bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 368: Detection of circulating tumor cells in peripheral blood of renal cell carcinoma patients is correlated with survival.
Authors: Goebel Steffen , Bluemke Karen , Bilkenroth Udo , Meye Axel , Fuessel Susanne , Lautenschlaeger Christine , Melchior Andres , Heynemann Hans , Fornara Paolo , Taubert Helge
Published: August 2014

Purpose:

The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood [so called circulating tumor cells (CTCs)] for renal cell carcinoma (RCC) patients after a median follow-up time of 81 months.

Methods:

233 peripheral blood samples from 154 RCC patients were investigated for the presence of disseminated tumor cells (DTCs) by autoMACS technique and immunocytochemical (ICC) staining of cytokeratin. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data.

Results:

Two kinds of tumor cells were detected: Those with expression of cytokeratin 8/18 (CK+), but also cells without a detectable CK+ expression, which we called large blue stained cells (Bl+) with a tumor-like morphology. We hypotesized, that the latter cells could be “sleeping” tumor cells.

After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients.

An update to follow up the RCC patients was conducted in 2012. Besides a significant correlation between the detection of CTCs before and after primary tumor resection a significant association with prognosis was found (p < 0.002; Chi-squared test). RCC patients with CK+ CTCs in their peripheral blood had a 5 times risk of tumor-related death compared to patients without these cells. In a Kaplan-Meier-Analysis these results were confirmed (p < 0.001).

Patients with large blue stained cells (Bl+) also had a shorter survival time.

Conclusion:

In summary, for RCC patients, the presence of CTCs was associated with a poor overall survival and significantly correlated with an increased risk of tumor-related death. It is important to evaluate CK+ tumor cells to determine the role of CTCs in tumor behavior and disease progression. Detection of CK+ CTCs in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma patients.

Citation Format: Steffen Goebel, Karen Bluemke, Udo Bilkenroth, Axel Meye, Susanne Fuessel, Christine Lautenschlaeger, Andres Melchior, Hans Heynemann, Paolo Fornara, Helge Taubert. Detection of circulating tumor cells in peripheral blood of renal cell carcinoma patients is correlated with survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 368. doi:10.1158/1538-7445.AM2013-368
Abstract 3684: Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53.
Authors: Gupta Karishma , Thakur Vijay S. , Bhaskaran Natarajan , Nawab Akbar , Babcook Melissa A. , Jackson Mark W. , Gupta Sanjay
Published: August 2014

Tumor suppressor gene p53 is frequently inactivated in a variety of human cancers including prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induces apoptosis in prostate cancer cells irrespective of p53 status (Carcinogenesis 33:377-84, 2012). However, the molecular mechanisms underlying these observations are not properly understood. In the present study we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer cells with and without functional p53. Human prostate cancer LNCaP cells were stably transfected with short hairpin-53 (LNCaPshp53) and control vector (LNCaPshV), exposed to GTP in dose and time dependent manner. GTP exposure induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through the activation of c-jun N-terminal kinase resulting in FADD phosphorylation, caspase-8 activation and truncation of BID leading to apoptosis was common in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases resulting in PARP cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in the inhibition of class I HDAC protein, accumulation of acetylated histone H3 in total cellular chromatin resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax irrespective of p53 status of cells, consistent with the effects elicited by HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces prostate cancer cell death by two distinct mechanisms irrespective of p53 status, thus identifying for the first time through defined molecular targets for their chemopreventive and/or therapeutic actions.

Citation Format: Karishma Gupta, Vijay S. Thakur, Natarajan Bhaskaran, Akbar Nawab, Melissa A. Babcook, Mark W. Jackson, Sanjay Gupta. Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2013-3684
Abstract 3699: Chemoprevention of skin cancer by benzophenone sunscreen agents in mice.
Authors: Kapadia Govind J. , Rao G Subba , Takasaki Midori , Iida Akira , Suzuki Nobutaka , Konoshima Takao , Tokuda Harukuni
Published: August 2014

Our recent study demonstrated the skin cancer chemoprevention potential of commonly used benzophenone sunscreen agents using the Epstein-Barr virus early antigen in vitro assay (Int. J. Cosmet. Sci. 2012: doi:10.1111/ics.12015). We now report the in vivo anti-tumor activity of two of the benzophenone sunscreens tested positive in the above in vitro assay, octabenzone (UV-1) and dioxybenzone (UV-2), in the two-stage mouse skin carcinogenesis test model utilizing (±)-(E)-4-methyl-2-[-(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) as inducer and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter. Studies were conducted in pathogen-free, female hairless mice of HOS-HR-1 strain, 15 animals per control and test groups. Tumor was induced by a single dose of NOR-1 (390 nmol in 100 μl of acetone). One week later, TPA (1.7 nmol in 100 μl of acetone) was applied to skin twice weekly for 20 weeks as a tumor promoter. The test compounds, 0.0025% of UV-1 or UV-2 were administered to the animals through drinking water ad libitum, starting 1 week prior to tumor initiation and stopping 1 week after tumor initiation. All animals were examined weekly for the development of skin papillomas and the following were recorded: the week papillomas appeared, number of mice bearing papillomas and number of papillomas per mouse. In both UV-1 and UV-2 treated mice, 2-week delay in tumor appearance and significant inhibition of tumor incidence (50% and 60%, respectively) and burden (papilloma inhibition/mouse, 50% and 70%, respectively) were observed when compared to the positive control group. Also, treatment with both benzophenone sunscreens produced marked reduction in the average number of tumors formed per mouse (5 and 3 papillomas/mouse for UV-1 and UV-2, respectively compared to 10 papillomas in positive control group) at the end of the 20-week experimental period. These in vivo results in mice further affirm the skin cancer chemoprevention potential of benzophenone sunscreen use in humans.

