bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3024: FGFR1 amplification is linked to the squamous cell carcinoma subtype in esophageal carcinoma.
Authors: von Loga Katharina , Kohlhaussen Jule , Marx Andreas H. , Sauter Guido , Grob Tobias , Quaas Alexander
Published: August 2014

Introduction

Stimulation of Fibroblast-growth-factor-receptor-1 (FGFR1) plays an important role in cell differentiation and cell growth. Amplification of the FGFR1 gene was described in various cancer types. Clinical phase I trials are currently performed to address the potential applicability of anti-FGFR1-drugs in squamous cell carcinoma of the lung. Prevalence and clinical significance of FGFR1 amplification are unknown in esophageal carcinoma.

Material and Methods

In this study, six hundred formalin-fixed paraffin-embedded tissue samples from patients with esophageal carcinoma were analysed, including 346 patients with adenocarcinoma and 254 patients with squamous cell carcinoma. Dual-labeling Fluorescence in situ hybridization (FISH) was applied using probes for FGFR1 and centromere 8. The initial analysis was in a tissue microarray (TMA) format. All available tumor blocks from amplified tumors were subsequently analysed for heterogeneity on large sections. When available, lymph node metastases and tumor-adjacent dysplasia were also analysed on large sections from these tumors,

Results

FGFR1 amplification was more frequently seen in squamous cell carcinoma (9.4% of 202 interpretable cases) than in adenocarcinoma (1.6% of 308). High polysomy was found in an additional 5.9% of squamous cell carcinoma and 0.6% of adenocarcinoma. FGFR1 amplification was significantly associated with histologic grade (p=0.02) but was unrelated to tumor type, pT, pN, pM, UICC stage and survival. FGFR1 amplification was homogeneous across the tumor in most cases. FGFR1 heterogeneity was observed in only 3 of 24 cases. FGFR1 amplification was similarly observed in the primary tumor and in 6 cases each of lymph node metastases and dysplasia.

Summary

In conclusion, FGFR1 amplification in squamous cell carcinoma of the esophagus occurs at similar frequencies as in squamous cell carcinoma of the lung. The high homogeneity of FGFR1 in the primary tumor and the lack of discordant results in dysplasia argue for FGFR1 amplification, representing an early alteration in a subgroup of esophageal cancers. The high homogeneity of FGFR1 amplification provides further arguments for FGFR1 representing an interesting drug target in these cancers.

Citation Format: Katharina von Loga, Jule Kohlhaussen, Andreas H. Marx, Guido Sauter, Tobias Grob, Alexander Quaas. FGFR1 amplification is linked to the squamous cell carcinoma subtype in esophageal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2013-3024
Abstract 3026: Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma.
Authors: Chen Zirong , Gu Yumei , Hu Chengbin , Li Jian-Liang , Lin Shuibin , Shen Huangxuan , Cao Chunxia , Chen Jie , Li Jiancheng , Ha Patick K. , Kaye Federic J. , Griffin James D. , Wu Lizi
Published: August 2014

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 gene fusion, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a highly specific genetic alteration in more than 50% of mucoepidermoid carcinoma (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth ad survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and the in vivo. CRTC1-MAML2 induced the up-regulation of the EGFR ligand AREG by co-activating the transcription factor CREB and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with unresectable CRTC1-MAML2-positive MEC.

Citation Format: Zirong Chen, Yumei Gu, Chengbin Hu, Jian-Liang Li, Shuibin Lin, Huangxuan Shen, Chunxia Cao, Jie Chen, Jiancheng Li, Patick K. Ha, Federic J. Kaye, James D. Griffin, Lizi Wu. Targeting aberrantly activated AREG-EGFR signaling in CRTC1-MAML2-positive mucoepidermoid carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2013-3026
Abstract 304: Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway.
Authors: Qu Yi , Liu Runhui , Zhang Jigang , Popa Mihaela , Hua Yaping , Lin Biaoyang , McCormack Emmet , Zhang Weidong , Oyan Anne Margrete , Kalland Karl-Henning , Ke Xisong
Published: August 2014

Background: Previously we have established a stepwise prostate carcinogenesis model including human primary prostate epithelial EP156T cells, non-malignant mesenchymal EPT1 cells and pre-malignant EPT2-D5 cells. Aims and methods: To further achieve malignant cells, EPT2-D5 cells were grown in protein-free medium and the tumorigenesis was tested in vivo. Results: 1) Lots of spheres were generated in monolayer culture of D5 cells in protein free medium. The sphere forming cells were named D5HS. 2) Different from EPT2-D5 cells, D5HS formed large subcutaneous tumors (EPT3) and metastasis (EPT3M) in SCID mice. 3) Gene expression profiling showed inflammation signatures in D5HS. 4) Cellular Reactive Oxygen Species Detection Assay (DCFDA) showed high reactive oxygen species (ROS) in D5HS, and ELISA and Western blotting showed high secreted IL6 and high STAT3 expression in D5HS. 5) Blocking of ROS or STAT3 signaling reduced the sphere formation. 6) Immunohistochemistry staining showed high pSTAT3 level in the EPT3 tumor. Conclusions: Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway.

