bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5553: In vivo and in vitro effect of Withania somnifera extract on the proliferation of side population (CD44HIGH /CD24LOW) cells sorted from MDA-MB-231 breast cancer cells
Authors: Khazal Kamel F.
Published: August 2015

Invasive breast cancer is considered a great challenge for treatment of cancer in women. In 2013, an estimated 232,340 new cases of invasive breast cancer were expected to be diagnosed in the U.S., along with 64,640 new cases of non-invasive breast cancer. For women under 45, breast cancer is more common in African-American women than white women, and, overall, African-American women are more likely to die of breast cancer. CD44high/CD24low cells have been associated with invasiveness of breast cancers, in particular basal-like breast cancers. In nude mice xenografted with MDA-MB-231 breast cancer cells, CD44high/CD24low cells are the side population associated with metastasis to the bones and are considered to be cancer stem cells. The side population cells are not only involved in invasiveness of breast cancer but are resistant to chemotherapeutic drugs such as mitoxantrone and doxorubicin.

The effects of an alcoholic extract of the roots of Withania somnifera (WS), a traditional herbal remedy, was tested in the sorted side population, CD44high/CD24low, of MDA-MB-231 breast cancer cells. WS caused a concentration-dependent inhibition of the sorted cells, with 95, 65, and 35% inhibition of the proliferation of cells after incubation with concentrations of 100, 50, and 25 μg/ml, respectively. Two groups of nude mice (10 mice ineach group) were xenografted with luciferase transfected CD44high/CD24low cells. Group1 served as control has been dosed with 10% DMSO via drinking water for 8 weeks. Group 2 has been given WS (300 mg/kg) via drinking water for 8 weeks. This experiment is on going and will end after 8 weeks. Tumoror size will be assessed each week using an in vivo imaging system. In conclusion, on, Withania somnifera extract showed a concentration-dependent inhibition of CD44high/CD24low cells and it is anticipated that the extract will be effective in inhibition of cell proliferation in treated mice. These findings serve as preliminary data for further preclinical studies evaluating the effects of WS.

Grant support: Research supported by NIH-NCI-U54-118948 at Tuskegee University and the University of Alabama at Birmingham.

Citation Format: Kamel F. Khazal. In vivo and in vitro effect of Withania somnifera extract on the proliferation of side population (CD44HIGH /CD24LOW) cells sorted from MDA-MB-231 breast 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 5553. doi:10.1158/1538-7445.AM2015-5553
Abstract 5556: Enhancing response of Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast cancer cells
Authors: Feliz-Mosquea Yismeilin , Suarez-Arroyo Ivette , Loperena Yaliz , Cubano Luis A. , Martinez-Montemayor Michelle M.
Published: August 2015

Inflammatory Breast Cancer (IBC) is an aggressive and unique form of breast cancer that develops rapidly. IBC, like non-IBC breast cancers, is a heterogeneous disease and can occur as any of the six molecular breast cancer subtypes. However, they are most commonly either triple negative; that is presenting with an absence of estrogen receptor (ER-), progesterone receptor (PR-) and the epidermal growth factor receptor 2 (HER2-) or ER-, PR- and HER2+ overexpressed. There is no specific IBC treatment; thus, it seems possible to find a therapeutic for this deadly disease. Our published data demonstrates that Ganoderma lucidum (Reishi) inhibits the viability of the triple negative IBC SUM149 cell line, but not of MCF10A noncancerous mammary epithelial cells. Subsequently, in this study, we aimed to investigate Reishi effects in the viability and molecular signaling of the ER-, PR-, HER2+ IBC cell lines SUM190, KPL-4 and IBC-3. Furthermore, we studied the effects of Reishi on IBC cells when treated in complement with the HER2 Tyrosine Kinase Inhibitor, lapatinib. The hypothesis for this study is that Reishi inhibits cancer cell viability and sensitizes IBC cells to lapatinib therapy and downregulates key proteins overexpressed in IBC. Herein, SUM190, KPL-4 and IBC-3 cells were treated with increasing concentrations of lapatinib and/or Reishi for 24 or 72h. The combination of lapatinib plus Reishi reduced cell viability of KPL-4 cells to 80% when treated with a concentration of 0.25 mg/mL of Reishi for 72h. Additionally, our data shows a reduction in cell viability of 70% in SUM190 cells treated with lapatinib plus Reishi for 72h. Also, Reishi and lapatinib downregulate the expression HER2 in KPL-4 cell after 72h of treatment. Our results provide evidence that Reishi inhibits cancer cell viability, highlighting the potential of Reishi as a natural anti-IBC-therapeutic. This project was sponsored by NIH/NCI #1F31 CA174307 to ISA, Title V PPOHA US Department of Education #P031M105050 to UCC/LAC, NIH/RCMI #G12 MD007583 to UCC/MMM, NIH/INBRE #5P20 GM103475 to UPR/UCC/MMM, NIH/NIMHD U54 MD008149 UH/MMM.

