bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2231: The prevalence of EGFR and KRAS mutations in a population of African immigrants in France is closer to that of Asian populations than to white Europeans
Authors: Lemoine Antoinette , Saffroy Raphael , Bosselut Nelly , Lecorche Emmanuel , Etessami Reza , Allard Marc A. , Tredaniel Jean , Balaton Andre , Validire Pierre , Morere Jean F.
Published: May 2015

The choice of treatment for patients with NSCLC relies on the EGFR and KRAS mutational status. Previous studies have reported differences in prevalence of mutations between Asiatics, Hispanics and Caucasians. However, there is few data with respect to the frequencies of EGFR and KRAS mutations in patients originated from Africa, who represent the largest immigrant population in France.

Between May 2011 and November 2013, a mutational analysis for KRAS and EGRF has been performed in tumor samples from 1,973 consecutive patients with NSCLC at a single oncogenetic platform. Demographic and clinical data of all these patients were extracted from their electronic medical record and analyzed.

Our study population included 1,739 (88%) white Europeans, 204 (10%) patients from Africa and 30 (2%) patients from Asia (mostly from Vietnam or Cambodge).

As expected, the higher prevalence of EGFR mutations was found in Asiatic women (50%), the lower was observed in Caucasian women population (17%, p<0.001). This low frequency of EGFR mutations has already been reported in other European populations. Surprisingly, EGFR mutations rate was as high as 32% in African women patients (p<0.001), whereas African men exhibited a very low frequency of EGFR mutations (5%, p<0.001) as well as the French non immigrant patients (6%).

KRAS mutation rate was higher among white European (26%) than Asiatic (10%) or African (10%) patients. Although KRAS mutation rate did not differ by sex in white Europeans (men 25% versus women 27%, respectively), it was 3 fold more frequent in men than in women among African (19% versus 5%, respectively, p<0.001) and Asiatic patients.Therefore EGFR or KRAS mutation rates in African immigrant population in France are closer to those observed in Asians or even Hispanics, than in Caucasians. These frequencies also differ from those reported in African Americans. The ancestral and recent migration of populations as well as social habits could explain these results.

Citation Format: Antoinette Lemoine, Raphael Saffroy, Nelly Bosselut, Emmanuel Lecorche, Reza Etessami, Marc A. Allard, Jean Tredaniel, Andre Balaton, Pierre Validire, Jean F. Morere. The prevalence of EGFR and KRAS mutations in a population of African immigrants in France is closer to that of Asian populations than to white Europeans. [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 2231. doi:10.1158/1538-7445.AM2014-2231
Abstract 2234: Combined analysis of TERT, EGFR and IDH status define distinct prognostic classes of GBM
Authors: Labussiere Marianne , Boisselier Blandine , Mokhtari Karima , Rahimian Anais , Saulnier Olivier , Marie Yannick , Sanson Marc
Published: May 2015

Background. The importance of telomerase reverse transcriptase (TERT) in gliomagenesis has been recently strengthened by the frequent occurrence of TERT promoter mutations in glioblastomas.

Methods. We sequenced TERT promoter mutation in 395 glioblastomas DNA and confronted the results with histology, genetic profile (IDH1 mutation, EGFR amplification, CDKN2A homozygous deletion, loss of chromosome 9 and 10, TP53 mutation), and overall survival (OS).

Results. TERT promoter mutations (TERTp-mut) were found in 299/395 glioblastomas (75.7%) and were associated with an older age (median 59.6 vs. 53.6 years p<0.0001). TERTp-mut was an independent factor of poor prognosis (OS= 13.8 months vs. 18.4 months), in both IDH mutated (OS= 13.8 months vs. 37.6., p= 0.022) and IDH wild type (IDH-wt) GBM (OS= 13.7 vs. 17.5 months, p= 0.006). TERTp-mut was associated with IDH-wt and EGFR amplification (EGFR-amp) status. In TERTp-wt group, OS was twice longer in EGFR wt compared to EGFR-amp GBM (OS= 26.6 months vs. 13.3 months; p= 0.005). In the EGFR-wt group, TERTp-wt patients had a much better outcome (OS= 26.3 vs. 12.5 months; p< 0.0001), whereas in the EGFR-amp group, TERTp-mut patients did better (OS = 15.8 vs. 13.3 months; p= 0.05). Taken together the absence of both EGFR-amp and Tertp-mut is associated with better survival (26.5 months for IDH-wt, 36.7 months for IDH-mut patients).

Conclusions. The analysis of TERT promoter mutations, in combination with EGFR amplification and IDH mutation status, refines the prognostic classification of GBM.