Citation Format: Yi Qu, Runhui Liu, Jigang Zhang, Mihaela Popa, Yaping Hua, Biaoyang Lin, Emmet McCormack, Weidong Zhang, Anne Margrete Oyan, Karl-Henning Kalland, Xisong Ke. Reactive oxygen species induce prostate cancer cells via activation of the IL6/STAT3 pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2013-304
Abstract 3047: The PML isoform IV is a negative regulator of nuclear EGFR's transcriptional activity in lung cancer.
Authors: Kuo Hong-Yi , Chen Yi-Chen , Chang Hsiang-Yi , Jeng Jen-Chong , Lin Erh-Hsuan , Pan Chih-Ming , Wang Mong-Lien , Chou Yu-Ting , Shih Hsiu-Ming , Wu Cheng-Wen
Published: August 2014

EGF receptor (EGFR) is a membrane-bound receptor tyrosine kinase which can transduce intracellular signals responsible for cell proliferation. It is frequently overexpressed and/or constitutively activated in non-small cell lung cancer (NSCLC) and thus is considered as a major cause of this disease. Recently, EGFR has been found in the nucleus where the nuclear EGFR (nEGFR) can function as a transcription factor activating the transcription of genes such as cyclin D1 gene (CCND1) which is essential for cell proliferation. Nevertheless, how nEGFR's transcriptional activity is regulated remains unclear. Promyelocytic leukemia protein (PML) is a tumor suppressor which is lost in various cancers including lung cancer. However, the role of PML in the suppression of lung cancer growth is still unclear. When we investigated the role of PML in the regulation of lung cancer cell growth, we found that PML isoform IV (PMLIV) preferentially represses the growth of lung cancer cells bearing constitutively active EGFR. Besides, when growing in the EGFR activating conditions, the growth of EGFR wild-type bearing A549 cells has been repressed by PMIV overexpression. We also found that PMLIV has protein-protein interactions with nEGFR and represses the transcription of nEGFR target genes. We showed that PMLIV is recruited by nEGFR to the target promoters and reduces the promoter histone acetylation level via HDAC1. Together, our results suggest that PMLIV interacts with nEGFR upon EGFR activation and represses the transcription of nEGFR target genes such as CCND1 and thus leading to inhibition of the lung cancer cell growth.

Citation Format: Hong-Yi Kuo, Yi-Chen Chen, Hsiang-Yi Chang, Jen-Chong Jeng, Erh-Hsuan Lin, Chih-Ming Pan, Mong-Lien Wang, Yu-Ting Chou, Hsiu-Ming Shih, Cheng-Wen Wu. The PML isoform IV is a negative regulator of nuclear EGFR's transcriptional activity in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2013-3047
Abstract 3060: MicroRNA expression profiling in African American and Caucasian prostate cancer.
Authors: Theodore Shaniece , Davis Melissa , Rhim Jhong , Grizzle William , Turner Timothy , Yates Clayton
Published: August 2014

PURPOSE: African American men have almost twice the incidence and death rates related to prostate cancer compared to Caucasian men. Several studies have suggested that some of these differences may be attributed to the elevated expression of different genes. Recently, a new class of genetic regulators has emerged known as microRNAs. Many reports have found microRNAs to be implicated in cell growth, cell differentiation and the onset of many diseases including prostate cancer. We sought to investigate whether microRNAs are differentially expressed in African American and Caucasian prostate samples.

METHODS: We analyzed the expression of over 300 microRNAs in 11 African American and Caucasian cell lines utilizing Affymetrix Microarray Chip. Validations of the most significant miRNAs were done by qRT-PCR. Methylation analysis of miR-152 promoter region was carried out by performing bisulfite modification and DNA sequencing. Characterization of the miRNA was carried out by performing MTT assay, Flow Cytometry, Western Blot, and Imunoflourescence.

RESULTS: We identified 15 microRNAs that were differentially expressed between the African American and Caucasian cell lines. Further validations led to the identification of miR-152 which is significantly expressed among both groups. Bisulfite modification and DNA sequencing revealed that miR-152 is methylated in aggressive prostate cancer cell lines. 5-Aza-2’/TSA treatment restored miR-152 levels in these cell lines. MiR-152 is responsible for inhibiting DNA methyltransferase 1, Rictor and TGF-β. Our results showed that miR-152 reduced cellular proliferation in PC-3, DU-145 and LNCaP prostate cancer cell lines. In addition, miR-152 initiated G2-M cell cycle arrest. Expression of miR-152 in 40 normal-tumor paired prostate tissue samples indicated that its expression was down-regulated in 67% of tumor samples. Analysis of African American and Caucasian prostate normal-tumor paired sample revealed that both normal and tumor prostate samples had lower miR-152 expression in the African American prostate samples compared to the Caucasian prostate samples.

