approaches, such as EGFR-targeted antibody treatments, notably EGFR-targeted cetuximab and nimotuzumab. This class of antibodies has shown modest activity in pediatric GBM and we propose that adjuvant immune stimulatory treatment, to reverse inherent immunosuppression, will be required for improved efficacy. Here we investigate the effect of cetuximab in GBM cell line/immune effector cell co-cultures with and without immune stimulating adjuvants, IL-2 and GM-CSF. Different GBM cell lines were co-cultured with HLA-matched allogenic peripheral blood mononuclear cells and treated with cetuximab, GM-CSF, IL-2, or combinations of these. Cetuximab alone had no effect on cytotoxicity. However, addition of immune stimulants to cetuximab resulted in cytotoxicity in GBM cells as measured by accumulation of cleaved caspase 3/7. Combination of cetuximab with both GM-CSF and IL-2 resulted in the greatest cytotoxicity. Additionally, and ex-vivo pediatric GBM organotypic slice culture model was used to better evaluate immunotherapeutic strategies. Organotypic slice cultures contain patient, therefore autologous, infiltrating myeloid cells and t-cells, providing a clinically relevant model of GBM. The slices were treated for 10 days and followed by flow cytometric measurement of proliferation and cell death. Cetuximab alone reduced growth of tumor cells and also reduced cell death. Combination therapy of GM-CSF, IL-2, and cetuximab, however, increased immune cell proliferation, and reduced tumor cell proliferation and increased tumor cell cytotoxicity. We suggest that cetuximab, along with any other therapeutic antibody approach, requires combination immune stimulation in the treatment of GBM.

Integrin β3 and LKB1 are independently involved in the inhibition of proliferation by lovastatin in human intrahepatic cholangiocarcinoma.

Human intrahepatic cholangiocarcinomas are one of the most difficult cancers to treat. In our study, Lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme-CoA (HMG-CoA) reductase inhibitor, demonstrated anticancer properties by inhibiting cancer cell proliferation, cell migration and cell adhesion. Lovastatin inhibited the expressions of transforming growth factor (TGF)-β1, cyclooxygenase (COX)-2, and intercellular adhesion molecule (ICAM)-1. Furthermore, lovastatin inhibited the expressions of integrin β1 and integrin β3 but not integrin αv or integrin β5. While Lovastatin's inhibitory effects on TGFβ1, COX2, and ICAM-1 expression were independently controlled by the tumor suppressor LKB1, integrin β3 expression was not affected. Lovastatin's inhibitory effect on cell adhesion was associated with the decreased expression of integrin β3 and cell surface heterodimer integrin αvβ3. Quantitative real time PCR, fluorescent microscopy, and cell migration assays all confirmed that Lovastatin inhibits integrin αvβ3 downstream signaling including FAK activation, and β-catenin, vimentin, ZO-1, and β-actin. Overall, Lovastatin reduced tumor cell proliferation and migration by modifying the expression of genes involved in cell adhesion and other critical cellular processes. Our study highlights novel anti-cancer properties of Lovastatin and supports further exploration of statins in the context of cholangiocarcinoma therapy.

Deletion of cyclooxygenase-2 inhibits K-ras-induced lung carcinogenesis.

