bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2666: PI3K inhibition induces homologous repair defects in endometrioid endometrial cancer patient derived xenografts leading to synergistic anti-tumor activity with PARP inhibitors
Authors: Rueda Bo , Digloria Celeste , Hall Tracilyn , Foster Rosemary , Borger Darrell , Growdon Whitfield
Published: August 2015

Inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to induce defective homologous repair in breast carcinoma leading to heightened sensitivity to poly-ADP ribose polymerase (PARP) inhibitors. Our objective was to assess the effects on DNA repair pathways following single agent and combined PI3K and PARP inhibition utilizing endometrial cancer (EnCa) patient derived xenografts (PDX) with and without identified PIK3CA mutations. Under an institutional protocol, tumors from consenting patients with endometrioid carcinoma were collected at the time of primary surgery and genotyped for common oncogenic mutations utilizing the SNaPshot® platform. NOD/SCID mice bearing xenografts derived from two primary human endometrioid endometrial tumors with and without PIK3CA gene mutations (ENCA1 & ENCA2 respectively) were divided into four arm cohorts with equivalent tumor volumes. Vehicle or NVP BKM-120 (30 mg/kg) was administered alone and in combination with veliparib (25 mg/kg) and endometrial xenograft tumor volumes were assessed. Immunoblotting was used to assess downstream PI3K pathway and DNA repair pathway alterations in post treatment samples at specific time points. While NVP BKM120 as a single agent failed to induce significant anti-tumor activity compared to vehicle in either model, decreased pAKT, RAD51 and increased γ-H2AX levels were observed in response to NVP BKM120 alone. PIK3CA mutation status did not affect these molecular alterations. Dual NVP BKM-120 and veliparib therapy significantly impeded tumor growth (p < 0.01) in the ENCA1 model, while in ENCA2, significant tumor regression was observed (p < 0.001). No changes in mouse weight or premature death were observed in the various treatment arms. Combination therapy was associated with reconstitution of baseline RAD51 and increased γ-H2AX levels. In conclusion, these results suggest that PI3K inhibition induces defects in homologous repair mediated by RAD51 and increases reliance on PARP mediated DNA repair mechanisms. Our in vivo data suggest that PI3K inhibition sensitizes endometrial tumors with and without PIK3CA mutations to a PARP inhibitor.

Citation Format: Bo Rueda, Celeste Digloria, Tracilyn Hall, Rosemary Foster, Darrell Borger, Whitfield Growdon. PI3K inhibition induces homologous repair defects in endometrioid endometrial cancer patient derived xenografts leading to synergistic anti-tumor activity with PARP inhibitors. [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 2666. doi:10.1158/1538-7445.AM2015-2666
Abstract 2672: Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models
Authors: Edgar Kyle A. , Nannini Michelle , Hong Rebecca , Eigenbrot Charlie , Schmidt Stephen , Young Amy , Sampath Deepak , Wallin Jeffrey J. , Friedman Lori S.
Published: August 2015

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis; thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The current study investigates preclinical mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of PI3K alpha sparing inhibition of PI3K beta. In preclinical studies, taselisib demonstrates greater potency in cancer cell lines harboring activating PI3K alpha mutations vs wild-type lines. Taselisib induces tumor growth arrest and regressions at tolerated doses in xenograft and patient-derived xenograft (PDX) tumor models bearing PI3K alpha mutations. Notably, taselisib is distinguished from other PI3K inhibitors by enhanced potency in PI3K alpha mutant isogenic cells compared to parental cells. Pathway inhibition and apoptosis are associated with the enhanced activity of taselisib in PI3K alpha mutant cells. Other PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not achieve the same level of activity in PI3K alpha mutant cell lines. Taselisib is more effective at maintaining suppression of the signaling pathway upon activation of Receptor Tyrosine Kinases (RTKs), such as after alleviation of negative feedback within the pathway. Additionally, a screen to evaluate secreted factors contributing to pan-PI3K inhibitor resistance was carried out in SW48 parental and PI3Kα H1047R isogenic cells. Growth factor ligands for the EGFR family were found to reduce the activity of PI3K inhibitors. Taken together, taselisib offers greater preclinical activity in PI3K alpha mutant cells when compared to other PI3K inhibitors, which may indicate a wider therapeutic index.

Citation Format: Kyle A. Edgar, Michelle Nannini, Rebecca Hong, Charlie Eigenbrot, Stephen Schmidt, Amy Young, Deepak Sampath, Jeffrey J. Wallin, Lori S. Friedman. Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models. [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 2672. doi:10.1158/1538-7445.AM2015-2672
Abstract 2680: Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition
Authors: Rodriguez Carlos I. , Setaluri Vijayasaradhi
Published: August 2015

Melanoma, which arises from malignant transformation of melanocytes, has proven to be difficult to treat due its intrinsic and acquired resistance for chemotherapy. Although polymorphisms in the melanocortin receptor 1 (MC1R), which result in impairment of cyclic AMP (cAMP) signaling, are associated with increased risk for melanoma development, the role of cAMP signaling in melanoma, specifically in drug resistance, is not fully understood. Given the importance of cAMP signaling in melanocytes proliferation and differentiation, we investigated the role of cAMP signaling in melanoma cell proliferation, survival and drug resistance. We found that upregulation of cAMP signaling, through activation of adenylate cyclase with forskolin (FSK), has both positive and negative effects on proliferation and survival of melanoma cells. The effect of FSK on melanoma proliferation and survival was not dependent on oncogenic (RAS vs. BRAF) mutation. Moreover, upregulation of cAMP conferred resistance to Selumetinib (AZD6244)-mediated MAPK inhibition by restoring the pERK levels. We also show that FSK-mediated resistance is paradoxically related to a downregulation of phosphorylated cAMP Responsive Element Binding Protein (pCREB), suggesting a CREB-independent mechanism of FSK-induced MAPK reactivation in acquired drug resistance.

