Growth Of Xenograft Tumors In Mice Research Articles

BAP1 suppresses lung cancer progression and is inhibited by miR-31

BRCA1-associated protein-1 (BAP1) is an important nuclear-localized deubiquitinating enzyme that serves as a tumor suppressor in lung cancer; however, its function and its regulation are largely unknown. In this study, we found that BAP1 protein levels were dramatically diminished in lung cancer tissues while its mRNA levels did not differ significantly, suggesting that a post-transcriptional mechanism was involved in BAP1 regulation. Because microRNAs (miRNAs) are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for miRNAs that could potentially bind BAP1. We predicted and experimentally validated miR-31 as a direct regulator of BAP1. Moreover, we showed that miR-31 promoted proliferation and suppressed apoptosis in lung cancer cells and accelerated the development of tumor growth in xenograft mice by inhibiting BAP1. Taken together, this study highlights an important role for miR-31 in the suppression of BAP1 in lung cancer cells and may provide insights into the molecular mechanisms of lung carcinogenesis.

Engineering of bacterial exotoxins for highly efficient and receptor-specific intracellular delivery of diverse cargos.

The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.

In vitro and in vivo studies on the inhibitory effects of myocardial cell culture medium on growth of a human lung adenocarcinoma cell line, A549.

Although the heart is one of the body's vital organs, with an abundant blood supply, metastasis to the heart is considered rare. In a previous study, we found that the myocardial microenvironment might contain a low molecular weight natural tumour suppressor. The present study was designed to investigate the inhibitory effect of cardiac myocyte-conditioned medium (cmcm) on the growth of A549 human lung adenocarcinoma cells in vitro and in vivo. An mtt assay was used to detect the inhibition ratio with respect to A549 proliferation. Human lung adenocarcinoma cells (A549 cell strain) were transplanted subcutaneously into nude mice to produce tumours. The xenograft tumour growth in mice was observed after selected drug administration. After treatment with cmcm and cisplatin (Cis), A549 cell viability significantly declined (p < 0.001). The cell viability in the cmcm and Cis groups were 53.42% ± 3.45% and 58.45% ± 6.39% respectively. Growth of implanted tumour cells in vivo was significantly inhibited in the cmcm group, the group treated with recombinant human adenovirus-p53, and the Cis-treated group compared with a control group. The inhibition rates were 41.44% in the cmcm group, 41.34% in the p53 group, and 64.50% in the Cis group. Lung metastasis capacity was significantly reduced in the presence of cmcm (p < 0.05). Lung metastasis inhibition rates in mice were 56.52% in the cmcm group, 47.83% in the p53 group, and 82.61% in the Cis group. With cmcm, the lives of A549-tumour-bearing mice could be significantly prolonged without any effect on weight loss. Use of cmcm has the effect of reducing A549 cell viability, tumour volume, and lung metastasis rate, while prolonging survival duration without severe toxicity.

Cell membrane capsules as biomimetic drug carriers

Event Abstract Back to Event Cell membrane capsules as biomimetic drug carriers Lijie Han1, Yuanhong Zhang1 and Zhengwei Mao1 1 Zhejiang University, Department of Polymer Science and Engieering, China Introduction: Systemic administration of chemotherapeutic agents results in indiscriminate drug distribution and severe toxicity. Until now, encapsulation and targeting of drugs have typically relied on synthetic vehicles, which cannot minimize clearance by the renal system and may also increase the risk of chemical side effects. Cell membrane capsules (CMCs) provide a generic and far more natural approach to the challenges of drug encapsulation and delivery in vivo[1],[2]. Results and Discussion: Here aptamer AS1411, which can recognize and bind over-expressed nucleolin on cancer cell membrane, was chemically conjugated onto CMCs. As a result, AS1411 modified CMCs showed enhanced ingestion in certain cancer cells in vitro and accumulation in mouse cancer xenografts in vivo. Chemotherapeutics and contrast agents with therapeutically significant concentrations can be packaged into CMCs by reversible permeating their plasma membranes. The systematic administration of cancer targeting CMCs loaded with doxorubicin hydrochloride can significantly inhibit tumor growth in mouse xenografts, with significantly reduced toxicity compare to free drug. These findings suggest that cancer targeting CMCs may have considerable benefits in drug delivery and cancer treatment. Conclusion: In this study, biocompatible cell membrane capsules were prepared from human endothelial cells with preserved membrane structure and functions. Cancer cell targeting aptamer AS1411 was conjugated onto the CMCs, resulted in enhanced ingestion of CMCs in certain cancer cells and accumulation of CMCs in mouse tumor xenografts. Chemotherapeutics and contrast agents can be packaged into the CMCs by reversible permeating their plasma membranes. The systematic administration of cancer targeting CMCs loaded with doxorubicin can inhibit tumor growth in mouse xenografts, with significantly reduced toxicity compare to free drug. These findings suggest that targeting CMCs may have considerable benefits in drug delivery and cancer treatment. Natural Science Foundation of China (51120135001); Key Science Technology Innovation Team of Zhejiang Province (2013TD02)

