bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5220: Reciprocal regulation of MYC and LKB1 modulates cell survival and differentiation in lung cancer
Authors: Cortez Maria A. , Yang Chao , Byers Lauren Averett , Diao Lixia , Allen Thaddeus , Giri Uma , Wang Jing , Welsh James , Heymach John Victor
Published: May 2015

MYC and LKB1 are important regulators of cell survival and differentiation. Although recent studies have been shown oncogenic cooperation between MYC and LKB1 loss, their relationship is unclear. In this study, we report a reciprocal regulation between MYC and LKB1 which modulates cell survival and differentiation in non-small cell lung cancer (NSCLC). We found that MYC regulates LKB1 expression via miR-17. Specifically, luciferase activity was reduced in cells transfected with miR-17, LKB1 3′ UTR constructs compared with scrambled control. Mutation of miR-17 interaction sites rescued the luciferase activity, thus confirming that miR-17 directly interacts with the LKB1 3´ UTRs. In agreement with our reporter gene assay results, enforced overexpression of miR-17 decreased the expression of LKB1 mRNA and protein levels compared with scrambled control. Conversely, miR-17 inhibition promoted upregulation of LKB1 expression in NSCLC cells. Furthermore, miR-17 expression was inversely correlated with LKB1 expression in 532 patients with NSCLC from The Cancer Genome Atlas data (TCGA). MYC and miR-17 overexpression promoted mTOR pathway activation, increased cell proliferation and colony formation in NSCLC cells. In line with these findings, similar results were obtained when we knockdown LKB1 expression, suggesting that MYC promotes mTOR pathway and cell survival, at least in part, via LKB1 regulation. Reciprocally, we found upregulation of MYC expression in NSCLC cell lines with LKB1 mutation or low expression compared to LKB1 wild-type (total n=78) via reverse phase protein array (RPPA) analysis. We validated these findings by analyzing MYC expression via western blotting in LKB1-knockdown and LKB1-ovexpressing stable cell lines. Mechanistically, we found that LKB1 downmodulates MYC expression via Yes-Associated protein 1 (YAP) phosphorylation at Ser127 site, which results in YAP cytoplasmic retention inhibiting transcription activation of its targets. In addition, we found that LKB1 also regulates ID2, a negative regulator of cell differentiation, via YAP/MYC axis. Taken together, our study reveals a reciprocal regulation between MYC and LKB1 offering additional mechanistic insights how MYC promotes cell survival and differentiation in lung cancer.

Citation Format: Maria A. Cortez, Chao Yang, Lauren Averett Byers, Lixia Diao, Thaddeus Allen, Uma Giri, Jing Wang, James Welsh, John Victor Heymach. Reciprocal regulation of MYC and LKB1 modulates cell survival and differentiation in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5220. doi:10.1158/1538-7445.AM2014-5220
Abstract 5223: Prognostic value of microRNA signature in non-small-cell lung cancer: A microRNA expression analysis
Authors: Pu Heng-Ying , Fu Da , Zhang Mei-Yin , Cha Yin-Lian , Peng Hai-Shan , Zhang Lan-Jun , Jia Wei-Hua , Zeng Yi-Xin , Liu Wan-Qing , Zheng Steven X.F. , Hu Guo-Hong , Wang Hui-Yun
Published: May 2015

Purpose: MicroRNA (miRNA) plays significant role in the development and progression of cancer. We aimed to identify miRNA signatures to be used as diagnostic and prognostic biomarkers in non-small-cell lung cancer (NSCLC).

Experimental Design: Patients with NSCLC who underwent surgical resection and didn't receive any therapy before surgery were randomly divided into training set (198 patients) and test set (199 patients). The miRNA expression levels were detected by microarray and selected miRNAs were verified by quantitative real-time PCR (qRT-PCR). The other 302 patients from another center were used as an independent set to verify the miRNA signature.

Results: A 37-miRNA signature consisting of 15 downregulated miRNAs and 22 upregulated miRNAs was established for distinguishing NSCLC specimens from noncancerous lung tissues in the training set with 98.2% accuracy. In the test set, the classification accuracy of the signature was 98.5%. Then three miRNAs were chosen to construct prognostic signature in the training set. A formula based on the 3 miRNAs was built to calculate prognostic index. Kaplan-Meier analysis showed that patients with a high prognostic index had a significant poorer survival than those with a low index. The 3-miRNA signature was validated in test set with the same formula for computing the prognostic index. The expression levels of 4 miRNAs including the 3 miRNA in the prognostic signature were verified by RT-PCR in 33 paired NSCLC and corresponding noncancerous lung tissues. The expression level of the 3 miRNA was detected by qRT-PCR in the independent set. Survival analysis showed that the 3-miRNA signature was associated with survival in the independent set. Multivariate analysis indicated that the 3-miRNA signature was an independent prognostic biomarker. A combination of the 3-miRNA signature and TNM stage had much better prognostic ability than did TNM stage alone in the training, test and independent sets.

