bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3198: The RNA-binding protein Musashi-1 stabilizes TAC1 mRNA in breast cancer cells.
Authors: Nahas George R. , Murthy Raghav G. , Greco Steven J. , Rameshwar Pranela
Published: August 2014

Breast cancer (BC) remains a major public health challenge in the United States. Prognosis for metastatic cancer is poor with nearly all deaths from BC due to metastatic disease. Upon discovery in bone, the five year survival rate of BC patients is approximately eight percent. The tachykinin gene, TAC1, has been implicated in facilitating metastasis of BC to the bone marrow. The predominant product of TAC1 is the neuropeptide, substance P (SP). We have previously demonstrated that in non-malignant cells, TAC1 translation is suppressed by miRNAs. Stimulation with specific cytokines decreased these miRNA to allow synthesis of SP. BC cells (BCCs) lack endogenous expression of the TAC1-specific miRNA, which is consistent with the increased levels of SP in these cells. Herein the present study investigated the translational control of TAC1 mRNA in BCCs. Cytoplasmic extracts from BCCs showed an increase in TAC1 translation and stabilization of its mRNA. These findings led us to bioinformatic analyses of the secondary structure of TAC1 mRNA, which predicted candidate interacting sites in Exons 3 and 7. These predictions were further refined by results from an RNA shift assay that indicated an RNA-binding protein specifically targeting Exon 7. Through western blots and proteomic analysis we identified the RNA-binding protein, Musashi-1 (Msi1), as a putative candidate. Identity was confirmed by RNA supershift assay utilizing an antibody specific for Msi1. Gain-of-function and reporter gene assays utilizing the Msi1 expression vector co-transfected with a luciferase reporter gene linked to the 3′UTR of TAC1, including Exon 7, confirmed a stabilizing role for Msi1 in TAC1 mRNA regulation, concomitant with an increase in substance P. In summary, we identified Msi1 as a novel binding protein for the oncogenic TAC1 mRNA. These findings have implications for the identification of novel targets to treat BC.

Citation Format: George R. Nahas, Raghav G. Murthy, Steven J. Greco, Pranela Rameshwar. The RNA-binding protein Musashi-1 stabilizes TAC1 mRNA in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2013-3198
Abstract 3201: PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls.
Authors: Arslan Ahmet Dirim , Asztalos Szilard , Stampfer Martha , Tonetti Debra , He Xiaolong , Beck William T.
Published: August 2014

PTBP1 (polypyrimidine tract-binding) protein is a splicing factor that is frequently upregulated in ovarian and breast tumors. Knockdown (KD) of PTBP1 expression by shRNA impairs ovarian tumor cell growth in vivo and in vitro, as well as colony formation and invasiveness in vitro (He et al, Oncogene 26:4961-8, 2007). We have also found that PTBP1 is progressively upregulated in immortalized human mammary epithelial cells (HMECs) and human ovarian surface epithelial (HOSE) cells, correlating with their transformation state. These results prompted us to determine the factors that play a role in the upregulation of PTBP1 protein expression. We first asked whether the increased PTBP1 protein in human ovarian and breast cell lines is accompanied by proportional increases of PTBP1 mRNA. We found that there was a discordance between PTBP1 mRNA and protein levels, as measured by RT-PCR and western blot, in these cancer cell lines in relation to the control cells (finite lifespan HMEC or HOSE, respectively). Accordingly, we attempted to understand the basis for this discrepancy, and measured PTBP1 protein half-life. We treated A2780 (ovarian cancer), MCF7 (breast cancer) cells, and “finite lifespan” HMEC (184A1) with cycloheximide to terminate protein synthesis and followed the fate of PTBP1 protein over time by western blot. We found that the half-life of PTBP1 protein is ∼3-5 h in the HMECs with finite lifespan, whereas it ranges from ∼9-12h in human epithelial ovarian and breast cancer cells. These results demonstrate that PTBP1 expression is regulated in part by events that control the stability of the protein and suggest that the normal regulation of PTBP1 protein turnover is altered in cancer cell lines. Bioinformatics analysis identified one possible SUMO-binding motif site within the PTBP1 protein (SUMOsp 2.0 - SUMOylation Site Prediction, http://sumosp.biocuckoo.org/), suggesting that SUMOylation may play a role in PTBP1 stability; we are currently examining this possibility. Findings presented herein will enhance our understanding of the regulation of PTBP1 expression and the mechanisms underlying its variation in protein and mRNA levels in ovarian and breast tumors.

(Support in part by NCI grants R01 CA40570 and R01 CA138762 [to WTB], by OCRF [to XH], and by UIC.)

