bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3220: The effects of cholesterol treatment drugs alone and in combination on prostate tumor xenograft growth
Authors: Masko Elizabeth M. , Solomon Keith R. , Valilis Nikolaos A. , Gaines Alexis R. , Muehlbauer Michael J. , Newgard Christopher B. , Dewhirst Mark W. , Pizzo Salvatore V. , Freedland Stephen J.
Published: August 2012

Background: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer (PC), but not the incidence of the disease. This may occur by lowering levels of low-density lipoproteins (LDL). Statins and cholesterol-uptake inhibitors like ezetimibe lower serum LDL and are commonly prescribed for patients with high cholesterol. In this study, we determined the therapeutic role of simvastatin, ezetimibe, or both in vitro using several PC cell lines and in vivo using a LAPC-4 xenograft model. Methods: We treated 4 PC cell lines (LAPC-4, CWR22rv1, PC-3, and DU145) with varying concentrations of simvastatin (0-1000nM) or ezetimibe (0-100μM) for 0-5 days. MTS assays were performed to determine cell proliferation, and the IC50 was calculated using BioDataFit 1.02. For our in vivo study, a total of 90 athymic male nude mice were fed a high-fat, high-cholesterol diet (HFHC; 40% fat, 17% protein, 43% carbohydrates, 1.25% cholesterol), subcutaneously injected with 1 x 10^5 LAPC-4 cells, and randomized 2 weeks later to 1 of 4 treatments: vehicle control, 11mg/kg/day simvastatin (delivered via osmotic pump), 30mg/kg diet ezetimibe (delivered in the food), or simvastatin + ezetimibe. Mice remained on study for 42 days post-randomization, at which point they were harvested and samples analyzed. Results: In vitro, simvastatin directly reduced PC cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no inhibitory effect. In vivo, treatment of low continuous dosing of simvastatin or ezetimibe for 42 days had no effect on tumor growth compared to control. However, relative to control, the combination of simvastatin and ezetimibe accelerated tumor growth (p=0.01). The simvastatin-ezetimibe mice also showed lower fat infiltration in the liver and lower serum cholesterol, but higher tumor cholesterol levels than control. Conclusions: Our results suggest that low continuous dosing of simvastatin or ezetimibe may have no effect on PC growth in a slow-growing LAPC-4 xenograft model, but the combination of simvastatin and ezetimibe may accelerate PC growth, possibly by stimulating cholesterol uptake within the tumor. This novel finding may be important in implementing new treatments for men with slow growing PC and high cholesterol.

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 3220. doi:1538-7445.AM2012-3220
Abstract 324: The integrin-linked kinase (ILK) / Rictor complex is required for TGFβ-1-induced epithelial to mesenchymal transition (EMT)
Authors: Serrano-Martinez Isabel , McDonald Paul C. , Dedhar Shoukat
Published: August 2012

Epithelial to mesenchymal transition (EMT) causes fibrosis, cancer progression and metastasis. Integrin-linked kinase is a focal adhesion adaptor and serine/threonine protein kinase that regulates cell proliferation, survival, and EMT. Elucidating the molecular mechanisms necessary for the development and progression of human malignancies is critical to predict the most appropriate targets for cancer therapy. Here we used transforming growth factor beta-1 (TGFβ-1) to promote EMT and migration in mammary epithelial cells. We demonstrate a requirement of ILK activity for TGFβ-1 mediated EMT in mammary epithelial cells. In addition to nuclear translocation of Snail and Slug, TGFβ-1 treatment also induced expression of the mTORC2 component Rictor as well as its phosphorylation on Thr1135. Interestingly, TGFβ-1 treatment also induced an interaction between ILK and Rictor. All of these TGFβ-1-induced processes were significantly suppressed by inhibiting ILK activity or by disrupting the ILK-Rictor complex using siRNA-mediated knockdown. Furthermore, we identified formation of the ILK/Rictor complex formation in cancer but not normal cell types, and this was accompanied by ILK-dependent phosphorylation of Rictor on residue Thr1135. Inhibition of ILK partially reversed the basal mesenchymal phenotype of MDA-MB-231 cells and prevented EMT in MCF10A cells following TGFβ-1 treatment. These data demonstrate a requirement for ILK function in TGFβ-1-induced EMT in mammary epithelial cells and identify the ILK/Rictor complex as a potential molecular target for preventing/reversing EMT.

