bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5298: Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer
Authors: KOMATSU HIROAKI , Oishi Tetsuro , Itamochi Hiroaki , Kudoh Akiko , Nonaka Michiko , Sato Seiya , Chikumi Jun , Sato Shinya , Shimada Muneaki , Kigawa Junzo , Harada Tasuku
Published: August 2015

Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including epithelial ovarian cancer (EOC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis, and VEGF expression is activated by hypoxia-inducible factor (HIF)-1α. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as prognostic biomarkers in patients with EOC.

Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2012 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and HIF-1α were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and HIF-1α and clinicopathologic variables. We also determined the protein expression of VEGF-A, -C, and HIF-1α by immunohistochemical staining in paraffin-embedded tumors from EOC patients

Results: The protein expressions of VEGF-A, -C, and HIF-1α were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and HIF-1α were 280, 4866 and 125 pg/ml, respectively. The levels of VEGF-A in patients at stage IV were significantly higher than those in patients at stage I. Both histologic subtypes and lymph node involvement were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the ascitic volume. The overall survival rate was significantly lower in FIGO stage III or IV patients with EOC. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (61.7% vs. 41.8%, P = 0.0147). Both HIF-1α and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors.

Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC.

Citation Format: HIROAKI KOMATSU, Tetsuro Oishi, Hiroaki Itamochi, Akiko Kudoh, Michiko Nonaka, Seiya Sato, Jun Chikumi, Shinya Sato, Muneaki Shimada, Junzo Kigawa, Tasuku Harada. Serum angiogenic factors may predict prognosis in patients with epithelial ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2015-5298
Abstract 5304: A serum protein test for improved prognostic stratification of patients with myelodysplastic syndrome (MDS)
Authors: Roder Joanna , Löffler-Ragg Judith , Stauder Reinhard , Germing Ulrich , Sperr Wolfgang R. , Valent Peter , Roder Heinrich , Steingrimsson Arni , Zwierzina Heinz
Published: August 2015

While existing scoring systems provide physicians with methods of assessing prognosis of patients with MDS and help with the choice of appropriate treatment regimens, some patients defined as lower risk still have poor outcomes and tests which can refine current stratifications are of clinical interest.

Serum samples, clinical and survival data were available from 106 MDS patients with refractory anemia (RA) (n = 46), RA with excess blasts (n = 29), RA with ring sideroblasts (n = 14), and chronic myelomonocytic leukemia (n = 17). The international prognostic scoring system (IPSS) score distribution 0/0.5/1/>1 was n = 36/23/24/12. Matrix assisted laser desorption/ionization (MALDI) mass spectra were acquired from the samples using the deep MALDI method1, allowing a deep probing of the proteome. The spectra were preprocessed and spectral features defined. The integrated intensities of these features were combined with the survival data using deep learning based machine learning techniques to create classifiers able to stratify patients into groups with better and worse survival.

Patients in the good prognosis group had median survival of 52 months, compared with 23 months in the poor prognosis group (hazard ratio = 0.37, p < 0.001). Within the subgroup of patients with refractory anemia (n = 46) it was possible to identify a group of patients with no death events before 30 months, while median survival in the other patients was only 25 months. Multivariate analysis stratified by MDS subtype found that the mass spectral (MS) classification was a significant independent predictor of survival even when adjusted for international prognostic scoring system (IPSS) score and cytogenetic status. MS classification was able to separate low to intermediate risk (IPSS 0-1) and good karyotype subgroups into groups with significantly better and worse survival.

If validated in an independent dataset, this serum test would provide complementary information for physicians to consider in addition to existing scoring systems to aid in planning treatment for MDS patients.

1. M Duncan et al, Proceedings of the 61st ASMS Conference on Mass Spectrometry and Allied Topics. Minneapolis, MN June 8-9, 2013 MP 181.

Citation Format: Joanna Roder, Judith Löffler-Ragg, Reinhard Stauder, Ulrich Germing, Wolfgang R. Sperr, Peter Valent, Heinrich Roder, Arni Steingrimsson, Heinz Zwierzina. A serum protein test for improved prognostic stratification of patients with myelodysplastic syndrome (MDS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5304. doi:10.1158/1538-7445.AM2015-5304
Abstract 531: lncRNA profiling and its potential usage as thyroid cancer biomarker
Authors: Tian Song , Rulli Samuel , Fahey Thomas J. , Lader Eric
Published: August 2015