Citation Format: Marianne Labussiere, Blandine Boisselier, Karima Mokhtari, Anais Rahimian, Olivier Saulnier, Yannick Marie, Marc Sanson. Combined analysis of TERT, EGFR and IDH status define distinct prognostic classes of GBM. [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 2234. doi:10.1158/1538-7445.AM2014-2234
Abstract 2258: Tempol diet provides chemoprotection against UV-induced skin cancer in mice with epidermal Smad4 deletion
Authors: Mitra Doyel , Li Bian , Zhang Qinghong , Mitchell James B. , Wang Xiao-Jing
Published: May 2015

Solar ultraviolet (UV) radiation is the primary cause of skin cancer. Non-melanoma skin cancer, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common form of skin cancer and accounts for 2-3 million worldwide cases of cancer each year. Smad4 loss occurs frequently in human skin squamous cell carcinoma (SCC), and we have shown that keratinocyte specific Smad4 deletion in K14.Smad4-/- mice leads to reduced expression of DNA repair genes, increased DNA damage and increased susceptibility to UV-induced SCC. Since UV-irradiation leads to oxidative damage to DNA, and unrepaired DNA damage may lead to cancer, so we tested if administration of an antioxidant, 4-hydroxy-tempo (tempol) in the diet of WT and K14.Smad4-/- mice reduces UV-induced skin carcinogenesis. In the present study, we divided WT and K14.Smad4-/- mice into two groups each, one group was fed tempol chow while the other group was fed control chow. All mice were exposed to chronic UV irradiation for 38 weeks from 3 months of age. Indeed, the tempol diet led to delayed onset and reduced number of skin tumors in K14.Smad4-/- mice. Tempol diet led to reduced 8-hydroxyguanosine staining, indicative of reduced oxidative DNA damage, as well as reduced cox-2 expression in the skin of K14.Smad4-/- mice compared to K14.Smad4-/- mice on control diet. It is plausible that reduced inflammation in these mice led to reduced carcinogenesis, these and other mechanisms are being investigated.

Citation Format: Doyel Mitra, Bian Li, Qinghong Zhang, James B. Mitchell, Xiao-Jing Wang. Tempol diet provides chemoprotection against UV-induced skin cancer in mice with epidermal Smad4 deletion. [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 2258. doi:10.1158/1538-7445.AM2014-2258
Abstract 2262: Elevated peroxiredoxin expression in breast cancer and its protective role in doxorubicin-resistance
Authors: Muhlbauer Jillian , Perlmutter Gregg , McDonald Caitlin , Cintineo Harry , Aiello Caterina , Phelan Shelley A.
Published: May 2015

Peroxiredoxin (Prdx) proteins are thiol-specific antioxidants that protect cells from oxidative stress in various normal and disease states. There are six different Prdx proteins expressed in mammals, each possessing a characteristic tissue and subcellular distribution. Recent studies have revealed elevated Prdx levels in many cancers, suggesting a protective role for these proteins in cancer cell survival. In the present study we analyzed Prdx levels in several human breast tumor samples, and also investigated the function of Prdx proteins in the MCF-7 breast cancer cell line. Analysis of breast tumor and adjacent normal breast tissue from almost 20 patients showed elevated levels of most Prdx proteins in tumor tissue from the majority of patients. To address the role of Prdxs in breast cancer, we used transient siRNA transfections of MCF-7 cells and examined resulting cell viability in the absence and presence of doxorubicin treatment. Our data show that individual suppression of four of six of the Prdx proteins leads to increased cell death, as well as increased sensitivity to doxorubicin-induced toxicity. Finally, we show that clonal selection of doxorubicin-resistant MCF-7 cells by two-week culture in 0.1uM doxorubicin resulted in a marked elevation in the expression of several Prdx proteins. Similar studies are currently underway in the highly metastatic MDA cell line, and possible upstream regulators of Prdx overexpression in these lines are being investigated. Together, these data show a strong protective role for Peroxiredoxins in breast cancer cell survival, and suggest that Prdx overexpression in breast cancer may play a role in chemotherapy-resistance.

Citation Format: Jillian Muhlbauer, Gregg Perlmutter, Caitlin McDonald, Harry Cintineo, Caterina Aiello, Shelley A. Phelan. Elevated peroxiredoxin expression in breast cancer and its protective role in doxorubicin-resistance. [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 2262. doi:10.1158/1538-7445.AM2014-2262
Abstract 2282: Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer
Authors: Fancy Romone M. , Wang Hong , Zhou Tong , Song Yuhua
Published: May 2015

Molecular classification of breast cancers into receptor-based subtypes, including estrogen positive or triple negative breast cancers, establish targets that guide individualized treatment for patients (Journal of clinical oncology:27, 1153-1154, 2009). However, drug resistance observed for breast cancer raises the challegens for the breast cancer treatment (CA: a cancer journal for clinicians 59, 303-313, 2009). Targeting the death receptor-5 (DR-5) to induce breast cancer apoptosis is a promising strategy for breast cancer treatment (Immunity 3, 673-682, 1995, Breast Cancer Res Treat 113, 217-230, 2009). TRA-8 is a DR-5 specific agonistic antibody (Nature medicine 7, 954-960, 2001), and TRA-8 can induce apoptosis in breast cancer cells (Clinical cancer research: 9, 3731-3741, 2003). TRA-8 signal DR-5-mediated apoptotic signal via formation of the death inducing signaling complex (DISC) at the DR-5 death domain (Immunity 12, 599-609, 2000). Calmodulin (CaM) is a mediator of calcium signaling and has been shown to play a role in regulating DR-5 mediated apoptosis (Biochem Cell Biol 87, 919-926, 2009). Understanding the mechanism of DR-5-CaM interaction and the role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation, may unveil CaM-DR-5 binding as a potential target to modulate DR-5 mediated apoptosis. In this study we characterize an interaction between CaM and DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. Complex-immunoprecipitation of DR-5 from MCF-7 and MDA-MB-231 lysates demonstrated endogenous CaM and DR-5 form a complex. Native PAGE of recombinant 6xHis-SUMO-CaM incubated with MCF-7 and MDA-MB-231 lysates showed the formation of a CaM and DR-5 complex. CaM pull down of DR-5 from MCF-7 and MDA-MB231 lysates in the presence of 1 mM Ca2+ or 2 mM EGTA showed CaM/DR-5 interaction is calcium dependent. TRA-8 activation of DR-5 resulted in CaM recruitment into the DR-5 mediated DISC. Additionally, Either EGTA or CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancers. We present evidence for the Ca2+ dependent CaM and DR-5 interaction and a role of CaM-DR-5 binding in regulating DR-5 mediated DISC formation in breast cancer cells. Results from this study provide the basis for the further characterization of CaM-DR5 interactions and its important role in cellular activities, which could lead to novel strategies for effectively regulating DR-5-mediated apoptosis in breast cancer.