Citation Format: Carlos I. Rodriguez, Vijayasaradhi Setaluri. Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition. [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 2680. doi:10.1158/1538-7445.AM2015-2680
Abstract 2685: Paracrine effect of NRG1 and HGF drives resistance to MEK inhibitors in metastatic uveal melanoma
Authors: Cheng Hanyin , Terai Mizue , Sato Takami , Aplin Andrew
Published: August 2015

Metastatic uveal melanoma (UM) patients usually die within one year, emphasizing an urgent need to develop new treatment strategies. Mitogen-activated protein kinase kinase (MEK) small molecule inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in UM patients. In this study, we identified compensatory mechanisms that provide resistance to MEK inhibition in metastatic UM. We screened for growth factors that were able to induce resistance in three newly characterized metastatic UM cell lines to MEK inhibitors, trametinib and selumetinib, with colony formation assay and viability assay. Cellular signaling pathway analysis was carried out to determine the molecular mechanisms of resistance to MEK inhibition in metastatic UM cells. Clinical-grade antibody and inhibitors targeting these pathways were used to determine their capability to overcome the resistance mediated by these growth factors. Conditioned medium collected from fibroblasts were used to evaluate whether stromal effect from fibroblasts could drive resistance to metastatic UM cells. We demonstrate that Neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Mechanistically, trametinib enhances the responsiveness to NRG1, whereas HGF induces sustained activation of AKT in the presence of trametinib. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, overcomes resistance to trametinib provided by these growth factors and by conditioned medium from fibroblasts, which produce NRG1 and HGF. Analysis of metastatic liver biopsies showed that ERBB2, which is the co-receptor for NRG1, and cMET were phosphorylated in some of these clinical specimens. Our data suggest that combination of MEK inhibitors with ERBB3 and cMET targeting agents may provide novel and effective treatment strategies in metastatic UM.

Citation Format: Hanyin Cheng, Mizue Terai, Takami Sato, Andrew Aplin. Paracrine effect of NRG1 and HGF drives resistance to MEK inhibitors in metastatic uveal melanoma. [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 2685. doi:10.1158/1538-7445.AM2015-2685
Abstract 2686: In vivo ERK1/2 pathway reporting during acquired resistance to combined RAF/MEK inhibition
Authors: Sanchez Ileine , Aplin Andrew
Published: August 2015

The MEK-ERK1/2 signaling pathway is constitutively activated in BRAF V600E/K melanomas. Concurrent BRAF and MEK inhibitor treatment was recently FDA-approved for BRAF V600E melanoma; however, acquired resistance to the drug combination limits durable responses. Our lab has developed a luciferase reporter system that temporally and non-invasively quantitates ERK1/2 activity in mutant BRAF melanoma xenografts. We used this reporter system to examine the temporal kinetics of ERK1/2 activity in vivo upon treatment with combined BRAF/MEK inhibition. Using a 1205Lu reporter system, all established tumors regressed to levels that were not detectable visually. After prolonged treatment with BRAF and MEK inhibitors, only 2 out of 8 mice regrew resistant tumors. In similar experiments with A375 xenografts, only 1 out 12 tumors developed resistance. In 1205Lu tumors, re-activation of the ERK1/2 pathway was evident prior to re-growth. Additionally, cell lines generated from these resistant tumors exhibited elevated levels of ERK1/2 suggesting that resistance to BRAF + MEK inhibitors involves re-activation of ERK1/2 signaling. In comparison to parental cells, resistant cells in vitro displayed reduced levels of apoptosis, increased proliferation, and increased viability in the presence of BRAF plus MEK inhibitor. Furthermore, when removed from drugs, combination-resistant cells showed increased apoptosis and reduced proliferation and viability, suggesting that the resistant lines may be addicted to the dual therapy. RPPA analysis and whole exome sequencing to elucidate mechanisms of resistance are ongoing.

Citation Format: Ileine Sanchez, Andrew Aplin. In vivo ERK1/2 pathway reporting during acquired resistance to combined RAF/MEK inhibition. [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 2686. doi:10.1158/1538-7445.AM2015-2686
Abstract 2692: High allele frequency of KRAS functional mutations predicts resistance to MEK inhibitors: Evidence from cell lines and human tumor xenograft models
Authors: Reddy Anupama , Pinzon-Ortiz Maria , Derti Adnan , Korn Joshua , Ruddy David , Yang Guizhi , Green John , Gao Hui , Lehar Joseph , Caponigro Giordano , Cao Z. Alexander
Published: August 2015

Introduction

KRAS mutant tumors represent a high unmet need for effective therapy. Even though there is no targeted therapy for KRAS, inhibitors of downstream targets like MEK have shown efficacy in preclinical studies. However, clinical trials of MEK inhibitors in KRAS-mutant tumors have not been successful, although there have been some responders. We sought to understand resistance to MEK inhibition in KRAS-mutant tumors and to develop biomarkers for patient stratification.

