bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2381: The effect of ERK5 inhibition in clear cell renal cell carcinoma
Authors: Kanno Hidenori , Naito Sei , Ichiyanagi Osamu , Tsuchiya Norihiko
Published: July 2017

Introduction Clear-cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer and is characterized by von Hippel-Lindau tumor suppressor gene (VHL) mutation. VHL protein acts in a ubiquitin ligase complex, mediating the proteasomal degradation of ubiquitinized proteins, including hypoxia-inducible factor (HIF). Therefore, HIF accumulates in VHL-defective cell lines. A recent study revealed that extracellular signal-regulated kinase 5 (ERK5) is degraded through the ubiquitin-proteasome system, in a process mediated by VHL and HIF. Our objectives were to examine the effects of ERK5 in ccRCC and to investigate ERK5 as a potential therapeutic target. Methods ERK5 expression was investigated in surgically resected ccRCC specimens using immunohistochemistry. To confirm that ERK5 is degraded by the VHL pathway, ERK5 levels were examined by western blotting in Caki-1 VHL wildtype renal cell carcinoma (RCC) cells and in A498 and A704 VHL-mutant RCC cells with or without the MG132 proteasome inhibitor. Furthermore, VHL-mutant cell lines were examined, with or without siRNA and XMD8-92-mediated ERK5 inhibition, to investigate the role of ERK5 in RCC cells using flow cytometry, MTS assays, and western blotting . Results In surgical specimens, RCC cells showed higher levels of ERK5 expression than did normal cells. In addition, MG132 enhanced ERK5 expression in wildtype cell lines, but not in VHL-mutant cell lines. Therefore, ERK5 degradation involves the VHL pathway. Furthermore, ERK5 inhibition resulted in increase of the sub-G1 population as observed by flow cytometry and cleaved RARP expression as seen by western blotting, indicating an increase in apoptotic cells. ERK5 inhibition also suppressed cell viability and downregulated p16 and Bcl-2 expression. Conclusion Our results show that ERK5 inhibition contributes to the downregulation of anti-apoptotic and cell cycle proteins and suggest that ERK5 is a promising therapeutic target for ccRCC.

Citation Format: Hidenori Kanno, Sei Naito, Osamu Ichiyanagi, Norihiko Tsuchiya. The effect of ERK5 inhibition in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2381. doi:10.1158/1538-7445.AM2017-2381
Abstract 2382: RACK1 regulates PKA activity to drive tumorigenesis in colon cancer
Authors: Hayes Sheri L. , Dowling Catríona M. , Coffey John C. , Kiely Patrick A.
Published: July 2017

RACK1 (Receptor for Activated C Kinases) is a scaffolding protein with 7 WD repeats that interacts with the Insulin-like Growth Factor I receptor (IGF-IR), integrins, and other signalling proteins. RACK1 functions as a scaffolding protein and regulator of many key biological processes and it is highly expressed in most tissue. We recently reported that RACK1 is essential for IGF-I-mediated regulation of PP2A activity and AGAP2 activity at focal adhesions. Downstream of IGF-1R signaling in cancer cells, the scaffolding properties of RACK1 are altered providing distinct migratory advantages to the cell and suggesting that RACK1 is an important regulator of IGF-I signalling in cancer progression. It is important to further characterise the role of RACK1 in cancer and to delineate the molecular mechanisms by which it regulates key signalling pathways. We have identified Protein Kinase A (PKA) as RACK1 interacting protein. PKA is a complex, multicomponent enzyme which is a fundamental protein that functions in cell survival, proliferation, and cytoskeletal remodelling among others. Our hypothesis is that RACK1 regulates the PKA axis in cancer cells leading to Akt and MAP Kinase activation downstream of IGF-1R signalling. We have demonstrated that RACK1 is required to mediate PKA activity in colon cancer cells. Although we show that PKA expression is not altered in colon cancer tissue, we believe that PKA activity is altered significantly in the disease state. Using PKA inhibitors, we have shown that PKA regulates IGF-I-mediated MAP Kinase activation in colon cancer cell lines. In conclusion, this research project will help elucidate the role of PKA in colon cancer. Inhibiting or promoting specific protein interactions with RACK1 will provide very novel therapeutic opportunities and anti-cancer drug targets.

