bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3688: Leptin contributes to prostate cancer progression.
Authors: De Velasco Marco A. , Yamamoto Yutaka , Hatanaka Yuji , Kura Yurie , Ando Naomi , Fukushima Emiko , Nozawa Masahiro , Shimizu Nobutaka , Yoshimura Kazuhiro , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: August 2014

Obesity is a complex chronic disorder that can result in a number of metabolic and endocrine disturbances and may thus account for the disparity seen in epidemiologic studies investigating the association between obesity and prostate cancer. Although the association between dietary factors and the occurrence of prostate cancer has been studied extensively, there is yet to be a published study on the relationship between diet and disease progression among prostate cancer patients. Circulating levels of most adipokines are positively correlated with obesity and exert a variety of biologic effects on prostate cancer cells modulating a variety of cellular processes. This would suggest that the presence of obesity may promote the progression of established prostate cancer rather than being a risk factor for the development of prostate cancer; however, a direct link has yet to be established. Recognizing adipose tissue as a metabolically active endocrine organ and identifying tumor promoting adipokines would support such an association.

To better understand the effects of increased dietary fat in prostate cancer, we used the prostate-specific PTEN conditional knockout mouse model to show that consumption of a high-fat diet (HFD) promoted prostate-specific cancer progression and decreased survival. Molecular analysis identified elevated levels of leptin as a likely molecule involved in driving tumor progression. In this study we provide data from in vitro experiments show that leptin promoted invasiveness, motility and adhesion of PC3, human prostate cancer cells. Together, our findings show that increased levels of leptin, induced by dietary fat, contributes to promote prostate cancer progression by upregulating STAT3 signal cascades.

Citation Format: Marco A. De Velasco, Yutaka Yamamoto, Yuji Hatanaka, Yurie Kura, Naomi Ando, Emiko Fukushima, Masahiro Nozawa, Nobutaka Shimizu, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Leptin contributes to prostate cancer progression. [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 3688. doi:10.1158/1538-7445.AM2013-3688
Abstract 3690: p53 stabilizing agent CP-31398 prevents urinary bladder tumor growth and invasion in transgenic UPII-SV40T mice.
Authors: Madka Venkateshwar , Zhang Yuting , Li Qian , Mohammed Altaf , Lightfoot Stan , Xue Wu Re , Kopelovich Levy , Rao Chinthalapally V.
Published: August 2014

The United States will have approximately 73,510 new cases of bladder cancer in 2012 with 14,880 deaths. More than 50% of the invasive (lamina and muscularis) bladder tumors have mutations in p53 and/or pRB. p53 appears to play a major role in the etiology of human bladder cancer, particularly in the high grade invasive type. Structural and functional defects in p53 are found in majority of human transitional cell carcinomas (TCC). The high prevalence of bladder cancer and the frequency of tumor recurrence make it an important target for chemoprevention. In UPII-SV40T transgenic mice, induced bladder TCC bears strong resemblance to that of human TCC in phenotype and in the mode of progression. In the present study, we determined the chemopreventive efficacy of a p53 stabilizing agent, CP-31398, using transgenic UPII-SV40T mouse model. After genotyping, six-week old UPII-SV40T mice (n=32/group) were fed control (AIN-76A) or experimental diets containing 150 or 300 ppm CP-31398 for ∼34 weeks. Progression of bladder cancer was monitored by MRI imaging. At 40 weeks of age, all mice were euthanized; urinary bladders were collected to determine weights, tumor incidence and multiplicity; and histopathology. There was a significant increase in bladder weights of transgenic vs. wild type mice (male 140.2 mg vs. 27.3 mg, p<0.0001; female 34.2 mg vs, 14.8 mg p<0.0001, respectively). A significant decrease in the tumor growth as evidenced by decreased bladder weights (by 52-65%, p<0.000 in males; by 32 %, p<0.001 in females) was observed in CP-31398 treated mice. Invasive papillary TCC incidence was 100 % in transgenic mice fed with control diet. The mice exposed to 150 and 300 ppm CP-31398 showed dose dependent inhibition (29%, p<0.01; 57%, p<0.005) of invasive TCC with 28% and 53% carcinoma In situ, respectively, suggesting suppression of invasive tumor growth. Kidney abnormalities like ureter obstruction and nephrohydrosis were observed in almost all control mice but significantly less frequent in CP-31398-fed mice. Molecular analysis of the bladder tumors showed an increase in apoptosis markers (p53, p21, Bax) with a decrease in angiogenic markers (CD31 and VEGF) in transgenic mice fed with CP-31398. These results suggest that p53 modulating agents can serve as potential chemopreventive for bladder TCC. (Supported in part by NCI-CN53300)

