bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3833: Ceramide/tamoxifen nanoliposomal combination - A promising strategy for treatment of chemoresistant breast cancer
Authors: Morad Samy A.F. , Levin Jonathan C. , Kester Mark , Shanmugavelandy Sriram S. , Cabot Myles C.
Published: August 2012

Drug resistance in breast and other cancers results from multiple gene interactions. Ceramide, a key intermediate in the sphingolipid pathway, can act as a powerful tumor suppressor. The employ of ceramide-based agents as opposed to the administration of ceramide generators such as daunorubicin, could be a smart strategy for cancer therapy. Polychemotherapy remains the best option for treatment of breast cancer at the aggressive, metastatic stage. The aim of our study was to investigate the cytotoxic and antiproliferative effects of ceramide (short-chain C6-ceramide) administered in combination with tamoxifen, used here as an inhibitor of ceramide glycosylation. We previously demonstrated the cytotoxicity of this combination in MDA-MB-231 cells; however, little regarding mechanism was established. The present study employs nanoliposomal formulations of both agents, a strategy to enhance efficacy. Four triple-negative chemoresistant breast cancer cell lines, MDA-MB-231, MDA-MB-468, BT-20 and Hs578T were used. Cytotoxicity assays revealed that C6-ceramide/tamoxifen synergistically reduced viability, compared to single agents in all four cell lines. For example, in MDA-MB-468 cells, nanoliposomal C6-ceramide (2.5 µM), tamoxifen (5 µM), and the combination reduced cell viability to 70, 85, and 10% of control, respectively (96 hr). A hallmark of cancer, implicated in tumor growth as well as metastasis, is resistance to induction of apoptosis. Assessment of cellular apoptosis showed that C6-ceramide/tamoxifen treatment promoted DNA fragmentation in a dose-dependant manner, attaining a value that was 36% over control at 24 hr. Introduction of the pan-caspase inhibitor, z-VAD-fmk, completely reversed DNA fragmentation, showing that apoptosis was mediated by caspase activation. The aggressive and metastatic behavior of any tumor is dependent on the ability of the cells to proliferate. Interestingly, the lower concentrations of our drug combination (2.5 µM) induced cell cycle arrest at G1, the first growth phase. In summary, this approach has potential to circumvent limitations of ceramide-based therapies, such as rapid ceramide clearance due to metabolism, the inability to attain therapeutic levels when ceramide-generating agents are employed, and inherent insolubility that is encountered. These findings demonstrate the in vitro efficacy of combination C6-ceramide/tamoxifen nanoliposomal formulations for suppressing growth of breast cancer cells, and suggest that tamoxifen may be an effective adjuvant for enhancing ceramide-driven cell death cascades. Supported by NIGMS 77391 and ABC's, Los Angeles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3833. doi:1538-7445.AM2012-3833
Abstract 3835: Identification and characterization of inhibitors of 5′-nucleotidase cN-II issued from virtual screening
Authors: Jordheim Lars P. , Marton Zsuzsanna , Rhimi Moez , Chaloin Laurent , Cros-Perrial Emeline , Lionne Corinne , Peyrottes Suzanne , Aghajari Nushin , Dumontet Charles
Published: August 2012

Clinical and preclinical observations have lead to the hypothesis that 5′-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se, either to increase the activity of cytotoxic nucleoside analogues. We performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant full length cN-II, soaking experiments with crystals of truncated cN-II and biological evaluation of inhibitors alone or in combination with cytotoxic nucleoside analogues on cancer cells both in vitro and in vivo. The top ranked compounds from virtual screening contained an anthraquinone-derivative (A) and a trisubstituted triazine (B) that were selected for further studies. In vitro enzymatic assay experiments with recombinant protein showed that compound A is a competitive inhibitor with Ki of approximately 3 mM, whereas derivative B is a non-competitive inhibitor with Ki of 1.1 mM. We also obtained crystallographic data at a resolution of 2.0 Å after soaking experiments with crystals of truncated cN-II. These showed interaction between A and F354/N154 situated in the effector site 1 of cN-II, whereas B was not found in crystallographic data even though crystals were degraded in presence of B over a longer time. Derivative A showed different levels of cytotoxicity in vitro on several cancer cell lines such as Raji (IC50=782 µM), RL (IC50=210 µM), CCRF-CEM (IC50=824 µM), MCF-7 (IC50=654 µM), A549 (IC50=569 µM) and HCT-116 (IC50=793 µM), whereas B was much less cytotoxic. When used at 1 mM, A and B did not modify IC50 of the nucleoside analogues cladribine, clofarabine or fludarabine in CCRF-CEM cells. When used at 100 µM, compound A increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5 µM cladribine, 0.05 µM clofarabine or 30 µM fludarabine. The administration of A (200 mg/kg, 5d/w, 4w) to mice xenografted with RL cells, induced a delay of tumor development that was higher than in mice treated with fludarabine (50 mg/kg, 1d/w, 4w). We showed that virtual screening can be used for the identification of potent cN-II inhibitors, and we produced clear evidences for the interaction of one inhibitor with the enzyme. Our biological evaluation indicated interesting activity for one lead compound that will be developed further in order to identify candidates with higher biological activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3835. doi:1538-7445.AM2012-3835
Abstract 3985: Senescent cells in growing tumors: population dynamics and cancer stem cells
Authors: LaPorta Caterina Anna Maria , Zapperi Stefano , Sethna James
Published: August 2012