Citation Format: Romone M. Fancy, Hong Wang, Tong Zhou, Yuhua Song. Calmodulin binding to DR-5 and the role of CaM-DR-5 binding in DR-5-mediated DISC formation in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2014-2282
Abstract 2285: PKC activation sensitizes certain basal breast cancer cell lines to the Smac mimetic LBW242
Authors: Cornmark Louise , Holmgren Christian , Masoumi Katarzyna , Larsson Christer
Published: May 2015

There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, such as LBW242, which antagonize inhibitor of apoptosis proteins (IAP)s and facilitates cell death. Recent studies have shown that combinations of Smac mimetics and chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with LBW242 induce cell death in two basal breast cancer cell lines (MDA-MB-468 and BT549), that are resistant to LBW242 as a single agent. Eleven other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα which can induce death in the presence of LBW242. The cell death, coinciding with caspsae-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with Caspase-8, as seen in complex II mediated apoptosis. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with LBW242 induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers.

Citation Format: Louise Cornmark, Christian Holmgren, Katarzyna Masoumi, Christer Larsson. PKC activation sensitizes certain basal breast cancer cell lines to the Smac mimetic LBW242. [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 2285. doi:10.1158/1538-7445.AM2014-2285
Abstract 2287: Nuclear localization of N-myc downstream regulated gene 4: A novel therapeutic target in meningioma
Authors: Kotipatruni Rama P. , Ren Xuan , Vanderwaal Robert P. , Thotala Dinesh , Jaboin Jerry J.
Published: May 2015

NDRG4 is a member of the N-Myc downregulated gene family (NDRG) belonging to the alpha/beta hydrolase super family. The roles of NDRG4 in meningioma tumor development have not previously been evaluated. Effective local therapies include surgery and radiation, but there is a need for novel molecular targets to improve survival and reduce morbidity for meningioma patients. We have recently identified the N-Myc down regulated gene 4, NDRG4, protein as being overexpressed in aggressive meningiomas and demonstrated that NDRG4 downregulation results in decreased cell viability, migration and invasive properties. NDRG4 was initially identified as a cytoplasmic protein. In the present study, we proposed that nuclear localization of NDRG4 and the role of NDRG4 in cell proliferation. Subcellular distribution of NDRG4 was identified by western blotting in nuclear and cytosolic extracts. Immunocytofluoresence analysis revealed the NDRG4 nuclear localization. Here we demonstrate that depletion of NDRG4 induces the predominant form of cell death is apoptosis, utilizing DNA laddering and Annexin V/APC flow cytometry assay. We show that apoptosis caused by transduction of lentiviral shNDRG4 involves of p53 phoshorylation at ser 15, Bax activation. Flow cytometry analysis revealed changes in mitochondrial membrane potential (Ψm), which was observed by JC-1. Sub-cellular distribution of Bax and Cytochrome c indicated the mitochondrial-mediated apoptosis. In conclusion, we show that NDRG4 is also a nuclear protein expressed in response to cell proliferation and demonstrated the suppression of NDRG4 induces the apoptosis through p53 and Bax mediated intrinsic apoptotic pathway.

Citation Format: Rama P. Kotipatruni, Xuan Ren, Robert P. Vanderwaal, Dinesh Thotala, Jerry J. Jaboin. Nuclear localization of N-myc downstream regulated gene 4: A novel therapeutic target in meningioma. [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 2287. doi:10.1158/1538-7445.AM2014-2287
Abstract 2291: Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia cells
Authors: Kumar Sanjay , Yedjou Clement , Tchounwou Paul B.
Published: May 2015

