bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3988: Aberrant NFKBIA expression in lung adenocarcinomas arising from never smokers
Authors: Furukawa Masashi , Soh Junichi , Toyooka Shinichi , Shien Kazuhiko , Maki Yuho , Muraoka Takayuki , Tanaka Norimitsu , Ueno Tsuyoshi , Asano Hiroaki , Tsukuda Kazunori , Yamane Masaomi , Oto Takahiro , Miyoshi Shinichiro
Published: August 2012

[Background] Lung cancer in never-smoker (NS) is known as a seventh cause of cancer-related death. Activating mutation of epidermal growth factor receptor (EGFR) gene is frequently observed in non-small-cell lung cancers (NSCLCs) with never smoking history and it is significantly associated with the sensitivity of EGFR tyrosine kinase inhibitor (EGFR-TKI). Recently, NFKBIA gene, a tumor suppressor gene, has been reported to be silenced by deletion in glioblastomas, and in NSCLCs, its expression level is associated with clinical outcome of EGFR-TKI therapy. In this study, we examined NFKBIA expression and the genetic alterations of EGFR, K-ras, EML4-ALK to investigate the inter-relationship between these genetic alterations and clinicopathological factors among lung adenocarcinomas in NS. [Material and Method] We obtained ninety seven lung adenocarcinomas samples in NS which were resected at our institution. Four NSCLC cell lines (NCI-H1299, NCI-H1650, HCC827, NCI-H1819) were also investigated. We examined NFKBIA expression using immunohistochemistory (IHC) in tumor samples and real time reverse transcriptional PCR (RT-PCR) in cell lines, respectively. Mutational statuses of EGFR and K-ras genes were determined using mutant-enriched PCR assay and direct sequencing, respectively. EML4-ALK fusion was screened by ALK IHC and confirmed using RT-PCR assay. The methylation status of NFKBIA gene is investigated by methylation specific PCR assay and bisulfite sequencing. [Result] NFKBIA expression was silenced in 36 out of 97 samples (37.1%). EGFR mutation, K-ras mutation, and EML4-ALK fusion were detected in 61.8%, 2.1%, and 1.0% of 97 samples, respectively. Of note, the silencing of NFKBIA expression was significantly frequent in EGFR wild-type patients than in EGFR mutant patients (p=0.0024), while there was no correlation between NFKBIA expression level and K-ras mutation or EML4-ALK fusion. Although NFKBIA expression was silenced in NCI-H1299 and NCI-H1650, NFKBIA expression was not restored after 5-Aza treatment in these cell lines. In addition, NFKBIA methylation was found in these two cell lines. [Conclusion] Our findings suggest that the degradation of NFKBIA expression is a frequent event in lung adenocarcinomas of NS and may mediate an important non-tobacco carcinogenic pathway especially among lung adenocarcinoma with wild-type EGFR.

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 3988. doi:1538-7445.AM2012-3988
Abstract 3994: Loss of STAT2 enhances the establishment of B16 melanoma tumors
Authors: Yue Chanyu , Gamero Ana M.
Published: August 2012

STAT2 is a key component of type I interferon (IFN-I) induced activation of the JAK/STAT signaling pathway. Several studies including ours have shown that STAT2 is a critical signaling mediator in the apoptotic and cell growth inhibitory effects of IFN-I. These findings suggest that STAT2 has tumor suppressive function. To investigate this further, the B16-F1 mouse melanoma cell line was used in a tumor transplantation model to interrogate the role of STAT2 in tumorigenesis. We found that STAT2 null mice injected subcutaneously with B16-F1 melanoma tumor cells showed accelerated tumor growth rate and grew larger tumors compared to those formed in wild type mice. We next compared the antitumor effects of IFN-I in wild type and STAT2 null mice administered intraperitoneally 3 days after tumor injection and given three times weekly until day 21 of the study. IFN-[[Unsupported Character - Symbol Font ]] therapy of STAT2 null mice was ineffective as tumors continued to grow at the same rate as in saline treated STAT2 null mice. In contrast, wild type mice that received IFN-[[Unsupported Character - Symbol Font ]] developed smaller tumors compared to saline treated control mice. Evaluation of B16 tumors established in wild type mice by Western blot analysis showed a marked reduction in the levels of STAT2 and STAT1 proteins. Similarly, B16 tumors established in STAT2 null mice also showed low levels of STAT1 protein. To determine whether tumor derived soluble factors were involved in decreasing STAT2 protein; supernatants collected from B16-F1 tumors incubated overnight with serum free medium were used to treat B16-F1 tumor cells grown in vitro. A decrease in STAT2 protein was evident when tumor supernatants derived from tumors established in wild type and STAT2 null mice were used. Taken together our findings reveal that host STAT2 restricts the establishment of melanoma tumors and soluble factors secreted by melanoma tumors reduce STAT2 protein levels thereby diminishing the antitumor effects of IFN.