Thyroid nodules are detected in as high as 67% of the population. Distinguishing thyroid cancers from benign lesions is crucial for determining appropriate and timely treatment. Fast and accurate detection using a simple screening method could greatly contribute to minimizing unnecessary surgery while increasing the accuracy of cancer detection. Several long non-coding RNAs (lncRNAs) have been linked to cancer development as either oncogenes or tumor suppressors, and their potential use as cancer biomarkers has been suggested. Unfortunately, limited analyses have been performed on the expression of lncRNA in thyroid cancer. Here, we explored the possible use of lncRNA as a specific thyroid cancer biomarker. The expression of 84 human cancer-related lncRNAs were evaluated in RNA samples from 9 thyroid cancer (PTC and FVPTC) patients and 13 non-cancer controls (NH and FA) with a simple real-time RT-PCR strategy. PTCSC1, an lncRNA previously linked to thyroid cancer, was found to be down regulated in patient 3.4 fold (p<0.005). In addition, we observed dysregulation of other lncRNA such as BANCR (down 3 fold, p<0.005) and SNHG16 (down 2 fold, p<0.005). We next used the lncRNA expression data for signature panel development to classify benign and malignant thyroid patient tissue samples. With a random forest classification model, a fine tuned classification signature of 16 target genes was reasonably successful (Accuracy 95%, sensitivity 92%, PPV = 90% and NPV = 100%). Our results suggested that in addition to the role that lncRNA plays in thyroid cancer, analysis of lncRNA expression represents a promising path towards a simple test for thyroid cancer detection.

Citation Format: Song Tian, Samuel Rulli, Thomas J. Fahey, Eric Lader. lncRNA profiling and its potential usage as thyroid cancer biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 531. doi:10.1158/1538-7445.AM2015-531
Abstract 5330: Methioninase-loaded erythrocytes: a promising drug for L-methionine restriction therapy in cancer
Authors: Gay Fabien , Bes Julie , Bourgeaux Vanessa , Godfrin Yann
Published: August 2015

Several studies reported a complete dependence of different tumor cells on L-methionine (Met), an essential amino acid, for maintaining their high proliferation rate while normal cells keep their ability to grow on Met-deprived medium (Cavuoto and Fenech, 2012).

Met restriction appearing as an important strategy for cancer treatment, different therapeutic approaches using either Met restricted regimens or Met-catabolizing enzymes were investigated.

Phase I and II clinical trials associating dietary Met restriction with chemotherapy were conducted with cancer patients (Thivat et al., 2009; Durando et al, 2010). A 40% reduction of plasmatic methionine was achieved with a 1-day Met-free diet per month. Interesting long-duration of disease stabilization was observed in some patients. However this diet is extremely difficult to maintain.

In order to obtain longer Met depletion in plasma, enzyme therapy using Methionine gamma-lyase (MGL), a pyridoxal-dependent enzyme that specifically degrades Met into alpha-ketobutyrate, ammonia and thiols, was explored. MGL enzyme isolated from Pseudomonas putida was selected as preferred candidate for the development of therapy. However, preclinical studies conducted in primates showed a very short half-life of the active form of MGL enzyme (2h) due to a rapid in vivo dissociation of the pyridoxal (PLP) cofactor (Yang et al., 2004).

To overcome this problem of PLP-dissociation, we encapsulated the active MGL into erythrocytes, natural pool of PLP in the body and evaluated PK-PD parameters of product over a 5-day period in a mouse model. Once entrapped into erythrocytes, the MGL enzyme maintained more than 30% of its specific activity after 5 days in circulation. A 60-70% Met depletion was achieved 15 min post-injection and then depletion was stabilized at 40% over the 5 day-period of the study. In comparison, administration of the free MGL at similar doses led to a rapid loss of the active form in several hours. Met was totally deprived 15 min post-injection but plasmatic Met level returned to baseline by 24h. Increasing two-fold the concentration of free methioninase did not improved the in vivo Met depletion.

In conclusion, we report that methioninase-loaded-erythrocytes can lower Met concentration in mouse plasma for at least 5 days and may be an innovative strategy to kill tumors by met-starvation.

Citation Format: Fabien Gay, Julie Bes, Vanessa Bourgeaux, Yann Godfrin. Methioninase-loaded erythrocytes: a promising drug for L-methionine restriction therapy in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2015-5330
Abstract 5336: Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma
Authors: Lin Hsiao-Hui , Feng Wen-Chi , Lu Li-Chun , Shao Yu-Yun , Cheng Ann-Lii , Hsu Chih-Hung
Published: August 2015

[Background] Sorafenib is currently the only approved systemic agent for advanced hepatocellular carcinoma (HCC); however, its efficacy is limited. Aberrant activation of WNT/β-catenin signaling pathway has been shown to mediate resistance to various types of anti-cancer therapy. We hypothesized that inhibition of WNT/β-catenin signaling pathway would improve the anti-tumor effect of sorafenib in HCC.

[Materials and Methods] Human HCC cell lines, including Huh7, HepG2, PLC5, and Hep3B, were included. Inhibition of WNT/β-catenin pathway was achieved by ICG-001, a small molecule disrupting the interaction of cAMP-responsive element binding (CREB)-binding protein (CBP) and β-catenin and inhibiting the β-catenin-mediated transcription activities, or by RNA interference (RNAi)- downregulation of β-catenin. The efficacy of sorafenib combined with WNT/β-catenin pathway inhibition in HCC cells was determined by MTT for cell viability, and by flow cytometry and Western blotting for apoptosis. The in vivo efficacy was evaluated in a subcutaneous xenograft mode of Huh7 cells.

