bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1998: Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH)
Authors: Johansson Hans E. , Yin Raymund H. , Mandal Swati , Park Myung-Hee
Published: July 2016

Normal expression of the minor oncogene EIF5A2 in the hypusine pathway is restricted to brain and testis. However aberrant expression has been noted in multiple cancers, including ovarian gastric, colorectal and pancreatic, and artificial overexpression causes tumor growth in naked mice. Unlike cDNAs from the homologous gene EIF5A1, which are readily translated into protein upon transient transfection, EIF5A2-mRNAs only associate with monosomes without production of eIF5A-2 protein.

Here we present data from multiple EIF5A2-positive cell lines on the steady state levels of the four different EIF5A2-mRNAs that arise through differential polyadenylation. While considering qPCR and RNA-Seq, we chose to apply Stellaris single molecule RNA fluorescence in situ hybridization with four unique probe sets, each reporting on the dependent A, B, C, or D segments. The probe sets were designed with multiple tiled singly labeled 18-22-mer oligonucleotides with balanced GC content, and hence similar hybridization characteristics, which in turn enabled the simultaneous quadruplex smRNA FISH.

Stellaris RNA FISH leaves the cell intact, and also does not require RNA isolation or nucleic acid amplification that may introduce an otherwise hidden experimental bias. The single molecule spots revealed by widefield microscopy and subsequent image analysis co-localized with a high degree of confidence. Taking advantage of the added spatial dimension that RNA FISH affords, we also determined the localization of each mRNA variant and recorded the cell-to-cell variability and the actual range of mRNA copy numbers per cell.

Our data is consistent with previous gene expression analysis data, but also provide actual counts of each mRNA variant.

The results presented here have direct applications for the expression of the minor oncogene EIF5A2, and the regulated protein synthesis of its mRNAs, as well as of other mRNAs in polyamine and hypusine pathways. Further, the techniques and probes developed here can be directly transferred to the analysis of other mRNA variants that may arise through alternative transcription start site use, alternative splicing or alternative 3’ end processing in normal and aberrant cells.

Citation Format: Hans E. Johansson, Raymund H. Yin, Swati Mandal, Myung-Hee Park. Alternative polyadenylation of eukaryotic initiation factor 5A-2 (eIF5A-2) mRNAs examined by quadruplex single molecule RNA fluorescence in situ hybridization (smRNA-FISH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1998.
Abstract 20: Outlier overexpression of genes involved in fatty acid metabolism and processing define two aggressive prostate cancer phenotypes
Authors: Gaston Sandra M. , Dehimer Kerry , Kearns James , Chinsky Kyle-Ravi , Otali Dennis , Oelschlager Denise , Rais-Bahrami Soroush , Nix Jeffrey W. , Kolettis Peter , Adams George W. , Grizzle William E.
Published: July 2016

Major shifts in fatty acid metabolism are part of the malignant phenotype of many types of cancer, including prostate cancer. We have used a biopsy based approach to characterize high grade prostate cancers in both African American and European American patients. This approach allows us to analyze prostate cancers from patients whose disease is too advanced for radical prostatectomy and who are thus not represented in the study specimens available from conventional biorepositories. mRNA gene expression profiling of high grade prostate cancers from biopsies revealed cancer subtypes with very high (>10 fold) levels of overexpression of fatty acid binding protein 5 (FABP5) and fatty acid synthase (FASN). Interestingly, these prostate cancer subtypes showed an either-or phenotype, with “super-overexpression” of either FABP5 or FASN but not both. These findings suggest two “outlier” prostate cancer subtypes with striking differences in the pathways that drive a shift in tumor fatty acid metabolism; one is a fatty acid synthase (FASN) dominant phenotype and the other a previously unrecognized fatty acid binding protein (FABP5) dominant phenotype. In contrast, prostate cancers with lower levels of FABP5 and/or FASN overexpression (2-4 fold) often co-overexpressed both of these genes. Immunohistochemical analyses confirmed prostate cancer FABP5 and FASN overexpression; both showed strong cytoplasmic staining. FABP5 also showed robust nuclear staining consistent with its proposed role in regulating fatty acid mediated gene expression. At both the mRNA and protein levels, the high grade prostate cancers with the highest levels of FABP5 over-expression were more common in African Americans, while the highest levels FASN overexpression were more common in prostate cancers from European Americans. These findings may provide the basis for more effective dietary interventions and targeted therapies for African American and European American patients with these two different subtypes of high grade prostate cancer.

Citation Format: Sandra M. Gaston, Kerry Dehimer, James Kearns, Kyle-Ravi Chinsky, Dennis Otali, Denise Oelschlager, Soroush Rais-Bahrami, Jeffrey W. Nix, Peter Kolettis, George W. Adams, William E. Grizzle. Outlier overexpression of genes involved in fatty acid metabolism and processing define two aggressive prostate cancer phenotypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 20.
Abstract 200: PDLIM2 phosphorylation in determining breast cancer phenotype
Authors: Cox Orla T. , Berghoff Janina , Buckley Deirdre A. , O’Connor Rosemary
Published: July 2016

Several genes that are involved in cancer progression are induced by Insulin-like Growth Factor-1 (IGF-1). One of them encodes the PDZ-LIM protein PDLIM2, which is localized at the cytoskeleton and nucleus in epithelial and hematopoietic cells. PDLIM2 is repressed in certain cancers including breast cancers, but it is highly expressed in invasive cancer cells in triple negative breast cancer (TNBC), where it has also been associated with poor survival. PDLIM2 regulates the stability and activity of transcription factors including beta catenin, STATs, NFκB and IRFs and has been associated with ubiquitination and destabilization of proteins such as NF-κB, E-cadherin, and p27KIP1. However, PDLIM2 itself is also regulated by posttranslational modifications, but how this controls PDLIM2 activity is still largely unclear.