Results

Using DNA and RNA sequencing data from cell lines, we found that high variant allele frequency (>50%) of KRAS functional mutations (G12, G13, Q61) was associated with baseline resistance to MEK inhibitors AZD6244 and MEK162 across multiple tumor types. Low allele frequency of KRAS mutation was found to be necessary but not sufficient for sensitivity to MEK inhibitors, suggesting the existence of additional resistance mechanisms. Comparison of copy number and variant allele frequency of KRAS showed that MEK inhibitor-resistant cell lines have either amplified mutant alleles or lost WT alleles.

This prediction of high KRAS mutant allele frequency being associated with MEKi resistance was successfully validated in vivo in 19 primary colorectal tumor xenograft models treated with MEK162. High KRAS mutant allele frequency and high KRAS expression were also associated with acquired resistance to BKM120+MEK162 combination treatment in an in vivo model of HeyA8, an ovarian cell line with a KRAS G12D mutation.

This prediction was also supported by the observation in a Phase II clinical trial with MEK inhibitor refametinib + gemcitabine in pancreatic cancer [1]. Patients with disease progression had a significantly higher KRAS mutant allele frequency compared to partial responders and stable disease.

Conclusion

High allele frequency of KRAS functional mutations is associated with resistance to MEK inhibitors. Excluding patients with high KRAS mutant allele frequency has the potential to improve clinical trial outcomes and benefit patients, with the added benefit that it may not require any additional screening or novel biomarkers.

References

[1] Reiss et al., J Clin Oncol 32:5s, 2014 (suppl; abstr 4129)

Citation Format: Anupama Reddy, Maria Pinzon-Ortiz, Adnan Derti, Joshua Korn, David Ruddy, Guizhi Yang, John Green, Hui Gao, Joseph Lehar, Giordano Caponigro, Z. Alexander Cao. High allele frequency of KRAS functional mutations predicts resistance to MEK inhibitors: Evidence from cell lines and human tumor xenograft models. [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 2692. doi:10.1158/1538-7445.AM2015-2692
Abstract 2693: Overcoming MITF-conferred resistance through dual AURKA/MAPK-axis targeting
Authors: Pathria Gaurav , Garg Bhavuk , Garg Kanika , Borgdorff Viola , Wagner Christine , Superti-Furga Giulio , Wagner Stephan N.
Published: August 2015

MITF is a frequently amplified lineage-specific oncogene in human melanoma. Current drug discovery effort has, however, paradoxically ignored its criticality in melanoma cell biology and has failed to address MITF-associated intrinsic and acquired drug resistance. Here, to search the kinase targets able to circumvent the MITF-conferred intrinsic drug resistance, we used a multi-kinase inhibitor-directed chemical proteomics-based differential target affinity screen in immortalized human melanocytes carrying ectopic MITF overexpression. Through a subsequent methodical ‘gene expression-disease stage’ associations and a detailed functional interrogation in molecular-genetically diverse melanoma cellular systems, we identify a mitotic Ser/Thr kinase, Aurora Kinase A (AURKA) as a potential drug target in human melanoma that successfully overcomes MITF-engendered drug resistance phenotype. Critically, assessing the long-term efficacy of the phase II clinical trial approved highly specific AURKA-inhibitor MLN-8237, we pre-emptively witness a robust MAPK signalling-mediated immediate acquired resistance program involving MITF and Cyclin D1 induction through activity and expression changes in CREB and c-Jun respectively in both the BRAF- and NRAS-mut cells. Notably, identification of these ERK-directed acquired resistance mechanisms informed combinatorial AURKA/BRAF or AURKA/MEK targeting as an intelligent therapeutic strategy in BRAF- and NRAS-mut melanomas respectively.

Citation Format: Gaurav Pathria, Bhavuk Garg, Kanika Garg, Viola Borgdorff, Christine Wagner, Giulio Superti-Furga, Stephan N. Wagner. Overcoming MITF-conferred resistance through dual AURKA/MAPK-axis targeting. [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 2693. doi:10.1158/1538-7445.AM2015-2693
Abstract 2703: Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106
Authors: Alwarawrah Yazan , Hughes Philip , Safi Rachid , McDonnell Donald P. , Spector Neil L. , Haystead Timothy A.
Published: August 2015

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that is overexpressed in about 20% of breast cancers and most often in the more aggressive inflammatory breast cancer (IBC). Lapatinib is one of the most potent small molecule inhibitors of HER2. Although Lapatinib has shown to be an effective targeted therapy, development of drug resistance stands as a major problem. Here we show that the anti-cancer activity of the novel fatty acid synthase inhibitor HS-106 can re-sensitize resistant cell lines to Lapatinib.