Conclusions: The 37-miRNA and 3-miRNA signatures identified in our study may be useful biomarkers for diagnosis and prognosis of NSCLC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Heng-Ying Pu, Da Fu, Mei-Yin Zhang, Yin-Lian Cha, Hai-Shan Peng, Lan-Jun Zhang, Wei-Hua Jia, Yi-Xin Zeng, Wan-Qing Liu, Steven X.F. Zheng, Guo-Hong Hu, Hui-Yun Wang. Prognostic value of microRNA signature in non-small-cell lung cancer: A microRNA expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2014-5223
Abstract 5225: MicroRNA profiling in serum samples from donors with germ cell cancer
Authors: Lee Kathy Y. , Patel Sunali , Hayashibara Kathleen , Chu Shirley , Gillis Ad J. M , Rijlaarsdam Martin , Dorssers Lambert C.J , Looijenga Leendert H. J
Published: May 2015

MicroRNAs are short non-coding RNA molecules (∼21 bases) that have been identified as important regulators of gene expression at the translational and transcriptional level. They are known to play a crucial role in cell development, differentiation, and disease. Dysregulation of miRNAs has been linked to cancer development and tumor progression. In addition, miRNAs have been identified as tumor classifiers and disease biomarkers. Recent studies have shown that miRNAs are present in body fluids (serum, saliva, semen, urine) thus providing a non-invasive tool to study and monitor disease states. Earlier research studies identified specific miRNAs as characteristic for germ cell tumors, such as testicular cancer (miR-372, miR-373). miRNA Profiling (∼760 miRNAs) was performed to identify additional miRNAs as candidate biomarkers in serum samples for testicular cancer (seminoma and non-seminoma types), from normal and cancer tissue in parallel with their matched serum samples collected from patients with testicular cancer. For this research study we used a miRNA specific bead capture system to isolate miRNAs from serum and TaqMan® Array Card platform for profiling. We will present results from this research study comparing the miRNA expression patterns in matched normal, cancer tissue, and serum.

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Kathy Y. Lee, Sunali Patel, Kathleen Hayashibara, Shirley Chu, Ad J. M Gillis, Martin Rijlaarsdam, Lambert C.J Dorssers, Leendert H. J Looijenga. MicroRNA profiling in serum samples from donors with germ cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2014-5225
Abstract 5227: The microRNA profile is altered in fibrosis-associated hepatocarcinogenesis in mice
Authors: Marrone April K. , Tryndyak Volodymyr , Chappell Grace , Beland Frederick A. , Rusyn Ivan , Pogribny Igor P.
Published: May 2015

Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is rising worldwide. The molecular mechanisms associated with the development of HCC are complex and include multiple interconnected alterations driven by both genetic and epigenetic events. Mounting evidence indicates an important role of microRNAs (miRNAs), post-transcriptional negative regulators of gene expression, in the pathogenesis of HCC. In humans, the development of HCC is commonly associated with liver cirrhosis. To mimic the molecular pathogenesis of human HCC, we used a mouse model of fibrosis-associated liver cancer, which was designed to emulate cirrhotic liver. Tumor and non-tumor liver samples from B6C3F1 mice treated with N-nitrosodiethylamine (DEN; a single ip injection of 1 mg/kg at 14 days of age) and carbon tetrachloride (CCl4; 200 µl/kg, 2 times/week ip for 14 weeks starting at 8 weeks of age) were analyzed. Using TaqMan® array miRNA cards, we identified a total of 52 and 45 miRNAs that were differentially expressed in tumor and non-tumor liver tissue, respectively, from DEN/CCl4-treated mice as compared to those in livers of vehicle-treated control B6C3F1 mice. Importantly, 27 differentially expressed miRNAs were common for both tumor and non-tumor samples from DEN/CCl4-treated mice. These miRNAs are involved in the major cancer pathways including apoptosis, cell proliferation, angiogenesis, and epithelial-to-mesenchymal transition. These results suggest that changes in miRNA expression during hepatocarcinogenesis may occur at very early stages of the carcinogenic process, preceding the formation of detectable neoplastic lesions. Most notably, our results showing 27 miRNAs common for tumor and non-tumor tissue suggest that a miRNA profile associated with the development of HCC could potentially be used to decipher the precise molecular mechanisms of disease progression and/or be used as early markers for detecting neoplastic lesions in the liver.

