bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4051: MiR-181b expression levels decreases during the progression of the Chronic Lymphocytic Leukemia: a new potential prognostic tool
Authors: Visone Rosa , Veronese Angelo , Rassenti Laura Z. , Balatti Veronica , Taccioli Cristian , Kipps Thomas J. , Croce Carlo M.
Published: February 2011

In patients with Chronic Lymphocytic Leukemia (CLL) the prognosis is extremely variable, ranging from a very short to a normal lifespan. Classic clinical stages, based on biologic and clinical parameters, have been the most important factor for assessing prognosis in CLL patients; however, they are not devoid of limitations. Therefore, investigations are ongoing for searching new parameters in an attempt to add prognostic power to clinical stages. In this study we performed a genome-wide microRNA expression profiling to investigate the role of microRNA genes in the progression of CLL and to develop better prognostic indicators of these disease. We compared paired samples from CLL patients having an indolent and stable disease with their progressed aggressive form. We found that miR-181b was consistently down-regulated in the aggressive form of the disease compared to its previous stable counterpart. Further studies on serial time points from the same patient demonstrated that the strong decrease of the miR-181b expression levels occurs before the disease is clearly aggressive Moreover, we observed that the miR-181b expression remain constant over the time in two time points from CLL patients having a stable course; entailing a decreased levels of the miR-181b in the aggressive form of the CLL as a specific effect due to the progression of the disease. Finally in this study we investigate the biological role of the putative prognostic indicator related to the TCL1A, which is a critical gene in the pathogenesis of the CLL. In conclusion our data hint a prognostic role of the miR-181b in CLL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4051.
Abstract 4052: Analysis of the matrix metalloproteinase family reveals MMP8 is often mutated in melanoma
Authors: Wei Xiaomu
Published: February 2011

Matrix Metalloproteinases (MMPs) have long been associated with multiple biological processes and a variety of pathological conditions, including cancer because of their ability to degrade extracellular matrices, which facilitates invasion and metastasis. We performed a mutational analysis of the Matrix Metalloproteinase gene family in human melanoma and identified 28 somatic mutations in eight MMPs, affecting 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP-8 enzyme activity. Expression of wild-type but not mutant MMP-8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression. Our findings emphasize the need to test the role of each MMP in an individual manner and to precisely define its functional role in cancer. This may allow the development of individualized therapy based on the mutant MMP present in specific tumors. To extend our knowledge of fundamental roles of proteases in cancer progression, we will compliment the studies by performing a systematic sequence analysis of all ADAMs (A Disintegrin And Metallproteinase) and the related ADAMTS family. Similar to MMPs, ADAM/ADAMTS belong to a super family of zinc-based proteinases, the metzincins. Many members of the ADAM/ADAMTS family have been associated with tumorigenesis and tumor progression. Therefore, our comprehensive genetic studies will provide insight into melanoma pathogenesis. In-depth illustrations of the biochemical and biological effects of the mutated genes will permit the design of personalized treatments for melanoma patients.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4052.
Abstract 4064: Development of ALN-VSP: An RNAi therapeutic for solid tumors
Authors: Toudjarska Iva , Buck Tim , Brodsky Joshua , Akinc Akin , Racie Timothy , MacLachlan Ian , Sah Dinah W. , Gollob Jared , Bumcrot David
Published: February 2011

Malignancies of the liver, including primary (hepatocellular carcinoma) and secondary (metastatic) tumors, represent a significant unmet medical need. In most patients with liver metastases, extra-hepatic tumors are also present. We are developing a therapeutic for solid tumors that is comprised of lipid particle (SNALP)-formulated small interfering RNAs (siRNAs) targeting VEGF and the mitotic kinesin, KSP (Eg5). For each target, potent siRNA duplexes were selected following extensive screening in tissue culture cells. A SNALP-formulated combination of the KSP and VEGF siRNAs (referred to as ALN-VSP) was tested in orthotopic liver tumor models, as well as models of extra-hepatic tumors. To generate orthotopic liver tumors, human hepatoma (Hep3B) or colorectal carcinoma (HCT116) cells were implanted directly into the livers of immunocompromised mice. These cell lines were also used to establish tumors at extra-hepatic sites including the lymph nodes and peritoneal cavity.

Studies in which tumor-bearing mice were treated with ALN-VSP demonstrated that each siRNA makes a distinct contribution to efficacy. ALN-VSP treatment led to accumulation of aberrant mitotic figures (monoasters), a hallmark of KSP inhibition, in both types of orthotopic liver tumors, as well as in extra-hepatic tumors of different origin. Evidence of therapeutic VEGF inhibition was shown by marked reductions in tumor microvessel density and intratumoral hemorrhage in orthotopic tumors. Similar effects on tumor vasculature were obtained with a SNALP formulation of the VEGF siRNA alone. Finally, we demonstrated that multi-dose administration of ALN-VSP significantly prolongs survival of mice harboring advanced orthotopic liver tumors. A Phase 1 clinical trial of ALN-VSP is ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4064.
Abstract 4074: Alterations of NDP kinase A/ NM23-H1 deregulate c-myc transcription
Authors: Chan Kai-Hui , Paris Larry , Ma Lin-Jen , Wei Renn-Shiuan , Tan Choon-Yee , Chang Christina L.
Published: February 2011

