bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4139: Clinical evaluation of microRNA expression profiling in non-small cell lung cancer
Authors: Markou Athina N. , Vorkas Panagiotis A. , Sourvinou Ioanna , Yousef George M. , Lianidou Evi S.
Published: August 2012

CONTEXT: MicroRNAs (miRNAs) represent a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level. miRNAs play an important role in tumorigenesis since they have functions similar to oncogene or tumor suppressors and represent a new class of powerful tools for cancer prevention and therapeutics. Especially circulating miRNAs in plasma seem to be very promising novel tumor biomarkers, since their expression is correlated to diagnosis, prognosis and prediction of response to treatment. OBJECTIVE: The aim of our study was to evaluate mature miRNAs as novel tumor biomarkers in NSCLC, by exploring global expression profile of miRNAs in NSCLC paired fresh tissues and corresponding plasma samples. METHODS: FlexmiR bead array (Luminex) assay was utilized for miRNA expression profiling in 21 surgically removed NSCLC fresh tissues and their corresponding adjacent non-cancerous tissues. According to our FlexmiR results, evaluated by three different statistical approaches, 23 miRNAs were found to be differentially expressed. Our FlexmiR experiments were validated for four of these microRNAs in 40 paired NSCLC and matched corresponding adjacent non-cancerous tissues by Taqman RT-qPCR microRNA assays (Applied Biosystems). Moreover, total RNA was extracted from 37 corresponding plasma samples and miR-21 expression was quantitated by RT-qPCR. RESULTS: 23 miRNAs were found to be differentially expressed in NSCLC by the FlexmiR assay. Four of these, miR-21, miR-126*, miR-30d and miR-451, were further evaluated by quantitative RT-qPCR. The expression level of miR-21 was significantly higher in NSCLC tissues than in adjacent normal tissues (P=0.002); while miR-126* (P=0.000), miR-30d (P=0.000) and miR-451 (P=0.000) were down-regulated in NSCLC. Interestingly, high miR-21 expression and low miR-126* and miR-30d expression were associated with disease free survival (P=0.027, P= 0.047 and P=0.048 respectively). Levels of circulating miR-21 in plasma of NSCLC patients were significantly higher in NSCLC than in healthy volunteers (P=0.003). Circulating miR-21 in plasma was found to be an independent prognostic factor for NSCLC (P=0.019).

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 4139. doi:1538-7445.AM2012-4139
Abstract 4155: Comprehensive glucan profile exploration of mutated K-ras knockdown in colorectal cancer
Authors: Kawamata Hiroshi , Yamashita Keishi , Kuno Atsushi , Shirasawa Seiji , Narimatsu Hisashi , Watanabe Masahiko
Published: August 2012

(Background) In colorectal cancer (CRC), K-ras mutation is found in nearly 40% of the patients, and it is of prognostic significance (Onozato W et al, J Surg Oncol, 2010). Moreover, its knockdown in CRC cell lines with mutated K-ras gene results in robust reduction of ability of cell proliferation and anchorage independent growth (Shirasawa S, Science, 1993), both of which reflect metastatic ability. On the other hand, robust alterations of glycan structures have been reported in CRC promotion steps, accompanied by remarkable phenotypic changes, however there is no report that mentions the relationship of K-ras mutation and glycan change. (Materials and Methods) We examined glycan change of CRC cell lines (DLD1 and HCT116) somatically knocked out for K-ras gene by lectin array including 41 lectins to elucidate whether K-ras mutation-induced glycan changes play a critical role in CRC promotion. (Result) (1) In DLD1, we compared DLD1 wild type genotype cells (DLD1, DKS-5) with those that were knocked out for mutated K-ras gene (DKO-3, DKS-8), and the signals of MAL, MPA, UEA-I, and TJA-II were remarkably decreased in the knockdown cells. (2) In HCT116, we compared HCT116 wild type genotypic cells (HCT116, Hk2-10) with those that were knocked out for the mutated K-ras gene (Hke-3), and both MAL and MPA were remarkably declined. (3) In both cell lines, expression changes of glycans which can bind with MAL and MPA were consistent with the results of the lectin blotting, and both lectin signals were confirmed to be commonly altered by removing the mutated K-ras gene. (Conclusion) Mutated K-ras may be involved in critical phenotype change of CRC, accompanied by abnormal sialic acid recognition.