[Results] In multiple HCC cell lines, ICG-001 enhanced the anti-proliferative effect of sorafenib dose-dependently; the combination of ICG-001 and sorafenib showed a synergistic antitumor effect. Downregulation of β-catenin by RNAi increased sorafenib sensitivity, whereas overexpression of β-catenin decreased sorafenib sensitivity in Huh7 cells. Furthermore, the sorafenib-sensitization effect by short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was offset by β-catenin overexpression. Mechanistically, sorafenib in combination of ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a more prominent downregulation of Mcl-1 in HCC cells. Finally, ICG-001 in combination of sorafenib showed a more significantly growth-retarding effect in Huh7 xenograft than either drug alone.

[Conclusion] Our data indicate that inhibition of WNT/β- catenin signaling pathway improves the antitumor effect of sorafenib against HCC in vitro and in vivo.(This study was supported by grants NRPB-100CAP1020-2 and NSC-101-2314-B-002-141, 100CAP1020-2).

Citation Format: Hsiao-Hui Lin, Wen-Chi Feng, Li-Chun Lu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. Improved antitumor effect of combining WNT/beta-catenin inhibition with sorafenib in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2015-5336
Abstract 5345: Enhanced inhibition of PC-3 xenograft prostate tumor growth by combination of green tea and quercetin with docetaxel
Authors: Wang Piwen , Henning Susanne , Heber David , Vadgama Jaydutt
Published: August 2015

Chemotherapy with docetaxel (Doc) is a standard treatment for metastatic and castration-resistant prostate cancer. However, the development of chemoresistance and its side effects limit the clinical success of Doc. Previously we demonstrated that the combined use of natural products green tea (GT) and quercertin (Q) with Doc synergistically enhanced the anti-proliferative effect in androgen-independent prostate cancer PC-3 cells. The objective of the present study was to confirm the combined effect of GT, Q and Doc in xenograft prostate cancer mouse models and determine underlying mechanisms. Male severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with 5×105 PC-3 cells subcutaneously. When tumors reached a size of about 100mm3 the intervention started. Mice were administered with GT+Q, Doc 5mg/kg (LD), GT+Q+LD Doc, Doc 10mg/kg (HD) or control. The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.4%. Doc was given through tail vein once a week for 4 weeks. There was a nonsignificant inhibition of tumor growth by GT+Q or LD Doc alone compared to control. However the combination treatment with GT+Q+LD Doc significantly inhibited the tumor growth by 62% compared to LD Doc alone after 7 weeks intervention. The combined effect was comparable to that by HD Doc treatment. Western blot analysis demonstrated a significantly increased ratio of Bax to Bcl-2 protein expression by GT+Q+LD Doc treatment compared to GT+Q or LD Doc. ELISA analysis of blood samples demonstrated that the combination treatment significantly decreased the blood concentrations of several growth factors, including VEGF, EGF, TGF-β, FGF-β, and TNF-α compared to GT+Q or LD Doc alone. A liver toxicity evaluation revealed that the blood alanine aminotransferase (ALT) level was significantly elevated with HD Doc treatment compared to control, while not changed by other groups. Further mechanistic investigations using protein array and tissue microarray assays are ongoing. This study provides a promising regimen by combining GT and Q with Doc to enhance chemotherapeutic effect in advanced prostate cancer in a less toxic manner. The results warrant future clinical trials to confirm the combined effect of the mixture in humans.

Citation Format: Piwen Wang, Susanne Henning, David Heber, Jaydutt Vadgama. Enhanced inhibition of PC-3 xenograft prostate tumor growth by combination of green tea and quercetin with docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5345. doi:10.1158/1538-7445.AM2015-5345
Abstract 5357: Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin for cancer therapy
Authors: Ali-Rahmani Fatima G. , Fitzgerald David , Martin Scott , Patel Paresma , Thomas Craig , Pastan Ira
Published: August 2015