Citation Format: Venkateshwar Madka, Yuting Zhang, Qian Li, Altaf Mohammed, Stan Lightfoot, Wu Re Xue, Levy Kopelovich, Chinthalapally V. Rao. p53 stabilizing agent CP-31398 prevents urinary bladder tumor growth and invasion in transgenic UPII-SV40T 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 3690. doi:10.1158/1538-7445.AM2013-3690
Abstract 3706: Intervention of human breast cell carcinogenesis chronically induced by 3, 4, 4’-trichlorocarbanilide.
Authors: Sood Shilpa , Choudhary Shambhunath , Wang Hwa-Chain R.
Published: August 2014

The purpose of this study was to investigate the ability of antimicrobial agent 3,4,4’-trichlorocarbanilide (TCC) to induce breast cell carcinogenesis, and then identify preventive agents to intervene in TCC-induced breast cell carcinogenesis. TCC is widely used in personal care products such as bath soaps, detergents, and cleansing lotions. Recent studies suggest that TCC acts as an endocrine disruptor to enhance the ability of steroid hormones (estrogen or testosterone) to induce estrogen and androgen receptor-mediated gene expression. Due to its environmental persistence and wide use, there are concerns about TCC's possible impact on human health. To determine whether TCC exposure may contribute to the development of breast cancer, we used our chronically-induced breast cell carcinogenesis model to determine the ability of TCC to induce progressive carcinogenesis of human breast epithelial MCF10A cells to acquire cancer-associated properties. We found that chronic exposure of MCF10A cells to TCC at physiologically-achievable nanomolar levels resulted in cellular acquisition of the cancer-associated properties of reduced dependence on growth factors, anchorage independent growth, and increased cell proliferation. These cellular changes were accompanied by upregulation of the ERK pathway, Nox1-induced ROS production, and DNA damage. We also found that curcumin, a component of turmeric extracted from the rhizome of the Indian herb Curcuma longa, was able to suppress TCC-induced cellular carcinogenesis. Our study revealed, for the first time, that chronic exposure to TCC was able to induce breast cell carcinogenesis, and TCC-induced cellular carcinogenesis was preventable by curcumin.

Citation Format: Shilpa Sood, Shambhunath Choudhary, Hwa-Chain R. Wang. Intervention of human breast cell carcinogenesis chronically induced by 3, 4, 4’-trichlorocarbanilide. [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 3706. doi:10.1158/1538-7445.AM2013-3706
Abstract 3708: Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues.
Authors: Chen Kun-Ming , Zhang Shang-Min , Aliaga Cesar , Sun Yuan-Wan , Berg Arthur , Sharma Arun , Amin Shantu , Stoner Gary , El-Bayoumy Karam
Published: August 2014

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. DMBA, however, is not present in the environment and the hamster cheek pouch model may not be applicable to humans. Recently, we developed a relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. In the current study, we evaluated the effects of BRB and related components including kaempferol and resveratrol on DNA adducts induced by DB[a,l]P in mouse oral cavity as an initial step to explore the potential preventive activity of BRB and its related components on DB[a,l]P-induced oral cancer. The DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We demonstrated that the administration of BRB (5%), kaempferol (0.025%) or resveratrol (0.002%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) resulted in 27%, 36% or 65% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively. In conclusion, our results support that dietary consumption of BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P.

Citation Format: Kun-Ming Chen, Shang-Min Zhang, Cesar Aliaga, Yuan-Wan Sun, Arthur Berg, Arun Sharma, Shantu Amin, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [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 3708. doi:10.1158/1538-7445.AM2013-3708
Abstract 371: PGI2 analog enhances endothelial-pericyte interactions and suppresses lung metastasis.
Authors: Minami Yoshinori , Sasaki Takaaki , Endo Satoshi , Shibukawa Kiyoko , Ohsaki Yoshinobu
Published: August 2014

Background: Endothelial cells (ECs) express IP (prostaglandin I2 (PGI2) specific receptor) and are playing important role in tumor angiogenesis. We have reported that the PGI2-IP system is necessary for vascular remodeling and angiogenesis. Additionally, we have reported that the knockdown of IP increases tumor metastasis in mouse models.

Objectives: In this study, we examined whether the activated PGI2-IP signaling could enhance EC-PC interactions and suppress tumor metastasis.

Materials & Methods: Mouse-derived Lewis lung carcinoma (LLC) cells were used for a mouse lung metastatic model, and were injected from the tail vein of mice (c57BL/6J). Beraprost sodium (BPS; PGI2 analog) was continuously administered for 3 weeks. Tumor metastasis to lung was assessed by using hematoxylin-eosin staining. The a-SMA and the NG2 as a pericyte marker and the endomucin as an endothelial cell marker were analyzed by immunofluorescence to evaluate angiogenesis in metastatic lung tumors. The structure of the tumor blood vessels was analyzed by scanning electron microscopy (SEM).