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 4155. doi:1538-7445.AM2012-4155
Abstract 4157: DACH1 Inhibited proliferation and tumor growth of lung adenocarcinoma cells
Authors: Li Zhaoming , Cai Shaoxin , Hu Junbo , Pestell Richard G. , Wu Kongming
Published: August 2012

Lung cancer is the leading cause of cancer death worldwide. A key driver to the proliferation of lung cancer includes increased activity of the gene encoding the epidermal growth factor receptor (EGFR) and mutation of K-ras oncogene. Target therapies through small molecules to inhibit the tyrosine kinase domain of EGFR (Gefitinib) or monoclonal antibody to EGFR (Cetuximab) have demonstrated clinical benefit. Unfortunately, development of resistance to those treatments occurs in most patients within one year. Studies of the hyperactive EGFR in drosophila identified a genetic inhibitor of EGFR signaling, known as dachshund (dac). Activate ras inhibits DACH1 expression in malignant peripheral nerve sheath tumor. Dachshund can activate or represses transcription, and regulates cell proliferation and differentiation in multiple context-dependent manners. Our studies have proved that DACH1, human homolog of dac, functions as a potential tumor suppressor of breast and prostate cancer. Expression of DACH1 is lost during cancer progression and re-expression of DACH1 gene inhibited cellular proliferation, migration and tumor formation. The expression levels and the role of DACH1 in lung cancer are largely unknown. As both EGFR and Ras signaling interact with DACH1, we hypotheses that loss of endogenous DACH1 may contribute to the lung tumorigenesis. The mRNA expression of DACH1 was decreased in lung adenocarcinoma compared with normal lung tissues. Ectopic expression of DACH1 by stably transduction inhibited cellular proliferation of lung cancer cell lines A549 and SKLU, cell cycle progression and clone formation rate in contact-dependent and -independent growth. ELISA using conditional medium from A549 cells expressing vector control or DACH1 demonstrated remarkable inhibition of secreting factors, such as CXCL1, CXCL2, CXCL5 and CXCL8. Those chemokines have already been proved to positively regulating angiogenesis and proliferation. To test in vivo tumorigenesis, 2x106 A549 cells expressing DACH1 or vector control were injected subcutaneously into nude mouse and tumor size was monitored weekly. Tumor growth was dramatically blocked by DACH1. Tumor weight was 297.8 ± 38.6mg for vector control and 31 ± 6.1mg for cells expressing DACH1(p value <0.001). Importantly, over-expression of CXCL5 can partly rescue tumor growth inhibition by DACH1 in A549 cells. Together, our study demonstrated that DACH1 is a potentially tumor suppressor of lung cancer though repression of chemokines secretion.

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 4157. doi:1538-7445.AM2012-4157
Abstract 4160: DEPTOR, an mTOR inhibitor, is a novel substrate of SCFαTrCP E3 ubiquitin ligase upon S6K1/RSK1 phosphorylation and regulates survival and autophagy
Authors: Zhao Yongchao , Xiong Xiufang , Sun Yi
Published: August 2012

DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCFαTrCP E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F-box protein, αTrCP via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by αTrCP; being decreased or increased upon αTrCP overexpression or knockdown, respectively. DEPTOR half-life is shortened by αTrCP but extended by its dominant negative mutant, by RSK1/S6K1 inhibition, or upon αTrCP degron site mutations. Biologically, DEPTOR, accumulated upon αTrCP knockdown, inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR, accumulated upon glucose deprivation and mTOR inhibition, induces autophagy. Thus, αTrCP-DEPTOR-mTOR intertwines to regulate cell survival and autophagy.

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 4160. doi:1538-7445.AM2012-4160
Abstract 4162: PTPRO silencing by MAPK pathway in colorectal cancer
Authors: Vecchione Loredana , Narzo Antonio Fabio di , Asbagh Layka Abbasi , Tejpar Sabine
Published: August 2012

Background The analysis of gene expression profile of a total of 688 primary colorectal (CRC) cancers revealed PTPRO to have an intriguing bimodal distribution. When looking for variables associated to it, such as clinical variables, mutation status, gene copy number variation and methylation pattern, a significant association was found with the BRAF mutated (BRAF mut) tumors and a not perfect association with the CIMP high methylation subtype. Based on these findings, we postulated that the Ras/Raf mitogen activated protein kinase (MAPK) pathway and the methylation could concomitantly or independently be involved in the downregulation of the gene. Material and methods Basal PTPRO mRNA was evaluated in a total of 13 CRC cell lines (7 wild type for BRAF and KRAS, 2 BRAF mut and 4 KRAS mut) by RT-qPCR. PTPRO promoter region (366bp, 15475534 - 15475900 - Human Genome browser gateway NCBI36/hg18) was studied by sequencing analysis after bisulfite conversion of DNA extracted from these cells. Treatments with 10 microM of 5-azacytidine (5-AZA), 1 microM of Mek inhibitor (AS703026) and the combination of 10 microM of 5-AZA with 1 microM of Meki were performed in SW480 and Lim1215. After 72 hrs of treatment samples were profiled for gene expression and methylation. Results 10 out of 13 cell lines showed low PTPRO expression at mRNA level independently from the methylation status. The expression of the gene was induced after treatment with 5-AZA in SW480 and Lim1215. PTPRO expression was also induced after Mek inhibition of 5,6 fold and 44 fold in SW480 and Lim1215, respectively, without any modifications of the methylation pattern. Moreover, Mek inhibitor could further increase the expression of the gene in cells treated with 5-AZA Conclusion Our clinical and preclinical data confirm the MAPK pathway to be involved in PTPRO silencing independently from its methylation status. PTPRO loss might play a role in mediating the oncogenic function of the MAPK activation in CRC cells either by deregulating receptor type kinanses (RTKs) or by directly modulating Erk activity. On going experiments will answer to this question