Discoidin domain receptor 1 (DDR1) is an emerging anti-cancer target that belongs to the family of tyrosine kinases that are activated by collagen, the most abundant component of extracellular matrix (ECM). Collagen mediated induction of DDR1 facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially can affect tumor response to therapy. Mesothelin is a cell-surface tumor-associated antigen over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a clinically optimized recombinant immunotoxin (RIT) being developed in collaboration with Roche that should enter clinical trials late this year. It consists of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A in which immunogenic B cell epitopes are silenced. Activation of tyrosine kinase mediated signaling in tumor cells has been shown to modulate the activity of RITs. Therefore, we hypothesized that DDR1 regulates RIT activity and its inhibition might enhance the activity of RG7787. Knockdown of DDR1 by siRNA or its inhibition with a novel and specific inhibitor, ‘7rh’, synergistically enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed the knockdown of DDR1 increased mesothelin expression, internalization of RG7787, and enhanced inhibition of protein synthesis. Furthermore, stimulation of DDR1 activity by collagen treatment protected cancer cells from killing by RG7787. However, this collagen mediated resistance of cancer cells to RIT therapy can be overcome by addition of 7rh. Combination of 7rh with RG7787 synergistically inhibited growth of A431/H9 tumors in nude mice. In conclusion, we report that collagen protects cells from killing by RITs through DDR1 and that lowering DDR1 protein or inhibiting DDR1 kinase activity enhances the cytotoxic activity of RITs. Our data suggest that the combination of ‘7rh’ and RG7787 represents a novel therapeutic strategy to target mesothelin-expressing cancers. These data also provide insight into how tumor stroma might be protecting cancer cells from therapy.

Citation Format: Fatima G. Ali-Rahmani, David Fitzgerald, Scott Martin, Paresma Patel, Craig Thomas, Ira Pastan. Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) tyrosine kinase enhances cytotoxicity of anti-mesothelin immunotoxin for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2015-5357
Abstract 5379: A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity
Authors: Ott Heidi , van Aller Glenn , Ward Jessica , Le BaoChau , Rominger Cynthia , Foley James , Korenchuk Susan , McHugh Charles , Butticello Michael , Blackledge Charles , Brackley James , Burgess Joelle , Duquenne Celine , Johnson Neil , Kasparec Jiri , LaFrance Louis , Li Mei , McNulty Kenneth , Newlander Kenneth , Romeril Stuart , Schmidt Stanley , Schulz Mark , Su Dai-Shi , Suarez Dominic , Tian Xinrong , Carpenter Christopher , Luengo Juan , Kruger Ryan , Knight Steven , McCabe Michael T.
Published: August 2015

The EZH2 histone methyltransferase is frequently mutated in diffuse large B-cell lymphoma leading to increased trimethylation of histone H3 lysine 27 (H3K27me3). Drug discovery efforts have previously identified a pyridone-based chemical series of EZH2 inhibitors that potently and selectively inhibit EZH2 catalytic activity. These compounds are capable of globally decreasing H3K27me3 levels, de-repressing EZH2 target genes, and inducing growth inhibition of many lymphoma cell lines both in cell culture and in vivo. Through medicinal chemistry optimization, we have developed EZH2 inhibitors with significantly improved potency in both biochemical and cellular assays. These compounds exhibit a prolonged enzyme residence time that can be further extended in vitro through the addition of an H3K27me3 peptide. Herein, we report the biochemical and cellular activity of these new EZH2 inhibitors.

Citation Format: Heidi Ott, Glenn van Aller, Jessica Ward, BaoChau Le, Cynthia Rominger, James Foley, Susan Korenchuk, Charles McHugh, Michael Butticello, Charles Blackledge, James Brackley, Joelle Burgess, Celine Duquenne, Neil Johnson, Jiri Kasparec, Louis LaFrance, Mei Li, Kenneth McNulty, Kenneth Newlander, Stuart Romeril, Stanley Schmidt, Mark Schulz, Dai-Shi Su, Dominic Suarez, Xinrong Tian, Christopher Carpenter, Juan Luengo, Ryan Kruger, Steven Knight, Michael T. McCabe. A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5379. doi:10.1158/1538-7445.AM2015-5379
Abstract 5389: Novel kinase inhibitor for imatinib-resistant chronic myeloid leukemia with T315I mutation
Authors: Hong Soon-Sun , Kim Soo Jung , Jung Kyung Hee , Yan Hong Hua , Fang Zhenghuan , Lim Joo Han , Ryu Jeong Seon
Published: August 2015

Chronic myeloid leukemia (CML) is characterized by a constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl/T315I is the predominant mutation that causes resistance to Imatinib. In the present study, we synthesized a novel Bcr-Abl inhibitor, HS-543, and investigated its effect on cell survival or apoptosis in CML cells bearing Bcr-Abl/T315I (BaF3/T315I) or wild-type Bcr-Abl (BaF3/WT). HS-543 showed anti-proliferative effects in the BaF3/WT cells as well as the BaF3/T315I cells with resistance to Imatinib and strongly inhibited the Bcr-Abl signaling pathways in a dose dependent manner. Furthermore, it significantly increased the sub G1 phase associated with early apoptosis, with increased levels of cleaved PARP and caspase-3, as well as the TUNEL-positive apoptotic cells. In addition, we found that HS-543 induced apoptosis with the loss of mitochondrial membrane potential by decreasing the expression of Mcl-1 and survivin, together with increasing the expression of Bax. In BaF3/T315I xenograft models, HS-543 significantly delayed tumor growth, unlike Imatinib. Our results demonstrate that HS-543 exhibits the induction of apoptosis and anti-proliferative effect by blocking the Bcr-Abl signaling pathways in the T315I-mutated Bcr-Abl cells with resistance to Imatinib. We suggest that HS-543 may be a novel promising agent to target Bcr-Abl and overcome Imatinib resistance in CML patients.