Citation Format: Govind J. Kapadia, G Subba Rao, Midori Takasaki, Akira Iida, Nobutaka Suzuki, Takao Konoshima, Harukuni Tokuda. Chemoprevention of skin cancer by benzophenone sunscreen agents in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2013-3699
Abstract 3707: Early stage inhibition of murine mammary carcinogenesis by ultraviolet radiation.
Authors: Makarova Anastasia M. , Wang Grace , Wang Lynn , Wang Joy , Khaimskiy Yefim , Epstein Ervin H.
Published: August 2014

Epidemiological studies of geographical variations in breast cancer mortality rates have found increased death rates at higher latitudes in USA and other countries 1. This breast cancer latitudinal gradient is hypothesized to be mediated by ultraviolet radiation (UVR) - produced vitamin D3. We first have tested this latitudinal hypotheses using the well-described C3(1)/Tag transgenic murine mammary carcinogenesis model2. We have found that UVR significantly prolongs palpable tumor - free survival (135±23 vs.118±18 days) and reduces the tumor burden and multiplicity (2.9±1.6 vs. 7.2±2.3 tumors), but does not alter the time between the appearance of the first palpable tumor and death in Tag female mice. Therefore we have focused on the effects of UVR on earlier stage carcinogenesis. Tag transgenic female mice have a time-dependent increase in the incidence of ductal carcinoma in situ (DCIS) - like incidence. Thus, Tag mice develop ductal epithelial atypia in 50% of mice at age 8 wks, with progression to intraepithelial neoplasia (resembling human DCIS) in 70-90% of mice (in at least one gland) at 10-13 wks, and DCIS-like changes and early invasion in 100% of mice at age 14-15 wks. We compared weekly the similarity of histological changes between the pairs of mammary glands and found no significant gland site-to-gland site variations. UVR treatment, 350mJ/cm2 thrice weekly from age 6 wks to 15 wks, reduced both the percentage of DCIS-bearing mice (65, 60, 51, 32 and 20% reduction at week 8, 9, 11, 13 and 14) and the percentage of DCIS-bearing glands in each animal (82, 55, 66, 20 and 19% reduction at week 9, 11, 13, 14 and 15). The mechanism of the UVR-mediated prevention of the murine mammary carcinogenesis is unclear but there is evidence favoring a Hedgehog (HH) signaling-dependent mechanism, since D3 is a potent inhibitor of the HH signaling pathway, which is upregulated in many cancers including breast cancer3-5. Consistent with this idea we found that UVR reduces DCIS-like changes in Ptch+/- mice. Taken together our results support our hypothesis that inhibition of early stage mammary carcinogenesis by UVR-produced vitamin D3 underlies the latitudinal gradient. 1. Garland, F. C. et all. Prev Med 1990, 19, 614-22. 2. Green, J. E. et all. Oncogene 2000, 19, 1020-7. 3. Bijlsma, M. F. et all. PLoS Biol 2006, 4, e232. 4. Moraes, R. C. et all. Development 2009, 136, 1423-32. 5. Lewis, M. T. et all. Development 1999, 126, 5181-93.

Citation Format: Anastasia M. Makarova, Grace Wang, Lynn Wang, Joy Wang, Yefim Khaimskiy, Ervin H. Epstein. Early stage inhibition of murine mammary carcinogenesis by ultraviolet radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2013-3707
Abstract 3714: Metabolomics and proteomics reveal inhibition of glycolysis in withaferin A-mediated prevention of mammary carcinogenesis in MMTV-neu mice.
Authors: Singh Shivendra V. , Hahm Eun-Ryeong , Lee Joomin , Kim Su-Hyeong , Sehrawat Anuradha , Arlotti Julie A.
Published: August 2014