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 4162. doi:1538-7445.AM2012-4162
Abstract 4163: Determination of synthetic lethal interactions in KRAS oncogene dependent cancer cells reveals novel therapeutic targeting strategies
Authors: Molina-Arcas Miriam , Hancock David , Steckel Michael , Downward Julian
Published: August 2012

Oncogenic mutations in RAS genes are very common in many human cancers, including those affecting the lung and the gastro-intestinal tract. RAS mutant cancers tend to be resistant to most targeted therapies, emphasising a clear need for the development of more effective treatments. Oncogenic KRAS deregulates numerous downstream pathways, resulting in cells with well-characterised selective advantages, but also less well understood vulnerabilities. In a panel of twenty-six non-small cell lung cancer (NSCLC) cell lines, half of which carry an activating KRAS mutation, knock-down of KRAS using siRNA indicates that KRAS mutant cells require KRAS expression to maintain viability. Moreover, KRAS depletion produces a clear inhibition of ERK and AKT signaling. As RAS proteins cannot be inhibited directly at present, we tested small molecule inhibitors targeting pathways directly controlled by RAS, such as RAS/RAF/MEK or PI3K/AKT/mTOR, as well as drugs directed against other targets (such as HSP90, topoisomerases or the proteasome) in order to affect tumour-specific vulnerabilities associated with the oncogenic state. Viability assays showed that cells harbouring an activated KRAS oncogene were more sensitive to MEK, RAF and IGF-1R inhibitors than cells carrying only wild-type KRAS alleles. In contrast, no differential loss of cell viability was observed when cells were treated with either PI3K or AKT inhibitors. Combinations of IGF-1R inhibitors with MEK or RAF inhibitors resulted in a synergistic anti-proliferative effect in KRAS mutant cell lines. Similar effects were observed upon simultaneous treatment with PI3K and MEK inhibitors. However, in this case, wild-type KRAS cells were also affected by PI3K inhibition, resulting in a less-pronounced differential effect. Pathway inhibition analysis showed that, whereas MEK and PI3K inhibitors respectively decreased ERK and AKT phosphorylation in all cell lines, IGF1R inhibitors decreased AKT phosphorylation only in KRAS mutant cell lines. Inhibition of AKT phosphorylation correlates with effectiveness of IGF-1R inhibitor in blocking cell proliferation. Mechanistically, it appears that mutant KRAS activation of PI3K requires coordinate input from IGF-1R. These findings suggest potential therapeutic strategies for NSCLC tumours harbouring KRAS mutations.

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 4163. doi:1538-7445.AM2012-4163
Abstract 4164: Regulatory mechanism for Ras degradation and its role in the suppression of cellular transformation and tumorigenesis
Authors: Jeong Woo-Jeong , Choi Kang-Yell
Published: August 2012

Ras is a well-characterized proto-oncogene, and its various mutations contribute to transformation and tumorigenesis in a wide variety of human cancers. Although the GTP/GDP loading switch is known as a major regulatory mechanism of Ras, other mechanisms affecting Ras activity, especially those related to cancer, are poorly understood. Here, we report a novel regulatory mechanism for Ras degradation and its role in suppression of cellular transformation and tumors induced by Ras mutations. We show that the negative regulators of Wnt/β-catenin signaling are associated with polyubiquitin-dependent degradation of Ras following phosphorylation and subsequent recruitment of α-TrCP-E3 ligase. The interaction of Ras with the WD40 domain of α-TrCP targeted Ras for polyubiquitination and degradation. This process stimulated by Axin or adenomatous polyposis coli (Apc) and was inhibited by Wnt3a via modulating the phosphorylation status of Ras. Ras-mediated cellular transformation was also inhibited by the expression of α-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/β-catenin signaling was indicated via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The Ras deregulation by negative Wnt/β-catenin signaling was also revealed in human cancers such as colorectal cancer and HCCs etc. Together, our results indicate an essential role for phosphorylation and degradation of Ras and suppression of Ras-induced transformation via the Wnt/β-catenin pathway and provide insight into the role of this novel regulatory mechanism for Ras in colorectal tumorigenesis.

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 4164. doi:1538-7445.AM2012-4164
Abstract 4168: Akt pathway in human breast cancer: A comparison between estrogen positive and estrogen negative tumors using reverse phase protein array
Authors: Carniello Jose Victor Scarpa , Assadi Maziar , Mello Fernanda M. , Rocha Rafael M. , Nogoceke Everton , Soares Fernando A. , Andrade Victor P.
Published: August 2012