The purpose of this study was to identify the role COX-2 plays in K-ras-induced lung carcinogenesis. We crossed COX-2-homozygous knockout mice with K-rasLA1 (G12D) expressing mice to obtain COX-2-deficient mice with K-ras expression (K-ras/COX-2(-/-) mice) and COX-2 wild type mice with K-ras expression (K-ras mice). At 3.5 months of age, the K-ras/COX-2(-/-) mice had significantly fewer lung adenocarcinomas and substantially smaller tumors than K-ras mice. K-ras/COX-2(-/-) mice also had significantly fewer bronchioalveolar hyperplasias than K-ras mice. Compared with lung tumors from K-Ras mice, the levels of prostaglandin E2 (PGE2) were significantly lower, whereas levels of the PGE2 metabolite 13,14-dihydro-15-keto-PGE2 were significantly higher, in lung tumors from K-ras/COX-2(-/-) mice. In addition, K-ras/COX-2(-/-) mice had strikingly lower rates of tumor cell proliferation and expressed less MEK and p-Erk1/2 protein than K-ras mice did. In line with this, knocking down COX-2 in mutant K-ras non-small cell lung cancer A549 cells reduced colony formation, PGE2 synthesis and ERK phosphorylation compared to that of vector control cells. Taken together, these findings suggest that COX-2 deletion contributes to the repression of K-ras-induced lung tumorigenesis by reducing tumor cell proliferation, decreasing the production of PGE2, and increasing the production of 13,14-dihydro-15-keto-PGE2, possibly via the MAPK pathway. Thus, COX-2 is likely important in lung tumorigenesis, and COX-2 and its product, PGE2, are potential targets for lung cancer prevention.

Abstract 1711: SLC6A15, a novel potential metabolic target for non-small cell lung cancer

Abstract Current therapeutic targets for non-small cell lung cancer consist only of signaling pathways targeting VEGF, EGFR or ALK. Recent researches have put focus on understanding cancer metabolism (such as 2-hydroxyglutarate accumulation in gliomas with mutant IDH1) and have opened a new avenue in cancer therapeutics which can complement the current practices. Here we introduce a new potential metabolic target for non-small cell lung cancer, SLC6A15. It is a trans-membrane protein known to facilitate uptake of branched chain amino acids (BCAA) (leucine, isoleucine and valine) and proline into the cells. BCAA was recently reported to increase cellular proliferation by regulating m-TOR in normal cells like beta-pancreatic cells. Here we show that these BCAA are highly enriched in lung tumor cell lines (H1299 &amp; H460) as well as in human lung tumor tissues as a result of overexpression of SLC6A15. We performed a transient knockdown of this transporter using siRNA for SLC6A15 and observed its effect on reduced clonogenicity of the two cancer cell lines by 3.3 and 1.6 fold, respectively. Further, gas chromatography mass spectrometry (GC-MS) analysis of the cells treated with si(SLC6A15) revealed selective reduction of BCAA levels by 1.3 to 1.5 fold but not in Proline, which can be synthesized in the cells unlike BCAA. These results support the role of these amino acids in tumor cell proliferation. Overexpression of SLC6A15 protein was confirmed by western blot analysis in 8 NSCLC cell lines and is indicative of its tumor specific nature. This finding is also supported by SLC6A15 gene expression data available on TCGA (The Cancer Genome Atlas), where 6 out of 10 patients show high expression of this gene. Therefore, we conclude that SLC6A15 is a new candidate of therapeutic target for NSCLC. Targeted inhibition may open new possibilities to specific reduction of tumor cell proliferation and possibly, increase life expectancy of patients suffering from NSCLC. Citation Format: PALLEVI SRIVASTVA, Hyuntae Yoo. SLC6A15, a novel potential metabolic target for non-small cell lung cancer. [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 1711. doi:10.1158/1538-7445.AM2015-1711

Abstract 895: Genetic deletion of cyclooxygenase-2 suppresses K-ras induced lung tumorigenesis