Tumors are defined by their intense proliferation, but sometimes cancer cells turn senescent and stop replicating. In the stochastic cancer model in which all cells are tumorigenic, senescence is seen as the result of random mutatations, suggesting that it could represent a barrier to tumor growth. In the hierarchical cancer model a subset of the cells, the cancer stem cells, divide indefinitely while other cells eventually turn senescent. Here we formulate cancer growth in mathematical terms and obtain predictions for the evolution of senescence. We perform experiments in human melanoma cells which are compatible with the hierarchical model and show that senescence is a reversible process controlled by survivin. We conclude that enhancing senescence is unlikely to provide a useful therapeutic strategy to fight cancer, unless the cancer stem cells are specifically targeted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3985. doi:1538-7445.AM2012-3985
Abstract 3995: C-terminal fragment of prostate apoptosis response-4 (Par-4) generated by caspase-3 cleavage induces apoptosis in cancer cells
Authors: Chaudhry Parvesh , Singh Mohan , Parent Sophie , Asselin Eric
Published: August 2012

Prostate apoptosis response-4 (Par-4) is a ubiquitously expressed pro-apoptotic tumor suppressor protein. Previously, we have shown that Par-4 undergoes caspase-3 mediated cleavage in normal and cancer cells. In the present study, we demonstrated that caspase-3 generated cleaved fragment of Par-4 displays apoptotic activity. Hela cells were transiently transfected with empty or Myc tagged C-terminal fragment of Par-4. The results from immunofluorescence studies revealed that the cleaved fragment of Par-4 is predominantly localized in the nuclear compartment of the cells. Further, cells expressing C-terminal Par-4 fragment underwent significant apoptosis as compared to empty vector transfected cells as measured by flow cytometry using annexin V/PI staining. Next, we tested whether C-terminal fragment of Par-4 follows similar mechanism of apoptosis induction as the full length Par-4 by the inhibition of protein kinase C (PKC)/NF-κB pathway. Western blot analysis revealed that the overexpression of C-terminal Par-4 fragment reduced the protein levels of Bcl-2 and FLIP as compared to empty vector transfected cells. Overexpression of the C-terminal Par-4 fragment also reduced the levels of phosphorylated IαBβ, however, there was no change in the levels of total IαBβ. Interestingly, C-terminal fragment of Par-4 prevented the nuclear mobilization of NF-κB (p65) compared to empty vector transfected cells. On the other hand, NF-κB (p50) protein levels were found to be decreased in cells expressing the C-terminal Par-4 fragment suggesting that C-terminal fragment of Par-4 possesses pro-apoptotic activity and regulates apoptotic induction by inhibiting the nuclear localization and activity of NF-κB in a manner similar to wild type full length Par-4. To conclude, our data suggest that caspase-3 mediated cleavage of Par-4 results in nuclear localization of C-terminal Par-4 fragment which possesses pro-apoptotic activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3995. doi:1538-7445.AM2012-3995
Abstract 3998: The role of serum amyloid A1 (SAA1) in nasopharyngeal carcinoma (NPC)
Authors: Lung Hong Lok , Cheung Arthur Kwok Leung , Chung Yeung Man , Liu Edison Tak-Bun , Chua Daniel , Lung Maria Li
Published: August 2012

Background and aims: Nasopharyngeal carcinoma (NPC) is a cancer which occurs in high frequency in Southern China. The identification of diagnostic and prognostic markers will be highly beneficial for early stage NPC detection. Previous microcell-mediated chromosome transfer (MMCT) studies showed that the transfer of an intact human chromosome 11 suppresses the in vivo tumor growth of a NPC cell line (HONE1) in nude mouse tumorigenicity assays. We aim to identify candidate genes which are associated with tumor suppression in NPC. Methods: Differential expression analysis of 19K genes was performed in oligonucleotide microarray hybridization for the chromosome 11 microcell hybrids (MCHs)/ tumor segregants (TSs) pairs to hunt for some other unknown NPC genes. The gene expression of the candidate gene was examined in the NPC cell lines and tumor tissues. Functional analysis of restoration of the candidate gene expression was studied. Results: Using oligonucleotide microarray analysis, Serum Amyloid A 1 (SAA1), mapping close to 11p15.1, was identified as showing consistent down-regulated expression in the TSs, as compared to their parental tumor-suppressing MCHs. Gene expression and protein analyses show that SAA1 was not expressed in the NPC HONE1 recipient cells, tumor segregants, and other NPC cell lines; SAA1 was exclusively expressed in the non-tumorigenic MCHs. The mechanism of SAA1 gene inactivation in these NPC cell lines was attributed to hypermethylation. The clinical relevance of SAA1 in NPC was examined by RT-PCR; 54.8% (23/42) of NPC specimens showed either down-regulation or loss of SAA1 gene expression. After transfection of SAA1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. Conclusions: These findings suggest that SAA1 is a candidate tumor suppressor gene in NPC. Acknowledgments Grant support: Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China grant HKU772309 (H.L. Lung)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3998. doi:1538-7445.AM2012-3998
Abstract 4014: The gp130/Stat3 pathway enables Wnt induced regeneration and tumorigenesis in the mouse intestine
Authors: Phesse Toby J.
Published: August 2012