Therefore, this study aimed to investigate PDLIM2 phosphorylation and its effect on PDLIM2 sub-cellular location and function in transcription factor regulation. The PDLIM2 protein sequence includes multiple potential phosphorylation sites and it migrates as several distinct phosphatase-sensitive species in SDS PAGE and 2D electrophoresis, indicating phosphorylation at several residues. Using cellular fractionation and immunofluorescence we show that PDLIM2 accumulates in the nucleus in response to cell stimulation with IGF-1 or TGFβ. Moreover, we show that inhibitors of Rho Kinases suppress PDLIM2 phosphorylation in response to these stimuli. In addition, we demonstrate that mutation of the predicted Rho Kinase phosphorylation sites in PDLIM2 reduce protein stability, an effect that can be reversed by application of proteasomal inhibitor.

Overall, our results indicate that phosphorylation by Rho kinases plays an important role in the subcellular location and regulation of PDLIM2 function, which opens a new avenue in targeting PDLIM2 activity in invasive cancer cells.

Citation Format: Orla T. Cox, Janina Berghoff, Deirdre A. Buckley, Rosemary O’Connor. PDLIM2 phosphorylation in determining breast cancer phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 200.
Abstract 2014: Alternative splicing is a frequent event in mouse PTEN-deficient prostate cancer
Authors: De Velasco Marco A. , Kura Yurie , Sakai Kazuko , Fujita Yoshihiko , Togashi Yosuke , Terashima Masato , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: July 2016

Castration-resistant prostate cancer (CRPC) remains an incurable disease and presents a major challenge for the development of novel treatment strategies. Alternative splicing occurs as a common natural phenomenon and promotes diversity, however, cancer-related alternative splicing events have been shown to contribute to disease progression and promote therapeutic escape. In prostate cancer, key genes required for normal biological function are frequently alternatively spliced resulting in altered phenotypes. Novel therapeutic strategies targeting the alternative splicing machinery are being developed and tested. Here, characterized the conditional PTEN-deficient mouse model of prostate cancer to determine its relevancy as preclinical tool for the development and efficacy determination of novel therapeutic strategies targeting aberrant alternative splicing. For this, we used the Affymetrix GeneChip mouse transcriptome assay to perform comparative analysis of the transcriptomes of normal prostate tissue and PTEN-deficient castration-naïve, castration-sensitive (4 weeks post-castration), and castration-resistant (10 weeks post-castration) prostate cancers. Clustering analysis revealed genes enriched with the functions involved in mRNA splicing and processing in mice with prostate tumors. Moreover, alternative splicing events were more prevalent in castration-sensitive tumors (>2.5-fold) compared to castration-naïve and castration resistant tumors. Exon skipping (cassette exon), was the most common splicing event observed in all conditions. Additionally, alternative splicing was observed in several genes frequently associated with human prostate cancer including CD44, AR, p53, Bcl2l1 and Klf6 among others. Collectively, our data shows that alternative mRNA splicing is a frequent event in mouse PTEN-deficient prostate cancer and supports a role for alternative splicing dysregulation and the pathogenesis of the disease. Furthermore, this mouse model may also provide a suitable platform to study current therapeutic approaches targeting alternative splicing.

Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Yoshihiko Fujita, Yosuke Togashi, Masato Terashima, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Alternative splicing is a frequent event in mouse PTEN-deficient prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2014.
Abstract 203: Single cell imaging of kinase inhibitor-induced effects in breast cancer cell lines
Authors: Mills Caitlin , Andrews David , Sorger Peter
Published: July 2016

There is need for improved targeted therapies for the treatment of breast cancer. Kinase inhibitors are candidates to be used in this context. With the goal of uncovering specific vulnerabilities in differing cell lines, we treated five breast cancer cell lines representing triple negative (BT20, MDAMB231, and Hs578T), hormone receptor positive (MCF7), and Her2 amplified (SKBR3) disease and the non-malignant MCF10A line with a panel of 105 kinase inhibitors covering a broad range of targets. Cells were treated with doses between 0.1 and 10 μM for 24 hours, at which time they were stained with DRAQ5 (DNA) and TMRE (mitochondrial membrane potential). Live-cell images were acquired using a high throughput, confocal Opera microscope. Cell segmentation, based on the DRAQ5 staining, and feature extraction (intensity, morphology, and texture) were performed with Acapella software. Over 300 features were extracted for ∼1.5 million cells. Analytical methods have been applied to identify those treatments that induced significant changes to the cells. Over half of the kinase inhibitors queried had a significant effect within 24 hours in all cell lines at 10 μM. Cell cycle inhibitors had the most common effects across cell lines. Cell line specific effects were also uncovered, for example, MDAMB231 cells were the most sensitive to MAPK inhibitors. These, early time point, results have been compared with the effects of the same inhibitors on cell growth to better understand the mechanisms leading to cell line and pathway specific effects.

