Nicole L. Seiler

Notch 1 signaling is active in ovarian cancer.

OBJECTIVE.: Despite advances in chemotherapy and radical surgery, most advanced stage ovarian cancer patients die from their disease, highlighting the need for the development of novel treatment strategies. The Notch signaling pathway plays an important role in cellular differentiation, proliferation and apoptosis. We hypothesized that the active form of Notch 1, the Notch 1 intracellular domain (NICD), would be overexpressed in ovarian cancer cells and that depletion of NICD would lead to growth reduction. METHODS.: Following institutional review board approval, NICD expression was analyzed in human ovarian cancer specimens as well as the ovarian cancer cell lines OVCAR3, SKOV3, and CaOV3. In addition, the effects of Notch 1 depletion on ovarian cancer cell growth were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay for 6 days following transfection with siRNA against Notch 1. RESULTS.: Western blot analysis revealed abundant NICD expression in all 3 ovarian cancer cell lines, as well as in 16 of 21 (76%) human ovarian cancer samples. Following treatment with Notch 1 siRNA, expression of NICD was greatly reduced in all three cell lines. Furthermore, this depletion of NICD was associated with significant growth inhibition of all three ovarian cancer cell lines. CONCLUSIONS.: NICD was frequently expressed in ovarian cancer cell lines and human ovarian cancer specimens. Importantly, depletion of Notch 1 led to growth inhibition of ovarian cancer cells. These findings support the hypothesis that Notch 1 plays a role in ovarian cancer proliferation, encouraging the investigation of this pathway as a therapeutic target.

Xanthohumol decreases Notch1 expression and cell growth by cell cycle arrest and induction of apoptosis in epithelial ovarian cancer cell lines

OBJECTIVE Notch1 signaling is active in ovarian cancer and is a promising pathway for new therapies in ovarian cancer. We have previously detected high Notch1 expression in ovarian tumors. Xanthohumol has been shown to inhibit cancer cell growth and invasion, including Kaposis sarcoma, which also highly expresses Notch1. We hypothesized that the Notch1 signaling pathway is targeted by xanthohumol leading to decreased ovarian cancer cell growth. METHODS SKOV3 and OVCAR3 cells were utilized. MTT growth assays were conducted following treatment with xanthohumol. Quantitative RT-PCR and Western blot analyses were conducted to assess Notch1 down-regulation. Luciferase reporter assays were performed to assess functional down-regulation of Notch1. Cell cycle analysis was performed by flow cytometry. RESULTS Significant growth inhibition and down-regulation of Notch1 transcription and protein expression were found following xanthohumol treatment. In addition, xanthohumol increased Hes6 transcription and decreased Hes1 transcription, known downstream targets of Notch 1. These observations were associated with cell cycle inhibition as demonstrated by an increase in p21 expression and S and G2/M cell cycle arrest confirmed by an increase in phosphorylated cdc2. Furthermore, an increase in the apoptotic markers, cleaved caspase-3 and cleaved PARP were observed. CONCLUSION Xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol may be influencing the Notch1 pathway. These findings suggest that xanthohumol could be useful as a therapeutic agent in ovarian cancer.

Mapping of Three Genetic Determinants of Susceptibility to Estrogen-Induced Mammary Cancer within the Emca8 Locus on Rat Chromosome 5

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer has gained wide use in the study of breast cancer etiology, prevention, and genetics. Emca8, a QTL that determines susceptibility to E2-induced mammary cancer, was previously mapped to rat chromosome 5 (RNO5) in an intercross between resistant Brown Norway (BN) and susceptible ACI rats. In this study, a panel of congenic rat strains, each of which carries BN alleles across a defined segment of RNO5 on the ACI genetic background, was generated and used to map more precisely the Emca8 determinants of mammary cancer susceptibility. Three distinct genetic determinants were localized within Emca8, and two of these were mapped to intervals of less than 15 megabases. Emca8.1 harbors Cdkn2a, Cdkn2b, and other genes and is orthologous to the 9p21 breast cancer locus identified in genome-wide and candidate gene association studies. Emca8.2 harbors Cdkn2c and other genes and is orthologous to the 1p32 locus in humans that is frequently deleted in breast cancers. Both Emca8.1 and Emca8.2 harbor copy number variants that are orthologous to copy number variant regions in humans. Gene expression profiles were defined for mammary tissues from E2-treated ACI and ACI.BN-Emca8 rats to define the impact of Emca8 on gene expression and identify differentially expressed genes residing within Emca8.1 and Emca8.2. This study further illustrates the relevance of the ACI rat model of E2-induced mammary cancer for identifying novel genetic determinants of breast cancer susceptibility and defining the mechanisms through which estrogens contribute to breast cancer development. Cancer Prev Res; 6(1); 59–69. ©2012 AACR.