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 3994. doi:1538-7445.AM2012-3994
Abstract 40: Targeting MLCK expression in cancer-associated fibroblasts disrupts tumor-stromal interactions in gastric cancer
Authors: Su Liping , Chen Lu , Chen Xuehua , Li Jianfang , Liu Bingya
Published: August 2012

Aim: Accumulating evidence has indicated that microenvironment consisting of stromal affects cancer progression. However, the mechanism by which cancer cells and fibroblasts, the major cell type in stromal, interact with each other during tumor development remains to be elucidated. Our aim is to identify the molecular mechanism involved in tumor-stromal cell interactions in human gastric cancer. Methods: The expression of Myosin Light Chain Kinase (MLCK) in gastric cancer-associated fibroblasts (CAF) was detected by quantitative RT-PCR. The effects of CAF, which was interfered with MLCK, on cell proliferation, apoptosis, migration and invasion in vitro were measured. Results: Primary fibroblast cultures were established from human gastric cancer. MLCK was upregulated in CAF compared with NF (fibroblasts from patient matching non-cancerous tissues). Small interfering RNA knockdown of MLCK in CAF impaired the augmenting effect of CAF on gastric cancer cell proliferation, and induced the apoptosis of gastric cancer cells (9.1+0.8 vs 22.3+1.4, p<0.05). Moreover, the ability of stimulating gastric cancer cells migration (77.7+4.3 vs 61+4.2, p<0.05) and invasion (60.9+1.5 vs 48.3+1.4, p<0.05) in vitro by CAF was also inhibited when MLCK was knocked down. Furthermore, gastric cancer cells can also activate CAF in a MLCK-dependent manner. Conclusions: These findings indicate that MLCK mediates the cross-talk between tumor and stromal in gastric cancer, and suggest that targeting stromal cells for MLCK interference may be a therapeutic target in human gastric cancer.

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 40. doi:1538-7445.AM2012-40
Abstract 4000: Restoring LSAMP expression decreases the proliferation rate in osteosarcomas
Authors: Barøy Tale , Kresse Stine H. , Hopmeier Dahlia , Stabell Marianne , Skårn Magne , Myklebost Ola , Meza-Zepeda Leonardo A.
Published: August 2012

Osteosarcomas are the most common primary malignant tumors of bone. The tumors are highly aggressive with poor prognosis, and display complex genomic aberrations. Previously, our group has identified a frequently deleted region in 3q13.31 in osteosarcoma clinical samples and cell lines [1]. The deleted region contains non-coding RNA genes, pseudogenes and the gene encoding the limbic-system associated membrane protein (LSAMP). The latter has previously been reported to be a candidate tumor suppressor gene in other cancer types, and more recently in osteosarcomas. Interestingly, our data show that low expression of LSAMP is statistically correlated with shorter patient survival. To examine the possible function of LSAMP in osteosarcomas, the expression was restored in an osteosarcoma cell line with a homozygous deletion of the gene. Characterization of the cell line with restored LSAMP expression showed decreased proliferation rate, which is consistent with its suggested role as a tumor suppressor gene. The migration rate and colony forming capability of the cells were unaffected. Interestingly, gene expression profiling of the restored lines, showed up-regulation of three genes, all proposed to have a role in cancer biology. The results indicate that the LSAMP protein might up-regulate the transcription of these three genes, and that LSAMP alone, or in conjunction with other genes, suppresses tumors by reducing their proliferation rate. Together, our data strengthens the hypothesis of LSAMP being a tumor suppressor gene in osteosarcomas. 1. Kresse, S.H., et al., LSAMP, a Novel Candidate Tumor Suppressor Gene in Human Osteosarcomas, Identified by Array Comparative Genomic Hybridization. Genes Chromosomes & Cancer, 2009. 48(8): p. 679-693.

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 4000. doi:1538-7445.AM2012-4000
Abstract 4006: Biochemical characteristics of functional domain of KITENIN: Its implication in AP-1 signal activation and colorectal cancer cell motility
Authors: Bae Jeong A , Sun Eun Gene , Park So Yeon , Kim Hangun , Kim Kyung Keun
Published: August 2012

Background & aims: KITENIN (KAI1 C-terminal interacting tetraspanin) is not only a membrane-associated protein but also a functional protein promoting the invasiveness of colorectal cancer (CRC) cells. Recent studies about the mechanism of KITENIN function in the CRC cells indicate that KITENIN plays a pivotal role in tumor initiation and promotion through activating the activator protein-1 (AP-1) signaling. However, the contribution of each functional domain of KITENIN to AP-1 signaling is still unknown and the biochemical properties of C-terminal region of KITENIN were investigated. Methods: To compare the biochemical and functional properties of KITENIN deletion mutants with wild-type KITENIN, the various mutant constructs of C-terminal region (238-512 aa) were generated and being expressed in the 293T and Caco2 cells. AP-1 activity was measured via AP-1 luciferase reporter assays, and the cellular phenotypes of these cells expressing KITENIN mutants were examined via in vitro cell invasion and wound-healing assays. Results: KITENIN mutant with deletion of C-terminal 63 amino acids (DCKIT) markedly increased the AP-1 activity in contrast to other deletion mutants and wild-type KITENIN. Also, DCKIT increased the degradation of Dvl2 and stability of c-Jun, which are responsible for AP-1 activation by KITENIN. The C-terminal fragment of KITENIN (KICD, roughly 30 kDa) was generated and preferentially translocated to the nucleus after PMA treatment, but its functional significance is under investigation. A mutant construct of 408ALRA411-KITENIN was obtained by mutation of YXXΨ motifs, which directs the clathrin-dependent endocytosis of membrane-spanning proteins. This mutation did not influence the AP-1 activity, cell invasiveness, and cell migration by KITENIN but disrupted the interaction of KITENIN with Dvl2. Moreover, co-expression of this mutant with ErbB4 exhibited more attenuated AP-1 activity than that of forced expression of wild-type KITENIN and ErbB4. These results indicate that endocytosis of KITENIN and subsequent interaction with Dvl2 is essential to activating AP-1 signal and modulating cell motility by KITENIN. Conclusion: These biochemical properties of deletion mutants of KITENIN suggest that the C-terminal region of KITENIN is an important component for modulating AP-1 signal and CRC cell motility by KITENIN.