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 324. doi:1538-7445.AM2012-324
Abstract 3241: Analysis of transcription factor activation by the embryonic microenvironment in metastatic breast cancer cells
Authors: Tarasewicz Elizabeth , Aviles Misael , Shea Lonnie D. , Jeruss Jacqueline S.
Published: August 2012

Introduction: The embryonic microenvironment is an important source of signals that program multipotent cells to adopt a particular fate. Numerous studies have shown that components of the embryonic stem cell microenvironment can induce a reversion of cancer cell phenotype from a more aggressive to less aggressive state through mechanisms that are largely unknown. We have examined the phenotypic transformation associated with the culture of metastatic breast cancer cells in mouse embryonic stem cell conditioned media and have implemented a novel cell array technology to characterize the signaling pathways associated with this transformation. Methods: We used phase contrast microscopy to determine the time course of structure formation in MDA-MB-231 cells cultured in 3D with control or conditioned mouse embryonic stem cell media. Cell proliferation of study cells stably expressing Gaussia Luciferase was quantified by measuring Gluc production over time. Cell migration was measured for treated and untreated cells using a transwell migration assay. A live cell array was implemented to quantify changes in activity of 19 cancer-related transcription factors over a period of 8 days. Results: MDA-MB-231 cells cultured in stem cell conditioned media showed decreased area covered by spindle-like projections over a period of 10 days. Study cells cultured in stem cell conditioned media, but not in control media or mouse embryonic fibroblast conditioned media, had decreased proliferation over time. Cells cultured in stem cell conditioned media had decreased cell migration relative to cells cultured with regular media. Using a cell array to report on TF activity, cells cultured in conditioned stem cell media, when compared to control media, had varied activity in 7 of the 19 constructs investigated, including metastasis-related factors SP1 and GATA1. Conclusion: Our findings support the normalizing influence provided by the stem cell microenvironment through decreased MDA-MB-231 spindle-like projections, cell proliferation, and cell migration. Of the 19 transcription factor reporter constructs tested using the novel live cell array, GATA1 and SP1 emerged as 2 of the transcription factors impacted by the embryonic microenvironment. These factors may reflect potential paracrine signaling pathways stimulated by the microenvironment that can influence transformation and may be targets for therapeutic intervention.

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 3241. doi:1538-7445.AM2012-3241
Abstract 3250: Chondroitin sulfate-mediated regulation of Wnt/β-catenin signaling in breast cancer
Authors: Willis Catherine M. , Klüppel Michael
Published: August 2012

Breast cancer is among the leading causes of cancer related deaths in women in the U.S. Aberrant activation of the Wnt/β-catenin signaling pathway plays a critical role in breast cancer progression; however, activating mutations in core pathway components are not frequently observed. Molecules that reside in the cellular microenvironment, including glycosaminoglycans, are known to regulate tumor progression. The glycosaminoglycan chondroitin sulfate, as part of proteoglycan molecules, resides at the cell surface or in the extracellular matrix, and has been proposed to regulate the activities of several signaling pathways. We show here that the balance of chondroitin sulfation is a critical regulator of Wnt/β-catenin signaling in breast cancer cells. Overexpression of the chondroitin sulfotransferase C4ST-1 in vitro can significantly reduce several parameters of Wnt/β-catenin signaling activity, including Wnt3a-stimulated total β-catenin levels, TOPFLASH reporter activity, and proliferation, suggesting that the specific chondroitin sulfation pattern conferred by C4ST-1 can negatively regulate Wnt/β-catenin signaling in breast cancer cells. Interestingly, we show that Wnt3a stimulation increases C4ST-1 mRNA and protein levels, thus forming a negative feedback loop to attenuate Wnt/β-catenin signaling output. Accordingly, treatment with the C4ST-1 product CS-E interferes with Wnt/β-catenin signaling activity, LRP6 phosphorylation, β-catenin nuclear localization, and Wnt3a-mediated biological events in vitro. These data identify a novel chondroitin sulfate-based control mechanism for the Wnt/β-catenin pathway, and provide evidence for a potential therapeutic use of CS-E as an inhibitor of Wnt/β-catenin signaling in 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 3250. doi:1538-7445.AM2012-3250
Abstract 3252: Divergence of activin and TGFβ-induced SMAD-independent signaling in colon cancer
Authors: Bauer Jessica , Jung Barbara H.
Published: August 2012

Introduction: Activin and TGFβ are members of the TGFβ superfamily with growth suppressive and pro-migratory properties. While growth suppressive SMAD 2/3/4 signaling is shared, we have evidence of diverging SMAD-independent migratory signaling involving the CDK inhibitor p21. Here, we dissect the participation of respective mitogenic pathways in activin and TGFβ-specific SMAD-independent signaling in colon cancer. Methods: Colon cancer cell lines with and without SMAD4 were assessed for SMAD, PI3K and MEK pathway influences on activin or TGFβ signaling and associated effects using pharmacologic inhibition, knock-down, co-immunoprecipitation, Western blotting, and functional assays of migration. Results: Irrespective of SMAD4 status, activin-induced migration was PI3K-dependent and associated with PI3K-induced p21 downregulation, while TGFβ-induced migration was MEK1/2-dependent. Activin but not TGFβ lead to colocalization of ACVR1, ACVR2's primary binding partner, with p85, the regulatory subunit of PI3K, in an ACVR1-dependent SMAD-independent manner. Further, activin-induced p21 downregulation was PI3K dependent and associated with proteasomal degradation, while TGFβ stabilized p21 via MEK/ERK. Inhibition of proteasomal degradation of p21 lead to a decrease in migration with activin implicating p21 downregulation in activin-induced migration but not in TGFβ-induced migration. Conclusion: Activin and TGFβ diverge in their pro-migratory SMAD-independent signaling in colon cancer affecting distinct targets to include p21 and employing diverse mitogenic signals. Dissecting ligand-specific functions in colon cancer may be exploited for risk stratification or therapeutic intervention in the near future.