Citation Format: Sheri L. Hayes, Catríona M. Dowling, John C. Coffey, Patrick A. Kiely. RACK1 regulates PKA activity to drive tumorigenesis in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2382. doi:10.1158/1538-7445.AM2017-2382
Abstract 2384: Aberrant expression of PDZ binding kinase modulates human pancreatic cancer cell invasion
Authors: Hinzman Charles , Trevino Jose , Banerjee Partha P.
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths and projected to be the second leading cause of cancer deaths by 2030. Incidence to mortality ratio for PDAC is almost one suggesting its aggressive phenotype. The poor survival rate of PDAC patients is due to metastasis to distant organs, and therefore, recent focus has been aimed towards the identification of molecular targets that drives metastatic disease. We are investigating an under-studied kinase, PDZ-domain binding kinase (PBK) as a target for pancreatic cancer (PanCa) therapy. PBK is a dual-specificity serine/threonine kinase involved in mitosis and invasive behavior of cancer cells. It is expressed physiologically in testis, but aberrant expression results in an aggressive phenotype in a variety of cancers. Our results demonstrate that PBK expression varies across a panel of human PanCa cell lines, and directly correlates with their invasive ability. Similarly, a panel of patient-derived primary PanCa shows higher PBK levels associated with aggressive behavior and invasion. To assure specificity of PBK to this invasive phenotype, we measured the effect of PBK overexpression or knockdown on the PanCa lines’ invasive ability. Overexpression of PBK in low PBK expressing cells increased PanCa invasion ~10-fold, while PBK knockdown reduced invasive ability ~5-fold. Immunohistochemical analyses of resected human PanCa specimens revealed that PBK is present in the majority of human PanCa tissue samples tested but not in the adjacent normal pancreatic tissue. Interestingly, aberrant PBK localization was found in even early stages of PanCa development and was sustained throughout its progression. In search of molecular effectors of invasion regulated by the PBK, we examined various genes/proteins that are involved in cancer cell invasion and metastasis. RelB a well-known metastatic inducer in various cancers was found to be a major payer in PanCa and regulated by PBK. These data indicate a novel molecular pathway of PBK where its presence plays a role in invasion of human PanCa cells. Therefore, the role of PBK in promoting cancer cell invasion, combined with its general lack of expression in normal cells, nominates PBK as a potentially important therapeutic target for pancreatic cancer.

Citation Format: Charles Hinzman, Jose Trevino, Partha P. Banerjee. Aberrant expression of PDZ binding kinase modulates human pancreatic cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2384. doi:10.1158/1538-7445.AM2017-2384
Abstract 2386: HDAC6 regulates ERK1’s kinase activity via deacetylation
Authors: Wu Jheng-Yu , Zhang Xiaohong
Published: July 2017

Extracellular signal-regulated kinases 1/2 (ERK1/2) are important kinases regulating cell proliferation and cell migration, and have been established as therapeutic targets for cancer treatment. Previously, we found that ERK1 phosphorylates histone deacetylase 6 (HDAC6), a class IIb HDAC, to regulate its enzymatic activity. HDAC6 plays an important role in several types of cancers. HDAC6 governs cancer cells’ migration by cooperating with cytoskeleton proteins and their associated proteins, and HDAC6 is arising as a determinant for cisplatin-resistance in non-small cell lung cancer (NSCLC) and ovarian cancer. Here, we have shown that HDAC6 manipulates ERK1’s kinase activity via deacetylation. We demonstrated that both ERK1 and ERK2 interact with HDAC6 physically via GST pull-down assays. To examine whether ERK1/2 are acetylated, we transfected GST-ERK1 or GST-ERK2 into HEK293T cells, and treated the cells with a pan-HDAC inhibitor, Trichostatin A. We showed that the level of acetylated GST-ERK1/2 increased in a dose- and time-dependent manner. Furthermore, when GST-ERK1 or GST-ERK2-expressed cells were treated with an HDAC6-specific inhibitor, ACY-1215, the level of acetylated GST-ERK1/2 increased. These results suggest that HDAC6 is a deacetylase for ERK1/2. In addition, we determined that acetyltransferases CBP and p300 acetylate ERK1/2. We found a novel acetylation site located in ERK1 N-terminus by mass-spectrometry analysis. We then mutated this lysine (K) site with either Glutamic acid (Q) or Arginine (R) to mimic the acetylation or deacetylation status, and performed kinase assays using ELK1 as a substrate. The acetylation mimicking mutant exhibits a decreased kinase activity toward ELK1, while the deacetylation mimicking mutant exhibits a similar level of kinase activity as the wild-type ERK1 does, suggesting that acetylation/deacetylation alters ERK1 enzymatic activities. Therefore, HDAC6 may regulate ERK1’s kinase activity via deacetylation of this site. Furthermore, we have shown that HDAC6 is overexpressed in lung cancer tissue samples and plays an important role in chemo-sensitivity in lung cancer. We predict that inhibition of the HDAC6-ERK1 pathway may provide clinical benefit for lung cancer patients.

Citation Format: Jheng-Yu Wu, Xiaohong Zhang. HDAC6 regulates ERK1’s kinase activity via deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2017-2386
Abstract 2389: LINE-1 tumor hypomethylation is associated with shorter recurrence-free survival in localized clear-cell renal cell carcinoma
Authors: Malouf Gabriel G. , Mouawad Roger , Allanic Frederick , Compérat Eva , Roupret Morgan , Khayat David , Spano Jean-Philippe
Published: July 2017

Background: Cancer cells are characterized by alterations of DNA methylation patterns involving global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation is thought to play a role in genetic instability and tumor aggressiveness. Long interspersed nucleotide element-1 (L1/LINE-1) repetitive elements represent 40% of the genome and their methylation is a good indicator of the global DNA methylation level. Although LINE-1 methylation has been previously shown to be associated with prognosis of patients with cancer, the value of LINE-1 methylation in predicting recurrence of patients with localized clear-cell renal cell carcinomas remains unknown.