Altered expression and mutations of NDP kinase A (NDPK-A), encoded by nm23-H1, have been detected in patients with metastatic tumors. One of new functions of NDPK-A is to participate in gene regulation. However, its role in regulating c-myc transcription remains unclear. Here we demonstrate for the first time that NDPK-A specifically bound nuclease hypersensitive element (NHE) III1 of the c-myc promoter in vitro and in vivo. Metastasis-associated S120G mutation of NDPK-A (NDPK-AS120G), but not S120G and N82S double mutations (NDPK-AS120G/N82S), retained the DNA-binding activity. In human NB69 neuroblastoma and HeLa cervical cancer cells, a high level of ectopic NDPK-A or NDPK-AS120 suppressed c-myc transcription based on the CAT reporter and RT-PCR. In contrast, shRNA-mediated knockdown of NDPK-A enhanced c-myc transcription. Without the DNA-binding activity, however, NDPK-AS120G/N82S did not display an effect on c-myc transcription. NDPK-A appears to exert transcriptional suppression via not only NHE III1 but also upstream cis-element(s) of c-myc promoter. The role of NDPK-A in regulating c-myc transcription was supported by nuclear localization of NDPK-A in cells and by negative correlation of NDPK-A and c-myc mRNA levels in several human cancer subtypes. Our findings provide a link between NDPK-A alterations and c-myc transcriptional deregulation in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4074.
Abstract 4081: Antisense against U6 induces cytotoxicity in cancer cells
Authors: Lin Xiaoqin , Hwang Larn , Desai Neil , Trieu Vuong
Published: February 2011

Background: The U6 snRNA is one of the most conformationally dynamic RNAs in the spliceosome. In preparation for spliceosome assembly, the U6 3′ intramolecular stem-loop unwinds and the exposed sequences pair with U4 RNA to form the U4/U6 small nuclear ribonucleoprotein complex. After assembly of the spliceosome on an intron, spliceosome activation occurs, during which U4 RNA is unwound from U6 RNA. U6 then base-pairs with U2 RNA to form the catalytically active form of the spliceosome. Upon activation of the spliceosome the “U6-ISL” structure forms to position important nucleotides of the U6 snRNA at the 5′splice site so they can base-pair with the pre-mRNA at the 5′ prime splice site. Here we report the cytotoxic activity against cancer cells treated with antisense DNAs against U6, especially those targeting U6-ISL.

Methods: The functional activity of U6 antisense oligos was tested by cytotoxic and apoptosis assays. In the cytotoxic assay, U6 antisense oligos were reverse transfected into various mammalian cell lines, and the cytotoxic activity was measured by cell-titer blue 72 hr post-transfection. In the apoptosis assay, U6 antisense oligos are reverse transfected into various mammalian cell lines, and the apoptosis was measured by the release of caspase 3/7 72 hr post-transfection. To evaluate the knockdown ability of antisense oligos, the expression level of U6 snRNA was measured by Taqman Gene Expression Assay. The localization of U6 antisense oligos in tissue assay was studied by ISH. The effects of U6 antisense oligos on cellular protein and mRNA profile were also examined using microarray technology.

Results: About 100 U6 antisense oligos were synthesized. Several U6 antisense oligos were identified to significantly induce cytotoxic activity of HT29 and other cancer cell lines but not in normal cells, such as hepatocytes. No significant apoptosis activity was induced by any U6 antisense oligos. The identified U6 antisense oligos localized at the nucleus and knocked down the U6 snRNA in vitro, suggesting that U6 antisense oligos target to U6 snRNA and lead to the downregulation of U6 snRNA. mRNA microarray and antibody array showed that U6 oligos regulate various mRNA and protein expression in cancer cells. The most effective U6 antisense oligos were those targeting the U6-ISL.

Conclusions: Antisense against U6, especially those targeting U6-ISL region, were effective in U6 knockdown and cytoxicity against cancer cells in vitro.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4081.
Abstract 4084: Alteration of BC200 RNA biogenesis in cancer cells
Authors: Lee Jungmin , Kim Youngmi , Kim Minseok S. , Lee Younghoon
Published: February 2011