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 4006. doi:1538-7445.AM2012-4006
Abstract 4009: Hypoxia potentiates Runx2-mediated increased Bcl-2 expression in prostate cancer resulting in apoptosis resistance
Authors: Browne Gillian , McCook Heather , Ming Louise , Stein Janet L. , Stein Gary S. , Lian Jane B. , McKeown Stephanie R. , Worthington Jenny
Published: August 2012

Hypoxia is known to act as a selective pressure in the tumor microenvironment, often resulting in sub-populations of cells that have a propensity for more aggressive behaviour. More recently, Runx2 (an osteogenic transcription factor) has been associated with tumor growth, invasion and metastasis. Previous results in our laboratory have indicated that hypoxia selects for more invasive, apoptosis-resistant prostate cancer cells. Microarray analysis revealed that both Runx2 and Bcl-2 (an anti-apoptotic protein) were up-regulated in the hypoxia-selected cells (Butterworth et al, 2008). Bcl-2 is known to be up-regulated in various malignancies, and is associated with more aggressive disease and resistance to chemotherapy. Subsequent analysis of the Bcl-2 promoter revealed two binding sites for Runx2. In the current study we confirmed regulation of Bcl-2 by Runx2 though ChIP binding and reporter assays in LNCaP prostate cancer cells. Runx2 over-expressing LNCaP cells showed increased cell viability which was inhibited by Runx2 siRNA knockdown. A variety of assays demonstrated that Runx2 over-expressing cells were more resistant to apoptosis than controls. We further addressed the effect of hypoxia on Runx2 expression and the potential role Runx2 might play in the regulation of Bcl-2 expression and thus apoptosis in prostate cancer. When LNCaP cells were exposed to hypoxia, both Runx2 and Bcl-2 mRNA and protein levels were significantly increased over oxic controls. In a separate in vivo study, treatment of LNCaP tumors grown on the backs of immuno-compromised mice with the anti-androgen bicalutamide resulted in a profound decrease in tumor oxygenation during the first 14 days. Over the following 14 days, oxygen levels gradually increased. Further investigation of the excised tumors showed an increase in Runx2 mRNA levels from day 7, which was further confirmed by western and immunohistochemical analyses. In summary, this study shows that hypoxia promotes over-expression of Runx2 in prostate cancer cells. The cells exhibit a survival advantage through up-regulation of Bcl-2 leading to apoptosis resistance. These results, taken together with the in vivo data, suggest that Runx2 expression (either through microenvironmental pressures or ectopic expression) promotes progression to a more apoptosis-resistant and thus malignant phenotype, highlighting Runx2 as a potential therapeutic target in prostate cancer.

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 4009. doi:1538-7445.AM2012-4009
Abstract 4012: Pyruvate kinase M2 promotes the growth and migration of gastric cancer cell via nuclear factor-kappa B and its target genes
Authors: Kwon Oh-Hyung , Kang Tae-Wook , Kim Jeong-Hwan , Kim Mirang , Noh Seung-Moo , Song Kyu-Sang , Yoo Hyang-Sook , Kim Woo-Ho , Xie Zhi , Pocalyko David , Kim Seon-Young , Kim Yong Sung
Published: August 2012

Tumor cells take up more glucose than normal cells and convert large amounts of glucose to lactate even in the presence of oxygen. This phenomenon is known as aerobic glycolysis, and is important for cell growth and metastasis. PKM2 is an isoenzyme of the glycolytic enzyme pyruvate kinase, and promotes aerobic glycolysis. Here, we describe an important role for PKM2 in regulating the survival and migration of gastric cancer cells. Using qRT-PCR and tissue microarray, we showed that PKM2 was overexpressed in gastric tumor tissues compared to normal tissues. Furthermore, PKM2 expression levels were inversely correlated with gastric cancer patient survival. Using shRNA-mediated knockdown of PKM2, we showed that PKM2 regulated apoptosis-related gene, Bcl-xL, at transcriptional levels, and affected cell survival. PKM2 also regulated migration-related gene, MMP-9, at transcriptional levels, and affected cell migration. The effect of PKM2 on cell death and migration was mediated by the p65 stability resulting in binding activity on its target gene promoter. These results provide new insight that PKM2 is an up-stream molecule regulating p65 function and that PKM2 may be a useful target for gastric cancer therapy.