Material and Methods: We quantified the LINE-1 methylation using bisulfite pyrosequencing in cohort of 200 patients with resected clear-cell renal cell carcinomas (AJCC stage I-III). LINE-1 methylation of adjacent normal kidney was also assessed in 128 cases. Threshold of tumor LINE-1 hypomethylation was defined as LINE-1 methylation in normal samples minus three standard deviation.

Results: Median methylation of tumor samples was 59.01% versus 61.61% for normal adjacent kidney samples (p<10-5). LINE-1 methylation level of normal adjacent kidney was associated with tumor size (Spearman R=0.21, P=0.02) but not with age (p=0.24) or Leibovitch score (p=0.05). Tumor LINE-1 hypomethylation was observed in 20 samples (n=10%). Strikingly, those were enriched for tumors occurring in female (60% versus 27.7%) (p=0.005); of note, no association was found between LINE-1 tumor hypomethylation and tumor grade (p=0.31), stage (p=1), age (p=0.24) and Leibovitch score (p=0.82). In multivariate analysis, only Leibovitch score and LINE-1 hypomethylation were independently associated with poor recurrence-free survival.

Conclusion: LINE-1 hypomethylation is associated with shorter recurrence-free survival in patients with resected clear-cell renal cell carcinomas, suggesting the possibility of using it as predictive biomarker of recurrence.

Citation Format: Gabriel G. Malouf, Roger Mouawad, Frederick Allanic, Eva Compérat, Morgan Roupret, David Khayat, Jean-Philippe Spano. LINE-1 tumor hypomethylation is associated with shorter recurrence-free survival in localized clear-cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2017-2389
Abstract 2390: Clinical validation of an epigenetic field of susceptibility to detect significant prostate cancer from 2 non-tumor biopsies
Authors: Yang Bing , McCormick Johnathon , Schultz Adam , Leverson Glen , Sonn Geoffrey , Magi-Galluzi Cristina , Klein Eric A. , Fumo Michael , Jarrard David F.
Published: July 2017

Background: An epigenetic field of cancer susceptibility exists for prostate cancer that gives rise to multifocal disease in the peripheral zone. In a previous studies (Neoplasia 2013, J Urol 2014) we identified using methylation arrays over 50 altered regions in normal prostate tissue of men with prostate cancer. In the current multicenter study, a validation was performed to determine the predictive strength of this approach.

Methods: We evaluated 2 archived, cancer negative prostate biopsy core tissue from 129 subjects from 4 urological centers. All negative cases (controls) underwent 2 or more repeat negative biopsies within 24 mo with a central review of all histopathology. Cancer cases were negative biopsies selected from patients who ultimately went prostate removal to confirm a final grade >Gleason Score 7. Biopsies were analyzed using pyrosequencing for DNA methylation changes at multiple CpGs surrounding the genes EVX1, CAV1, PLA2G16, FGF1, SPAG4 and NCR2. Analyses used multiplex logistic regression modeling with backward elimination.

Results: Patients diagnosed with GS>7 cancer (77) and the control group (52) were similarly matched except for PSA (7 vs 5.8; p<0.03) and prostate size (46 vs 36; p<0.02). We observed robust methylation differences for all genes (p<0.05) (Table). A multiplex regression model for biomarker performance incorporating a gene combination of EVX1, NCR2, PLAG2 and SPAG4 discriminated non-tumor from tumor-associated tissues (AUC 0.744, p<0.0001). A multiplex model incorporating 3 genes (EVX1, PLA2G16, NCR2) and clinical information (PSA, age) identified patients with GS>7 prostate cancers (AUC 0.808, p=0.001).

Conclusions: A widespread epigenetic field defect can be used to detect GS>7 prostate cancers in patients with histologically negative biopsies. This assay is sensitive in requiring only 2 biopsies and detects alterations in nontumor cells at distance from the cancer. This has the potential to decrease the need for repeat prostate biopsies, a procedure associated with cost and complications.

Support: UW Ignitor; Gen X foundation

Citation Format: Bing Yang, Johnathon McCormick, Adam Schultz, Glen Leverson, Geoffrey Sonn, Cristina Magi-Galluzi, Eric A. Klein, Michael Fumo, David F. Jarrard. Clinical validation of an epigenetic field of susceptibility to detect significant prostate cancer from 2 non-tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2390. doi:10.1158/1538-7445.AM2017-2390
Abstract 2392: A multiplex histone H3 PTM assay for epigenetic biomarker discovery in tissue biopsy and archived clinical samples
Authors: Jelinek Mary Anne , Ritland Melissa , Westergren AJ
Published: July 2017

Dysregulation of epigenetic mechanisms is known to play an important role in the development and progression of cancer. One such mechanism is manifested as altered levels of histone modifications involved in regulating gene transcription. N-terminal histone tails can have a variety of modifications, such as phosphorylation, methylation and acetylation at specific amino acid residues which are conserved throughout eukaryotes and function by altering chromatin structure and creating binding sites for chromatin readers, writers or erasers.