INTRODUCTION: Estrogen receptor status is crucial for breast cancer treatment planning. Some estrogen receptor (ER) negative tumors carry a worse prognosis and lack more specific therapies. The AKT pathway is a promising target in breast cancer and was reported hyper activated in ER- tumors cells using in vitro models. We aimed to compare the level of Akt pathway activation between ER+ and ER- tumors using frozen human breast tumor samples. METHODS: 40 cases of breast cancer (20 ER+, 20 ER-) were identified from the frozen tumor bank. Patients were between 40 and 70 years old, with Stage I or II, node negative disease. About 20.000 cells from each tumor cells were purified by laser capture microdissection (LCM) and the protein lysate applied to the Reverse Phase Protein Array (RPPA). Antibodies tested by RPPA included Src, p-Src, Akt, p-Akt Thr308, p-Akt Ser473, mTOR, p-mTOR, PDGFRα, p-PDGFRα Tyr751. We also compared these results to patient matched non-microdissected samples and to immunohistochemistry (IHQ) using digital quantification. RESULTS: 6 out of 9 antibodies showed higher mean expression in ER- negative tumors but only p-Src had statistically significant different levels (0.043). The coordinated high levels (above median) of p-Src/p-Akt/p-mTOR was observed in 1 (5%) of ER+ and in 8 (40%) of ER- tumors. All 8 cases from the ER- group were triple negative (ER-/PR-/Her2-). Correlation coeficients of LCM-RPPA samples and non-microdissected/RPPA samples varied from 0.02 to 0.47. Only p-Akt Ser473 showed a significant correlation (r=0.47, p=0.003). Correlation coeficients between LCM-RPPA and IHQ for antibodies Akt, p-Akt and mTOR were 0.03, –0.22 and 0.05, respectively. CONCLUSION: Protein quantification using LCM-RPPA in human breast tumors is feasible and can detect significant differences between ER+ and ER- tumors. Triple negative tumors showed a high Akt pathway activation profile compared to ER+ tumors. Triple negative tumors are more likely to benefit from targeted therapies directed against the Akt pathway. LCMed and non-LCMed matching samples analyzed by RPPA showed discrepant results. The same happened to the comparison between LCM-RPPA and IHQ with digital quantification.

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 4168. doi:1538-7445.AM2012-4168
Abstract 417: Inflammation initiates OCT4/NANOG expression through IL-6-IGF-1R signaling activation and is associated with early recurrence of HBV-related HCC
Authors: Chang Te-Sheng , Lee Kam-Fai , Chi Ching-Chi , Yang Ya-Ting , Lin Hsin-Yi , Chang Pey-Jium , Tung Shui-Yi , Kuo Liang-Mou , Ling Thai-Yen , Huang Yen-Hua
Published: August 2012

Inflammatory microenvironment is a well-known promoter of hepatocellular carcinoma (HCC), a cancer with high rates of postoperative recurrence and poor prognosis. However, mechanisms underlying the inflammation-induced HCC promotion remain largely unknown. Here we demonstrate the effect of proinflammatory cytokine IL6 on IGF-1R signaling activation which initiates OCT4/NANOG expression and is associated with HBV-related HCC (HBV-HCC) recurrence. In this study, real-time quantitative PCR analysis demonstrated a high correlation between IGF-1R and OCT4/NANOG transcriptional expression in human HCC frozen tissues (N=119, R=0.8097 for OCT4; R= 0.8375 for NANOG). Interestingly, the OCT4, NANOG, or IGF-1R level in HBV-HCC was significantly higher than that in non HBV-HCC. Cultivation of cells in inflammation condition medium (Inflam-CM) increased OCT4/NANOG level in HBV+HBsAg+ cells (HepG2.2.15 and Hep3B), but not in HBV+HBsAg− (HA22T) or HBV−HBsAg− cells (HepG2 and Huh7). Epifluorescence/luciferase assay further demonstrated that Inflam-CM increased the GFP+ cell population as well as the luciferase activity of OCT4 promoter-GFP/luciferase-HepG2.2.15 cells. Besides, Inflam-CM significantly increased the IGF-1/IGF-1R transcriptional level and activated the IGF-1R signaling (phospho-IGF-1R and phospho-Akt) in HepG2.2.15 and Hep3B. Blockage of IGF-1R phosphorylation by picropodophyllin (PPP) dramatically decreased the OCT4/NANOG transcriptional activity. IL-6 stimulated the autocrinal IGF-I and IL-6 expression whereas AG490 (a phospho-STAT inhibitor) decreased the IGF-1R phosphorylation. The significant clinical association between early and overall postoperative recurrence and IGF-1R/OCT4/NANOG expression in both gene (real time RT-PCR) and protein (immunohistochemical staining) levels were demonstrated. When etiologic differences were considered, HBV-positive patients have the trend towards early HCC recurrence. Conclusion: Niche inflammatory stress might activate an autocrinal IL-6-IGF-I/IGF-1R-Akt signaling and OCT4/NANOG expression which is associated with early HBV-HCC recurrence. These results provide potential targets for individualized adjuvant therapy for HBV-HCC.