Results: The size and number of lung metastatic nodules were significantly decreased in BPS group compared with in control group assessed by the mice lung metastatic model. Immunofluorescence analysis revealed that the number of vessel-associated a-SMA+ cells was significantly increased by BPS administration. Scanning electron microscopy revealed that pericytes covering the tumor blood vessels increased in BPS group.

Conclusions: The present study demonstrated that BPS suppresses lung metastasis in our model. These results also suggested that the maturation of tumor blood vessels by pericytes may decrease tumor metastasis. Finally, we propose that BPS would be a novel therapeutic target in lung metastasis.

Citation Format: Yoshinori Minami, Takaaki Sasaki, Satoshi Endo, Kiyoko Shibukawa, Yoshinobu Ohsaki. PGI2 analog enhances endothelial-pericyte interactions and suppresses lung metastasis. [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 371. doi:10.1158/1538-7445.AM2013-371
Abstract 3711: Human colon mucosal crypts in culture: a model to assess colon chemoprevention.
Authors: Dame Michael K. , Jiang Yan , Turgeon Danielle Kim , Appelman Henry , Aslam Muhammad N. , Copley Kelly , Attili Durga , Brenner Dean E. , Varani James
Published: August 2014

Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps.

Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system.

Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization.

Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives.

Epithelial Cell Proliferation and Differentiation in Cultured Human Crypts (fold changes) Markers Zero-time 24 hours 48 hours 72 hours Ki67 1.00 0.36 0.18 0.00 E-cadherin 1.00 2.00 2.89 3.22

Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [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 3711. doi:10.1158/1538-7445.AM2013-3711
Abstract 3713: Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis.
Authors: Bommareddy Ajay , Eggelston William , Prelewicz Stacy , Antal Andrea , VanWert Adam L. , Patel Hiral , Chinikaylo Aleona , Gutierrez Linda S. , Sreevidya Santha , Dwivedi Chandradhar
Published: August 2014

α-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. The present study was undertaken to determine the in vivo efficacy of α-santalol using TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice as a model. The i.p administration of α-santalol (50 mg/kg) did not cause any side effects or weight loss throughout the study. Administration of α-santalol (50 mg/kg) showed a trend in decrease in average wet weights of urogenital weights compared to control mice (∼34% decrease in α-santalol group compared to control). Furthermore, the dorsolateral sections of prostate from α-santalol-treated mice exhibited decreased cell proliferation in association with induction of apoptosis. Western blotting analysis of prostate tumor samples from α-santalol-treated group revealed an increase in the expression levels of cleaved PARP, phospho p53 (ser 15), cleaved caspase-3, cyclin B, p21 and a modest reduction in antiapoptotic protein Bcl-xL compared to control samples. In agreement with these results, α-santalol treatment resulted in G2/M cell cycle arrest in cultured prostate cancer cells. In conclusion, the present study indicates that α-santalol administration inhibits the development of prostate cancer in TRAMP mice by decreasing cell proliferation, causing cell cycle arrest, and inducing apoptosis. This study was supported by Wilkes University Type I grant, American Association of Colleges of Pharmacy and by Translational Cancer Research Center funded by South Dakota Governor's Office of Economic Development.

Citation Format: Ajay Bommareddy, William Eggelston, Stacy Prelewicz, Andrea Antal, Adam L. VanWert, Hiral Patel, Aleona Chinikaylo, Linda S. Gutierrez, Santha Sreevidya, Chandradhar Dwivedi. Chemoprevention of prostate carcinogenesis in TRAMP mice by α-santalol by causing cell cycle arrest and induction of apoptosis. [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 3713. doi:10.1158/1538-7445.AM2013-3713
Abstract 3715: Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma.
Authors: Lee Sung Hee , Kim Reuben , Kang Mo , Park No-Hee , Shin Ki-Hyuk
Published: August 2014

Zoledronic acid (ZA), a third generation of bisphosphonates, is known to have both anti-osteolytic and anti-tumor activities. It exerts anti-osteolytic function by interfering with mevalonate pathway and preventing protein prenylation required for growth and survival in osteoclast cells. Also, similar function has been found to induce cancer apoptosis and inhibit cancer growth in bone-related cancer, but its effect on other cancer types, including head and neck squamous cell carcinoma (HNSCC) is still unclear. In this study, we first monitored the effect of ZA on cancer growth in HNSCC cell lines in vitro. ZA significantly suppressed cancer growth, shown by decreased anchorage independent growth in soft agar assay and diminished cell growth in organotypic raft culture. Because cancer stem cell (CSC; also called tumor-initiating cell) plays a significant role in cancer growth, we further investigated the effect of ZA on CSC in HNSCC. ZA-treated HNSCC cells significantly decreased CSC phenotypes such as sphere forming ability and migratory ability. In SCC4, a tongue squamous cell carcinoma cell line, side population and expression of pluripotency marker genes were significantly decreased by ZA. Also, ZA inhibited the CSC-related Sonic Hedgehog pathway in SCC4 as demonstrated by decreased mRNA expression of transcriptional factor, Gli-1. Taken together, our data suggest that ZA targets CSC and hence inhibits cancer growth in HNSCC.