BC200 RNA was initially identified as a noncoding RNA of about 200 nt in somatodendritic domains of human nerve cells. It is thought to act as a translational modulator for local protein synthesis. Since BC200 RNA was not observed in somatic cells other than neurons, it was regarded as a neural-specific RNA. However, the neuron-only expression of BC200 does not hold true because it is highly expressed in a number of tumors that are of non-neuronal origin. In this study, we examined biogenesis of BC200 RNA in tumors in detail using cancer cell lines. BC200 RNA was expressed at high levels in several cancer cell lines, but at low levels in normal cell lines. Primer extension analysis and self-ligation RACE revealed that the size of BC200 RNA isolated from cancer cells was heterogeneous, implying the difference of BC200 RNA biogenesis between cancer cells and neural cells. The treatment of actinomycin D, a transcription inhibitor, had little effect on the cellular level of BC200 RNA in cancer cells, but led to the rapid degradation of BC200 RNA in normal cells. This result suggests that BC200 RNA is metabolically unstable in normal cells, while it becomes stable in cancer cells. The increase of stability of BC200 RNA in cancer cells could explain why BC200 RNA was observed at high levels in cancer cells. For cancer cells to maintain high levels of BC200 RNA, therefore, they should have a strategy for RNA protection that normal cells cannot provide. If BC200 RNA plays analogous functional roles in cancer cells as in nerve cells, BC200 RNA may provoke tumorigenesis by repressing translation of tumor suppressing genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4084.
Abstract 4090: RASSF1A gene specific methylation by overexpression of miR-200c in lung cancer
Authors: Chun Sung-Min , Jin Min-Young , Choi Jene , Lee Je-Hwan , Hwang Jung Jin , Kim Chung-Soo , Jang Se Jin
Published: February 2011

DNA methylation, which is an important epigenetic mechanism of gene regulation during cellular differentiation, is mediated by enzymatic actions of DNA methyl transferase family (DNMT). In pathologic conditions, however, certain critical genes such as tumor suppressor genes of various human cancers are aberrantly methylated resulting in inactivation of the genes regardless of DNMT amount. In this process, what induces aberrant methylation of a specific gene is currently unknown. We focused actions of microRNA because of their natural function of sequence recognition. As a model, we selected miR-200c and RASSF1A since those are frequently overexpressed and hypermethylated in lung cancer, respectively, and have partial sequence homology between miR-200c and promoter region of RASSF1A. In this study, we tested expression of miR-17, 21, 200c and 205 by quantitative real-time PCR and methylation status of RASSF1A, CDH13, p16ink4a, DAPK1, and APC1 by pyrosequencing assay in 3 lung cancer cell lines and 10 fresh frozen normal and tumor lung tissues. Fluorescent In Situ Hybridization (FISH) using anti-miR-200c probe labeled with Cy-3 dye was performed to confirm expression and localization of mature miR-200c. Interestingly, we found A549 and H460 cells were completely hypermethylated in RASSF1A promoter region and showed high level of miR-200c expression. In contrast, Calu6 cells showed opposite results. In vitro inhibition of miR-200c by transfection of anti-miR-200c oligonucleotide induced gradual decrease in methylation level of RASSF1A in A549 and H460 cells without change in expression of DNMT. Decrease of CpG methylation by anti-miR-200c transfection was associated with increase of RASSF1A gene expression in A549 and H460 cells. Calu6 cell showed no change in methylation level of RASSF1A. This effect was not seen in anti-miR-17 transfected cells. Transfection of anti-miR-200c did not affect methylation level of other frequently hypermethylated genes including CDH13, p16ink4a, DAPK1, and APC1. The results suggest that overexpression of miR-200c induces sequence specific methylation of RASSF1A and provide a clue to gene specific reactivation of epigenetically silenced genes. (This work was supported by the Korea Research Foundation Grant funded by the Korean Government. [KRF-2008-357-E00006], This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (A062254))

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4090.
Abstract 4092: Aberrant miRNA methylation in tumors from surgically resected lung cancer patients and association with survival
Authors: Tan Weiqi , Gu Jian , Hildebrandt Michelle , Huang Maosheng , Wistuba Ignacio I. , Roth Jack A. , Spitz Margaret R. , Wu Xifeng
Published: February 2011

MicroRNAs (miRNAs) are small non coding RNAs of about 22 nucleotides in length, which regulate their target gene expression by binding to the 3′ UTR of mRNA. Recent studies have shown that some miRNAs are regulated by DNA methylation and aberrant miRNA gene methylation contributes to human tumorigenesis. In this study, we used real-time quantitative methylation-specific PCR to detect a panel of miRNA methylation in tumor and adjacent normal tissues from surgically resected lung cancer patients. We found significantly higher methylation levels of miR-124a family, miR-126, miR-193a, miR-9 family, and miR-34b/c in tumor tissues (N=107) than in normal tissues (N=19, P<0.05), but no significant difference was found for miRNA-137 and miRNA-148a. The methylation levels of four miRNAs (miRNA-124a3, miRNA-148a, miR-9-3, and miR-34b/c) were higher in tumors from older patients than those from younger patients. MiR-9-1 (29.56±29.51) and miR-9-3 (30.49±28.50) had higher methylation in tumors from current smokers than never smokers (15.77±15.28 and 10.71±11.32, P=0.036 and 0.021, respectively). There were no significant differences in methylation levels of these four miRNAs between tumors from men and women. Hypermethylation of miR-9-1, miR-9-3, miR-34b/c and miR-148a was associated with significantly better survival with hazard rations of 0.35 (95% CI, 0.17-0.72), 0.48 (95% CI, 0.25-0.91), 0.52 (95% CI, 0.27-0.99), and 0.52 (95% CI, 0.28-0.98), respectively. Our results demonstrated that selected miRNAs are hypermethylated in lung cancer and miRNA hypermethylation might be a useful biomarker in detection and prognosis of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4092.
Abstract 4093: miR-193b is an epigenetically regulated putative tumor suppressor in prostate cancer
Authors: Rauhala Hanna E. , Jalava Sanni E. , Isotalo Jarkko , Bracken Hazel , Lehmusvaara Saara , Oja Hannu , Tammela Teuvo L. , Visakorpi Tapio
Published: February 2011

miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in six prostate cancer cell lines and non-malignant prostate epithelial cells. 38 miRNAs showed increased expression in any prostate cancer cell line after 5-aza-2′-deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR-193b was methylated in 22Rv1 cell line at a CpG island ∼1kb upstream of the miRNA locus. Expressing miR-193b in 22Rv1 cells using pre-miR-193b oligonucleotides caused a significant growth reduction (p<0.001) resulting from a decrease of cells in S-phase of the cell cycle (p<0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR-193b -expressing 22Rv1 cells (p<0.01). Altogether, our data suggest that miR-193b is an epigenetically silenced putative tumor suppressor in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4093.
Abstract 4094: Pancreatic cancers epigenetically silence ZFHX1B, hypomethylate and overexpress miR-200a/−200b, in association with elevated circulating miR-200a and miR-200b levels
Authors: Li Ang , Omura Noriyuki , Hong Seung-Mo , Vincent Audrey , Walter Kimberly , Griffith Margaret , Borges Michael , Goggins Michael
Published: February 2011

Aberrant DNA methylation and miRNA expression play important roles in the pathogenesis of pancreatic cancer. While interrogating differentially methylated CpG islands in pancreatic cancer we identified two members of miR-200 family, miR-200a and miR-200b that were hypomethylated and overexpressed in pancreatic cancer. We also identified prevalent hypermethylation and silencing of one of their downstream targets, ZFHX1B (SIP1, ZEB2), whose protein product suppresses E-cadherin expression and contributes to epithelial mesenchymal transition. In a panel of 23 pancreatic cell lines we observed a reciprocal correlation between miR-200, ZFHX1B and E-cadherin expression with pancreatic cancer associated fibroblasts showing the opposite expression pattern to most pancreatic cancers. In Panc-1cells, which express ZFHX1B, have low E-cadherin expression and do not express miR-200a or miR-200b, exogenous expression of miR-200a and miR-200b down regulated ZFHX1B mRNA and increased E-cadherin expression. However, most pancreatic cancers express miR-200a and miR-200b, but this expression does not affect ZFHX1B expression as the ZFHX1B promoter is silenced by hypermethylation, and in these cancers E-cadherin is generally expressed at or above normal levels. Both miR-200a and miR-200b were significantly elevated in the sera of pancreatic cancer and chronic pancreatitis patients compared to healthy controls (p<0.0001), yielding receiver operating characteristic curve areas of 0.861 and 0.85, respectively. In conclusion, most pancreatic cancers display hypomethylation and overexpression of miR-200a and miR-200b, silencing of ZFHX1B by promoter methylation and retain expression of E-cadherin. The elevated serum levels of miR-200a and miR-200b in most patients with pancreatic cancer could have diagnostic utility.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4094.
Abstract 4097: Protein tyrosine phosphatase SHP-2 regulates human pancreatic cancer cell biology on collagen I
Authors: Connelly Sarah , Isley Beth A. , Baker Cheryl H. , Summy Justin M.
Published: February 2011

With a five year survival rate of less than 5%, pancreatic cancer stands as one of the deadliest human malignancies. There is a great need for novel therapeutic strategies for this illness, and improving our understanding of pancreatic cancer biology will be a key step in developing these therapies. The purpose of these studies is to investigate the regulation and function of the Src Homology 2 (SH2) domain containing protein tyrosine phosphatase SHP-2 in human pancreatic cancer cells and to determine its potential value as a therapeutic target for this disease. In other cellular systems, SHP-2 is crucial for integrin and receptor tyrosine kinase-mediated activation of diverse signaling pathways, regulating many of the cellular events necessary for oncogenesis, including cell division, motility, adhesion, and survival. We hypothesized that inhibition of SHP-2 function would negatively impact integrin-mediated cell signaling and biology in response to the extracellular matrix protein collagen I, an abundant component of pancreatic tumor stroma in vivo. Initial results demonstrate that SHP-2 expression and tyrosine phosphorylation/activity are elevated in pancreatic cancer cells, relative to immortalized normal human pancreatic cells. Phosphorylation of SHP-2 is further elevated in the presence of collagen I. SHP-2 tyrosine phosphorylation in pancreatic cancer cells occurrs in a Src family kinase-dependent fashion. Initial results using miRNA-mediated knock-down of SHP-2 expression indicate the importance of SHP-2 for the ability of pancreatic cancer cells to proliferate, migrate and heal cell culture wounds on collagen I. Additionally, SHP-2 knockdown cells grown on collagen I demonstate altered tyrosine phosphorylation patterns of several signal transduction proteins, including focal adhesion kinase (FAK), in comparison to controls. These data suggest, for the first time, an importance for SHP-2 in pancreatic cancer cell biology and the potential value of SHP-2 as a therapeutic target for this disease. Ongoing and future experiments will compare the effects of SHP-2 inhibition on pancreatic tumor growth in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4097.
Abstract 4105: Nitric-oxide-mediated inhibition of Na+/H+ exchange (NHE) is a novel mechanism of bile acid induced damage: Relevance to esophageal carcinogenesis
Authors: Goldman Aaron , Shahidullah Mohammad , Chen Hwudaurw , Delamere Nicholas , Dvorak Katerina
Published: February 2011