Citation Format: Yismeilin Feliz-Mosquea, Ivette Suarez-Arroyo, Yaliz Loperena, Luis A. Cubano, Michelle M. Martinez-Montemayor. Enhancing response of Ganoderma lucidum (Reishi) and lapatinib in HER2+ inflammatory breast 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 5556. doi:10.1158/1538-7445.AM2015-5556
Abstract 5566: Carnosic acid induces apoptosis through inhibition of STAT3 signaling and production of ROS in human colon cancer HCT116 cells
Authors: Chae In Gyeong , Yu Mi-Hee , Park Ki-Woong , Chun Kyung-Soo
Published: August 2015

Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been shown to display anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. This study revealed that treatment with CA significantly reduced the cell viability and induced apoptosis in HCT116 cells as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, -7 and -3 and PARP and the inhibition of Bcl-2 and Bcl-xl expression. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of signal transducer and activator of transcription-3 (STAT3) in HCT116 cells by blocking the phosphorylation of upstream Janus-activated kinase-2 (JAK2) and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cylcin D1, D2, and D3. Since ROS is a universal entity mediating apoptosis, we examined whether CA induced apoptosis via ROS formation. Treatment with CA generated ROS and pretreatment with ROS scavenger N-acetyl cysteine (NAC) rescued cells from apoptosis by abrogating the inhibitory effect of CA on the activation of JAK2-STAT3 and Src-STAT3 signaling pathways and rescued cells from CA-induced apoptosis by blocking the induction of p53, and the cleavage of caspase-3 and PARP. In conclusion, CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases and inhibition of STAT3 signaling pathway.

Citation Format: In Gyeong Chae, Mi-Hee Yu, Ki-Woong Park, Kyung-Soo Chun. Carnosic acid induces apoptosis through inhibition of STAT3 signaling and production of ROS in human colon cancer HCT116 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 5566. doi:10.1158/1538-7445.AM2015-5566
Abstract 5567: Anti-angiogenic activity of Thio-Cl-IB-MECA, a novel A3 adenosine receptor agonist, by regulating of PI3K/AKT/mTOR and ERK signaling pathways in endothelial cells
Authors: Oh Jedo , Kim Gi Dae , Jeong Lak Shin , Lee Sang Kook
Published: August 2015

Angiogenesis is a complex process that involves sprouting of new capillaries from pre-existing blood vessels. Angiogenesis is also involved in many pathological conditions including tumor growth, diabetic retinopathy, psoriasis, rheumatoid arthritis, and atherosclerosis. In cancer, newly formed vessels not only promote tumor growth but also cause the tumor cells to become more malignant and metastatic. Therefore, angiogenesis is an attractive target for the development of a wide variety of therapies, including antitumor agents. A3 adenosine receptor (A3AR) agonists have exhibited anti-proliferative activity against cancer cells. However, anti-angiogenic effects of A3AR agonists in endothelial cells have not been investigated yet. In the present study, the potential anti-angiogenic activity and precise mechanism of action of the A3AR agonist thio-Cl-IB-MECA (TCIM) were investigated in cultured HUVECs and mouse embryonic stem cells/embryonic body (mES/EB) derived endothelial cells. TCIM inhibited migration and tube formation of endothelial cells and markedly decreased ex vivo microvessel sprouting in cultured mouse aortic rings. TCIM also suppressed the expression of the endothelial biomarker platelet endothelial cell adhesion molecule (PECAM) via regulation of PI3K/AKT/mTOR and ERK signaling in mES/EB-derived endothelial cells.

Citation Format: Jedo Oh, Gi Dae Kim, Lak Shin Jeong, Sang Kook Lee. Anti-angiogenic activity of Thio-Cl-IB-MECA, a novel A3 adenosine receptor agonist, by regulating of PI3K/AKT/mTOR and ERK signaling pathways in endothelial 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 5567. doi:10.1158/1538-7445.AM2015-5567
Abstract 5569: p73 dinucleotide polymorphism genotyping of human cancer cell lines
Authors: Carastro L. Michael , Hampton Kaia K. , Cordova Estupinan Ricardo A. , Park Hyun Y. , Kim Dongwha , Park Jong Y.
Published: August 2015

The p73 gene is a member of the p53 tumor suppressor family. The p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and ΔNp73 isoforms, respectively. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer (PCa) and (2) increased TAp73/ΔNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize the presence of the p73 DNP allele causes altered p73 promoter usage resulting in increased TAp73/ΔNp73 protein isoform ratios, which could explain the observed decreased risk for PCa aggressiveness. Our ultimate goal is to assess the potential of p73 DNP as a biomarker for lower aggressive PCa risk. Therefore, our initial aim in this study was to determine the p73 DNP genotype in ten additional human cancer cell lines (DU 145, JEG-3, K-562, LNCaP, MDA-MB-231, MDA-MB-468, MRC-5, PC-3, SW48 and U-2OS) not yet characterized for p73 DNP status. We previously reported the p73 DNP genotype of three human cancer cell lines that we used as genotyping controls: Caco-2 (homozygous polymorphic), NCI-H1299 (homozygous wild type), and HepG2 (heterozygous). Cell lines were cultured and genomic DNA was isolated and quantitated. Cell line genomic DNA samples were used in TaqMan Real Time-PCR allelic discrimination assays to determine the p73 DNP genotypes. Our data conclusively determined that DU 145, JEG-3, K-562, LNCaP, MDA-MB-468, MRC-5, SW48 and U-2OS were p73 DNP homozygous wild type, while PC-3 and MDA-MB-231 werw p73 DNP heterozygous.