Citation Format: Sung Hee Lee, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma. [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 3715. doi:10.1158/1538-7445.AM2013-3715
Abstract 3718: Taxane-induced hedgehog activation promotes stemness of breast cancer cells.
Authors: Opdenaker Lynn , Sims-Mourtada Jennifer
Published: August 2014

Recurrence of breast cancer after chemotherapy is thought to arise from resistant breast cancer stem cells which are eventually able to repopulate the tumor. The hedgehog (HH) signaling pathway has been shown to regulate the proliferation and survival of breast cancer stem cells, and has been shown to promote resistance to chemotherapy through the activation of multi-drug resistance and prosurvival pathways. Here we report that exposure of heterogenous breast cancer cell lines (T47D and SKBr3) to low doses (1-10nM) of docetaxel resulted in activation of HH signaling. Increased levels of sonic HH were detected in supernatants of breast cancer cells at 6- hours post-treatment. This release was accompanied by increased expression and nuclear translocation of Gli-1, peaking at 24 hours after exposure. Additionally, increased expression of the PTCH receptor was observed by western blotting in these cells 24 hours after treatment, indicating activation of a Gli-1 induced negative feeback loop. Increased mammosphere formation was observed in cells after preincubation with low dose docetaxel. Secondary sphere forming efficiency (SFE) was also increased in pretreated cells. The increase in SFE was similar to that of cell lines cultured in the presence of recombinant SHH. Additionally, the increase in SFE observed after docetaxel treatment could be blocked by the addition of sublethal doses of the hedgehog inhibitor cyclopamine. These results suggest that hedgehog pathway activation induced by docetaxel treatment could result in increased stemness of breast cancer cell lines. We propose that chemotherapy may activate stem cell signaling pathways, resulting in expansion of cancer stem cells and increased drug resistance.

Citation Format: Lynn Opdenaker, Jennifer Sims-Mourtada. Taxane-induced hedgehog activation promotes stemness of 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 3718. doi:10.1158/1538-7445.AM2013-3718
Abstract 372: Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling.
Authors: Chen Hong , Satish Pasula , Cai Xiaofeng , Dong Yunzhou , Chang Baojun
Published: August 2014

To examine the angiogenic role of endothelial epsins, a family of ubiquitin binding endocytic clathrin adaptors, we generated mice with constitutive or inducible deletion of epsins 1 and 2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 does not affect normal blood vessels. However, adult mice lacking endothelial epsins 1 and 2 exhibited highly disorganized tumor vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promotes binding of epsin to ubiquitinated VEGF receptor 2 (VEGFR2). Loss of epsins 1 and 2 specifically impairs endocytosis and degradation of VEGFR2 which results in excessive VEGF signaling that compromises tumor vascular function by exacerbating non-productive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development, and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Thus, promoting excessive VEGF signaling within tumors via a block of epsin 1 and 2 function, may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

Citation Format: Hong Chen, Pasula Satish, Xiaofeng Cai, Yunzhou Dong, Baojun Chang. Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling. [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 372. doi:10.1158/1538-7445.AM2013-372

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 3720: Rad51 is involved in PARP inhibitor resistance in triple-negative breast cancer stem cells (TNBCSCs).
Authors: Liu Yajing , Martin Rachel , Bai Shoumin , Wicha Max , Liu Suling
Published: August 2014