Citation Format: Soon-Sun Hong, Soo Jung Kim, Kyung Hee Jung, Hong Hua Yan, Zhenghuan Fang, Joo Han Lim, Jeong Seon Ryu. Novel kinase inhibitor for imatinib-resistant chronic myeloid leukemia with T315I mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2015-5389
Abstract 5407: T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression
Authors: Dziegielewska Barbara , Casarez Eli V. , Yang Wesley Z. , Dziegielewski Jaroslaw , Slack-Davis Jill K.
Published: August 2015

Recent studies by our group and others have shown that T-type Ca2+ channels play a reinforcing role in cancer cell proliferation, cell cycle progression and apoptosis evasion. We have demonstrated drug cooperativity between T-type Ca2+ channel inhibitor, mibefradil and carboplatin in vivo in ovarian cancer xenografts. To investigate the molecular mechanism by which T-type Ca2+ channels affect tumor growth, we used a panel of ovarian cancer cell lines together with mibefradil or small interfering RNA (siRNA) to silence the T-channels gene expression. Inhibition of T-type Ca2+ channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer with simultaneous increase in apoptosis and decrease in expression of the anti-apoptotic gene survivin (BIRC5). Combining carboplatin with mibefradil resulted in increased apoptosis. Analysis of intracellular signaling revealed T-type Ca2+ channel inhibitors induced changes in the levels of FOXO proteins, which regulate BIRC5 gene expression. Mibefradil treatment caused greater nuclear retention of FoxO1 and FoxO3a, two transcription factors that repress BIRC5 expression, while protein expression of the transcription factor that activates BIRC5 expression, FoxM1, decreased. Chromatin immunoprecipitation of FOXO proteins from mibefradil treated ovarian cancer cells demonstrated binding of FoxM1 and FoxO1 within the BIRC5 (survivin) promoter. Together, the data show that T-type Ca2+ channels support ovarian cancer cell cycle, proliferation and regulate pro-survival pathways through the FOXO-survivin signaling axis, which has been reported to be deregulated in ovarian cancer. Moreover, our results provide the rationale for future use of T-type antagonists as sensitizing agents in combination with standard chemotherapeutics.

Citation Format: Barbara Dziegielewska, Eli V. Casarez, Wesley Z. Yang, Jaroslaw Dziegielewski, Jill K. Slack-Davis. T-type Ca2+ channel inhibitors sensitize ovarian cancer to carboplatin through downregulation of survivin gene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5407. doi:10.1158/1538-7445.AM2015-5407
Abstract 5423: Identification of immunotargets in Hedgehog medulloblastoma for immunotherapy
Authors: Tan Grace S. , Curran Tom
Published: August 2015

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Although approximately 75% of patients survive with current therapies, the associated complications drastically affect their quality of life, including severe neurological impairment and secondary cancers.

Approximately 30% of MB cases exhibit activated hedgehog signaling pathway (HH-MB). Recent clinical trials using small molecular HH inhibitors exhibit promising results. Unfortunately, there are also indications of high rates of resistance and relapses, affirming the pressing need to develop complimentary therapies.

Although immunotherapy has previously been proposed to treat MB, unique antigens are rare and monoclonal antibody therapies may not ensure tumor cytotoxicity, without additional adjuvants and associated severe side effects. However, recent successful developments of various cancer immunotherapies, especially the chimeric antigen receptor modified T-cell therapy, have rekindled interest in MB immunotherapies.

To address the heterogeneity of MB and to develop a precision immunotherapy, we propose to identify uniquely or differentially overexpressed cell surface proteins in HH-MB, using human MB microarray and RNA-Seq data. Our preliminary studies have identified 39 potential candidates that are differentially expressed in HH-MB. We are currently confirming the presence and cellular localization of these candidate proteins using human MB tissue microarrays.

This work is supported by NIH (T32 HL07915 [GST]) and Stand Up To Cancer - St. Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.