Cell Line p73 DNP GenotypesCELL LINE NAMEP73 DNP GENOTYPEDU145CCJEG-3CCK-562CCLNCaPCCMDA-MB-231CTMDA-MB-468CCMRC-5CCPC-3CTSW48CCU-2OSCC

Citation Format: L. Michael Carastro, Kaia K. Hampton, Ricardo A. Cordova Estupinan, Hyun Y. Park, Dongwha Kim, Jong Y. Park. p73 dinucleotide polymorphism genotyping of human cancer cell lines. [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 5569. doi:10.1158/1538-7445.AM2015-5569
Abstract 5577: Text-mining methods applied to clinical records support an association between androgen deprivation therapy and subsequent cardiometabolic disease
Authors: Nead Kevin T. , Gaskin Gregory L. , Chester Cariad , Shah Nigam H. , Leeper Nicholas J.
Published: August 2015

We conducted this study to examine the association of androgen deprivation therapy in the treatment of prostate cancer with future metabolic and cardiovascular disease.

Here we utilize electronic medical record data from 1.8 million subjects in the Stanford Translational Research Integrated Database Environment. Specifically, we extracted ICD-9 diagnostic codes, patient medication lists and data from clinical notes using a previously validated text-analytics pipeline. Through this approach, we identified a cohort of 4,578 individuals with prostate cancer containing data on all defined covariates including Gleason score, age, race and use of antiplatelet, anticoagulant, antihypertensive and lipid lowering agents. We used multivariable logistic regression techniques to examine the association of androgen deprivation therapy use with diabetes, peripheral arterial disease, and stroke.

Following prostate cancer diagnosis, there were 80 new diagnoses of diabetes, 39 new diagnoses of peripheral arterial disease and 72 new diagnoses of stroke with 409 individuals receiving androgen deprivation therapy. The use of androgen deprivation therapy to treat prostate cancer was associated with an increased risk of diabetes (odds ratio [OR] = 2.37; 95% confidence interval [CI] 1.40-4.03; P = 0.001), peripheral arterial disease (OR = 2.15; 95% CI 1.07-4.32; P = 0.032) and stroke (OR = 2.88; 95% CI 1.72-4.80; P<0.001).

Here we further demonstrate the utility of clinical data obtained during routine care to investigate clinical hypotheses. Additionally, we add evidence to the growing body of literature linking the use of androgen deprivation therapy to future cardiovascular and metabolic disease.

Citation Format: Kevin T. Nead, Gregory L. Gaskin, Cariad Chester, Nigam H. Shah, Nicholas J. Leeper. Text-mining methods applied to clinical records support an association between androgen deprivation therapy and subsequent cardiometabolic disease. [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 5577. doi:10.1158/1538-7445.AM2015-5577
Abstract 5582: Survey of oral cancer screening participants reveals widespread misconceptions about oral cancer risk factors, independent of educational background
Authors: Chester Cariad , Sabile Jean , Cho Grace , Hu Annie , Hamaguchi Ryoko , Ellis Lauren , Erdman Jonathan S. , Praharaj David , Golla Abhinav , Sunwoo John
Published: August 2015

Introduction: To develop more effective strategies to screen for oral cancer, we surveyed participants in a free oral cancer-screening event held at Stanford University to assess knowledge and understanding of oral cancer risk factors and behaviors.

Method: The survey was administered by trained members of the oral cancer screening team in accordance with guidelines approved by the Stanford Research Compliance Office.

Results: Participants (n = 51) were racially diverse with varied educational levels: approximately 43% of participants had completed high school only or attended some college, and 57% of participants had obtained a bachelor’s, master’s, or doctoral degree. A majority of the participants (62%) reported an annual income below $50,000, and a minority of participants (35%) had active dental insurance. Most of the participants (84%) had never previously been screened for oral cancer. All participants, regardless of educational level, demonstrated limited knowledge of the risk factors that contribute to oral cancer. Approximately 50% of the lower-education cohort and 31% of the higher-education cohort incorrectly believed that dental fillings increase the risk of developing oral cancer. Only 23% and 34% of the lower- and higher-education cohorts, respectively, correctly identified heavy alcohol consumption as a risk factor for oral cancer. Encouragingly, 85% of all participants recognized that behavioral changes can decrease one's risk of developing cancer.

Conclusions: Knowledge of oral cancer risk factors was low in all participants despite the majority of responders having obtained post-secondary degrees. Our work highlights the need to expand the target audience of oral cancer educational efforts to include community members from all educational backgrounds.

Note: This abstract was not presented at the meeting.

Citation Format: Cariad Chester, Jean Sabile, Grace Cho, Annie Hu, Ryoko Hamaguchi, Lauren Ellis, Jonathan S. Erdman, David Praharaj, Abhinav Golla, John Sunwoo. Survey of oral cancer screening participants reveals widespread misconceptions about oral cancer risk factors, independent of educational background. [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 5582. doi:10.1158/1538-7445.AM2015-5582
Abstract 5593: Cigarette smoke and therapeutic response to chemotherapy and radiotherapy in cancer cells
Authors: Warren Graham W. , Romano Michelle , Sobus Samantha , Balasubramanian Sundaravadivel
Published: August 2015

Background: The 2014 Surgeon General's Report concludes that cigarette smoke (CS) causes increased cancer specific mortality. Clinical data support that the effects of CS are reversible, but there are no extant biologic models that have shown therapeutic response profiles for cancer cells exposed to CS.