Abstract A75: Translational studies of a first-in-class FASN Inhibitor, TVB-2640, linking preclinical studies to clinical laboratory observations in solid tumor patients

Abstract Dysregulated expression of FASN is a central mediator of neoplastic lipogenesis. FASN catalyzes the production of palmitate, the building block of long chain fatty acids, providing a mechanism to convert glucose and other carbon sources into lipids needed for production of phospholipids, formation of lipid rafts and the subsequent assembly of receptor tyrosine kinases and other signaling molecules in microdomains juxtaposed to the membrane. Inhibition of FASN in tumor cells leads to disruption of PI3K/Akt/mTOR and other important pathways leading to apoptosis. Targeting this critical nexus between lipogenesis and cellular survival/proliferation signaling pathways makes inhibition of FASN a novel therapeutic approach with a strong biological rationale and 3-V Biosciences has initiated clinical studies with its first-in-class FASN inhibitor. 3-V Bio has developed a series of orally available, reversible, potent, and selective FASN inhibitors that remodel tumor cell membranes, disrupt pAkt and Wnt/beta-catenin pathways, and reprogram gene expression. These effects lead to apoptosis of tumor cells in culture, inhibition of anchorage-independent cell growth under lipid-rich conditions and inhibition of in vivo xenograft tumor growth in mice and rats. We are currently using transcriptomic and metabolomic analyses, including lipidmomic profiling, to explore the fundamental mechanisms resulting in sensitivity to the FASN inhibitor, TVB-3166. These same platforms are used to examine the changes in response to FASN inhibition in tumor and surrogate tissues of xenograft models as well as surrogate tissues of cancer patients. A panel of tumor and normal cell lines was assembled and lipids were assessed prior to and 72 hours after treatment with TVB-3166. A reduction of saturated fatty acids was observed in all cell lines indicative of FASN inhibition. A similar extent of reduction was observed in media in the presence of 1% or 10% serum; these results demonstrated that the tumor cells did not compensate for FASN inhibition by increasing uptake of saturated fatty acids from the extracellular milieu. An inverse correlation was observed between the pretreatment quantities of saturated fatty acids and the induction of apoptosis resulting from FASN inhibition with TVB-3166; low levels of saturated fatty acids in cell lines such as COLO-205, correlated with a high level of cell killing. Normal, untransformed cells had significantly higher levels of pretreatment saturated fatty acids and a commensurate insensitivity to TVB-3166. These in vitro observations were extended to a COLO-205 rat xenograft model. Significant tumor growth inhibition was measured in these animals in response to FASN inhibition by TVB-3166. Tumor tissues were isolated and a reduction in pAkt and c-Myc expression were observed, the latter consistent with a reduction of Wnt/beta-catenin driven transcription. Further metabolomic analyses of surrogate and tumor tissues from this model continue. The power of the metabolomic/lipidomic and transcriptomic analyses to evaluate drug responsiveness was applied to surrogate tissues of a small number of cancer patients treated with the FASN inhibitor, TVB-2640. Following 8 days of oral dosing, a preliminary evaluation of aqueous metabolites and lipids in patients' sera demonstrated changes consistent with the mechanism of FASN inhibition. Larger numbers of patients are being evaluated in order better characterize the extent of these changes. Preclinical evaluations of the molecular transformations caused by 3-V Bio's FASN inhibitors are being translated into clinical assessments that may ultimately lead to a more complete understanding of the impact of FASN inhibition in cancer patients and potentially the identification of tumor types that are likely to respond to treatment. Citation Format: Doug Buckley, Tim Heuer, Marie O'Farrell, Bill McCulloch, George Kemble. Translational studies of a first-in-class FASN Inhibitor, TVB-2640, linking preclinical studies to clinical laboratory observations in solid tumor patients. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A75.

Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression.

Bromodomain 4 (BRD4) is an epigenetic regulator that, when inhibited, has anti-cancer effects. In this study, we investigated whether BRD4 could be a target for treatment of human hepatocellular carcinoma (HCC). We show that BRD4 is over-expressed in HCC tissues. Suppression of BRD4, either by siRNA or using JQ1, a pharmaceutical BRD4 inhibitor, reduced cell growth and induced apoptosis in HCC cell lines while also slowing HCC xenograft tumor growth in mice. JQ1 treatment induced G1 cell cycle arrest by repressing MYC expression, which led to the up-regulation of CDKN1B (P27). JQ1 also de-repressed expression of the pro-apoptotic BCL2L11 (BIM). Moreover, siRNA knockdown of BIM attenuated JQ1-triggered apoptosis in HCC cells, suggesting an essential role for BIM in mediating JQ1 anti-HCC activity.

Fibrinogen-derived fibrinostatin inhibits tumor growth through anti-angiogenesis.

Angiogenesis is a prerequisite of tumor growth and metastasis and, thus, anti-angiogenesis treatment has become an important part of cancer therapy. A 15-amino acid peptide of the fibrinogen α chain, fibrinostatin, was previously found in serum samples of gastric cancer patients. Herein we demonstrated that fibrinostatin has anti-angiogenesis activity in several angiogenesis models and it reduces tumor growth in mouse xenografts and allografts. Increased tumor necrosis and reduced microvessel density in tumors were observed in mouse xenograft models. Fibrinostatin inhibited proliferation and induced apoptosis in HUVEC, but not in cancer cells. In addition, fibrinostatin specifically entered HUVEC. Fibrinostatin also prevented migration, adhesion and tubule formation of HUVEC in vitro. A single-dose acute toxicity testing and a repeated-dose chronic toxicity study in the mouse, rat and monkey indicated that fibrinostatin had a wide margin of safety. Taken together, fibrinostatin shows promise as a potential anti-angiogenesis therapeutic agent.

ZIC2-dependent OCT4 activation drives self-renewal of human liver cancer stem cells.

Liver cancer stem cells (CSCs) have been identified and shown to have self-renewal and differentiation properties; however, the biology of these hepatic CSCs remains largely unknown. Here, we analyzed transcriptome gene expression profiles of liver CSCs and non-CSCs from hepatocellular carcinoma (HCC) cells lines and found that the transcription factor (TF) ZIC2 is highly expressed in liver CSCs. ZIC2 was required for the self-renewal maintenance of liver CSCs, as ZIC2 depletion reduced sphere formation and xenograft tumor growth in mice. We determined that ZIC2 acts upstream of the TF OCT4 and that ZIC2 recruits the nuclear remodeling factor (NURF) complex to the OCT4 promoter, thereby initiating OCT4 activation. In HCC patients, expression levels of the NURF complex were consistent with clinical severity and prognosis. Moreover, ZIC2 and OCT4 levels positively correlated to the clinicopathological stages of HCC patients. Altogether, our results indicate that levels of ZIC2, OCT4, and the NURF complex can be detected and used for diagnosis and prognosis prediction of HCC patients. Moreover, these factors may be potential therapeutic targets for eradicating liver CSCs.