Arsenic trioxide (ATO) has recently been successfully used in the treatment of all-trans retinoic acid resistant relapsing acute promyelocytic leukemia (APL) patients both alone or combination with other drugs. Its use as induction and consolidation therapy has resulted in complete remission rate of both de novo and relapsed APL patients. However, the detailed molecular mechanisms of its therapeutic action are poorly known. We have used human leukemia (HL-60) cells as a model to elucidate the anti-cancer properties of arsenic trioxide. We hypothesized that ATO arrests cell cycle progression of HL-60 cells at G1 phase and leading to cell death by intrinsic pathway of apoptotic signaling. To test the hypothesis, we used western blotting, confocal imaging and spectrofluoremetric techniques to identify detailed cellular and molecular mechanisms of ATO action in HL-60 cells. We found that the expression levels of pro-apoptotic molecules (Bax) and cytochrome C were up-regulated, while that of anti-apoptotic Bcl2 protein was down-regulated at 4 and 6mg/mL ATO doses significantly. We also found that ATO stimulates signaling molecules like P38, JNK in a dose dependent manner in HL-60 cells. Oxidative stress and related imbalance of pro- and anti-apoptotic molecules lead to change of mitochondrial membrane potential and opening of the mitochondrial membrane transition pores. Our confocal imaging data shows a clear translocation of Bax from cytosol to mitochondria and of cytochrome c from mitochondria to cytosol in ATO-treated cells. A dose-dependent decrease/drop-down in mitochondrial membrane potential was also observed in ATO-treated cells, as evidenced by JC-1 leveling and spectrofluoremetric potential recording. On the basis of these findings, we conclude that mitochondrial pathway of apoptosis plays a key role in ATO pharmacology and should be further assessed as a drug target in APL chemotherapy.

Citation Format: Sanjay Kumar, Clement Yedjou, Paul B. Tchounwou. Arsenic trioxide induces cell cycle arrest, apoptosis and MAPKinase signaling cascade in acute promyelocytic leukemia 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 2291. doi:10.1158/1538-7445.AM2014-2291
Abstract 2300: Silencing of the transcription repressor HOXA2 by aberrant hypermethylation enhances invasion via MMP-9 activation in NPC
Authors: Peng Chen-Ching
Published: May 2015

Nasopharyngeal carcinoma (NPC) is notorious for its high invasiveness and metastatic ability. In this study, we identified a differential hypermethylated transcription repressor, Homeobox A2 (HOXA2), which may render NPC cells invasive and metastatic. Aberrant hypermethylation of HOXA2 led to low RNA expression in NPC tumors and cells. Addition of methylation inhibitor 5′Aza restored HOXA2 RNA expression in NPC cells. Methylated HOXA2 promoter reduces the binding affinity of the transcriptional co-activator p300, causing transcriptionalrepression of HOXA2. In NPC cells, re-expression of ectopic HOXA2 wascorrelated with decreased invasive ability and reduced metalloproteinase MMP-9 RNA and protein expression. Promoter, ChIP and DNA-pull down assays indicated that HOXA2 competes with the transcription activator, TATA-box binding protein (TBP) for a recognition sequence near the MMP-9 transcription start site, and suppresses MMP-9 transcription. Thus, HOXA2 acts as a suppressor or TBP-antagonist to inhibit MMP-9 expression; while methylation-mediated inactivation of HOXA2 in NPC derepresses MMP-9 production and increases invasion of NPC cells. In NPC plasma samples, increased plasma EBV copy number was correlated with increased in cell-free HOXA2 hypermethylation and elevated MMP-9 levels. Plasma EBV DNA and methylated cell-free HOXA2 can be used as biomarkers for monitoring NPC treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Chen-Ching Peng. Silencing of the transcription repressor HOXA2 by aberrant hypermethylation enhances invasion via MMP-9 activation in NPC. [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 2300. doi:10.1158/1538-7445.AM2014-2300
Abstract 2301: Cross validation of NGS methylated targets using droplet digital PCR (ddPCR)
Authors: Shelton Dawne N. , Litterst Claudia , Regan John F. , Moinova Helen R. , Markowitz Sanford D.
Published: May 2015

DNA methylation is one of the most studied epigenetic modifications. DNA methylation plays an important role in a number of physiological processes as well as common diseases such as cancer and neurodegenerative disorders. In mammals, DNA methylation occurs at the C-5 position of cytosine in CpG dinucle¬otide sequences, which are mainly concentrated in regions known as CpG islands. Methylation in CpG islands within gene promoters usually leads to gene silencing.

Recent data have shown the correlation of methylation and disease status. However, there are significant limitations in both biology and technology. Biologically, methylation status is highly heterogeneous in nature. In normal controls there is never a complete lack of methylation marks, and conversely, never a completely saturated methylation signature. In addition methylation sites of interest are often located in CpG islands, regions where multiple methylation sites occur side by side, making assay design and discrimination challenging. Technological hurdles add significant variability as well. The typical method for determining methylation is bisulfite conversion, which uses harsh chemicals to convert cytosines to uracil. When a cytosine is methylated, it is largely, though not completely, protected from the conversion process. However, bisulfite conversion also causes a great deal of DNA damage, causing the strands to separate and fragment. In most cases of research, the DNAs that researchers want to examine are from FFPE or cfDNA samples, samples which are already both limited and damaged.

Because of these challenges, there is an unmet need for a sensitive yet robust method to analyze DNA methylation in clinical samples in a rapid, inexpensive, and high throughput manner.

Herein we showcase the use of ddPCR for detection of DNA methylation in bisulfite converted gDNA samples. First we demonstrate the superior sensitivity, linearity and robustness of ddPCR methylation detection in the SNRPN promoter, a model system for an imprinted gene involved in neurological disorders. Secondly we show the detection of DNA methylation in the vimentin promoter, which is known to be methylated in cancer (Li et al. Nat. Biotech. 2009). We will demonstrate reproducible detection in replicate bisulfite converted samples and sensitivity.