Background: Barrett's esophagus (BE) is a premalignant condition that is associated with gastroesophageal reflux disease (GERD). Gastric acid and bile acids are two components of refluxate that induce proliferation, DNA damage and oxidative stress. In combination, the effects of gastric acid and bile acids are amplified and synergistic. The major goal of this study was to evaluate the effect of bile acids alone or in combination with acid on intracellular pH (pHi). A mechanism responsible for bile acid-induced pHi alterations was examined.

Methods: CP-A cells (non-dysplastic Barrett's esophagus cells) were treated with medium acidified to pH 5.5 and/or 0.5mM bile acid cocktail in the presence/absence of inhibitors of nitric oxide synthase (NOS) or NHE (sodium-hydrogen exchanger). pHi was measured by BCECF microfluorimetry in perfused cells, nitric oxide by DAF-FM fluorescence and DNA damage by the comet assay. Western blot analysis was used to detect the expression of phosphorylated (activated) eNOS, iNOS and NHE1.

Results: A dose-dependent decrease in pHi was observed in CP-A cells exposed to bile acids. Bile acid-induced intracellular acidification was abolished when NO production was inhibited by a nonspecific NOS inhibitor L-NAME or by thea specific iNOS inhibitor 1400W. In contrast the NHE inhibitor DMA, significantly decreased the bile acid-mediated pHi response. Exposure of the cells to acidified medium (pH5.5) containing a 0.5mM bile acid cocktail, markedly decreased pHi pHi to a value below extracellular pH. Importantly, medium acidified to pH5.5 containing 0.5mM bile acids induced a similar decrease in pHi as medium acidified to pH 4.5 without bile acids. This The same treatment resulteds in a 250% increase in acid-mediated DNA damage compared to medium acidified to pH5.5 in CP-A cells.

Conclusion: In summary, a new mechanism of bile acid induced damage was identified. Bile acids alone or in combination with acidic pH significantly decrease intracellular pH in esophageal cells. The results suggest the effect ofresponse tpo bile acids is the consequence of NOS activation and that leads to NHE inhibition. The mMarked increase in DNA damage after suggests exposure to bile acids in combination with acid may play a crucial role in esophageal carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4105.
Abstract 4116: Relationship between K-ras 4A and superoxide in lung adenocarcinoma cell lines
Authors: Calvert Richard J. , Maciag Anna E. , Anderson Lucy M.
Published: February 2011

The K-ras gene is mutated in 30-50% of human lung adenocarcinomas, and even more frequently in cancers of colon and pancreas. It exists in two protein isoforms, K-ras 4A and 4B. Their amino acid sequence is very similar, except for marked differences in the hypervariable domain near the carboxyl terminus. K-ras 4B, with a polylysine tail, is unique among the ras gene products and is the dominant K-ras isoform in most tissues. Wild-type K-ras 4B has tumor suppressor activity (Zhang et al. Nature Gen. 29:25, 2001), whereas K-ras 4A was recently found to be necessary for lung tumor development in mice (To et al. Nature Gen. 40:1240, 2008). In search of an oncogenic function for K-ras 4A in lung cancer, we previously demonstrated a highly significant (P<0.01) positive correlation between K-ras 4A expression, at both mRNA and protein levels, and superoxide amounts in a panel of 11 human lung adenocarcinoma cell lines (Proc. Am. Assoc. for Cancer Research 50: (#437)). Superoxide has been widely implicated in cancer causation and progression. In the present study, we test whether there is a cause-effect relationship between expression of K-ras 4A and superoxide production. Cell line H441 expresses high levels of both superoxide and K-ras 4A. Superoxide levels were determined using a nitroblue tetrazolium (NBT) assay and K-ras 4A protein levels quantified by immunoblot with a specific antibody. Results are means ± SEMs. The effect of reduced levels of K-ras 4A on superoxide production was determined using a K-ras 4A silencing siRNA, which reduced K-ras 4A protein by 60.2 ± 3.0% after 72 hours of siRNA treatment. This reduction had no effect on superoxide (control 1.13 ± 0.08, siRNA 1.17 ± 0.08 nmol NBT reduced/ug protein). The effect of reduced levels of superoxide on K-ras 4A expression was determined using 5 mM Tiron, an electron scavenger. A 36% decrease in superoxide did not affect K-ras 4A protein levels (control 26.90 ± 0.89 vs. Tiron-treated 26.89 ± 1.91, arbitrary units). If these preliminary results are confirmed with additional cell lines and reagents, they may suggest that both expression of K-ras 4A and production of superoxide are controlled by another, pro-oncogenic factor or set of conditions. In view of the importance of both K-ras and superoxide in cancer, discovery of this putative central pro-oncogenic regulator might offer an important preventive and therapeutic target in lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4116.
Abstract 4119: Cytoplasmic location of CXCR4 is correlated to loss of EMT marker and activation of downstream signaling pathway in non-small cell lung cancer
Authors: Wang Yuxiang , Huang Donghai , Fu Hongpeng , Wang Dongsheng , Koenig Lydia , Khuri Fadlo R. , Shin Dong M. , Chen Zhuo (Georgia)
Published: February 2011