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 4012. doi:1538-7445.AM2012-4012
Abstract 4018: DNA methylation controls CHST11 gene expression in breast cancer
Authors: Herman Damir , Leakey Tatiana I. , Jousheghany Fariba , yao-Borengasser Aiwei , Cooney Craig A. , Kieber-Emmons Thomas , Monzavi-Karbassi Behjatolah
Published: August 2012

Introduction: We have previously demonstrated that chondroitin sulfates (CS) play a crucial role in metastatic potential of breast cancer cells. We have shown that the expression level of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with CS-A expression and aggressiveness of human breast cancer cells. The current study was mainly performed to determine whether the expression of CHST11 gene in breast cancer cells is controlled by DNA methylation. Methods: Quantitative real-time PCR was used to detect the expression of CHST11 in human breast cancer cell lines. MCF7 cells were treated with 5-aza-2′-deoxycytidine (5AzadC) for DNA demethylation and expression analysis. Methylation in MCF7, MDA-MB-231 cells and patient breast cancer specimens was analyzed using bisulfite genomic sequencing (BGS). Reduced representation bisulfite sequencing (RRBS) was performed in the CpG island of CHST11 with a minimum coverage of 10. Digital gene expression was used to estimate the number of transcribed copies of the CHST11 gene from the second-generation mRNA-sequencing data. Results: Treatment of MCF7 cells with 5-AzadC increased the expression of CHST11 and its product CS-A in a dose-dependent manner. We observed very low levels of DNA methylation in a CpG island of CHST11 in estrogen receptor-negative basal-like cell lines MDA-MB-231, MDA-MB-468, and BT-20, but very high levels in the same region in estrogen receptor-positive luminal MCF7, ZR-75-1, and T47-D cells. We found that the CpG island can also be hypomethylated in triple negative (negative for estrogen and progesterone receptors and Her2/neu) patient breast cancer tissues. Conclusions: The data suggest that DNA hypomethylation significantly contributes to CHST11 and surface CS-A expression in aggressive breast cancer cells. The data suggest that therapeutic use of DNA demethylating agents may promote more aggressive forms of breast cancer.

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 4018. doi:1538-7445.AM2012-4018
Abstract 4037: Downregulation of ZNF516 and INTS1 is related with promoter methylation in cervical uterine cancer cell lines
Authors: Ili Carmen G. , Brebi Priscilla , Andana Alejandra , Leal Pamela , Garcia Patricia , Guerrero-Preston Rafael , Roa Juan C.
Published: August 2012

Introduction: One of the most common neoplasia in developing countries is cervical uterine carcinoma (CC). HPV infection is recognized as the major risk factor for carcinogenesis of cervix. However, not all infected women will develop a cervical cancer. Several studies have proposed DNA methylation as early biomarker in CC. A higher promoter methylation is associated with lower gene expression. Aim: To evaluate the association between DNA methylation and gene expression of ZNF516 and INTS1 in cervical cancer cell lines. Materials and Methods: DNA isolated from 12 normal and 7 cervical cancer tissue samples was enriched by Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K CpG Islands plus Promoter arrays (MeDIP-chip). Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions by Methylation Specific PCR in a cohort of 221 normal, premalignant, and cervical cancer samples. Relative expression analysis was performed in a normal cervical epithelium cell line (ECT1 E6/E7) and three cervical cancer cell lines (C-4I, SiHa and C-33A) using real time PCR. MSP was also used to examine promoter methylation in normal cervical and cervical cancer cells lines. Results: ZNF516 and INTS1 were differential methylated in the promoter region of normal and cervical cancer samples in the MeDIP-chip discovery project. The methylation frequency was 100% in cervical cancer cell lines. Relative expression of ZNF516 was significantly lower in C-4I and SiHa (p <0.05) in relation with the normal cell line. C-33A showed elevated promoter methylation and a low expression of ZNF516, however, this result was not significant. INTS1 expression was significantly lower in C-33A and SiHa (p <0.05) in relation with the normal cell line. C-4I showed a high promoter methylation and a low expression of INTS1, however, this result was not significant. Conclusions: We have identified ZNF516 and INTS1 as potentials biomarkers for cervical cancer. These genes may be a useful tool for diagnosis and clinical management of cervical cancer. These results need to be confirmed in clinical samples, from an independent laboratory before the gene panel is examined in a Phase II Biomarker Development Trial. Work sponsored by CORFO Project 09CN14-5960 and Postdoctoral Fondecyt Research Project 3120141.