Citation Format: Ahmet Dirim Arslan, Szilard Asztalos, Martha Stampfer, Debra Tonetti, Xiaolong He, William T. Beck. PTBP1 stability is increased in ovarian and breast cancer cell lines compared to matched controls. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3201. doi:10.1158/1538-7445.AM2013-3201
Abstract 3207: Application of an antibody-free, highly sensitive PRISM-SRM proteomics approach for monitoring low abundance proteins and protein isoforms in cell lines and tumor tissue.
Authors: He Jintang , Sun Xuefei , Shi Tujin , Schepmoes Athena , Fillmore Thomas , Petyuk Vladislav , Xie Fang , Gritsenko Marina , Yang Feng , Kitabayashi Naoki , Chae Sung Suk , Rubin Mark , Siddiqui Javed , Wei John , Chinnaiyan Arul , Qian Wei-Jun , Smith Richard , Kagan Jacob , Srivastava Sudhir , Liu Tao , Rodland Karin , Camp David
Published: August 2014

Stable-isotope dilution coupled to targeted mass spectrometry, applying selected reaction monitoring (SRM), provides good selectivity, reproducibility, and sensitivity rendering it highly suitable for robust and high-throughput protein quantification. Historically, SRM sensitivity for measuring protein concentration in biofluids such as blood plasma has been limited to the low μg/mL level. Application of front-end sample preparation methods such as immunoaffinity depletion and strong cation exchange (SCX) chromatographic fractionation has improved the SRM detection limit to low ng/mL level. The ability to detect and quantify low abundance proteins present in blood plasma/serum at sub-ng/mL levels, however, relies on the availability of specific affinity reagents (e.g., antibodies that have the associated disadvantage of an expensive, lengthy, and high failure rate de novo process).

To address this limitation, we have recently developed an antibody-free, targeted quantification capability that enables protein quantification of prostate-specific antigen and other exogenous proteins at 50-100 pg/mL levels in plasma/serum. Unlike the traditional SCX fractionation approach where all resulting fractions are analyzed by the second dimension LC-SRM, this new capability, PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing), uses high resolution.

Citation Format: Jintang He, Xuefei Sun, Tujin Shi, Athena Schepmoes, Thomas Fillmore, Vladislav Petyuk, Fang Xie, Marina Gritsenko, Feng Yang, Naoki Kitabayashi, Sung Suk Chae, Mark Rubin, Javed Siddiqui, John Wei, Arul Chinnaiyan, Wei-Jun Qian, Richard Smith, Jacob Kagan, Sudhir Srivastava, Tao Liu, Karin Rodland, David Camp. Application of an antibody-free, highly sensitive PRISM-SRM proteomics approach for monitoring low abundance proteins and protein isoforms in cell lines and tumor tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2013-3207
Abstract 3213: A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays.
Authors: Ryner Lisa , Fourie Aimee , Tsai Siao Ping , Scales Suzie , Kozak Kathy , Firestein Ron , Guan Yinghui , Do An , Wang Yulei
Published: August 2014

MUC16 and MSLN are tumor-specific antigens highly prevalent in ovarian and pancreatic cancers and have been used as targets in developing antibody-drug conjugates (ADCs). Development of a sensitive method to quantitatively measure these protein biomarkers in archival clinical samples may enable potential companion diagnostics to these novel therapeutic agents. Here we report the development of a method to quantitatively measure these biomarkers from formalin fixed paraffin embedded (FFPE) tumor samples using TaqMan proximity ligation assays (PLA). Monoclonal antibodies that recognize the extracellular domains of MUC16 and MSLN were conjugated to amplicon forming oligonucleotides to generate proximity ligation probes for TaqMan quantification. Using purified MUC16 and MSLN recombinant protein the assay sensitivities were determined to be lower than single-digit ng/mL concentrations for both MUC16 and MSLN and the dynamic ranges were >3 logs and ∼2 logs, respectively. In addition, the assays were demonstrated to be highly selective by testing lysates from expressing and non-expressing cell lines as determined by qRT-PCR. For clinical application of these assays, we further developed and optimized a simple two-step method for extraction and antigen retrieval of protein from archival FFPE tissues. FFPE extracts were prepared from a set of 10um sections of ovarian tumor tissue blocks and analyzed to determine relative MUC16 and MSLN protein expression levels. We benchmarked these results from the FFPE protocol with PLA data generated from matched fresh frozen tumor tissue as well as expression level determined by immunohistochemistry (IHC) and gene expression level determined by qRT-PCR assays from the same FFPE tissue blocks. All three comparisons with FFPE PLA were remarkably well correlated. In conclusion, we have established the technical feasibility and clinical utility of TaqMan PLA-based protein assays for sensitive and quantitative measurement of protein biomarkers in archival clinical samples. The methodology developed in this study represents a promising approach in companion diagnostic development for quantitative protein biomarker assessment.