Citation Format: Grace S. Tan, Tom Curran. Identification of immunotargets in Hedgehog medulloblastoma for immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2015-5423
Abstract 5439: Development of a robust reporter-based T-cell activation assay for bispecific therapeutic antibodies in immunotherapy
Authors: Stecha Pete , Grailer Jamison , Cheng Zhi-jie Jey , Hartnett Jim , Fan Frank , Cong Mei
Published: August 2015

Bispecific T-cell Engager (BiTE), which simultaneously targets CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, has emerged as a promising immunotherapy approach to treat cancer. Current methods for bispecific antibody potency determination measure T-cell proliferation or cytokine release using primary peripheral blood mononuclear cells. They can be complex and highly variable. Here we report the development of a reporter-based T cell activation assay using two Jurkat cell lines stably expressing luciferase reporter driven by IL-2 promoter or NFAT-response element. Both Jurkat reporter cell lines showed robust reporter signal upon stimulation of crossed-linked CD3 antibody. These cell lines were developed in Thaw-and-Use format and showed similar assay performance as that from the cells fresh-from-culture. When tested with bispecific therapeutic antibody catumaxomab, we showed specific reporter response by co-culturing Jurkat reporter cells with cancer target cells endogenously expressing EpCAM, such as MDB-MA-231 and SK-BR-3 cells. No signal was observed without target cells or with EpCAM negative Raji cells. The assay can measure the relative potency for catumaxomab with good precision. It also can detect changes in biological activity for catumaxomab in stressed stability study, and therefore has appropriate stability-indicating property. In summary, the reporter-based T cell activation assay provides a simple and robust approach to quantitatively measure antibody potency for bispecific antibody. It can potentially serve as a potency bioassay for bispecific therapeutic antibodies during drug development and manufacture.

Citation Format: Pete Stecha, Jamison Grailer, Zhi-jie Jey Cheng, Jim Hartnett, Frank Fan, Mei Cong. Development of a robust reporter-based T-cell activation assay for bispecific therapeutic antibodies in immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2015-5439
Abstract 5446: Continuous real-time measurement of live and dead cells in culture over multiple days
Authors: Riss Terry , Vidugiriene Jolanta , Duellman Sarah , Zhou Wenhui , Vidugiris Gediminas , Valley Mike , Osterman Jean , Arbit Ruslan , Bernad Laurent , Meisenheimer Poncho , Cali James J.
Published: August 2015

Recently developed assay technologies have made it possible to use a multi-well plate reader to estimate the number of live or dead cells in culture in real time over a period of days to monitor the effects of long term exposure to test substances. An advantage of the real time assay chemistries is that the cells remain viable and available for other measurements. The purpose of this study was to determine if the real time assay methods could be multiplexed with other assay endpoints and applied to three dimensional (3D) culture models. The measurement of the number of live cells in real time is accomplished by providing a pro-substrate that viable cells (but not dead cells) can convert into a substrate for a luciferase derived from a marine shrimp. The pro-substrate and luciferase can be added with a single reagent addition step and because they are not toxic, they can be incubated with cells in culture for up to three days. Live cell conversion of the pro-substrate into a substrate for luciferase results in a luminescent signal that is proportional to the number of live cells. The signal diminishes immediately upon cell death. Detection of dead cells in real time is accomplished by using a fluorogenic DNA binding dye that is non-permeant and thus non-toxic to live cells. The dye enters dead cells that have a compromised membrane, binds to DNA, and becomes fluorescent. We have demonstrated that the real time assays can be multiplexed to measure both live and dead cells in the same sample. We further demonstrate the use of these assays to measure viability of cells in 3D spheroids formed using the hanging drop culture technique. We also demonstrate multiplexing of real time assays with subsequent measurement of apoptosis or extraction of RNA for expression studies. Utilization of real time assay chemistries that are non-toxic enabled repeated recording of data in a kinetic mode from the same sample of cells which eliminated the need for duplicate cultures and provided flexibility during assay protocol development. The ability to multiplex real time assays improved overall lab efficiency by eliminating the need for duplicate cultures for each experiment.

Citation Format: Terry Riss, Jolanta Vidugiriene, Sarah Duellman, Wenhui Zhou, Gediminas Vidugiris, Mike Valley, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Continuous real-time measurement of live and dead cells in culture over multiple days. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2015-5446
Abstract 5463: Role of the endothelin axis in astrocyte and endothelial cell mediated chemoprotection of cancer cells
Authors: Kim Mark S. , Choi Hyun Jin , Lee Ho-Jeong , He Junqin , We Qiuyu , Langley Robert R. , Kim Sin-Jin , Fidler Isaiah J.
Published: August 2015

Background. Recent evidence suggests that astrocytes protect cancer cells from chemotherapy by stimulating the upregulation of anti-apoptotic genes in cancer cells. We investigated the possibility that activation of the endothelin axis orchestrates survival gene expression and chemoprotection in MDA-MB-231 breast cancer cells and H226 lung cancer cells.

Methods. Cancer cells, murine astrocytes, and murine fibroblasts were cultured in isolation and expression of endothelin (ET) peptides and ET receptors (ETAR and ETBR) was compared with expression on cancer cells and astrocytes (or cancer cells and fibroblasts) that were co-incubated for 48 hours. Type-specific endothelin receptor antagonists were used to evaluate the contribution of ETAR and ETBR to astrocyte-induced activation of the protein kinase B (AKT)/mitogen-activated protein kinase (MAPK) signal transduction pathways, anti-apoptotic gene expression, and chemoprotection of cancer cells. We also investigated the chemoprotective potential of brain endothelial cells and microglial cells.