Cell Membrane Capsules for Encapsulation of Chemotherapeutic and Cancer Cell Targeting in Vivo.

Systemic administration of chemotherapeutic agents can cause indiscriminate drug distribution and severe toxicity. Until now, encapsulation and targeting of drugs have typically relied on synthetic vehicles, which cannot minimize the clearance by the renal system and may also increase the risk of chemical side effects. Cell membrane capsules (CMCs) provide a generic and far more natural approach to the challenges of drug encapsulation and delivery in vivo. Here aptamer AS1411, which can recognize and bind overexpressed nucleolin on a cancer cell membrane, was chemically conjugated onto CMCs. As a result, AS1411 modified CMCs showed enhanced ingestion in certain cancer cells in vitro and accumulation in mouse cancer xenografts in vivo. Chemotherapeutics and contrast agents with therapeutically significant concentrations can be packaged into CMCs by reversible permeating their plasma membranes. The systematic administration of cancer targeting CMCs loaded with doxorubicin hydrochloride can significantly inhibit tumor growth in mouse xenografts, with significantly reduced toxicity compared to free drug. These findings suggest that cancer targeting CMCs may have considerable benefits in drug delivery and cancer treatment.

Abstract 4446: Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis

Abstract Inhibition of de novo palmitate synthesis by fatty acid synthase (FASN) is a novel cancer therapeutic approach with strong biological rationale. Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. FASN expression increases with tumor progression in human tumors and associates with chemoresistance, metastasis, and diminished patient survival in many tumor types. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences that exhibit anti-tumor activity in diverse preclinical tumor models. FASN inhibition induces tumor cell apoptosis by remodeling tumor cell membranes, blocking signal transduction, and reprogramming gene expression. These effects lead to inhibition of anchorage-independent tumor cell growth under lipid-rich conditions and inhibition of in vivo xenograft tumor growth in mice and rats. Studies to understand the mechanisms of action and biological consequences of FASN inhibition are guiding the discovery of tumors highly dependent on FASN activity and biomarkers for assessment of pharmacodynamic activity and patient selection. Using quantitative genomics and a variety of directed analytical approaches, we identified lipid, mRNA, and protein profiles in tumor cells that change in response to FASN inhibition. Subsets of the changes show correlation with FASN inhibitor sensitivity. Marker candidates, such as gene expression signatures that classify in vitro sensitivity to FASN inhibition, are being investigated for detection in clinical human tumor data sets. Our studies also identified FASN inhibitor-mediated mechanisms of action that function in specific tumor types and biomarkers with potential utility for selecting responsive patients and measuring tumor response. Inhibition of the Wnt/b-catenin pathway and expression of TCF-promoter-regulated genes such as c-Myc are examples. Tumors dependent on Wnt/b-catenin activity or with lower intracellular palmitate stores prior to drug treatment may be susceptible to FASN inhibitor treatment. Pairing MYC expression with additional markers improves the performance of MYC as a marker to select tumor cell lines highly sensitive to FASN inhibition. Biomarker candidates from in vitro studies have been examined in vivo using xenograft tumor studies. These preclinical data support 3-V Biosciences’ ongoing Phase I clinical study of a first-in-class FASN inhibitor TVB-2640, and help guide the next steps in its development. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Glenn Hammonds, Marina Fridlib, Russell Johnson, Lily Hu, Allan Wagman, Marie O' Farrell, Douglas Buckley, George Kemble. Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis. [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 4446. doi:10.1158/1538-7445.AM2015-4446

Sprouty2 Drives Drug Resistance and Proliferation in Glioblastoma.