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 417. doi:1538-7445.AM2012-417
Abstract 4174: Identification of PRPF4 as a novel cancer promoter through AKT signaling in lung cancer
Authors: Choi So-Young
Published: August 2012

We recently constructed a large-scale gene expression database containing more than 33,000 tumors samples by applying cancer outlier profile analysis, and identified many candidate genes as therapeutic targets (Shin G et al., 2011). One of potential target candidates is PRPF4, which is over-expressed in a variety of human cancer, particularly in the breast, colon, ovary, skin and lung cancer patients. PRPF4 (PRP4 pre-mRNA processing factor 4 homolog), also called PRP4, HPRP4, Prp4p, or HPRP4P, is involved in pre-mRNA splicing and interacts with U4/U6-U5 tri-snRNP complex. Moreover, there have been a few studies on the roles of PRPF4 in diseases including cancer. In one study, PRPF4, along with MDR1 (ABCB1) and survivins, was involved in drug resistance in ovarian cancer patients (Duan Z et al., 2008), but its role in cancer is mostly unknown. In this study, we found that PRPF4 was over-expressed in breast, colon, and lung cancers, and involved in cancer progression, invasion, and migration, and interacted with AKT. Knock-down of PRPF4 by small interfering RNA in both lung and breast cancer cell lines dramatically reduced invasion, migration, and cell proliferation in vitro. We confirmed the up-regulation of PRPF4 in about 30% of lung and breast cancer specimens by RT-PCR. We also found that PRPF4 interacts with AKT and thus activates PI3K/AKT signaling pathway and increases proliferation, invasion, and migration of lung cancer cells. Collectively, we suggest PRPF4 is a novel up-regulated gene involved in various cancer progression and propose it as a novel cancer biomarker for targeted therapeutics.

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 4174. doi:1538-7445.AM2012-4174
Abstract 4178: Tcl1 enhances keratinocytes’ survival/proliferation by promoting erk and jnk/sap phosphorylation at the expense of p38 and by controlling c-fos expression through miR-29b and miR-181a-1
Authors: Bresin Antonella , Narducci Maria Grazia , Picchio Cristina , Cristofoletti Cristina , Citterich Mauro Helmer , Caprini Elisabetta , Tocco Valeria , Lazzeri Cristina , Russo Giandomenico , Ragone Gianluca
Published: August 2012

The Tcl1 oncogene has been initially isolated for its involvement in chromosomal translocations of T-prolymphocytic leukemias and overexpression in B chronic lymphocytic leukemia by enhancing AKT nuclear translocation and allowing the Ser-473 transphosphorylation. Tcl1, also, acts as AP-1 transcriptional inhibitor, by interacting with c-fos and c-jun. More recent works have associated Tcl1 to proliferation and self-renewal ability of ES cells under the direct activation of OCT3/4, Zfx, KLF5 transcription factors, being Tcl1 expressed in preimplantation embryos where it allows the progression beyond the 4-cells stage. We have recently shown that the Tcl1 oncogene is expressed in epidermis and defines secondary hair germ (transient-amplifying, TA) cells differentiation at catagen-telogen (the degenerative-resting phase of the hair follicle (HF)) transition, allowing the proliferation of TA cells in anagen (regenerative phase of the HF), giving the slow-cycling stem cells, the ability to incorporate BrdU. In fact, Tcl1 mutant (Tcl1-/-) affects stem-cell marker CD34 expression and BrdU incorporation in the bulge and TA cells, resulting in skin defects in adults with the onset of alopecia followed by skin wounding. Phenomena that are almost completely rescued by K14-TCL1 transgenic expression, in vivo. Since Tcl1 has a role in maintenance of a normal skin homeostasis in mice, involving both hair growth and epidermis, we used the approach of the expression chip analysis to unravel the pathways that are affected by loss of function and overexpression of Tcl1 in epidermal keratinocytes, by using Tcl1-/- and K14-TCL1;Tcl1-/- mice models. Our findings show that Tcl1 function involves the MAPK pathway, since Tcl1-/- shows increasing in p38MAPK phosphorylation linked to terminal differentiation/senescence/apoptosis of keratinocytes, while K14-driven overexpression shows increasing of p-Erk and p-Sapk/p-Jnk phosphorylations, linked to proliferation/commitment of keratinocytes. These signals flow through the MAPK cascade lead to altered AP1 factor function. In particular, the phosphorylation of AP-1 subunit c-Jun and c-fos transcriptional regulation and cellular localization result also affected.

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 4178. doi:1538-7445.AM2012-4178
Abstract 4182: Overexpression of Mig-6 suppresses endometrial cancer development and progression in ablation of Pten mice
Authors: Kim Tae Hoon , Kim Hong Im , Large Michael J. , Broaddus Russell R. , Lydon John P. , Jeong Jae-Wook
Published: August 2012