CONCLUSION: These results give evidence that microRNAs may play a role in the advance progression seen in prostate cancer in African Americans. Future studies will focus on validating these results in larger patient sample sizes. Research supported by G12 RR03059-21A1(NIH/RCMI), and CA118623 (NIH/NCI).

Citation Format: Shaniece Theodore, Melissa Davis, Jhong Rhim, William Grizzle, Timothy Turner, Clayton Yates. MicroRNA expression profiling in African American and Caucasian prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3060. doi:10.1158/1538-7445.AM2013-3060
Abstract 3064: Epstein-Barr virus encoded BART microRNAs facilitate nasopharyngeal carcinoma development and progression via deregulation of Wnt signaling.
Authors: Wong Alissa Michelle G. , Chen Jinna , Ma Stephanie , Tsang Janice Wing Hang , Guan Xin Yuan
Published: August 2014

Epstein-Barr Virus (EBV) encoded BamHI Rightward Transcript (BART) microRNAs are frequently upregulated in Nasopharyngeal Carcinoma (NPC) specimens as previously reported by our group and also several others. However, very few studies have linked the potential role of these BARTs with commonly deregulated signaling pathways in NPC, particularly Wnt Signaling. Herein, we demonstrate that functional clustering of overexpressed BART microRNA target genes confer high enrichment within the Wnt signaling pathway, whereby BARTs 6-3p, 7, 14, 17-5p and 19-3p appear to extensively target key Wnt inhibitors such as WIF1, GSK3B, APC (forms destruction complex), BTRCP (Ubiquitin mediated proteolysis of B-catenin) and NLK (interfere with B-catenin/TCF/LEF binding). Although these 5 candidate BARTs were able to enhance TCF/LEF activity to induce downstream factors (e.g. CMYC, CCND1, MMP7, OCT4, SLUG), the extent and underlying mechanisms of Wnt activation conferred by these BARTs are not entirely the same. BARTs 17-5p and 19-3p can efficiently activate Wnt signaling marked by significantly enhanced levels of total and active B-catenin via post-translational downregulation of GSK3B, APC, BTRC and NLK. Meanwhile, the more conservative increase in active B-catenin and TCF/LEF activity induced by BARTs 6-3p, 7 and 14 could be attributable to their inability to inhibit the ubiquitin mediated proteolysis effects of B-TRC, despite partial downregulation of GSK3B and NLK. Upon demonstrating the wide influence of BART19-3p in Wnt deregulation, in vitro and in vivo functional assays also demonstrate the ability of BART19-3p to acquire oncogenic properties associated with Wnt activation (e.g. enhanced proliferation, invasion, EMT and stemness).

Citation Format: Alissa Michelle G. Wong, Jinna Chen, Stephanie Ma, Janice Wing Hang Tsang, Xin Yuan Guan. Epstein-Barr virus encoded BART microRNAs facilitate nasopharyngeal carcinoma development and progression via deregulation of Wnt signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2013-3064
Abstract 3095: Oncogenic miRNA-182-5p targets Smad4 and RECK in human bladder cancer.
Authors: Hirata Hiroshi , Ueno Koji , Shahryari Varahram , Tanaka Yuichiro , Tabatabai Z. Laura , Hinoda Yuji , Dahiya Rajvir
Published: August 2014

Abstract

Introduction and Objective: Onco-miR-182-5p has been reported to be over-expressed in bladder cancer (BC) tissues however a detailed functional analysis of miR-182-5p has not been carried out in BC. Therefore the purpose of this study was to: 1. conduct a functional analysis of miR-182-5p in bladder cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in BC. Methods: Initially we found that miR-182-5p expression was significantly higher in bladder cancer compared to normal tissues and high miR-182-5p expression was associated with shorter overall survival in BC patients. To study the functional significance of miR-182-5p, we over-expressed miR-182-5p with miR-182-5p precursor. Results: Cell proliferation, migration and invasion abilities were increased in BC cells. However cell apoptosis was inhibited by miR-182-5p. We also identified Smad4 and RECK as potential target genes of miR-182-5p using several algorithms. 3’UTR luciferase activity of these target genes was significantly decreased and protein expression of these target genes was significantly up-regulated in miR-182-5p inhibitor transfected bladder cancer cells. MiR-182-5p also increased nuclear beta-catenin expression and while Smad4 repressed nuclear beta-catenin expression. Conclusions: Our data suggests that miR-182-5p plays an important role as an oncogene by knocking down RECK and Smad4, resulting in activation of the Wnt-beta-catenin signaling pathway in bladder cancer.