The transcription factor Stat3 is frequently activated in human intestinal tumors. Using a hyperactive gp130 mutant mouse, gp130Y757F (in which the Socs3 site of the gp130 receptor is mutated leading to constitutive activation of Stat3), we have recently demonstrated a key role for Stat3 during inflammation associated colonic cancer (1). However, although gp130Y757F mice are predisposed to gastric adenomas due to hyper Stat3 activation, they do not develop intestinal tumors. Deregulated Wnt signaling is also a key event in intestinal tumorigenesis. Mice which are heterozygous for the negative Wnt regulator Apc (ApcMin/+) develop multiple intestinal neoplasia, and in humans, constitutive heterozygosity for Apc underlies familial adenomatous polyposis (FAP) syndrome. To investigate a possible interaction between Stat3 and Wnt signaling in intestinal cancer we generated compound ApcMin/+ mutant mice to modulate expression and/or activation of Stat3. Compared to ApcMin/+ or gp130Y757F mice, corresponding gp130Y757F ApcMin/+ mice have a significantly reduced lifespan associated with an increase in the number of intestinal tumors. Conversely, when Stat3 is reduced in ApcMin/+Stat3+/− mice the number of polyps in both the small and large intestine is significantly reduced. Tumor initiation, but not tumor growth, is promoted in gp130Y757F ApcMin/+ mice compared to ApcMin/+ littermates. These results suggest that Stat3 is rate limiting for Wnt induced intestinal tumourigenesis. In support of this data we found that the in vitro colony forming potential of human colonic cancer cells (SW480 cells) was reduced when gp130/Stat3 signaling was inhibited. Importantly, inhibition of gp130/Stat3 did not reduce the colony forming potential of SW480 colon cancer cells expressing full-length APC (by stable transfection of APC), suggesting that Stat3 is only rate limiting during cellular conditions of high Wnt signaling. We have recently demonstrated that intestinal regeneration is characterized by high Wnt signaling and requires an intact Wnt signaling pathway (2). Stat3 is also rate limiting for intestinal regeneration in vivo and for intestinal organoid assays in vitro. Together these data provide a novel role for gp130/Stat3 signaling, which becomes rate limiting in situations of high Wnt signaling in the intestinal epithelium including regeneration and tumourigenesis. Mechanistically we find that Stat3 up-regulates Bcl2 (an apoptosis inhibitor protein) expression and allows cells to evade apoptosis, providing a survival advantage. Thus, compound gp130Y757F Bcl2+/− mice have reduced regenerative capacity in vitro and in vivo. (1) Bollrath and Phesse et al. 2009. Cancer Cell (2) Ashton and Phesse et al. 2010. Dev Cell

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4014. doi:1538-7445.AM2012-4014
Abstract 4020: The Sox17 gene hypermethylation is useful as a molecular diagnostic application in early stage of gastric cancer
Authors: Hama Ryota , Watanabe Yoshiyuki , Shinada Kanako , Yamada Yosuke , Oishi Yoshichika , Yoshida Yoshihito , Hiraishi Tetsuya , Oikawa Ritsuko , Maehata Tadateru , Suzuki Hiromu , Itoh Fumio
Published: August 2012

Although minimal invasive treatment is widely accepted for early stage of gastric cancer (GCa), we still do not have any appropriate risk markers to detect residual neoplasia and some potential of recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia. Our goal is to find and identify some candidate genes as a treatment marker using genome wide DNA methylation analysis for early stage of gastric cancer (EGC). We studied Methylated CpG Island Amplification Microarray (MCAM) analysis using 12 gastric washes (each 6 before (Pre) and after (Post) endoscopic treatment in same patients). One of them are Sox17. We examined the DNA methylation status of Sox17 in a validation set consisting of 128 washes samples (Pre, 64: Post, 64) at EGC. We next identified functional studies about Sox17 as a putative tumor suppressor gene. Sox17 showed significantly differential methylation between before and after treatment in EGC patient (Sox17, p<0.0001). Moreover, treating GCa cells that lacked Sox17 expression with a methyltransferase inhibitor, 5-aza-2′-deoxycytidine, restored the gene's expression. Moreover, introduction of exogenous Sox17 into silenced cells suppressed colony formation. Gastric washes based DNA methylation analysis is easy and useful for early detection of recurrence after ER in EGC patients. Our data suggest that silencing of Sox17 occurs frequently in EGC and may play a key role in development and progression of the disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4020. doi:1538-7445.AM2012-4020
Abstract 4021: Breaking the dogma: AWT1 hypermethylation in myeloid leukemia despite high expression
Authors: Guillaumet Amy , Sandoval Juan , Esteller Manel , Issa Jean-Pierre , Sibert Reiner , Monk David
Published: August 2012