Citation Format: Dawne N. Shelton, Claudia Litterst, John F. Regan, Helen R. Moinova, Sanford D. Markowitz. Cross validation of NGS methylated targets using droplet digital PCR (ddPCR). [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 2301. doi:10.1158/1538-7445.AM2014-2301
Abstract 2306: Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors
Authors: Kinders Robert J. , Hollingshead Melinda , Thottassery Jaideep , Parker William B. , Pfister Thomas D. , Anderson Lawrence W. , Tomaszewski Joseph E. , Collins Jerry M. , Doroshow James H.
Published: May 2015

Targeting cancer epigenetic control of cell growth via DNA methylation has been successful in treating hematologic diseases, such as Decitabine (DAC) and Azacitidine for Myelodysplastic Syndrome including Acute Myelomonocytic Leukemia. This success has not extended to solid tissue tumors. The Division of Cancer Treatment and Diagnositcs of NCI has initiated pre-clinical development of TdCyd as an agent for treating solid tumors after promising early results in a lung adenocarcinoma xenograft model (NCI-H23). IP dosing at 5MKG (0.56 MTD) in nu/nu mice on a Q5D x 3 cycle schedule resulted in tumor stasis with no accompanying weight loss in the mice. A DAC-treated control arm treated at MTD resulted in tumor growth delay but not stasis, and a 10% weight loss was noted. Intratumoral levels of DNMT1 were reduced to undetectable levels in xenografts post administration of TdCyd by ELISA and Western Blot assays, but were unaffected by DAC treatment. Mass Spectrometry analysis demonstrated incorporation of both TdCyd and thiothymidine (TdThd) into H23 DNA. In vitro experiments on a selected panel of cancer cell lines confirmed the conversion of TdCyd to the triphosphate and re-expression of tumor suppressor proteins p15 and p16. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: Robert J. Kinders, Melinda Hollingshead, Jaideep Thottassery, William B. Parker, Thomas D. Pfister, Lawrence W. Anderson, Joseph E. Tomaszewski, Jerry M. Collins, James H. Doroshow. Pre-clinical development of 4′-thio-2′-deoxycytidine (TdCyd) as a DNA-demethylating agent for use in treating solid tissue tumors. [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 2306. doi:10.1158/1538-7445.AM2014-2306
Abstract 2324: Phospho-MED1 mediates the transcriptional regulation of AR splice variants in castration-resistant prostate cancer
Authors: Liu Gang , Sprenger Cynthia , Wu Pin-Jou , Sun Shihua , Uo Takuma , Haugk Kathleen , Epilepsia Kathryn Soriano , Plymate Stephen
Published: May 2015

The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). Prior to treatment with enzalutamide or abiraterone, around 20-30% of CRPC patients already have high levels of AR-Vs in their metastases, especially ARv567es, and this portends a rapid progression and shorter survival. However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation still remains unclear. A distinct transcriptome enriched with cell cycle genes (like UBE2C) has been associated with AR-Vs, which indicates the possibility of a different transcriptional mechanism than that of wild type AR. In our study, we performed co-immunoprecipitation, ChIP and luciferase reporter assays to explore the components involved in ARv567es activated UBE2C transcription. We found that (i) MED1 is necessary for ARv567es induced UBE2C up-regulation and subsequent prostate cancer cell growth; (ii) p-MED1 is recruited to ARv567es independent of full-length AR; (iii) p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of ARv567es, (iv) ARv567es enhanced UBE2C transcription could be blocked by silencing MED1; (v) ARv567es/p-MED1 signaling cross talks with the PI3K-AKT pathway but not the MAPK pathway, and (vi) FoxA1 and histone methylation are involved in ARv567es/p-MED1 induced UBE2C long range chromatin interactions. In summary, these data indicate that p-MED1 serves as a key mediator in ARv567es transcriptional regulation and suggests a mechanism by which AR-Vs promote the development and progression of CRPC.

Citation Format: Gang Liu, Cynthia Sprenger, Pin-Jou Wu, Shihua Sun, Takuma Uo, Kathleen Haugk, Kathryn Soriano Epilepsia, Stephen Plymate. Phospho-MED1 mediates the transcriptional regulation of AR splice variants in castration-resistant prostate 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 2324. doi:10.1158/1538-7445.AM2014-2324
Abstract 2327: Defining the determinants of sensitivity and resistance to BET bromodomain inhibition
Authors: Follmer Nicole , Mertz Jennifer , Conery Andrew , Bryant Barbara , Hatton Charlie , Centore Richard , Huang Hon-Ren , Spillane Kerry , Sims Robert
Published: May 2015

Selective inhibition of BET bromodomains by small molecule inhibitors has emerged as a promising therapeutic strategy for the treatment of cancer. Accordingly, we have recently initiated clinical trials to assess this mechanism in patients. Recent evidence demonstrates that BET bromodomain inhibition leads to anti-proliferative activity in pre-clinical models of hematological malignancies and solid tumors. This anti-proliferative activity involves down-regulation of the transcription factor MYC, among other cancer-relevant pathways. However, heterogeneity exists in the response to BET bromodomain inhibition at both the molecular and phenotypic level. Given this complexity, the molecular determinants of sensitivity and resistance to BET bromodomain inhibition remains poorly understood. Current efforts are aimed at addressing this key issue by integrating the following: (1) phenotypic endpoints observed upon BET bromodomain inhibition with transcriptional changes, including down-regulation of MYC and other transcription factors important for regulating proliferation and cell death, such as MYB and BCL-2,(2) transcriptional response with BET protein chromatin binding and eviction, and (3) phenotypic endpoints and transcriptional response with genomic context. Our data potentially stratify indications within hematologic malignancies and solid tumors, and provide insight into patient selection strategies.

