bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1238: Apigenin suppresses inhibitor of apoptosis family protein expression and disrupts Ku70-bax interaction in prostate cancer cells in culture and in vivo
Authors: Shukla Sanjeev , Fu Pingfu , Gupta Sanjay
Published: May 2015

Apigenin (4′,5,7-trihydroxyflavone), an anticancer agent, selective toxic to cancer cells induces cell cycle arrest and apoptosis. The molecular mechanism(s) of this effect has not been fully elucidated. It is well known that dysfunction of the apoptotic pathway confers apoptosis resistance through inhibitor of apoptosis family protein (IAP) and deregulation of Bax/Bcl2 pathway in prostate cancer cells. In the present study we provide evidence for the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with 5-40µM apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment (20µM) resulted in significant decrease in cell viability and apoptosis induction with the release of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. In in vivo studies, per-oral intake of apigenin at doses of 20 and 50µg/mouse/day, two weeks after PC-3 cell implantation, continued for 8 weeks resulted in significant inhibition of tumor volume by 32.5% and 51.3% (P<0.001) and decrease in wet weight tumor by 33.3% and 55.1% (P<0.001), respectively. The animals did not exhibit any signs of toxicity or reduced food consumption. Compared to vehicle-treated controls, induction of apoptosis was significantly increased in tumor xenografts in apigenin fed mice (P<0.001) along with marked reduction in the protein expression of XIAP and survivin in PC-3 tumor xenografts. A dose-dependent decrease in HDAC1 expression, increase in Bax and PARP cleavage was observed in apigenin-treated mice. Taken together, these results suggest that apigenin targets IAP proteins and Ku70-Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.

Citation Format: Sanjeev Shukla, Pingfu Fu, Sanjay Gupta. Apigenin suppresses inhibitor of apoptosis family protein expression and disrupts Ku70-bax interaction in prostate cancer cells in culture and in vivo. [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 1238. doi:10.1158/1538-7445.AM2014-1238
Abstract 1244: Licofelone, a dual COX-LOX inhibitor prevents transitional cell carcinoma of urinary bladder in UPII-SV40T transgenic mice
Authors: Madka Venkateshwar , Mohammed Altaf , Li Qian , Zhang Yuting , Biddick Laura , Lightfoot Stanley , Wu Xue-Re , Kopelovich Levy , Steele Vernon , Rao Chinthallapally V.
Published: May 2015

Urinary bladder cancer is the sixth most commonly diagnosed cancer and significant proportion of the patients diagnosed with bladder cancer have high-grade tumors that are at potential risk for post-surgical recurrence and metastasis, where 5-year survival is very low. Hence it is important to identify and develop regimens that prevent urothelial tumorigenesis. Inflammatory pathways particularly, cyclooxygenase (COX-2) and lipoxygenase (5-LOX) mediated metabolites are known to play important role in bladder tumor progression. Consistent with that epidemiological and clinical observations suggest that use of anti-inflammatory agents is associated with reduced risk for bladder cancer. Here we determined the chemopreventive efficacy of a Licofelone, a dual COX-LOX inhibitor, in a transgenic UPII-SV40T mouse model of bladder transitional cell carcinoma (TCC). Six-week old transgenic UPII-SV40T mice (n=30/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm licofelone for 34 weeks. At 40 weeks of age, all mice were euthanized; urinary bladders were collected to determine urothelial tumor weights and histopathological grading. Results suggest that bladders of the control diet fed transgenic mice weighed 3-5 fold more than that of the wild type mice. However, treatment of transgenic mice with licofelone led to a significant, dose dependent inhibition of the urothelial tumor weights (65.2 - 82.7%, p<0.0001 in males; 35.0 - 49.0%, p<0.0001 in females) compared to the control group. Importantly, mice fed licofelone diet significantly reduced development of invasive tumors. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 50%; p<0.01) and females mice (up to 46%; p<0.003). Molecular analysis of urothelial tumors by immuno-histochemisty, qPCR and/or western immunoblotting for the expression of various markers that effect tumor growth and progression showed an increase in apoptosis (p53, p21, Bax, Caspase3, Annexin V) with a decrease in proliferation and angiogenesis (PCNA, COX2, 5LOX, mPGES2, VEGF) in the licofelone diet fed mice tumors. These results suggest that licofelone can serve as potential chemopreventive for bladder TCC. (Supported in part by NCI-CN53300)

Citation Format: Venkateshwar Madka, Altaf Mohammed, Qian Li, Yuting Zhang, Laura Biddick, Stanley Lightfoot, Xue-Re Wu, Levy Kopelovich, Vernon Steele, Chinthallapally V. Rao. Licofelone, a dual COX-LOX inhibitor prevents transitional cell carcinoma of urinary bladder in UPII-SV40T transgenic 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 1244. doi:10.1158/1538-7445.AM2014-1244
Abstract 1252: 6-c-(e-phenylethenyl)-naringenin suppresses colorectal cancer growth by inhibiting cyclooxygenase-1
Authors: Li Haitao , Zhu Feng , Chen Hanyong , Cheng Ka Wing , Zykova Tatyana , Oi Naomi , Lubet Ronald A. , Bode Ann M. , Wang Mingfu , Dong Zigang
Published: May 2015

Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors therefore COX-1 is now being reconsidered as a target for chemoprevention. Our aim was to examine whether selective COX-1 inhibition could delay or prevent cancer development and clarify the underlying mechanisms. Data clearly showed that COX-1 was required for maintenance of malignant characteristics of colon cancer cells or tumor promoter(s)-induced transformation of pre-neoplastic cells. We also successfully applied a ligand docking computational method to identify a novel selective COX-1 inhibitor, 6-C-(E-phenylethenyl)-naringenin (designated herein as 6CEPN). 6CEPN could bind to COX-1 and specifically inhibit its activity both in vitro and ex vivo. In colorectal cancer cells, it suppressed anchorage-independent growth by inhibiting COX-1 activity. 6CEPN also effectively suppressed tumor growth in a 28-day colon cancer xenograft model without any obvious systemic toxicity. Taken together, COX-1 plays a critical role in human colorectal carcinogenesis, and the specific COX-1 inhibitor merits further investigation as a potential preventive agent against colorectal cancer.

Keywords: colon cancer; natural product; cyclooxygenases; signal transduction

Note: This abstract was not presented at the meeting.

Citation Format: Haitao Li, Feng Zhu, Hanyong Chen, Ka Wing Cheng, Tatyana Zykova, Naomi Oi, Ronald A. Lubet, Ann M. Bode, Mingfu Wang, Zigang Dong. 6-c-(e-phenylethenyl)-naringenin suppresses colorectal cancer growth by inhibiting cyclooxygenase-1. [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 1252. doi:10.1158/1538-7445.AM2014-1252
Abstract 1277: The association between seasonality, vitamin D and calcium intake and mammographic density in Norwegian postmenopausal women
Authors: Ellingjord-Dale Merete , Silva Isabel dos Santos , Grotmol Tom , Sakhi Amrit Kaur , Qureshi Samera , Hofvind Solveig , Markussen Marianne Skov , Couto Elisabeth , Andersen Lene Frost , Ursin Giske , Ursin Giske
Published: May 2015

Background:

Mammographic density (MD) is one of the strongest known breast cancer risk factors. The role of vitamin D in breast cancer etiology is unclear. The evidence of an association between Vitamin D and MD is limited and inconsistent, as is the seasonal variation of MD. We evaluated the association between MD and season/month the mammogram was obtained, as well as vitamin D intake, energy and calcium intake in a population of women from Norway, a country with limited sunlight exposure for a large part of the year.

Methods:

We studied 2662 postmenopausal women aged 50-69 who participated in the Norwegian Breast Cancer Screening Program (NBCSP) in 2004 or 2006/07. Women completed risk factor and food frequency questionnaires (FFQ). Vitamin D and calcium intake was assessed with and without taking supplements into consideration. Month of mammogram was recorded from the mammogram. MD was assessed using a computer assisted method (Madena, University of Southern California). We used linear regression to determine the association between month of mammogram and MD, as well as vitamin D and calcium intake (assessed from the FFQ) and MD, adjusting for age, education, body mass index (BMI), parity and postmenopausal estrogen and progestin therapy. We also examined the association between vitamin D, calcium and MD by season (month) the mammogram was obtained.

Results:

Age, BMI and parity were inversely associated with MD, while education and postmenopausal estrogen and progestin therapy were positively associated\ with MD. There was no association between quartiles of vitamin D or calcium intake and MD, regardless of whether the nutrients were estimated with our without supplements. We found no association between season (month) and MD. There was no evidence for an inverse association between vitamin D intake and MD when women were stratified by the month the mammogram was obtained.

Conclusion:

Our results provide no support for a protective effect of dietary vitamin D intake against increased MD. We found no evidence that season or month the mammogram was obtained, was associated with MD.

Citation Format: Merete Ellingjord-Dale, Isabel dos Santos Silva, Tom Grotmol, Amrit Kaur Sakhi, Samera Qureshi, Solveig Hofvind, Marianne Skov Markussen, Elisabeth Couto, Lene Frost Andersen, Giske Ursin, Giske Ursin. The association between seasonality, vitamin D and calcium intake and mammographic density in Norwegian postmenopausal women. [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 1277. doi:10.1158/1538-7445.AM2014-1277
Abstract 1287: Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population
Authors: Wong Edward , Yap Yoon Sim , Cheng Ying Ying , Chua Delia , Hong Lewis Zuocheng , Burkholder William F. , Ho Gay Hui , Tan Min Han , Ang Peter , Lee Ann S.G.
Published: May 2015

INTRODUCTION

Although mutations in BRCA1 and BRCA2 are associated with familial hereditary breast and ovarian cancer, mutations are only found in 20 to 25% of these patients. This suggests that additional genes may be mutated. The Casitas B-lineage lymphoma (CBL) gene encodes a RING finger E3 ubiquitin ligase. Recent studies have shown that mutations in the CBL gene occurs in human acute myeloid leukemia and lung cancers. The relationship of the CBL gene with breast cancer, however, is not well characterized. The aim of this study was to identify mutations in the CBL gene in Asian patients with familial or early-onset breast cancer.