Methods: An acute, subacute, and chronic dose escalated CS exposure model was developed to test the hypothesis that CS decreases therapeutic response to chemotherapy (CT, cisplatin) and radiotherapy (RT, 0, 2, 4, and 6 Gy) in a reversible manner in cancer cells.

Results: H460, A549, and FaDu cancer cells were treated with single and multiple exposure CS for up to 12 weeks. Subacute and prolonged exposure to CS significantly decreased response to CT and RT. CS increased survival fraction following CT or RT between 30-157% across all cell lines. Exposure of cancer cells to CS for 6 or 9 weeks followed by removal of CS for 3 and 6 weeks resulted in a partial to complete restoration of baseline therapeutic response in a cell specific manner; however, all cell lines required several weeks to achieve a partial or full restoration of therapeutic response. Dose response curves for CS exposure demonstrate that H460 and A549 cells had a decreased therapeutic response profile at higher CS concentrations while FaDu cells were more sensitive to lower concentrations to CS. Dose response curves further demonstrated that high dose CS exposure was cytotoxic in all cell lines. Results were verified in all cell lines using 2 additional repeated experiments with freshly thawed cancer cells purchased from ATCC over 10 months.

Conclusions: CS exposure reproducibly decreases therapeutic response to CT and RT in a dose dependent and reversible manner, reflective of clinical observations. Dose specific effects may vary between cancer cells. Data suggest that the biologic effects of CS removal, such as with acute smoking cessation in cancer patients, may not manifest in an immediate restoration of cancer cell response to CT or RT.

Citation Format: Graham W. Warren, Michelle Romano, Samantha Sobus, Sundaravadivel Balasubramanian. Cigarette smoke and therapeutic response to chemotherapy and radiotherapy in 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 5593. doi:10.1158/1538-7445.AM2015-5593
Abstract 568: Fibroblast growth factor receptors 2 is a novel therapeutic target in esophagogastric junction adenocarcinoma
Authors: Tokunaga Ryuma , Imamura Yu , Nakamura Kenichi , Ishimoto Takatsugu , Iwagami Shiro , Kurashige Junji , Izumi Daisuke , Kosumi Keisuke , Higashi Takaaki , Taki Katsunobu , Hiyoshi Yukiharu , Baba Yoshifumi , Sakamoto Yasuo , Miyamoto Yuji , Yoshida Naoya , Hiroshi Saeki , Oki Eiji , Maehara Yoshihiko , Baba Hideo
Published: August 2015

Background: The prognosis of esophagogastric junction (EGJ) adenocarcinoma remains poor, though it is increasing worldwide. The fibroblast growth factor receptors (FGFRs) play critical roles in various human cancers, especially FGFR2 amplification has been implicated as a key genetic alteration in EGJ adenocarcinoma. In this study, we confirmed that FGFR2 can be a therapeutic target for EGJ adenocarcinoma.

Methods: Utilizing 200 cases with EGJ adenocarcinoma (Siewert types I-III), FGFR2 copy number was assayed by Real-time PCR (using RNaseP as a referent), and FGFR2 expression was detected by immunohistochemistry. We examined whether FGFR2 amplification correlates with FGFR2 expression. The associations between FGFR2 expression and clinicopathological factors, and prognostic impact of FGFR2 expression were examined. We investigated the role of FGFR2 in cell proliferation, invasion, apoptosis and cell cycle, using OACM 5.1C (FGFR2 overexpressing cell line), by siRNA technique targeting FGFR2.

Results: FGFR2 amplification was detected in 33 (22%) cases, and FGFR2 expression was observed in 112 (58%) cases. FGFR2 amplification significantly correlated with FGFR2 expression (p = 0.045). FGFR2 expression was significantly associated with tumor depth of invasion, nodal involvement, distant metastasis, lymphatic invasion, and vascular invasion (all, p < 0.001). In survival analysis, tumors harboring FGFR2 expression experienced significantly unfavorable outcome (p = 0.039). We observed that si-FGFR2 significantly suppressed phosphorylation of Akt and Erk, resulting in suppressing cell proliferation and invasion; and inducing apoptosis and cell-cycle arrest (p<0.001). We also confirmed that FGF 7 promoted the proliferation and AZD4547 inhibited the proliferation through RAS-ERK and PI3K-AKT pathways.

Conclusion: FGFR2 can be a therapeutic target for EGJ adenocarcinoma.