Although the mortality rate in breast cancer (BC) has been reduced by the advancing technology, the outcome of patients with “triple-negative” (TN) BC (accounts for 10-17% of invasive BC) has achieved little progression. Recently, inhibitors of poly (adenosine-disposphate-ribose) polymerase (PARP) have been developed to target cells with defective DNA repair molecules such as BRCA1-deficient cells. Despite the tumor regression induced by these agents in a subset of BCs, their ability to effect patient outcome remains inconclusive. According to the CSC hypothesis, a hierarchical tumor organization exists whereby dysregulated self-renewing CSCs drive tumorigenesis. Our preliminary results showed that PARP inhibitor significantly decreased cell number in BRCA1-deficient SUM149 cells in a dose-dependent manner, whereas BRCA1 wild type SUM159 cells were relatively resistant. However, there was little change in absolute CSC number in SUM149 cells after PARP inhibitor treatment, suggesting PARP inhibitor only targets the non-CSC population in SUM149 cells. More interestingly, the resistant population expressed higher active Rad51 as measured for Rad51 foci formation. Our pervious study also showed that Rad51 mRNA level was significantly higher in ALDH+ cells of some breast cancer cell lines. Therefore, in this study, we established an inducible knock-down system using lentivirus and found that knocking down Rad51 sensitizes SUM159 (both CSC and non-CSC) as well as ALDH+ SUM149 cells to PARP inhibitor, suggesting the expression of Rad51 is associated with PARP inhibitor resistant population. This system was also tested in mouse model where cells carrying Rad51 shRNA were injected into the mammary fat pads of female NOD/SCID mice. Knock-down of Rad51 alone and combination treatment with PARP inhibitor dramatically inhibited tumor growth in the xenografts of both SUM149 and SUM159 cells, whereas, tumor grew back after Rad51 expression was restored. However the inhibitory effect remained in the combined treatment group after the treatment was terminated. Furthermore, the combination treatment decreased ALDH+ cells by IHC staining. It also showed a significant decrease in tumor-initiating cells in the combined treatment group by limiting dilution transplantation. Our results suggest that Rad51 plays an important role in resistance to PARP inhibitor in TNBCSCs thus Rad51 inhibitor may be a novel therapeutic approach in addition to PARP inhibition for treating TNBC patients.

Citation Format: Yajing Liu, Rachel Martin, Shoumin Bai, Max Wicha, Suling Liu. Rad51 is involved in PARP inhibitor resistance in triple-negative breast cancer stem cells (TNBCSCs). [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 3720. doi:10.1158/1538-7445.AM2013-3720
Abstract 3724: Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4.
Authors: An Hyun Sook , Kang Han Na , Kang Myoung Hee , Kim Jung Lim , Kim Jun Suk , Seo Jae Hong
Published: August 2014

Salinomycin isolated from Streptomyces albus and a potassium ionophore. It has been used to eliminate bacteria, fungi and parasites. Also, Cancer stem cells (CSCs) are a subpopulation of cells within the tumor that has ability to self-renew and undergo differentiation to phenotypically diverse cell populations. So, CSCs are involved in metastasis, chemoresistance and even cancer relapse. Recently, there are many evidences to CSCs subpopulations enhance capability of DNA repair and reduce the apoptosis. Furthermore, salinomycin has been reported to act as selective breast cancer stem cell inhibitor based on its property of reducing the cancer stem cell and to induce the apoptosis in cancer cell. Therefore, we investigated the effect of salinomycin on breast cancer stem cells.

First, Mammosphere formation assay was performed to observe stem cell-like property after salinomycin treat in anchorage-independent state. We confirmed salinomycin reduces number and size of mammosphere at 3, 6, 9 day. Also, we compared mRNA expressing level of stem cell marker such as SOX2 and OCT4 by RT-PCR. Consequently, salinomycin was the down-regulated stem cell marker mRNA level as well as protein level.

To confirm the reduction of the side population level, we performed FACS analysis using CD44+ and CD24- markers. Results by FACS analysis revealed that salinomycin treated with breast cancer stem cell reduced stemness by downregulating CD44+/CD24- population. Consistently, when mammosphere implanted into the mammary fat pads of BALB/c female nude mice cleared of endogenous epithelium, the tumor size was decreased. In conclusion, we concluded that effect of salinomycin to reduce stemness is mediated by downregulation of SOX2 and OCT4 in human breast cancer cell.

Citation Format: Hyun Sook An, Han Na Kang, Myoung Hee Kang, Jung Lim Kim, Jun Suk Kim, Jae Hong Seo. Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4. [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 3724. doi:10.1158/1538-7445.AM2013-3724
Abstract 3729: Targeting breast cancer stem cells via oxidative stress with inhibitors of hydroperoxide metabolism and decyl-triphenylphosphonium.
Authors: Conley Sarah J. , Baker Trenton , Theisen Rebecca , Gheordunescu Elizabeth , Zhu Yueming , Aykin-Burns Nukhet , Fath Melissa , Spitz Douglas , Wicha Max
Published: August 2014