Glioblastoma multiforme (GBM) is notoriously resistant to therapy, and the development of a durable cure will require the identification of broadly relevant regulators of GBM cell tumorigenicity and survival. Here, we identify Sprouty2 (SPRY2), a known regulator of receptor tyrosine kinases (RTK), as one such regulator. SPRY2 knockdown reduced proliferation and anchorage-independent growth in GBM cells and slowed xenograft tumor growth in mice. SPRY2 knockdown also promoted cell death in response to coinhibition of the epidermal growth factor receptor (EGFR) and the c-MET receptor in GBM cells, an effect that involved regulation of the ability of the p38 mitogen-activated protein kinase (MAPK) to drive cell death in response to inhibitors. Analysis of data from clinical tumor specimens further demonstrated that SPRY2 protein is definitively expressed in GBM tissue, that SPRY2 expression is elevated in GBM tumors expressing EGFR variant III (EGFRvIII), and that elevated SPRY2 mRNA expression portends reduced GBM patient survival. Overall, these results identify SPRY2 and the pathways it regulates as novel candidate biomarkers and therapeutic targets in GBM. SPRY2, counter to its roles in other cancer settings, promotes glioma cell and tumor growth and cellular resistance to targeted inhibitors of oncogenic RTKs, thus making SPRY2 and the cell signaling processes it regulates potential novel therapeutic targets in glioma.

Abstract 1916: Proanthocyanidins from grape seeds inhibit growth of head and neck squamous cell carcinoma by decreasing dna methylation and reactivation of silenced tumor suppressors

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in diagnostic and therapeutic methods, survival of HNSCC patients remains unchanged over the last 30 years. Epigenetic alterations are commonly associated with several types of cancers including HNSCC. Studies have shown that DNA hypermethylation plays important roles in the risk of cancers by inactivating the tumor suppressor genes/proteins. Thus, epigenetic changes are considered as promising therapeutic targets for cancer treatment. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. In the present study, we investigated the effect of grape seed proanthocyanidins (GSPs) on DNA methylation and growth of HNSCC cells in vitro and in vivo models. Our analytical data revealed that the levels of global DNA methylation and DNA methyltransferase (DNMT) activity were higher in HNSCC cells compared with normal human bronchial epithelial cells (NHBEC). Treatment of HNSCC cells (FaDu and SCC-1) with GSPs for 6 days results in significant decrease in global DNA methylation (70-80%, P&amp;lt;0.001) level, DNMT activity (60-80%) as well as in levels of DNMT proteins (DNMT 3A and DNMT 3B) compared to non-GSPs-treated control cells. The observation on DNA methylation levels was verified by analyzing the levels of 5-methylcytosine (5-mC) using dot-blot analysis. Additionally, GSPs treatment also reactivated tumor suppressor proteins, such as Cip1/WAF1/p21 and p27 in HNSCC cells. Treatment of HNSCC cells with 5-Aza-dc, an inhibitor of DNA methylation, also resulted in suppression of DNA methylation as well as DNMT protein expression and DNMT activity in these cells. Tumor xenograft studies were performed to further determine the effect of GSPs on the growth of HNSCC cells using athymic nude mouse model. Our in vivo study demonstrated that administration of GSPs (0.5%, w/w) in supplementation with AIN76A control diet resulted in inhibition of xenograft growth of FaDu cells in nude mice (80%; P&amp;lt;0.01) compared to the tumor xenograft growth in mice fed with non-GSPs-supplemented control diet. The growth inhibitory effect of dietary GSPs on the HNSCC tumor xenografts was associated with the inhibition of DNA methylation, DNMT activity and reactivation of silenced tumor suppressor proteins in tumor tissues. Together, this preclinical study provides evidence that GSPs act as a DNA demethylating agent and able to reactivate an epigenetically silenced tumor suppressors to inhibit growth of HNSCC cells. Citation Format: Santosh K. Katiyar, Ram Prasad. Proanthocyanidins from grape seeds inhibit growth of head and neck squamous cell carcinoma by decreasing dna methylation and reactivation of silenced tumor suppressors. [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 1916. doi:10.1158/1538-7445.AM2015-1916

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR's role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

5-Fluorouracil preferentially sensitizes mutant KRAS non-small cell lung carcinoma cells to TRAIL-induced apoptosis