METHODS

All patients (n=75) were accrued at the risk assessment clinic at the National Cancer Centre Singapore, and presented with a family history of breast and/or ovarian cancer or early-onset breast cancer. Screening for mutations in the BRCA1 and BRCA2 genes was performed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Next generation sequencing was done using the Agilent SureSelect target-enrichment technology on the Illumina MiSeq platform, followed by validation using Sanger sequencing.

RESULTS

Two mis-sense variants in the CBL gene were identified. They are CBL c. 560C>T (p. A187V) and c. 1858C>T (p.L620F) occurring in 2/75 (3%) and 8/75 (11%) of the patients, respectively. Both mis-sense mutations were predicted to be damaging using Polyphen-2 and SIFT. The p.A187V alteration was predicted to interrupt the N-terminal tyrosine kinase binding domain (TKB) that is important for ligand-induced ubiquitination of receptor tyrosine kinases (RTKs). The CBL c. 1858C>T variant has previously been reported in the NCBI database (rs2227988). Three individuals, who carried this mutation, had early-onset breast cancer. In addition, the p.L620F alteration within the proline-rich region, may disrupt the interactions of binding partners to the c-CBL protein, which are required to recruit c-CBL to RTKs. All patients, except one, with germline CBL mutations were negative for BRCA1 and BRCA2 mutations.

CONCLUSION

This preliminary study has identified damaging CBL mutations in an Asian population with breast cancer and warrants further investigations in other breast cancer populations.

Citation Format: Edward Wong, Yoon Sim Yap, Ying Ying Cheng, Delia Chua, Lewis Zuocheng Hong, William F. Burkholder, Gay Hui Ho, Min Han Tan, Peter Ang, Ann S.G. Lee. Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population. [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 1287. doi:10.1158/1538-7445.AM2014-1287
Abstract 1292: Identification of BRCA1 and BRCA2 founder mutations in population isolates from Colombia
Authors: Tuazon Anna Marie D. , Ramírez Carolina , Bohorquez Mabel , Prieto Rodrigo , Castro Jorge , Mateus Gilbert , Velez Alejandro , Echeverry Magdalena , Carvajal-Carmona Luis G.
Published: May 2015

Breast cancer (BC) is the most common malignancy in women. Germline mutations in breast cancer risk genes, such as BRCA1 and BRCA2, account for a large portion of hereditary breast cancer families. The contribution of mutations in breast cancer susceptibility genes has been poorly investigated in minority populations, such as in Hispanics from South America. While mutations in BRCA1 and BRCA2 display a heterogeneous mutation spectrum, specific mutations often occur in population isolates. Using the KASP genotyping technology, we screened 476 unselected Colombian breast cancer patients for three common BRCA1 and one BRCA2 founder mutations in the Hispanic population. The BRCA2 c.2806_2809delAAAC founder mutation was identified in 3 of the 476 BC cases (0.6%). Strikingly, 24 out of 476 cases (5.0%) harbored the BRCA1 founder mutation c.3331_3334delCAAG, which has also been reported in Southern European populations, including Spain and Portugal. The clinical data and family history of these patients have highlighted a distinct geographic region from where these cases originate. This finding potentially represents one of the most remarkable founder effects reported in human populations. To date the arrival of the BRCA1 c.3331_3334delCAAG mutation to Colombia, we are completing haplotype analysis of the patients and their families, along with BC cases from Spain and Portugal that harbor the same mutation. Interestingly, haplotype analyses of our BC cases have revealed a haplotype larger than 3.5 cM. In the future, we aim to perform haplotype analysis of family members of the mutation carriers and BC cases from Portugal and Spain to further study the haplotype around this BRCA1 mutation, which will allow us to date the origin of the mutation. By understanding the prevalence of mutations in known breast cancer risk genes in minority populations, cancer disparities can be better addressed and breast cancer screening can be improved. Furthermore, the ability to identify such strong founder effects highlights an advantage of using this Colombian population isolate in facilitating the identification of novel BC risk genes.