Citation Format: April K. Marrone, Volodymyr Tryndyak, Grace Chappell, Frederick A. Beland, Ivan Rusyn, Igor P. Pogribny. The microRNA profile is altered in fibrosis-associated hepatocarcinogenesis in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5227. doi:10.1158/1538-7445.AM2014-5227
Abstract 5240: Epigenetic dysregulation of miR-196b in breast cancer
Authors: Marquez Rebecca T. , Smith Amber R. , Tsao Bryan , Xu Liang
Published: May 2015

Dysregulation of microRNA (miRNA) expression levels is a major cause of cancer development. miRNA expression profiles can determine tumor type, patient prognosis, reoccurrence and treatment outcome, with greater accuracy as compared to mRNA expression profiles. The expression of miRNAs can be epigenetically dysregulated by aberrant DNA methylation and histone deacetylation in breast cancer cells. Furthermore, one third of miRNA promoters are targets for aberrant methylation in breast cancer. Identifying miRNAs that are epigenetically silenced in breast cancer will provide new insight into breast cancer development as well as define novel signatures for predicting therapeutic response to epigenetic modifying agents. New studies have shown that using transient low doses of demethylating agents, such as decitabine (DAC), result in sustained changes in major cancer cell signaling pathways, therefore reducing tumorigenicity and cancer stem cell self-renewal. The goal of this project is to identify epigenetically-silenced miRNAs that can be reactivated upon low dose treatment of DAC. miR-196b has been shown to inhibit metastasis by targeting HOXC8. Using online datasets, we determined miR-196b is hypermethylated in breast cancer patient samples compared to normal breast samples. We used Taqman miRNA assays to measure the expression of miR-196b in breast cancer patient samples and found it was down-regulated in tumor samples compared to paired normal breast tissue. miR-196b is also down-regulated in MDA-MB-231 cells. We treated MDA-MB-231 cells with low dose (100 nM) DAC for 3 days and found miR-196b was upregulated 5-fold compared to DMSO control. The methylated CpG content of miR-196b-associated CpG island was measured using bisulfite sequencing DNA purified from DMSO and DAC treated cells. The miR-196b CpG loci in the DAC treatment was demethylated by 33% compared to DMSO control. Therefore, reactivation of miR-196b is associated with demethylation of miR-196b CpG loci. Interestingly, fourteen days after ending DAC treatment, miR-196b re-expression continued to increase up to 13-fold above DMSO control. In addition, we found that exogenously re-introduced mature miR-196b into MDA-MB-231 breast cancer cells resulted in upregulation of endogenous primary miR-196b. This suggests a novel positive feedback loop that allows miR-196b to regulate its own expression, possibly through epigenetic mechanisms. Future studies will involve identifying the mechanism associated with this re-expression, determining whether miR-196b regulates additional gene promoters, and the affect this has on breast cancer metastatic phenotype.

Citation Format: Rebecca T. Marquez, Amber R. Smith, Bryan Tsao, Liang Xu. Epigenetic dysregulation of miR-196b in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2014-5240
Abstract 5241: Accurate quantitation of micro RNA by chip-based digital PCR
Authors: Wu Yalei , Dahlhoff Victoria , He Lin , Chen Caifu
Published: May 2015

MicroRNAs (miRNAs) are small non-coding RNAs that function to regulate gene expression at transcriptional and post-transcriptional level. Mature miRNAs are short, usually 18-23 nucleotides (nt) in length, with high homology in the seed region (position 2-8 from the 5′ end of a mature miRNA) within each family. This presents a great challenge when designing assays for quantitation by real-time polymerase chain reaction (PCR). Existing miRNA assays often trade in sensitivity and/or assay performance for specificity. Comparison between two different mature miRNAs with quantitative real-time PCR (qPCR) requires generating standard curves and characterizing PCR efficiency and sensitivity. Here we report a chip-based digital PCR technology. With this technology, we are able to accurately quantitate mature miRNAs without characterizing and optimizing the assay.

Briefly, we input the same amount (ratio=1:1) of synthetic hsa-miR-19b and hsa-miR-92a. We then quantitated the miRNAs using reverse transcription followed by quantitative RT-PCR or chip-based QuantStudio™ 3D digital PCR (dPCR). Data from qPCR showed a delta Ct=2.58 between the cDNAs. This converted to a 3.84 fold difference between the two mature miRNAs, which deviated 284% from the input ratio, which equals 1. However, the data from dPCR indicated that the ratio between the amounts of the two mature miRNAs was 0.94 ± 0.02 (SD). Although statistically different, the results from dPCR deviated only 5% from the input ratio. Compared to qPCR, dPCR significantly enhanced the precision in sample quantitation.

In summary, our study demonstrated that the chip-based dPCR technology has greatly increased precision in quantitating miRNA samples. The simplified workflow and improved output of dPCR are very useful features in broad applications such as revealing small changes in the miRNA expression levels.

Citation Format: Yalei Wu, Victoria Dahlhoff, Lin He, Caifu Chen. Accurate quantitation of micro RNA by chip-based digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2014-5241
Abstract 525: Automated microRNA detection in formalin-fixed, paraffin-embedded tissue samples
Authors: Jiang Zeyu (David) , Pedata Anne , Shingler Taylor , Behman Lauren , Chafin David , Day William
Published: May 2015

The human genome encodes more than one thousand short (17-24 base) non-coding microRNA molecules which regulate diverse cellular functions. Several of these functions are also involved in tumorigenesis including cell growth, motility, and immune surveillance. Not surprisingly, numerous clinical studies suggest that microRNAs could serve as potent prognostic and/or predictive biomarkers for a variety of diseases including breast, colorectal, lung, and prostate cancers. However, due to their short sequence length, technical obstacles exist for detection of these potential biomarker targets particularly in FFPE tissue samples: (i) limited target sequence limits probe length and subsequent target specificity as well as (ii) detection sensitivity since short probes require significant amplification technologies to generate a visible signal; lastly, (iii) short microRNA targets may be less stable in tissue (increased ribonuclease sensitivity and/or limited crosslinking to cellular components). In the current study, new RNA detection technologies, which include hapten-labeled oligonucleotide probes and sensitive tyramide-hapten/Silver detection designed for detection of longer RNA targets, were adopted for automated detection of microRNA targets in formalin-fixed, paraffin-embedded (FFPE) tissue.