Citation Format: Ryuma Tokunaga, Yu Imamura, Kenichi Nakamura, Takatsugu Ishimoto, Shiro Iwagami, Junji Kurashige, Daisuke Izumi, Keisuke Kosumi,, Takaaki Higashi, Katsunobu Taki, Yukiharu Hiyoshi, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Saeki Hiroshi, Eiji Oki, Yoshihiko Maehara, Hideo Baba. Fibroblast growth factor receptors 2 is a novel therapeutic target in esophagogastric junction adenocarcinoma. [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 568. doi:10.1158/1538-7445.AM2015-568
Abstract 58: 5-HT promotes hepatocellular carcinoma by influencing β-catenin
Authors: Fatima Sarwat , Xiaoki Shi , Zesie Lin , Guo Chen , Ho John W. , Lee Nikki P. , Zhao Xiang Bian
Published: August 2015

5-hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived human hepatocellular carcinoma cells (HCC) but the detailed intracellular mechanism is still unknown. As Wnt/β-catenin signaling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signaling by 5-HT. 5-HT promoted proliferation of serum-deprived HuH-7 and HepG2 cells and also increased total β-catenin levels, and active β-catenin protein levels compared to just control cells under serum free medium without 5-HT. Furthermore, 5-HT increased β-catenin levels in the presence of cycloheximide, a protein synthesis inhibitor, suggesting increased β-catenin levels due to inhibition of β-catenin degradation. Quantitative real-time polymerase chain reaction (qPCR) showed increased β-catenin downstream target genes, Axin1, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS), in serum-deprived HCC cell lines treated with 5-HT. We next studied the expression of various 5-HT were receptors (5-HT1D, 5-HT2A, 5-HT2B, 5-HT5 and 5-HT7) by qPCR in 33 pairs of HCC tumors and corresponding adjacent non-tumor tissues. Receptors 5-HT1D (21/33, 64%), 5-HT2B (12/33, 36%) and 5-HT7 (15/33, 45%) were overexpressed in HCC tumour tissues whereas receptor 5-HT5 was reduced (30/33, 91%) in HCC tumour tissues. Receptor 5-HT2A did not show any significant statistical difference between HCC tumour and corresponding non-tumour tissues. We further investigated whether antagonists of the 5-HT receptors attenuated the activity of 5-HT both in vitro and in vivo and we narrowed our study to 5-HT7 antagonist as the expression of 5-HT7 was found to be significantly associated to liver histology and venous infiltration. Antagonist of receptor 5-HT7, SB258719, attenuated growth of serum-deprived HCC cell lines and primary tumor tissues in the presence of 5-HT. Furthermore, SB258719 reduced tumor growth in a xenograft mouse model accompanied by reduced β-catenin and increased GSK-3β levels as observed by immunohistochemical analysis. Conclusion: This study provides evidence of Wnt/β-catenin signaling activation in 5-HT induced proliferation of serum-deprived HCC cells. The proliferation is inhibited by 5-HT7 antagonist, SB258719, which may represent a potential therapeutic target for hepatocarcinogenesis.

Citation Format: Sarwat Fatima, Shi Xiaoki, Lin Zesie, Chen Guo, John W. Ho, Nikki P. Lee, Xiang Bian Zhao. 5-HT promotes hepatocellular carcinoma by influencing β-catenin. [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 58. doi:10.1158/1538-7445.AM2015-58
Abstract 581: Mutational landscape of PIK3CA gene and its association with oral squamous cell carcinoma in Indian population
Authors: Shah Sejal , Shah Siddharth , Padh Harish , Kalia Kiran
Published: August 2015

Oral Squamous Cell Carcinoma (OSCC) - the subset of Head and Neck Cancer is the sixth most common cancer worldwide and leading cancer in India. Western part of India (Gujarat) is a prime tobacco growing belt, henceforth, has a high tobacco consumption and increased occurrence of oral cancer. Phosphatidylinositol- 3- kinase (PI3K) is heterodimeric protein kinase composed of catalytic subunit, p110α, and a regulatory subunit, p85α; which regulates cell proliferation, apoptosis, and metastasis. The PIK3CA gene, encodes p110α catalytic subunit, when somatically mutated gets highly deregulated in number of cancers. Canonical mutations E542K, E545K and H1047R in p110α have proven to have higher oncogenic potential, subsequently activating downstream pathways. However, this observation has been reported in limited ethnicity in OSCC. Therefore, we have analyzed mutation profile of exon9 and 20 of PIK3CA gene in 50 OSCC primary tumors and corresponding control samples by direct sequencing in Indian population. We have observed two hot spots mutations E542K and E545K in exon 9, which were earlier reported to activate downstream signaling pathways and two synonymous mutations C3075T and C2985T in exon 20 in fourteen and one OSCC patients, respectively. Patients harboring oncogenic mutations were at advanced stage of OSCC having tobacco chewing history more than ten years. Additionally, we observed two Single Nucleotide Polymorphism rs114587137 (C>T) and rs17849071 (T>G) in intron 9 of PIK3CA gene, which was found germ line mutated in fifteen and three OSCC patients respectively.

In summary, we found 4% (2/50) oncogenic mutation frequency of PIK3CA gene, which may play a crucial role through PI3K downstream signaling pathway in oral neoplasia. Our study supports the notion that lower oncogenic mutation frequency of PIK3CA gene has been found in Asian populations but comparable to that seen in western countries. The knowledge of the involvement of PIK3CA mutations in OSCC is of importance, because a specific kinase inhibitor of PIK3CA may potentially serve as an effective therapeutic target against OSCC as a personalized medicine.