Endometrial cancer is the most common gynecological cancer. Estrogen-dependent endometrioid carcinoma is the most common type of endometrial cancer. In previous studies, we have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of Progesterone Receptor (Pgr) action in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. These results suggest that Mig-6 act as a tumor suppressor in endometrial cancer. To assess the effects of the overexpression Mig-6 and the PTEN/PI3K/AKT signaling pathways on uterine tumorigenesis, mice with Pten floxed (Ptenf/f) and LoxP-STOP-LoxP Mig-6 (R26Mig-6LSL) were bred to the PRCre mouse model to generate ablation of Pten and overexpression of Mig-6 in the uterus (PRcre+ R26Mig-6LSL Ptenf/f). PRcre+ R26Mig-6LSL Ptenf/f showed significantly increased survival time compared to PRcre/+ Ptenf/f mice. Double mutant mice showed significantly decreased uterine weight compared to PRcre/+Ptenf/f at 3 months of age. Gross morphology at 3 months of age showed that double mutant mice dramatically retarded the development of endometrial cancer compared to ablation of Pten alone. Histological analysis showed that endometrial adenocarcinoma with invasion into the myometrium was observed in the PRcre/+Ptenf/f mice at 3 months of age. However, double mutant mice did not develop endometrial cancer. Immunohistochemstry analysis showed significantly increased apoptosis and decreased proliferation at epithelial cells in PRcre+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice at 3 months of age. The expression of pERK1/2 was significantly decreased in PRcre+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice at 2 and 3 months of age. The expression of Pgr and its tartget genes was significantly increased in PRcre+ R26Mig-6LSL Ptenf/f mice compare to PRcre/+ Ptenf/f mice. However, the expression of Estrogen Receptor ≤ (Esr1) and its target genes was significantly decreased. The ability of Mig-6 to repress cancer development indicates that it exerts a tumor suppressor function in endometrial cancer. These results will contribute to the understanding of the molecular mechanism of tumorigenesis and to the development of therapeutic approaches for endometrial cancer.(Supported by NIH, R01 HD057873)

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 4182. doi:1538-7445.AM2012-4182
Abstract 4188: Whole transcriptome analyses of platinum-resistant ovarian cancer cells
Authors: Miller David F. , Lui Yunlong , Yan Pearlly , Rodriguez Benjamin , Lin Hai , Goel Shokhi , Jani Edra , Bhattrai Anurag , Koekesh Kevin , Guo Cong , Huang Tim H-M , Nephew Kenneth P.
Published: August 2012

Women with advanced stage ovarian cancer have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant ovarian cancer, which is uniformly fatal. Similar to other malignancies, ovarian cancer progression is associated with transcriptional deregulation and such changes contribute to ovarian cancer progression and development of resistance to chemotherapy. In an effort to better understand the causes of acquired platinum resistance in ovarian cancer, we performed whole transcriptome analysis of platinum sensitive and resistant ovarian cancer cell lines. We used RNA-sequencing to identify candidate genes and pathways that contribute to platinum-resistant ovarian cancer. We detected altered mRNA expression of transcription factors (DMRT3, DMRT1, GATA1, EMX2, LHX1, SRY, SOX9, FOXL2, LHX9) and signaling pathway genes (RXFP2, AR, WT1, FGF9, RSPO1, TUSC3, NROB1, WNT4). Additionally, significant up regulation (>20 fold increase) of the long non-coding RNAs (lncRNAs) HOTAIR and HOTTIP, which direct polycomb group and trithorax complexes to HOX promoters, was detected. We observed elevated HoxA gene expression (> 100 fold in some cases) associated with increased HOTTIP lncRNA expression in the platinum resistant cells. Altered expression of genes associated with DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L MECP2, MBD3L1, MBD6, MBD2, MBD3L2, MBD5, MBD1, MBD3, MBD4) was also observed. Furthermore, the splicing patterns of BRCA1 and BRCA2 genes in platinum resistant cells were altered, resulting in splice variants lacking functional coding regions. Altered BRCA1 and BRCA2 splicing patterns were correlated with reduced expression levels for numerous splicing factors (A2BP1, PTBP1, RBM9, SFRS3, ARID3A, ARID3B, SF1). DNA methylation analysis indicated epigenetic derepression consistent with increased expression for gonad developmental genes (HOTAIR, HOTTIP, HOXA genes). Our data suggest that genes associated ovarian development and differentiation contribute to the development of platinum resistant ovarian cancer. We are currently conducting integrated data and bioinformatics analyses to further investigate changes found in platinum resistance.

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 4188. doi:1538-7445.AM2012-4188
Abstract 4191: HECT-type ubiquitin ligase ITCH targets lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) and prevents LAPTM5-mediated cell death
Authors: Inoue Jun , Ishihara Takaya , Kozaki Ken-ichi , Imoto Issei , Inazawa Johji
Published: August 2012

Lysosomal-associated protein multispanning transmembrane 5 (LAPTM5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. While the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is negatively regulated by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death.