Numerous studies have reported aberrant global levels of several histone H3 and H4 post-translational modifications (PTMs) in a wide range of solid tumor types. These changes have been shown to be predictive of clinical outcome, raising the possibility that histone modifications have potential as epigenetic biomarkers. Clinical samples have immense potential for biomarker identification since they are often accompanied with valuable information pertaining to patient history, treatment courses and disease outcome. The preferred method for clinical sample preservation is formalin-fixation followed by paraffin embedding (FFPE). While extraction and downstream analysis of DNA and RNA from FFPE samples is now routine, proteomic studies of FFPE samples is hampered by extensive protein cross-linking generated by formalin fixation. Analysis of histone PTMs in patient archival samples is limited to low-throughput immunohistochemical staining, and is not an ideal approach for mining large sample cohorts for biomarker identification. Western blot or ELISA methods, which have large sample requirements, are simply not an option for FFPE samples.

We have developed a multiplex bead-based ELISA assay which enables simultaneous interrogation of thirteen histone H3 PTMs. The assay is performed in 96-well plates and is ideally suited for profiling histone modification levels in limiting or small samples. We will present the application of this technology in a variety of sample types including frozen tissue, formaldehyde-cross linked cells and tissues, and in small molecule inhibitor screens with as few as 2,000 cells per well. The ability to simultaneous detect up to thirteen histone H3 PTMs provides a unique feature that enables determination of for on-target and off-target effects within the same sample.

Citation Format: Mary Anne Jelinek, Melissa Ritland, AJ Westergren. A multiplex histone H3 PTM assay for epigenetic biomarker discovery in tissue biopsy and archived clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2392. doi:10.1158/1538-7445.AM2017-2392
Abstract 2393: Decreased expression of 5-hydroxymethylcytosine is associated with grade of malignancy in lung cancer
Authors: Tominaga Yoshiaki , Ito Ken-ichi , Nakayama Jun
Published: July 2017

Purpose: The ten-eleven translocation (TET) proteins are critical regulators in epigenetic modification. It is well known that TET proteins catalyze the oxidation of 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), and 5-hmC levels reflect diverse functions of TET proteins. It has been reported that the expression of 5-hmC in cancer tissues is decreased compared with that in normal tissues in several organs. However, 5-hmC expression in lung cancer has not been fully analyzed. To examine whether or not 5-hmC was associated with phenotypes or progression of lung cancer, we evaluated 5-hmC expression in a variety of histological types of lung cancers.

Materials and methods: The expression of 5-hmC was analyzed by immunohistochemistry in 109 paired specimens of lung cancer and adjacent normal tissues obtained from the patients underwent operation at Shinshu University Hospital. Eighty-nine adenocarcinomas, 17 squamous cell carcinomas, two large cell carcinomas, and one small cell carcinoma were analyzed in the study.

Results: 5-hmC expression in female was significantly lower than that in male, and was lower in smokers than non-smokers. 5-hmC expression was positive in 34.1±22.0% of lung cancer tissues and 69.4±18.8% of normal tissues. The expression of 5-hmC was significantly decreased in the lung cancer tissues compared with that in the adjacent normal tissues (p<0.001). With regard to the histological types, the positive ratios of 5-hmC expression were 36.0±21.7% in adenocarcinoma, 24.0±20.3% in squamous cell carcinoma, 8.0±7.3% in large cell carcinoma, and 13.0% in small cell carcinoma. Thus, the frequency of positive expression of 5-hmC was decreased in accordance with the increase of grade of malignancy of lung cancer, and a negative correlation between 5-hmC expression and the grade of malignancy of lung cancer was observed. On the other hand, no difference of the expression of 5-mC was observed between lung cancer tissues and normal tissues.

Conclusions: Our results indicate a possibility that 5-hmC may be related to carcinogenesis and progression or differentiation of lung cancer.

Citation Format: Yoshiaki Tominaga, Ken-ichi Ito, Jun Nakayama. Decreased expression of 5-hydroxymethylcytosine is associated with grade of malignancy in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2017-2393
Abstract 2396: Methylation of subtelomeric repeat D4Z4 in peripheral blood leukocytes is associated with biochemical recurrence in localized prostate cancer patients
Authors: Han Yuyan , Kim Jeri , Wu Xifeng , Gu Jian
Published: July 2017

Background: Global DNA methylation may affect chromosome structure and genomic stability and is involved in carcinogenesis. In this study, we aimed to investigate whether methylation of pericentromeric repeat (NBL2) and subtelomeric repeat (D4Z4) in peripheral blood leukocytes was associated with the aggressiveness of prostate cancer (PCa).