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 3252. doi:1538-7445.AM2012-3252
Abstract 3387: Secreted exosomes from cultured bladder cells are enriched for distinct miRNAs detected in circulation of metastatic bladder cancer patients
Authors: Ostenfeld Marie S. , Jeppesen Dennis , Morth Jens P. , Khanh Huy B. , Theodorescu Dan , Borre Michael , Dyrskjøt Lars , Ørntoft Torben
Published: August 2012

Exosomes are small nanovesicles (30-100 nm) secreted from the cell surface of most cell types. The vesicles are secreted at increased levels by cancer cells and can be detected in body fluids of cancer patients. Exosomes function in paracrine delivery of proteins, RNA, and DNA into recipient cells. By this, exosomes are speculated to be involved e.g. in pre-metastatic niche formation at distant sites. Here we present a characterization of exosome vesicles isolated from bladder cancer cells as examined by electron microscopy, dynamic light scattering and by western blotting of common exosome protein markers. The expression of 671 unique human miRNAs by qPCR was analyzed in both exosomes and donor cells from: 1) cultivated normal urothelial cells (NHU), 2) T24 bladder carcinoma cell line (non-metastatic) and its metastatic isogenic derivates FL3 and SLT4 and 3) UMUC3 bladder carcinoma cell line (moderate metastatic) and its highly metastatic isogenic derivative LUL2. Several miRNAs were detected in the vesicles yet absent in their parental donor cell indicating a possible preferential vesicular packaging. miRNAs miR92a, -106a, -20a, -21, -16, and let-7a/b/d/e were detected among the top100 ranked miRNAs in all vesicles. Among the top30 enriched exosomal miRNAs, miR-223 and-383 were detected in all cancer vesicles, and miR-921 in the highly metastatic vesicles (FL3, SLT4, LUL2). The release of miRNAs (miR-21, -16, and let7b) from FL3 cells into the culture media was time-dependent and a fraction of these miRNAs were stable to RNaseOne degradation indicating possible protection inside the vesicles. Examination of miRNAs in human plasma samples also suggested resistance towards endogenous RNAse degradation as well as towards repetitive freeze/thawing. The profiling of miRNA expression by Affymetrix microarray analysis of crude exosome-containing fractions of human plasma samples (10 healthy controls and 10 cases of metastatic bladder cancer) showed significant upregulation in cancer plasma of e.g. miR-92a, -106a, -23a, -20a, -21, some of which were the same as the miRNAs highly expressed in the tumor vesicles. The significant increase was confirmed by q-RT-PCR for miR-92a and -23a and validated on an independent set of plasma samples (8 healthy controls and 8 metastatic cancer cases). Our results suggest that specific miRNAs are encapsulated into exosomes in bladder cancer and that these may be detected in circulation in advanced stages of bladder 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 3387. doi:1538-7445.AM2012-3387
Abstract 3388: Progression of ductal carcinoma in situ to invasive breast cancer is associated with gene expression programs of EMT and myoepithelia
Authors: Witkiewicz Agnieszka , Ertel Adam , Knudsen Erik S.
Published: August 2012

Ductal carcinoma in situ (DCIS) is a precursor lesion that can give rise to invasive breast cancer (IBC). It has been proposed that both the nature of the lesion and the tumor microenvironment play key roles in progression to IBC. Here laser capture microdissected tissue from pure DCIS and pure IBC were employed to define key gene expression profiles in either the epithelial or stromal compartment associated with disease progression. Each tissue had distinct gene expression profiles, and a DCIS/IBC classifier accurately distinguished DCIS versus IBC in multiple independent data sets. However, contrary to other studies that profiled DCIS associated with IBC, we found that the most significant alterations in gene expression were observed in the epithelial compartment rather than in the stroma. In particular, genes associated with epithelial to mesenchymal transition (EMT) and myoepithelial cell specific genes were enriched in invasive cancer relative to pure DCIS. Such alterations in transcript levels were associated with all subtypes of breast cancer, but were particularly indicative of poor outcome in ER-negative breast cancer. Together, these studies indicate that lesion specific differences in gene expression associated with invasive phenotype are particularly relevant in the progression of DCIS to IBC.