Hematological malignancies are the sixth leading cause of cancer related death worldwide. The Wilms tumor 1 (WT1) gene is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. WT1 has been shown to be detectable at low levels only in CD34+/CD38- hematological stem cells but is undetectable after differentiation. WT1 is reported to be an excellent marker for diagnosis and minimal residual disease in acute myeloid leukemia, but the underlying epigenetic alterations are unknown. Using 23 hematological cancer cell lines we confirm the high expression of WT1 and the transcripts derived the from alternative promoter regions (AWT1) in myeloid associated cancer cell lines. To determine if this expression profile is associated with lineage specific DNA methylation changes we performed bisulphite analysis across the loci. Surprisingly we observe hypermethylation of the AWT1 promoter region in despitehigh levels of expression. This hypermethylation is associated with the expected canonical histone modification signature, with high levels of H3K9me3 and low H3K4me3 or H3K9ac. Subsequent bisulphite pyrosequencing of the WT1 and AWT1 promoters in a 356 cytogenetically characterized hematological malignancies revealed that AWT1 was hypermethylated in 63% and WT1 in 8% of primary myeloid cancers at diagnosis, whereas only 26% and 27% where hypermethylated in primary lymphoid cancers. A subset of these patients were treated with decitabine (5-aza-2′-deoxycytidine), a DNA demethylation agent commonly used to treat myeloid leukemia and myeloproliferative disease. The AWT1 methylation state did not change during treatment suggesting that AWT1 methylation state does not correlate with drug response. Our data suggests that combining (A)WT1 expression and AWT1 methylation analyses will be a useful tool for diagnose and confirmation of myeloid-lineage malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4021. doi:1538-7445.AM2012-4021
Abstract 4024: Aberrant DNA methylation of Shisa3 is a novel prognostic biomarker for colorectal cancer
Authors: Jao Tzu-Ming , Tsai Ming-Hong , Chen Wen-Chi , Chen Chun-Chieh , Yu Sung-Liang , Yang Ya-Chien
Published: August 2012

Shisa3, a newly identified tumor suppressor, inhibits invasion and metastasis via regulation of epithelial-mesenchymal transition in non-small cell lung cancer. We first screened 11 colorectal cancer (CRC) cell lines as well as 10 pairs of primary tumors and matched normal mucosas for Shisa3 expression by semi-quantitative RT-PCR. Shisa3 gene was silenced or down-regulated in 8 (72.7%) cell lines and 8 (80%) CRC tumors, suggesting that Shisa3 might be also involved in colorectal tumorigenesis. Accordingly, we further confirmed Shisa3 expression was significantly decreased in 59.6% (31/52) of colorectal carcinomas compared with corresponding normal mucosas by real-time RT-PCR (P < 0.001). Using gene copy number assay, we ruled out chromosomal instability pathway as the major mechanism resulting in Shisa3 silencing in CRC. While Shisa3 mRNA expression was restored in 66.7% (4/6) of CRC cell lines as exposure to DNA methylation inhibitor 5-Aza-2′-deoxycytidine. CpG islands spanning around Shisa3 gene were predicted via MethPrimer program. Aberrant methylation of 5 CpG loci in Shisa3 intron 1 was identified in CRC cell lines and primary tumors by bisulfite sequencing, and their methylation levels determined by pyrosequencing were inverse correlation with Shisa3 mRNA expression. In addition, among 127 pairs of CRC tissues, Shisa3 methylation levels in tumors were much higher than that in corresponding normal mucosas (P < 0.001), and Shisa3 hypermethylation was significantly associated with disease recurrence in stages B and C patients (P = 0.008). Kaplan-Meier survival analysis showed that patients with Shisa3 hypermethylation revealed significantly shortened overall survival (P < 0.001) and disease-free survival (P = 0.006). In conclusion, Shisa3 gene hypermethylation could serve as a molecular predictor of disease recurrence and poor prognosis in CRC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4024. doi:1538-7445.AM2012-4024
Abstract 4032: Consequences of NEIL1 promoter hypermethylation in head and neck squamous cell carcinoma
Authors: Schmezer Peter , Chaisaingmongkol Jittiporn , Warta Rolf , Dyckhoff Gerhard , Oakes Christopher C. , Kuhmann Christine , Geiselhart Lea , Waas Peter , Herold-Mende Christel , Plass Christoph , Popanda Odilia
Published: August 2012