Citation Format: Caitlin Mills, David Andrews, Peter Sorger. Single cell imaging of kinase inhibitor-induced effects in breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 203.
Abstract 2034: Personalized chemotherapy regimen selection for breast cancer
Authors: Zhang Jinfeng , Yu Kaixian , Sang Qingxiang Amy , Tan Winston , Dargham Mayassa B. , Liu Jun S. , Lively Ty , Sheffield Cedric
Published: July 2016

Despite the rapid progress in personalized cancer therapy (PCT) for breast cancer, no previous studies have used genomic predictors to choose among multiple chemotherapy regimens. It is unclear that given the current regimens how much PCT can improve the response rate for patients who will receive chemotherapy. In this study, we reanalyzed data from published studies of 1111 breast cancer patients who were treated with neoadjuvant chemotherapies. Those patients were divided into three regimen groups: an anthracycline alone, anthracycline plus paclitaxel, and anthracycline plus docetaxel. We developed a new strategy called PRES (Personalized REgimen Selection) to reassign the optimal regimen to each of the patients. First, a variable selection scheme was developed to identify significant genetic predictors for chemotherapy response. The selected genetic variables were then combined with clinical variables to build random forest models to predict the response of a patient to each regimen using pCR (pathological complete response) as the measure of response. The models were used to assign an optimal regimen to each patient to maximize the chance of pCR. We found that the expected rate of pCR was improved from 21.2% to 39.6% (95% CI: 34.6% - 43.0%). We also found that 31.1% of the patients may have been overtreated and 8.2% patients undertreated. A validation study on 21 cell lines showed that our prediction agrees with their paclitaxel-sensitivity profiles. We performed additional analysis on the Cancer Genome Atlas (TCGA) data and found that 18 of the 19 genes identified are significantly differentially expressed between normal and tumor tissues, and 2 of them, TAF6L and METRN (meteorin), are associated with overall survival. In conclusion, PRES could substantially increase response rates for breast cancer patients who will receive one of the widely-accepted chemotherapy regimens at present.

Citation Format: Jinfeng Zhang, Kaixian Yu, Qingxiang Amy Sang, Winston Tan, Mayassa B. Dargham, Jun S. Liu, Ty Lively, Cedric Sheffield. Personalized chemotherapy regimen selection for breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2034.
Abstract 204: Disruption of the RACK1/PP2A complex results in a decrease in the adhesion, proliferation, migration and invasion capabilities of breast cancer cells
Authors: Kiely Maeve , O’Connor Rosemary , Adams David , Baillie George S. , Kiely Patrick A.
Published: July 2016

Conflicting reports implicate the scaffolding protein RACK1 in the progression of breast cancer. PP2A has a well-established role as a tumour suppressor within signalling pathways but is also known to play a pro-carcinogenic role in certain circumstances [1]. RACK1 has been identified as a direct binding partner of PP2A to regulate cell migration and stabilize PP2A activity [2]. Our objective was to further characterise the interaction between PP2A and RACK1 in breast cancer cells. The PP2A holoenzyme is assembled in MCF-7 cells and we found that both the C subunit and A subunit of PP2A are assembled on the RACK1 scaffold. We used immobilized peptide arrays representing the entire PP2A-Catalytic protein to identify amino acids on the C subunit of PP2A that are required for the binding of RACK1. Once identified, these sites were mutated and expressed in the context of the full length PP2A C subunit protein and stable cell lines were generated. When the RACK1/PP2A interaction was disrupted, cells exhibited reduced PP2A phosphatase activity, confirming the role for RACK1 in stabilizing PP2A activity. We used the stable cell lines to determine that disruption of the RACK1/PP2A complex also reduces the adhesion, proliferation, migration and invasion of this breast cancer cell model. The work has significantly advanced our understanding of the RACK1/PP2A complex and indicates a pro-carcinogenic role for the RACK1/PP2A complex. This work has highlighted a novel mechanism to target PP2A activity and may provide a potential therapeutic target in the treatment of breast cancer.

1. Kiely, M. and P.A. Kiely, PP2A: The Wolf in Sheep's Clothing? Cancers, 2015. 7(2): p. 648-669.

2. Kiely, P.A., et al., Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration. Journal of Biological Chemistry, 2008. 283(34): p. 22952-22961.