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 3388. doi:1538-7445.AM2012-3388
Abstract 3393: Antibody-based array proteomics identify proteins differentially expressed in human metastatic and non-metastatic triple negative breast cancer cells
Authors: Roberti Maria P. , Arriaga Juan M. , Bianchini Michele , Quintá Héctor R. , Bravo Alicia I. , Levy Estrella M. , Mordoh José , Barrio María M.
Published: August 2012

Triple negative breast cancers (TNBC) lacking hormone receptors (ER, PR) and HER-2 amplification are very aggressive tumors. The IIB-BR-G cell line isolated from a primary TNBC, and its spontaneous metastatic variant, IIB-BR-G-MTS6 were compared using an antibody-based protein array to characterize their expression profile. We also analyzed their growth kinetics, migration, invasiveness, lymphangiogenesis and cytoskeleton structure. Doubling times in vitro were shorter for IIB-BR-G although in vivo IIB-BR-G-MTS6 tumors grew significantly faster than IIB-BR-G. IIB-BR-GMTS6 showed higher anchorage independent growth in a clonogenic assay. IIB-BR-G-MTS6 cells showed 100% incidence of lymph node metastasis at 5-6 weeks, although no metastasis was observed for IIB-BR-G even 19 weeks after inoculation. CCL3, IL1α, CXCL1, CSF2, CSF3, IGFBP1, IL1α, IL6, IL8, CCL20, PLAUR, PlGF and VEGF were strongly up-regulated in IIB-BR-G-MTS6 compared to IIB-BR-G, while CCL4, ICAM3, CXCL12, TNFRSF18, FIGF, were the more down-regulated proteins in the metastatic cell line. IIB-BR-G-MTS6 protein expression profile was compatible with higher NF-κB activation. In vitro, IIB-BR-G displayed higher migration but IIB-BR-G-MTS6 had a higher matrigel invasion. Accordingly, IIB-BR-G-MTS6 had an up-regulated expression of MMP1, MMP9, MMP13, PLAUR and HGF as compared to IIB-BR-G. IIB-BR-G-MTS6 tumors presented higher local lymphatic invasion than IIB-BR-G but similar lymphatic vessel densities. VEGFC and VEGFA/B expression were higher both in vitro and in vivo for IIB-BR-G-MTS6. Higher vimentin expression was mainly localized in the cellular extremities in IIB-BR-G-MTS6.Our results suggest that IIB-BR-G-MTS6 have acquired an epithelial-to-mesenchymal transition phenotype, modulating tumor stroma to favor tumor growth, increase angiogenesis and promote their metastatic dissemination.

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 3393. doi:1538-7445.AM2012-3393
Abstract 3396: Predictive value of metastatic lymph node ratio in diagnosis of recurrence after surgical resection of gastric cancer
Authors: Tanaka Hiroaki , Muguruma Kazuya , Yoshii Mami , Lee Tomohiro , Kimura Kenjiro , Nagahara HIsashi , Noda Eiji , Amano Ryosuke , Kubo Naoshi , Yashiro Masakazu , Yamada Nobuya , Maeda Kiyoshi , Sawada Tetsuji , Ohira Masaichi , Hirakawa Kosei
Published: August 2012

Peritoneal meatastasis is the most important pattern in postoperative recurrence of gastric cancer. The purpose of this study was to investigate the predictive factors of peritoneal recurrence in gastric cancer patients who underwent surgical resection. We retrospectively analyzed clinical data of 137 consecutive patients diagnosed with peritoneal recurrence after curative gastrectomy at Department of Osaka City University Hospital. We evaluated association of clinicopathological features with relapse free survival rate. Parameters analyzed in this study included age, sex, tumor diameter, histological type, tumor infiltration, number of lymph node metastasis, TNM stage, lymphatic invasion, venous invasion, and metastatic lymph node ratio (MLR). Median survival time of entire patients was 12 months and mean MLR was 34%. For statistical analysis, we divided patients into two groups based on relapse free survival time. 78 patients who had peritoneal recurrence within one year after gastrectomy were defined as early recurrence group. The remaining 59 patients were stratified to late recurrence group. In comparison of both groups, univariate analysis showed statistically significant predictive factors associated with recurrence, for example, peritoneal lavage cytology, lymphatic invasion, tumor diameter, number of lymph node metastasis, and MLR. In multivariate analysis, MLR was identified as an independent predictive factor. Also the patients with 20% or higher MLR had poorer prognosis in overall survival. We demonstrated that lymph node metastasis was associated with peritoneal recurrence. Our findings suggested that MLR should be considered for postoperative adjuvant therapy to impede progression of peritoneal metastasis for 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 3396. doi:1538-7445.AM2012-3396
Abstract 3403: E-cadherin and vimentin patterns by immunohistochemistry identify epithelial and mesenchymal phenotypes in lung adenocarcinoma
Authors: Pool Mark D. , Kaiser Kelly A. , Basu Sanjib , Borgia Jeffrey A. , Fidler Mary J. , Bonomi Philip
Published: August 2012