Citation Format: Lisa Ryner, Aimee Fourie, Siao Ping Tsai, Suzie Scales, Kathy Kozak, Ron Firestein, Yinghui Guan, An Do, Yulei Wang. A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2013-3213
Abstract 3238: A novel natural product disrupts androgen receptor signaling in prostate cancer cells.
Authors: Amin Karishma , Jagadeesh Shankar , Barua Nabin , Bhattacharya Samir , Banerjee Partha P.
Published: August 2014

Continued reliance on androgen receptor (AR) signaling is a hallmark of prostate cancer, including the development of castration-resistant prostate cancer (CRPC), making it an attractive therapeutic target for prostate cancer treatment. Mahanine is a novel carbazole alkaloid derived from the leaves of Murraya koenigii, commonly known as the curry leaf plant, which grows widely across East Asia. We have reported previously that mahanine possesses the ability to inhibit prostate cancer cell growth. Since sustained AR signaling is associated with the development of CRPC, we investigated the effect of mahanine treatment on AR signaling in prostate cancer cells. Using reverse transcriptase polymerase chain reaction, we observed that AR signaling was disrupted by mahanine treatment, leading to a prominent decline in the expression of a number of AR target genes. Androgen response element-luciferase and PSA promoter-luciferase assays showed that androgen-induced AR transcriptional activity diminished by approximately 85% upon mahanine treatment. Furthermore, mahanine induced the export of the AR from the nucleus, resulting in its accumulation in the cytoplasm. Cellular levels of the AR were found to decrease in a time- and dose-dependent manner following mahanine treatment, without any noticeable change in its message levels, suggesting that mahanine affected AR protein levels post-translationally, without any effect on its gene expression. Concomitant administration of a proteasome inhibitor, MG132, significantly rescued the decline in AR levels observed upon mahanine treatment, indicating that mahanine induced proteasomal degradation of the AR. Collectively, these data demonstrate that mahanine treatment disrupts AR signaling in prostate cancer cells, leading to its cytoplasmic accumulation and subsequent degradation by proteasomal mechanisms, and suggest a potential therapeutic role for mahanine in prostate cancer treatment. This work is supported by NIH R01 CA131123.

Citation Format: Karishma Amin, Shankar Jagadeesh, Nabin Barua, Samir Bhattacharya, Partha P. Banerjee. A novel natural product disrupts androgen receptor signaling in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3238. doi:10.1158/1538-7445.AM2013-3238
Abstract 3248: Oral administration of FAK inhibitor TAE226 inhibits the progression of peritoneal dissemination of colorectal cancer.
Authors: Takaoka Munenori , Hao Hui-fang , Bao Xiao-hong , Tomono Yasuko , Sakurama Kazufumi , Ohara Toshiaki , Fukazawa Takuya , Yamatsuji Tomoki , Naomoto Yoshio
Published: August 2014

Peritoneal dissemination is one of the most terrible types of colorectal cancer progression. Focal adhesion kinase (FAK) plays a crucial role in the biological processes of cancer, such as cell attachment, migration, proliferation and survival, all of which are essential for the progression of peritoneal dissemination. Since we and other groups have reported that the inhibition of FAK activity exhibited a potent anticancer effect in several cancer models, we hypothesized that TAE226, a novel ATP-competitive tyrosine kinase inhibitor designed to target FAK, can prevent the occurrence and progression of peritoneal dissemination. In vitro, TAE226 greatly inhibited the proliferation and migration of HCT116 colon cancer cells, while their adhesion on the matrix surface was minimally inhibited when FAK activity and expression was suppressed by TAE226 and siRNA. In vivo, when HCT116 cells were intraperitoneally inoculated in mice, the cells could attach to the peritoneum and begin to grow within 24 h regardless of the pretreatment of cells with TAE226 or FAK-siRNA, suggesting that FAK is not essential, at least for the initial integrin-matrix contacts. Interestingly, the treatment of mice before and after inoculation significantly suppressed the cell attachment to the peritoneum. Furthermore, oral administration of TAE226 greatly reduced the size of disseminated tumors and prolonged survival in tumor-bearing mice. Taken together, a possible strategy for inhibiting peritoneal dissemination by targeting FAK, including TAE226, appears to be applicable through anti-proliferative and anti-invasion/anti-migration mechanisms.

Citation Format: Munenori Takaoka, Hui-fang Hao, Xiao-hong Bao, Yasuko Tomono, Kazufumi Sakurama, Toshiaki Ohara, Takuya Fukazawa, Tomoki Yamatsuji, Yoshio Naomoto. Oral administration of FAK inhibitor TAE226 inhibits the progression of peritoneal dissemination of colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2013-3248
Abstract 3267: Bortezomib sensitizes human acute myeloid leukemia cells to all-trans-retinoic acid-induced differentiation by modifying the RARα/STAT1 axis.
Authors: Ying Meidan , Zhang Lei , Zhong Like , Shao Xuejing , Luo Peihua , Yang Xiaochun , Yang Bo , He Qiaojun
Published: August 2014

All-Trans-Retinoic Acid (ATRA) has held great promise for differentiation-based therapy, but reportedly down-regulates retinoic acid receptor alpha (RARα) in a proteasome-dependent manner, which leads to decreased AML cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond APL are key investigative avenues. Here, we demonstrate that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation, without cell death. Additionally, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (p<0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy.