Plants used in Ayurvedic medicine, which has been practiced for centuries in India for the treatment of different ailments, continue to draw attention for identification of novel small molecule cancer chemopreventive agents. Withaferin A (WA) is one such promising small molecule constituent of the medicinal plant Withania somnifera (also known as Ashwagandha) with in vivo activity against xenografted human breast cancer cells. We have also shown recently that mammary cancer development and pulmonary metastasis in MMTV-neu transgenic mice is inhibited significantly by WA administration. In the present study, we performed metabolomics profiling using plasma and tumor tissues (n=8) and proteomics profiling using tumor tissues (n=3) from control and WA-treated MMTV-neu mice to gain insights into the mechanism underlying in vivo preventive effect of WA. A total of 320 and 328 biochemicals of known identity were detectable in the plasma and tumor tissue, respectively. The WA-mediated mammary cancer prevention in MMTV-neu mice was associated with a statistically significant alteration (P≤0.05) in the levels of 76 biochemicals in the plasma (24 increase, 52 decrease). Metabolomics using tumor tissues revealed alterations in the levels of 24 biochemicals (2 increase, 24 decrease) upon WA administration. One change that was highly consistent in the plasma and tumor tissue from WA-treated mice compared to controls was a decrease in glucose utilization, glycolysis, and TCA cycle activity. Proteomics using tumor tissues confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase A isoform 2. Downregulation of TCA cycle-related proteins in the tumor of WA-treated mice compared with control included pyruvate carboxylase and isocitrate dehydrogenase. Other altered biochemicals were reflective of changes in arginine metabolism, polyamine biosynthesis, glutathione metabolism, lipid metabolism, and catabolism of branched-chain amino acids. These observations indicate that WA treatment prevents mammary cancer in MMTV-neu mice by suppressing glycolysis (Warburg effect) and TCA cycle. This study was supported by the grant CA142604 awarded by the National Cancer Institute.

Citation Format: Shivendra V. Singh, Eun-Ryeong Hahm, Joomin Lee, Su-Hyeong Kim, Anuradha Sehrawat, Julie A. Arlotti. Metabolomics and proteomics reveal inhibition of glycolysis in withaferin A-mediated prevention of mammary carcinogenesis in MMTV-neu mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3714. doi:10.1158/1538-7445.AM2013-3714
Abstract 3725: Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts.
Authors: Yen Wan-Ching , Fischer Marcus M. , Shah Jalpa , Wei Jie , Cain Jennifer , Yeung Pete , Beviglia Lucia , Cancilla Belinda , Kapoun Ann , Lewicki John , Gurney Austin , Hoey Timothy
Published: August 2014

Ovarian cancer in a major cause cancer related death in women and this disease is often characterized by resistance to chemotherapy and tumor recurrence. Cancer stem cells (CSCs) have been shown to be relatively insensitive to chemotherapeutic agents. Several lines of evidence indicate that aberrant Notch signaling plays a major role in ovarian cancer progression and resistance to standard therapies. DLL4 is a key ligand that activates the Notch pathway and has been shown to regulate both CSC function and tumor angiogenesis. DLL4 expression in tumor and endothelial cells has also been associated with resistance to bevacizumab treatment. In these studies, we investigated the role of anti-DLL4 in a panel of patient-derived ovarian cancer xenograft models. We utilized anti-human DLL4 (OMP-21M18) and anti-murine DLL4 to block Notch signaling in both the tumor and stromal/vascular cells in the xenografts. We found that anti-DLL4 treatment was broadly efficacious in these ovarian cancer models, significantly inhibiting tumor growth both as a single agent and in combination with paclitaxel. We carried out serial transplantation studies to investigate the effect on cancer stem cells and found that anti-DLL4 in combination with paclitaxel profoundly reduced the tumor initiating cell frequency. In contrast, treatment with paclitaxel alone had the opposite effect and increased ovarian CSC frequency. These data suggest that anti-DLL4 treatment sensitizes chemoresistant ovarian tumorigenic cells to therapy and provide compelling evidence that anti-DLL4 (demcizumab) is a promising strategy for the clinical treatment of ovarian cancer.

Citation Format: Wan-Ching Yen, Marcus M. Fischer, Jalpa Shah, Jie Wei, Jennifer Cain, Pete Yeung, Lucia Beviglia, Belinda Cancilla, Ann Kapoun, John Lewicki, Austin Gurney, Timothy Hoey. Anti-DLL4 (demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3725. doi:10.1158/1538-7445.AM2013-3725
Abstract 3731: Targeting ovarian cancer stem cells using a novel ALDH inhibitor.
Authors: Chefetz Ilana , Yang Kun , Larsen Scott D. , Buckanovich Ronald J.
Published: August 2014

Background: Cancer cells with stem cell-like properties (CSC) are resistant to chemotherapy and are believed to be the source of cancer recurrence. Aldehyde dehydrogenase enzymatic activity (ALDH), alone and in combination with the stem cell marker CD133, can be used to define a hierarchy of Ovarian Cancer Stem Cells (OvCSC). ALDH is functionally important for stem cells, regulating motile cellular metabolic functions and the biosynthesis of retinoic acid. ALDH agonists have been demonstrated to enhance ‘stemness’ in some tissues. We therefore sought to determine the impact of ALDH antagonist on OvCSC.

Methods and Results: We screened a variety of novel derivatives with confirmed or potential ALDH activity based on the molecular homology to the known ALDH inhibitor DEAB. Using the ALDEFLUOR assay and A2780 cancer cell line as a source of ALDH+CD133+ cells, we identified a lead compound 673 which effectively inhibits ALDH activity. Interestingly this compound also potently depletes CD133+ ovarian cancer cells. Supporting functional depletion of OvCSC, treatment of primary human ovarian tumor ascites cells with 673 resulted in 4-20 fold decrease tumor sphere formation. 673 treatment is highly synergistic with cisplatin both in-vitro and in-vivo as assessed by cell growth curves and tumor growth. Importantly, pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Finally, 673 is non-toxic to normal stem cells.