Source of Funding: Grants RO1CA138642, RO1CA130860, RO1CA160079, VA Research Enhancement Award Program (REAP) and Merit Review grants

Citation Format: Hiroshi Hirata, Koji Ueno, Varahram Shahryari, Yuichiro Tanaka, Z. Laura Tabatabai, Yuji Hinoda, Rajvir Dahiya. Oncogenic miRNA-182-5p targets Smad4 and RECK in human bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3095. doi:10.1158/1538-7445.AM2013-3095
Abstract 3099: Radiation-induced angiogenesis is associated with MMP-9-miR-494-Syndecan-1 regulatory loop in medulloblastoma cells.
Authors: Asuthkar Swapna , Velpula Kiran Kumar , Gondi Christopher , Nalla Arun Kumar , Gogineni Venkateswara Rao , Rao Jasti S.
Published: August 2014

Matrix metalloproteinase-9 (MMP-9) is one of the most prominent proteins associated with tumorigenesis and is a modulator of the tumor microenvironment during angiogenesis. Recently, it has been speculated that Syndecan-1, a transmembrane heparan sulfate-bearing proteoglycan, has a critical role in contributing to angiogenesis when associated with MMP-9. However, the mechanism behind their synergistic regulation is not fully understood. In the current study, we report that ionizing radiation (IR)-induced MMP-9 enhances Syndecan-1 shedding and tube-inducing ability of medulloblastoma cells. Furthermore, tumor angiogenesis is associated with higher MMP-9-Syndecan-1 interactions on both the cell surface and extracellular medium. Our results also revealed the existence of a novel regulatory mechanism where MMP-9 drives the suppression of miR-494, thereby resulting in enhanced Syndecan-1 shedding and angiogenesis. By employing bioinformatics and functional luciferase experiments, we demonstrated that 3’-UTR of Syndecan-1 mRNA is a direct target of miR-494. Moreover, from the in situ hybridization analysis, we found that MMP-9-specific shRNA (pM) treatment of mouse intracranial tumors resulted in elevated expression of miR-494. This negative correlation between MMP-9 and miR-494 levels was found to be dependent on the methylation status of the miR-494 promoter-associated CpG island region (-186 to -20), which was confirmed by bisulfite-sequencing and methylation-specific-PCR (MSP) analysis. Overall, our results demonstrate a previously unrecognized mechanism of angiogenesis regulation in medulloblastoma through which miR-494 modulates the transcription of radiation-induced angiogenesis promoting molecules and that MMP-9-Syndecan-1 activity creates a negative feedback loop by regulating the expression of miR-494.

Citation Format: Swapna Asuthkar, Kiran Kumar Velpula, Christopher Gondi, Arun Kumar Nalla, Venkateswara Rao Gogineni, Jasti S. Rao. Radiation-induced angiogenesis is associated with MMP-9-miR-494-Syndecan-1 regulatory loop in medulloblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3099. doi:10.1158/1538-7445.AM2013-3099
Abstract 3104: HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway.
Authors: Liu Shou , Zhang Hongmei , Chen Hexin
Published: August 2014

Our previous studies have shown that overexpression of Hoxb7 delays tumors onset, but promotes tumor progression and metastasis in oncogene Her2/neu-induced mammary tumorigenesis, which is reminiscent of the dual role of TGF-β in breast cancer. To investigate whether HOXB7 directly or indirectly regulates the TGF-β signaling, we examined the phosphorylation levels of SMAD3, an important mediator of TGF-beta signaling, in primary mammary tumors derived from the Hoxb7 and Her2/neu double-transgenic mice and the Her2/neu mono-transgenic mice. Phosphorylation of SMAD3 is detected in a higher percentage of primary tumor tissues from double transgenic mice than that from Her2/neu mono-transgenic mice (69.2% versus 15.4%, p < 0.05), suggesting that Hoxb7 may activate TGF-β/Smad signaling in breast tumor tissues. In line with this finding, overexpression of HOXB7 induces expression of TGF-β ligands in both mouse and human breast cancer cell lines, leading to increased cell motility and invasiveness. The acquainted phenotypes in HOXB7-overepressing cells can be reversed by pharmacologic inhibition of TGF-β signaling. Promoter deletion analysis and Chromotin-immunprocipiration assay further demonstrated that HOXB7 can directly bind to and activate the promoter of TGFβ2. More importantly, expression of HOXB7 and TGFβ2 is strongly correlated in primary breast cancer tissues and associated with advanced stages of tumor progression and reduced survival rate. Taken together, our results suggest that HOXB7 may promote tumor migration, invasion and possibly metastasis through activation of the TGF-β signaling pathway.

Citation Format: Shou Liu, Hongmei Zhang, Hexin Chen. HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2013-3104

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3147: Cancer-specific mutations in adult soft tissue sarcomas.
Authors: Abaan Ogan D. , Francis Princy , Pineda Marbin , Walker Robert L. , Zhu Yuelin J. , Bilke Sven , Davis Sean R. , Meltzer Paul S.
Published: August 2014