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 4037. doi:1538-7445.AM2012-4037
Abstract 4042: Loss of imprinting at the 14q32 locus contributes to osteosarcoma
Authors: Shu Jingmin , Li Lihua , Moriarity Branden , Thayanithy Venugopal , Spector Logan G. , Largaespada David A. , Steer Clifford J. , Subramanian Subbaya
Published: August 2012

Osteosarcoma (OS) is the most common primary bone malignancy affecting children and young adolescents. Downregulation of microRNAs at 14q32 locus is implicated in the pathogenesis of OS. However, array comparative genome hybridization did not show any DNA copy number loss at this locus. We hypothesized that epigenetic alterations may regulate genes and microRNAs present in this locus and contribute to the development and progression of OS. Here, we show that OS is characterized by a selective loss of imprinting (LOI) at 14q32 locus. LOI at the 14q32 locus was unique to OS, compared with 10 other cancer cell lines representing 6 tissue types. Furthermore, the average demethylation at 14q32 locus was 24%, but the global demethylation as determined by LINE-1 retrotransposons was only 7%. We also found LOI in H19, MEST, and PEG3 loci in OS samples, albeit less frequently. LOI at the 14q32 locus is highly prevalent (82.6%, 19 out of 23) only in OS patients with early-onset (12-30 years of age) of the disease. Imprinted genes at 14q32 locus were downregulated in OS due to histone modifications and the locus was controlled by bivalent domain enriched with both K4-3m and K27-3m. We determined the tumor suppressor function of this locus by overexpression of 3 imprinting genes, DLK1, RTL1 or DIO3 in SaoS2 cells. We found that all 3 genes inhibited cell proliferation and enhanced apoptosis. We also tested methylation patterns using matched buccal DNA samples obtained from OS patients and their parents. Interestingly, loss of imprinting at 14q32 locus was found in most of the OS patients and their parents, suggesting that children might be predisposed to developing OS by inheriting the instability of imprinting at this genomic site. To determine if loss of imprinting at 14q32 locus is the driving mechanism, or a secondary genetic event, we evaluated tumor tissues obtained from p53LSLR270H/;Osx-Cre mice OS models. These mice developed OS at 8-12 months (potentially representing the late-onset (> 30yrs) of human OS). LOI was found only in 33% of mice OS samples, which is in agreement with the low rate of LOI detected in late-onset human OS. In conclusion, our findings suggest that LOI at the 14q32 genomic locus is likely a contributing event in the pathogenesis of OS.

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 4042. doi:1538-7445.AM2012-4042
Abstract 4059: Development of a point-of-care device for circulating tumor cell/cluster detection
Authors: Zhou Yang , Harb Wael A. , Shao Guoqiang , Hong Bin
Published: August 2012

Circulating tumor cell (CTC) has recently grabbed numerous headlines with the promise for personalized cancer diagnostics and treatment. Its clinical value, in terms of presence and the number, has been demonstrated in accumulated results for prognosis of cancer progression and mortality, and prediction of therapy response. CTCs, the leukemic phase of solid tumor, further give us the hope for a rapid and affordable optical imaging tool as CTCs are readily accessible and optically observable. Fluorescence based cell imaging has been widely spread to detect CTCs. However, it is still a challenge to accomplish a point-of-care test (POCT) due to many limitations such as the cost, fluorescence stability, variations in fluorescence detection and image processing, and data interpretation. Chromogenic labeling may offer exciting perspectives in POCT for CTC detection along with the application of metallic signal enhancement. Based on this concept, TeloVISION LLC developed a palm-sized, manually operated, POCT device for CTC detection. The device, which bears a membrane with rod-shaped, micron-sized pores, processes the whole blood without vigorous manipulation. By manually rotating the device counter-clockwise, the entire CTC assay is split into 7 steps, performed inside the device, and completed in 40 minutes. To validate the device, whole blood was withdrawn from the animals bearing metastatic tumors. Due to the restriction of the membrane, CTCs larger than 5µm size were retained while majority of the normal cells were removed due to the discrepancy in deformability. In addition, the filtration was driven by gravity so that the mechanical damage from external force was avoided. After filtration, enriched CTCs were identified by DAB stain in the presence of horseradish peroxidase (HRP) tagged anti-cytokeratin (CK) and the signal was intensified 50-fold by metal precipitation. CTCs were found in blood as well as the circulating tumor microemboli (CTM) by both chromogenic labeling and fluorescence staining (control). Heterogeneity in the CK expression was seen for both CTCs and CTM. The recovery of the device was 94% compared to that by hemocytometer. We conclude that chromogenic labeling with the signal enhancement by in situ metal precipitation can provide high-resolution, sensitive CTC detection, and prevent the false positive results as seen in fluorescent labeling due to autofluorescence and fluorescence ghost in the blood. This new portable device may provide an efficient low-cost CTC detection in clinical settings. Adjustment of the antibodies will expand the field of application such as sarcoma and melanoma, non-epithelial derived CTCs.