Results. Gap junction signaling between astrocytes and MDA-MB-231 cancer cells stimulates upregulation of interleukin 6 (IL-6) and IL-8 expression in cancer cells, which increases ET-1 production from astrocytes and ET receptor expression on cancer cells. ET-1 signals for activation of AKT/MAPK and upregulation of survival proteins that protect cancer cells from taxol. Brain endothelial cell-mediated chemoprotection of cancer cells also involves endothelin signaling. Dual antagonism of ETAR and ETBR is required to abolish astrocyte- and endothelial cell-mediated chemoprotection.

Conclusions. Bidirectional signaling between astrocytes and cancer cells involves upregulation and activation of the endothelin axis, which protects cancer cells from cytotoxicity induced by chemotherapeutic drugs.

Citation Format: Mark S. Kim, Hyun Jin Choi, Ho-Jeong Lee, Junqin He, Qiuyu We, Robert R. Langley, Sin-Jin Kim, Isaiah J. Fidler. Role of the endothelin axis in astrocyte and endothelial cell mediated chemoprotection of cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5463. doi:10.1158/1538-7445.AM2015-5463
Abstract 5475: Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy
Authors: Wu Chiahao , Silvers Chris , Guancial Elizabeths , Hsu Jong-Wei , Messing Edward , Lee Yi-Fen
Published: August 2015

Exsosomes are nano-vesicles secreted by most types of cells, and substantially increased secretion in cancer cells. Their role in tumorigenesis/cancer progression have been extensively studied but their role in regulating therapy response have been suggested without in depth investigation. We are particularly interested in exosome's roles in cisplatin resistant, since cisplatin is one of the most widely used chemodrugs for many cancer types.

To investigate the role of exosomes in cisplatin resistance in bladder cancer, we examined the cisplatin sensitivity among five bladder cancer cell lines. The expression levels of exosome associated genes that are involved in exosome biogenesis and secretion/internalization were examined by quantative PCR. Interestingly, cisplatin resistant bladder cancer cells express relative high level of exosome associated genes as compared to cisplatin sensitive cells. More importantly, cisplatin treatment induces the expression of exosome associated genes’ expression in cisplatin resistant cells, but not in sensitive cell. Among all those genes, we are particularly interested in Rab gene family, and mechanistic study of targeting those Rab proteins is undergoing. The correlative exosome genes profiles with cisplatin resistance suggested exosome production might facilitate cancer cells escaping from cisplatin-induced cell death; thereby, inhibition of exosome might sensitize cancer cells response to cisplatin. To test this, we have screened several compounds that are known to inhibit exosome biogenesis and uptake, and. found that inhibition of exosomes by dynasore + NSC23766, indeed can sensitize bladder cancer cells to cisplatin. In summary, we concluded that cancer exosome play a critical role of promoting cisplatin resistance in bladder cancer, and inhibition of exosomes sensitizes bladder cancer cell response to cisplain. The exosome related profiles potentially can serve as therapy biomarkers

Citation Format: Chiahao Wu, Chris Silvers, Elizabeths Guancial, Jong-Wei Hsu, Edward Messing, Yi-Fen Lee. Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2015-5475
Abstract 548: Exosome as biomarkers and diagnostics in bladder cancer
Authors: Liu Yu-Ru , Silver Christopher , Lee Yi-Fen
Published: August 2015

Esosomes are small membrane vesicles secreted by most cell types. They are first found in reticulocytes and play important roles in cell-to-cell communication. Recent studies suggest that exosomes secreted by tumor cells containing tumor specific proteins and miRNAs.

Previous study shows that exosomes purified from high grade bladder cancer promote cancer progression. This study aims to characterize the genetic contents in bladder cancer exosomes through mass spectrometry and miRNA array. Exosomes were purified from bladder cancers, immortalized bladder cells, and urine of healthy volunteers and muscle invasive bladder cancer patients. Via proteomics analysis, we have identified several cancer associated exosomal proteins that were found only in bladder cancer, including EDIL-3, an anagiogenic protein in our previous report. Functional characterization of those proteins is currently undertaken with a focus on periostin an extracellulear matrix protein.

In addition to protein, exosomes also contain miRNAs that regulate cellular behaviors. Thus, we performed miRNA expression profiling microarray on exosomes from high grade bladder cancer cells, and immortalized bladder cells (LC Sciences). The differences in miRNA expression were compared and differences were determined by a student T test (P<0.05). Results showed significant difference miRNAs between cancer cells vs normal cells. Some of cancer-specific miRNAs which have shown to play critical roles in tumorigenesis and cancer progression will be study priority. Ultimately our work should contribute to the understanding of how exosomes contribute to bladder cancer biology and may elucidate novel urine exosomes markers for bladder cancer diagnosis.