Abstract Lung cancer is the leading cause of cancer-related death worldwide. Proto-oncogene K-ras is mutated in 30-50% of lung adenocarcinomas, the most common histological subtype of the non-small cell lung cancer. Cyclooxygenases are the key enzymes in the conversion of arachidonic acid to prostaglandins (PGs) which are thought to promote tumor growth, angiogenesis and metastasis in various tumors including lung cancer. However, the role of cyclooxygenase 2 (COX-2) in lung tumorigenesis in mice carrying the K-ras oncogene remains unclear. We evaluated the contribution of COX-2 in lung tumorigenesis in K-rasLA1(G12D) mice and its relevant molecular mechanisms. We used a genetic knockout approach by crossing COX-2 homozygous mice (COX-2-/-) with a mouse strain which develops lung tumorigenesis driven by mutant K-ras (K-rasLA1) to obtain COX-2 deficient mice with K-ras expression (K-ras/COX-2-/-) and COX-2 wild type mice with K-ras expression (K-ras). Loss of COX-2 was associated with a significant reduction in both multiplicity and tumor volume of lung adenocarcinoma examined by ex-vivo MRI. The average number of lung nodules in K-ras/COX-2-/- mice was 6.8 per mouse which was significantly less than that of K-ras mice (19.8) (p&amp;lt;0.05). Similarly, tumor size in K-ras mice (65.6 ± 31.1 mm3) was larger than that of K-ras/COX-2-/- mice (25.5 ± 8.7 mm3). Histological examination of mouse lung demonstrated that there was a significantly higher incidence of bronchioalveolar hyperplasia in lung tissues of the K-ras group (9.5±3.2) than in the K-ras/COX-2-/- group (5.3±2.5) (p&amp;lt;0.05). In line with this, studying tumor cell proliferation by Ki67 staining suggested that COX-2 deletion in K-ras mice was associated with less lung tumor cell proliferative potential evidenced by Ki67 positive cells being significantly decreased from 6.2% in K-ras mice to 2.9% in K-ras/COX-2-/- mice. The level of PGE2 was notably lower in lung tumor tissue derived from K-ras/COX-2-/- mice than that of K-ras mice. Similarly, the urinary metabolite of PGE2, PGEM, was also significantly reduced by 3-fold in K-ras/COX-2-/- mice in comparison with K-ras mice. In contrast, level of 13,14-dihydro-15-keto-PGE2, a metabolite of PGE2 in K-ras/COX-2-/- mouse lung tumor tissue was significantly increased by almost 3-fold. Furthermore, MEK and p-ErK1/2 expressions significantly decreased in lung tissues of K-ras/COX-2-/- mice compare to that of K-ras mice. Together, our data suggest that COX-2 deletion contributes to repression of K-ras-induced lung tumorigenesis through reducing tumor cell proliferation rate, decreasing production of PGE2, and increasing 13,14-dihydro-15-keto-PGE2 in the tumors, which potentially involves the MAPK pathway. These observations suggested the importance of COX-2 in lung tumorigenesis, especially under K-ras mutation and further identified COX-2 as a potential target in lung cancer prevention. The study is supported by NIH Grant R01CA144053. Citation Format: Yong Pan, Yan Jiang, Lin Tan, Murali K. Ravoori, Mihai Gagea-Iurascu, Mihai Gagea-Iurascu, Vikus Kundra, Susan M. Fischer, Peiying Yang. Genetic deletion of cyclooxygenase-2 suppresses K-ras induced lung tumorigenesis. [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 895. doi:10.1158/1538-7445.AM2015-895

Abstract 2800: Dietary black raspberries (BRBs) inhibit tumor progression in PTEN-deficient mouse model of prostate cancer