Methods: We measured the methylation status of different CpG sites of NBL2 and D4Z4 in 900 PCa patients via pyrosequencing after bisulfite treatment. We then used multivariate logistic regression to estimate the odds ratio (ORs) and 95% confidence intervals (CIs) for analyzing the association of NBL2 and D4Z4 methylation with the aggressiveness of PCa at diagnosis. We then analyzed the association of CpG sites with the risk of biochemical recurrence (BCR) in patients receiving radical prostatectomy or radiotherapy using a multivariate Cox proportional hazards model adjusting for age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. In addition, we used the Kaplan-Meier survival function and log-rank tests to assess BCR-free survival associated with these markers.

Results: There was no significant differences in the methylation level of NBL2 and D4Z4 between clinically defined aggressive and non-aggressive PCa at diagnosis. However, the methylation of subtelomeric region D4Z4 was associated with BCR, while the methylation of NBL2 was not associated with BCR. In tertile analysis, we found that patients with higher methylation of D4Z4 exhibited an increased risk of BCR in localized patients receiving definitive therapy. Because of the relatively low rate of BCR among patients, we combined the highest and second tertiles to increase statistical power. Patients in the second and third (highest) tertiles had an increased risk of BCR (HR=1.74, 95% CI, 1.14-2.65; p= 0.0096) compared to patients in the lowest tertile after adjustment of age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. We assayed 4 CpG sites in this region. In detailed analysis of each CpG site, the association was mostly attributable to the methylation of the 2nd methylation site of D4Z4 (HR=1.80, 95% CI, 1.19 - 2.72).

Conclusion: These data suggest that methylation in the subtelomeric region D4Z4 may be able to predict worse prognosis of localized PCa patients.

Citation Format: Yuyan Han, Jeri Kim, Xifeng Wu, Jian Gu. Methylation of subtelomeric repeat D4Z4 in peripheral blood leukocytes is associated with biochemical recurrence in localized prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2017-2396
Abstract 2399: Metabolic and epigenetic targets in multiple myeloma therapy
Authors: Maneix Laure , Shih Fu-Yuan , Iakova Polina , Hsu Joanne I. , Catic Andre
Published: July 2017

The dynamic interaction of transcription factors (TFs) with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of TFs at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are being removed. We previously devised a method to detect genomic locations that are associated with nuclear protein turnover mediated by the ubiquitin-proteasome system (UPS).

Multiple myeloma, the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Velcade, for instance, has become first-line treatment in myeloma. Yet, our knowledge of how myeloma cells are killed by this drug is incomplete. Our research is focused on defining how proteolysis regulates transcriptional dynamics in this disease and how this impacts Velcade sensitivity.

Following proteasome inhibition, we are utilizing next generation sequencing to identify sites of nuclear protein turnover and evaluate epigenetic changes. Our aim is to identify TFs and epigenetic marks that may qualify as more specific targets for treatment compared to blunt proteasome inhibition. Our findings reveal that certain TFs, including metabolic and cell growth regulators, are particularly sensitive to Velcade. Among these transcriptional regulators, the co-repressor NCoR1 plays a key role in controlling cell metabolism by adjusting mitochondrial gene expression. We are currently validating the mechanism of NCoR1 degradation, with a particular emphasis on the new role of its E3-ubiquitin ligase Seven in absentia 2 (Siah2) in mitochondrial regulation, to better understand how this degradation pathway impacts myeloma proliferation and metabolism.

This research project will contribute to our understanding of epigenetic and transcriptional dynamics in multiple myeloma. With the focus on gene programs that are continuously adapting or changing, we seek to unlock new attractive targets for molecular therapy.

Citation Format: Laure Maneix, Fu-Yuan Shih, Polina Iakova, Joanne I. Hsu, Andre Catic. Metabolic and epigenetic targets in multiple myeloma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2399. doi:10.1158/1538-7445.AM2017-2399
Abstract 2407: Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation
Authors: Dreval Kostiantyn , Conti Aline de , Orisakwe Orish Ebere , Beland Frederick A. , Pogribny Igor P.
Published: July 2017

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers and the incidence is steadily increasing worldwide. The molecular mechanisms leading to the development of HCC consist of genetic and epigenetic aberrations, including changes in global and gene-specific DNA methylation and altered expression of several classes of non-coding RNAs. While changes in DNA methylation and microRNA expression during the development of HCC are well-studied, the role of aberrant post-translational histone modifications remains unexplored. Using a mouse Stelic Animal Model (STAM) of NASH-associated liver carcinogenesis, we have investigated alterations in hepatic histone modifications at steatotic (6 weeks), fibrotic (12 weeks), and full-fledged HCC (20 weeks) stages of the HCC development. NASH-related liver carcinogenesis was characterized by a progressive decrease in the levels of histone H4 lysine 20 trimethylation (H4K20me3) and histone H4 lysine 16 acetylation (H4K16ac), with the greatest decrease occurring in full-fledged tumors. Mechanistically, the deacetylation of H4K16ac was associated with nuclear protein 1 (Nupr1)-mediated inhibition of histone lysine acetyltransferase KAT8. In addition to a global loss of hepatic H4K16ac, there was a significant decrease of gene-specific H4K16ac, as indicated by a reduced level of H4K16ac in 16 out of 25 critical cancer-related genes. More importantly, the changes in the extent of gene-specific H4K16 deacetylation were positively correlated with the level of gene transcription (r = 0.965, p < 0.01). These results indicate that a reduction of global and gene-specific of H4K16ac is a key pathophysiological mechanism contributing to the development of NAFLD-derived HCC.