Citation Format: Maeve Kiely, Rosemary O’Connor, David Adams, George S. Baillie, Patrick A. Kiely. Disruption of the RACK1/PP2A complex results in a decrease in the adhesion, proliferation, migration and invasion capabilities of breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 204.
Abstract 2057: Dual peptide-mediated targeted delivery of siRNAs for the treatment of oral cancer
Authors: Alexander-Bryant Angela A. , Zhang Haiwen , Pugh William , Dinh Lu , Attaway Christopher , Eggart Laurence , Sansevere Robert , Cantini Liliana , Jakymiw Andrew
Published: July 2016

Two major hurdles for small interfering RNA (siRNA)-mediated therapies are cell/tissue-type targeted delivery and endosomal entrapment of siRNAs, resulting in inefficient gene silencing. As a result, the objective of this study was to examine the feasibility of utilizing two peptides, one with cancer cell-targeting specificity and the second with endosome-disruptive properties, to co-deliver bioactive siRNAs into oral cancer cells overexpressing the epidermal growth factor receptor (EGFR) and induce silencing of the targeted oncoprotein, CIP2A. Previously, we designed a novel endosome-disruptive peptide, termed 599, that was demonstrated to mediate intracellular delivery of bioactive siRNAs targeting CIP2A (siCIP2A) in vitro. Furthermore, we recently demonstrated that 599 peptide-mediated delivery of siCIP2A into tumor tissues via intratumoral injections reduced the expression of CIP2A and significantly inhibited tumor growth. In order to induce targeted uptake via systemic administration, we designed the EGFR targeting peptide, GE11R9, which was found to deliver siRNAs specifically to EGFR-overexpressing oral cancer cells; however, it was incapable of mediating the delivery of bioactive siRNAs due to endosomal entrapment. Consequently, we developed a novel dual peptide delivery strategy, combining the 599 peptide, with the GE11R9 peptide and examined their ability to co-deliver siRNAs. Our results demonstrated that the dual peptide complex exhibited effective binding and specific uptake of siRNAs into EGFR-overexpressing cells compared to low-EGFR-expressing cells. The co-addition of the 599 peptide with the GE11R9 peptide also restored siRNA functionality. Furthermore, when administered systemically to mice bearing xenograft oral tumors, the dual peptide complex mediated increased targeted delivery of siRNAs into tumor tissues in comparison to the 599 peptide alone. Together, these data demonstrate the clinical potential of the dual peptide strategy for RNAi-based therapeutics by synergistically mediating the effective targeting and delivery of bioactive siRNAs into EGFR-overexpressing cancer cells.

Citation Format: Angela A. Alexander-Bryant, Haiwen Zhang, William Pugh, Lu Dinh, Christopher Attaway, Laurence Eggart, Robert Sansevere, Liliana Cantini, Andrew Jakymiw. Dual peptide-mediated targeted delivery of siRNAs for the treatment of oral cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2057.
Abstract 206: Cytochrome c oxidase subunit 5a (COX5A) is identified as a potential therapeutic target for lung cancer with high therapeutic index through a pooled shRNA screen
Authors: Kato Toshio , Sato Mitsuo , Kondo Masashi , Kakumu Tomohiko , Yogo Naoyuki , Hase Tetsunari , Morise Masahiro , Hasegawa Yoshionori , Minnna John D. , Girard Luc
Published: July 2016

[Background]Recent advances in high throughput analysis of genetic and epigenetic alterations revealed numerous changes in lung cancer cells. It is vital to sort out genes that substantially contribute to oncogenic properties of cancer cells from these numerous altered genes by performing a functional screening because such genes could serve as valuable therapeutic targets. To this end, we performed a screening with a pooled shRNA library for genes critical for survival and/or proliferation in lung cancer cells. [Methods and Results]A pooled shRNA library comprising 5,000 genes, with 3 to 7 constructs per gene, were transduced in NCI-H460 lung cancer cell line. Subsequently, genomic DNA was extracted from the transduced cells and numbers of shRNA for each gene were quantified by next generation sequencing. Through a stringent filtering process we narrowed down genes to a list of 24 candidates as potentially target genes. Next, we integrated data of genome-wild gene expression and copy number analyses of a panel of lung cancer cell lines to narrow down to genes more relevant to lung cancer. Through these processes, we listed up several candidate genes. Among them, we focused on Cytochrome c oxidase subunit 5a (COX5A), one of the nuclear encoded subunits of cytochrome c oxidase, the terminal protein of the mitochondrial electron transport chain. Importantly, COX5A knockdown didn't suppress growth of HBEC3, a normal control cell line, suggesting that therapeutics of targeting COX5A may have high therapeutic index.

[Conclusions]Our results suggest that inhibiting COX5A may be an attractive strategy for the treatment of NSCLC with high therapeutic index.

Citation Format: Toshio Kato, Mitsuo Sato, Masashi Kondo, Tomohiko Kakumu, Naoyuki Yogo, Tetsunari Hase, Masahiro Morise, Yoshionori Hasegawa, John D. Minnna, Luc Girard. Cytochrome c oxidase subunit 5a (COX5A) is identified as a potential therapeutic target for lung cancer with high therapeutic index through a pooled shRNA screen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 206.
Abstract 2060: Polymer mediated RNAi-based combination therapy for treating prostate cancer
Authors: Danquah Michael
Published: July 2016

Purpose. To determine whether RNAi-based combination therapy targeting androgen receptor (AR) and XIAP signaling pathways using polymeric micelles can treat prostate cancer.