Citation Format: Anna Marie D. Tuazon, Carolina Ramírez, Mabel Bohorquez, Rodrigo Prieto, Jorge Castro, Gilbert Mateus, Alejandro Velez, Magdalena Echeverry, Luis G. Carvajal-Carmona. Identification of BRCA1 and BRCA2 founder mutations in population isolates from Colombia. [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 1292. doi:10.1158/1538-7445.AM2014-1292
Abstract 13: Color-coded imaging of vessel anastomosis in vivo using RFP and CFP transgenic mice
Authors: Uehara Fuminari , Tome Yasunori , Maehara Hiroki , Tanaka Kazuhiro , Kanaya Fuminori , Miwa Shinji , Hiroshima Yukihiko , Yano Shuya , Yamamoto Mako , Matsumoto Yasunori , Hoffman Robert M.
Published: May 2015

We previously report the development of a color-coded imaging model that can visualize the anastomosis between blood vessels of red fluorescent protein (RFP)-expressing vessels in Gelfoam® previously vascularized in RFP transgenic mice and blood vessels of nestin-driven green fluorescent protein (ND-GFP) mice that express GFP in nascent vessels (Anticancer Res. 33: 3041-3046, 2013). In the present study, fourteen days after transplantation into RFP transgenic nude mice, the Gelfoam® was removed and re-transplanted into the subcutis on the flank of cyan fluorescent protein (CFP) transgenic mice. Skin flaps were made and anastomosis between the CFP-expressing vessels and RFP blood vessels in the Gelfoam® was imaged 14 and 21 days after re-transplantation. The results presented in this and our previous report suggest a new paradigm of visualizing blood vessel anastomosis and its implications for tumor angiogenesis.

Citation Format: Fuminari Uehara, Yasunori Tome, Hiroki Maehara, Kazuhiro Tanaka, Fuminori Kanaya, Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Mako Yamamoto, Yasunori Matsumoto, Robert M. Hoffman. Color-coded imaging of vessel anastomosis in vivo using RFP and CFP transgenic 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 13. doi:10.1158/1538-7445.AM2014-13
Abstract 1304: Cyclin D:Cdk4/6 activates RB by mono-phosphorylation during early G1 phase
Authors: Dowdy Steven F. , Kaulich Manuel
Published: May 2015

The retinoblastoma tumor suppressor protein (Rb) functions to transcriptionally regulate multiple critical cellular activities, including early G1 phase, the DNA damage response, cell cycle exit and differentiation, but is infrequently mutated or deleted in cancer. To account for the lack of Rb mutations, a large body of work over the last 20+ years has led to a widely accepted model of G1 cell cycle progression that proposes cyclin D:Cdk4/6 complexes partially inactivate Rb during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, on 15 Cdk sites resulting in release of E2F transcription factors that drive cells across the late G1 phase checkpoint and ultimately undergo cellular division. Surprisingly given the importance of G1 cell cycle progression in cancer, the core tenet of the model, namely cyclin D:Cdk4/6 inactivation of Rb by progressive multi-phosphorylation, remains largely unproven biochemically. Consequently, it remains entirely unknown if hypo-phosphorylated Rb contains one, two, three, five, seven or more phosphates, at what phosphate number hypo-phosphorylated Rb releases E2F transcription factors, what the phosphorylation kinetics are and what the biologically active form(s) of Rb during early G1 phase is. Here using highly synchronized normal and tumor cells combined with two-dimensional isoelectric focusing (2D IEF) to quantify the number of phosphates on Rb, we find no evidence to support the 20 year old notion that cyclin D:Cdk4/6 complexes progressively hypo-phosphorylate Rb. In contrast, we discovered that cyclin D:Cdk4/6 complexes exclusively mono-phosphorylates Rb throughout early G1 phase, and generates fourteen independent mono-phosphorylated Rb isoforms. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 complexes to generate mono-phosphorylated Rb that regulated global transcription, whereas non-phosphorylatable Rb remained non-functional. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6 complexes, is the functionally active isoform of Rb in early G1 phase.

Citation Format: Steven F. Dowdy, Manuel Kaulich. Cyclin D:Cdk4/6 activates RB by mono-phosphorylation during early G1 phase. [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 1304. doi:10.1158/1538-7445.AM2014-1304
Abstract 1310: Increased cytoplasmic CDC25A phosphatase in cutaneous squamous cell carcinoma leads to 14-3-3 relocalization and suppression of apoptosis
Authors: Almatouq Jenan , Ho Kristen , Hansen Laura A.
Published: May 2015

Non-melanoma skin cancer, the most common cancer in the United States, frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the Cell Division Cycle 25 (CDC25) phosphatases, which are over-expressed and, thus, deregulate cell cycle control in many human cancers. To investigate a role for CDC25 in skin cancer, CDC25 levels were examined in both UV-induced mouse tumors and human skin cancer. CDC25A and CDC25C protein and transcripts were increased in benign and malignant skin tumors from UV-exposed v-rasHa transgenic Tg.AC mice. CDC25B protein but not transcripts was increased in mouse skin tumors compared to normal skin. Similarly, CDC25A and CDC25C but not CDC25B were elevated in human squamous cell carcinoma (SCC) and in the premalignant precursors actinic keratosis and Bowen's disease when compared to skin. While CDC25A was localized primarily to nuclei in normal epidermis, both the premalignant precursors and SCC exhibited strong cytoplasmic CDC25A localization. To determine whether increased CDC25A stimulates proliferation in skin SCC, CDC25A levels were modulated by siRNA-targeting or forced expression in cultured SCC cells, but no difference in proliferation resulted. Because cytoplasmic CDC25A can bind to the apoptotic regulator 14-3-3, we hypothesized that the increase in cytoplasmic CDC25A may suppress apoptosis. Forced over-expression of CDC25A in SCC cells suppressed cell death, whereas CDC25A silencing increased apoptotic cell death. Phosphorylation of CDC25A at serine178, a docking site for 14-3-3 proteins, was elevated in the cytoplasmic compartment of SCCs, and cytoplasmic 14-3-3ϵ was increased in cutaneous SCC.Mutation of the nuclear export sequence on CDC25A relocalized both CDC25A and 14-3-3ϵ to the nucleus, while mutation of 14-3-3 binding sites on CDC25A led to a decrease in 14-3-3ϵ levels. Furthermore, silencing of 14-3-3ϵ increased cell death by 60%. Taken together, these data suggest that increased cytoplasmic CDC25A in cutaneous SCC may suppress apoptotic cell death through its interaction with 14-3-3ϵ.