Citation Format: Sejal Shah, Siddharth Shah, Harish Padh, Kiran Kalia. Mutational landscape of PIK3CA gene and its association with oral squamous cell carcinoma in Indian population. [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 581. doi:10.1158/1538-7445.AM2015-581
Abstract 589: Expression levels of ABCA6 or ABCA7 predict primary Ewing sarcoma progression at diagnosis
Authors: Pasello Michela , Fanelli Marilù , Mularoni Valentina , Ciotti Sonia , Picci Piero , Serra Massimo , Scotlandi Katia
Published: August 2015

Introduction:

Ewing Sarcoma (EWS), the second most common bone tumor occurring in children and young adults, is a clinically very aggressive tumor with high metastatic tendency. EWS therapy is still firmly based on chemotherapy and development of chemoresistance is a major cause of disease progression. The mechanisms of chemoresistance are, however, still poorly defined. In this study, we analyzed the prognostic value of 21 ABC transporters with different mechanisms of action.

Materials and methods:

Gene expression of 21 ABC transporters was determined by RT-PCR in two different series of EWS patients with primary localized tumor at diagnosis (training set: 30 pts; validation set: 109 pts) and in human EWS cell lines. Association with patient outcome was determined by log-rank test and Kaplan-Meier survival curves (EFS and OVS). Sensitivity to conventional chemotherapeutic agents, such as Doxorubicin, Vincristine, Etoposide, Actinomicin-D and Ifosfamide, was tested in a representative panel of human EWS cell lines.

Results:

Among the 21 ABC transporters that were analyzed in the training set, expression of ABCA2, ABCA6, ABCA7, ABCB9 and ABCC4 was found to be associated with differential prognosis. Of these genes, only ABCA6 and ABCA7 were confirmed to be significant indicators of EWS progression when the analysis was extended to a new series of 109 patients with localized primary tumor (validation set): high expression of ABCA6 or ABCA7 was associated with a better prognosis. Correlative in vitro studies on EWS cell lines showed that the high expression of ABCA7 was not associated with higher response to conventional drugs, indicating a more complex role in the regulation of EWS aggressiveness.

Conclusions:

The study validated the impact of ABCA6 or ABCA7, the still poorly understood members of ABC transporters, on EWS progression. However, since high expression of these two transporters is related to better prognosis rather that worse, a different role from the canonical function of drug extruders should be supposed. We are currently testing the association between ABCA7 expression and ceramide levels, which are known to induce cell apoptosis, and macrophage-mediated phagocytosis as indicated in the literature (Kielar D. et al. J Invest Dermatol. 2003 Sep;121(3):465-74; Jehle AW et al. J Cell Biol. 2006 Aug 14;174(4):547-56).

Funded by: AIRC to KS IG 14049.

Citation Format: Michela Pasello, Marilù Fanelli, Valentina Mularoni, Sonia Ciotti, Piero Picci, Massimo Serra, Katia Scotlandi. Expression levels of ABCA6 or ABCA7 predict primary Ewing sarcoma progression at diagnosis. [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 589. doi:10.1158/1538-7445.AM2015-589
Abstract 6: Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199)
Authors: Jin Sha , Tapang Paul , Osterling Donald J. , Gao Wenqing , Albert Daniel H. , Souers Andrew J. , Leverson Joel D. , Phillips Darren C. , Chen Jun
Published: August 2015

The BCL-2-selective inhibitor venetoclax (ABT-199) binds with high affinity to the BH3-binding groove of BCL-2, thereby competing for binding with the BH3-only protein BIM (Souers et al., 2013). Venetoclax is currently being evaluated in clinical trials for CLL, AML, multiple myeloma and NHL. To facilitate these studies, we developed and validated a 384-well electrochemiluminescent ELISA (MSD, Gaithersburg, MD,USA) that quantifies expression of BCL-2, BCL-XL, and MCL-1protein alone or in complex with BIM. We subsequently quantified expression of BCL-2 and BCL-2:BIM complexes in 16 hematologic tumor cell lines. We found the EC50 of venetoclax in these tumor cell lines to correlate strongly with baseline BCL-2:BIM complex levels. This correlation was superior to the correlation between venetoclax EC50 and absolute BCL-2 expression. We also applied the assay to measure disruption of BCL-2:BIM complexes in vivo. Treatment of the Non-Hodgkin's Lymphoma (NHL) xenograft model SU-DHL-4 with a BCL-2-selective inhibitor resulted in disruption of tumor BCL-2:BIM complexes that aligned with serum and tumor concentrations of inhibitor. Collectively, these data demonstrate that quantifying BCL-2:BIM complexes offers an accurate means of assessing target engagement by venetoclax and, potentially, predicting its efficacy. The utility of this assay is currently being assessed in clinical trials.

Citation Format: Sha Jin, Paul Tapang, Donald J. Osterling, Wenqing Gao, Daniel H. Albert, Andrew J. Souers, Joel D. Leverson, Darren C. Phillips, Jun Chen. Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199). [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 6. doi:10.1158/1538-7445.AM2015-6
Abstract 633: Amanitin-based ADCs with an improved therapeutic index
Authors: Hechler Torsten , Müller Christoph , Pahl Andreas , Anderl Jan
Published: August 2015

Antitumoral activity of monoclonal antibodies can be dramatically enhanced by conjugation to toxic small molecules. Beside the recent approval of Kadcyla (T-DM1) and Adcetris (SGN-35) more than 30 antibody-drug conjugates (ADC) have entered clinical trials, promising to strengthen the therapeutic capabilities for cancer treatment in the next decade. Surprisingly most ADCs are based on one of few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansins. Toxins that operate through such a mechanism could suffer from limited activity in different cancer indications and in cells expressing resistance mechanisms. Accordingly the use of new drugs that function via alternative toxicity mechanisms could enhance the therapeutic potential of ADCs.