Methods. Potency of siRNAs targeting AR and XIAP genes was assessed using real time RT-PCR, Western Blot and ELISA. Cell viability was determined by MTT assay. A library of polymers designed to degrade via hydrolysis and enhance siRNA delivery was synthesized by conjugating hexylamine, pentaethylenehexamine and 3-morpholinopropylamine to poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) backbone using EDC/HOBT coupling chemistry. Polymer structure and molecular weight were verified with proton NMR and Gel Permeation Chromatography, respectively.

Results. Three siRNA sequences targeting different regions of the AR and XIAP genes were designed, blasted against the human genome database to eliminate cross-silencing of nontarget genes and their potency screened in LNCaP and C4-2 cells following transfection. All siRNAs targeting AR (start site: 1172, 1648 and 1768) used in this study silenced AR mRNA and protein expression in both cell lines. Furthermore, 1172 was identified as the most potent sequence resulting in approximately 75% knockdown regardless of cell type. Also, the three siRNAs targeting XIAP (start site: 264, 297 and 458) down-regulated XIAP mRNA and protein levels in both cells and were generally more potent in C4-2 cells compared to LNCaP cells. C4-2 cells transfected with siRNA targeting AR (start site: 1172) and XIAP (start site: 264) alone or in combination and assayed after 96 hours revealed XIAP and AR down-regulation resulted in approximately 20% and 50% growth inhibition in C4-2 cells, respectively, compared to control. However, combined silencing of XIAP and AR genes decreased cell growth by 62% and was more potent in inhibiting cell proliferation compared to monotherapy. Additionally, combination of siRNA targeting AR (start site: 1172) and XIAP (start site: 264) significantly increased percentage of cells undergoing apoptosis (85%) compared to control or monotherapy. Poly(ethylene glycol)-block-poly(5-methyl-5-carboxyl-propylene carbonate) copolymer was synthesized by ring opening polymerization and conjugated with various ratios of hexylamine, pentaethylenehexamine and 3-morpholinopropylamine moieties. The resulting copolymer (PEG-b-P(CC-g-PHEXYL-g-PMORPH-g-PPHEA) were non-toxic even at concentration of 1500 μg/mL and successfully condensed siRNA at N/P of 25 with average hydrodynamic diameter of approximately 175 nm.

Conclusions. Combination based therapy using siRNA targeting AR and XIAP can potentially treat prostate cancer.

Citation Format: Michael Danquah. Polymer mediated RNAi-based combination therapy for treating prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2060.
Abstract 2072: Inhibition of breast cancer stem cells by Hedgehog-inhibitory peptide conjugated with Elastin-like biopolymers
Authors: Ryu Jung Su , Raucher Drazen
Published: July 2016

The Hedgehog(Hh) signaling pathway has been reported to play a pivotal role in cancer. Studies have shown that suppression of this aberrantly activated Hh pathway may be effective in controlling the development and proliferation of many cancers. However, to date, there is no drug in clinical application that can be used to inhibit the Hh signaling pathway and suppress cancer growth.

In attempt to address this problem, we developed a thermally responsive polypeptide inhibitor of the Hh pathway. This polypeptide is derived from a mammalian tropo-elastin protein which will aggregate and accumulate at the tumor site where local hyperthermia is applied. For this study, ELP was fused to a peptide that inhibits Hh signaling by blocking interaction of Sonic Hh ligand (Shh) with the Patch-1 docking site. To improve bioavailability, ELP was further modified by adding the cell penetrating peptide, Tat.

The anti-proliferative activity of Tat-Hhi-ELP was examined in three breast cancer cell lines: MCF7, MDA-MB-231, and SKBR-3. Treatment of the cells with 20 uM of peptide for 2 days resulted in maximally 40% inhibition of cell proliferation. Observed cytotoxicity was further increased two fold by application of hyperthermia.

To validate that Tat-Hhi-ELP is targeting the Hh pathway, we determined the levels of GLI-1, which is a downstream target in the Hh pathway. Treatment of the breast cancer cells with Tat-Hhi-ELP resulted in reduction of GLI-1 levels, indicating that the cytotoxicity is based on hedgehog pathway inhibition. Furthermore, the formation of mammospheres was significantly reduced by treatment with Tat-Hhi-ELP in SKBR-3 cells. These results suggest that thermal targeting of ELP-based Hedgehog inhibitory peptides to breast cancer cells may be an effective and promising treatment strategy against breast cancer stem cells.

Citation Format: Jung Su Ryu, Drazen Raucher. Inhibition of breast cancer stem cells by Hedgehog-inhibitory peptide conjugated with Elastin-like biopolymers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2072.
Abstract 2074: Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell characteristics by suppressing sonic hedgehog pathway
Authors: Yu Wei , Ma Yiming , Shankar Sharmila , Srivastava Rakesh K.
Published: July 2016