DNP-labeled oligonucleotide probes, directed against four microRNA targets (miR-21, miR-200c, miR-25, and miR-200b) plus sequence-scrambled negative control were synthesized and combined with a modified VENTANA OptiView amplification kit and silver detection. The probes and new detection system catalyzed sensitive and specific detection of each microRNA target in MCF-7 xenograft FFPE tissues, which correlates well with microarray profiling data from MCF-7 cells by Fix et. al. (2010)*.

In summary, this study demonstrates a robust and fully automated ISH assay for microRNA targets in FFPE tissue and enables potential microRNA biomarkers being analyzed within tissue context.

* Fix et. al. Cancer Genomics & Proteomics (2010) 7:261-278.

Citation Format: Zeyu (David) Jiang, Anne Pedata, Taylor Shingler, Lauren Behman, David Chafin, William Day. Automated microRNA detection in formalin-fixed, paraffin-embedded tissue samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 525. doi:10.1158/1538-7445.AM2014-525
Abstract 5250: The effect of introducing a C118S mutation into the endogenous Kras gene on early tumorigenesis
Authors: Huang Lu , Counter Christopher M.
Published: May 2015

The Ras family of small GTPase, comprised of HRAS, NRAS, and KRAS, have been found mutated to remain in an active oncogenic state in many different human cancers, which is well established to promote tumorigenesis. All three RAS isoforms contain a conserved C118, of which the thiol group can be post-translationally modified by processes such as S-nitrosylation, which have been reported to promote RAS activation. To investigate the effect of blocking such modifications on RAS function in vivo, a C118S mutation, which lacks the reactive thiol group, was knocked into the endogenous Kras gene in mice. We now report that KrasC118S/C118S mice were born at the expected ratio with no overt phenotypes. To explore the effect of this mutation on early tumorigenesis, Kras+/+, KrasC118S/+, and KrasC118S/C118S mice were treated with the carcinogen urethane to induce oncogenic mutations in Kras and lung lesions. Interestingly, both KrasC118S/+ and KrasC118S/C118S mice developed fewer and smaller lung tumors than control Kras+/+ mice, suggesting that KrasC118S impairs lung tumorigenesis. Such a reduction in tumorigenesis could be the consequence of the C118S mutation on either the unaffected Kras allele or the Kras allele with the oncogenic mutation. To evaluate the first possibility, we tested and found that mice harboring a Cre-activated LSL-KrasG12D allele formed a similar number and size of lung tumors after activation of this allele when the remaining Kras allele was wild-type or C118S mutant. These data indicate that the reduction in tumor lesions observed in KrasC118S/+ and KrasC118S/C118S mice treated with urethane was unlikely to be the consequence of the C118S mutation on the non-oncogenic Kras allele. To evaluate the second possibility, we tested and found that oncogenic mutations occurred preferentially in the native compared to the C118S mutant Kras allele in KrasC118S/+ mice treated with urethane, suggesting a selection against oncogenic mutations on the KrasC118S allele. These results are consistent with the hypothesis that post-translational modifications such as S-nitrosylation on C118 enhance the oncogenic activity of Kras in vivo, and demonstrate for the first time that blocking the ability of a protein to be modified by processes like S-nitrosylation has a biological consequence in vivo.

Citation Format: Lu Huang, Christopher M. Counter. The effect of introducing a C118S mutation into the endogenous Kras gene on early tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5250. doi:10.1158/1538-7445.AM2014-5250
Abstract 5251: ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation
Authors: Kosari Farhad , Ida Cristiane M. , Aubry Marie Christine , Yang Lin , Kovtun Irina V. , Klein Janet L. Schaefer , Tomaszek Sandra C. , Murphy Stephen J. , Yang Ping , Wigle Dennis , Vasmatzis George
Published: May 2015

ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine cell development, but its value as a biomarker of neuroendocrine differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1expression in lung cancer samples of varied histologic subtype, clinical outcome, and smoking status and compared to expression of traditional neuroendocrine markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers. ASCL1 protein expression by immunohistochemistry (IHC) correlated best with synaptophysin compared to chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1 positive tumors (ASCL1+) compared to ASCL1- tumors (q-value < 10 - 9). High levels of RET expression in ASCL1+ but not in ASCL1- tumors was associated with significantly shorter overall survival in stage 1 (p = 0.007) and in all AD (p = 0.037). RET protein expression by IHC had an association with overall survival in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression define a clinically relevant subgroup of approximately 10% of AD characterized by neuroendocrine differentiation.