Purpose: To examine markers of epithelial-mesenchymal transition (EMT) in lung adenocarcinoma (ADC) defining epithelial-like (EPT) and mesenchymal-like (MES) phenotypes and correlate these with outcomes, in addition to other EMT markers. This study examined 33 patients with lung adenocarcinoma (ADC) who underwent surgical resection and later at disease progression received erlotinib. Experimental Design: All slides with tumor from resection specimens were reviewed (mean 4.8/case; range 1-9). Tumors were histologically subtyped according to recent IASLC classification. Tumor budding was graded per Ueno et al. (2002). A block containing the invasive front was selected for IHC staining with antibodies to e-cadherin (ECAD), vimentin (VIM), β-catenin (α-CAT), and SNAI1. Pattern-specific staining was evaluated at the invasive tumor front: E-cadherin-membranous, vimentin-cytoplasmic, α-CAT-membranous, SNAI1-nuclear, cytoplasmic. Complete circumferential membranous staining for e-cadherin was scored as present/absent; other markers were scored by the Allred system (0-8). PFS and OS were calculated from time of diagnosis to disease progression or death. Results: Two distinct phenotypes were identified with ECAD and VIM: EPT showing preserved membrane ECAD staining with low VIM (≥4) (N=19) and MES showing weak/absent membrane ECAD staining with high VIM (Δ5) (N=10). Median PFS for EPT group was 4.2 mo vs MES 1.6 mo; median OS for EPT group 15.5 mo vs 6.5 mo. These differences were not statistically significant. No association was found between high grade tumor budding and EPT vs MES phenotype p=0.221). Membranous α-CAT expression and nuclear SNAI1 expression showed a statistically significant correlation with EPT vs MES (β-catenin mean score 7.8 vs 6.1, p=0.0184) and (nuclear SNAI1 mean score 7.3 vs 6.1, p=0.0364). Conclusions: Epithelial and mesenchymal phenotypes can be identified by IHC for e-cadherin and vimentin. These phenotypes correlate with other EMT markers β-catenin and SNAI1 but not with tumor budding. Although differences in median PFS and OS were seen between the two groups, the number of cases is too small to identify a statistically significant difference. The results do support the rationale for defining EPT and MES groups for prognosis in a larger group of early-stage surgically resected patients.

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 3403. doi:1538-7445.AM2012-3403
Abstract 3405: MicroRNA expression characterizes oligometastasis(es)
Authors: Xing Rosie Hongmei , Lussier Yves A. , Salama Joseph K. , Khodarev Nikolai N. , Huang Yong , Zhang Qingbei , Khan Sajid A. , Yang Xinan , Hasselle Michael D. , Darga Thomas E. , Malik Renuka , Fan Hanli , Perakis Samantha , Filippo Matthew , Corbin Kimberly , Lee Younghee , Posner Mitchell C. , Chmura Steven J. , Hellman Samuel , Weichselbaum Ralph R.
Published: August 2012

Background: Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by β5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy. Methods: Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy. Results: Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression. Conclusions: These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.

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 3405. doi:1538-7445.AM2012-3405
Abstract 3432: HLJ1 serves as an inhibitor of epithelial-mesenchymal transition by inactivating β-catenin signaling
Authors: Chen Ching-Hsien , Lin Sheng-Yi , Chen Hsuan-Yu , Su Kang-Yi , Yuan Ang , Chen Huei-Wen , Yu Sung-Liang , Chen Jeremy
Published: August 2012

HLJ1 (DnaJ-like heat shock protein), also known as DNAJB4, was classified as a tumor suppressor gene. The expression of HLJ1 inversely associates with invasive ability and can predict the clinical outcomes of non-small-cell lung cancer (NSCLC) patients. However, the role of HLJ1 in suppressing cancer metastasis is still unclear. Here, we identified HLJ1 as a novel inhibitor of epithelial-mesenchymal transition (EMT). Our functional assays in vitro and in vivo demonstrated that Knock-down of HLJ1 promotes invasion capabilities of lung cancer cells and causes liver macrometastasis; meanwhile, the crucial domain of HLJ1 in suppression of cancer invasion was also confirmed. We found that the HLJ1-overexpressing cells maintain the well-organized cell adhesion and polarity. Inversely, an increase in actin polymerization and the formation of actin stress fibers were observed in the HLJ1-silencing cells. HLJ1 down-regulates the transcriptional activities of slug promoter and facilitates E-cadherin transcription, leading to the inhibition of epithelial to mesenchymal transition. Further, we identified the Wnt/β-catenin signalling pathway in which HLJ1 involved by cDNA microarray and pathway analysis. The subcellular distribution of β-catenin is predominant in the nucleus and the tyrosine-phosphorylated levels of β-catenin are up-regulated in the HLJ1-silenced clones. Finally, we showed a decreased accumulation of β-catenin and an enhanced degradation of β-catenin in absence of HLJ1 expression. Taken together, our study reveals that HLJ1 has anti-metastatic activity by repressing β-catenin signaling and provides a molecular model for the development of new anticancer agents.