Citation Format: Meidan Ying, Lei Zhang, Like Zhong, Xuejing Shao, Peihua Luo, Xiaochun Yang, Bo Yang, Qiaojun He. Bortezomib sensitizes human acute myeloid leukemia cells to all-trans-retinoic acid-induced differentiation by modifying the RARα/STAT1 axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2013-3267

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3269: Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery .
Authors: Rodriguez-Aguayo Cristian , Shen Haifa , Gonzalez-Villasana Vianey , Zhang Guodong , Deng Xiaoyong , Aslan Burcu , Liu Xuewu , Sakamoto Jason , Chavez-Reyes Arturo , Han Hee-Dong , Sood Anil K. , Ferrari1 Mauro , Lopez-Berestein Gabriel
Published: August 2014

RNA interference has the potential to specifically knock down the expression of target genes, and thereby transform cancer therapy. However, lack of effective delivery of small inhibitory RNA (siRNA) has dramatically limited its in vivo applications. We have developed a multistage vector (MSV) system, composed of discoidal porous silicon particles loaded with siRNA oligos packaged in dioleoyl phosphatidylcholine (DOPC) nanoliposomes, that directs effective delivery and sustained release of siRNA in tumor tissues. Tumor accumulation of siRNA was observed after i.v. administration of MSV/siRNA, and sustained release of siRNA from DMSV maintained high levels of siRNA in tumor for over one week after a single dose of DMSV/siRNA. Treatment of SKOV3ip2 tumor mice with DMSV carrying siRNA oligos specific for the human EphA2 gene (MSV/EphA2) biweekly for 6 weeks resulted in dose-dependent (5, 10 and 15 μg/mice) reduction of tumor weight (36%, 64%, and 83%) and number of tumor nodules compared with the control groups. In addition, tumor growth was completely inhibited when mice were treated with MSV/EphA2 in combination with paclitaxel. Furthermore, combination treatment with MSV/EphA2 and docetaxel inhibited growth of HeyA8-MDR tumors, which were otherwise resistant to docetaxel treatment. Taken together, MSV/EphA2 merits further development as a therapeutic approach for ovarian cancer.

Citation Format: Cristian Rodriguez-Aguayo, Haifa Shen, Vianey Gonzalez-Villasana, Guodong Zhang, Xiaoyong Deng, Burcu Aslan, Xuewu Liu, Jason Sakamoto, Arturo Chavez-Reyes, Hee-Dong Han, Anil K. Sood, Mauro Ferrari1, Gabriel Lopez-Berestein. Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3269. doi:10.1158/1538-7445.AM2013-3269
Abstract 3323: E7449, a potent inhibitor of PARP 1 and 2, is active in acute myeloid leukemia.
Authors: McGonigle Sharon , Chen Zhihong , Wu Jiayi , Kolber-Simonds Donna , McGrath Shannon , TenDyke Karen , Wang Zichun , Nomoto Kenichi
Published: August 2014

Leukemic cells, like many other cancer cells, are often deficient in DNA repair capacity. PARP inhibitors have demonstrated synthetic lethality in cancer cells with defective DNA repair. E7449 is an orally bioavailable, potent, small molecule inhibitor of poly(ADP-ribose) polymerase (PARP)1 and PARP2. In this study we describe the therapeutic potential of the PARP inhibitor E7449 in acute myeloid leukemia (AML). E7449 inhibited proliferation of leukemia cells in a long term in vitro growth inhibition assay in a panel of leukemic cell lines. The MV-4-11 AML cell line was identified as particularly sensitive to E7449 PARP inhibition, and induction of apoptosis was observed in these cells. An in vivo survival model of acute myeloid leukemia (AML) was established in NSG mice using MV-4-11-luc2/AcGFP human AML cells. Daily dosing of E7449 at 100 mg/kg resulted in statistically significant anticancer activity in this model. Reduced disease burden as measured by luciferase signal was observed. This decrease in leukemic load also translated into a statistically significant survival benefit in E7449 treated mice. In AML cells, high P-glycoprotein (P-gp) expression is often observed, particularly in older patients, and this often leads to resistance to typical therapies. E7449 anti-proliferative activity was assessed in paired murine leukemia cells with basal (P388/S) and overexpressed P-gp protein (P388/VMDRC.04). E7449 effectively inhibited growth of the high P-gp expressing cells as well as the parent P388/S cells, with no significant difference in the presence or absence of verapamil, in contrast to data obtained for the P-gp substrate doxorubicin. These data demonstrate that E7449 is not a substrate for P-gp transport. Importantly, E7449 also significantly inhibited proliferation of primary patient AML cells ex vivo. In addition, E7449 proved highly potent at inhibiting colony formation in the majority of primary patient AML specimens assessed in long term (14 day) clonogenic survival assays. Studies to discern the mechanism underlying sensitivity to E7449 in AML cells are underway. Conclusion: In this study, E7449 possessed potent anti-cancer activity against AML cell lines and primary patient AML cells in vitro and ex vivo, it was not a substrate for P-gp transport, and it reduced disease burden and increased survival in an AML in vivo model. These data support the assessment of E7449 as a therapy for AML in the clinic.