Lapatinib resistant SKBR3 and BT474 breast cancer cell lines were treated with increasing concentrations of HS-106 either alone or in combination with Lapatinib and their effects on cell proliferation were assessed using the DNA dye Hoechst 33342. The ability of HS-106 and Lapatinib drug combination to induce apoptosis were measured in Caspase 3/7 activity assay using the fluorogenic Caspase substrate (DEVD)2 R110. Their effect on apoptosis was further confirmed in Lapatinib resistant BT474 cells using flow cytometry based Annexin V assay. In addition, the effect of HS-106 on the expression levels and phosphorylation of the kinases involved in Her2 pathway including Her2, AKT and ERK were determined. The fatty acid synthase inhibitor HS-106 significantly inhibits the proliferation of Lapatinib resistant cell lines with concentrations. Interestingly, when combined with Lapatinib, the drugs synergize to dramatically affect cell growth. This anti-proliferative activity of HS-106 was found to be the result of induction of apoptosis, Caspase 3/7 activity was found to increase in a dose dependent manner following HS-106 treatment. Low concentrations of Lapatinib and HS-106, which does not induce Caspase activity when administrated separately, were found to significantly induce Caspase activity when combined together. These results were confirmed with the Annexin V assay. By examining the expression levels of HER2 pathway proteins we found a decrease in HER2 levels which was reflected on the levels of the downstream kinases AKT and ERK when treating with high concentrations of HS-106.

In a previous work, HS-106 showed a good tolerability and anti-cancer activity in the MMTV neu+ mouse model. By showing potent efficacy against Lapatinib resistant breast cancer cell lines, our data suggest that HS-106 is an excellent drug candidate to advance into translational studies for the treatment of Lapatinib resistant breast cancer.

Citation Format: Yazan Alwarawrah, Philip Hughes, Rachid Safi, Donald P. McDonnell, Neil L. Spector, Timothy A. Haystead. Overcoming Lapatinib resistance by the fatty acid synthase inhibitor HS-106. [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 2703. doi:10.1158/1538-7445.AM2015-2703
Abstract 2705: AKT inhibition sensitizes tumor cells to rapamycin by enhancing the repressive function of PRS40 on mTORC1/4E-BP1 axis
Authors: Mi Wenting , Ye Qing , Liu Side , She Qing-Bai
Published: August 2015

The mTORC1 inhibitors, rapamycin and its analogs, have been used for the treatment of a variety of cancers, but the clinical performance of these drugs has fallen short of expectations due to only modest efficacy in tumors. The underlying mechanisms of rapamycin resistance remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 shows incomplete inhibition of 4E-BP1 phosphorylation, and is functionally manifest as cap-dependent translation, cell growth and motility. However, treatment with both drugs provides profound effects in vitro and in vivo. Mechanistically, we demonstrate that the combination treatment is required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex is enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduces the dependence of tumor cells on AKT/mTORC1 signaling for translational control of cell proliferation, survival and motility. Together, these findings reveal a critical role of PRAS40 as a key integrator of mTORC1 and AKT signaling for the translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential surrogate marker to evaluate the therapeutic response to mTOR inhibitors.

Citation Format: Wenting Mi, Qing Ye, Side Liu, Qing-Bai She. AKT inhibition sensitizes tumor cells to rapamycin by enhancing the repressive function of PRS40 on mTORC1/4E-BP1 axis. [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 2705. doi:10.1158/1538-7445.AM2015-2705
Abstract 2709: The role of the phosphorylated RXRα on cellular proliferation and liver tumorigenesis in mice
Authors: Sakai Hiroyasu , Shirakami Yohei , Shimizu Masahito
Published: August 2015

We have reported that a malfunction of RXRα due to aberrant phosphorylation at serine 260 by MAPK is associated with the development of hepatocellular carcinoma (HCC) (Cancer Res 2001). The phosphorylated RXRα impairs normal receptor functions of either RXR or RAR, which are respective homodimeric or heterodimeric partner of RXRα, in a dominant-negative manner. Besides, these impaired receptor functions result in the downregulation of their target gene expression and inducing hepatocarcinogenesis by promoting cell proliferation and the inhibition of apoptosis, which suggesting that phosphorylated RXRα plays a crucial role in the development of HCC. However, these findings were revealed primarily using HCC cell lines that express the phosphorylated-RXRα (p-RXRα) proteins and it still remains unclear whether p-RXRα may impact in the process of hepatocarcinogenesis in vivo. Therefore, in order to investigate the biological functions of the p-RXRα in vivo, we generated doxycycline-inducible ES cell line and transgenic mouse, both of which overexpress phosphomimic mutant of RXRα (T82D/S260D). We found that the T82D/S260D inducible ES cells kept undifferentiated state in differentiating media, which indicating that the phosphorylated form of RXRα plays an important role to maintain pluripotency of ES cells. On the other hand, the development of liver tumors induced by liver carcinogen diethylnitrosamine (DEN) was enhanced in the transgenic mice that overexpress T82D/S260D protein in the liver. Besides, the increased liver tumors in the transgenic mice resulted from the promotion of cell cycle progression, not the inhibition of apoptosis. Interestingly, expression of β-catenin and its target genes, cyclin D1 and c-myc, were increased in transgenic liver tumors, although the expression of retinoid-related genes, such as RARβ, p21 and p27, were not altered compared with control mice. These results suggest that a retinoid nuclear receptor, RXRα may prevent the development of DEN-induced liver tumors by regulating the canonical Wnt/β-catenin signaling pathways in mice.