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 3432. doi:1538-7445.AM2012-3432
Abstract 3433: Molecular regulation of RKIP, a tumor metastasis suppressor in breast cancer
Authors: Lee Jiyoung , Yun Jieun , Yeung Kam C. , Rosner Marsha R.
Published: August 2012

Breast cancer mortality is primarily due to metastasis to organs including bone, lungs, liver and brain. Tumor metastasis suppressors are metastasis inhibitors but do not affect the ability of the transformed cells to produce a tumor at the primary sites. Raf kinase inhibitory protein (RKIP) has been identified as a metastasis suppressor for a number of cancers including breast, prostate, liver, colon, skin, and nasopharyngeal carcinoma. RKIP overexpression in hepatoma, melanoma, and prostate metastatic cell lines diminished their invasiveness and motility in vitro. The molecular mechanism by which RKIP transcription is regulated in breast cancer is largely unknown. Interestingly, RKIP expression was highly and positively correlated with patient's metastasis-free survival after radiotherapy. Likewise, when RKIP was reduced in human prostate cancer cells, radiation-induced apoptosis was reduced in xenograft tumors. Moreover, RKIP expression levels have been induced upon chemotherapeutic drug treatment in human breast cancer cells, sensitizing the tumor cells to apoptosis. These studies indicate that chemotherapy and radiotherapy are more effective when RKIP is present in the metastatic cancer cells. Therefore, high RKIP levels are desirable to prevent metastasis of breast tumor cells and to sensitize tumor cells for chemotherapy. Our goal is to understand the mechanism by which RKIP is regulated in order to restore its levels. Previously, we have observed that let-7 negatively regulates two target genes, BACH1 and HMGA2 that promote breast cancer metastasis in a xenograft mouse model. Here we demonstrate that BACH1 and HMGA2 are themselves negative regulators of RKIP. Specifically, we have delineated molecular mechanisms by which BACH1 and HMGA2 inhibit RKIP expression in breast cancer cell lines using both luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays. We are also analyzing whether BACH1 and HMGA2 are dysregulated by chemotherapy in highly metastatic breast cancer cells and whether suppression of these RKIP regulators can potentiate sensitivity of the breast cancer cells to chemotherapy. These studies provide valuable mechanistic insights for potentiating chemotherapeutic sensitivity as well as potential biomarkers for metastatic 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 3433. doi:1538-7445.AM2012-3433
Abstract 3447: Tetrac (tetraiidothyroacetic acid) decreases in vitro x-ray survival of a human medullary thyroid cancer cell line under oxic and hypoxic conditions
Authors: Hercbergs Aleck A. , Davis Faith B. , Davis Paul J. , Leith John T.
Published: August 2012

In 2009 we showed that 2 µM tetrac given for 1 hour at 37oC prior to 250 kVP x-irradiation sensitized GL261 murine brain tumor cells. Radiosensitization was accomplished by increasing the alpha component in the linear-quadratic equation of cell survival (Cell Cycle, 8:2586-91). In 2011, we showed that tetrac also sensitized U87MG human brain tumor cells to 250 kVp x-rays (Cell Cycle, 10: 352-57). Again radiosensitization was accomplished by an increase in the αx-ray component. Tetrac also inhibited the repair of DNA double strand breaks in U87MG cells. However, tetrac which binds to the αvβ3 integrin receptor and inhibitssignaling from the αvβ3 receptor, penetrates the cell and has a thyromimetic effect which offsets radiosensitization. In this regard, we synthesized a nanoparticle tetrac formulation termed Nano-T. Nano-T also binds to the αvβ3 receptor but does notpenetrate the cell. Nano-T causes radiosensitization in vitro in H522 lung cancer cells, producing an added benefit compared to tetrac. We have also shown that daily treatment of in vivo human-MTC (h-MTC) solid tumors in mice with Nano-T for 20 days produced significant growth inhibition. It is our intent to combine Nano-T with ionizing radiation in vivo, and we have begun by studying the effects of tetrac on h-MTC cells in vitro. These h-MTC cells are show an in vitro cell culture doubling time of 96.2 hours and a clonogenicity of 3.16%. We irradiated cells in exponential growth with 250 kVp X-rays. Below we give the LQ survival parameters, and the surviving fraction at 2 Gy (SF2). Table 1. Initial Results of 250 kVp Single Dose X-irradiation on h-MTC CellsIn Vitro without and with Tetrac ≤ α SF2 Tetrac Oxygen (Gy-1 x 10-1) (Gy-2 x 10-2) Enhancment Enhancement Factor Factor Without Tetrac Oxic Cells - 0.033 0.0256 0.901 2.55 Hypoxic Cells 0.030 0.0023 0.922 With Tetrac Oxic Cells 0.472 0.0495 0.330 2.84 2.60 Hypoxic Cells 0.013 0.0256 0.803 2.84 First, h-MTC cells irradiated as oxic or hypoxic, exponentially growing cells are very radioresistant suggesting a large capacity to repair radiation damage. Second, tetrac sensitized both oxic and hypoxic h-MTC cells. These characteristics speak to our proposed Nano-T in treatment of h-MTC tumors in vivo. Nano-T should increase the radiosensitivity of both oxic and hypoxic cells and should also decrease the repair of radiation damage in conventional fractionated radiation 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 3447. doi:1538-7445.AM2012-3447
Abstract 3452: The role of CX3CR1 in resistance of ovarian carcinoma cells to radiation therapy
Authors: Xie Jia , Barbolina Maria
Published: August 2012