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 4059. doi:1538-7445.AM2012-4059
Abstract 4061: Computer-assisted Gleason grading of prostate cancer: Two novel approaches using nuclear shape and texture feature to classify pathologic Gleason grade patterns 3 and 4
Authors: Veltri Robert W. , Christudass Christhunesa , Epstein Jonathan I. , Ali Sahirzeeshan , Yoon Hong-Jun , Li Ching-Chung , Madabhushi Anant
Published: August 2012

Introduction: We used two novel computer-assisted imaging applications that incorporate nuclear shape and texture analysis to differentiate Gleason grade patterns 3 and 4 of H&Ein a tissue microarray (TMA). The two approaches utilized a variational adaptive segmentation scheme (AdACM) (Rutgers) and the cardinal multiridgelet transform (CMRT) based texture analysis (University of Pittsburgh). Methods: The TMA include 0.6 mm cores from blocks that included Gleason patterns of CaP radical prostatectomy cases from 40 patients. The TMA comprised 13 Gleason patterns 6 (3+3), 8 pattern 7 (3+4), 7 pattern 8 (4+4) and 5 pattern 9 (4+5) Gleason grade (GG) patterns. The AdACM uses 200 × 200 pixel images extracted for nuclear & architectural textural features. All features were based on mathematical energy terms with a shape prior in a multi-level set formulation. The CMRT is an image transform that is translation- and rotation-invariant, and provides a means of displaying texture contents for selection of discriminative features. With a 3-scale decomposition, it gives 8 ridgelet packets and 16 statistical texture features in a 256 x 256 pixel image patch that are averaged over 9 non-overlapping patches in a given image for separation of GG patterns 3 and 4. Results: The CMRT study utilized 3 of 16 texture features extracted from images and trained a Gaussian kernel support vector machine (SVM), a leave-one-out cross-validation showed an accuracy of 94% and AUC of 0.97. For AdACM, nuclear features and architectural features were extracted. Linear Discriminant Analysis (LDA) was employed for feature selection and best features from all three classes could discriminate Gleason grade 3 vs. 4 with an accuracy of 86% via a SVM classifier. Conclusions: Unique quantitative imaging of tissue nuclear shape and texture features can accurately differentiate Gleason patterns 3 and 4. Our research will next evaluate CaP outcomes.

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 4061. doi:1538-7445.AM2012-4061
Abstract 4066: Early assessment of the XL184 treatment by Diffusion-Weighted MRI in metastatic prostate cancer to the bone murine model
Authors: Brisset Jean-Christophe , Hoff Benjamin , Galban Stefanie , Lemasson Benjamin , Rehemtulla Alnawaz , Galban Craig , Ross Brian Dale
Published: August 2012

Ninety percent of patients with advanced prostate cancer develop metastases to the bone1. At present, there is no definitive means for assessing response to therapy, with RECIST identifying tumors in the bone as “unmeasureable”. Cabozantinib (XL184) is an oral small molecular pan-tyrosine kinase inhibitor of primarily MET and vascular endothelial growth factor receptor 2 (VEGFR2). MET is known to play key roles in cellular proliferation, migration and invasion, which contribute to the survival and metastasis of cancer cells. XL184 is already in Phase 2 trials to evaluate the therapeutic benefit on castration-resistant prostate cancer metastatic to the bone. Diffusion-weighted MRI (DW-MRI) is highly sensitive to microscopic changes in tumor tissue. Our goal was to evaluate DW-MRI for detection and quantification of changes in tumor tissue following one-week XL184 treatment. MR experiments were performed on a 9.4T MRI. Human prostate cancer (PC3) cells were implanted by direct intra-tibial injection into male SCID mice, which serves as a model for prostate bone metastases2. Upon measuring tumor volume of ∼10mm3 on anatomical scans, mice were distributed into 2 groups: XL184 at 30 mg/kg (N=7) and vehicle (N=5). Treatments were delivered by oral gavage once a day for 7 days. DW-MRI was performed at days 0 (pre-treatment), 1, 4, 7, 11, 14 and twice a week after. Apparent diffusion coefficient (ADC) maps were calculated analytically from DW-MR images acquired at b-value 120 and 1200 s/mm2. End point of the study was when tumor volume reached 5x the initial measure. Unpaired Student t-test was performed to compare MRI metrics. XL184 treated mice had smaller tumor volume growth rate than control mice. Significant differences in tumor volume were not observed until 14 days post-treatment initiation (p<0.05). In contrast, significantly higher ADC values (p<0.0.5) for the treated group compared to control were observed at day 7. These results demonstrate the use of DW-MRI as a early biomarker of tumor response to XL184 in a metastatic prostate cancer tumor model. DW-MRI may serve as a surrogate endpoint for assessing patient response to treatment in clinical trials. References: 1. Carlin BI et al. Cancer. 2000;88(12):2989-2994. 2. Lee KC et al. Cancer Res. 2007;67(8):3524-3528.