Conclusions: We have identified a novel ALDH inhibitor, 673, which depletes the ovarian cancer cells expressing CSC markers. CSC marker independent functional assays, with both cell lines and primary human ovarian cancer cells, indicate 673 is depleting OvCSC activity. Thus this may be a powerful new therapeutic approach to target CSC. Future studies will elucidate the molecular mechanism that underlies 673 OvCSC depletion activities as well as to identify a specific ALDH isoforms targeted in ovarian cancer.

Citation Format: Ilana Chefetz, Kun Yang, Scott D. Larsen, Ronald J. Buckanovich. Targeting ovarian cancer stem cells using a novel ALDH inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2013-3731
Abstract 3758: RIG-I plays a critical role in negatively regulating stemness properties in hepatoma cell lines.
Authors: Chen Ping-Yu , Lai Chao-Kuen , Kuo Min-Linag
Published: August 2014

Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide due to its high rate of recurrence. Cancer stem cells are considered to generate tumors through the stem cell processes including self-renewal and differentiation into multiple cell types. And these distinct populations of cells are suggested to cause relapse and metastasis by eliciting new tumors. Retinoic acid-inducible gene I (RIG-I) has been identified as a pattern recognition receptor in innate immune defense for sensing intracellular viral RNAs, like Hepatits C virus which is one of the major risks of HCC, and inducing type I interferons production. In previous studies, RIG-I-like receptors recognizing some potentially oncogenic agents may have functional consequences that can lead to association of such single nucleotide polymorphisms with risk of some diseases or even cancers. Thus these gene polymorphisms may affect cancer risk indirectly. On the other hand, a previous study have demonstrated that RIG-I is an essential regulator of physiological myelopoiesis suggesting RIG-I possesses the potential of modulating stem cell differentiation. In our present study, we examined Huh7 and its RIG-I-mutant cell line by sphere formation assay and found solely the mutated cells possessed the self-renewal ability. After transfection of dominant-negative mutant of RIG-I to Huh7, the self-renewal ability were rescued as expected. We also injected 1000 cells for each two cell lines into NOD/SCID mice subcutaneously and observed the mutated cells formed tumors whereas Huh7 didn't. Hence, our study reveals the potential of RIG-I to suppress the stemness properties in the HCC cell lines and provides a new possible remedy target of liver cancer.

Citation Format: Ping-Yu Chen, Chao-Kuen Lai, Min-Linag Kuo. RIG-I plays a critical role in negatively regulating stemness properties in hepatoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3758. doi:10.1158/1538-7445.AM2013-3758
Abstract 3762: The role of aldehyde dehydrogenase isoenzymes in cancer cell proliferation, migration and drug resistance.
Authors: Moreb Jan S. , Mona Mahmoud , Chang Lung-Ji , Yeh Yu-Ling , Amory John , Goldstein Alex
Published: August 2014

Aldehyde dehydrogenase (ALDH) proteins are a superfamily of 19 human genes that play crucial role in epithelial cell homeostatsis. Several of these enzymes have been studied in cancer and were shown to protect cells against cytotoxic drugs and oxidative stress in addition to the promotion of cancer cell proliferation and invasion. Our study focuses on 5 less studied isoenzymes (ALDH1B1, ALDH3B1, ALDH4A1, ALDH5A1, and ALDH7A1) using lentiviral mediated overexpression of the enzymes in H1299 lung cancer cell line which is previously known to have negligible amount of ALDH activity. H1299 wild type cells (WT) and cells expressing GFP or RFP were used as controls. Expression of the proteins was confirmed by Western blots and ALDH enzymatic activity assay. We used cell culture, colorimetric MTT assay, wound healing assay, aldefluor assay, colony-forming assay, and in vitro treatment with doxorubicin (Doxo) and 4-hydroperoxycyclophosphaimde (4-HC). All the proteins were highly expressed and resulted in increased ALDH activity detected by using propionaldehyde or benzaldehyde as substrates with NAD+ as a co-factor. The activity of ALDH4A1, 5A1 and 7A1 was not detected by aldefluor flow cytometry assay. Cells expressing ALDH1B1, ALDH4A1 or ALDH7A1 had delayed wound healing at 6 hr, while overall cell proliferation was depressed for ALDH1B1, ALDH3B1 and ALDH5A1 and normal for the other isoenzymes. Cells overexpressing ALDH4A1 or ALDH7A1 showed higher clonal efficiency as well. Only ALDH1B1 and ALDH7A1 showed significant protection against the toxicity of 4-HC (10 μM for 72 hrs) as measured by MTT assay with an increase in average cell survival of 52 and 23%, respectively. Cells overexpressing ALDH5A1 showed significantly increased sensitivity to Doxo (1 and 10 μM for 72 hrs). There was a trend for ALDH7A1 to be protective against Doxo toxicity but did not reach statistical significance. In conclusion, the overexpression of ALDH isoenzymes in this manner may contribute to better understanding of their role in cancer biology and may lead to new cancer treatment approaches that incorporate specific inhibition or stimulation of these enzymes. This is an important goal especially in view of the high ALDH activity in cancer stem cells and their potential contribution to cancer recurrence.