Ovarian carcinoma is a leading cause of death from gynecologic malignancies. The current standard of care, a combination of chemotherapy and radiation, fails to keep patients in remission. Thus, modifications of the therapy and the combinations with the new molecular biological technologies are crucial for the development of the modernized approaches to combat this disease. Previous attempts to use radiotherapy as an additional treatment was not successful mainly due to the dose-related side effects, as well as radioresistance. Therefore, mechanistical understanding of the biology of ovarian carcinoma could offer ways to overcome current problems. Fractalkine receptor (CX3CR1) belongs to a chemokine family of G protein-coupled receptors, the most frequently and successfully drug-targeted group of proteins. Interaction of chemokines with their receptors plays a pivotal role in homing distant metastasis, as well as promoting migration, adhesion, and proliferation in many types of cancer. This project is aimed at understanding the role of CX3CR1 in sensitization of epithelial ovarian cancer cells to radiotherapy. Our data indicate that downregulation of CX3CR1 in ovarian carcinoma cells leads to approximately 50% sensitization to x-ray radiation, as determined by the clonogenic ability assay. Our Western blotting and immunofluorescence analysis data also show that downregulation of CX3CR1 results in accumulation of DNA damage at lower doses. Thus, our data indicate that disruption of the CX3CR1 signaling could sensitize ovarian carcinoma to radiation 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 3452. doi:1538-7445.AM2012-3452
Abstract 3456: Nicotine and the alpha-7 nicotinic acetylcholine receptor in the regulation of response to radiotherapy
Authors: Sobus Samantha L. , Romano Michelle A. , Mackowiak Janelle , Warren Graham W.
Published: August 2012

Purpose: Nicotine is a systemically available component of tobacco known to decrease the effectiveness of chemotherapy and/or radiotherapy (RT) however, the mechanisms of decreased RT response are not well characterized. The purpose of this study is to evaluate the effects of nicotine and activation of the alpha-7 nicotinic acetylcholine receptor (a7-nAChR) on regulating cell survival following RT and to identify potential mechanisms of regulation. Methods: Human lung cancer (A549, H460) and oropharyngeal cancer (FaDu) cell lines were treated with nicotine alone or in combination with nAChR inhibitors, MEK inhibitors, PI3K inhibitors, or mTOR inhibitors followed by treatment with RT to evaluate cell survival using clonogenic assays. The effects of a7-nAChR agonists on RT were also evaluated. Western blots were performed to evaluate nAChR subunit expression profiles in a broad spectrum of cancer cell lines and to evaluate the effects of nicotine (alone or with inhibitors listed above) or agonists on downstream signal transduction. Results: Nicotine administration 2 hours prior to RT increased survival in all cell lines. nAChR expression was confirmed in several cell lines including A549 and H460 cells. Inhibition of specific nAChRs in A549 and H460 cells demonstrated that the a7-nAChR prevented the effects of nicotine on RT in both cell lines. Nicotine administration increased p-ERK 1/2, p-Akt (Ser-473), mTOR (S2448), activated matrix metalloproteinase 2 (MMP2), and hypoxia inducible factor 1-alpha (HIF-1a). Inhibition of MEK prevented ERK 1/2 phosphorylation, but did not prevent the effects of nicotine on RT. Inhibition of PI3K prevented Akt phosphorylation, MMP2 activation, and HIF-1a induction with concomitant inhibition of the effects of nicotine on RT. Administration of an mTOR inhibitor (rapamycin) significantly sensitized cells to RT, but did not prevent the pro-survival effects of nicotine. Administration of selective a7-nAChR agonists (LY 2087101, PHA 54613, PNU 282987, TC 1698) demonstrated that all increased p-ERK 1/2 and p-Akt; however, only LY 2087101 and PHA 54613 increased cell survival with RT. Further analysis demonstrates that only LY 2087101 and PHA 54613 increased mTOR (S2448) while there was no change in mTOR (S2448) with PNU 282987 or TC 1698. Conclusions: Data demonstrates that activation of the a7-nAChR can decrease the effectiveness of RT. Results suggest that PI3K and mTOR may play a central role in conferring resistance to radiation by nicotine or during a7-nAChR activation. Further analyses of critical mechanisms are required to understand the adverse systemic effects of tobacco on cancer treatment.