Citation Format: Kostiantyn Dreval, Aline de Conti, Orish Ebere Orisakwe, Frederick A. Beland, Igor P. Pogribny. Inhibition of gene expression during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis is mediated by histone H4 lysine 16 deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2407. doi:10.1158/1538-7445.AM2017-2407
Abstract 242: The anti-tumor effect and the impact on gut microbiota of gypenosides Rd and Rb3 in ApcMin/+ mice
Authors: Hsiao W.L. Wendy , Huang Guoxin , Khan Imran , Chen Lei , Li Xiaoang
Published: July 2017

Gut commensal microbiota (GM) is closely associated with health and diseases, including cancer. In our previous studies, we have demonstrated that Gynostemma pentaphyllum saponins (GpS) exert anticancer effects in both athymic and ApcMin/+ mice. Our recent study also showed that GpS exhibited prebiotic effect by up regulating the beneficial bacteria and improving the epithelial microenvironment of the gut. Gypenoside IV (Rb3) and gypenoside VII (Rd) are the two constituents of GpS. The Rd exhibits stronger anticancer effect than Rb3 in an in vitro cell model system established in our lab. Our aim is to confirm the antitumor effect of these two compounds, as well as their impact on GM in the ApcMin/+ mouse model. In the study, single daily dose of Rb3 or Rd at 20mg/kg, and solvent control were orally fed to the mice for 8 weeks, started at 6 weeks of age before the appearance of spontaneous intestinal polyps. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing technique. We found that Rd was more effective than Rb3 in reducing polyp formation, and improving the intestinal lining by up-regulating anti-inflammatory cytokines IL-4 and IL-10. Rd, not Rb3, might facilitate intestinal tissue repair by enhancing M2 marker while suppressing M1 markers expression. Moreover, Rd treatment greatly enhanced the ratio of Bacteroidetes vs Firmicutes, while profoundly reduced the abundance of cancer-cachexia-associated bacteria, such as Parabacteroides goldsteinii (22.25%), Dysgonomonas wimpennyi (48.54%) and Blautia coccoides (36.77%). These findings suggested that the level of antitumor effect of the gypenoside might be correlated with its ability to modulate the gut microbiota from dysbiosis state to symbiosis.

This study was supported by Macau Science and Technology Development Fund 015/2014/A1.

Citation Format: W.L. Wendy Hsiao, Guoxin Huang, Imran Khan, Lei Chen, Xiaoang Li. The anti-tumor effect and the impact on gut microbiota of gypenosides Rd and Rb3 in ApcMin/+ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 242. doi:10.1158/1538-7445.AM2017-242
Abstract 2434: LEF1 negatively regulates differentiation of HCC by activation of Notch signaling pathway
Authors: FANG Shuo , LI Lei , LIU MIng , Tsang Janice , GUAN Xinyuan
Published: July 2017

Lymphoid enhancer binding factor-1 (LEF1), an essential regulatory protein, plays a crucial role on self-renewal and differentiation properties of several cancers, contributing to tumor malignancy degree and prognosis. However, its molecular mechanisms to promote tumor differentiation still keep unknown, especially in hepatocellular carcinoma (HCC). In this study, by analyzing the transcriptome datasets and our qPCR result of clinical HCC samples, we found that LEF is frequently overexpressed in HCCs and significantly associated with HCC differentiation (p=0.015). In addition, functional study showed that LEF1 could enhance the cell proliferation, matrigel invasion, transwell migration, as well as self-renewal abilities both in vivo and in vitro experiments. We also demonstrated LEF1 is closely related to Notch intracellular domain (NICD), and revealed several putative sites as the targets for LEF1 activating Notch signaling pathway. LEF1 could induce the Notch signaling via transcriptionally up-regulate Notch expression through direct DNA binding. The expressions of several downstream members in Notch signaling pathway including Notch1 and Notch2 were subsequently increased. Moreover, LEF1 could down-regulate various differentiation markers and over-express tumor stemness related genes. Together, LEF1 is a novel oncogene that show a crucial role in the tumorigenesis and progression of HCC via activating Notch signaling pathway and inhibiting the differentiation and stemness of HCC, resulting in poor pathologic differentiation and leads to high degree of malignant. All the findings suggest a novel treatment possibility and a new target for liver stem cell eradication. LEF1 could be investigated for clinical early diagnosis and prognosis prediction of HCC patients and improving the development of new therapy against HCC.

Note: This abstract was not presented at the meeting.