Citation Format: Farhad Kosari, Cristiane M. Ida, Marie Christine Aubry, Lin Yang, Irina V. Kovtun, Janet L. Schaefer Klein, Sandra C. Tomaszek, Stephen J. Murphy, Ping Yang, Dennis Wigle, George Vasmatzis. ASCL1 and RET expression define a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2014-5251
Abstract 5253: PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin
Authors: Benitez Jorge A. , Cavenee Webster K. , Furnari Frank F.
Published: May 2015

One of the most mutated and deleted gene in cancer is the tumor suppressor gene pten (phosphatase and tensin homology, deleted on chromosome ten). To further understand how PTEN inactivation contributes to cancer predisposition, we investigated how PTEN regulates gene expression. We found that the histone chaperon protein Daxx (death domain-associated protein) associates with PTEN, in vivo and in vitro, and regulates PTEN stability through acetylation and ubiquitination. We observed that PTEN regulates Histone 3.3 (H3.3 variant) loading in the chromatin by limiting Daxx interactions, and thereby controls expression of several tumor driver genes. Furthermore, when Daxx expression is disrupted in pten-deficient glioma stem cells, we observed a dramatic diminution of pluripotency transcription factors, a decrease in differentiation and sphere-forming capacity, and an increase in radiation sensitivity. In conclusion, this work supports a novel chromatin-associated function for PTEN that regulates H3.3 deposition, and gene expression, through Daxx interaction. This nominates Daxx as a therapeutic target to treat patients with gliomas and other types of cancers in which PTEN is mutated.

Citation Format: Jorge A. Benitez, Webster K. Cavenee, Frank F. Furnari. PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2014-5253
Abstract 5263: Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness
Authors: Yong Hae-Young , Kim Eun-Sook , Koh Minsoo , Son Hwajin , Jeon You Rim , Hwang Jin-Sun , Kim Myeong-Ok , Cha Yujin , Choi Wahn Soo , Noh Dong-Young , Lee Kyung-Min , Kim Ki-Bum , Lee Jae-Seon , Kim Hyung Joon , Kim Hong-Hee , Kim Eun Joo , Park So Yeon , Kim Hyeong-Reh Choi , Moon Aree
Published: May 2015

In an attempt to identify biomarkers and/or therapeutic targets for malignant breast cancer, the present study showed a comparative proteome profiling of invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein DS-20, a crucial regulator of H-Ras activation, as a potential marker for invasive breast cancer. DS-20 (amino acid residues 1-38) interacted with H-Ras (residues 166-189) in lipid rafts and their interaction was important for H-Ras activation. DS-20 knockdown inhibited H-Ras activation and invasion in Hs578T triple-negative breast cancer cells (TNBC) and T24 bladder carcinoma cells in which H-Ras is endogenously activated, suggesting a crucial role of DS-20 in the invasive program which relies on the activation of H-Ras. We further showed that DS-20 was required for epidermal growth factor-induced H-Ras activation, but not that of N-Ras, in MDA-MB-231 TNBC cells. Intravasation of MDA-MB-231 cells treated with shRNA DS-20 in the chick chorioallantoic membrane model was markedly reduced, indicating that DS-20 is required for an invasive capacity in vivo. In a xenograft mice tumor model, DS-20 was essential for in vivo tumor aggressiveness of Hs578T cells, suggesting DS-20 as a potential target for the treatment of breast cancer. Using human breast cancer samples, we provide clinical evidence for the tumorigenic potential of DS-20 and its association with H-Ras. Taken together, our findings provide a new insight into the molecular basis of Ras isoform-specific interplay with plasma membrane leading to cell invasion.

Citation Format: Hae-Young Yong, Eun-Sook Kim, Minsoo Koh, Hwajin Son, You Rim Jeon, Jin-Sun Hwang, Myeong-Ok Kim, Yujin Cha, Wahn Soo Choi, Dong-Young Noh, Kyung-Min Lee, Ki-Bum Kim, Jae-Seon Lee, Hyung Joon Kim, Hong-Hee Kim, Eun Joo Kim, So Yeon Park, Hyeong-Reh Choi Kim, Aree Moon. Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5263. doi:10.1158/1538-7445.AM2014-5263
Abstract 5265: S100P increases Twist expression and triggers FAK/AKT signaling in lung cancer
Authors: Liang Yung-Yu , Kuo Po-Lin , Hsu Ya-Ling
Published: May 2015

Lung cancer accounts for 12% of all new cases of cancers worldwide and is the leading cause of cancer-related death. S100P, a calcium binding protein, has been reported to involve in cancer metastasis. However, the role of S100P is poorly known in lung cancer. This study first reports that S100P enhances the migration and invasion ability of lung cancer cells through the Twist1 upregulation and FAK (focal adhesion kinase)/Src/AKT signaling pathway. Compared to low invasive CL1-0, the expression of S100P is greater in that of high invasive CL1-5 cells. Overexpression of S100P increases the migration and invasion in CL1-0 cells, whereas knockdown of S100P expression decreases the migration and invasion of CL1-5 cells. Enhancement of S100P expression triggers FAK activation, which in turn increases the activation of Src and AKT signaling. Blockade of FAK by specific inhibitor prevents S100P-mediated cell migration and invasion. In the other hand, S100P also increases Twist1, which is responsible for epithelial-mesenchymal transition in lung cancer. More important, elevated expression of S100P protein was also found in human lung cancer specimen. This study demonstrates that S100P may be a novel anticancer target gene for the prevention of non-small cell lung cancer metastasis. In other way, it also can be a biomarker for prognosis of lung cancer metastasis.