Abstract Black raspberries (BRBs) a natural food demonstrated to have anti-oxidant, anti-inflammatory and anti-cancer activities,have been shown to inhibit oral, esophageal, mammary gland and colon cancers in rodents. Several human trials have been completed to date to assess the efficacy of BRB formulations for cancer prevention. However, the chemopreventive potential of BRBs against prostate cancer, the most commonly diagnosed malignancy and the second leading cause of death among men in the United States, is yet to be reported. Among the murine models, PTEN-mutant mice develop tumors in situ that faithfully mimic the intratumor heterogeneity observed during the progression of human prostate cancer. Therefore, we utilized this clinically relevant PTEN-mutant mouse model to evaluate the chemopreventive potential of BRBs against prostate cancer. Genotyped 5-week-old male PTEN-mutant mice, randomly assigned to control and treatment groups (n = 12 mice/group), were fed AIN-93G diet (control) or AIN-93G diet supplemented with BRBs (5 or 10%) for 23 weeks. Age-matched non-transgenic mice (wild-type) served as experimental controls (n = 6). Animal weight and food consumption were measured during the treatment period, and the mice were euthanized at 28 weeks of age. Prostate tissues were harvested, weighed and fixed in 10% formalin for histopathological analysis. Histological, cell proliferation (Ki-67 staining) and apoptosis (TUNEL) analyses were performed to determine the chemopreventive potential of BRBs. PTEN-mutant mice fed control or BRB (5 or 10%) diets had steady body weight gain, 16 to 18 g during the 23-week treatment. BRB diets were well tolerated as none of the animals fed BRBs exhibited any observable toxicity. PTEN-mutant mice (control) had increased prostate weight (ave. = 248 mg) relative to wild-type mouse prostate (ave. = 90 mg), and 5 and 10% BRB diets significantly (p&amp;lt;0.01) decreased the prostate weights to ave. = 116 and 162 mg, respectively, compared to control. In addition, 5% BRBs significantly (p&amp;lt;0.05) reduced the prostate weights compared to 10% BRBs. Histological examination of dorsolateral prostate (DLP) revealed that 98% of PTEN-mutant mice fed control diet developed invasive adenocarcinomas, whereas both 5 and 10% BRB diets significantly reduced tumor incidence and the progression of PIN lesions to invasive adenocarcinomas by 64% (p&amp;lt;0.0001) and 43% (p&amp;lt;0.001), respectively. Interestingly, 5% BRBs appear to be more effective than 10% BRBs (p&amp;lt;0.01). Both 5 and 10% BRBs significantly reduced tumor cell proliferation rates and induced apoptosis, p&amp;lt;0.0001 and p&amp;lt;0.001, respectively. Furthermore, mice fed both 5 and 10% BRBs contained more normal appearing prostates (free from PIN lesions and adenocarcinomas), when compared to control mice (54 and 33% compared to 3%, respectively). In conclusion, our data indicate that BRBs may have significant potential for prostate cancer prevention and/or treatment. Citation Format: Narayanan K. Narayanan, Gary D. Stoner, Daniel S. Peiffer, Karen Galdanes, Eric Larios, Alu Mark, Lisa Maziniski, Luis Chiriboga, Maarten C. Bosland. Dietary black raspberries (BRBs) inhibit tumor progression in PTEN-deficient mouse model of prostate cancer. [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 2800. doi:10.1158/1538-7445.AM2015-2800

Antineoplastic effects of Rhodiola crenulata treatment on B16-F10 melanoma.

Melanoma is an aggressive form of skin cancer with limited treatment options for advanced stage disease. Early detection and wide surgical excision remain the initial mode of treatment for primary tumors thus preventing metastases and leading to improved prognosis. Through this work, we have evaluated the antineoplastic effects of Rhodiola crenulata (R. crenulata) root extracts on the B16-F10 melanoma cell line, both in vitro and in vivo. We observed that R. crenulata treatment resulted in increased cell death as well as a reduction in tumor cell proliferation and migration in vitro. Additionally, we observed that R. crenulata decreased the expression of integrin β1 and vimentin and increased the expression of E-cadherin. Further, in mice treated with a topical R. crenulata-based cream therapy, tumors were more likely to have a radial growth pattern, a reduction in mitotic activity, and an increase in tumor necrosis. We also observed that mice drinking water supplemented with R. crenulata displayed a reduction of metastatic foci in disseminated models of melanoma. Collectively, these findings suggest that R. crenulata exhibits striking antitumorigenic and antimetastatic properties and that this extract may harbor potential novel adjuvant therapy for the treatment of melanoma.