Introduction: Although epidermal growth factor receptor (EGFR) targeted therapy initially proved to be effective in some lung cancer patients, emerging resistance to EGFR- tyrosine kinase inhibitor (TKI) became an urgent issue to solve. Some research has shown that cancer cells undergoing epithelial mesenchymal transition (EMT) acquired resistance to EGFR-TKI, though the underlying mechanism remains unknown. Our preliminary study in non-small cell lung cancer (NSCLC) cell lines showed that G-protein coupled chemokine receptor CXCR4 could activate both p-AKT and p-ERK pathways, which have been reported to induce EMT. In this study, we hypothesize that CXCR4 may be an alternative route for cancer cells to bypass EGFR to activate the downstream pathway under an EMT phenotype. Methods: Immunohistochemistry (IHC) was used to detect the expressions of CXCR4, p-AKT, p-ERK, and E-cadherin in 94 clinical NSCLC samples and a weighted index (WI = % expression x intensity score) was used to quantify the expression level of these markers. Western blot was used to detect the expression of CXCR4 and activation of its downstream pathways in NSCLC cell lines. Results: CXCR4 was detected in 98.9% (93/94) of lung cancer samples, however, of greater interest were the 3 distinctive expression patterns for CXCR4 including cytoplasmic, nuclear, and cytoplasmic/nuclear expressions. Since it seems unreasonable to see a transmembrane receptor located in nuclei, we confirmed nuclear localization of CXCR4 by western blot using nuclear extractions from NSCLC cell lines. Furthermore, we evaluated the correlation of different locations of CXCR4 with p-AKT, p-ERK, and E-cadherin by ANOVA statistical analysis. The result showed the expression level of p-AKT and p-ERK are both significantly higher (p=0.004 and p=0.014, respectively) in CXCR4 cytoplasmic and cytoplasmic/nuclear localization groups than those in CXCR4 nuclear localization group. The expression level of E-cadherin in the CXCR4 cytoplasmic and cytoplasmic/nuclear localization groups was also significantly lower (p=0.021) than that in CXCR4 nuclear staining group. Conclusions: Our data suggested that cytoplasmic CXCR4 may contribute to activation of AKT and ERK signaling pathways under EMT, while nuclear localization of CXCR4 may be an inactive form. The functional cytoplasmic CXCR4 may contribute to EGFR-TKI resistance by offering an alternative pathway for tumors to maintain progression. (Supported by grants from NIH R21 CA125062, DOD W81XWH-07-1-0306 Project 5 to ZGC, and GCC Distinguished Scholar Award to ZGC and DMS).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4119.
Abstract 4121: Hyaluronan promotes Rho kinase and PI-3 kinase signaling in head and neck cancer
Authors: Wang Steven J. , Torre Carlos , Xia Weiliang , Bourguignon Lilly YW
Published: February 2011

Objectives: Hyaluronan (HA) is a ligand for the transmembrane receptor CD44, which acts through multiple signaling pathways to influence cellular behavior. The objective of this study was to investigate whether HA and CD44 promote Rho kinase and phosphatidyl inositol-3 (PI-3) kinase mediated oncogenic signaling to alter cisplatin sensitivity and stimulate tumor cell proliferation, migration, and matrix metalloproteinase (MMP) secretion in head and neck squamous cell carcinoma (HNSCC).

Design/Subjects: Laboratory investigation utilizing a HNSCC cell line, HSC-3.

Interventions: Rho kinase and PI-3 kinase activity, myosin phosphatase and AKT phosphorylation, tumor cell growth, migration, and MMP secretion were measured in the presence or absence of HA, cisplatin, and inhibitors of Rho kinase and PI-3 kinase.