Citation Format: Hiroyasu Sakai, Yohei Shirakami, Masahito Shimizu. The role of the phosphorylated RXRα on cellular proliferation and liver tumorigenesis in mice. [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 2709. doi:10.1158/1538-7445.AM2015-2709
Abstract 2710: Tumorigenic activity of a novel KIF5B-RET fusion gene
Authors: Shin Jung-Young , Kim Min-Young , Son Kyoung-Hwa , Kim Jeong-Oh , Kang Jin-Hyoung
Published: August 2015

The KIF5B-RET gene is a novel fusion gene resulting from pericentric inversion between the kinesin family member 5B (KIF5B) and the rearranged during transfection (RET) gene, which recently isolated as an oncogene in non-small cell lung cancers. In the present study, we investigated the mechanisms associated with tumorigenic activity of KIF5B-RET fusion gene. We used a HEK-293T cell line stably expressing the KIF5B-RET fusion gene in pLOC lenti-viral vector. We investigated the tumor formation in a xenograft mouse models after subcutaneous injection and confirmed the expression of KIF5B-RET by PCR and western blot analysis. We also explored the mechanism of KIF5B-RET fusion gene in stable cell line. The KIF5B-RET stable cells showed upregulation of the cell growth, migration and colony formation. Furthermore, the KIF5B-RET stable cells induced the activation of BRAF, AKT and ERK compared with the parental cells. These results suggest that the KIF5B-RET fusion gene promotes the cell proliferation and tumorigenic activity though BRAF, AKT and ERK activation. These findings may also provide the possible that KIF5B-RET fusion gene as a ‘driver’ in some subgroups of lung cancers.

Citation Format: Jung-Young Shin, Min-Young Kim, Kyoung-Hwa Son, Jeong-Oh Kim, Jin-Hyoung Kang. Tumorigenic activity of a novel KIF5B-RET fusion gene. [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 2710. doi:10.1158/1538-7445.AM2015-2710
Abstract 2711: Regulation of the IGF-1R signaling by angiotensin signaling pathway
Authors: Boo Hye-Jin , Jang Hyun-Ji , Jung Yujin , Min Hye-Young , Lee Ho-Young
Published: August 2015

Lung cancer is one of the most commonly diagnosed cancers in the world. Tobacco smoking is the most predominant risk factor for the development of lung cancer. Carcinogenic compounds in tobacco smoke are thought to be responsible for lung cancer, at least in part, by activating G-protein coupled receptor (GPCR)-mediated signaling pathways. GPCR signaling has been suggested to cross-talk with the IGF-1R signaling pathway, a key pathway for cell survival, transformation and growth. However, the mechanisms underlying the crosstalk between GPCR and IGF-1R signaling pathways remain poorly understood. Here we show that tobacco carcinogen (TC) contributes to the lung carcinogenesis by activating the angiotensin signaling and subsequent IGF-1R signaling pathways. We observed that a potent tobacco carcinogen, nitrosamine 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone), induced transformed phenotypes, including foci formation, viability in the absence of growth factors, and anchorage-dependent and -independent colony formation, and activation of the IGF-1R in normal human bronchial epithelial cells. The NNK treatment also induced lung tumor formation in mice. Losartan, an angiotensin type-1 receptor blocker, and captopril, an angiotensin converting enzyme (ACE) inhibitor, disrupted NNK-induced IGF-1R activation and transformed phenotypes in normal human bronchial epithelial cells and reduced lung tumor formation in mice. These findings indicated that TC-induced angiotensin signaling pathway can enhance lung carcinogenesis by activating the IGF-1R signaling. Our results suggest that targeting IGF-1R by using angiotensin signaling inhibitors may be an effective strategy for the chemoprevention of lung cancer.

Citation Format: Hye-Jin Boo, Hyun-Ji Jang, Yujin Jung, Hye-Young Min, Ho-Young Lee. Regulation of the IGF-1R signaling by angiotensin signaling pathway. [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 2711. doi:10.1158/1538-7445.AM2015-2711
Abstract 2714: Effects of endosulfan on COX-2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways
Authors: Han Eun Hee , Jun Sang Mi , Chung Young Ho
Published: August 2015

Endosulfan is correlated with endocrine disruption, reproductive, and immune dysfunctions. Recently, endosulfan was shown to have an effect on inflammatory pathways, but its influence on cyclooxygenase-2(COX-2) expression is unclear. This study investigated the effects of COX-2 and molecular mechanisms by endosulfan in murine macrophage RAW 264.7 cells. Endosulfan significantly induced COX-2 protein and mRNA levels, as well as COX-2 promoter-driven luciferase activity and the production of prostaglandin E2, a major COX-2 metabolite. Transfection experiments with several human COX-2 promoter constructs revealed that endosulfan activated NF-KB, C/EBP, AP-1, and CREB. Moreover, Akt and mitogen-activated protein kinases (MAPK) were significantly activated by endosulfan. Moreover, endosulfan increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2, and NOX3. These results demonstrate that endosulfan induces COX-2 expression via NADPH oxidase, ROS, and Akt/MAPK pathways. These findings provide further insight into the signal transduction pathways involved in the inflammatory effects of endosulfan.