Citation Format: Nicole Follmer, Jennifer Mertz, Andrew Conery, Barbara Bryant, Charlie Hatton, Richard Centore, Hon-Ren Huang, Kerry Spillane, Robert Sims. Defining the determinants of sensitivity and resistance to BET bromodomain inhibition. [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 2327. doi:10.1158/1538-7445.AM2014-2327
Abstract 2335: Thymoquinone induces the MUC4 mRNA-destabilizing activity of Tristetraprolin
Authors: Lee Hyun Hee , Lee Se-Ra , Leem Sun-Hee
Published: May 2015

Tristetraprolin (TTP) is an AU-rich element binding protein that destabilizes ARE-containing mRNAs. We previously reported that TTP suppresses several target genes such as those encoding proto-oncogenes and cytokines in various cancer cells. However, the signaling pathway to induce TTP expression is not well known. In this study, we investigate that Tymoquinone (TQ) which is an active ingredient derived from the medicinal plant Nigella satia induces the TTP mRNA and protein expression in AGS and MDA-MB231. TQ treatment destabilized a luciferase mRNA containing cIAP2, VEGF and MUC4 ARE. Transfected cells were treated with various kinds of MAPK inhibitors, treatment with Sorafenib (RAF inhibitor) and U0126 (MEK-1/2) reduced the TTP induction activity of TQ in a dose-dependent manner. We confirmed that co-treatment with sorafenib/U0126 and TQ also reduced TTP expression. Following TQ downregulated the expression of MUC4 in previous study, we next examined whether the changes of TTP expression affects the expression of MUC4. Overexpression of TTP decreased the stability of MUC4 mRNA and expression level of MUC4. On the contrary, inhibition of TTP using siRNA increased the MUC4 expression. MUC4 mRNA contains two AREs within the 3′UTR and TTP destabilized a luciferase mRNA containing MUC4 mRNA 3′UTR. Analyses of point mutants of MUC4 mRNA 3′UTR demonstrated that ARE2 were mostly responsible for the TTP mediated destabilization of MUC4 mRNA. Our data suggest that TQ enhances the mRNA and protein level of TTP via the modulation of the Raf-MEK MAPK signaling and suppressed cancer cell growth and promotion through destabilizing MUC4 mRNA as TTP novel target gene.

Citation Format: Hyun Hee Lee, Se-Ra Lee, Sun-Hee Leem. Thymoquinone induces the MUC4 mRNA-destabilizing activity of Tristetraprolin. [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 2335. doi:10.1158/1538-7445.AM2014-2335
Abstract 2336: Interplay of Fra-2/AP-1 and Ezh2/PRC2 orchestrates epidermal differentiation
Authors: Wurm Stefanie , Guinea-Viniegra Juan , Zhang Jisheng , Ezhkova Elena , Wagner Erwin Friedrich
Published: May 2015

Disrupted epidermal differentiation characterizes numerous inflammatory skin diseases and skin cancer that impact >25% of the population. In search for a key mediator of differentiation, we identified Fra-2/AP-1 as a dominant regulator of terminal epidermal differentiation. Loss of Fra-2 in suprabasal keratinocytes was sufficient to give rise to epidermal differentiation defects, keratinocyte hyperproliferation as well as skin inflammation. Additionally, the epithelial-restricted, ectopic expression of Fra-2 in papilloma prone K5-SOS-F transgenic mice resulted in reduced tumor burden due to precocious differentiation.

Fra-2 transcriptionally controlled genes located within the Epidermal Differentiation Complex (EDC), encoding structural proteins, which form the skin barrier as well as keratinocyte derived chemokines. We found that Fra-2 induced EDC genes by binding to conserved AP-1 consensus sites in promoter regions. These consensus sites were also bound by the transcriptional repressor Ezh2. Besides binding to the same consensus sites, the methyl-transferase Ezh2 methylated Fra-2 on lysine residues in basal epithelial progenitor cells. Interestingly, the interaction of Fra-2 and Ezh2 was disrupted in the time course of keratinocyte differentiation, suggesting that the transcriptional activity of Fra-2 is modulated by Ezh2.

Collectively, we identify Fra-2 as an effector of terminal keratinocyte differentiation and uncover a distinct mechanism of how Ezh2 regulates epidermal differentiation by methylation of the transcription factor Fra-2.

Citation Format: Stefanie Wurm, Juan Guinea-Viniegra, Jisheng Zhang, Elena Ezhkova, Erwin Friedrich Wagner. Interplay of Fra-2/AP-1 and Ezh2/PRC2 orchestrates epidermal differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2014-2336
Abstract 2339: Transcriptional basis for loss of cellular differentiation in colon cancer
Authors: Luk Ian Y. , Tran Hoanh , Tse Janson W. , Chueh Anderly C. , Chionh Fiona , Clemons Nicholas J. , Mariadason John M.
Published: May 2015

Background:

Colon cancer is a leading cause of cancer related death. Colon cancer arises due to uncontrolled cell proliferation, failure of cells to undergo apoptosis and loss of cellular differentiation. While the pathways which drive the uncontrolled proliferation of colon cancer cells are relatively well understood, the mechanistic basis for loss of cell differentiation is less well characterized. The aim of this study was to identify novel transcription factors that regulate the differentiation status of colon cancer cells.