Cancer stem cells (CSCs) exhibit lower intracellular reactive oxygen species (ROS) levels than non-CSCs, which may be due to the increased expression of free radical scavenging systems. Exogenous agents may be useful to increase ROS and selectively kill CSCs by oxidative stress. Here we tested a combination approach to increase ROS as an effective strategy to eradicate breast CSCs and metastases. Buthionine sulfoximine (BSO) and auranofin (AU) were used to deplete glutathione (GSH) and inhibit thioredoxin reductase (TR), respectively, while mitochondrial-targeted decyltriphenylphosphonium (dTPP) was used to elevate ROS levels. In vitro clonogenicity assays using SUM159 cells showed that treatment with dTPP alone resulted in <30% survival, while AU+BSO, BSO+dTPP and AU+BSO+dTPP all resulted in <1% survival. These effects were reversible with N-acetylcysteine pre-treatment. The Aldefluor+ (CSC) population was also measured following drug treatment in vitro. dTPP or AU treatment alone resulted in <50% of CSCs remaining, while BSO+AU or BSO+dTPP resulted in <25% of CSCs remaining, and treatment with all three compounds resulted in <1% of CSCs remaining. In a preclinical model of breast cancer metastasis, long-term adjuvant treatment with each individual compound significantly reduced the amount of metastases and increased survival, with dTPP treatment alone resulting in 100% survival rate. Interestingly, the combination of all three compounds resulted in increased metastases compared to the single agents. This may potentially be a stress-induced effect resulting from drug toxicity. In conclusion, the approach of increasing ROS in breast cancer cells may be an effective way to target CSCs and useful for adjuvant therapy to reduce metastases in patients.

Citation Format: Sarah J. Conley, Trenton Baker, Rebecca Theisen, Elizabeth Gheordunescu, Yueming Zhu, Nukhet Aykin-Burns, Melissa Fath, Douglas Spitz, Max Wicha. Targeting breast cancer stem cells via oxidative stress with inhibitors of hydroperoxide metabolism and decyl-triphenylphosphonium. [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 3729. doi:10.1158/1538-7445.AM2013-3729
Abstract 3733: Defining and targeting a Glioblastoma cancer stem cell population with EGF Receptor Variant III.
Authors: Emlet David R. , Vecchio Catherine Del , Gupta Puja , Mitra Siddhartha , Han Shuang-Yin , Holgado-Madruga Marina , Li Gordon , Jensen Kristen , Vogel Hannes , Skirboll Stephen , Wong Albert J.
Published: August 2014

The relationship between mutated proteins and the relatively rare cancer stem cell population is unclear since driver mutations are present throughout a tumor. Glioblastoma (GBM) tumors frequently express EGFRvIII, an EGFR variant that arises via gene rearrangement and amplification. The expression of EGFRvIII is restricted despite the prevalence of the alteration. Here we show that EGFRvIII is expressed in a population of human GBM cancer stem cells. EGFRvIII positive cancer stem cells demonstrate greater self-renewal and tumor initiation than the more abundant EGFRvIII negative cells. EGFRvIII positive cells are associated with stem/progenitor markers while markers of differentiation are found in EGFRvIII negative cells. EGFRvIII is highly co-expressed with CD133, and the EGFRvIII positive/CD133 positive sub-population defines the highest self-renewal and tumor initiating population. Surprisingly, wildtype EGFR was infrequently co-expressed with EGFRvIII but present in the majority of cells, both in primary GBM and cultured GBM neurospheres. Elimination of the EGFRvIII positive or the EGFRvIII/CD133 dual positive population with an engineered anti-EGFRvIII/CD133 bi-specific antibody reduced tumorigenicity of implanted tumor cells. This work demonstrates that a mutated oncogene can have CSC specific expression and be used to specifically target this population.

Citation Format: David R. Emlet, Catherine Del Vecchio, Puja Gupta, Siddhartha Mitra, Shuang-Yin Han, Marina Holgado-Madruga, Gordon Li, Kristen Jensen, Hannes Vogel, Stephen Skirboll, Albert J. Wong. Defining and targeting a Glioblastoma cancer stem cell population with EGF Receptor Variant III. [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 3733. doi:10.1158/1538-7445.AM2013-3733
Abstract 3737: Identifying novel therapeutic targets for cancer stem cell differentiation.
Authors: Reyes Germán L. Vélez , Yang Haidi , Wilson Aaron , Henault Martin , Frias Elizabeth , Schlabach Michael , Mclaughlin Margaret , Ross Nathan , Hoffman Gregory , Chen Amy , Hild Marc , Jaffe Aron
Published: August 2014

A growing body of evidence supports a role for cancer stem cells in the initiation and maintenance of a wide range of tumors. Cancer stem cells appear to be less sensitive to current treatments, necessitating the discovery of novel strategies for targeting this cell type. We hypothesize that driving cancer stem cells to differentiate will be an effective approach for inhibiting tumor growth and recurrence. Indeed, this strategy is used for the treatment of Acute Promyelocytic Leukemia, which presents with an accumulation of promyelocytes. Treatment with all-trans retinoic acid drives these cells to differentiate into neutrophils and leads to complete remission in over 90% of patients.