Citation Format: Jenan Almatouq, Kristen Ho, Laura A. Hansen. Increased cytoplasmic CDC25A phosphatase in cutaneous squamous cell carcinoma leads to 14-3-3 relocalization and suppression of apoptosis. [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 1310. doi:10.1158/1538-7445.AM2014-1310
Abstract 1318: Mps1 is modified by sumoylation during the cell cycle
Authors: Restuccia Agnese , Yang Feikun , Yixin Yao , Dai Wei
Published: May 2015

Mps1 is a dual specificity protein kinase with crucial roles in regulating the spindle assembly checkpoint and chromosome microtubule attachment. Molecular mechanisms that regulate Mps1 levels and its activity during mitosis remain unclear. Given that sumoylation plays an important role in mitotic progression, we investigated if Mps1 could be modified by SUMO conjugation. Endogenous sumoylated Mps1 was detected by nickel affinity pull-down from HeLa cells that constitutively expressed His6-SUMO1 or His6-SUMO2. When HEK293T cells transfected with a plasmid construct expressing Myc-Mps1, along with plasmids expressing His-SUMO-1 and FLAG-UBC9, were analyzed sumoylated Mps1 was also detected in both asynchronized and mitotic cell populations. Expression of wild-type FLAG-SENP1 completely abrogated sumoylated Mps1 compared with cells transfected with enzymatically defective FLAG-SENP1, confirming that Mps1 is modified by sumoylation. We also identified potential lysine residues of Mps1 that were sumoylated by the mutagenesis approach. Our study reveals that Mps1 is modified via sumoylation and that future experiments will be centered on understanding the biological function of this modification during the cell cycle.

Citation Format: Agnese Restuccia, Feikun Yang, Yao Yixin, Wei Dai. Mps1 is modified by sumoylation during the cell cycle. [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 1318. doi:10.1158/1538-7445.AM2014-1318
Abstract 1325: Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells
Authors: Jiang Chang , Veon William , Roy Partha
Published: May 2015

Profilin-1 is a ubiquitously expressed actin-binding protein that is downregulated in human breast cancer. Overexpression of profilin-1 in human breast cancer cells suppresses their tumorigenic potential. We had previously shown that profilin-1 overexpression elevates the expression level of p27kip1 leading to cell-cycle arrest in G1 phase. In the present work, we explore the underlying mechanism. Our studies showed that profilin-1-induced accumulation of p27kip1 in breast cancer cells occurs most dramatically in the nuclear compartment and this is due to impaired protein degradation. Consistent with these observations, we found that phosphorylation of p27kip1 at T187 (a prerequisite for p27kip1 degradation by SCF-Skp2 complex in the nucleus) and S10 (a post-translational event that promotes nuclear export of p27kip1) residues are both significantly inhibited by profilin-1 overexpression. Elevating profilin-1 level suppresses AKT activation, and hyper-activating AKT through overexpression of a constitutively active mutant relieves profilin-1-induced inhibition of T187 phosphorylation and elevation of p27kip1. These data demonstrate that profilin-1 can control the cellular level of p27kip1 in breast cancer cells through affecting its post-translational modification in an AKT-dependent pathway. These findings may provide mechanistic insights underlying the tumor-suppressive action of profilin-1.

Citation Format: Chang Jiang, William Veon, Partha Roy. Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast 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 1325. doi:10.1158/1538-7445.AM2014-1325
Abstract 1349: Chemosensitization of breast cancer cell lines by Ell3
Authors: Ahn Hee-Jin , Heo Sun-Hee , Kim Gwangil , Park Kyung-Soon
Published: May 2015