Citation Format: Eun Hee Han, Sang Mi Jun, Young Ho Chung. Effects of endosulfan on COX-2 expression via NADPH oxidase and the ROS, MAPK, and Akt pathways. [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 2714. doi:10.1158/1538-7445.AM2015-2714
Abstract 2721: Induction of aberrant crypt foci in the colon of mice exposed to tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole
Authors: Kim Sangyub , Guo Jingshu , Trudo Sabrina P. , Gallaher Daniel D. , Turesky Robert J.
Published: August 2015

Epidemiological studies indicate that tobacco smoking is a risk factor for colon cancer. However, the etiological agents in tobacco smoke remain uncertain. Heterocyclic aromatic amines (HAAs) that are produced during high-temperature cooking of the proteinaceous food are associated with the elevated risk of colon cancer. Tobacco smoking also produces HAAs, the most abundant one being 2-amino-9H-pyrido[2,3-b]indole (AαC). The carcinogenic potency of AαC is unknown in humans, but AαC forms DNA adducts in multiple organs, including liver and colon, as well as being carcinogenic in rodents. In this study, AαC-induced colon cancer risk was assessed by determining DNA adducts and putative colonic precancerous lesions, aberrant crypt foci (ACF) and dysplastic ACF. For comparison, the HAA, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), a liver and colon carcinogen in mice, and azoxymethane (AOM), a potent colon carcinogen in rodents, were also administered. AαC (400 ppm) or MeIQ (300 ppm) were fed in a high-fat diet to A/J mice (n = 12 per group) for 4 weeks. AOM was injected intraperitoneally to another 12 mice at 5 mg/kg body weight weekly for 6 weeks. At the end of carcinogen treatment, liver and colon were harvested from 4 mice from each group for DNA adduct measurement. dG-C8-AαC, dG-C8-MeIQ, and O6-methyl-dG DNA adducts were measured by liquid chromatography/mass spectrometry. The remaining 8 mice from each group were then fed a carcinogen-free diet for additional 8 weeks, and then colons harvested. Colonic total and dysplastic ACF were counted by light microscopy after appropriate staining. AαC feeding induced the greatest number of DNA adducts in liver, followed by AOM and MeIQ. AOM administration and AαC feeding induced similar numbers of DNA adducts in the colon, both of which were significantly greater than MeIQ. However, AOM administration induced a significantly greater number of total and dysplastic ACF in the colons than did feeding either AαC or MeIQ, which had similar numbers of ACF and dysplastic ACF. The high levels of AαC DNA adducts and induced dysplastic ACF in this animal model, provide biological plausibility for a causal role for AαC in tobacco-induced colorectal carcinogenesis. However, DNA adduct numbers induced by these three carcinogens was not proportional to the colonic ACF or dysplastic ACF number, suggesting that different types of DNA adducts differ in their ability to promote colonic precancerous lesions.

Citation Format: Sangyub Kim, Jingshu Guo, Sabrina P. Trudo, Daniel D. Gallaher, Robert J. Turesky. Induction of aberrant crypt foci in the colon of mice exposed to tobacco carcinogen 2-amino-9H-pyrido[2,3-b]indole. [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 2721. doi:10.1158/1538-7445.AM2015-2721
Abstract 2722: Characterization of human lung enzymes involved in the reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
Authors: Ashmore Joseph , Liu Ying , Peterson Amity , Fechtner Sabrina , Chen Gang , Lazarus Philip
Published: August 2015

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the primary tobacco-specific nitrosamine (TSNA) related to cancer. The major metabolic pathway for NNK is carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) which, like NNK, is a potent lung and pancreatic carcinogen in rodents, with it estimated that 39-100% of NNK is converted to NNAL in cigarette smokers. NNAL is formed as both (R)- and (S)-NNAL enantiomers; while (S)-NNAL is more readily formed from NNK, it is also more readily glucuronidated and detoxified by the phase II UGTs than is (R)-NNAL in monkeys and probably in humans. Therefore, both (S)- and (R)-NNAL are likely to be important metabolites in the NNK carcinogenic process. In the present study, we demonstrated that while (S)-NNAL is the major form (>90%) produced using lung cytosolic fractions, lung microsomes formed primarily (R)-NNAL (>95%). Consistent with previous studies using placenta-fractionated cytosolic enzymes, we demonstrated that several cytosolic enzymes including the aldo-keto reductases (AKRs) 1C1, 1C2, and 1C4, as well as carbonyl reductase I (CBR1) were active against NNK, with all of them forming primarily (>90%) (S)-NNAL. This pattern is consistent with that observed for lung cytosolic fractions. Using HEK293 cells over-expressing microsomal enzymes HSD11β1, HSD11β2, HSD17β12, SDR5A1, SDR5A3, HSD17β1, HSD17β2 or HSD3β7, only HSD11β1 and HSD17β12, all of which were reported to be expressed in lung, exhibited activity against NNK. Most interestingly, both enzymes formed primarily (R)-NNAL (>80%), which is consistent with that observed in lung microsomes. Real-time PCR showed that HSD17β12 was expressed at a level that, on average, was three-fold higher than that observed for HSD11β1 in a screening of normal lung tissue specimens from five individuals. These data suggest that both cytosolic and microsomal enzymes are active against NNK and that HSD11β1 and HSD17β12 are the active microsomal reductases in human lung microsomes, contributing to the exclusive formation of (R)-NNAL