The goal of our work is to identify druggable nodes to drive epithelial cancer stem cells to differentiate, leading to inhibition of tumor growth. We are currently focused on colorectal cancer, for which there is a high unmet medical need. Each year, more than 1.2 million new cases of colorectal adenocarcinoma are reported. It is estimated that 80% of colon tumors have mutations in the adenomatous polyposis coli (APC) gene, which results in constitutive activation of the Wnt pathway and nuclear accumulation of β-catenin. Wnt signaling activates the transcription of genes that drive cell proliferation. Recent studies have shown that β-catenin is necessary for the maintenance of colorectal adenocarcinomas with APC mutations. Indeed, β-catenin knockdown in colon cancer cells drives them to differentiate and decreases their proliferative capacity, reducing tumor size in vivo. We have developed a number of methods to monitor this process in vitro, and will present our efforts to screen for novel targets that can drive colon tumor stem cells to differentiate.

Citation Format: Germán L. Vélez Reyes, Haidi Yang, Aaron Wilson, Martin Henault, Elizabeth Frias, Michael Schlabach, Margaret Mclaughlin, Nathan Ross, Gregory Hoffman, Amy Chen, Marc Hild, Aron Jaffe. Identifying novel therapeutic targets for cancer stem cell differentiation. [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 3737. doi:10.1158/1538-7445.AM2013-3737
Abstract 374: KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors.
Authors: Nilsson Monique B. , Cascone Tina , Gudikote Jayanthi , Roarty Emily , Diao Lixia , Koo Andrew , Ramachandran Sumankalai , Riquelme Erick , Tran Hai , Wistuba Ignacio , Carbone David P. , Heymach John
Published: August 2014

Targeted therapies designed to inhibit the vascular endothelial growth factor (VEGF) pathway have been extensively evaluated in the treatment of malignancies including Non-Small Cell Lung Cancer (NSCLC). VEGF pathway inhibitors such as bevacizumab, or the multitargeted receptor tyrosine kinase inhibitors (TKIs) vandetanib and sorafenib, have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, seen only in subsets of patients. Thus, predictive markers for identifying which patients are likely to benefit are critically needed. Although expression of VEGF receptor-2 (VEGFR-2, also known as KDR) was initially thought to primarily occur in endothelial cells, VEGFR-2 has been detected on malignant cells, including lung cancer cells, and in NSCLC, overexpression of VEGFR-2 on tumor cells is associated with a poor clinical outcome. Amplification of KDR has been detected in lung cancer specimens at a relatively high frequency (9% and 32%). The consequences of KDR copy number gains (CNGs) are not yet understood. Recently, we have shown that NSCLC cell lines with KDR copy number gains (CNGs) were associated with in vitro resistance to platinum chemotherapy, and KDR CNG predicted worse overall survival in patients who received platinum adjuvant therapy but not in untreated patients. We investigated the hypothesis that NSCLC tumor cells with KDR CNG display increased sensitivity to VEGFR TKIs compared to tumor cells without KDR CNG. In tumor cell lines with KDR CNG, treatment with exogenous VEGF ligand enhanced cell motility and this was inhibited by VEGFR blockade with TKIs. Multiple receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxic conditions compared to NSCLC cell lines without KDR CNG. Here, we show that in NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α- regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG as determined by SNP array had a partial response to VEGFR inhibition with sorafenib.

Citation Format: Monique B. Nilsson, Tina Cascone, Jayanthi Gudikote, Emily Roarty, Lixia Diao, Andrew Koo, Sumankalai Ramachandran, Erick Riquelme, Hai Tran, Ignacio Wistuba, David P. Carbone, John Heymach. KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors. [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 374. doi:10.1158/1538-7445.AM2013-374
Abstract 3743: Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines.
Authors: Farias Nathan , Coomber Brenda
Published: August 2014