Citation Format: Sharon McGonigle, Zhihong Chen, Jiayi Wu, Donna Kolber-Simonds, Shannon McGrath, Karen TenDyke, Zichun Wang, Kenichi Nomoto. E7449, a potent inhibitor of PARP 1 and 2, is active in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3323. doi:10.1158/1538-7445.AM2013-3323
Abstract 337: A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib.
Authors: Cai Jie , Yang Mengmeng , Deng Jianyun , Song Xiaoming , Huang Xuesong , Chen Taiping , Wery Jean Pierre , Chen Yiyou , Li Henry QX
Published: August 2014

Non-small cell lung cancer (NSCLC) has the highest mortality among all malignances while with limited treatment options. Among the most significant treatment advancement in recent years are the introduction of target therapies of EGFR-TKIs (Erlotinib and Gefitinib) for adenocarcinoma with EGFR activating mutations, and Crizotinib for NSCLC with ALK activation (EML4-ALK fusion)1. ALK activation are associated with a subset of NSCLC patients (3∼5%).Crizotinib is considered not a particularly potent ALK inhibitor, with IC50 at range of tens of nanomoles. It may not be an ideal agent for treating NSCLC with activating ALK. Therefore, a new ALK inhibitor with better potency and specificity could be of particularly medical importance. Currently, the industry is actively searching for such new agents. A relevant experimental model could also greatly facilitate evaluating and validating these new drug candidates.

Patient derived xenografts (PDX) (including NSCLC PDX) mirror patients’ histopathology and genetic profiles2, thus known as “patient avatar”. It has improved predictive power as preclinical cancer models, and enables discovery of predictive biomarkers for targeted therapeutics. We have established a large collection of NSCLC PDX models (NSCLC-HuPrime®) by engrafting naïve patient tumor tissues into immune-compromised mice2.

Among these models, we have identified one model, LU1656, of NSCL carcinosarcoma histology and with EML4-ALK fusion mutation. This mutation apparently causes over-expression of ALK as measured at mRNA level. This model has wild-type KRAS, c-MET and EGFR genes. Treatment of LU1656 with Crizotinib caused significant but not complete tumor response, suggesting ALK activation is one of the oncogenic drivers of this model. In addition, LU1656 also responded to Cetuximab, but respond poorly to Erlotinib. It would be interesting to examine the combination of these treatments. This model is, to our knowledge, the first reported PDX model with EML4-ALK fusion mutation. We believe that LU1656 would be a useful model to evaluate investigational ALK inhibitors, and new combination treatment regimens of Crizotinib and standard of care chemotherapies.

Citation Format: Jie Cai, Mengmeng Yang, Jianyun Deng, Xiaoming Song, Xuesong Huang, Taiping Chen, Jean Pierre Wery, Yiyou Chen, Henry QX Li. A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 337. doi:10.1158/1538-7445.AM2013-337
Abstract 3385: Increased eIF4E phosphorylation prevents response of prostate cancer cells to mTOR inhibitors.
Authors: D'Abronzo Leandro S. , Beggs Ryan , Bose Swagata , Ghosh Paramita
Published: August 2014

Inhibitors of the mammalian target of rapamycin (mTOR) had been used in various clinical trials, but with limited efficacy. The overall goal of the present studies was to identify predictive markers of response to mTOR inhibitors in prostate cancer (PCa) cells. We previously showed that the mTOR pathway is activated in castration resistant PCa (CRPC), while its inhibition upregulates androgen receptor (AR) signaling, resulting in resistance to mTOR inhibitors (Wang et al, Oncogene, 2008). mTOR forms two complexes, mTORC1 and mTORC2: in this study we compared the effects of RAD001, an mTORC1 inhibitor, BEZ235, a dual mTOR/PI3K inhibitor, and Torin1, which inhibits both mTORC1 and mTORC2. Comparison of the effects of RAD001, alone, or in combination with the AR inhibitor bicalutamide in various CRPC cell lines showed that although C4-2 cells responded well to the combination, other cell lines, such as LNCaP-AI, PC-346C and CWR-R1, did not. The resistance of LNCaP-AI and CWR-R1 was partially overcome by treatment with BEZ-235 or Torin1, alone or in combination with bicalutamide; however, PC-346C cells were resistant to all three drugs. In an effort to identify the mechanism of resistance to mTOR inhibitors, therefore, the molecular effects of the drugs in downstream targets p70S6 kinase, 4E-BP1, eIF4G, Akt and eIF4E were compared in C4-2 (least resistant) and PC-346C (most resistant) cell lines. Western blots revealed that p70S6 kinase was downregulated by all the drugs whereas 4E-BP1 phosphorylation was effectively downregulated by BEZ235, but not Torin1, or RAD001. Phosphorylation of both p70S6K and 4E-BP1 results in activation of the translation initiation complex: a complex of eIF4E and eIF4G, therefore, inactivation of the translation initiation complex requires inhibition of eIF4E phosphorylation, which is independent of mTOR activation. In C4-2 cells, eIF4E phosphorylation was increased by RAD001 but suppressed by the other two drugs. However, in PC-346C cells, despite inhibition of 4E-BP1 phosphorylation, eIF4E phosphorylation was increased by all 3 drugs. Our data indicate eIF4E phosphorylation as a likely cause of resistance to mTOR inhibitors in PCa.