Citation Format: Joseph Ashmore, Ying Liu, Amity Peterson, Sabrina Fechtner, Gang Chen, Philip Lazarus. Characterization of human lung enzymes involved in the reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). [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 2722. doi:10.1158/1538-7445.AM2015-2722
Abstract 2727: Molecular markers and pathway analysis of Middle Eastern colorectal carcinoma
Authors: Beg Shaham , Prabhakaran Sarita , Bu Rong , Al-Assiri Mohammed , Sairafi Rami , Al-Dayel Fouad , Al-Sanea Nasser , Siraj Abdul K. , Uddin Shahab , Al-Kuraya Khawla
Published: August 2015

Colorectal cancer (CRC) is one of the common cancers in the world. In Saudi Arabia CRC is the most common cancer in males and third most common in females. A newly proposed integrated pathways comprising traditional, alternate and serrated by genetic and epigenetic factors was defined recently and hypothesized to role in the pathogenesis of CRC; however there is a paucity of information about these proposed molecular pathways in different ethnic group. Hence we analyzed a large cohort of Saudi colorectal tumors to understand the role of these molecular pathways. 770 archival CRCs specimen were evaluated for Microsatellite Instability (MSI), BRAF, KRAS and 500 cases for CpG Island Methylator Phenotype (CIMP). Traditional pathway constituted 33.4% of CRC cases; the alternate pathway comprised 11.6% of cases. Strikingly, the serrated molecular pathway accounted for only 0.8% of Middle Eastern CRC that is extremely below compared to publish data. 54.2% CRC cases did not qualify to fit into any pathway and thus were designated an unassigned group. Molecular pathways were significantly associated with tumor site and grade. In our attempt to further classify unassigned group a subset of the uncategorized pathway showed a significant survival difference (p = 0.0079). The Unassigned group that accounted for the majority of our cases reflects the heterogeneity of colorectal cancers and warrants the need to unravel the molecular genetic basis of this disease to further subcategorize these CRC cases. It also identifies the need to do further studies on different populations for better understanding of their exact role and incidence.

Keywords: colorectal; molecular pathways; serrated

Citation Format: Shaham Beg, Sarita Prabhakaran, Rong Bu, Mohammed Al-Assiri, Rami Sairafi, Fouad Al-Dayel, Nasser Al-Sanea, Abdul K. Siraj, Shahab Uddin, Khawla Al-Kuraya. Molecular markers and pathway analysis of Middle Eastern colorectal 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 2727. doi:10.1158/1538-7445.AM2015-2727
Abstract 2733: Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids
Authors: Dyba Marcin , Da Silva Brandon , Pan Jishen , Chung Fung-Lung
Published: August 2015

Dietary polyunsaturated fatty acids (ω-3 and ω-6 PUFAs) play a role in certain human cancers. The oxidation of PUFAs produces reactive α,β-unsaturated aldehydes (enals) that can modify DNA producing mutagenic lesions. Of particular interest, 7-(1′,2′-dihydroxyheptyl)-1,N6- ethenodeoxyadenosine (DHHϵdA) is a novel DNA adduct in vitro and in vivo derived from lipid peroxidation of ω-6 PUFAs. Despite its recent identification in vivo, the role of DHHϵdA in tumorigenesis is not yet known. Previously, the endogenously formed DHHϵdA was detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). Due to its low levels in vivo (1-30 adducts/109 DNA bases), a relatively high quantity of DNA is needed for its detection and quantification. In this study we developed a monoclonal antibody against DHHϵdA in order to detect and quantify this adduct in cells and tissues. Two antigens were synthesized by conjugating activated hapten: 5′-carboxy-7-(1′,2′-dihydroxyheptyl) adenosine (5′-carboxy DHHϵA) to BSA and KLH carrier proteins. KLH-conjugated hapten was used for mice immunization, whereas BSA-based hapten was used to test for an immune response. After immunization and several screenings, 6 monoclonal cell lines were chosen for further tests. Normal and competitive ELISA was performed to characterize the antibodies from all cell lines. Competitive ELISA revealed no cross-reactivity towards normal nucleosides and α- and γ-OHPdG, the cyclic 1,N2-propano adduct derived from acrolein, and 8-oxo-dG for all of the tested antibodies. However, we observed a weak (< 1:100) specificity for HNE-dG, an adduct derived from (E)-4-hydroxy-2-nonenal produced from oxidized ω-6 PUFAs. Because of the relatively low level of endogenous HNE-dG compared to DHHϵdA, this weak cross-reactivity should not affect the specificity of the developed antibody to detect DHHϵdA. Two cell lines were discarded after antibodies from them showed moderate competitive effects against 1,N6-etheno-2′-deoxyadenosine (ϵdA). From the remaining 4 cell lines the best one was chosen to produce purified antibody. We believe that this antibody will be a useful tool in the studies of the role of DHHϵdA as an endogenous DNA lesion in cancer development associated with ω-6 PUFAs. This work was supported by the NCI grant CA134892.