Citation Format: Jan S. Moreb, Mahmoud Mona, Lung-Ji Chang, Yu-Ling Yeh, John Amory, Alex Goldstein. The role of aldehyde dehydrogenase isoenzymes in cancer cell proliferation, migration and drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3762. doi:10.1158/1538-7445.AM2013-3762
Abstract 3778: Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.
Authors: Wilson Ashley L. , Schrecengost Randy S. , Guerrero Michael S. , Thomas Keena S. , Bouton Amy H.
Published: August 2014

Metastatic breast cancer is currently incurable and associated with a 5-year survival rate of only 23%. Thus it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility in order to improve patient survival. The adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) functions in cellular processes that contribute to cell motility. Previous work from our group has shown that elevated BCAR3 protein levels enhance breast cancer cell motility, while depletion of BCAR3 decreases cell motility and invasion in vitro. Here, we show BCAR3 controls membrane protrusion, Rac1 activity, and adhesion disassembly in invasive breast cancer cells in response to adhesion signals. Conversely, RhoA signaling pathways appear to predominate when BCAR3 is depleted from these cells, as evidenced by an increase in ROCK-mediated myosin light chain phosphorylation and the presence of stress fibers and large ROCK/mDia1-dependent focal adhesions. Thus, through its ability to tip the balance in favor of Rac1 signaling, BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells, thereby promoting a more invasive, pro-migratory phenotype. Interestingly, we demonstrate that BCAR3 also controls actin cytoskeletal and adhesion remodeling in invasive breast cancer cells in response to epidermal growth factor. Considering that BCAR3 protein levels are elevated in advanced breast cancer cells lines, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process. BCAR3 function is intimately linked to two other proteins, the adaptor molecule p130Cas (Cas) and the non-receptor tyrosine kinase c-Src. While BCAR3 protein levels have yet to be thoroughly assessed in human breast tumors, high expression of Cas and/or c-Src is associated with more aggressive breast cancer behaviors. Future work will determine whether this BCAR3/Cas/c-Src signaling network serves as a useful biomarker for invasive disease, and whether BCAR3 promotes breast tumor progression and metastasis in mouse models of human breast cancer.

Citation Format: Ashley L. Wilson, Randy S. Schrecengost, Michael S. Guerrero, Keena S. Thomas, Amy H. Bouton. Breast Cancer Antiestrogen Resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3778. doi:10.1158/1538-7445.AM2013-3778
Abstract 378: The significance of vascular endothelial growth factor C in the lymphangiogenesis and lymph node metastasis of ovarian cancer.
Authors: Hisamatsu Takeshi , Mabuchi Seiji , Sasano Tomoyuki , Takahashi Ryoko , Kawano Mahiru , Matsumoto Yuri , Hashimoto Kae , Isobe Aki , Sawada Kenjiro , Morishige Kenichiro , Kimura Tadashi
Published: August 2014

Purpose: Vascular endothelial growth factor-C (VEGF-C) is commonly expressed in malignant cells, creating a favorable environment for generation of new lymphatic vessels. It has been recently reported that mammalian target of rapamycin (mTOR) stimulates tumor-lymphangiogenesis by inducing the production of VEGF-C. In the current study, we investigated the significance of VEGF-C in tumor lymphangiogenesis and lymph node metastasis of ovarian cancer. Moreover, we investigated whether mTOR complex 1 (mTORC1) is involved in the VEGF-C-induced lymphangiogensis and lymph node metastasis in ovarian cancer.

Experimental design: Using human ovarian cancers, we first performed immunostaining for VEGF-C, and examined the correlation between VEGF-C expressions and the extent of lymphvascular space invasion (LVSI) or the number of lymph node metastasis. Using two pairs of ovarian cancer cell lines producing different level of VEGF-C (VEGF-C-low and VEGF-C-high cells), we next investigated the role of VEGF-C in tumor-lymphangiogenesis in a mouse xenograft model. We then examined whether mTORC1 is involved in the production of VEGF-C in ovarian cancer cells as well as in tumor-lymphangiogenesis in a mouse xenograft model. Finally, by using a mouse model of paraaortic-node metastasis, we investigated whether inhibition of mTORC1 suppress lymph node metastasis.

Results: VEGF-C expression was significantly associated with the extent of LVSI or the number of lymph node metastasis in ovarian cancer specimens. In a mouse xenograft model, tumors derived from VEGF-C-high cells showed greater lymphatic vessel density than that observed in tumors from VEGF-C-low cells. Inhibition of mTORC1 by everolimus decreased the expression of VEGF-C in ovarian cancer cells in vitro, lymphangiogenesis in tumors, and paraaortic-node metastasis in mouse models.