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 4066. doi:1538-7445.AM2012-4066
Abstract 4069: Determination of single cell surface protein expression using a tagless microfluidic method
Authors: Vellanki Sri HariKrishna , Smith Robin , Kumar Rajan , Wieder Robert
Published: August 2012

Tumors are often inaccessible to biopsy and fine needle aspirates are often the only source of cellular material from tumors either at diagnosis or during therapy for determination of pharmacodynamic response. The few cells that needle aspirates yield are not sufficient for more than visual diagnostic determination of malignancy or potential tumor category. Most processing would result in the loss of these cells and is impractical. A need exists for a different diagnostic tool that can obtain biologically relevant information from cells when only few are available without the need for processing them. We describe a method to detect the expression of a surface protein in single cells without prior labeling or manipulation using a microfluidic device. When the protein is expressed, it undergoes transient bond formation with a ligand immobilized on a patch as the cell is pumped through a microfluidic device, resulting in a specific decrease in the cell's velocity. We sterile sorted LM2 cells, a subline of MDA-MB-231 human breast cancer cells, with a unique lung metastatic capability, into fractions expressing none (P1), medium (P2) and high levels (P3) of interleukin13α2 receptor (IL13Rα2). We used variable chamber heights (25, 50, 75 μm), width 7 mM and 1%, 1.5%, 2.5% glycerol in buffer with variable flow rates. We were able to get specific flow retardation with reduction in mean velocity 15.1 % with shear rate 62.86 (s−1) and shear stress 0.63 (dynes/cm2) in the fraction expressing high levels (P3) of IL13Rα2 receptor compared to controls LM2 (P1), MDA-MB-231, MCF-7 cells. The detection of cells with high expression of the protein (P3) was 100% sensitive and 100% specific. Later we asked the following questions: can cells be reanalyzed after pumping through microfluidic device, can we indentify IL13Rα2 positive cells in mixture and whether specificity of flow retardation is due to antigen and antibody binding. The method is non-destructive and P3 cells can be collected for reanalysis and we got reduction in mean velocities in first and second run 9.7% and 10.0%. The system could identify P3 positive cells with mean velocity reduction 5.5% when mixed with MDA-MB-231 cells in 1:1 ratio and the interaction between the antigen/antibody is highly specific as the mean velocity reduction in P3 cells is 0.2%. Further needle aspirates from mice xenograft were able to detect cells expressing IL13Rα2 receptor. This method will have a potential impact in analyzing cancer cells when only few are available, such as the case with needle aspirates, when labeling and manipulation results in cell loss.

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 4069. doi:1538-7445.AM2012-4069
Abstract 4075: Aerosol azacytidine inhibits the orthotopic lung cancer in mice by its demethylation function but not cytotoxic effect
Authors: Qiu Xuan , Perez-Soler Roman , Zou Yiyu
Published: August 2012

Introduction: Silencing of key tumor suppressor genes by promoter hypermethylation plays an important role in lung cancer carcinogenesis. Reversing the hypermethylation in the early lung cancer cells adjacent to the airway surface may have a potential to inhibit lung cancer development. This study was to explore the therapeutic potential of aerosol administered demethylating agent azacytidine (Aza) for the treatment of bronchial epithelial premalignancy or lung cancer. Methods: The demethylation and gene reactivation functions of Aza administered by aerosol were detected by qPCR methylation array and western blotting assay in the tumor tissues from the orthotopic human non-small cell lung cancer (NSCLC) xenograft models in nude mice. The therapeutic efficacy and toxicity of the aerosol Aza were also evaluate in the same lung cancer models. An intravenous administered Aza with clinical equivalent dose and schedule was used as a control. Results: The aerosol Aza is an appropriate pharmaceutical inhalation formulation with about 80% of droplets size distributing between 0.1 to 5 micron. This aerodynamic size range ensures the aerosol droplets depositing in the lower airway. In animal studies, aerosol administration of Aza significantly prolonged the survival of nude mice with intratracheally inoculated human NSCLC tumors. The %-increased life span was 5- to 10-fold higher than that of a systemic treatment of Aza at a clinical equivalent dose. The aerosol treatment did not cause any detectable lung toxicity or myelosuppression (systemic toxicity of Aza), since the treatment dose was >100-fold lower than the toxic dose. The lung tumors were resected after the aerosol Aza treatment and the methylation levels of the 24 promoters that drive the lung cancer related tumor suppressor genes (TSG) were detected. We found that aerosol Aza significantly reduced the methylation level in 9 of these 24 promoters. The dementhylating aerosol also resulted in the gene reactivation in the protein level of several tested genes. Conclusions: Aerosol administration of Aza has significant therapeutic advantages over the systemic Aza. It can be given at a non-cytotoxic dose, thus its main therapeutic efficacy is the demethylation function of Aza and the TSG reactivation. In our case, the therapeutic index of aerosol Aza is >800-fold higher than that of IV Aza. The clinical trial of aerosol Aza is worth to be conducted. Acknowledgement: Supported by a NIH (NCI) grant 5R01CA154755-02

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 4075. doi:1538-7445.AM2012-4075
Abstract 4089: Identification of a DNA methylation marker to detect the presence of lymph node metastases of gastric cancers
Authors: Shigematsu Yasuyuki , Niwa Tohru , Yamashita Satoshi , Taniguchi Hirokazu , Kushima Ryoji , Katai Hitoshi , Ichinose Masao , Ushijima Toshikazu
Published: August 2012