Src family kinases differentially influence glioma growth and motility

Src-family kinase (SFK) signaling impacts multiple tumor-related properties, particularly in the context of the brain tumor glioblastoma. Consequently, the pan-SFK inhibitor dasatinib has emerged as a therapeutic strategy, despite physiologic limitations to its effectiveness in the brain. We investigated the importance of individual SFKs (Src, Fyn, Yes, and Lyn) to glioma tumor biology by knocking down individual SFK expression both in culture (LN229, SF767, GBM8) and orthotopic xenograft (GBM8) contexts. We evaluated the effects of these knockdowns on tumor cell proliferation, migration, and motility-related signaling in culture, as well as overall survival in the orthotopic xenograft model. The four SFKs differed significantly in their importance to these properties. In culture, Src, Fyn, and Yes knockdown generally reduced growth and migration and altered motility-related phosphorylation patterns while Lyn knockdown did so to a lesser extent. However the details of these effects varied significantly depending on the cell line: in no case were conclusions about the role of a particular SFK applicable to all of the measures or all of the cell types examined. In the orthotopic xenograft model, mice implanted with non-target or Src or Fyn knockdown cells showed no differences in survival. In contrast, mice implanted with Yes knockdown cells had longer survival, associated with reduced tumor cell proliferation. Those implanted with Lyn knockdown cells had shorter survival, associated with higher overall tumor burden. Together, our results suggest that Yes signaling directly affects tumor cell biology in a pro-tumorigenic manner, while Lyn signaling affects interactions between tumor cells and the microenvironment in an anti-tumor manner. In the context of therapeutic targeting of SFKs, these results suggest that pan-SFK inhibitors may not produce the intended therapeutic benefit when Lyn is present.

Combining radiation with autophagy inhibition enhances suppression of tumor growth and angiogenesis in esophageal cancer.

Radiotherapy is an effective treatment for esophageal cancer; however, tumor resistance to radiation remains a major biological problem. The present study aimed to investigate whether inhibition of autophagy may decrease overall tumor resistance to radiation. The effects of the autophagy inhibitor 3-methyladenine (3-MA) on radiosensitivity were tested in the EC9706 human esophageal squamous cell carcinoma cell line by colony formation assay. Furthermore, the synergistic cytotoxic effects of 3-MA and radiation were assessed in a tumor xenograft model in nude mice. Mechanistic studies were performed using flow cytometry, immunohistochemistry and western blot analysis. The results of the present study demonstrated that radiation induced an accumulation of autophagosomes and 3-MA effectively inhibited radiation-induced autophagy. Inhibition of autophagy was shown to significantly increase the radiosensitivity of the tumors in vitro and in vivo. The enhancement ratio of sensitization in EC9706 cells was 1.76 when the cells were treated with 10 mM 3-MA, alongside ionizing radiation. In addition, autophagy inhibition increased apoptosis and reduced tumor cell proliferation. The combination of radiation and autophagy inhibition resulted in a significant reduction in tumor volume and vasculature in the murine model. The present study demonstrated in vitro and in vivo that radiation-induced autophagy has a protective effect against cell death, and inhibition of autophagy is able to enhance the radiosensitivity of esophageal squamous cell carcinoma.

Optimal treatment scheduling of ionizing radiation and sunitinib improves the antitumor activity and allows dose reduction.

The combination of radiotherapy with sunitinib is clinically hampered by rare but severe side effects and varying results with respect to clinical benefit. We studied different scheduling regimes and dose reduction in sunitinib and radiotherapy in preclinical tumor models to improve potential outcome of this combination treatment strategy. The chicken chorioallantoic membrane (CAM) was used as an angiogenesis in vivo model and as a xenograft model with human tumor cells (HT29 colorectal adenocarcinoma, OE19 esophageal adenocarcinoma). Treatment consisted of ionizing radiation (IR) and sunitinib as single therapy or in combination, using different dose-scheduling regimes. Sunitinib potentiated the inhibitory effect of IR (4 Gy) on angiogenesis. In addition, IR (4 Gy) and sunitinib (4 days of 32.5 mg/kg per day) inhibited tumor growth. Ionizing radiation induced tumor cell apoptosis and reduced proliferation, whereas sunitinib decreased tumor angiogenesis and reduced tumor cell proliferation. When IR was applied before sunitinib, this almost completely inhibited tumor growth, whereas concurrent IR was less effective and IR after sunitinib had no additional effect on tumor growth. Moreover, optimal scheduling allowed a 50% dose reduction in sunitinib while maintaining comparable antitumor effects. This study shows that the therapeutic efficacy of combination therapy improves when proper dose-scheduling is applied. More importantly, optimal treatment regimes permit dose reductions in the angiogenesis inhibitor, which will likely reduce the side effects of combination therapy in the clinical setting. Our study provides important leads to optimize combination treatment in the clinical setting.

MTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma.

Background:Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models.Methods:Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used.Results:In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg−1) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis.Conclusions:Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.

Combination treatment with doxorubicin and microRNA-21 inhibitor synergistically augments anticancer activity through upregulation of tumor suppressing genes.

Doxorubicin (DOX) is a key chemotherapeutic drug for cancer treatment. The antitumor mechanism of DOX is its action as a topoisomerase II poison by preventing DNA replication. Our study shows that DOX can be involved in epigenetic regulation of gene transcription through downregulation of DNA methyltransferase 1 (DNMT1) then reactivation of DNA methylation-silenced tumor suppressor genes in glioblastoma (GBM). Recent evidence demonstrated that microRNA (miR or miRNA) can mediate expression of genes through post-transcriptional regulation and modulate sensitivity to anticancer drugs. As one of the first miRNAs detected in the human genome, miR-21 has been validated to be overexpressed in GBM. Combination treatment of a chemotherapeutic and miRNA showed synergistically increased anticancer activities which has been proven to be an effective strategy for tumor therapy. In our study, co-treatment of DOX and miR-21 inhibitor (miR-21i) resulted in remarkably increased expression of tumor suppressor genes compared with DOX or the miR-21i treatment alone. Moreover, we demonstrate that combining DOX and miR-21i significantly reduced tumor cell proliferation, invasion and migration in vitro. Our study concludes that combining DOX and miR-21i is a new strategy for the therapy of GBM.

Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles

Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not β-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

Paclitaxel-loaded PEG-PE-based micellar nanopreparations targeted with tumor-specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors.

In an effort to improve the therapeutic index of cancer chemotherapy, we developed an advanced nanopreparation based on the combination of landscape phage display to obtain new targeting ligands with micellar nanoparticles for tumor targeting of water-insoluble neoplastic agents. With paclitaxel as a drug, this self-assembled nanopreparation composed of MCF-7-specific phage protein and polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles showed selective toxicity to target cancer cells rather than nontarget, non cancer cells in vitro. In vivo, the targeted phage micelles triggered a dramatic tumor reduction and extensive necrosis as a result of improved tumor delivery of paclitaxel. The enhanced anticancer effect was also verified by an enhanced apoptosis and reduced tumor cell proliferation following the treatment with the targeted micellar paclitaxel both in vitro and in vivo. The absence of hepatotoxicity and pathologic changes in tissue sections of vital organs, together with maintenance of overall health of mice following the treatment, further support its translational potential as an effective and safe chemotherapy for improved breast cancer treatment.

Abstract 4545: Anticancer effects and mechanism of VR23, a novel chloroquine derivative

Abstract Chloroquine, a worldwide used drug in treatment of malaria, rheumatoid and other human diseases, has recently been focused as a potential anti-cancer drug as well as a chemo-sensitizer when given in combination with other anti-cancer drugs. In cancer treatment, chloroquine possesses a major potential therapeutic advantage in which it specifically targets cancer cells. Unfortunately, however, its anticancer activity is rather low. To further improve its efficacy and specificity in killing cancer cells, we created and characterized several chloroquine derivatives using a hybrid pharmacophore approach, which led us to identify VR23 as the most promising compound. Our in-vitro model of cancer cells shows that VR23 functions as a novel proteasome inhibitor. It also deregulates activity of cyclin E and other centrosomal proteins, resulting in induction of multiple centrosome amplification, abnormal spindle formation, uneven cytokinesis, irreversible mitotic arrests, and eventually apoptosis. These VR23-induced phenomena occur in cancer-specific manner and cause massive death in a variety of cancer cell types. The treatment of engrafted animals with VR23 (30 mg/kg) twice per week for three weeks effectively inhibits tumor growth. Interestingly, administration of VR23 following 24 hours pre-treatment with paclitaxel (20mg/kg/week) enhances paclitaxel's anti-tumor activity by 70%. Our histological data shows that VR23 causes substantial decrease in mitotic index, inhibition of tumor infiltration into surrounding tissues and remarkable reduction of tumor cell proliferation and angiogenesis. Most importantly, VR23 shows very low toxicity to vital organs (liver, spleen, kidney and lung) of non-tumor-bearing nude mice. In addition, VR23 seems to reduce paclitaxel-induced toxicity when used in combination. Taken together, VR23, our novel chloroquine derivative and newly discovered proteasome inhibitor, appears to be an effective and safe anticancer therapeutic alone or in combination with other anticancer agents. Citation Format: Hai-Yen Thi Vu, Sheetal Pundir, Raja V. Solomon, Hoyun Lee. Anticancer effects and mechanism of VR23, a novel chloroquine derivative. [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 4545. doi:10.1158/1538-7445.AM2014-4545