Conclusion: Inhibition of mTORC1 may represent a novel molecular-targeted approach for lymph node metastasis prevention in patients with ovarian cancer.

Citation Format: Takeshi Hisamatsu, Seiji Mabuchi, Tomoyuki Sasano, Ryoko Takahashi, Mahiru Kawano, Yuri Matsumoto, Kae Hashimoto, Aki Isobe, Kenjiro Sawada, Kenichiro Morishige, Tadashi Kimura. The significance of vascular endothelial growth factor C in the lymphangiogenesis and lymph node metastasis of ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 378. doi:10.1158/1538-7445.AM2013-378
Abstract 3791: Secreted plectin-rich exosomes from pancreatic cancer cells promote tumor aggressiveness.
Authors: Shin Soo J.
Published: August 2014

Background & Aims: We recently demonstrated that the aberrant extracellular localization of the cytoskeletal linker protein plectin is a robust biomarker for the progression of pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive malignancies. Exosomes are nanometer-sized particles containing cytoplasm and plasma membrane that are secreted by both normal and cancerous cells. Recently the importance of exosomes in cross-talk in the tumor microenvironment has been demonstrated. Fusion of exosomes to the cellular membrane in either a paracrine or autocrine manner can result in aberrant protein localization. Here, we assess the mechanism for plectin mislocalization and its functional role in PDAC.

Methods: A plectin-binding assay was developed to assess the presence of cell-surface localized plectin. To examine their potential contribution to aberrant extracellular localization of plectin, we determined whether plectin was present in PDAC-secreted exosomes collected by ultracentrifugation and evaluated by immunoblotting. Based on these observations, we then determined whether exosomes with or without plectin altered gross phenotypic tumor characteristics in vitro in tumor stromal and endothelial cells. Finally, we tested the ability to alter outgrowth kinetics by directly injecting exosomes with or without plectin in tumors in vivo.

Results: Plectin was localized on the cell surface as well as present in exosomes derived from PDAC cell lines but not from non-transformed human pancreatic ductal epithelial (HPDE) cells. Plectin presence in the exosomes hinged on the expression of integrin β4, a protein known to interact with cytosolic plectin. shRNA and overexpression studies collectively demonstrated a positive role of plectin and its localization in the exosomes in proliferation, migration, and invasion of PDAC cells and migratory capacity of microenvironment cells. Finally, plectin-rich exosomes enhanced in vivo tumor growth.

Conclusions: Plectin is expressed in PDAC and is aberrantly localized to the cell surface via exosomes in an integrin β4-dependent manner. Additionally, plectin-rich exosomes induce pronounced increase in tumor growth in vitro and in vivo further they increased invasion and migration in vitro. Plectin-positive exosomes had profound effects on stromal and endothelial cells. Because cell surface plectin has been shown to be a biomarker for transition of pre-invasive to invasive PDAC, the elucidation of the mechanisms of its deregulation may provide important insights for successful detection and management of PDAC.

Citation Format: Soo J. Shin. Secreted plectin-rich exosomes from pancreatic cancer cells promote tumor aggressiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2013-3791
Abstract 3813: Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients.
Authors: Robbins Katherine M. , Stabley Deborah L. , Conard Katrina , Wu David J. , Stitik Hunter , Kolb Edward A. , Napper Andrew , Mason Robert W. , Gripp Karen W. , Sol-Church Katia
Published: August 2014

Embryonal rhabdomyosarcoma (ERMS) is the most prevalent pediatric soft tissue tumor and is characterized at the molecular level by loss of heterozygosity (LOH) at 11p15.5. We aimed to study ERMS in the context of Costello syndrome (CS), a rare syndromic predisposition to ERMS. Because CS arises from a typically paternal germline mutation in the proto-oncogene HRAS, it is considered a rasopathy. CS results in failure to thrive, intellectual disabilities, short stature, coarse facial features, skeletal abnormalities, congenital heart disease, and a predisposition for cancer, most commonly ERMS. We molecularly characterized seven ERMS tumors from six unrelated CS patients, carrying paternally derived HRAS p.G12S (5) or p.G12A (2) mutations. LOH was evaluated in all CS ERMS tumors by microarray and/or using short tandem repeat (STR) markers along both arms of chromosome 11. One patient developed a second primary ERMS tumor showing the ERMS hallmark LOH limited to 11p15.5. The six other CS ERMS tumors displayed paternal uniparental disomy (UPD) of the entire chromosome 11. Paternal UPD was also observed in a pure ERMS cell line, established using fresh tumor sample from one of the patients with a p.G12A germline mutation. This represents, to our knowledge, the first CS ERMS cell line. Additional cell lines derived from non CS patients were used in this study and included two ERMS (RD and RH36), one Alveolar RMS (RH30), and one mixed A/ERMS (RH18). RH36 cells carried an HRAS mutation at position 61 (p.Q61K). Unexpectedly, both ERMS cell lines, RD and RH36, displayed a complete LOH for chromosome 11. The ARMS RH30 cells carried no mutation in HRAS or LOH. RH18 cells derived from an A/ERMS had no HRAS mutation and showed LOH on both, the p and q arm of chromosome 11, except at locus D11S1338. Cytogenetic analysis with fluorescence in situ hybridization (FISH) on three CS tumors revealed multiple copies of chromosome 11 leading to trisomies and tetrasomies in the tumor and cultured cells. Gene expression revealed loss of imprinting in the growth regulating genes H19, IGF2, and CDKN1C on chromosome 11 not only in CS tumors, but also in nonsyndromic ERMS.