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 3456. doi:1538-7445.AM2012-3456
Abstract 3463: The role of Atg13 in response to radiotherapy in cervical cancer cells
Authors: Roossink Frank , Boers Aniek , Wisman Bea , Schuuring Ed , van der Zee Ate , de Jong Steven
Published: August 2012

Introduction The role of autophagy protecting cancer cells against anti-cancer therapy, especially via the Beclin-1 axis, has become more clear. In cervical cancer, functionality of the Beclin-1 axis can be questioned, since promoter methylation and silencing of two components of this axis, C13ORF18 (Rubicon-like) and DAPK, is frequently observed. The involvement of another autophagy pathway, the ULK1 axis is less well studied in this tumor type. The ULK1 axis, consisting of ULK1, Atg13 and FIP200 is under control of mTOR. Here, we investigated the role of the essential component of the ULK1 complex, Atg13 in the response to (chemo)radiation in cervical cancer. Methods and Results Using immunohistochemistry we first analyzed expression of Atg13 in a large cohort of 375 pretreatment tissue samples of advantaged stage cervical cancer patients primarily treated with (chemo)radiation. Positive expression of Atg13 was more frequently observed in adenocarcinoma, known to have a poor response to chemoradiation, compared to squamous carcinoma (75% versus 56 % positive tumors, p< 0.03). Transient overexpression of GFP-tagged Atg13 in HEK293T kidney cells increased radiotherapy resistance (1.5-2 fold). Western blot showed that Atg13 was expressed in several cervical cancer cell lines, including the adenocarcinoma model HeLa. Downregulation of Atg13 expression with siRNA, resulted in a 2-fold increase in radiotherapy sensitivity in HeLa cells as determined with clonogenic assays. In HeLa-GFP-LC3 cells, we found that irradiation caused a strong increase in LC-3 containing autophagosomes, which co-localized with Atg13. The autophagy inhibitor bafilomycin A1 blocks the conversion of autophagosomes into autolysosomes. Bafilomycin A1 treatment indeed resulted in an accumulation of radiation-induced autophagosomes, demonstrating that autophagy was occurring after irradiation. In addition, HeLa cells were sensitized to irradiation, when autophagy was inhibited with bafilomycin A1. These results were confirmed using the less specific autophagy inhibitor 3-methyladenine and the autophagy inducer rapamycin (an mTOR inhibitor). Conclusion We showed that the Atg13 expression is related to the presence of cervical adenocarcinoma. Our in-vitro results indicate that the autophagic ULK1 pathway plays an important role in protecting cervical cancer cells to irradiation. Thus, inhibition of autophagy may be an interesting strategy to sensitize cervical adenocarcinoma to radiotherapy.

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 3463. doi:1538-7445.AM2012-3463
Abstract 3470: HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: Implications for efficacy of adjuvant trastuzumab
Authors: Malik Fayaz , Ithimakin Suthinee , Day Kathleen , Ignatoski Kathy , Zen Qin , Thomas Dafydd , Bersano Tom , Dawsey Scott , Hall Christopher , Davis April , Quraishi Ahmed A. , Tawakkol Nader , Clouthier Shawn G. , Day Mark , Hayes Daniel F. , Korkaya Hasan , Wicha Max S.
Published: August 2012

Although current breast treatment guidelines limit the use of HER2 blocking agents such as trastuzumab to women whose tumors display HER2 gene amplification, recent retrospective analysis by Paik et al., have suggested that a wider group of patients may benefit from this therapy. Utilizing breast cancer cell lines, mouse xenograft models and primary human tumors and metastatic tissues, we provide a potential molecular explanation for these unanticipated clinical finding by demonstrating that HER2 is selectively expressed in the cancer stem cell (CSC) population in luminal ER+ breast cancers that do not display HER2 amplification. Furthermore, we demonstrate that the effects of trastuzumab on mouse tumor xenografts are highly dependent on the timing of administration. While the effects of trastuzumab on established tumors are limited to those with HER2 amplification, when this HER2 blocker is given immediately after tumor inoculation simulating an adjuvant setting, it significantly inhibits the growth of luminal tumors in the absence of HER2 amplification. In addition, HER2 expression is increased in luminal tumors propagated in mouse tibias as compared to primary tumors grown in mammary fat pads. Utilizing co-culture experiments with human osteocytes, we provide evidence that this regulated expression of HER2 is mediated by RANK ligand present in the bone microenvironment. The clinical relevance of these studies is demonstrated in breast cancer tissues by the co-expression of HER2 and CSC marker ALDH1 in a subset of cells which are selectively found at the invasive tumor front in luminal breast cancers. Recapitulating the mouse xenograft studies, we demonstrate increased expression of HER2 in bone metastasis compared to matched primary tumors in 8 patients with luminal breast cancer. Our results have significant clinical implications since they suggest that the benefits of adjuvant HER2 targeting therapies may extend to patients with luminal breast cancers who are currently classified as HER2-negative. Furthermore, these results support the cancer stem cell hypothesis and emphasize the clinical importance of targeting cancer stem cells in the adjuvant setting.

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 3470. doi:1538-7445.AM2012-3470