Note: This abstract was not presented at the meeting.

Citation Format: Yu-Ru Liu, Christopher Silver, Yi-Fen Lee. Exosome as biomarkers and diagnostics in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 548. doi:10.1158/1538-7445.AM2015-548
Abstract 5482: CYP2D6 genotype and response to neoadjuvant tamoxifen therapy: a prospective study in Japan
Authors: Zembutsu Hitoshi , Nakamura Seigo , Akashi Sadako , Kuwayama Takashi , Watanabe Chie , Takei Hiroyuki , Ishikawa Takashi , Hasegawa Yoshie , Lee Soo Chin , Yu Tan Ern , Matsukata Ayami , Matsumoto Hiroshi , Kutomi Goro , Nakamura Yusuke
Published: August 2015

Although lots of CYP2D6-Tamoxifen studies have been reported, the results of the association are still controversial. Because prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy, we have initiated the prospective study to clarify the relationship between CYP2D6 genotype and response to preoperative Tamoxifen therapy using a surrogate marker of Ki-67 labeling index in cancer tissue (C-GENT study) since July, 2012. Of the 342 patients who have been recruited in this study, we analyzed 173 cases whose genotyping and measurement of Ki-67 Labeling Index were completed. We observed significant Ki-67 decrease after 2-4 weeks preoperative Tamoxifen therapy (P = 0.000052). Moreover, the patients with CYP2D6 *V/*V is unlikely to show Ki-67 decrease compared with those with CYP2D6 Wt/Wt or Wt/*V (P = 0.00046). Although further analysis should be continuing using a larger number of samples registered in this study, our results suggested that polymorphisms in CYP2D6 could be a key predictor for the prognosis of patients with breast cancer treated with tamoxifen.

Citation Format: Hitoshi Zembutsu, Seigo Nakamura, Sadako Akashi, Takashi Kuwayama, Chie Watanabe, Hiroyuki Takei, Takashi Ishikawa, Yoshie Hasegawa, Soo Chin Lee, Tan Ern Yu, Ayami Matsukata, Hiroshi Matsumoto, Goro Kutomi, Yusuke Nakamura. CYP2D6 genotype and response to neoadjuvant tamoxifen therapy: a prospective study in Japan. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5482. doi:10.1158/1538-7445.AM2015-5482
Abstract 5484: FGFR2 amplification is predictive of sensitivity to regorafenib in gastric and colorectal cancers
Authors: Cha Yongjun , Kim Hwang-Phill , Han Sae-won , Yun Jiyeon , Song Sang-Hyun , Kim Tae-You
Published: August 2015

Purpose Regorafenib is an orally bioavailable, small molecular multikinase inhibitor of VEGFR, PDGFR, FGFR, KIT, RET, BRAF, BRAFV600E, CRAF, and TIE2. Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, there is no proven biomarker predicting sensitivity to regorafenib. We investigated preclinical activity of regorafenib in gastric and colorectal cancer cell-lines to identify genetic alterations associated with sensitivity to regorafenib.

Methods Fourteen gastric cancer cell lines and 18 colorectal cancer cell lines were screened for regorafenib sensitivity with cell proliferation assays. We performed targeted exome and transcriptome sequencing of the cell lines to identify genetic alterations associated with sensitivity to regorafenib. Mechanisms of regorafenib sensitivity were investigated in regorafenib sensitive cell lines with immunoblotting and flow cytometry.

Results Two gastric (SNU-16, KATO-III) and 1 colorectal (NCI-H716) cancer cells were sensitive to regorafenib with GI50 values of less than 1 μM. Mutation profiles and mRNA expression levels of regorafenib target molecules in regorafenib sensitive and insensitive cell lines demonstrated that amplification of FGFR2 was the only genetic alteration associated with in vitro sensitivity to regorafenib. FGFR2 amplified gastric (SNU-16, KATO-III) and colorectal (NCI-H716) cancer cell lines revealed activated signaling through FGFR2 pathway, and knock-down of FGFR2 with siRNA significantly repressed cellular growth rate and decreased sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in dose-dependent manner selectively in FGFR2 amplified cells. In cell cycle analysis, regorafenib induced G1/S arrest (SNU-16, KATO-III) and apoptosis (NCI-H716), however no significant changes were seen in cell lines without FGFR2 amplification.

Conclusions Signaling through FGFR2 pathway plays a key role in cellular growth and survival in FGFR2 amplified cancers. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2 amplified gastric and colorectal cancer cells, and therefore should be considered for integration into treatment in patients with FGFR2 amplified gastric and colorectal cancers.

Citation Format: Yongjun Cha, Hwang-Phill Kim, Sae-won Han, Jiyeon Yun, Sang-Hyun Song, Tae-You Kim. FGFR2 amplification is predictive of sensitivity to regorafenib in gastric and colorectal cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5484. doi:10.1158/1538-7445.AM2015-5484