Understanding of how cancer initiates and progresses is a fundamental goal of cancer research. Identification of cancer mutations is of profound importance for both understanding of the disease and for developing novel therapies. Soft-tissue sarcomas include multiple uncommon histotypes with features of diverse connective tissue lineages. Although some of their underlying genetic changes have been elucidated, for example in those types which contain etiologic chromosome translocations, in general, the genetic basis of these tumors is incompletely described. To investigate mutations in 8 subtypes of adult soft tissue sarcomas, we resequenced coding exons from about 1300 cancer relevant genes in 66 tumor samples. Data was analyzed for somatic variants in samples with matched normals while samples without normal were analyzed individually. The most frequent events were TP53 mutations (26%) followed by RB1 (9%) and APC (8%) mutations. Even though, well-defined oncogene activating mutations were present, they were very rare. Given the prevalence of tumor suppressor mutations in these samples we are currently integrating CGH results to look for chromosomal deletions to support loss of tumor suppressor genes. In addition, we are investigating low-frequency mutations in lesser-known genes that may be of potential interest. Our preliminary results suggest that these subtypes of soft-tissue sarcomas depend on tumor-suppressor loss predominantly, and may have gained multiple complex sets of mutations in a number of other pathways. In conclusion, our work will be important in cataloging novel cancer mutations in rare types of adult sarcomas, with potential implications for understanding their biology and the development of new therapies.

Citation Format: Ogan D. Abaan, Princy Francis, Marbin Pineda, Robert L. Walker, Yuelin J. Zhu, Sven Bilke, Sean R. Davis, Paul S. Meltzer. Cancer-specific mutations in adult soft tissue sarcomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2013-3147
Abstract 3192: Lin28 promotes growth of prostate cancer cells and activates the androgen receptor.
Authors: Tummala Ramakumar , Nadiminty Nagalakshmi , Zhu Yezi , Lou Wei , Evans Christopher P. , Gao Allen C.
Published: August 2014

Purpose: Lin28 is essential for stem cell viability and pluripotency. Recently Lin28 expression has been linked to several malignancies and its mechanisms of action proposed to be both let-7-dependent and -independent. It is a master regulator of let-7 miRNA biogenesis as well as a regulator of translation of a multitude of eukaryotic proteins. This study tested the hypothesis that Lin28 is overexpressed in prostate cancer (CaP) and that it activates the androgen receptor (AR) and promotes growth of CaP cells. Experimental Design: Human clinical CaP samples were analyzed for the expression of Lin28 by qRT-PCR, Western blotting and immunohistochemistry. Lin28 was transfected into a panel of CaP cell lines and growth was monitored. LNCaP cells stably expressing Lin28 were generated and growth was examined. Clonogenic ability of CaP cells expressing Lin28 was determined by colony formation and soft agar assays. Increase in expression of the AR and subsequent increase in transcription of AR-target genes were analyzed by qRT-PCR, luciferase assays and ELISA. LNCaP cells stably expressing Lin28 were injected into nude mice and tumorigenesis was monitored. Results: Lin28 was found to be overexpressed in clinical CaP compared to the benign tissues. Overexpression of Lin28 enhanced while downregulation of Lin28 reduced growth of CaP cells. Lin28 enhanced the ability of CaP cells to form colonies in anchorage-dependent and -independent manners. LNCaP cells stably expressing Lin28 exhibited significantly higher tumorigenic ability in vivo. Furthermore, Lin28 induces expression of the AR and its target genes such as PSA and NKX3.1. Conclusions: Lin28 promotes growth of CaP cells in vitro and in vivo. Lin28 induces expression and activation of the AR. Our findings suggest an important role of Lin28 in CaP development and activation of the AR axis.

Citation Format: Ramakumar Tummala, Nagalakshmi Nadiminty, Yezi Zhu, Wei Lou, Christopher P. Evans, Allen C. Gao. Lin28 promotes growth of prostate cancer cells and activates the androgen receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3192. doi:10.1158/1538-7445.AM2013-3192
Abstract 3216: Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications.
Authors: Ma Donghui , Shu Youmin , Yuan Kehu , He Wei-Wu
Published: August 2014

Antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is a predictive biomarker for cisplatin based chemotherapy. High ERCC1 expression is linked to drug resistance to cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissue samples. By using high density protein microarray chip, we discovered that 8F1 not only reacts with its authentic target ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity to PCYT1A was further confirmed by other immunoassays, including Western blot, antigen absorption test, and immunohistochemistry (IHC) analyses. In this study, we also discovered that PCYT1A gene expression level is even significantly higher than ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. In summary, 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on protein microarray chip test and both worked in IHC application.

Citation Format: Donghui Ma, Youmin Shu, Kehu Yuan, Wei-Wu He. Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3216. doi:10.1158/1538-7445.AM2013-3216
Abstract 3218: Interrogating the role of NOX4 in colon cancer metastasis.
Authors: Bauer Kerry M. , Buechler Steven , Hummon Amanda B.
Published: August 2014

Colon cancer is a major cause of cancer-related deaths worldwide. Adjuvant chemotherapy significantly reduces mortality in stage III colon cancer; however, it is only marginally effective in stage II patients. Molecular biomarkers are used to partition tumors into a poor prognosis group that is likely to metastasize and a good prognosis group that has a higher probability to be relapse-free. These biomarkers for relapse risk are of clinical interest, and lead to insight into how a tumor progresses to metastasis.