Abstract 4702: Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic cancer cells

Abstract Caveolin-1 (Cav-1) is a 21kDa protein found in 50-100nm omega-shaped invaginations of the plasma membrane known as caveolae. It has been implicated in the processes of oncogenic cell transformation, tumorigenesis and metastasis. Cav-1 is upregulated in pancreatic cancer and has been shown to be correlated with poor prognosis. We hypothesized that Cav-1 promotes resistance to chemotherapy and radiation, and that downregulation may lead to sensitization to radiation and/or chemotherapy. We found that radiation and chemotherapy up-regulate Cav-1 expression, and that downregulating Cav-1 in multiple pancreatic cancer cell lines by small interfering RNA induces sensitization to chemotherapeutics such as gemcitabine and 5-fluorouracil. Furthermore, genetic knockdown causes reduction in tumor cell proliferation and colony formation. Knockdown of caveolin-1 also led to radiosensitization of tumor cells and resulted in delayed DNA repair as demonstrated by delayed pH2.AX foci resolution, increased mitotic catastrophe, as well as decreased formation of BRCA1 and DNAPK-cs foci in the nuclei of these cells. Furthermore, Cav-1 loss promoted cleavage of Caspase-9 and PARP after treatment with gemcitabine. These results indicate that knocking down Cav-1 in pancreatic cancer cells sensitizes them to radiation with delayed DNA damage repair and also to chemotherapeutics with the onset of apoptosis. In vivo, stable knockdown of Cav-1 attenuated the rate of tumor growth significantly compared to control tumors in subcutaneous xenograft pancreatic tumors. In summary we conclude that Cav-1 is responsible in promoting resistance to radiation and chemotherapy in pancreatic cancer cells and that downregulation of this protein leads to improved therapeutic response. Thus, it is possible that caveolin-1 may serve as a prognostic predictive biomarker in pancreatic cancer patients for response to radiation and chemotherapy. Citation Format: Moumita Chatterjee, Terence M. Williams. Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic 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 4702. doi:10.1158/1538-7445.AM2014-4702