Mutations in the KRAS gene are very common in non–small cell lung cancer (NSCLC), but effective therapies targeting KRAS have yet to be developed. Interest in tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a potent inducer of cell death, has increased following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and in xenograft models. However, results from clinical trials of TRAIL-based therapy are disappointingly modest at best and many have demonstrated a lack of therapeutic benefit. Current research has focused on selecting a subpopulation of cancer patients who may benefit from TRAIL-based therapy and identifying best drugs to work with TRAIL. In the current study, we found that NSCLC cells with a KRAS mutation were highly sensitive to treatment with TRAIL and 5-fluorouracil (5FU). Compared with other chemotherapeutic agents, 5FU displayed the highest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells. We also found that, on a mechanistic level, 5FU preferentially repressed survivin expression and induced expression of TRAIL death receptor 5 to sensitize NSCLC cells to TRAIL. The combination of low-dose 5FU and TRAIL strongly inhibited xenograft tumor growth in mice. Our results suggest that the combination of TRAIL and 5FU may be beneficial for patients with mutant KRAS NSCLC.

Boronic prodrug of endoxifen as an effective hormone therapy for breast cancer.

As a prodrug, tamoxifen is activated by the P450 enzyme CYP2D6 that is responsible for converting it to the active metabolites, 4-hydroxytamoxifen and endoxifen. Patients with genetic polymorphisms of CYP2D6 may not receive the full benefit of tamoxifen therapy. There is increasing evidence that poor metabolizer patients have lower plasma concentrations of endoxifen and suffer worse disease outcome, although some clinical studies reported no correlation between CYP2D6 polymorphism and tamoxifen therapy outcome. Endoxifen is currently undergoing clinical trials as a potentially improved and more potent SERM (Selective Estrogen Receptor Modulator) for endocrine therapy that is independent of CYP2D6 status in patients. However, direct administration of endoxifen may present the problem of low bioavailability due to its rapid first-pass metabolism via O-glucuronidation. We have designed and synthesized ZB483, a boronic prodrug of endoxifen suitable for oral administration with greatly enhanced bioavailability by increasing the concentration of endoxifen in mouse blood. Our study demonstrated that ZB483 potently inhibited growth of ER+ breast cancer cells in vitro and was efficiently converted to endoxifen in cell culture media by oxidative deboronation. This metabolic conversion is equally efficient in vivo as indicated in the pharmacokinetic study in mice. Moreover, when administered at the same dose, oral ZB483 afforded a 30- to 40-fold higher plasma level of endoxifen in mice than oral administration of endoxifen. The significantly enhanced bioavailability of endoxifen conferred by the boronic prodrug was further validated in an in vivo efficacy study. ZB483 was demonstrated to be more efficacious than endoxifen in inhibiting xenograft tumor growth in mice at equal dosage but more so at lower dosage. Together, these preclinical studies demonstrate that ZB483 is a promising endocrine therapy agent with markedly enhanced bioavailability in systemic circulation and superior efficacy compared to endoxifen.

Small-Molecule ONC201/TIC10 Targets Chemotherapy-Resistant Colorectal Cancer Stem-like Cells in an Akt/Foxo3a/TRAIL-Dependent Manner.

Self-renewing colorectal cancer stem/progenitor cells (CSC) contribute to tumor maintenance and resistance to therapy. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. ONC201/TIC10 is a first-in-class antitumor agent that induces TRAIL pathway-mediated cell death in cancer cells without observed toxicity. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. The Akt and ERK pathways serve as important targets in CSCs. Foxo3a is a key mediator of Akt and ERK-mediated CSC regulation. We hypothesized that the potent antitumor effect of ONC201/TIC10 in colorectal cancer involves targeting CSCs and bulk tumor cells. ONC201/TIC10 depletes CD133(+), CD44(+), and Aldefluor(+) cells in vitro and in vivo. TIC10 significantly inhibits colonosphere formation of unsorted and sorted 5-fluorouracil-resistant CSCs. ONC201/TIC10 significantly reduces CSC-initiated xenograft tumor growth in mice and prevents the passage of these tumors. ONC201/TIC10 treatment also decreased xenograft tumor initiation and was superior to 5-fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vitro and in vivo. ONC201/TIC10 inhibits Akt and ERK, consequently activating Foxo3a and significantly induces cell surface TRAIL and DR5 expression in both CSCs and non-CSCs. ONC201/TIC10-mediated anti-CSC effect is significantly blocked by the TRAIL sequestering antibody RIK-2. Overexpression of Akt, DR5 knockdown, and Foxo3a knockdown rescues ONC201/TIC10-mediated depletion of CD44(+) cells and colonosphere inhibition. In conclusion, ONC201/TIC10 is a promising agent for colorectal cancer therapy that targets both non-CSCs and CSCs in an Akt-Foxo3a-TRAIL-dependent manner.