There is increasing evidence that cancer of the ascending (right-side) colon is different from the descending (left-side) colon. There are site-specific differences in histological and molecular characteristics and different signaling pathways have been shown to dominate progression to relapse in right and left-side colon cancer. We have observed that the genes altered in expression between the poor prognosis and good prognosis tumors vary significantly depending on whether the malignancy originates on the right or left side of the colon. We have identified NADPH oxidase 4 (NOX4) to be highly predictive of relapse in stage II and stage III left-side colon cancer where elevated expression of NOX4 is correlated with relapse. Furthermore, NOX4 expression is greatly elevated in the human colon cancer derived metastatic cell line SW620 compared to human cell lines derived from primary adenoma or carcinoma colon tumors. NOX4 silencing using RNA interference results in decreased levels of reactive oxygen species (ROS). The NOX family of genes has been implicated in cancer development in several types of cancer while overproduction of ROS has been implicated in tumor metastasis.

We are exploring the newly identified role of NOX4-mediated ROS generation in metastatic colon cancer. To investigate the underlying molecular mechanism we are analyzing the effect of NOX4 silencing via RNA interference and NOX4 inhibition using pharmacological NOX inhibitors on global protein expression patterns in metastatic-derived SW620 cells. Proteomic changes are being assessed by iTRAQ labeling and mass spectrometry. Bioinformatics and cluster analysis will be used to identify the functional role of redox responsive and ROS regulative proteins in colon cancer metastasis. The role of NOX4 as a prognostic biomarker in colon cancer makes it an important target for research in cancer biology and therapeutics.

Citation Format: Kerry M. Bauer, Steven Buechler, Amanda B. Hummon. Interrogating the role of NOX4 in colon cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3218. doi:10.1158/1538-7445.AM2013-3218
Abstract 3219: DrIMS-3DCC: Drug imaging mass spectrometry of 3D cell culture systems.
Authors: Liu Xin , Weaver Eric , Hummon Amanda B.
Published: August 2014

3D cell cultures have increased complexity compared to simple monolayer and suspension cultures, recapitulating the cellular architecture in the body. For example, many colon cancer cell lines form rounded heterogeneous spheroid structures that mimic the pathophysiological properties of tumors when grown under appropriate cell culture conditions. Classical imaging methodologies, like immunohistochemistry, are commonly used to examine the distribution of specific species within the spheroids. However, there is a need for an unbiased discovery-based methodology that would allow examination of protein/peptide distributions in 3D culture systems, without a need for prior knowledge of the analytes. We have developed a MALDI imaging mass spectrometry approach to examine protein distributions in 3D culture models (MALDI IMS-3DCC), allowing us to map the spatiotemporal dynamics of proteins in these valuable model systems.

We are currently expanding our approach to image the distribution of drugs within 3D cell cultures. We are using our MALDI IMS-3DCC to analyze the penetration of drugs within 3D cell cultures. With our mass-spectrometric based approach, we have the ability to map the distribution of the 1) the drug, 2) the metabolites produced from the drug, and 3) the spatiotemporal changes in proteins following treatment. Thus far, we have successfully evaluated the effects of two clinically relevant species, oxaliplatin and Irinotecan, on spheroids. In the future, we will expand our approach to evaluate new therapeutic compounds. We believe that our novel imaging strategy will drastically alter how new therapeutic compounds are evaluated, reducing the need for animal models in the early stages of drug development, thereby speeding up the drug evaluation process.

Citation Format: Xin Liu, Eric Weaver, Amanda B. Hummon. DrIMS-3DCC: Drug imaging mass spectrometry of 3D cell culture systems. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3219. doi:10.1158/1538-7445.AM2013-3219
Abstract 3230: Targeted sequencing of 16 archived ovary cancers.
Authors: Li Shihong , Jongkam Narisra , Xia Wei , Tran Hong , Suh K. Stephen
Published: August 2014

We developed a method to extract up to 22 μg DNA from a single 10 μm FFPE slide. Although FFPE DNA is notoriously fragmented, our extraction method enabled us to consistently generate 150-250 bp amplicons. This DNA enables whole genome sequencing and targeted sequencing using various platforms. This poster will present targeted amplicon sequencing on the Ion Torrent PGM™ .

Single FFPE pathology slides from 16 ovary cancer specimens (5 cured with first line chemotherapy and 11 relapsed within 5 years), archived from 1999-2005, were processed. Amplicons targeting selected exons and hotspots of 54 cancer genes were sequenced using both traditional Sanger sequencing (28 amplicons covering 8 genes) and by Next Generation sequencing (190 amplicons covering 46 genes using Ion AmpliSeq™ Cancer Panel).

5 ng FFPE DNA (based on Qubit measurement) from each patient was subjected to 190-plex PCR. The resulting amplicon libraries were ligated with indexed adaptors. Each eight libraries were pooled and sequenced on one 318 Chip. Average read depth ranged from 1521 to 3138, with 87.3% to 99.6% targets covered at least 20 times. Quality scores were above 30 for the first 80 bps, above 25 for 100 bps, and above 19 for 200 bps.