Citation Format: Katherine M. Robbins, Deborah L. Stabley, Katrina Conard, David J. Wu, Hunter Stitik, Edward A. Kolb, Andrew Napper, Robert W. Mason, Karen W. Gripp, Katia Sol-Church. Uniparental disomy is associated with embryonal rhabdomyosarcoma in Costello Syndrome and nonsyndromic patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3813. doi:10.1158/1538-7445.AM2013-3813
Abstract 382: Profiling angiogenic factors, RTK phosphorylation and inhibition events in breast cancer and endothelial cells using antibody arrays.
Authors: Finkel David J. , James Amy E. , Wegner Greta J.
Published: August 2014

Tumor growth and progression in breast cancer is dependent on angiogenesis, the growth of new vasculature from pre-existing vessels. Since invasive and in situ breast cancers express many angiogenic factors, we examined the profiles of angiogenesis-related proteins in the conditioned media from multiple breast cancer cell lines and their effect on activating receptor tyrosine kinases (RTKs) in Human Umbilical Vein Endothelial Cells (HUVEC). Antibody arrays were used to measure the secretion of multiple angiogenic factors including VEGF, TIMP-1, IGFBP-2, IGFBP-3, THSP-1, Angiogenin, and CXCL16. We then tested the capacity of these breast cancer cell line supernatants to induce receptor tyrosine kinase (RTK) phosphorylation in endothelial cells. After treatment with conditioned media, an increase in the tyrosine phosphorylation of VEGF R1 and VEGF R2 was observed using both antibody arrays and ELISA. Select small molecule VEG FR inhibitors were then screened for their effect on tyrosine phosphorylation events. Axitinib, Ki8751, and Sunitib effectively reduced VEGFR phosphorylation induced in endothelial cells by angiogenic factors released from these breast cancer lines.

Citation Format: David J. Finkel, Amy E. James, Greta J. Wegner. Profiling angiogenic factors, RTK phosphorylation and inhibition events in breast cancer and endothelial cells using antibody arrays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 382. doi:10.1158/1538-7445.AM2013-382
Abstract 3820: Wnt signaling and telomerase activation in hepatoblastoma.
Authors: Hiyama Eiso , Ueda Yuka , Kamimatsuse Arata , Onitake Yoshiuki , Ogura Kaoru , Hiyama Keiko
Published: August 2014

Purpose: The abnormal Wnt/β-catenin signaling plays a key role in hepatoblastoma (HBL) development. In HBL, CTNNB1 coding ß-catenin protein with mutated or deleted at hot-spot regions involving exon 3 stimulates this pathway. Previously we reported that the levels of telomerase reverse transcriptase (TERT) mRNA and telomerase activities were correlated with outcomes of the patients with HBL. In this study, we examined the correlation between Wnt/β-catenin signaling and telomerase activation in HBL. And, we investigated the location of β-catenin and TERT in HBL and the expression levels of the Wnt signal genes downstream of ß -catenin.

Methods: Tumors derived from 212 HBL cases treated with the JPLT-2 protocol were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene encoding β-catenin and mutations of Wnt signal genes including APC, AXIN1, and 2. Moreover, these tumors were analyzed for the expression levels of TERT (telomerase reverse transcriptase) and the Wnt signal genes downstream of ß -catenin (MYC, cyclin D1 and MMP7) using Real time RT-PCR and immunohistochemistry.

Results: Oncogenic mutations of CTNNB1 were detected in 163 cases (76.4%) and more than 80% showed abnormalities of Wnt signal genes. The expression levels of TERT were significantly higher in 49 cases without mutation (P < 0.05). Interestingly, Wnt/β-catenin target genes including MYC, cyclin D1 and MMP7 were significantly activated in the tumors with telomerase activation (P = 0.01). ß -catenin staining distinguished the CTNNB1 mutated tumors from others but cyclin D1 and MMP-7 did not. In advanced HBLs, double stain analysis of TERT and β-catenin showed that both were located in the same site in nucleus, suggesting that both proteins were co-localized in nucleus in hepatoblastoma.Conclusions: Wnt/β-catenin signaling in the HBLs without CNNB1 mutations was activated by telomerase activation. The clinical courses in HBLs with mutated Wnt signal gens and high TERT expression seemed to be unfavorable due to highly activated Wnt/β-catenin signaling. This correlated pathway might become molecular targets for advanced hepatoblastoma.