Citation Format: Leandro S. D'Abronzo, Ryan Beggs, Swagata Bose, Paramita Ghosh. Increased eIF4E phosphorylation prevents response of prostate cancer cells to mTOR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3385. doi:10.1158/1538-7445.AM2013-3385
Abstract 3438: Selective inhibitors of Mcl-1 with potent activity against multiple myeloma patient cells and animal xenografts.
Authors: Azmi Asfar S. , Nikolovka-Coleska Zaneta , Abidi Muneer , Marsack Kelley , Masood Ashiq , Yano Hiroshi , Pregja Silvana , Zonder Jeffrey , Mohammad Ramzi M.
Published: August 2014

To date most of the studies targeting apoptotic machinery have focused on inhibiting Bcl2 and BclXL proteins that have shown limited clinical utility. This could be in part due to the over-expression of Myeloid Leukemia-1 (Mcl-1) that carries similar anti-apoptotic functions. Our recent evaluations of Multiple Myeloma (MM) patient derived samples have shown consistent over-expression of Mcl-1 suggesting that it is an attractive therapeutic target. We have discovered a novel druggable site in Mcl-1 protein. Using structure based drug design we have developed small molecule inhibitors (SMIs) of Mcl-1 that bind with high affinity to the BH-3 hydrophobic groove of the protein thereby displacing different pro-apoptotic partners such as Bax, Bak, Bok and Bid with activity against MM cells (IC50 5 μM) and anti-tumor activity in animal xenografts. The lead SMI (UMI-77) inhibits growth and induces apoptosis in MM cells. Molecular analysis of MM cells reveals activation of pro-apoptotic factors post UMI-77 treatment. Co-immunoprecipitation studies show that UMI-77 disrupts the Mcl-1-Bax and Mcl-1-Bak interactions. The drug synergizes with standard proteasome inhibitor Velcade (5 nM) and Carfilosimib (1 nM). In patient derived MM cells (n=45), UMI-77 inhibits growth and induces apoptosis that is concurrent with induction of Bax, Bak and enhancement in cleaved fractions of caspases 3 and 9. At similar concentrations UMI-77 does not affect normal peripheral lymphocytes. UMI-77 is well tolerated by animals at MTD (60 mg/kg i.v.). In a mice lymphoma model, oral administration of UMI-77 significantly reduced tumor growth and histological examination of tumors revealed suppression of Mcl-1 protein expression along with enhancement of pro-apoptotic markers. Our novel Mcl-1 inhibitors provide a new therapeutic option for cancers for MM that overexpress Mcl-1 that have failed standard therapies.

Citation Format: Asfar S. Azmi, Zaneta Nikolovka-Coleska, Muneer Abidi, Kelley Marsack, Ashiq Masood, Hiroshi Yano, Silvana Pregja, Jeffrey Zonder, Ramzi M. Mohammad. Selective inhibitors of Mcl-1 with potent activity against multiple myeloma patient cells and animal xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3438. doi:10.1158/1538-7445.AM2013-3438
Abstract 3447: Obatoclax analogue SC-2001 induced autophagy through SHP1/STAT3 pathway in hepatocelluar carcinoma.
Authors: Su Jung-Chen , Liu Chun-Yu , Tai Wei-Tien , Chen Kuen-Feng , Shiau Chung-Wai
Published: August 2014

Obatoclax induced cancer cells apoptosis through targeting anti-Bcl-2 family. Previously, we have generated 20 obatoclax analogues with three synthetic steps and tested cytotoxicity in HCC cell lines. Among these analogues, SC-2001 not only inhibited anti-Bcl-2 family activity but also reduced STAT3 phosphorylation and subsequently repressed transcriptional activity of STAT3 targeting genes. Here, we further revealed that SC-2001 induced LC3-II with time and dose dependent manner in HCC cell line. We also observed that autophogosomes were induced with SC-2001 treated cells under electron microscope detection. Conversely, sodium vanadate, a phosphatase inhibitor, abolished the LC3 induction and autophagic effect in SC-2001 treated cells. In addition, overexpression of STAT3 in PLC5 cells attenuate autophagic effect with SC-2001 treatment. Genetic knockdown of SHP-1, a negative regulator of STAT3, by siRNA reduced autophagy induced by SC-2001. In summary, our data suggested that SC-2001 can induce autophagic cell death through, at least in part, SHP-1/STAT3 signaling pathway. (Supported by NSC-100-2325-B-010-007)

Citation Format: Jung-Chen Su, Chun-Yu Liu, Wei-Tien Tai, Kuen-Feng Chen, Chung-Wai Shiau. Obatoclax analogue SC-2001 induced autophagy through SHP1/STAT3 pathway in hepatocelluar carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3447. doi:10.1158/1538-7445.AM2013-3447
Abstract 3451: Gramicidin-based nanopores induce cellular energy depletion and cell death in renal cell carcinoma.
Authors: David Justin M. , Rajasekaran Ayyappan K.
Published: August 2014