Accurate detection of the presence of lymph node metastases of gastric cancers (GCs) is clinically important. However, current methods to detect it are unsatisfactory. As a marker, DNA methylation is advantageous because it is biologically and chemically stable and the DNA methylation profile is not disturbed by a small population of cells. Therefore, we focused on aberrant DNA methylation in GCs as a possible marker to detect the presence of lymph node metastases. We first performed genome-wide methylation analyses by Illumina Infinium HumanMethylation450 BeadChip array using three metastatic lymph nodes (MLNs) and three primary GCs without MLNs, and isolated 31 differentially methylated CpG sites. Then, using primary GCs with and without MLNs (screening set; 28 GCs with MLNs and 10 GCs without MLNs), their methylation levels were analyzed by quantitative PCR-based methods. Among the 31 regions, 10 regions showed higher methylation levels in GCs with MLNs than in GCs without MLNs (P < 0.05). In further analysis of the 10 regions using another sample set (validation set; 129 GCs with MLNs and 20 GCs without MLNs), hypermethylation of a region (cg06436185) in GCs with MLNs was validated, and the presence of lymph node metastases was detected with sensitivity of 43% and specificity of 85%. These results indicated that the methylation status of the cg06436185 region is a good marker to detect the presence of lymph node metastases of GCs.

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 4089. doi:1538-7445.AM2012-4089
Abstract 4093: Investigating a role for menin and MLL-AF9 in suppressing C/EBPβ-mediated mixed lineage leukemia cell differentiation
Authors: Thiel Austin T. , Hua Xianxin
Published: August 2012

The Mixed Lineage Leukemia (MLL) gene forms chromosomal translocations with one of multiple partner genes, leading to the expression of MLL fusion proteins (MLL-FPs), which cause acute leukemias with particularly poor prognoses. MLL-FPs contain an N-terminal portion the MLL protein, which is responsible for MLL-FP recruitment to MLL target genes, through interactions with various proteins, including the MEN1 gene product menin. The ability of menin to recruit MLL-FPs to target genes is critical for leukemic transformation. A central hallmark of leukemia is oncogene-mediated suppression of differentiation. Many leukemic oncogenes, including Bcr-Abl and AML-ETO suppress differentiation through inhibiting the C/EBPα transcription factor, a driver of myeloid differentiation. However, a mechanism for MLL-FP-mediated inhibition of C/EBPα has not been reported. We have found that disruption of the MLL-AF9 pathway by acute depletion of its recruiting factor menin causes differentiation of MLL-AF9 leukemia cells in vivo. These results suggest that a major function of menin/MLL-AF9 is to maintain leukemia cells in a de-differentiated state. Further, cDNA microarray analysis of primary MLL-AF9 splenocytes with acute menin depletion showed induction of genes involved in hematopoietic differentiation, including transcriptional targets of C/EBPβ. These results suggest a mechanism for MLL-FP-mediated inhibition of C/EBPα and suppression of leukemic progenitor cell differentiation.

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 4093. doi:1538-7445.AM2012-4093
Abstract 4096: Correction of metabolically sensitive histone epigenetic marks mediates the drug sensitivity of MDA-MB-231 human breast cancer cells
Authors: Simpson Natalie E. , Pogribny Igor P. , Beland Frederick A.
Published: August 2012

Evidence is accumulating that epigenetic alterations are major determinants of breast cancer progression. In a previous study we demonstrated that specific patterns of histone modifications correlated with changes in histone modifying enzymes in human breast cancer cells. More importantly, these changes were metabolically sensitive and differed between cells with epithelial and mesenchymal phenotypes, suggesting that they can be used as diagnostic and prognostic epigenetic biomarkers for breast cancer progression. We have now investigated whether or not metabolic targeting of the epigenome may be used for the treatment of breast cancer. Exposure of breast cancer cells to Compound 968, an allosteric inhibitor of glutaminase (GLS1), which we demonstrated previously to increase global histone H4 lysine 16 acetylation levels in mesenchymal MDA-MB-231 cells versus epithelial T-47D cells, resulted in a marked inhibition of cancer cell growth, with the effects being more prominent in mesenchymal MDA-MB-231 cells than epithelial T-47D cells. The growth inhibitory effect of Compound 968 in MDA-MB-231 breast cancer cells was accompanied by modest changes in H4 lysine 16 acetylation and a decreased level of histone H4 lysine 20 trimethylation at the promoter of tumor suppressor genes (e.g., CDH1), with a concordant rise in expression of the transcriptionally silenced CDH1 gene. Although, Compound 968 did not restore fully CDH1 expression in MDA-MB-231 to the level observed in epithelial T-47D cells, the observed increase in expression may lead to formation of a less aggressive cancer phenotype in MDA-MB-231 cancer cells and increase their sensitivity to chemotherapeutic agents. Indeed, a combined treatment of Compound 968 and the conventional chemotherapeutic drug cisplatin substantially increased the sensitivity of MDA-MB-231 cells to cisplatin. These results indicate that correcting of cancer-related metabolic disturbances may be an effective method for the targeted treatment of the cellular epigenome in aggressive breast cancer and may be useful for improving of the clinical management of breast cancer.

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 4096. doi:1538-7445.AM2012-4096