Citation Format: Shuo FANG, Lei LI, MIng LIU, Janice Tsang, Xinyuan GUAN. LEF1 negatively regulates differentiation of HCC by activation of Notch signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2434. doi:10.1158/1538-7445.AM2017-2434
Abstract 2439: Establishment of six human colorectal cancer cell lines: identification of genes that have metastatic potential
Authors: Kim Soon-Chan , Hong Chang-Won , Jang Sang-Geun , Jeong Seung-Yong , Park Jae-Gahb , Ku Ja-Lok
Published: July 2017

A number of genes have been studied in accordance with their metastatic potential. Nevertheless, widely used metastatic colorectal cancer cell lines such as SW-620 or IS3 only show the influence of metastatic genes within limited mutational landscape. Here, three pairs of primary CRC (SNU-2335A, SNU-2335D, SNU-2404A, SNU-2404B, SNU-2414A, and SNU-2414B) and corresponding metastasis cell lines were established and analyzed by whole exome sequencing and microarray.

Three pairs of primary CRC (SNU-2335A, SNU-2404A, and SNU-2414A) and corresponding matched metastasis cell lines (SNU-2335D, SNU-2404B, and SNU-2414B) were analyzed for gene expression by microarray and whole exome sequencing. Selected target genes from microarray were analyzed again by real-time PCR in order to confirm the mRNA expression level in the paired CRC cell lines.

The genes selected for examination were calponin 3, acidic (CNN3); sorbin and SH3 domain containing 1(SORBS1); epithelial stromal interaction 1 (EPSTI1); bone marrow stromal cell antigen 2(BST2); kelch-like 5 (KLHL5); trypsinogen C (TRY6); and synaptotagmin-like 5 (SYTL5). We calculated the intersection of all three metastatic CRC cell lines. Five genes (BST2, SORBS1, CNN3, EPSTI1, and KLHL5) were up-regulated in metastatic cell lines compared to primary cell lines. Two genes (TRY6 and SYTL5) were down-regulated in metastatic cell lines compared to primary cell lines. Then, we ranked 15 genes that were up-regulated and 15 genes that were down-regulated in omental metastasis regardless of the 2-folds threshold. Of the 30 genes that were differently expressed, we selected three genes (CNN3, SORBS1, and TRY6) that showed the highest fold changes and are found in 2-fold change analysis.

We identified 7 genes that were differently expressed in metastatic CRC cell lines compared to primary CRC cell lines. These genes may serve as diagnostic markers and therapeutic targets for patients with metastatic CRC.

Note: This abstract was not presented at the meeting.

Citation Format: Soon-Chan Kim, Chang-Won Hong, Sang-Geun Jang, Seung-Yong Jeong, Jae-Gahb Park, Ja-Lok Ku. Establishment of six human colorectal cancer cell lines: identification of genes that have metastatic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2017-2439
Abstract 244: Eicosapentaenoic acid reduces effects of some adipocyte derived factors on breast cancer cell inflammation and glucose metabolism
Authors: Aljawadi Arwa , Alhaj Sara , Dharamawardhane Suranganie , Scoggin Shane , Gollahon Lauren , Gunaratne Preethi , Moustaid-Moussa Naima
Published: July 2017

Breast cancer is still the leading cause of death in women among all cancer types. Obesity, a chronic low grade inflammatory disease, is a key contributor to the progression of breast cancer especially in women, post-menopause. Given the current understanding of cancer-related inflammation and associated microvesicles in restructuring the microenvironment, we hypothesized that adipocyte-derived cytokines and exosomes negatively impact breast cancer progression. By contrast, eicosapentaenoic acid (EPA), a dietary omega 3 polyunsaturated fatty acid and a well-established anti-inflammatory compound will reduce adipocyte-secreted pro-inflammatory factors, thereby reducing breast cancer progression. To test these hypotheses, we investigated the effects of conditioned media or exosomes from 3T3-L1 adipocytes or human mesenchymal stem cells (HMSC), treated +/- 100uM EPA on MCF7 and MDA-MB231 breast cancer cells for 24 – 72 h. Following treatment, changes in breast cancer cell gene expression were measured using RT-qPCR and glycolytic rate was measured using XFe24 Seahorse extracellular flux analyze. We observed that conditioned medium from HMSC significantly increased the mRNA expression levels of oncogenic genes such as signal transducer and activator of transcription 3 (STAT3), baculoviral IAP repeat-containing protein3 (BIRC3) known as cIAP2, and the lipogenic fatty acids synthase (FASN). In contrast, conditioned media from EPA-treated human adipocytes reduced the expression levels of these genes. Similarly, exosomes isolated from EPA-treated adipocytes showed a significant reduction in mRNA expression levels of STAT3 and cIAP2 in both cancer cell lines. Furthermore, glycolysis was significantly reduced in MCF7 but not MDA-MB231 cells incubated with 3T3-L1 adipocyte-conditioned medium pretreated with EPA for 24 hrs. Taken together, our data suggest that adipocytes play a significant role in promoting breast cancer progression by providing a microenvironment that increases survival and inflammation. EPA is a promising anti-inflammatory nutrient that may help reduce breast cancer cell inflammation and survival, possibly by modulating adipocyte-derived cytokines and exosomes in obesity and thus warrants further investigations.