In the present study we evaluated the antitumoral potency of monoclonal antibodies conjugated to small molecules from the amatoxin family. Amanitin, the most well-known toxin of the amatoxin family, binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription at very low concentrations. In our experiments, we tested random- and site-specific strategies to covalently conjugate amanitin to antibodies and generated conjugates with low aggregation and high affinity for the target antigen. We compared the cytotoxic activity of stable and cleavable linker ADCs and the stability of such constructs in plasma. Overall we observed high stability in plasma after incubation for four days. Moreover we demonstrated superior efficacy of amanitin-based ADCs in cancer xenograft models compared to tubulin-inhibiting toxins. For site-specific conjugation antibodies were engineered at different locations to incorporate cysteines (Thiomabs). Thiomabs with different positions of the engineered cysteine were compared in vitro and in vivo to identify the best position for amanitin-thiomab-conjugates. Safety profiling of the two best Thiomabs in cynomolgous monkey revealed a substantial in improvement in tolerability and of the therapeutic index by using site-specific engineered amanitin-ADCs. The data support the development of amanitin-based ADCs by using a toxin with a new mode of action and with a favorable therapeutic index.

Citation Format: Torsten Hechler, Christoph Müller, Andreas Pahl, Jan Anderl. Amanitin-based ADCs with an improved therapeutic index. [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 633. doi:10.1158/1538-7445.AM2015-633
Abstract 638: Modulation of antibody-dependent cytokine release using Fc engineering
Authors: Kinder Michelle , Greenplate Allison , Strohl William , Jordan Robert , Brezski Randall
Published: August 2015

Antibodies can elicit multiple functions through its Fc region, including cell-killing through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxity (CDC) and antibody-dependent cellular phagocytosis (ADCP). In addition to the cell-killing functions, antibodies also mediate antibody-dependent cytokine release (ADCR). In this study, we demonstrate that ADCR is regulated differentially in PBMC and macrophages. ADCR is FcγRIII-dependent in PBMC and mediated by NK cells. In contrast, ADCR in macrophages is FcγRI-dependent. In PBMC, ADCR is characterized by expression of primarily pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1ß, TNF-α. Meanwhile in macrophages, ADCR results in a mixture of the anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines including IFN-γ, IL-1α, IL-1ß, TNF-α, and RANTES. Using an Fc engineering approach, we generated antibody variants lacking FcγRI binding while maintaining binding to FcγRIIa and FcγRIII. These variants retain ADCC and ADCR in PBMC, but modulate ADCP without eliciting cytokine release in macrophages, separating the two processes. Ultimately, a better understanding of how cytokine release is regulated by antibodies and of strategies to modulate cytokine release through Fc engineering could be used to influence immune cell fate decisions in vivo.

Citation Format: Michelle Kinder, Allison Greenplate, William Strohl, Robert Jordan, Randall Brezski. Modulation of antibody-dependent cytokine release using Fc engineering. [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 638. doi:10.1158/1538-7445.AM2015-638
Abstract 656: Discovery of MAbs against difficult GPCRs, ion channels, and transporters using the MPS Discovery Engine®
Authors: Willis Sharon H. , Mattia Kimberly , Payne Riley , Hall Moniquetta , Mabila Manu , Rettew Christine , Couto Joseph , Keshwara Rohan , Paes Cheryl , Doranz Benjamin J. , Rucker Joseph
Published: August 2015

Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for therapeutic purposes. However, the complex structure of membrane protein targets makes the discovery of these MAbs especially challenging. To address this need, we have developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for challenging membrane protein targets, including GPCRs, ion channels, and transporters. MPS harnesses the strength of Integral's 10+ years of expertise in membrane protein expression and analysis, as well as proprietary technologies including Lipoparticles for high concentration membrane protein presentation and Shotgun Mutagenesis for comprehensive epitope mapping and MAb optimization. Integral Molecular currently has a pipeline of several dozen MAbs against complex membrane proteins that are involved in a variety of diseases, including cancer, inflammation, pain, and infectious diseases. There are currently no FDA-approved MAbs that target GPCRs, ion channels, or transporters, and MPS provides a pathway to these new and previously intractable targets.