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 4191. doi:1538-7445.AM2012-4191
Abstract 4207: CCN5/WISP-2 upregulates p27Kip1 in breast cancer cell lines through AKT/FOXO3a signaling
Authors: Haque Inamul , Banerjee Snigdha , De Archana , Jha Saheli Sinha , Banerjee Sushanta K.
Published: August 2012

CCN5 is a member of the CCN (cysteine rich 61-connective tissue growth factor-nephroblastoma-overexpressed) family of growth factors. Our studies have shown that CCN5 is mainly expressed in less aggressive human breast cancer cells (i.e., MCF-7 and ZR-75-1) and noninvasive lesion (i.e., atypical ductal hyperplasia and ductal carcinoma in situ), whereas its expression is minimally detected in moderately aggressive breast cancer cell lines (i.e., SKBR-3), and it is undetected in triple negative highly aggressive breast cancer cell line (i.e., MDA-MB-231) and invasive lesion. Ectopic expression of CCN5 has been shown to inhibit tumorigenicity in MDA-MB-231-xenograft models therefore suggesting that CCN5 is an anti-invasive molecule. The objective of the present study was to determine how CCN5 overexpression affects cell-cycle progression of breast cancer cells. We found knocking down of CCN5 in non-invasive breast cancer cells significantly downregulated the expression of p27Kip1, a tumor suppressor molecule. In contrast, enforced/ectopic expression of CCN5 overexpression in MDA-MB-231 breast cancer cells caused cell cycle arrest at G1 phase and dramatically reduced proliferation of these cells. CCN5 overexpression results in increased the stability as well as the expression of p27Kip1 protein. The regulation of p27Kip1 by CCN5 is mediated through AKT/FOXO3a signaling. Collectively, the studies suggest that CCN5 exerts its anti-invasive action through the regulation of p27Kip1.

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 4207. doi:1538-7445.AM2012-4207
Abstract 4216: Protein kinase C ≤ overexpression is associated with loss of membrane-associated E-cadherin and β-catenin in T47D xenograft breast tumors
Authors: White Bethany E. Perez , Zhao Huiping , Kundu Madhuchhanda , Molloy Mary Ellen , Tonetti Debra A.
Published: August 2012

Expression of protein kinase C ≤ (PKC∈) has been shown to be a clinically relevant indicator of tamoxifen response, recurrence and overall survival in breast cancer (Tonetti, 2003; Assender 2007). To study this association, we developed a model of PKC∈-overexpressing breast cancer by stable transfection of the prkca gene into T47D breast cancer cells (T47D/PKC∈, Tonetti, 2000). We have shown that PKC∈ induces a hormone-independent, tamoxifen-resistant phenotype in vitro and in vivo (Chisamore, 2001). This model is further characterized in vitro by increased migratory and invasive capability, expression of mesenchymal markers and reduced expression of adherens junctions and epithelial proteins (Perez White, AACR 2011). β-Catenin and E-cadherin are components of adherens junctions in normal epithelia and loss of E-cadherin is associated with worse outcome in breast cancer (Prat, 2010). Further, the reduction or cytoplasmic relocalization of β-catenin has been associated with poor prognosis (Lopez-Knowles, 2010; Dolled-Filhart, 2006) though the processes upstream or downstream of these changes are not well characterized in breast cancer. Therefore we hypothesized that xenograft tumors derived from T47D/PKC∈ cells would have loss of membrane-associated β-catenin and E-cadherin. Immunofluorescence staining showed cytoplasmic localization of β-catenin and E-cadherin in T47D/PKC∈ tumors in sharp contrast to membrane association in T47D/neo (vector control) tumors. Western blot showed reduction in the level of E-cadherin in T47D/PKC∈ tumor samples but no change in the level of β-catenin compared to T47D/neo tumors. Based on this, we next wanted evaluate the β-catenin degradation pathway mediated by glycogen synthase kinase-3β (GSK-3β). Cellular extracts show an increase in the levels of the serine 9 inhibitory phosphorylation of GSK-3β in T47D/PKC∈ cells but no increase in total protein compared to T47D/neo cells. Treatment with the proteasomal inhibitor MG132 (10 μM) for 2 h did not increase the levels of β-catenin in vitro in T47D/PKC∈ cells. Current data indicate a correlation between PKC∈ expression and loss of membrane-associated β-catenin and E-cadherin. Further, β-catenin is stabilized in the cytoplasm of T47D/PKC∈ cells possibly allowing for nuclear localization and downstream gene transcription. Taken together with previous data, PKC∈ correlates with markers of poor outcome in addition to hormone-independence and tamoxifen-resistance suggesting that PKC∈ may play a central role in breast cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4216. doi:1538-7445.AM2012-4216
Abstract 4226: CD44 increases drug resistance by protecting FBXO21 mediated ubiquitination of P-glycoprotein
Authors: Ravindranath Abhilash K. , Kaur Swayamjot , Rodriguez Lorna
Published: August 2012