Human endonuclease VIII-like1 (NEIL1) encodes a DNA glycosylase which is suggested to be involved in the repair of oxidative damage in association with DNA replication and transcription. We have recently demonstrated that hypermethylation of the NEIL1 gene promoter occurs in more than two third of head and neck squamous cell carcinoma (HNSCC) patients, and that this promoter hypermethylation correlated with a decreased expression both on the mRNA and protein levels. Further experiments were performed to investigate the relevance and functional consequence of these observations. Demethylation experiments using 5-aza-2′-deoxycytidine and DNMT1 knockdown demonstrated an up to 15-fold re-expression of NEIL1 mRNA in cultured HNSCC cells which initially carried hypermethylated promoter regions. The relevance of promoter methylation for NEIL1 expression was further studied by a luciferase promoter assay using CpG-free pCpGL vector constructs in 293T cells. A promoter construct covering the 5′ region of NEIL1 was treated with CpG methyltransferase (M.SssI) or remained untreated. Luciferase expression of the methylated NEIL1 promoter construct was significantly reduced compared to the unmethylated construct (p<0.01, T-Test). Furthermore, NEIL1/2 glycosylase activity was measured in vitro using a bubble structure substrate containing 5-OHU and by quantifying the cleavage products. Nuclear extracts of HNSCC tissue samples which carried NEIL1 promoter hypermethylation showed a more than 2-fold decrease in NEIL1/2 activity when compared to extracts prepared from normal tissue samples without promoter hypermethylation. Taken together, the presented results strengthen the hypothesis that promoter hypermethylation can reduce NEIL1 gene expression and can lead to a significant loss of NEIL1/2 glycosylase activity in HNSCC tissue. NEIL1 hypermethylation may alter the cellular DNA repair capacity and thus play a role in modulating the response to DNA damaging therapies of HNSCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4032. doi:1538-7445.AM2012-4032
Abstract 4039: Loss of imprinting in PEG3, MEST and ARHI/DIRAS3 in invasive breast cancer
Authors: Barrow Timothy M. , Ellsworth Rachel E. , Harris Holly R. , Barault Ludovic , Valente Allyson , Shriver Craig D. , Michels Karin B.
Published: August 2012

Genomic imprinting is the epigenetic regulation of genes to enable monoallelic expression according to parental origin. Imprinting is established in the germline and is particularly important in the regulation of fetal development; loss of imprinting (LOI) has been implicated in the development of a variety of childhood disorders and several cancer types. We investigated loss of imprinting in PEG3, MEST and ARHI/DIRAS3 in breast cancer. Blood and tissue samples from women with benign breast disease or no abnormalities (n = 52) and patients with invasive breast cancer (n = 37) were provided by the Clinical Breast Care Project at the Walter Reed Army Medical Center (Washington, USA). DNA extracted from the samples was bisulfite converted and the methylation status of the three genes determined by pyrosequencing. Loss of PEG3 imprinting was observed in 2% of benign tissues and 34% of invasive cancers. LOI in MEST was similarly observed in 4% of benign tissues and 39% of invasive cancers, while in ARHI/DIRAS3 it was observed in 11% of benign samples and 54% of invasive ones. LOI was not observed in blood samples for any of the three genes, suggesting that these epigenetic alterations are highly tissue-specific. There was no correlation between the methylation profiles of the three genes (Spearman's rank correlation coefficient; p = >0.25). Loss of imprinting in PEG3, MEST and ARHI/DIRAS3 appears to be a frequent event in breast cancer, and it is more common in invasive forms than benign proliferative breast disease. The impact of the observed methylation changes upon allele-specific expression is currently investigated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4039. doi:1538-7445.AM2012-4039
Abstract 405: Innate immunity in taxol-mediated tumor regression
Authors: Kuhn Hallie E. , Orth James , Weissleder Ralph , Mitchison Timothy
Published: August 2012

Taxol is a common chemotherapeutic used against many tumor types, including breast, ovarian and lung tumors. However, the mechanism by which it promotes tumor regression is unclear. While in cell culture taxol kills dividing cells, its ability to promote regression of slow growing tumors in some patients suggests it may have additional actions in vivo. To better understand taxol drug response in vivo we profiled xenograft tumors 1, 4, and 7 days post-drug treatment for changes gene expression and cytokine levels. Enrichment of genes associated with immune response pathways, and an increase in chemotactic cytokines Rantes and Mip-1α, suggest a pro-inflammatory immune response. In accordance, immunohistochemistry confirms that tumor associated macrophages (TAMs) are recruited following treatment. While TAMs are generally considered pro-tumorigenic, our system suggests that macrophages may play a cytotoxic role under certain conditions. Understanding the mechanisms by which macrophages can be pro or anti-tumor growth following drug treatment will shed light on the heterogeneity in patient response and may suggest more efficacious, targeted treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 405. doi:1538-7445.AM2012-405
Abstract 4064: 3-D imaging of human colorectal cancer tissues with optical clearing: Spatial analysis of the tumor vasculature through transparent specimens
Authors: Liu Yuan-An , Pan Shien-Tung , Hou Yung-Chi , Tang Shiue-Cheng , Chung Yuan-Chiang
Published: August 2012