Ell3 is a RNA polymerase II transcription elongation factor that is enriched in testis. The C-terminal domain of Ell3 shows strong similarities to that of Ell (eleven-nineteen lysine-rich leukemia gene), which acts as a negative regulator of p53. Recent studies in our laboratory showed that Ell3 induces the differentiation of mouse embryonic stem cells by protecting differentiating cells from apoptosis by the promotion of p53 degradation. In this study, we evaluated the function of Ell3 in breast cancer cell lines. Contrary to our expectation, ectopic expression of Ell3 in MCF7 induces CDDP (cisplatin) susceptibility by enhancing p53 dependent apoptosis. CDDP treatments leads to the accumulation of p53 protein in the Ell3 overexpressing MCF7. We confirmed that Ell3 inhibits proteosomal degradation of p53 upon cisplatin treatment in MCF7. Ell3 also confer chemosensitivity to MCF7 to DNA damaging agent, camptothecin. The treatment of camptothecin to Ell3 overexpressing MCF7 results in the accumulation of cells at G2/M phase of cell cycle by the increases of p53 protein. Consistent with these results, Ell3 downregulates the expression of MDM2, which binds p53 and marks it for degradation using the proteasome. The underlying mechanism of Ell3 to regulate MDM2 expression is under investigation.

Citation Format: Hee-Jin Ahn, Sun-Hee Heo, Gwangil Kim, Kyung-Soon Park. Chemosensitization of breast cancer cell lines by Ell3. [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 1349. doi:10.1158/1538-7445.AM2014-1349
Abstract 1373: Comprehensive analysis of HBV DNA methylation in liver tissues of hepatitis B, cirrhosis and hepatocellular carcinoma patients
Authors: Jain Surbhi , Chen Sitong , Boldbaatar Batbold , Lin Selena Y. , Yan Ran , Hu Chi-Tan , Guo Haitao , Block Timothy M. , song Wei , Su Ying-Hsiu
Published: May 2015

Hepatitis B virus (HBV) is a hepatotropic virus causing hepatitis, cirrhosis and hepatocellular carcinoma. In patients, HBV DNA circulates in blood as virion DNA and exists in hepatocytes both as nuclear form (episomal cccDNA and integrated DNA) and as cytoplasmic core DNA form. It has been reported that HBV infection up-regulates DNMTs and thus induces epigenetic changes in the host cells. Understanding the methylation status of the HBV DNA in its different forms can potentially provide insight into the pathogenesis of HBV-related liver diseases including hepatocarcinogenesis. Previous studies conducted in patient serum and in cell cultures have demonstrated that the CpG islands of HBV virion DNA are in the unmethylated state, and cccDNA methylation has been extensively studied for its role in the regulation of transcription, but very few studies have investigated the methylation status of integrated HBV DNA in human liver tissues. To better understand the methylation status of HBV DNA in hepatitis B, cirrhosis and HCC, a comprehensive methylation profile of three CpG islands in the HBV genome was assessed by BS-sequencing, and methylation of each of the 3 CpG islands was compared by using bisulfite specific PCR and methylation specific PCR assays in a larger sample size. We observed that the methylation of CpG island 1 and 3 of HBV DNA from liver tissues was significantly higher in HCC as compared to hepatitis and cirrhosis. We also obtained, for the first time, the evidence of rare non-CpG methylation in the CpG island 1 and 2 of HBV genome in liver tissues. The overall co-relation of HBV genome methylation and disease progression was also investigated. In addition, no co-relation between the methylation levels of the HBV genome and host genome was found, suggesting independent mechanisms regulate the methylation of host and viral genomes. The mechanisms of HBV genome methylation in liver disease and its regulation are discussed.

Citation Format: Surbhi Jain, Sitong Chen, Batbold Boldbaatar, Selena Y. Lin, Ran Yan, Chi-Tan Hu, Haitao Guo, Timothy M. Block, Wei song, Ying-Hsiu Su. Comprehensive analysis of HBV DNA methylation in liver tissues of hepatitis B, cirrhosis and hepatocellular carcinoma patients. [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 1373. doi:10.1158/1538-7445.AM2014-1373
Abstract 1388: Biological role of novel NQO2 inhibitors in cancer therapeutics
Authors: Santina Elham , Chadwick Amy , Nabulsi Soraya A. , Freeman Sally , Demonacos Constantinos , Stratford Ian
Published: May 2015

Background: NRH: quinone oxidoreductase 2 (NQO2), is a cytosolic flavoprotein that catalyses the two-electron reduction of quinones into hydroquinones. The physiological role of NQO2 in cancer biology is still not well understood. Some studies have suggested that inhibiting NQO2 could have a great potential in chemoprevention. This is based on the observation that NQO2 gene knockdown promotes protection against the toxicity of quinones such as menadione.

Aim: To evaluate the biological role of novel NQO2 inhibitors in cancer therapeutics. Novel inhibitors will be used as pharmacological tools to study the physiological role of NQO2 in cancer cells, and to identify the consequences of inhibiting NQO2 on the tumor microenvironment.

Methods: Novel compounds with diverse structures have been tested for their inhibition of NQO2 enzymatic activity in a cell-free system using a pure enzyme assay. The functional activity of these compounds as NQO2 inhibitors in the MDA-MB 231 breast cancer cells was also assessed. The effect of these inhibitors on the cellular proliferation of MDA-MB 231 breast cancer cells was tested using an MTT assay. Off-target (DNA binding) effects were analyzed by measuring the DNA thermal temperature. Moreover, the effect of these inhibitors on modulating TNFα-mediated NF-kB transcriptional activity was tested and the mechanism behind this modulation was also examined.