Abstract 4605: STK17A is a potential therapeutic target in glioblastoma

Abstract Current targeted therapies for glioblastoma multiforme (GBM) fail to significantly improve clinical outcome. Identifying new molecular targets driving GBM tumorigenesis is imperative. Our previous study demonstrated that STK17A, a serine-threonine kinase in the death-associated protein family, is a bona fide p53 target gene. In silico analyses indicated that STK17A is highly overexpressed in GBM patients in a tumor grade-dependent manner. Furthermore, high STK17A expression correlated with poor clinical outcome and decreased survival of patients from multiple datasets. This correlation was independent of age, tumor subtype and known biomarkers such as EGFR, NF1, and IDH, suggesting STK17A may contribute to GBM development and progression. In vitro experiments confirmed increased mRNA and protein expression of STK17A in GBM cells compared to immortalized normal human astrocytes and other cancer types. ShRNA mediated STK17A knockdown in GBM cells decreased cell survival and sensitized cells to genotoxic stress. In addition, STK17A knockdown led to reduced tumor cell proliferation and clonogenicity, suggesting a tumor-promoting role for STK17A in GBM. Interestingly, STK17A depletion resulted in a cell morphological change from a spindle-like phenotype to a phenotype with a flattened, enlarged and more rounded shape that was associated with induction of actin stress fibers. This cytoskeleton remodeling was associated with impaired cell migration and invasion. In contrast STK17A overexpressing cells displayed a pronounced needle-like elongated phenotype. In addition genome-wide expression analysis of STK17A knockdown GBM cells revealed regulation of genes involved in glycolysis including PKM2, PGAM1 and HK2, suggesting that STK17A may also promote tumor growth and survival through regulating metabolism. Small molecule inhibitors that block the kinase activity of STK17A decreased cell survival of GBM cells cultured under both serum and serum-free conditions. Further investigation is required to understand the precise role of STK17A in GBM. Citation Format: Pingping Mao, Mary P. Jardine, Gilbert J. Rahme, Eric C. Yang, Janice Tam, Anita Kodali, Bijesh Biswal, Camilo E. Fadul, Arti B. Gaur, Mark A. Israel, Alexandre Pletnev, Michael Spinella. STK17A is a potential therapeutic target in glioblastoma. [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 4605. doi:10.1158/1538-7445.AM2014-4605

Abstract 926: Nitric oxide enhances tumor progression and disease aggressiveness in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is among the most malignant cancer, with 5-year survival of a mere 6%. Chronic inflammation is associated with an increased risk of PDAC. Nitric oxide (NO•) is an important mediator of immune and inflammatory responses and plays a role in tumorigenesis. The principal source of an increased and sustained level of NO• is inducible nitric oxide synthase (NOS2). In this study, we have tested the hypothesis that NO• enhances tumor progression in pancreatic cancer. We found that a higher NOS2 expression in tumors was associated with poor survival in resected PDAC patients (p=0.011). We then used a genetic strategy to investigate the role of NO• in the development and progression of PDAC by deleting NOS2 gene in a genetically engineered mouse model of pancreatic cancer (KPC mice) with pancreas specific activation of mutant KRAS and P53, which faithfully recapitulates the development and progression of human pancreatic cancer. NOS2-deficient KPC mice showed a longer survival as compared to the KPC littermates with wild type NOS2 (p&amp;lt;0.01). Tumors from KPC/NOS2-/- mice showed reduced tumor cell proliferation and increased apoptosis as indicated by a lower expression of Ki67 and a higher level of activated caspase-3, as compared with KPC mice. Furthermore, tumors from KPC/NOS2-/- mice were less vascularized as compared with KPC mice, as indicated by CD31 expression. Additionally, NOS2-deficiency led to a decreased inflammatory response by attenuation of macrophage recruitment in pancreatic tumors as indicated by a significantly lower expression of F4/80. However, in contrast FOXP3+ regulatory T-cells were increased in tumors from KPC/NOS2-/- mice. Further mechanistic analysis revealed a higher expression of ph-FOXO3 and nuclear ph-ERK in tumors from KPC mice as compared with KPC/NOS2-/- mice. Analysis of human PDAC samples showed a positive correlation between NOS2 and ph-FOXO3 expression (P=0.004). In summary, NOS2 gene expression in tumors is a candidate prognostic marker in resected pancreatic cancer patients, and NOS2/NO• signaling may contribute to the pancreatic tumor progression by inactivating FOXO3 in KPC mice. In this ongoing study, we are further investigating the mechanistic role of NO• in disease progression. Citation Format: Jian Wang, Peijun He, Matthias M. Gaida, Shouhui Yang, Aaron Schetter, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Harris G. Yfantis, Dong H. Lee, Jonathan M. Weiss, Jim Stauffer, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. Nitric oxide enhances tumor progression and disease aggressiveness in pancreatic cancer. [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 926. doi:10.1158/1538-7445.AM2014-926