Citation Format: Marcin Dyba, Brandon Da Silva, Jishen Pan, Fung-Lung Chung. Development of novel monoclonal antibodies for a cyclic DNA adduct derived from oxidation of ω-6 polyunsaturated fatty acids. [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 2733. doi:10.1158/1538-7445.AM2015-2733
Abstract 2735: Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune cells
Authors: Lawrence Shanieek T. , Whalen Margaret , Pellom Samuel , Shanker Anil
Published: August 2015

Tributyltin (TBT) is an environmental contaminant that has been used for several industrial, agricultural and household purposes. Despite limited bans on the usage of Tributyltin, the stability and toxicity of the compound may prove to have negative effects on immune function in terrestrial mammals. Studies show that there is an increase in the incidences of tumors and decreased immune cell function in TBT-exposed mammals. TBT interferes with the ability of human natural killer (NK) cells to lyse target cells; it also alters the secretion of the pro-inflammatory cytokines Interferon gamma (IFNγ) and Tumor necrosis factor alpha (TNFα) from human immune cells ex vivo. Any dysregulation of the cytokine communication network may have detrimental effects on immune function, as cytokines play a key role in the regulation of immune responsiveness. There have been no in vivo studies on the effects of exposure to TBT on the cytokine communication network. The effects of 24 hour exposure to TBT on the production of IFNγ and TNFα from human immune cells were analyzed in an ex vivo system using western blotting. The serum of TBT-exposed mice was analyzed for changes in the levels of cytokine secretion and production using a Milliplex mouse cytokine/chemokine magnetic bead premixed 32 plex kit and western blotting; the effects of TBT-exposure was assessed using a time curve of 6h, 12h, 24h, 48h and control. TBT increased the levels of the cytokines IFNγ, TNFα, IL1β, IL5, IL7, IL12βp40, IL13, IL15, MIP1β, MIP2 and RANTES in the serum at a minimum of one time point. IFNγ and TNFα secretion and production from human cells also increased, showing striking agreement in the response to TBT between the human and mouse systems.

Supported by: NIH grant 5U54CA163066-03

Citation Format: Shanieek T. Lawrence, Margaret Whalen, Samuel Pellom, Anil Shanker. Tributyltin alters the production and secretion of inflammatory cytokines from human and mouse immune 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 2735. doi:10.1158/1538-7445.AM2015-2735
Abstract 2756: Whole-exome sequencing reveals a novel germline variant in CEBPA-associated familial acute myeloid leukemia: 45-year follow-up of a large family
Authors: Pathak Anand , Seipel Katja , Pemov Alexander , Dewan Ramita , Brown Christina , Ravichandran Sarangan , Luke Brian T. , Yeager Meredith , Gatti Richard A. , Caporaso Neil , Mulvihill John J. , Goldin Lynn , Pabst Thomas Muller , McMaster Mary Lou , Stewart Douglas R.
Published: August 2015

Familial acute myeloid leukemia (AML) is rare and linked to germline mutations in RUNX1 or CCAAT/enhancer binding protein-α (CEBPA). We conducted whole-exome sequencing on a large family with AML originally evaluated at NIH in 1969 and uncovered a CEBPA Q311P mutation in all tested, affected members. The Q311P variant, located in the C-terminal bZip domain of CEBPA, was predicted to be highly deleterious by in silico algorithms; the Q311 position was highly conserved among CEBPA orthologs and paralogs. This variant has not been observed in the NHLBI's Exome Sequencing Project or the Broad Institute's Exome Aggregation Consortium. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mutant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation of a promoter with a tetramer of CEBP sites, consistent with a loss-of-function mutation. In addition, there was decreased colony survival in fibroblasts with CEBPA Q311P, associated with the clinical radiosensitivity postulated in one carrier. From 45 years of follow-up, we observed incomplete penetrance (46%) of CEBPA Q311P; we did not observe other cancers or leukemias segregating with the mutation. Thus, we present a comprehensive clinical and molecular characterization of a novel AML-associated variant in CEBPA in a NCI family with nearly half a century of follow-up. This study reveals that a germline mutation in the C-terminal bZip domain can alter the ability of CEBPA to bind DNA and reduces transactivation, leading to AML, though with lower penetrance than the canonical N-terminal frameshift germline mutations.

Citation Format: Anand Pathak, Katja Seipel, Alexander Pemov, Ramita Dewan, Christina Brown, Sarangan Ravichandran, Brian T. Luke, Meredith Yeager, Richard A. Gatti, Neil Caporaso, John J. Mulvihill, Lynn Goldin, Thomas Muller Pabst, Mary Lou McMaster, Douglas R. Stewart. Whole-exome sequencing reveals a novel germline variant in CEBPA-associated familial acute myeloid leukemia: 45-year follow-up of a large family. [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 2756. doi:10.1158/1538-7445.AM2015-2756