Citation Format: Sharon H. Willis, Kimberly Mattia, Riley Payne, Moniquetta Hall, Manu Mabila, Christine Rettew, Joseph Couto, Rohan Keshwara, Cheryl Paes, Benjamin J. Doranz, Joseph Rucker. Discovery of MAbs against difficult GPCRs, ion channels, and transporters using the MPS Discovery Engine®. [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 656. doi:10.1158/1538-7445.AM2015-656
Abstract 675: Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia
Authors: Kuenzi Brent M. , Remsing Rix Lily L. , Kunigal Sateesh S. , Kinose Fumi , Knezevic Claire E. , Wright Gabriela , Kroeger Jodi L. , Lancet Jeffrey E. , Padron Eric , Rix Uwe
Published: August 2015

GSK3 alpha has been shown to be a new target in the treatment of acute myeloid leukemia (AML); however current GSK3 inhibitors are unselective and target both GSK3 alpha and GSK3 beta. Interestingly, a known pan-GSK3 inhibitor LiCl has been previously investigated for the treatment of AML yet has met limited clinical success. This could be partly due to the fact that pan-GSK3 inhibition results in beta-catenin stabilization, which has been shown to mediate hematopoietic self-renewal and leukemogenesis. As beta-catenin stabilization requires inhibition of both kinases and most GSK3 inhibitors target GSK3 alpha and GSK3 beta with equal potency, these compounds may possess some significant limitations. We have previously shown GSK3 alpha to be a prominent target of the intended MET inhibitor tivantinib (ARQ197) and that tivantinib shows some specificity for GSK3 alpha over GSK3 beta. We thus hypothesized that tivantinib would be an effective therapy for the treatment of AML. Consistently, tivantinib potently inhibited cellular viability across several AML cell lines. Using an unbiased, mass-spectrometry based chemical proteomics approach; we confirmed GSK3 alpha (and to a slightly lesser extent GSK3 beta) to be targeted by tivantinib in AML. Tivantinib strongly induced apoptosis as compared to the pan-GSK3 inhibitor LiCl in these cells while LiCl showed larger effects on cell differentiation. Interestingly tivantinib caused less stabilization of beta-catenin as compared to LiCl. Subsequent drug combination studies identified the Bcl-2 inhibitor ABT-199 to synergize with tivantinib and to amplify apoptosis as seen by PARP1 cleavage. Furthermore, the combination of tivantinib with ABT-199 completely abrogated beta-catenin stabilization. Tivantinib was able to significantly inhibit colony formation of primary AML patient bone marrow mononuclear cells (BMNCs) and ABT-199 combination showed significant benefit over tivantinib or ABT-199 alone. In summary, tivantinib has potent anticancer activity in AML based on targeting GSK3 alpha; and tivantinib single agent or combination with ABT-199 may represent a novel and exciting opportunity for the treatment of AML.

Citation Format: Brent M. Kuenzi, Lily L. Remsing Rix, Sateesh S. Kunigal, Fumi Kinose, Claire E. Knezevic, Gabriela Wright, Jodi L. Kroeger, Jeffrey E. Lancet, Eric Padron, Uwe Rix. Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia. [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 675. doi:10.1158/1538-7445.AM2015-675
Abstract 682: Targeting heat shock protein 90 with ganetespib for molecularly targeted therapy of gastric cancer
Authors: Liu Hanqing , Lu Jian , Hua Ye , Zhang Peishan , Liang Zhiquan , Ruan Lingling , Lian Caixia , Shi Haifeng , Chen Keping , Tu Zhigang
Published: August 2015

Gastric cancer (GC) remains the fifth most common cancer worldwide. Heat shock protein 90 (HSP90) has become an attractive therapeutic target in treating cancers, due to its abnormally high expression in cancers. Several successful cases of HSP90 inhibitors capable of inhibiting GC inspired us to try ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor in GC treatment. In our study, we show that ganetespib dramatically reduced growth of MGC-803 and also significantly inhibited growth of SGC-7901 and MKN-28 in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in all three cell lines, together with the related markers affected significantly. Mechanistically, ganetespib caused pronounced decrease of expression of classic HSP90 client proteins. Specifically, it greatly affected EGFR signaling cascades by dramatically decreasing levels of total EGFR and EGFR on cell membranes. EGFR knockdown also induced cell-cycle arrest and apoptosis accompanied with a decrease of several EGFR downstream proteins. These results strongly support EGFR signaling greatly contributes to the ganetespib inhibitory effects. Besides, we found the responses of GC cell lines to ganetespib correlated well with their EGFR expression levels: MGC-803, as well as AGS and BGC-803, with higher EGFR expression responded to ganetespib better, while SGC-7901 and MKN-28 with lower EGFR levels were much less sensitive to ganetespib. Though SGC-7901 and MKN-28 were not very sensitive to ganetespib, ganetespib worked synergistically with radiation and cisplatin in killing them. Importantly, ganetespib significantly inhibited growth of xenograft tumors in vivo as a single agent or in combination with cisplatin. Results of H&E staining, TUNEL assays, and immunohistochemistry staining of pCDK1, EGFR, and Ki-67 revealed significant differences in ganetespib-treated tumors. Collectively, our data suggest that ganetespib, as a new potent treatment option, can be used for the molecularly targeted therapy of GC patients according to their expression profiles of EGFR.

Note: This abstract was not presented at the meeting.

Citation Format: Hanqing Liu, Jian Lu, Ye Hua, Peishan Zhang, Zhiquan Liang, Lingling Ruan, Caixia Lian, Haifeng Shi, Keping Chen, Zhigang Tu. Targeting heat shock protein 90 with ganetespib for molecularly targeted therapy of gastric 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 682. doi:10.1158/1538-7445.AM2015-682