Introduction: Microscopic analysis of tumor vasculature in the endoscopic and surgical biopsies is a valuable diagnostic tool to evaluate cancer treatment at different stages. However, the standard microtome-based 2-dimensional (2-D) histology provides only limited perspective of the dispersed vasculature. To provide a global view of the vascular network, in this research we employed optical clearing to generate transparent specimens for penetrative, 3-dimensional (3-D) visualization of the tumor vasculature in the human colorectal cancer tissues. Methods: Colorectal cancer tissues were obtained from colectomies and endoscopic procedures. Samples of normal mucosa were obtained at least 5 cm apart from the carcinoma. Nuclear and CD34 immuno-staining were used to reveal the mucosal microstructure and vasculature. Optical clearing – use of immersion solution to reduce random scattering as light travels across media (Liu et al., Neurogastroenterol Motil 23:e446, 2011; Fu & Tang, Gastroenterology, 139:p1100, 2010) – was applied to prepare transparent specimens for 3-D imaging via confocal microscopy. Afterward, the specimens were extensively washed with saline to reverse the clearing step and then underwent the standard microtome-based 2-D histology. Avizo projection algorithms were used for image rendering and analysis. Results: The improved photon transmission resulting from optical clearing led to penetrative imaging (up to 300 µm) of the mucosal microstructure and vasculature with high definition. In the normal mucosa, we observed the peri-cryptic capillaries being orderly arranged as a network embedded in the hexagonal honeycomb structure of stroma. In comparison, the vessels in the colorectal carcinoma became dilated and distorted with irregular and disoriented architecture embedded in the deformed mucosa. Significantly, our 3-D image data provided a continuous flow of the anatomic information for panoramic projection and spatial analysis of the vessel density to compare and distinguish the normal and the tumor vasculature. In addition, matched tissue information was observed between the 3-D confocal and the standard H&E and IHC images, indicating our optical method is reversible and can serves as an add-on to the current 2-D histological evaluation of the colorectal cancer. Conclusion: Our optical method connects 3-D microscopy of the human colorectal cancer tissues to the standard microtome-based 2-D histology. This approach is particularly useful in 3-D projection and spatial analysis of the vasculature for future clinical evaluation of the colorectal carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4064. doi:1538-7445.AM2012-4064
Abstract 4080: 5-Azacitydine augments the anti-glioma efficacy of Herpes-based oncolytic virotherapy
Authors: Okemoto Kazuo , Wagner Benjamin P. , Haseley Amy M. , Price Richard L. , Kaur Balveen , Chiocca Antonio E.
Published: August 2012

Oncolytic virotherapy (OV) using modified Herpes simplex virus (HSV) type 1, is currently being evaluated for safety and efficacy in numerous phase I-III clinical trials. However, the host innate immune response and a compromised ability to counter cellular defense responses in order to sustain efficient viral replication in tumor cells is still a major obstacle to efficacious virotherapy. Sodium valporate (VPA) is a simple branched-chain fatty acid FDA-approved medication used in treating epilepsy. We previously demonstrated that pretreatment of cultured glioma cells with VPA significantly increases OV replication. However, the mechanism underlying VPA mediated increased virus replication has not been fully elucidated. Understanding the mechanism of this effect can lead to the development of better pharmacologic sensitizers for OV therapy. We found that activation of STAT3 and inhibition of DNA methylation are involved in VPA's ability to enhance virus replication. Furthermore, we demonstrated that the combination of a DNA methylation inhibitor, 5-Azacitydine (5-Aza), at a 1-2 uM concentration coupled with IL-6-mediated (10ng/mL) STAT3 activation effectively enhanced OV replication 125 fold when compared with OV alone. This combination was comparable to what was previously observed when VPA alone was utilized to increase OV replication. The combination also showed increased anti-glioma activity (ED50 of 5-Aza: 2 uM; with IL-6, 0.3 uM) when compared to only VPA treatment. Given these in vitro results, we are currently performing experiments in mice assessing the effect of 5-Aza pretreatment on enhancing OV therapy of gliomas. Oncolytic HSV in combination with the DNA methylation inhibitor, 5-Aza, and activation of STAT3 represents a promising strategy for treating human gliomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4080. doi:1538-7445.AM2012-4080
Abstract 4083: Increased DNA methyltransferase activity and DNA methylation following epidermal growth factor stimulation in ovarian cancer cells
Authors: Samudio-Ruiz Sabrina L. , Hudson Laurie G.
Published: August 2012

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In patients with ovarian cancer, ascites fluid builds up and contains abundant levels of Epidermal-Growth-Factor-Receptor (EGFR) activators. This fosters a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as RAS, regulate DNA methylation and expression of DNA methyltransferases. We hypothesized that mimicking this microenvironment of chronic EGFR activation in culture could alter DNA methylation or expression of DNA methyltransferases in ovarian cancer cells. We found evidence that EGFR activation increased DNA methyltransferase activity by ∼3 fold acutely and by ∼7 fold following long term EGFR activation. Furthermore, this increase in activity was dependent on continued EGFR activity and resulted in a 50% increase in global DNA methylation. We also found that constitutive EGFR activation can decrease expression of DNMT3A. However, this decrease does not explain the observed increases in DNMT activity or methylation, thus future experiments will focus on a potential mechanism that mediates these EGF induced alterations to DNMT activity and DNA methylation. The data support a role for EGFR in the process of DNA methylation and accumulation during ovarian cancer progression; thus, suggesting that epigenetic therapy, used in combination with conventional chemotherapy, may be beneficial at preventing cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4083. doi:1538-7445.AM2012-4083
Abstract 4086: Location matters: The impact of the location of CpG sites on the specificity of methylation biomarkers for HCC
Authors: Jain Surbhi , Chen Sitong , Lin Selena Y. , Boldbaatar Batbold , Xie Lijia , Chang Ting-Tsung , Hu Chi-Tan , Hamilton James P. , Evans Alison A. , Song Wei , Meltzer Stephen J. , Block Timothy M. , Su Ying-Hsiu
Published: August 2012