Pancreatic cancer is the fourth leading cause of cancer-related deaths in US, with a 5-year survival rate of approximately 6%. Patients develop resistance to currently used anticancer drugs which are mainly toxic and also demonstrate severe side effects. Cancer stem cells (CSCs) / tumor initiating cells (TICs) have been proposed for tumor initiation, promotion, metastasis, and chemotherapy failure. Thus, there is an urgent need to develop novel strategies for the management of pancreatic cancer. Sonic hedgehog (Shh) pathway has been implicated in pancreatic carcinogenesis and is constitutively active in CSCs. The α-Mangostin is derived from the plant mangosteen (Garcinia mangostana) which is a tropical evergreen tree. Mangosteen contains Mangnoids, such as α-Mangostin, and other phytochemicals. The main objective of the study was to demonstrate the biological activities of α-Mangostin-encapsulated PLGA nanoparticles (Mang-NPs) in human pancreatic CSCs and cells lines. We have also performed docking of α-Mangostin on to the DNA binding sites of Gli to assess whether α-Mangostin disrupts Gli-DNA binding and acts as a Gli inhibitor. Mang-NPs inhibited cell proliferation, colony formation, and induced apoptosis in pancreatic cancer AsPC-1, PANC-1 and Mia-Paca-2 cell lines, and CSCs. However, Mang-NPs had no effect on human pancreatic normal ductal epithelial cells. In addition, Mang-NPs inhibited epithelial to mesenchymal transition. α-Mangostin disrupted Gli-DNA binding activity. Western blot analysis has demonstrated that Mang-NPs inhibited pluripotency maintaining factors Nanog and c-Myc. Furthermore, Mang-NPs inhibited the expression of Gli1, Gli2, Patched1 and Patched2, and Gli transcriptional activity in pancreatic CSCs. Our data demonstrate that Mang-NPs can inhibit pancreatic CSC characteristics by disrupting Gli-DNA binding activity and may act as a Gli transcription inhibitor.

Citation Format: Wei Yu, Yiming Ma, Sharmila Shankar, Rakesh K. Srivastava. Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic cancer stem cell characteristics by suppressing sonic hedgehog pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2074.
Abstract 2085: Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize tumor killing effect of an anti-VISTA monoclonal antibody in tumor bearing mice
Authors: Jiang Xiling , Leu Jocelyn , Singh Indrajeet , Snyder Linda A. , Wang Weirong
Published: July 2016

The V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a recently discovered co-inhibitory molecule that shares limited sequence homology with the IgV domain of immune regulators of the B7 family. A first-in-class monoclonal antibody (mAb) that targets VISTA is currently in development for evaluation in patients with non-small cell lung cancer and other types of cancer. Data from a preclinical mouse model (in human VISTA knock-in mice) showed that treatment with its surrogate antibody demonstrated tumor growth inhibition, which is believed to be associated with modulation of the myelomonocytic and T cell compartments. A mechanistic pharmacokinetic/ pharmacodynamic (PK/PD) modeling approach was applied that integrated the in vitro and in vivo information (PK, target occupancy (TO) and tumor growth), and incorporated key physiological processes. A one-compartment pharmacokinetic model with Michaelis-Menten elimination kinetics was used to characterize the nonlinear clearance of the surrogate antibody. The growth and killing of tumor cells was described with a cell distribution tumor growth/ killing model. Tumor killing effects can be described well by a linear relationship to VISTA TO. The developed PK/PD model captured the observed data in mice. This work also presents the potential of how mechanistic modeling and simulation can support first-in-human dose selection and dosing regimen optimization for immunomodulatory mAbs for anticancer immunotherapy.

Citation Format: Xiling Jiang, Jocelyn Leu, Indrajeet Singh, Linda A. Snyder, Weirong Wang. Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize tumor killing effect of an anti-VISTA monoclonal antibody in tumor bearing mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2085.
Abstract 2097: Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models
Authors: Yu Xin , Tsang Ashley T. , Withers Tracy , Gelleran John , Augeri David , Kimball S. David , Carpizo Darren R.
Published: July 2016

Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to KRAS as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. We identified ZMC1 as an allele specific mutant p53 reactivator and lead compound for mutant p53 targeted drug development. ZMC1 restores wildtype structure and function by functioning as a zinc metallochaperone to restore zinc binding to mutant p53 proteins with impaired zinc binding. Our aim was to translate this novel mechanism in vivo using murine pancreatic cancer models. We investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 and performed efficacy studies for allele specificity in nude mice with subcutaneous tumors from murine pancreatic cancer cell lines derived from a genetically engineered mouse model (KPC) expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Tumor growth inhibition became apparent only in KPCp53-R172H xenografts but not in KPCp53-R270H. We then performed efficacy studies in the autochthonous KPC model and found that, ZMC1 extended the median survival from 15 to 26 days in the KPCp53-R172H mice (p = 0.05) but not in the KPCp53-R270H mice. We sought to improve the efficacy of ZMC1 by synthesizing it complexed with zinc (Zn-1) in a 2:1 molar ratio. Zn-1 significanlty increased the median survival of KPCp53-R172H mice from 15 to 35 days (p = 0.0042). The apoptosis rate in the tumors (by Immunohistochemistry staining with Cleaved Caspase 3) was also increased by treatment of ZMC1 and Zn-1. These studies indicate that ZMC1 can function in vivo as a mutant p53 targeted anti-cancer drug at doses that are well tolerated. Furthermore, ZMC1 can be optimized by synthesizing it complexed with zinc.