Results: The novel inhibitors tested resulted in the inhibition of NQO2 activity in the cell free and the cellular system at nano-molar concentrations .Inhibition of MDA-MB 231 cell proliferation was also witnessed at low micro-molar concentrations. The inhibitors also displayed no DNA binding ability. In addition, the inhibitors were able to attenuate TNFα-mediated NF-kB transcriptional activity by blocking the phosphorylation of IkBα. This has resulted in the suppression of cell growth and promotion of cell apoptosis.

Conclusions: This study has demonstrated novel potent inhibitors of NQO2 that are functionally active in the cells at nano-molar concentrations. The effect of the inhibitors on TNFα-mediated NF-kB transcriptional activity raises the possibility that inhibiting NQO2 could modulate the tumor micro-environmental factors, such as metastasis and angiogenesis.

Citation Format: Elham Santina, Amy Chadwick, Soraya Al Nabulsi, Sally Freeman, Constantinos Demonacos, Ian Stratford. Biological role of novel NQO2 inhibitors in cancer therapeutics. [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 1388. doi:10.1158/1538-7445.AM2014-1388
Abstract 1389: Insights into the invasiveness of triple-negative breast cancer from genome-wide profiling of AP-1
Authors: Zhao Chunyan , Qiao Yichun , Dahlman-Wright Karin
Published: May 2015

Triple-negative breast cancer (TNBC) is an aggressive clinical phenotype, and accounts for 15% to 20% of all breast cancers. The molecular determinants of malignant cell behaviors in TNBC remain largely unknown. We show that the AP-1 transcription factor component Fra-1 is overexpressed in basal-like breast tumors, often referred to as TNBCs, and that its expression level has high prognostic significance. Depletion of Fra-1 or its heterodimeric partner c-Jun inhibits the proliferative and invasive phenotypes of TNBC cells in vitro. Furthermore, knockdown of Fra-1 or c-Jun also reduces cellular invasion in vivo in a zebrafish xenograft model. Exploring the AP-1 cistrome and the AP-1-regulated transcriptome, we provide insights into the transcriptional regulatory networks of AP-1 in TNBC cells. We identify direct targets of the Fra-1/c-Jun heterodimer involved in cell proliferation, cell adhesion, and cell-cell contact. Importantly, we show that AP-1 represses E-cadherin expression by transcriptional upregulation of ZEB2, thereby stimulating cell invasion. This work sheds light on mechanisms and pathways by which TNBCs acquire invasiveness and proliferative propensity.

Note: This abstract was not presented at the meeting.

Citation Format: Chunyan Zhao, Yichun Qiao, Karin Dahlman-Wright. Insights into the invasiveness of triple-negative breast cancer from genome-wide profiling of AP-1. [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 1389. doi:10.1158/1538-7445.AM2014-1389
Abstract 1391: SOX2-p63 interaction and genomic co-localization in squamous cell carcinomas
Authors: Watanabe Hideo , Ma Qiuping , Peng Shouyong , Adelmant Guillaume , Swain Danielle , Song Wenyu , Fox Cameron , Francis Joshua M. , Pedamallu Chandra Sekhar , Deluca David S. , Brooks Angela N. , Que Jianwen , Rustgi Anil K. , Wong Kwok-kin , Ligon Keith L. , Liu X. Shirley , Marto Jarrod A. , Meyerson Matthew , Bass Adam J.
Published: May 2015

SOX2 is a transcription factor essential for pluripotent stem cells and the development and maintenance of squamous epithelium. We previously reported SOX2 to be an oncogene subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs). Here, we aim to further characterize SOX2's function in SCC. We demonstrate that SOX2 binds to distinct genomic loci in SCCs than in embryonic stem (ES) cells through comparative ChIP-seq analysis. Through mass-spectrometric analysis following tandem-affinity immunopurification in SCCs we identify SOX2 interacts with another master squamous transcription factor, p63, instead of its partner in ES cells, OCT4. We find that genomic occupancy of SOX2 in SCC overlaps with that of p63 at a large number of loci and this SOX2-p63 coordinate binding is absent in ES cells. We further demonstrate that SOX2 and p63 jointly regulate gene expression, including the oncogene ETV4, which we find essential for SOX2-amplified SCC cell survival. Together, these findings demonstrate that SOX2's actions in SCC differ substantially from its role in pluripotency. The identification of novel SOX2-p63 interaction enables deeper characterization of SOX2's function in SCC and normal squamous epithelial physiology.