Results: The addition of HA, but not HA plus anti-CD44 antibody, resulted in increased Rho kinase and PI-3 kinase activity. Immunoblotting studies demonstrated that HA promotes Rho kinase mediated myosin phosphatase phosphorylation and PI-3 kinase mediated AKT phosphorylation. HA was shown to promote migration and increased MMP secretion through Rho kinase mediated signaling. HA treatment promoted increased tumor proliferation and resulted in a 12-fold reduced ability of cisplatin to cause HNSCC cell death. On the other hand, the presence of Y-27632, a Rho kinase inhibitor, and LY-294002, a PI-3 kinase inhibitor, blocked HA-mediated cisplatin resistance.

Conclusions: Our results suggest that HA and CD44 promote Rho kinase and PI-3 kinase mediated oncogenic signaling and cisplatin resistance. Perturbation of HA-CD44 mediated Rho kinase and PI-3 kinase signaling pathways may be a novel strategy to treat HNSCC.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4121.
Abstract 4131: Activation of PKC-delta inhibits apoptosis leading to mitomycin C resistance
Authors: Khatri Raju , Niture Suryakant K. , Jaiswal Anil K.
Published: February 2011

Antioxidant-induced protein kinase C delta (PKCdelta) is known to phosphorylate cell survival transcription factor Nrf2. Phosphorylated Nrf2 translocates to the nucleus leading to coordinated activation of detoxifying enzyme gene expression. This mechanism is critical for protection against chemical and radiation stress and cell survival. Human breast cancer MCF-7 cells expressing endogenous PKCdelta, human breast cancer BT549 cells deficient in PKCdelta expression (PKCdelta-null) and BT549-C1 cells expressing cDNA derived PKCdelta demonstrated differential survival and apoptotic death responses to anti-tumor drug mitomycin C. MCF-7 and BT549-C1 cells expressing PKCdelta showed increased survival and decreased apoptotic cell death as compared with PKCdelta-null BT549 cells. The studies also demonstrated that mitomycin C induced serine40 phosphorylation and nuclear translocation of Nrf2. Further studies determined that Nrf2 translocation in the nucleus led to an increase in Bcl2 and BclXL expression that contributed to the inhibition of apoptosis, increased cell survival and mitomycin C resistance. Interestingly, inclusion of antioxidant tert-butyl hydroquinone with mitomycin C further enhanced the PKCdelta effect on cell survival and apoptotic cell death. These results led to the conclusion that PKCdelta is endogenous factor in drug resistance and could serve as a therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4131.
Abstract 4137: Verification of antitumor effect of γ-secretase-inhibitor and radiotherapy
Authors: Mizugaki Hidenori , Konishi Jun , Kikuchi Junko , Kikuchi Eiki , Shinagawa Naofumi , Oizumi Satoshi , Nishimura Masaharu
Published: February 2011

[Background and Aim] Notch3 is a member of the Notch signaling pathway, which plays an important role in cancer development. We have already demonstrated that Notch3 is over-expressed in approximately 40% of lung cancer tumors and that inhibition of the Notch3 pathway by γ-secretase-inhibitor (GSI) induces apoptosis and suppresses tumor growth in lung cancer. Although a previous report has shown that radiation enhanced Notch1 expression in breast cancer, little is known about the relationship between radiation and Notch pathway. In this study, we attempt to elucidate the combined effect of GSI and radiotherapy in lung cancer. [Results] We used two Notch3-expressing lung cancer cell lines (HCC2429 and H460). In a MTT proliferation assay and clonogenic assay, combination of GSI and radiation suppressed the growth of lung cancer cells and colony formations compared to GSI or radiation alone. We also showed that GSI down-regulates Notch3 expression in a dose dependent manner, and radiation up-regulates Notch expression and downstream Notch target gene, Hey-1. Notch up-regulation by radiation is down-regulated by the combination. Combination of GSI and radiation reduced p-Bcl2, Bcl-XL and p-Erk compared to GSI or radiation alone, whereas no effect on p-Akt level was seen.[Conclusion] Our results suggest that combination of GSI and radiation may be clinical potential strategy in the treatment of patients with lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4137.
Abstract 4142: The effects of SBHA on Notch 1 expression and platinum sensitivity in ovarian cancer cells
Authors: Rose Stephen L. , Kunnimalaiyaan Muthusamy , Drenzek Jessica , Seiler Nicole
Published: February 2011

Objective: Despite advances in radical surgical debulking and intraperitoneal chemotherapy, ovarian cancer remains the most deadly gynecologic malignancy. The Notch signaling pathway plays an important role in cellular differentiation, proliferation, and apoptosis. We have previously found Notch 1 intracellular domain (NICD) to be highly expressed in the most platinum resistant ovarian cancer cell lines. Recent evidence has shown that histone deacetylase inhibitors (HDACi's) decrease platinum resistance in oral squamous cell cancers. In addition, the HDACi suberoyl bis-hydroxamic acid (SBHA) has been found to modify Notch 1 expression in other cancers. We hypothesized that SBHA would reduce ovarian cancer cell proliferation through inhibition of the Notch 1 signaling pathway and help sensitize ovarian cancer cells to platinum.