Citation Format: Xin Yu, Ashley T. Tsang, Tracy Withers, John Gelleran, David Augeri, S. David Kimball, Darren R. Carpizo. Translating a mutant p53 reactivating drug (ZMC1) in murine pancreatic cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2097.
Abstract 2098: Understanding the synergy between temozolomide and PARP inhibition in Ewing sarcoma
Authors: Carlson Ruth , Kurmasheva Raushan
Published: July 2016

Ewing sarcoma is the fourth most common highly malignant childhood cancer; it is defined by a tumor-specific chromosomal translocation. In approximately 85% of all tumors, the EWSR1 gene on chromosome 22 is fused to a member of E26 transformation-specific sequence family of transcription factors, the FLI1 gene on chromosome 11. DNA damage induced by expression of EWSR1-FLI1 fusion gene is potentiated by PARP1 inhibition in Ewing cells, where EWSR1-FLI1 genes act in a positive feedback loop to maintain the expression of PARP1. As single agents, PARP inhibitors have shown promising activity in vitro, but only modest activity in in vivo models. In our previous work (in PPTP), we showed that low level damage to DNA by temozolomide (TMZ) can be potentiated up to 40-fold through inhibition of PARP by BMN-673, leading to dramatic tumor regressions in 5 of the 10 Ewing sarcoma xenograft models. Therefore, we were aimed to determine the differences between the tumors that respond to treatment with BMN-673 and those intrinsically resistant to it, and to understand the underlying mechanisms of such resistance, with the ultimate goal to develop more effective therapy for Ewing sarcoma.

We have used the same cell lines to investigate biochemical differences between those Ewing sarcoma lines where there is synergy in xenograft models in mice, and those where the combination is inactive. Our studies using cell lines show that, 1) in vitro BMN-673 and TMZ are synergistic in all cell lines, irrespective of their response as xenografts; 2) In vivo synergy correlates with intrinsic sensitivity to BMN-673 and TMZ in vitro. For Ewing lines, where there is no in vivo synergy when they are grown as xenografts, they are ∼9-fold resistant to BMN-673, and ∼15-fold resistant to TMZ relative to lines where there is synergy as xenografts.

The resistance of the Ewing sarcoma xenografts to the synergy of the combination of BMN-673 and TMZ in mice is a consequence of intrinsic resistance to either or both of the drugs.

The combination of BMN-673 and TMZ in Ewing sarcoma xenografts is the most dramatic synergy between two drugs to be reported. The synergy in vivo is restricted to Ewing sarcoma, yet only half of the models respond to combination treatment with the other half completely resistant. Studies proposed in this application will potentially identify biomarkers that will allow identification of patients likely to benefit from such treatment, and spare toxicity for those unlikely to respond.

Citation Format: Ruth Carlson, Raushan Kurmasheva. Understanding the synergy between temozolomide and PARP inhibition in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2098.
Abstract 211: The mitochondrial protein MTP18 enhances chemosensitivity by promoting mitochondrial fission
Authors: Aung Lynn Htet Htet , Li Peifeng
Published: July 2016

Background: Chemotherapy plays a major role in cancer therapy. Yet, one of its greatest obstacles is the development of chemotherapy resistance. Hence, understanding the molecular basis of chemoresistance is crucial to improve its therapeutic outcome. A close relationship between mitochondrial dynamic and cellular apoptosis has recently been identified. Mitochondrial fission is necessary for initiation of cellular apoptosis, whereas mitochondrial fusion is able to inhibit apoptosis. However, the association of mitochondrial fission and chemo-sensitivity has not been widely explored. Recently, mitochondrial protein MTP18 has been shown to promote mitochondrial fragmentation in various cancer cell lines. However, its role in apoptosis and chemotherapeutic resistance remains elusive. Here, we aimed to detect 1) whether MTP18 is able to regulate mitochondrial fission and apoptosis in gastric cancer cells, and 2) by which mechanism it regulates these events.

Methods and Results: Doxorubicin is one of the most widely used chemotherapeutic agents in variety of cancers. Mitochondrial staining with MitoTracker Red CMXRos revealed doxorubicin was able to induce mitochondrial fission both in vitro and in vivo gastric cancer models. We observed doxorubicin led to a decreasing level of MTP18 expression in gastric cancer cell lines, and this effect was concentration- and time-dependent. To understand the role of MTP18 in doxorubicin-induced mitochondrial fission and apoptosis, endogenous MTP18 expression was knockdown using MTP18-shRNA. Strikingly, doxorubicin could not induce mitochondrial fission in gastric cancer cells transfected with MTP18-shRNA. On the other hand, upon enforced expression of MTP18, the same concentration of doxorubicin sensitized a significantly higher number of gastric cancer cells to undergo mitochondrial fission and apoptosis. These findings suggest that MTP18 is required for doxorubicin-induced mitochondrial fission and apoptosis. In exploring the molecular mechanism of MTP18, we found that MTP18 enriched dynamin-related protein Drp1 accumulation in mitochondria and mediated the signal of doxorubicin to induce mitochondrial fission. Drp1 accumulation in mitochondria caused the release of cytochrome-c to cytosol and subsequently promoted apoptosis.

Conclusion: Taken together, our findings suggest that MTP18 induces mitochondrial fission and enhances apoptosis in gastric cancer cells, by promoting Drp1 accumulation in mitochondria. Thus, it is compelling us to conclude that MTP18 enhances doxorubicin sensitivity of gastric cancer cells by targeting mitochondrial fission machinery.