Hepatocellular carcinoma (HCC) is the second fastest growing cancer and one of the top 10 causes of cancer deaths in the United States. The 5-year survival rate is 14%, ranging from 26% among early stage tumors to only 2% in late stage HCC. The only available biomarker for screening, serum alpha-fetoprotein (AFP), has a low sensitivity of detection (40%-60%), so the need for a better detection method is urgent. Given the heterogeneous nature of HCC, a panel of genetic and epigenetic biomarkers would be needed to obtain high sensitivity for the early diagnosis of HCC. In the course of assembling a panel of epigenetic markers for HCC, we noticed variable HCC specificity of three extensively studied potential methylation biomarkers, APC, GSTP1, and RASSF1A. We hypothesized that the specificity of promoter methylation of the tumor suppressor genes APC, GSTP1, and RASSF1A as a biomarker for HCC could depend upon the location of the CpG sites analyzed. To test this hypothesis, we used bisulfite-PCR sequencing to compare the methylation status of the promoter region of these three genes in DNA isolated from HCC, matched adjacent non-HCC, cirrhosis, hepatitis, and normal liver. We identified HCC-specific CpG methylation sites and unique liver-specific methylation patterns in the promoter region of the APC (Jain et. al, PloSOne 2011) and GSTP1 genes and in a more HCC-specific promoter region (p < 0.05 by Fisher's exact test) of the RASSF1A gene. Together, these results proved our hypothesis and provided an interpretation for the variability in the literature. Using PCR-based technology, we have developed assays for a panel of genetic and epigenetic markers for liver cancer screening. These assays were first tested in tissue to determine their ability to distinguish HCC (n=120) from cirrhosis (n=35) and hepatitis (n=35). The AUROC is 0.809 for mAPC, 0.756 for mGSTP1, 0.887 for mRASSF1, and 0.733 for TP53. When combined, this panel of DNA markers had an AUROC of 0.931 and identified 90% of AFP-negative tumors. This study clearly demonstrates (1) the importance of location of CpG site methylation for HCC specificity and the way in which liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC and (2) the potential of a sensitive DNA test for HCC screening. An analysis of the performance of this panel of markers in peripherals (blood and urine) for HCC screening is in progress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4086. doi:1538-7445.AM2012-4086
Abstract 4087: Microbiomic subprofiles and MDR1 promoter methylation in head and neck squamous cell carcinoma
Authors: Bebek Gurkan , Bennett Kristi L. , Funchain Pauline , Campbell Rebecca , Seth Rahul , Scharpf Joseph , Burkey Brian , Eng Charis
Published: August 2012

Background: Clinical observations and epidemiologic studies suggest that the incidence of head and neck squamous cell carcinoma (HNSCC) correlates with dental hygiene, implying a role for bacteria-induced inflammation in its pathogenesis. Differences in microbiota in HNSCC compared to matched normal oral mucosa, and whether these differences are accompanied by epigenetic modifications in selected HNSCC-associated genes and clinico-pathologic features has not yet been investigated. Standard microbiologic culturing techniques miss many fastidious organisms. Methods: Human genomic DNA and associated microbial DNA were isolated from 42 prospectively accrued consecutive HNSCC tumors and paired normal oral mucosae. Microbiomic profiling was carried out by serially characterizing bacteria-specific 16S rRNA and taxa classified by the presence of specific polymorphisms in the 16S rDNA genes. Promoter methylation and microbial populations of each sample was determined. Microbiomic profiles were compared between HNSCC and matched normal mucosa, to specific promoter methylation in four genes (MDR1, IL8, RARB, TGFBR2) previously linked to HNSCC or inflammation, and to clinico-pathologic features. Results: Specific microbiomic profiles significantly associate with HNSCC over normal mucosae. While methylation of both MDR1 (p<10-9) and IL8 (p<0.02) genes significantly associate with HNSCC, only MDR1 methylation associates with specific microbiomic profiles in HNSCC over normal mucosae. Furthermore, MDR1 methylation correlates with regional nodal metastases in the context of two specific bacterial taxa, Enterobacteriaceae and Tenericutes. Additionally, microbial populations can separate tumors by tobacco status (p<0.005), but not by alcohol status (p=0.37). Conclusions: Specific microbial populations may contribute to HNSCC pathogenesis, possibly by triggering inflammation and consequent aberrant DNA methylation. These associations could lead to better understanding of the pathogenesis and treatment of HNSCC, suggesting novel roles for demethylating agents and probiotic adjuncts particularly for patients with advanced or refractory disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4087. doi:1538-7445.AM2012-4087
Abstract 4088: Cotreatment with decitabine and paclitaxel is affected by the re-expression of drug-metabolising enzymes
Authors: Cosentino Laura , Masrour Nahal , Burns Julie , Fang Weishuo , Brown Robert , Burns Phil , Pors Klaus
Published: August 2012