Methods: Following treatment with SBHA, NICD expression in the ovarian carcinoma cell lines OVCAR3, SKOV3, and CaOV3 was analyzed by Western blot. The effects of SBHA and cisplatin on ovarian cancer cell growth were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay.

Results: NICD expression by Western blot was consistently reduced in all 3 cell lines following treatment with SBHA. Dose-dependent growth inhibition of ovarian cancer cells was demonstrated with SBHA treatment. Furthermore, the addition of SBHA to cisplatin resulted in additional reduction of NICD and further growth suppression in SKOV3, the most platinum resistant cell line tested.

Conclusions: SBHA treatment of ovarian cancer cells led to decreased NICD expression and dose dependent growth reduction in ovarian cancer cells. In addition, we found that SBHA in combination with platinum further reduced NICD expression and growth in SKOV3 cells. These findings provide a compelling argument for further study into the role that Notch 1 signaling may be playing in platinum resistance and ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4142.
Abstract 4143: Bz-423, a novel benzodiazpine, inhibits PI3 kinase signaling in ovarian cancer cells
Authors: Garg Gunjal , Liu Rebecca , Boitano Anthony , Tan Lijun , Kwok Roland , Glick Gary , Opipari Anthony
Published: February 2011

Objective: Bz-423 is a 1,4-benzodiazepine with immunomodulatory and cytotoxic properties. The effect of Bz-423 on survival signaling in lymphoid neoplasms predicts activity against similar pathways in ovarian cancer cells. Having previously established the cytotoxic activity of Bz-423 against ovarian cancer, we now characterize the mechanism of action.

Methods: Human ovarian cancer cell lines were maintained in culture. Cells were treated with Bz-423 or vehicle control. Cell survival and proliferation were evaluated, along with signaling mechanisms including the PI3-kinase-Akt-mTOR axis. DNA degradation (apoptotic fraction), mitochondrial function and caspase activation were evaluated by flow cytometry. Cytochrome c release and protein kinase activation were analyzed by immunoblotting. Apoptosome complex formation was monitored by gel filtration chromatography. Cellular ultrastructure was determined by electron microscopy (EM).

Results: Growth inhibition and apoptosis were observed in all ovarian cell lines following treatment with Bz-423. Caspase-9 and −3 cleavage, along with cytochrome c release were seen, consistent with activation of the intrinsic pathway of apoptosis. Early mitochondrial changes identified by ultrastructural evidence of mitochondrial swelling and the accumulation of superoxide within 1 hr of treatment were seen. Bz-423 induced cell death was associated with inhibition of Akt activation through a oxidant dependent mechanism. Antioxidants blocked Bz-423-induced signaling.

Conclusions: Bz-423 is a novel benzodiazepine being developed as a mitochondria targeted therapy, and displays anti-neoplastic activity against ovarian cancer cells in vitro. Results show that by acting on mitochondrial complex V, this compound inhibits PI3-kinase signaling, including the tumor survival factor Akt. PI3-kinase signaling is increasingly recognized as a survival and growth promoting mechanism in ovarian cancer. Since Akt inhibition by Bz-423 is dependent upon cellular bioenergetics and anti-oxidant balance, strategies employing this compound offer a selective new approach to target ovarian cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4143.
Abstract 4145: Increased anti-tumor effect of sorafenib by depleting tumor associated macrophages
Authors: Hui-Chuan Sun , Zhang Wei , Zhu Xiao-Dong , Tang Zhao-You
Published: February 2011

Background: Sorafenib prolonged survival in patients with unresectable hepatocellular carcinoma (HCC), but the effect is modest and tumor finally progressed even under continuous sorafenib therapy. The aim of this study is to investigate the mechanism of tumor progression under sorafenib treatment.

Methods: Tumor growth, local invasion and lung metastasis were observed in MHCCLM3-R, a human HCC xenograft nude mouse model, when treated by sorafenib (30 mg/kg qd, n=6 per group) and vehicle as control. Macrophage infiltration was measured in the peripheral blood and macrophage in tumor after sorafenib treatment by immunohistochemistry and flow cytometry with F4/80 antibody, and the effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drugs target macrophages, zoledronic acid (ZA) and clodrolip, in a mouse model of human hepatocellular carcinoma (HCC).

Results: Sorafenib treatment significantly inhibited tumor growth and lung metastasis, but also induced significant increase in peripheral F4/80-positive cells and intratumoral macrophages infiltration, which was associated with elevation of colony stimulating factor-1, stromal-derived factor 1α and vascular endothelial growth factor in tumor and plasma VEGF of mouse origin in peripheral blood, suggesting the role of macrophage in tumor progression during sorafenib treatment. Depletion of macrophage by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis and lung metastasis compared to that of mice treated with sorafenib alone. ZA was more effective than clodrolip.

Conclusions: Macrophage may have an important role in tumor progression under sorafenib treatment. Zoledronic acid is promising when combined with sorafenib to enhance its anti-tumor effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4145.