Citation Format: Yung-Yu Liang, Po-Lin Kuo, Ya-Ling Hsu. S100P increases Twist expression and triggers FAK/AKT signaling in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5265. doi:10.1158/1538-7445.AM2014-5265
Abstract 5266: Role of nuclear Met receptor in hepatocellular carcinoma
Authors: Yam Judy Wai Ping , Tey Sze Keong
Published: May 2015

Met is a receptor tyrosine kinase encoded by c-Met proto-oncogene which triggers a wide range of normal physiological signaling cascades upon activation by hepatocyte growth factor (HGF). However, aberrant Met expression and activity are commonly found in human cancers. Emerging evidence has shown the presence of nuclear Met (nMet) in some cancerous tissues and cell lines, suggesting that nMet could have unexplored functions in the nucleus. Met is an attractive therapeutic target for human cancers. Indeed, therapeutic antibodies and inhibitors that antagonize dysregulated Met are currently in clinical trials. However, the classic therapeutic strategies against the Met surface receptor will unequivocally exhibit limitations in acting against nMet. Despite the oncogenic role of Met in hepatocellular carcinoma (HCC), the existence and functions of nMet in HCC has never been reported. In this study, we aim to evaluate the clinical relevance and characterize the functions of nMet in HCC. We studied nMet expression in 103 human HCC paired samples by immunohistochemistry. Our data showed that nMet was overexpressed in 89.3% of HCC tissues and its expression was progressively increased along HCC development. Nonetheless, nMet overexpression was significantly associated with venous invasion and poorer overall survival. We found that nMet, which has a lower molecular weight than Met, could only be detected using an antibody against the carboxyl terminus of Met (C28) in tumorous tissues. This finding suggests that nMet only comprises the carboxyl cytoplasmic region of full length Met. Immunofluorescence microscopy showed that juxtamembrane deletion of Met accumulates in the nucleus whereas truncated tyrosine kinase domain accumulates in the cytoplasm, implying a region important for nuclear localization of the cytoplasmic fragment. Cellular fractionation also confirmed the existence of nMet in the nuclear fraction of HCC cells. To explore the functional role of nMet, expression vector was constructed to express the cytoplasmic protein of Met in cells. In vitro functional study showed that overexpression of nMet augments the invasiveness of HCC cells suggesting the unexplored functional effect of nMet exerted within nucleus in promoting HCC invasiveness. Furthermore, we showed that nMet activates NF-kappaB reporter activity which might enhance the pro-metastatic gene expression to promote tumor invasiveness and aggressiveness.

Citation Format: Judy Wai Ping Yam, Sze Keong Tey. Role of nuclear Met receptor in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2014-5266
Abstract 5276: Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway
Authors: Tsai Fu-Ming , Wu Chang-Chieh , Wang Chun-Hua , Tsai Tzung-Chieh , Shyu Rong-Yaun , Jiang Shun-Yuan
Published: May 2015

H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family that includes HREV107, Retinoid-inducible gene 1, and HRASLS. The HREV107 protein family contains an NC domain, with unknown function at the N-terminus, and a hydrophobic membrane-anchoring domain at the C-terminus. Previous study showed that the H-rev107 gene specifically expressed in H-ras resistant fibroblasts. However, the mode of action and the site of inhibition of H-rev107 are still unknown. Our results revealed that H-rev107 reduced the levels of activated RAS (RAS-GTP) or Elk1-mediated transactivation in epidermal growth factor stimulated HtTA cells that expressed H-RAS. Further, we found that H-rev107 reduced H-RAS palmitoylation determined by acyl-biotin exchange assay. Palmitoylation or S-acylation is the post-translational attachment of fatty acids to cysteine residues and continuous cycles of de- and reacylation reactions accounts for the specific subcellular distribution of RAS. H-rev107 was shown as a phospholipase A/Acyltransferase (PLA/AT) that involved in the phospholipid metabolism. We next determined whether H-rev107 affected H-RAS palmitoylation through deacylation/acylation by acyl-protein thioesterases (APT1) or PLA/AT using APT1 inhibitor (palmostatin B) or PLA/AT inhibitor (arachidonyl trifluoromethyl ketone, AACOCF3). Treating cells with AACOCF3 can increase H-rev107-induced acylated H-RAS suppression of HtTA cells. Also, AACOCF3 significantly increased the levels of activated RAS and Elk-mediated transactivation in H-rev107 expressed cells. However, treating cells with palmostatin B enhanced H-rev107-induced acylated H-RAS suppression in H-rev107 expressing cells. In conclusion, H-rev107 can inhibit H-RAS palmitoylation to reduce its GTP-form levels and further suppress the downstream signal pathway. Our study suggested that PLA/AT activity of H-rev107 might play an important role in H-rev107 mediated RAS suppression.