Citation Format: Lynn Htet Htet Aung, Peifeng Li. The mitochondrial protein MTP18 enhances chemosensitivity by promoting mitochondrial fission. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 211.
Abstract 2112: ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells
Authors: Oba Takaaki , Ito Ken-ichi , Taniguchi Shun’ichiro
Published: July 2016

Background: Eribulin is a key drug for the patients with metastatic breast cancer; however, the mechanisms of how breast cancer cells become resistant to eribulin have not been fully elucidated. The purpose of this study is to analyze the mechanisms of eribulin resistance in breast cancer cells.

Cell lines:

Eribulin resistant cell lines were established for three estrogen receptor (ER) positive cell lines (MCF7/E, BT474/E,ZR75-1/E), three triple negative cell lines (MDA-MB-231/E, Hs578T/E, MDA-MB-157/E), and one HER2 positive cell line (SKBR3/E) by exposing to stepwise increased doses of eribulin.

Results: The mRNA and protein expressions of both ABCC11 and ABCB1 were increased in all eribulin resistant cell lines regardless of their subtype. Inhibition of either ABCC11 or ABCB1 by small interfering RNA (siRNA) partially restored the sensitivity to eribulin in MCF7/E, BT474/E, and ZR75-1/E cells. Moreover, dual inhibition of ABCC11 and ABCB1 additively restored the sensitivity to eribulin in MCF7/E cells. On the other hand, overexpression of exogenous ABCC11 caused eribulin resistance in MCF7 and MDA-MB-231 cells (resistance ratio; MCF7; 13.8±0.85, MDA-MB-231; 11.4±0.74). Interestingly, inhibition of ER expression by siRNA or down regulation of ER by fluvestrant decreased ABCC11 expression and increased the sensitivity to eribulin in MCF7/E cells.

Discussion: Our data demonstrates that ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells regardless of their subtype; moreover, our data suggests the possibility of involvement of ER pathway in the regulation of ABCC11 expression in the ER positive breast cancer cells. Although further studies should be required, the expression of ABCC11/ABCB1 may be used as biomarkers for prediction of response to eribulin in breast cancer. In addition, the inhibition of ABCC11/ ABCB1 might be of service in a novel strategy to enhance the antitumor effect of eribulin.

Citation Format: Takaaki Oba, Ken-ichi Ito, Shun’ichiro Taniguchi. ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2112.
Abstract 2154: TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine
Authors: Gnanamony Manu , Stepanova Victoria , Criscione Lily , Boyineni Jerusha , Marshall Stephen J. , Holterman AiXuan , Gondi Christopher S.
Published: July 2016

Pancreatic Ductal Adeno Carcinoma (PDAC) is the fourth most common cause of cancer deaths in the United States and accounts for over 95% of all pancreatic cancers. The combined 1- and 5-year survival rates for PDAC are very poor, at 25% and 6% respectively. A major hallmark of pancreatic cancer is tumor recurrence and extremely poor response to chemotherapy. The current standard of care for patients with advanced pancreatic cancer is a chemotherapy regimen that includes gemcitabine. This treatment results in a modest benefit i.e., an increase in survival of only 5 weeks. The high mortality in patients diagnosed with this pancreatic cancer is primarily due to its drug-resistant nature and the lack of effective therapeutic strategies to overcome drug resistance. This disappointing situation strongly suggests that an improved and increased understanding of drug resistance mechanisms could lead to the development of novel therapeutic strategies for the successful treatment of patients with pancreatic cancer.

Our studies have shown that one of the contributing factors that lead to chemotherapy resistance is the protease activator urokinase plasminogen activator (uPA). To validate the contribution of uPA in chemoresistance, we overexpressed uPA in MIAPaCa-2 and PANC-1 cells and determined chemoresistance. We observed that uPA overexpressed cells showed increased chemoresistance in both Mia PaCa-2 (>25 fold) and PANC-1 (>6 fold). Mass-Spec analysis of nuclear uPA-IP from pancreatic cancer cells revealed that uPA interacts with Topoisomerase-1 (TOPO-1). This was further validated by western blot analysis of nuclear co-IP in pancreatic cancer cells. Further the binding domains of TOPO-1 to uPA were identified using overlapping a peptides array (PepSpot-JPT) followed by synthesis of these peptides. We observed that a TOPO-1 homologous peptide designated TX1111 was capable of significantly suppressing gemcitabine resistance in pancreatic cancer cells. Our observations show that the specific peptide TX1111 suppresses chemoresistance by 45% in Mia Paca-2 cells (p<0.001) and by 40% in PANC cells (p = 0.002).

These results demonstrate that:

1. Overexpression of uPA promotes gemcitabine resistance in pancreatic cancer cells

2. uPA and TOPO-1 interact in the nucleus of pancreatic cancer cells

3. The peptide homologue of TOPO-1, TX1111 sensitizes gemcitabine resistant pancreatic cancer cells.

Citation Format: Manu Gnanamony, Victoria Stepanova, Lily Criscione, Jerusha Boyineni, Stephen J. Marshall, AiXuan Holterman, Christopher S. Gondi. TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2154.