Citation Format: Eiso Hiyama, Yuka Ueda, Arata Kamimatsuse, Yoshiuki Onitake, Kaoru Ogura, Keiko Hiyama. Wnt signaling and telomerase activation in hepatoblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2013-3820
Abstract 3824: Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma.
Authors: Lo Angela Kwok-Fung , Wang Jian , Shen Jianhe , Yu Alexander , Chow Wing-Yuk , Su Jack , Adesina Adekunle , Dauser Robert , Whitehead William , Chintagumpala Murali , Guerra Rudy , Man Tsz-Kwong , Lau Ching C.
Published: August 2014

Medulloblastoma (MB) is the most malignant brain tumor in children and is characterized by marked biological and clinical heterogeneity. It includes four distinct molecular subtypes [wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4]. Previous attempts to identify prognostic markers applicable to all MBs were unsuccessful and we therefore hypothesize that each molecular subtype of MB may be associated with its unique prognostic markers. The aim of this study is to identify prognostic markers specific for SHH-active MB tumors. We analyzed SNP-array CGH data and gene expression profiling for 60 pediatric MBs and compared the molecular results with clinical outcome data. Using unsupervised hierarchical clustering, we identified 18 MBs that have activated SHH signaling. This subtype of SHH-active tumors also have frequent chromosome 9q loss which are associated with more favorable outcome. In an attempt to identify candidate genes on chromosome 9q with survival impact, we found that low expression of Heat Shock 70kDa Protein 5 (HSPA5) was significantly associated with better event free survival (EFS) in SHH MB patients independent of other clinical variables. This finding had been validated in two other independent MB cohorts. By immuohistochemistry and western blotting, we confirmed the negative correlation between patient survival and HSPA5 protein expression in SHH tumors. Furthermore, in vitro study indicated that knockdown of HSPA5 by siRNA caused dramatic growth suppression in SHH active MB cells. These findings suggest that HSPA5 is a potential prognostic marker in predicting survival of SHH active MB patients and this protein may also be exploited as therapeutic target for SHH MBs that do not harbor chromosome 9q loss.

Citation Format: Angela Kwok-Fung Lo, Jian Wang, Jianhe Shen, Alexander Yu, Wing-Yuk Chow, Jack Su, Adekunle Adesina, Robert Dauser, William Whitehead, Murali Chintagumpala, Rudy Guerra, Tsz-Kwong Man, Ching C. Lau. Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3824. doi:10.1158/1538-7445.AM2013-3824
Abstract 3825: Inhibitory effect of PID1 in medulloblastomas, gliomas and ATRT.
Authors: Erdreich-Epstein Anat , Ren Xiuhai , Zhou Hong , Sposto Richard , Ji Lingyun , Pfister Stefan , Kool Marcel , Asgharzadeh Shahab
Published: August 2014

The recently identified PID1 (Phosphotyrosine Interaction Domain containing 1) is a modulator of insulin signaling in adipocytes and muscle cells. Using a number of independent datasets we show that mean PID1 mRNA was lower in unfavorable medulloblastomas (subgroups 3 and 4) and in glioblastomas compared with favorable medulloblastomas (SHH and WNT subgroups) or normal brain, respectively. Higher PID1 mRNA correlated with longer progression-free survival and overall survival in medulloblastoma patients and with longer overall survival in glioma patients. Ectopic expression of PID1 in medulloblastoma, glioblastoma and ATRT (atypical teratoid rhabdoid tumor) cell lines inhibited colony formation by 68% ± 3.5 (p<0.0001 in total of 24 experiments in triplicates) compared with controls. PID1 effects on apoptosis and proliferation were demonstrated by increased sub-G0/G1 and annexinV staining, decreased S-phase. Additionally, PID1 induced mitochondrial depolarization in the medulloblastoma, glioblastoma, and ATRT cell lines. In keeping with PID1 having a PTB (phosphotyrosine binding) domain, endogenous PID1 co-immunoprecipitated with the low density lipoprotein receptor-related protein-1 (LRP1, which is a potential binding partner for PID1 via its C-terminal NPXY motif). However, PID1 with mutated PTB domain retained its inhibitory effects on colony formation, suggesting that other domains of PID1 mediated (or contributed to) PID1’s inhibitory effect. Analysis of PID1 domains comprising its N-terminal region (D1), C-terminal region (D3) and middle region (D2), each comprising approximately 1/3 of the protein, revealed that indeed, each 1/3 of PID1 inhibited colony formation at least as efficiently as full length PID1. Interestingly, although by itself D2 was more inhibitory than full length PID1, when it was combined with D3, which was also inhibitory by itself, we observed marked mitigation of the inhibitory effect of the combined D2+D5 fragment on colony formation, suggesting functional intramolecular interactions in PID1. These data suggest for the first time that PID1 has a tumor suppressor-like effect in brain tumors and shed light on the molecular mechanism of its inhibitory action.

Citation Format: Anat Erdreich-Epstein, Xiuhai Ren, Hong Zhou, Richard Sposto, Lingyun Ji, Stefan Pfister, Marcel Kool, Shahab Asgharzadeh. Inhibitory effect of PID1 in medulloblastomas, gliomas and ATRT. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3825. doi:10.1158/1538-7445.AM2013-3825