12 to 76 genetic variants were detected from each patient at frequency ranges from 0.7% to 100%. Both the genomic variation that occurred only in cured or relapsed group, and the genetic variations that were common to both groups will be presented.

Citation Format: Shihong Li, Narisra Jongkam, Wei Xia, Hong Tran, K. Stephen Suh. Targeted sequencing of 16 archived ovary cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2013-3230
Abstract 3231: Comprehensive profiling of actionab.
Authors: Wang Yulei , Bourgon Richard , Lu Shan , Yan Yibing , Guan Yinghui , Ryner Lisa , Partel Rajesh , Lackner Mark , Amler Lukas
Published: August 2014

Comprehensive characterization of genetic profiles in clinically actionable genes in each tumor promises to facilitate personalized approaches to cancer treatment. However, development of systematic genetic profiling of cancers in the clinical setting is facing unique technical challenges. More than 90% of tumor samples available for clinical sequencing are formalin-fixed and paraffin-embedded (FFPE). These samples are not only limited by quantities, the DNA quality in these samples is often compromised due to different degree of degradation, cross-links and chemical modifications. Targeted sequencing, in which genomic regions of interests are selectively enriched before sequencing to achieve high coverage and sensitivity, can potentially provide a cost-effective and efficient solution for clinical sequencing. However, very few studies have been reported on the development of methodology that is applicable and robust in analyzing “real-world” clinical FFPE samples. Here we report the development of a novel methodology that integrates microfluidic multiplex PCR-based target enrichment with massively parallel sequencing for high-throughput, high-content targeted sequencing of clinically actionable cancer genes in FFPE tumor tissues. This method enables amplifying 963 regions from 88 common cancer genes in 48 samples and simultaneously sequencing 48 pooled barcoded samples in a single sequencing reaction. Using this method, we sequenced 40 cancer cell lines and a well-defined Latin square study with dilution series of 8 known somatic mutations, to comprehensively assess the sensitivity, specificity, reproducibility in both target enrichment and variant detection. Furthermore, using paired fresh frozen/FFPE tissues obtained from 16 cancer patients, we developed and optimized a FFPE protocol that yields high-quality genetic profiles highly concordant to those derived from parallel fresh frozen samples. To demonstrate the clinical utility of this method, we sequenced a cohort of 73 FFPE tumor samples from endometrial cancer patients. Many clinically actionable mutations were identified in these patients’ samples and confirmed using orthogonal validations. Our results demonstrated the feasibility and clinical utility of our method in clinical sequencing, setting the stage for ultimately translating this method in clinical setting to inform and guide cancer treatment.

Citation Format: Yulei Wang, Richard Bourgon, Shan Lu, Yibing Yan, Yinghui Guan, Lisa Ryner, Rajesh Partel, Mark Lackner, Lukas Amler. Comprehensive profiling of actionab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3231. doi:10.1158/1538-7445.AM2013-3231
Abstract 3232: Oligonucleotide-selective sequencing (OS-Seq): a rapid and efficient targeting technology for ultra-deep detection of cancer mutations and rearrangements.
Authors: Hopmans Erik S. , Natsoulis Georges , Buenrostro Jason , Grimes Sue , Bell John , Ji Hanlee P.
Published: August 2014

Somatic mutations, chromosomal rearrangements and other genomic aberrations in cancer genomes are critical events in the development of cancer and increasingly used in identifying oncogenic events that may be indicators for targeted therapies. For rapid and high accuracy analysis of targeted candidate genes and regions from cancer genomes, we developed OS-Seq that can be used to simultaneously discover cancer point mutations, copy number variants and rearrangements. For this new sequencing technology, we re-engineer the immobilized oligonucleotide primer lawn on a solid phase support (e.g. Illumina flow cell) with capture “primer-probes” such that it functions for both genomic target capture and sequencing (Myllykangas et al. 2011, Nature Biotech). As a result, in a matter of days we can rapidly sequence target regions in cancer genomes. We designed an in-silico exome capture probe set, enabling rapid design of disease-specific targeted capture primer probe sets. Subsequently, we designed assays to cover 313 and 1,400 cancer genes. Using the 313-gene assay targeting 5,000 exons, we can routinely sequence cancer genomes with on-target coverage greater than 500X, including for the entire length of exons as large as 1 Mb regions. Using a smaller gene set we have established coverage depths over 8,000 that enables the investigation of tumor evolution via mutations with low allelic fractions. In addition, we have designed OS-Seq assays for characterizing loci that are involved in rearrangements. We design capture probes positioned on opposing strands upstream and downstream of suspected rearrangement breakpoints and by localized assembly were able to confirm certain rearrangements, including a long deletion and inversions. For our current studies, we are using OS-Seq to identify cancer mutations in colorectal adenocarcinoma and determine the extent of genetic heterogeneity prevalent in these tumors. OS-Seq provides high-depth, high quality cancer genome sequence data, opening the door to potential diagnostic analysis of cancer genetic heterogeneity as seen in tumor evolution and cancer rearrangements.