The literature describes the role of DNA methylation in regulation of tumour suppressor genes, DNA repair genes, but not much attention has been paid to drug metabolising enzymes (DMEs). Some evidence has been reported and includes methylation of the promoter regions of CYP450 and ABC transporters. Cotreatment with decitabine (DAC) and paclitaxel (PAC) has been reported in several studies, but few of these have reported in a focused manner on the effect on DMEs. As a consequence, the major DMEs involved in the detoxification of PAC (namely ABCB1, CYP2C8 and CYP3A4) were investigated. The aldehyde dehydrogenase enzymes (ALDH1A1, ALDH2 and ALDH3A1) were also explored, as they have recently been shown to play a chemo-protective role against taxanes. Analysis of the CpG island methylation level indicated that all the above-mentioned DMEs were densely methylated and that treatment of MCF-7 cells with DAC for 24 h resulted in a decrease in methylation within the gene promoter region. Furthermore, correlation of decrease in DNA methylation with increase in mRNA level of ABCB1, CYP3A4, ALDH2 and ALDH3A1 was observed, but not for CYP2C8 and ALDH1A1. However, only the ABCB1 protein level was shown to significantly increase. Chemosensitivity of DAC combined with PAC was tested in MCF-7 cells: treatment with DAC for 24 h was followed by PAC exposure on days 4, 6 or 8. Evaluation of the cotreatment on cell survival demonstrated that the cytotoxic potential of PAC was reduced proportionally to the exposure time to DAC before PAC treatment, suggesting that ABCB1 protein confers chemo-protection on the cells. To investigate whether this resistance was applicable to other taxanes with proven efficacy against MDR-resistant cells, the preclinical agent Lx2-32c (aziditaxel) was evaluated. Lx2-32c also lost potency similar to PAC, indicating that ABCB1 is responsible for loss of activity of these two taxanes. The same experiments were also carried out in HeLa cells as they were shown to contain low level of DNA methylation of the above-mentioned DME genes including the ABCB1 gene. DAC treatment did not lead to significant reactivation at protein level and cotreatment of DAC and PAC produced a synergistic effect on cell killing. In conclusion, our data demonstrate that cotreatment of DAC and PAC can produce a synergistic effect, but that it is affected by the re-expression of DMEs. Our observations provide useful information for clinical evaluation of combination strategies comprising DAC and PAC, and chemotherapeutics in general that are affected by DMEs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4088. doi:1538-7445.AM2012-4088
Abstract 4100: Aberrant methylation of the PTPRO gene in peripheral blood as a potential biomarker in esophageal squamous cell carcinoma patients
Authors: Zhang Hao , You Yanjie , Chen Yuping , Zheng Xiaoxuan , Meltzer Stephen J.
Published: August 2012

Purpose: Compared with tyrosine kinases, our understanding of protein-tyrosine phosphatases (PTPs) in cancer is limited, and further knowledge could provide new therapeutic targets and diagnostic markers. Inactivation of protein tyrosine phosphatase receptor-type O (PTPRO), a new member of the PTP family, by hypermethylation has been described in several forms of cancers. We evaluated PTPRO hypermethylation as a potential epigenetic biomarker in esophageal squamous cell carcinoma (ESCC). Experimental Design: In 36 primary ESCC tissue specimens and matched peripheral plasma and buffy coat samples, we determined the methylation status of PTPRO by performing methylation-specific PCR. Correlations with PTPRO methylation and clinicopathologic features were also examined. Results: PTPRO hypermethylation was observed in 27 (75%) of 36 primary tumors and correlated significantly with depth of invasion (T-stage, P = 0.013). Among matched peripheral blood samples from ESCC patients, 13 (36.1%) of 36 had detectable methylated PTPRO in plasma, and 15 (41.7%) of 36 had it in the buffy coat. No methylated PTPRO was observed in normal peripheral blood samples from 10 healthy individuals. In addition, promoter methylation correlated with loss of PTPRO mRNA expression, and demethylation by 5-aza-dC treatment led to gene reactivation in PTPRO-methylated and -silenced ESCC cell lines. Conclusions: This is the first report for detection of PTPRO as a non-invasive biomarker for solid tumors in peripheral blood. Our findings suggest that hypermethylated PTPRO occurs frequently in ESCC. Its detection in the peripheral blood of ESCC patients illustrates its potential clinical application as an epigenetic biomarker for noninvasive diagnosis and disease monitoring.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4100. doi:1538-7445.AM2012-4100