The plasma membrane physically separates the intracellular components of a cell from the extracellular environment and its integrity is absolutely essential to sustain cellular functions. Ionophores are lipid soluble organic molecules that disrupt cellular transmembrane potential by rendering biological membranes permeable to specific ions. They are classified on the basis of their mechanism of action; mobile carriers complex with metal cations to shield their hydrophilic charge from the interior of the lipid bilayer and channel-formers insert into the membrane to form hydrophilic pores that permit the rapid passage of selected ions through membranes. Research over the past decade has described an new role for mobile carrier ionophores as novel anticancer therapeutics yet the potential anticancer attributes of channel-forming ionophores have not been explored. In this study we utilized the channel-forming ionophore gramicidin A (GA) as a model nanopore to study its effects upon the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional chemotherapy. We found that disruption of plasma membrane integrity with GA reduced the in vitro viability of several RCC cell lines when treated at sub-micromolar concentrations (all IC50 < 1.0μM). Comparison of GA with the mobile carrier ionophore monensin revealed that cell viability was decreased by GA to a degree that was equal to or greater than monensin depending on the cell line. Mechanistic examination determined that GA induced cell death that was associated with cellular energy depletion. Importantly, GA exhibited similar toxicity in RCC cells regardless of histological subtype or the expression of either the von Hippel-Lindau (VHL) tumor suppressor or its downstream target hypoxia-inducible factor (HIF) -1α. Taken together, these results demonstrate the utility of nanopore-forming drugs as a new strategy for cancer therapy.

Citation Format: Justin M. David, Ayyappan K. Rajasekaran. Gramicidin-based nanopores induce cellular energy depletion and cell death in renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3451. doi:10.1158/1538-7445.AM2013-3451
Abstract 3562: Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like breast cancer cells.
Authors: Tavera Luz E. , Westerling Thomas , Brown Myles
Published: August 2014

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), regulates gene expression through the vitamin D receptor (VDR). Even though 1,25D3 is still best known as a regulator of bodily calcium homeostasis, 1,25D3 has a plethora of physiological actions which include immune and nervous system modulation, cellular proliferation and differentiation. Epidemiological and model studies have revealed the cancer-protective properties of 1,25D3 in colon, prostate and notably in breast. We are actively investigating the anti-proliferative properties of 1,25D3 in a panel of human breast cancer cell lines. We found that 1,25D3 induces autophagy in several luminal-like breast cancer cell models, but not in basal-like or mesenchymal-like models. We performed VDR and signaling partner RXR whole-genome ChIP-seq analysis to uncover all of the direct 1,25D3 target genes involved in regulation of metabolism and autophagy. Our studies suggest that energy regulation as an important aspect of 1,25D3 anti-proliferative properties in luminal-like breast cancer cells.

Citation Format: Luz E. Tavera, Thomas Westerling, Myles Brown. Genome-wide analysis of the vitamin D receptor (VDR) binding sites reveals vitamin D role modulating autophagy and metabolism in luminal-like breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2013-3562
Abstract 3776: Hypoxia-inducible factor 1α (HIF1α) has a prometastatic effect in uveal melanoma .
Authors: Asnaghi Laura , Lin Michael H. , Lim Kah Suan , Lim Kah Jing , Bar Eli E. , Eberhart Charles G.
Published: August 2014

Melanomas arising from the uveal tract of the eye represent the most frequent primary intraocular malignancy in adults and are the second most common type of melanoma. Uveal melanomas are prone to disseminate hematogenously to the liver, and have an overall survival rate of approximately 50%. Hypoxia-inducible factor 1α (HIF1α), a master transcriptional regulator of the hypoxic response, is strongly associated with aggressive uveal melanomas. Elevated expression of one of the HIF target gene lysyl oxidase (LOX) has been found at the invasive front of primary tumors, and is associated with shorter metastasis-free survival. Here we investigated the mechanism responsible for the prometastatic effect of HIF1α in uveal melanoma. Western blot examination of HIF1α and HIF2α proteins in uveal melanoma cell lines grown in normoxia (21% pO2) and in hypoxia (1% pO2) revealed HIF2α to be low. In contrast, HIF1α protein was relatively abundant in the OCM1, OCM3, OMM1, Mel290 and 92.1 uveal melanoma lines grown in normoxia, and was further induced by up to 5 fold in hypoxia. The mRNA levels of VEGF and LOX, two transcriptional targets of HIF1α, were also induced by 2 to 4 fold in hypoxia. Interestingly, while hypoxia reduced the growth of these lines as measured by MTS assay, low oxygen tension increased the ability of uveal melanoma cells to invade Matrigel by 2 to 5 fold. Genetic suppression of HIF1α expression through shRNA in OCM1 and 92.1 cells significantly decreased cellular growth in hypoxia (p<0.0001) and greatly impaired cellular invasion both in normoxia and in hypoxia (p<0.0001), suggesting that HIF1α is necessary for invasion even under normoxic conditions. In order to determine the downstream targets responsible for such profound effect of HIF1α on cellular invasion, we examined expression of Notch pathway members, which we have previously linked to uveal melanoma invasion. Interestingly, hypoxia induced mRNA expression of the Notch ligand Jag2 and target Hey1 by up to 3 fold. We also analyzed the expression of Snail, which is known to promote invasion and metastasis of tumors outside the eye, and which can be regulated by HIF1α. We observed more than 2 fold increase of Snail mRNA levels under hypoxic conditions in OCM1 cells, and this induction was abolished by suppressing HIF1α using shRNA. These data suggest that HIF1α plays a crucial role in promoting metastatic spread in uveal melanoma cells, possibly by regulating Notch activity or expression of the Snail transcription factor.