Methods:

Affymetrix microarrays were used to identify transcription factors which are significantly differentially expressed between well differentiated and poorly differentiated colon cancer cell lines. Differential gene expression was confirmed by q-RT-PCR in 40 colon cancer cell lines. The well differentiated colon cancer cell line SW948 was transfected with siRNAs using lipofectamine.

Results:

Microarray analysis of well and poorly differentiated colon cancer cell lines identified the EHF transcription factor to be significantly downregulated in expression in poorly differentiated cell lines. Q-RT-PCR analysis demonstrated that while both EHF variants were expressed in a panel of 40 colon cancer cells, variant 2 was the isoform differentially expressed between well and poorly differentiated cell lines. Basal expression of EHF correlated significantly with expression of the differentiation markers villin and KRT20 across the 40 cell line panel. Knockdown of EHF in the well differentiated colon cancer cell line SW948, reduced expression of villin and KRT20 by 82% and 64% respectively. Knockdown of EHF also reduces expression of Cdx1 suggesting it may be act upstream of this known differentiation regulator.

Conclusion:

This study identifies the transcription factor EHF as a novel regulator of cell differentiation in colon cancer.

Citation Format: Ian Y. Luk, Hoanh Tran, Janson WT Tse, Anderly C. Chueh, Fiona Chionh, Nicholas J. Clemons, John M. Mariadason. Transcriptional basis for loss of cellular differentiation in colon 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 2339. doi:10.1158/1538-7445.AM2014-2339
Abstract 2340: Disentangling the Myc:Max from the Mad:Max network
Authors: Rebocho Ana Paula , Littlewood Trevor , Evan Gerard I.
Published: May 2015

The Myc family comprises three basic helix loop helix leucine zipper (bHLHLZ) transcription factors - C-myc, N-myc and L-myc - which regulate a plethora of intra and extracellular processes. Myc, when bound to Max, another bHLHLZ transcription factor, is able to bind specific DNA sequences, recruit coactivator complexes and promote transcriptional activation. Max can also promote transcriptional repression by dimerizing with distinct transcription factors such as Mad. Previous work has shown that Myc expression promotes proliferation whereas Mad expression leads to differentiation, implying that Myc and Mad have opposing roles in cells. Myc and Mad share the same heterodimeric partner, Max, suggesting that the cell proliferation/differentiation fate is determined by a Myc:Max:Mad network. To understand how this equilibrium is maintained, we are investigating the effects of separating the Myc:Max from the Mad:Max network. To separate these networks we have made use of specific Myc and Max mutant proteins in which reciprocal exchanges at the e and g positions of the leucine zipper have been introduced. Whereas the MycEG and MaxEG proteins dimerize with each other they are unable to associate with their wild type counterparts. Using these mutants we will determine whether cell fate is dictated by a balanced Myc:Max:Mad network or whether Max is simply a common binding partner for both Myc and Mad. In order to validate the EG mutants in vitro, we have used mouse embryonic fibroblasts derived from a c-Myc ER/ER mouse model generated in our laboratory, in which ER is fused to endogenous c-terminus, allowing Myc to be toggled on and off at will. These cells have to be cultured in the presence of the ER ligand, 4-hydroxytamoxifen (4OHT), so that C-myc is functional and the cells proliferate normally. Whereas MycEG or MaxEG alone are unable to support growth of these cells in the absence of 4OHT, the proliferative arrest phenotype observed without 4OHT is rescued by co-expression of MycEG and MaxEG. We are currently generating mouse models where C-mycEG and MaxEG will replace the endogenous proteins in order to separate the roles of Myc:Max and Mad:Max in development and adult tissues.

Citation Format: Ana Paula Rebocho, Trevor Littlewood, Gerard I. Evan. Disentangling the Myc:Max from the Mad:Max network. [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 2340. doi:10.1158/1538-7445.AM2014-2340
Abstract 2347: PSA splice variants and their expression in prostate cancer
Authors: Yang Alice S. , Alahapperuma Thushari , Mubiru James N. , Garcia-Forey Magdalena , Shade Robert E.
Published: May 2015

Introduction

Prostate cancer (PCa) is the second most common cancer in American men, resulting in 32,000 deaths annually. Challenges for the management of PCa include the inability to distinguish indolent from aggressive and untreatable castration-resistant PCa. These challenges can be addressed by understanding the molecular basis of cancer initiation and progression. Prostate specific antigen (PSA) is widely used for screening, diagnosing and monitoring of PCa, although it is also elevated in other disorders and therefore lacks specificity and sensitivity. Alternative splicing is a common mechanism used in nature to enhance protein diversity. Alternative splicing of the PSA gene produces at least 15 transcripts of 0.7-6.1 kb (Heuzé-Vourc'h et al 2003). Several variants of PSA have been reported and are shown to be differentially expressed in PCa compared to normal prostates or to benign prostatic hyperplasia (BPH).

Here we describe the identification of four variants of PSA with the potential to improve on the specificity of the PSA test.