Citation Format: Arwa Aljawadi, Sara Alhaj, Suranganie Dharamawardhane, Shane Scoggin, Lauren Gollahon, Preethi Gunaratne, Naima Moustaid-Moussa. Eicosapentaenoic acid reduces effects of some adipocyte derived factors on breast cancer cell inflammation and glucose metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 244. doi:10.1158/1538-7445.AM2017-244
Abstract 2442: Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System
Authors: Ashby Meredith , Hon Ting , Tseng Elizabeth , Vedula Aparna , Clark Tyson A.
Published: July 2017

Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping.

The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 - 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples.

Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell. The results highlight the potential for broader application of the Iso-Seq method to more comprehensively characterize alternative splicing in cancer.

Citation Format: Meredith Ashby, Ting Hon, Elizabeth Tseng, Aparna Vedula, Tyson A. Clark. Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2017-2442
Abstract 2443: Massively parallel single-cell RNA-Seq identifies diverse subpopulations displaying EMT and stem-like features
Authors: Lau Billy , Chen Jiamin , Ji Hanlee P.
Published: July 2017

Single-cell transcriptome analysis enables a new paradigm for studying complex systems in cancer. As opposed to bulk sequencing, which averages genomic signals across thousands or millions of cells and obscures the presence of rare subtypes, single cell sequencing enables the interrogation of individual cells. In cancer, intratumoral heterogeneity is observed at both genomic and epigenomic levels, and its analysis enables the discovery of new actionable targets and treatment modalities tailored to individual subpopulations. As an example, many cancer cell lines and those derived from patients contain subpopulations marked by distinct patterns of surface markers such as CD44, and are linked to drug resistant and tumor initiating phenotypes. A complete characterization of such cellular populations ideally requires marker-free sampling, followed by clustering into distinct subgroups. In this study, we demonstrate the significant advantages of such an approach; we utilize a high-throughput single-cell RNA-Seq method to characterize the transcriptomic profiles of cellular populations.

We performed single-cell RNA-Seq on thousands of cells in the matched SW480 (primary) and SW620 (metastatic) colorectal cell lines using a microfluidic droplet barcoding technology that enables the tracking of single cells during library preparation. By focusing on genes with high inter-cell variability, we discovered a small subpopulation of cells that displayed a distinct gene expression signature from the major subpopulation. Differential gene expression analysis of this subpopulation yielded genes virtually all enriched in the epithelial-to-mesenchymal transition (EMT) pathway. These cells showed significant increases in canonical mesenchymal marker genes such as VIM, CD44, and SOX9. Gene expression profiles of these subpopulations also correlated with established EMT signatures. Remarkably, this subpopulation did not display mutual exclusivity in gene expression with the epithelial marker EPCAM, which possibly indicates an intermediate mesenchymal phenotype. We also observed in the major population cluster a small subset of cells totaling less than 1% of the population that were significantly enriched for LGR5 expression, a common stem-like marker in colorectal cancer. Overall, we demonstrate the use of single-cell RNA-Seq to discover and characterize a diversity of cellular states that would otherwise be impossible from bulk analysis.

Citation Format: Billy Lau, Jiamin Chen, Hanlee P. Ji. Massively parallel single-cell RNA-Seq identifies diverse subpopulations displaying EMT and stem-like features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2017-2443
Abstract 2462: Whole-exome sequencing reveals genetic variants in ERC1 and KCNG4 in complete hydatidiform moles from Chinese Han women
Authors: Yu Yan , Lu Bingjian , Lu Weiguo , Wang Xinyu , Liu Pengyuan , Lu Yan , Cheng Xiaodong , Xie Xing
Published: July 2017

Complete hydatidiform mole (CHM) is an uncommon pregnancy-related disease with an invasive potential. The genetic background of the sporadic CHM has not been addressed previously despite the possible mechanisms of maternal genetic variants to the development of this disease with biparental origin. We performed the whole-exome sequencing analysis on 51 CHM patients and 47 normal healthy women, then the second screening of the probable mutations by mass spectrometry in 199 CHM patients and 400 normal healthy women. Finally the candidate genetic polymorphisms were validated by direct Sanger sequencing in 247 cases and 599 controls with another 205 new controls. We eventually found that two SNPs c.G48C(p.Q16H) in ERC1 and c.G1114A(p.G372S) in KCNG4, were associated with an increased risk for CHM (p<0.05). These genetic variants will facilitate our understanding on the pathogenesis of CHM and related tumors as well as the oogenesis and embryonic implantation. They will provide beneficial information for the pregnant women both in physiology and psychology. Further multiple-disciplinary collaborations should be encouraged to clarify the accurate pathogenesis of CHM.

Citation Format: Yan Yu, Bingjian Lu, Weiguo Lu, Xinyu Wang, Pengyuan Liu, Yan Lu, Xiaodong Cheng, Xing Xie. Whole-exome sequencing reveals genetic variants in ERC1 and KCNG4 in complete hydatidiform moles from Chinese Han women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2017-2462