Drug resistance and metastases are major causes of treatment failure in cancer. These two important but not clearly related aspects in the biology of cancer have been extensively studied, but not until recently has there been evidence that these phenotypes are functionally linked. Two proteins, P-glycoprotein (P-gp) and CD44 are well known determinants of multidrug resistance and metastases, respectively. P-gp, the product of the ABCBC1 gene is a transmembrane ATP-dependent transporter that confers drug resistance to cancer cells. CD44 is a membrane protein implicated in metastases and has been identified as a “stem cell” marker for various types of cancer. Previously, we reported about a physical and functional interaction between P-gp and CD44. We found that the two proteins co-immunoprecipitate and co-localize within the cell membrane. When we introduced CD44 into CD44 (-) breast cancer cell lines, we found that P-gp expression was also induced both at the mRNA and protein level and the cells became drug resistant. In the present work we created a yeast-2-hybrid system that corroborates the physical interaction between the two proteins. We generated point mutants of the CD44 intracytoplasmic domain at two different serine residues known to be phosphorylation sites and show that when Ser 291 of CD44 is mutated, P-gp function is completely inhibited and the cells revert to drug sensitive in face of P-gp expression. We therefore hypothesized that CD44 could be stabilizing P-gp. We first determined that P-gp is ubiquitinated and degraded by the proteasomal pathway. We then screened a siRNA E3 ligase library and found several candidates for P-gp E-3 ligases. We tested FBXO21, which is in the FBOX family of ligases and does not have a known function yet. We demonstrated that FBXO21 ligase binds, ubiquitinates, and degrades P-gp. Furthermore, we show that CD44 inhibits FBXO21 directed ubiquitination of P-gp. Our findings are key to the understanding of P-gp induced drug resistance and reveal a new control mechanism of P-gp expression. Our work uncovers CD44 phosphorylation at Ser 291 as an excellent and very specific new drug target that would distinctively affect drug resistant cancer cells that are CD44 positive, such as cancer stem cells.

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 4226. doi:1538-7445.AM2012-4226
Abstract 4227: Sensitizing resistant ovarian cancer to chemotherapy through inhibition of small GTPases
Authors: Goncharov Kirill , Zeineldin Reema R.
Published: August 2012

Platinum- or paclitaxel-resistance is a major problem in women with advanced ovarian cancer (OVCA) and that confers a poor prognosis for patients. Consequently there is a great need to identify new drugs and drug targets for the management of residual and recurrent metastatic OVCA. About 90% of OVCA starts on the ovarian surface epithelium, but individual cells or multicellular aggregates (MCAs) shed into the peritoneal cavity and contribute to OVCA's metastasis into adjacent organs. These MCAs are resistant to apoptosis and to radio- and chemo-therapy. In OVCA, resistance to chemotherapy and apoptosis has been linked to the small GTPases, Rac1 and Cdc42, and their downstream effectors. Identifying compounds that disperse MCAs through disrupting adhesions would sensitize this resistant subpopulation to chemotherapy. We hypothesize that inhibition of one or both of these GTPases disrupts MCAs and sensitizes drug-resistant OVCA cells to respond to chemotherapeutic agents. We assessed if chemical inhibition of Rac1 or Cdc42 would sensitize drug-resistant OVCA monolayers and in vitro MCAs to cisplatin's cytotoxicity. The OVCA cell lines evaluated were OVCA 429 and SKOV-3ip. The inhibitors we used included: (1) NSC 23766, a Rac1-specific inhibitor, (2) ML141, a Cdc42-specific inhibitor, (3) Toxin B, an inhibitor of Rac, Cdc42 and Rho, and (4) R-naproxen, an inhibitor of Rac1 and Cdc42 (based on our data). We found that Rac1 and Cdc42 inhibitors disperse MCAs in a liquid overlay assay. Furthermore, these inhibitors sensitized OVCA cells to cisplatin's cytotoxic effect. Finding drugs that increase efficacy of chemotherapeutic agents against resistant OVCA cell populations is of particular importance. Thus far, GTPases have not been extensively examined as therapeutic targets, but they offer new opportunities for OVCA therapy. Our findings indicate that Rac1 and Cdc42 inhibitors disperse MCAs and improve their response to chemotherapeutic agents. This could increase the effectiveness of intraperitoneal chemotherapy and improve patient outcomes and survival. Since one of these inhibitors is R-Naproxen (an FDA approved off-patent drug), it has potential for rapid clinical application.

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 4227. doi:1538-7445.AM2012-4227
Abstract 4232: GFP imaging of DNA repair after UV irradiation of cancer cells
Authors: Tome Yasunori , Yano Shuya , Maehara Hiroki , Efimova Elena V. , Kanaya Fuminori , Tsuchiya Hiroyuki , Tomita Katsuro , Bouvet Michael , Hoffman Robert M.
Published: August 2012

The ability to image DNA repair in cancer cells after irradiation as well as its inhibition by potential therapeutic agents is important for the further development of radiotherapy of cancer. The relocalization of GFP fused to the chromatin-binding domain of a check-point-adapter protein 53BP1 was imaged after UV irradiation of breast cancer and MiaPaca-2 human pancreatic cancer cells. 53BP1 is a DNA repair protein that is over-expressed when double-stranded breaks occur in DNA. During live-cell imaging, 53BP1-GFP foci formation was observed within 15 minutes after UV irradiation. Most 53BP1 foci resolved by 90 min. To block UV-induced double stranded break (DSB) repair in cancer cells, we targeted poly (ADP-ribose) polymerase (PARP) with ABT-888 (veliparib). PAPR inhibition markedly enhanced UV-irradiation-induced foci persistence, even more than 90 min after UV irradiation, and decreased cell proliferation of breast and pancreatic cancer cells. The results suggest UV-irradiation and PAPR inhibition are a promising therapeutic combination for breast and pancreatic 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 4232. doi:1538-7445.AM2012-4232