Citation Format: Laura Asnaghi, Michael H. Lin, Kah Suan Lim, Kah Jing Lim, Eli E. Bar, Charles G. Eberhart. Hypoxia-inducible factor 1α (HIF1α) has a prometastatic effect in uveal melanoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3776. doi:10.1158/1538-7445.AM2013-3776
Abstract 410: Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers.
Authors: Zhu He , Zhou Xinchun , Redfield Samantha , Lewin Jack , Miele Lucio
Published: August 2014

Background: Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with prostate cancer progression and metastasis.

Methods: Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded tissue specimens with various prostatic disorders, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low and high grade prostate cancers as well as metastatic prostate cancers. The expression levels of Jagged-1 and Notch-1 were reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3); with a maximal score equal to 9. ANOVA and t-test were employed to analyze the difference of IHC scores among and between different pathologic categories

Results: Jagged-1 cytoplasmic IHC scores in both metastatic PCa (5.92 ± 2.12, Mean ± SD) and high grade PCa (5.74 ± 2.45) were significantly higher than those in low grade PCa (3.55 ± 2.02, p = 9.02E-07 and 4.65E-08, respectively), in HGPIN (4.50 ± 2.34, p = 0.023 and 0.030, respectively), and in prostatic tissues with benign changes (3.39 ± 1.85, p = 7.96E-07 and 2.47E-07, respectively). In addition, Jagged-1 membranous IHC scores in both metastatic PCa (3.05 ± 3.52) and high grade PCa (2.41 ± 2.90) were also significantly higher than those in low grade PCa (0.71 ± 1.42, p = 1.48E-05 and 3.40E-06, respectively) and in prostatic tissues with benign changes (0.82 ± 1.28, p = 3.19E-04 and 0.001, respectively). Similarly, Notch-1 cytoplasmic IHC scores in both metastatic PCa (2.22 ± 2.11) and high grade PCa (1.10 ± 1.68) were significantly elevated when compared with the IHC scores observed in low grade PCa (0.51 ± 1.15, p = 3.63E-06 and 0.023, respectively) and in prostate tissues with benign changes (0.48 ± 1.01, p = 3.37E-06 and 0.017, respectively). Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasians (vs. African Americans) and in positive surgical resection margin group (vs. negative surgical resection margin group).

Conclusions: Our results provide strong evidence that dysregulation of Jagged 1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive prostate cancers.

Citation Format: He Zhu, Xinchun Zhou, Samantha Redfield, Jack Lewin, Lucio Miele. Elevated Jagged-1 and Notch-1 expression in high grade and metastatic prostate cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 410. doi:10.1158/1538-7445.AM2013-410
Abstract 4181: Oncogenic microRNA-183 induces cell growth and motility through down-regulation of Dkk-3 and SMAD4 in bladder cancer.
Authors: Ueno Koji , Hirata Hiroshi , Shahryari Varahram , Tanaka Yuichiro , Tabatabai Z. Laura , Hinoda Yuji , Dahiya Rajvir
Published: August 2014

Introduction and Objective : The purpose of this study was to carry out functional analysis of oncogenic microRNA-183 and identify their specific target genes in bladder cancer.

Methods : Expression levels of miR-183 in bladder normal/cancer tissues and bladder cancer cell lines were confirmed using real-time PCR. To examine the function of miR-183, cell growth, and migration were performed using UM-UC-3 cells because the miR-183 expression level was highest among bladder cancer cell lines. To search for target genes of miR-183, we used a target scan algorithm and performed Dkk-3 and SMAD4 3’UTR luciferase assays. To analyze whether Dkk-3 and SMAD4 affect cell growth and motility, Dkk-3 and SMAD4 expressing plasmids were transfected into UM-UC-3 cells and cell growth and motility were performed.

Results: Expression of miR-183 in cancer bladder tissues was significantly up-regulated compared to normal bladder tissues. MiR-183 expression was significantly higher in the pT3+4 tissues than in pT1+2 tissues. Higher miR-183 expression was significantly associated with shorter survival. MiR-183 increased cell growth and migration abilities in UM-UC-3 cells. MiR-183 decreased 3’UTR luciferase activity of Dkk-3 and SMAD4. Cell growth and motility was significantly decreased in Dkk-3 and SMAD4 transfected cells compared with control cells.

Conclusions : MiR-183 is a oncogenic microRNA in bladder cancer through down-regulation of Dkk-3 and SMAD4.

Citation Format: Koji Ueno, Hiroshi Hirata, Varahram Shahryari, Yuichiro Tanaka, Z. Laura Tabatabai, Yuji Hinoda, Rajvir Dahiya. Oncogenic microRNA-183 induces cell growth and motility through down-regulation of Dkk-3 and SMAD4 in bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4181. doi:10.1158/1538-7445.AM2013-4181