Citation Format: Erik S. Hopmans, Georges Natsoulis, Jason Buenrostro, Sue Grimes, John Bell, Hanlee P. Ji. Oligonucleotide-selective sequencing (OS-Seq): a rapid and efficient targeting technology for ultra-deep detection of cancer mutations and rearrangements. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2013-3232
Abstract 3236: Molecular characterization of lung tumors based on metabolomic profiling.
Authors: Sappington Daniel R. , Helms Scott , Dhakal Ishwori B. , Siegel Eric R. , Bartter Thaddeus , Boysen Gunnar
Published: August 2014

Lung cancer is the leading cause of cancer-related deaths in US. For the majority of patients (70%), the extent of the disease precludes complete surgical resection and treatment relies solely on radiation, chemotherapy or a combination of both. Unfortunately, these treatments improve survival only minimally and are accompanied by considerable adverse side effects. Despite immense research efforts over the last 20 years, the mean survival of lung cancer patients has only increased by 13 days. This relatively poor mean survival is attributed to inadequate therapy selection. Current therapy selection is mainly based on the histopathologic examination of needle biopsies obtained during bronchoscopy. There is growing concern that these classifications and stratification are insufficient to predict treatment response of individual tumors. To complement current histopathological-based tumor classification, we investigated the suitability of metabolomics to improve tumor classification. Therefore, metabolomic profiles were obtained from various lung cancer cell-lines. Data were analyzed for molecular features characteristic for cell-lines derived from adenocarcinoma (AdenoCa) or squamous cell carcinoma (SqCCa). Data analyses revealed 78 features that are capable of clearly separating AdenoCa, SqCCa, and lung fibroblasts from each other. Subsequently, the studies were extended to specimen from 30 lung cancer patients. Similar to the cell-line experiments, metabolomic profiles were obtained and analyzed for characteristic features of patients with AdenoCa, SqCCa, or non-malignant specimen. The analysis of the human specimen revealed 21 unique features that are potentially suitable biomarkers for tumor classification. Our data suggest that metabolomic profiling is a promising approach to classify and distinguish between individual tumor cell-lines and to distinguish patients with AdenoCa from patients with SqCCa.

Citation Format: Daniel R. Sappington, Scott Helms, Ishwori B. Dhakal, Eric R. Siegel, Thaddeus Bartter, Gunnar Boysen. Molecular characterization of lung tumors based on metabolomic profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2013-3236
Abstract 3251: Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma.
Authors: Jayakumar Arumugam , Rauvolfova Jana , Bao Hanying , Fokt Izabela , Skora Stanislaw , Heimberger Amy , Priebe Waldemar
Published: August 2014

WP1066 was shown to exert its potent inhibitory activity against several types of human cancer including melanoma in vitro and in vivo via suppression of JAK2/STAT3/STAT5 activation. However, if there is any other additional molecular mechanism(s) by which WP1066 generates its anti-tumor effects remain largely unknown. Here, we report a novel function for WP1066 in inducing the degradation of both HIF-1α and HIF-1β subunits in human melanoma cells. We found constitutive activation of HIF-1α and an increased expression of hexokinase II, a down target of HIF-1α, in normoxia in 6/6 of melanoma cell lines, but not in other tumor types. Both hypoxia and cobalt chloride, a transition metal that mimics hypoxia, further increased HIF-1α protein accumulation in a time-dependent manner in WM35 and SKMEL-1 melanoma cells. WP1066 selectively decreased the constitutive and hypoxia and cobalt chloride induced increase in the level of HIF-1α in a dose-dependent manner with a half-life (T1/2) of ∼13, ∼10, and ∼10 min in WM35 cells. Moreover, WP1066 decreased the T1/2 of HIF-1α following cycloheximide treatment from ∼13 to ∼7 min suggesting that WP1066 induced HIF-1α instability is not mediated via inhibition of its translation. Further, inhibition of HIF-1α protein accumulation by WP1066 was not abolished in the presence of MG132 and proteasome inhibitor I, a potent inhibitor of the 26S proteasome, suggesting that the 26S proteasome-system is not responsible for WP1066-induced HIF-1α instability. Immunoprecipitation, confocal microscopy, and cellular fractionation analyses revealed that WP1066 induced ubiquitinylation, aggregation and degradation of HIF-1α in WM35 cells. Finally, WP1066 directly inhibits activity of rhUSP5 and rUCH-L1. Altogether, our study identified a novel function for WP1066 in inducing HIF-1 instability that may also contribute to the anti-cancer effects of WP1066 in human melanoma.

Citation Format: Arumugam Jayakumar, Jana Rauvolfova, Hanying Bao, Izabela Fokt, Stanislaw Skora, Amy Heimberger, Waldemar Priebe. Blockade of HIF-1 with a small molecule inhibitor WP1066 in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3251. doi:10.1158/1538-7445.AM2013-3251