Mandated fortification of folic acid, a B vitamin involved in nucleotide synthesis and DNA CpG methylation, has led to a subsequent increase in blood folate concentration as well as a simultaneous increase in colorectal cancer incidence in North America. Recent findings suggest that a proportion of tumor cells, termed cancer stem cells (CSC), share properties with native stem cells and are vital in the development and perpetuation of tumor metastasis. Both epigenetic DNA modifications as well as the activation of established stem cell signaling pathways are responsible for the activation and maintenance of (CSC). CSC share properties with native stem cells and are vital in the development of primary lesions and the perpetuation of tumor metastasis. We hypothesize that folate, a CpG methylation altering agent, and extracellular microenvironment factors can influence the development and maintenance of CSCs. By isolating and characterizing the CSC fraction in established human colorectal cancer cell lines HCT116, Caco2, and SW480, LS174T and PC7, a subline of DLD-1, we have assessed the effect of folate on CSC signaling pathways and global DNA methylation. Cell lines were grown in suspension and supplemented with specialized stem cell media to promote the growth and expansion of the CSC fraction. Western blot analysis and immunofluorescence were used to characterize the protein expression and nuclear localization of proteins associated with established CSC maintenance pathways, respectively. Global DNA methylation status was assessed using an ELISA-based assay. HCT116 and SW480 show an increased ability to form colonospheres in culture compared to Caco2 and IEC18 (normal rat intestinal cell line). Cells grown in suspension show variable activation of stem cell renewal pathways compared to monolayer. Low folic acid levels led to reduced methylation status and colonosphere yield while high folic acid levels led to increased global methylation and an increased in colonosphere yield. Thus, altered folic acid exposure in vitro can influence the methylation status and CSC self-renewal ability of human colorectal cancer cells. The relationship between folic acid and cancer stem cell maintenance may be an important factor in the apparent duel modulatory effect of folic acid on colorectal cancer and warrants further investigation.

Citation Format: Nathan Farias, Brenda Coomber. Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer 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 3743. doi:10.1158/1538-7445.AM2013-3743
Abstract 3751: Characterization of melanospheres from BRAFV600E mutated A2058 melanoma cell line.
Authors: Liao Yi-Hua , Deng Jhu-Yun , Kuo Min-Liang
Published: August 2014

Increasing evidence suggests that there is a unique cell subpopulation in melanomas that possess stem cell-like activities and play important roles in tumor maintenance, metastasis, and chemo- or radio-resistance. It has been found that a subset of cells in human melanomas can form nonadherent melanospheres under serum-free stem cell medium. These sphere-forming cells have been demonstrated to have the capabilities of self-renewal and multi-differentiation similar to cancer stem cells. The goal of the study was to understand the features associated with tumorigenesis and melanoma progression in melanospheres compared to that in the adherent parental melanoma cells. We demonstrated that melanospheres had a higher tumorigenic ability than parental melanoma cells in that as few as 100 cells injected subcutaneously could form tumors. The tumorigenic ability was more enhanced in serially transplanted xenografts in severe combined immunodeficient mice as revealed by soft agar colony formation assay. Melanospheres showed chemo-resistance to cisplatin-induced cytotoxicity, which was also enhanced in cells derived from serially transplanted xenografts. Moreover, upregulation of markers of epithelial to mesenchymal transition (EMT) was found in melanospheres. The sphere-forming cells showed decreased levels of E-cadherin and significantly increased expression of the transcription factor SLUG/SNAI2. Taken together, these results suggested that melanosphere cultures represent a useful methodology to enrich and investigate melanoma cells with higher tumorigenic and metastatic potentials.

Citation Format: Yi-Hua Liao, Jhu-Yun Deng, Min-Liang Kuo. Characterization of melanospheres from BRAFV600E mutated A2058 melanoma cell line. [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 3751. doi:10.1158/1538-7445.AM2013-3751
Abstract 3760: EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target.
Authors: Buckanovich Ronald J. , Bai Shoumei , Deng Ning , Ingram Patrick , Yoon Euisik
Published: August 2014

Cancer stem cells (CSC) are closely associated with tumor vasculature in the CSC niche. Tumor vascular cells are known to provide critical growth, differentiation, and survival cues for CSC. Unfortunately little is known about the specific factors produced by vascular cells. EGFL6 is a factor specifically secreted by ovarian tumor vascular cells. EGFL6 is reported to regulate the differentiation of normal stem cells. We examined the impact of EGFL6 on ovarian cancer cell growth. We found that EGFL6 significantly promoted ovarian cancer cell growth in vitro. Ovarian cancer cell proliferation was associated with a decrease in the percentage of ALDH+ ovarian CSC. Using a novel single cell microfluidic culture system we found that EGFL6 acts specifically on ALDH+ CSC to promote proliferation via asymmetric division. We used a in vivo novel model of human tumor vasculature to express EGFL6 in vivo and demonstrate that EGFL6 significantly promotes ovarian tumor growth. Finally we generated an EGFL6 blocking antibody and found that anti-EGFL6 antibodies could restrict tumor growth and delay disease recurrences. Thus EGFL6 is an import vascular regulator of ovarian CSC and EGL6 blocking antibodies represent a potentially important ovarian CSC targeting therapeutic.

Citation Format: Ronald J. Buckanovich, Shoumei Bai, Ning Deng, Patrick Ingram, Euisik Yoon. EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target. [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 3760. doi:10.1158/1538-7445.AM2013-3760