Flavokawain B inhibits the growth of acute lymphoblastic leukemia cells via p53 and caspase-dependent mechanisms

The development of novel chemotherapeutic drugs is needed for the treatment of patients with acute lymphoblastic leukemia (ALL). In this study, the anti-leukemic effect and the potential molecular mechanisms of action of flavokawain B on ALL were investigated. Flavokawain B was found to significantly inhibit the cellular proliferation of B-ALL and T-ALL cell lines in a dose-dependent manner. It also induced cellular apoptosis by increasing the expression of p53, Bax and Puma, and activating the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, the enhancement of p53-dependent apoptosis by flavokawain B could be rescued by pifithrin-α, a pharmacological inhibitor of p53 transcriptional activity. Moreover, the proliferation of leukemia blast cells from 16 patients with ALL was inhibited by flavokawain B, and tumor growth in xenograft mice was also suppressed by this drug. In conclusion, our results demonstrate the therapeutic potential of flavokawain B for the treatment of ALL.

Abstract A02: Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid

Abstract Introduction: Medulloblastomas (MB) are the most common malignant brain tumors in children and constitute 20% of all pediatric brain tumors. Significant developments in the areas of neurosurgical techniques, dosing and delivery of radiation and optimized chemotherapy have limited response on improving the five year survival rates. Because of the aggressive multimodality therapy, survivors frequently suffer numerous long-term side effects. There is an urgent need for novel strategies for enhancing and optimizing the therapeutic efficacy of current MB treatments. Studies have shown that the Specificity Protein (Sp) transcription factors regulate critical genes that are involved in regulation of proliferation, survival, angiogenesis and resistance to chemo-/radiation therapy. Rationale: Research from our laboratory and others showed the anti-cancer activity of Tolfenamic acid (TA), a small molecule and non-steroidal anti-inflammatory drug in several adult malignancies. Recently, we demonstrated for the first time that TA inhibits MB cell proliferation and tumor growth in mice xenografts. Mechanistic studies revealed that TA acts via downregulation of Sp proteins and its downstream targets like survivin and VEGF. In this study, we tested the efficacy of TA for enhancing the response of chemotherapeutic agents used as standard therapy for MB. We hypothesize that TA sensitizes MB cells to chemotherapeutic drugs via down-regulation of Sp1 and survivin. Methods: Human MB cell lines, DAOY and D283 were used to test the anti-proliferative response of TA, Irinotecan, Topotecan (TOPO), Temozolomide (TMZ) and Doxorubicin (DOXO). Subsequently, the combination of TA with TOPO was tested using the optimized doses of TA (10 µg/ml) or TOPO (20 nM for DAOY; 25 nM for D283). Cell viability was measured at 24, 48, and 72h post-treatment using CellTiter-Glo kit. To correlate the anti-proliferative response with the induction of apoptotic pathways we analyzed apoptosis by flow cytometric analysis of Annexin V stained cells, PARP cleavage, and the activation of caspase3/7. The expression of Sp1, survivin, and cleaved-PARP was determined by Western blot. qPCR analysis using TaqMan assays was used to detect microRNAs (miR-20a and miR-27a), which act as Sp repressor proteins. Results: TA and chemotherapeutic agents caused a dose and time-dependent inhibition of cell viability. The combination of TA and TOPO caused significantly higher inhibition when compared to either TA or TOPO alone confirming the efficacy of this combination therapy in pre-clinical models for MB. The effect on cell apoptosis was evaluated at 48 h post-treatment. Annexin V staining for apoptotic cells showed a significant increase in the apoptotic fraction (annexin V positive) following combination therapy when compared to individual treatment of TA or TOPO. These results were further supported by significantly upregulated caspase 3/7 activity when compared to individual treatment. DAOY and D283 were treated with vehicle (DMSO) or 10 or 20 µg/ml TA for 48 h. Whole cell lysates were prepared and analyzed by Western blot analysis. TA inhibited both Sp1 and survivin in MB cell lines. Consistent with Annexin V staining and caspase 3/7 results, TA augmented PARP cleavage confirming the activation of apoptotic pathways. TA treatment also downregulated mirR-20a and miR-27a. Conclusion: Overall, these results demonstrate that the combination of TA with standard chemotherapeutic agents effectively inhibits MB cell growth. This chemo-sensitization can be correlated with the downregulation of survivin by TA, in part due to its effects on Sp1 transcription factor either directly or through miRNAs. These preliminary results strongly demonstrate the potential of targeting Sp1 and survivin using TA to enhance the therapeutic response in pre-clinical model of MB. Financial Support: HyundaiHopeOnWheels; UFHCC, Orlando Health, Orlando, FL and Department of Pediatrics, UNT Health Science Center, Fort Worth, TX. Citation Format: Don Eslin, Umesh T. Sankpal, Jeffrey C. Murray, W. Paul Bowman, Robert Sutphin, Riyaz Basha. Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A02.