Note: This abstract was not presented at the meeting.

Citation Format: Fu-Ming Tsai, Chang-Chieh Wu, Chun-Hua Wang, Tzung-Chieh Tsai, Rong-Yaun Shyu, Shun-Yuan Jiang. Phospholipase A/acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signal pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5276. doi:10.1158/1538-7445.AM2014-5276
Abstract 5277: Latent transforming growth factor beta-binding protein-3 interacts with midkine
Authors: Weng Chunhua , Dong Haojie , Xu Zhengping
Published: May 2015

Midkine (MK) is a multifunctional heparin-binding growth factor with a molecular weight of 13 kDa, which plays important roles in carcinogenesis. In order to study the action mechanism of MK,we employed a yeast two-hybrid system to screen a human liver cDNA library to determine the putative MK-interacting proteins. cDNA clones containing nucleotide sequences which encode latent transforming growth factor -beta binding protein-3 (LTBP-3) were obtained. The MK/LTBP-3 interaction was confirmed by fluorescence resonance energy transfer analysis. Furthermore, the interaction was analyzed in detail by yeast two-hybrid assay and GST pull down assay. The binding site of MK in LTBP-3 was mapped to the center of the protein, a tandem array of EGF-like repeats, by a fragment deletion analysis in yeast two-hybrid system. The eighth EGF-like domain of LTBP3 plays a critical role in the MK/LTBP-3 interaction in GST pull down assay. On the other hand, four basic amino acids (K-79, R-81, K-86 and K-87) present within the heparin-binding cluster I and II of MK are necessary for the two proteins binding process. These results indicate that MK can bind to LTBP-3, and locate to the eighth EGF-like domain. The heparin-binding clusters of MK are a multifunctional domain, which provide a potential mechanism whereby MK may participate in the TGF-beta system to interfere in carcinogenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Chunhua Weng, Haojie Dong, Zhengping Xu. Latent transforming growth factor beta-binding protein-3 interacts with midkine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5277. doi:10.1158/1538-7445.AM2014-5277
Abstract 5288: PHLPP controls cell polarity by negatively regulating the activity of atypical PKC in colon cancer cells
Authors: Xiong Xiaopeng , li Xin , Gao Tianyan
Published: May 2015

The proper establishment of epithelial polarity allows cells to sense and to respond signals that arise from the microenvironment in a spatiotemporally controlled manner. Increasing evidence has suggested that disruption of polarity promotes the malignant progression of cancer cells. However, the molecular mechanism underlying polarity regulation in the initiation and progression of colon cancer remains largely unknown. PHLPP belongs to a novel family of Ser/Thr protein phosphatases that consists of PHLPP1 and PHLPP2 isoforms. Previously, we have identified conventional PKC isoforms as substrates of PHLPP. Here, we demonstrated that PHLPP controls phosphorylation levels of atypical PKC (aPKC) isoforms, including PKCζ and PKCι, as well. Overexpression of either PHLPP isoform resulted in a decrease in aPKC phosphorylation at both the activation loop (Thr403/410) and the turn motif (Thr555/560) sites in colon cancer cells; conversely, knockdown of PHLPP increased aPKC phosphorylation. In addition, in vitro dephosphorylation experiments revealed that both aPKC isoforms are direct substrates of PHLPP. Since aPKC has been widely implicated in the regulation of cell polarity in intestinal epithelial cells, we determined the function of PHLPP in establishing cell polarity using a 3-D Matrigel culture model. Whereas control Caco2 cells form well polarized cysts in 2 weeks, PHLPP depletion alters the acinar structure by inducing the formation of multiple lumens. Interestingly, knockdown of PKCζ, but not PKCι, results in similar disruption of the polarized lumen structure in Caco2 cells, suggesting that PHLPP may regulate cell polarity through dephosphorylating PKCζ. Taken together, our results identify both aPKC isoforms as novel substrates of PHLPP and downregulation of PHLPP may promote tumorigenesis in colon cancer by disrupting cell polarity.