Citation Format: Hideo Watanabe, Qiuping Ma, Shouyong Peng, Guillaume Adelmant, Danielle Swain, Wenyu Song, Cameron Fox, Joshua M. Francis, Chandra Sekhar Pedamallu, David S. Deluca, Angela N. Brooks, Jianwen Que, Anil K. Rustgi, Kwok-kin Wong, Keith L. Ligon, X. Shirley Liu, Jarrod A. Marto, Matthew Meyerson, Adam J. Bass. SOX2-p63 interaction and genomic co-localization in squamous cell carcinomas. [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 1391. doi:10.1158/1538-7445.AM2014-1391
Abstract 1393: Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal cancer
Authors: Dowling Catríona M. , Hanly Jennifer , Dalton Tara , Kiely Patrick A.
Published: May 2015

Protein Kinase C (PKC) is a family of serine/threonine kinases that are involved in almost every signal transduction pathway. Their regulation is mediated by several factors and by binding to a group of scaffolding proteins called RACKs (Adams et al. 2011). The development of PKC modulators with anti-cancer therapeutic value is a major target in cancer. However, this task is made difficult because PKC has an important role to play in normal processes and the PKC family consists of at least 12 different isozymes. In colon cancer, there is differential expression of the PKC isozymes, giving the cancer cells a migratory advantage and thus promote cancer progression. Our hypothesis is that PKC expression, activity and localisation are altered as colon epithelial cells switch from normal to the transformed state. We are confident that being able to recognise these changes has the potential to be used as a biomarker and prognostic marker in the early detection of colon cancer. Using novel 3D culture techniques, we examined the gene expression pattern of the 12 different PKC isozymes. We found that the isozymes were differentially expressed and that their expression pattern changed depending on whether the cells were grown under 2-dimensional or 3-dimensional culture systems. Using patient samples, we also document differential expression of several isozymes when compared to matched normal tissue. Using a series of PKC activators, together with a series of synergistic cellular and molecular approaches, we follow the migration of specific PKC isoforms to the membrane fraction in cells. This migration is dependent on the expression of RACK1 as when we use siRNA against RACK1, we disrupt PKC translocation in colon cancer cells. Taken together, our findings confirm that there is differential expression of PKC isozymes in cancer cells. We also confirm that specific PKC isozymes colocalise with RACK1 at the membrane supporting our hypothesis that RACK1 plays a central role in translocating PKC in the colon epithelium. Differential PKC expression and PKC translocation may be an important mechanism regulating the transformed phenotype of colon cancer cells.

Citation Format: Catríona M. Dowling, Jennifer Hanly, Tara Dalton, Patrick A. Kiely. Correlating the expression of protein kinase C isozymes with the transformed phenotype in colorectal 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 1393. doi:10.1158/1538-7445.AM2014-1393
Abstract 1394: Role of non-coding RNA in regulation of stemness and differentiation via notch signaling pathway in glioblastoma
Authors: Katsushima Keisuke , Shinjo Keiko , Ohka Fumiharu , Natsume Atsushi , Shibata Tatsuhiro , Kondo Yutaka
Published: May 2015

Multiple distinct subpopulations of cancer cells within tumors may derive from a limited source of cancer cells, called cancer stem cells (CSCs), which may have plasticity and respond to signals from their microenvironment. Recent studies demonstrated the fundamental roles of long non-coding RNA (lncRNA) in various biological processes. Here, we investigated the roles of lncRNA in glioma stem cells (GSCs) differentiation. Whole-genome RNA sequencing revealed that 180 and 171 lncRNAs were commonly upregulated and downregulated with a greater than 2-fold difference in GSC differentiation, respectively. Among those, linc-A was consistently downregulated during GSC differentiation. Linc-A, of which expression is regulated by the Notch signaling pathway, physically interacted with polycomb repressive complex 2 (PRC2) and conferred a recruitment of PRC2 and histone H3 lysine 27 trimethylation (H3K27me3) in its target loci. An RNA pull-down assay revealed that linc-A specifically bound to a number of neuronal differentiation-associated genes in GSC. These target genes were consistently silenced in GSCs. Inhibition of linc-A using antisense oligonucleotide reduced the H3K27me3 level and increased expression of its target genes, resulted in loss of stemness and induction of GSC differentiation into neuronal lineage. Intriguingly, in silico analysis revealed that linc-A shared a sequence of microRNA-response element (MRE) with mRNAs of stemness markers, such as MYC, SOX2, and NANOG. Indeed, downregulation of linc-A increased the miR-X expression together with repression of its target genes, MYC, SOX2, and NANOG, being consistent with an RNA sponge mechanism, in which lncRNAs contain complementary binding sites (MRE) to a miRNA of interest and prevent these mRNAs from degradation. Our data indicated that linc-A plays consistent roles in modulation of stemness properties in GSC in response to Notch-epigenetic regulatory pathway.

Citation Format: Keisuke Katsushima, Keiko Shinjo, Fumiharu Ohka, Atsushi Natsume, Tatsuhiro Shibata, Yutaka Kondo. Role of non-coding RNA in regulation of stemness and differentiation via notch signaling pathway in glioblastoma. [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 1394. doi:10.1158/1538-7445.AM2014-1394