Candidate tumor suppressor B-cell translocation gene 3 impedes neoplastic progression by suppression of AKT.

BTG3 (B-cell translocation gene 3) is a p53 target that also binds and inhibits E2F1. Although it connects two major growth-regulatory pathways functionally and is downregulated in human cancers, whether and how BTG3 acts as a tumor suppressor remain largely uncharacterized. Here we present evidence that BTG3 binds and suppresses AKT, a kinase frequently deregulated in cancers. BTG3 ablation results in increased AKT activity that phosphorylates and inhibits glycogen synthase kinase 3β. Consequently, we also observed elevated β-catenin/T-cell factor activity, upregulation of mesenchymal markers, and enhanced cell migration. Consistent with these findings, BTG3 overexpression suppressed tumor growth in mouse xenografts, and was associated with diminished AKT phosphorylation and reduced β-catenin in tissue specimens. Significantly, a short BTG3-derived peptide was identified, which recapitulates these effects in vitro and in cells. Thus, our study provides mechanistic insights into a previously unreported AKT inhibitory pathway downstream of p53. The identification of an AKT inhibitory peptide also unveils a new avenue for cancer therapeutics development.

FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells.

Chemoresistance to anti-cancer drugs substantially reduces survival in epithelial ovarian cancer. In this study, we showed that chemoresistance to cisplatin and paclitaxel induced the epithelial-mesenchymal transition (EMT) and a stem cell phenotype in ovarian cancer cells. Chemoresistance was associated with the downregulation of epithelial markers and the upregulation of mesenchymal markers, EMT-related transcription factors, and cancer stem cell markers, which enhanced invasion and sphere formation ability. Overexpression of FOXM1 increased cisplatin-resistance and sphere formation in cisplatin-sensitive and low FOXM1-expressing ovarian cancer cells. Conversely, depletion of FOXM1 via RNA interference reduced cisplatin resistance and sphere formation in cisplatin-resistant and high FOXM1-expressing cells. Overexpression of FOXM1 also increased the expression, nuclear accumulation, and activity of β-CATENIN in chemoresistant cells, whereas downregulation of FOXM1 suppressed these events. The combination of cisplatin and the FOXM1 inhibitor thiostrepton inhibited the expression of stem cell markers in chemoresistant cells and subcutaneous ovarian tumor growth in mouse xenografts. In an analysis of 106 ovarian cancer patients, high FOXM1 levels in tumors were associated with cancer progression and short progression-free intervals. Collectively, our findings highlight the importance of FOXM1 in chemoresistance and suggest that FOXM1 inhibitors may be useful for treatment of ovarian cancer.