Citation Format: Xiaopeng Xiong, Xin li, Tianyan Gao. PHLPP controls cell polarity by negatively regulating the activity of atypical PKC in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5288. doi:10.1158/1538-7445.AM2014-5288
Abstract 5292: The functional role of Tensin4 in hepatocellular carcinoma
Authors: Chan Lo Kong
Published: May 2015

The focal adhesion protein Tensin4 (also known as cten: c-terminal tensin like) is structurally distinct from the three other members in the Tensin family and has been reported to play critical roles in cancer cell migration and invasion in breast and colorectal cancers. However, its expression and potential function in other cancers including hepatocellular carcinoma are not well understood. By quantitative real time PCR and western blotting with Tensin4 specific antibodies, Tensin4 was found to be expressed in a panel of HCC cell lines with a number of cells showing high levels of expression. In these cells, treatment with growth factors (e.g. EGF) significantly induced Tensin4 protein expression; while treatment of pharmacological inhibitors against the kinases in the MAPK signaling cascade abolished this induction. These observations suggested that Tensin4 expression was Ras/MAPK signaling dependent. Intriguingly, the growth factor-induced Tensin4 expression could not be observed in HCC cell lines with low basal Tensin4 expression, implying that other regulatory mechanisms do exist. By immunofluorescence microscopy, the focal adhesion localization of Tensin4 was confirmed in HCC cells. Detail examination of a panel of Tensin4 deletion constructs revealed the requirement of the C-terminal SH2 domain for this specific localization. To examine the functional role of Tensin4, SMMC and BEL7402 HCC cells with high basal expression of Tensin4 were subjected to stable Tensin4 knockdown followed by cell proliferation and cell migration assays. Consistently, we observed that both stable Tensin4 knockdown cells proliferated and migrated in slower rates in vitro. However, the molecular mechanisms regarding this reduction remains to be explored. Taken together, our data suggested that Tensin4 may play a pro-oncogenic role in HCC, possibly function as a downstream effector of Ras/MAPK signaling.

The study was funded by a Hong Kong Research Grants Council GRF grant (775110).

Citation Format: Lo Kong Chan. The functional role of Tensin4 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5292. doi:10.1158/1538-7445.AM2014-5292
Abstract 5295: MELK enhances gastric cancer progression via the FAK/paxillin pathway
Authors: Liu Bingya , Du Tao , Qu Ying , Li Jian-fang , Su Liping , Zhu Zheng-gang
Published: May 2015

Elevated MELK expression is a characteristic of multiple tumors and has been correlated with tumorigenesis and tumor development. However, the precise mechanism underlying this correlation is unclear. Here, we report that there are pleiotropic effects associated with enhanced MELK expression in gastric cancer, and this are mediated via the FAK/paxillin pathway. We first show that MELK mRNA and protein expression are both elevated in human gastric cancer, and this is associated with chemoresistance to 5-FU. Knockdown of MELK significantly suppresses cell proliferation, migration, and invasion of gastric cancer both in vitro and in vivo, and decreases the percentages of cells in the G1/G0 phase and increases those in the G2/M and S phases. Moreover, depletion of MELK decreases the amount of actin stress fibers and reduces RhoA activity. Finally, knockdown of MELK decreases the phosphorylation of molecular components of the FAK/Paxillin pathway and prevents gastrin-mediated FAK/paxillin phosphorylation. By contrast, MELK overexpression produces the opposite results, although the differences are moderate. Together, our work suggests that MELK plays an important role in the occurrence and development of gastric cancer, implicates the FAK/Paxillin pathway as the means by which it promotes these effects, and identifies MELK as a potential target for treatment against gastric cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Bingya Liu, Tao Du, Ying Qu, Jian-fang Li, Liping Su, Zheng-gang Zhu. MELK enhances gastric cancer progression via the FAK/paxillin pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5295. doi:10.1158/1538-7445.AM2014-5295
Abstract 5298: Regulation of urothelial bladder cancer by nicotine and stress neurotransmitters
Authors: John Arokya M. Papu , Banerjee Jheelam , Schuller Hildegard M.
Published: May 2015

Urothelial bladder cancer (UBC) is the 7th most common cancer in men and the 17th most common cancer in women. Smoking is an established risk factor for UBC. However, the mechanisms how smoking induces bladder cancer are poorly understood. Using two established human urothelial bladder cancer cell lines, our data show that nicotine significantly increased the proliferation of both cell lines. Both cell lines produced the stress neurotransmitters norepinephrine and epinephrine and nicotine further enhanced this activity. The broad-spectrum beta-blocker propranolol strongly inhibited base level and nicotine-induced proliferation in both cell lines, identifying beta-adrenergic receptors as mediators. Proliferation in response to exogenous addition of epinephrine, norepinephrine or the selective β-adrenergic agonist isoproterenol and complete blockage of these responses by propranolol support this interpretation. Treatment with the inhibitory neurotransmitter γ-aminobutyric acid (GABA), that inhibits beta-adrenergic receptor-mediated proliferation of other cancer types via inhibition of cAMP formation, was less effective. These findings suggest that urothelial bladder cancer cells are stimulated in their growth via beta-adrenergic receptor signaling independent of cAMP by nicotine or psychological stress. Additional investigations are underway to further dissect the signal transduction pathways involved.

Supported by the State of Tennessee Center of Excellence in Livestock Diseases and Human Health.

Citation Format: Arokya M. Papu John, Jheelam Banerjee, Hildegard M. Schuller. Regulation of urothelial bladder cancer by nicotine and stress neurotransmitters. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2014-5298