Material and Methods

Human prostate cDNAs were from cryopreserved prostate tissues collected after radical prostatectomies and have been described previously (Mubiru et al. 2004). PCR was carried out using primers that amplify the complete PSA mRNA and also those specific for the most common splice variant (Heuzé-Vourc'h et al. 2001). The PCR products were inserted into the PCR2.1-TOPO vector (Life Technologies) and sequenced.

Results

We identified four novel splice variants of the PSA gene.

Novel PSA Splice VariantsTypeNameContaining Exons (Ex) and Introns (In)Molecular Weight (Da)pIPSA mRNAPSAEx3del246Ex1, Ex2, Partial Ex38,5949.61PSA mRNAPSA25RVEx1, Partial In111,1815.55PSA mRNAPSA Yang 28Ex1, Ex2, Ex514,1539.59PSA mRNAPSA Yang 21Ex1, Ex2, Partial Ex3, Ex4, Ex523,6986.64

Results also indicate that some transcripts are differentially expressed in tumors compared to non-tumors.

References

1.Mubiru JN, et al. (2004). Gene 327: 89-98.

2.Heuzé-Vourc'h N, et al. (2003) Eur J Biochem 270: 706-714.

3.Heuzé-Vourc'h N, et al. (2001) Eur J Biochem 268: 4408-4413.

Citation Format: Alice S. Yang, Thushari Alahapperuma, James N. Mubiru, Magdalena Garcia-Forey, Robert E. Shade. PSA splice variants and their expression in prostate 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 2347. doi:10.1158/1538-7445.AM2014-2347
Abstract 2350: Inhibition of Gli1 as a novel therapeutic target for lung squamous cell carcinoma
Authors: Shao Chunli
Published: May 2015

Purpose: It is clear that Hedgehog (Hh) signaling pathway can promote small cell lung cancer development but is not sufficient to drive tumor formation. However, the functional role of Hh pathway in non-small cell lung cancer remains unclear. We found that Gli1, a transcription factor of Hh pathway, is highly expressed in lung squamous cell carcinoma (SSC). The purpose of our study is to identify the functional importance of Gli1 in lung SCC.

Methods: We performed a genome-wide gene expression analysis to examine Hh pathway component expression in 275 lung cancer patient specimens. qRT-PCR and western blot were used to validate their expression in SCC lines. To further explore its biological function in lung SCC we manipulated Gli1 expression genetically or pharmacologically followed by colony formation and tumor formation assays.

Results: Microarray analysis revealed that Gli1, Gli2, Gli3, and Smoothened are significantly upregulated in lung SCC compared to lung adenocarcinoma (AD). Consistently we found the increased expression of Glis in lung SCC lines relative to AD lines by real-time PCR and western blot. Knocking down Gli1 by siRNA decreased liquid colony formation ability and Gli1-luciferase reporter activity in SCC lines. A significant reduction of cell proliferation and liquid colony formation was observed in shGli1 expressing SCC cells compared to control cells. Gli1 specific inhibitor GANT61 strongly inhibited SCC cell proliferation through inducing cell apoptosis. However, Gli1 expression or transcriptional activity is not regulated by classical Hh pathway upstream regulators in lung SCC. We found that blocking PI3K or ERK pathways by pharmacological inhibitors significantly reduced Gli1 expression and cell survival. In addition, TGF-β stimulation promoted Gli1 expression and transcriptional activity in SCC cells.

Conclusions: Our data suggest that Hh pathway downstream factor Gli1 expression is essential for cell proliferation and colony formation in lung SCC. Gli1 is not regulated by canonical Hh pathway regulators, whereas PI3K, ERK, and TGF pathways are involved in its regulation. Blocking Gli1 function is a novel therapeutic strategy for lung SCC treatment.

Citation Format: Chunli Shao. Inhibition of Gli1 as a novel therapeutic target for lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2350. doi:10.1158/1538-7445.AM2014-2350
Abstract 2366: Transcriptome analysis of human metastasis prostate cancer cells deciphers new function of Esrrb
Authors: Lu Yuan , Li Jilong , Cheng Jianlin , Lubahn Dennis B.
Published: May 2015

Orphan nuclear receptor family member, Estrogen Related Receptor β (esrrb) became a research hot topic in recent years. Although the function of esrrb is relatively well studied in stem cells and early embryonic development, little is known about its function in cancer. Esrrb is reported to arrest prostate cancer cell cell-cycle by upregulating p21. Here we investigate the transcriptome alteration induced by esrrb expression and its synthetic ligand DY131 in a human prostate cancer cell line. We distinguished 205 qualified differentially expressed (DE) genes regulated by esrrb alone, and 3202 genes with DY131 treatment with esrrb expression, indicating esrrb has both ligand independent and ligand dependent activity. From the esrrb-regulated transcriptome, we established a hypothetical esrrb transcription regulation model in prostate cancer cells by matching the DNA motif recognized by transcriptional factors (TFs) to the promoter sequences of differentially expressed genes. We found esrrb-regulated gene expression is mediated by TAGLN, HOXB2 and HOXB8 from our model. Our findings potentially provide multiple markers to characterize esrrb's function in prostate cancer.

Citation Format: Yuan Lu, Jilong Li, Jianlin Cheng, Dennis B. Lubahn. Transcriptome analysis of human metastasis prostate cancer cells deciphers new function of Esrrb. [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 2366. doi:10.1158/1538-7445.AM2014-2366