Validation of six genetic determinants of susceptibility to estrogen-induced mammary cancer in the rat and assessment of their relevance to breast cancer risk in humans.

When treated with 17β-estradiol, female ACI rats (Rattus norvegicus) rapidly develop mammary cancers that share multiple phenotypes with luminal breast cancers. Seven distinct quantitative trait loci that harbor genetic determinants of susceptibility to 17β-estradiol−induced mammary cancer have been mapped in reciprocal intercrosses between susceptible ACI rats and resistant Brown Norway (BN) rats. A panel of unique congenic rat strains has now been generated and characterized to confirm the existence of these quantitative trait loci, designated Emca3 through Emca9, and to quantify their individual effects on susceptibility to 17β-estradiol−induced mammary cancer. Each congenic strain carries BN alleles spanning an individual Emca locus, introgressed onto the ACI genetic background. Data presented herein indicate that BN alleles at Emca3, Emca4, Emca5, Emca6, and Emca9 reduce susceptibility to 17β-estradiol−induced mammary cancer, whereas BN alleles at Emca7 increase susceptibility, thereby confirming the previous interval mapping data. All of these Emca loci are orthologous to regions of the human genome that have been demonstrated in genome-wide association studies to harbor genetic variants that influence breast cancer risk. Moreover, four of the Emca loci are orthologous to loci in humans that have been associated with mammographic breast density, a biomarker of breast cancer risk. This study further establishes the relevance of the ACI and derived congenic rat models of 17β-estradiol−induced mammary cancer for defining the genetic bases of breast cancer susceptibility and elucidating the mechanisms through which 17β-estradiol contributes to breast cancer development.

Development and characterization of a novel rat model of estrogen-induced mammary cancer

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer is highly relevant for use in establishing the endocrine, genetic, and environmental bases of breast cancer etiology and identifying novel agents and strategies for preventing breast cancer. E2 treatment rapidly induces mammary cancer in female ACI rats and simultaneously induces pituitary lactotroph hyperplasia and adenoma. The pituitary tumors can result in undesired morbidity, which compromises long-term studies focused on mammary cancer etiology and prevention. We have defined the genetic bases of susceptibility to E2-induced mammary cancers and pituitary tumors and have utilized the knowledge gained in these studies to develop a novel inbred rat strain, designated ACWi, that retains the high degree of susceptibility to E2-induced mammary cancer exhibited by ACI rats, but lacks the treatment-related morbidity associated with pituitary lactotroph hyperplasia/adenoma. When treated with E2, female ACWi rats developed palpable mammary cancer at a median latency of 116 days, an incidence of 100% by 161 days and exhibited an average of 15.6 mammary tumors per rat following 196 days of treatment. These parameters did not differ from those observed for contemporaneously treated ACI rats. None of the E2-treated ACWi rats were killed before the intended experimental end point due to any treatment-related morbidity other than mammary cancer burden, whereas 20% of contemporaneously treated ACI rats exhibited treatment-related morbidity that necessitated premature killing. The ACWi rat strain is well suited for use by those in the research community, focusing on breast cancer etiology and prevention.

Abstract 951: Preclinical evaluation of Xanthohumol in carcinoid cancer growth in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Besides surgery, there is no potential curative treatment for carcinoid tumors. Because these cancers are metastatic in nature and produce excessive amounts of various bioactive hormones, patients diagnosed with this malignancy will have poor quality of life due to carcinoid syndrome. Therefore, new anticarcinogenic agents are required to improve the effectiveness of treatment. Xanthohumol (XN) (Tetrahydroxy-3′-prenylchalcone) is a prenylated chalconoid found in hops and beer that has been found to have potential anticancer and bioactive properties. Therefore, the purpose of this study was to evaluate the effectiveness of xanthohumol (XN) on carcinoid cancer growth in vitro and in vivo. Methods: To examine the anticancer activity of XN, we treated human gastrointestinal carcinoid BON and bronchopulmonary carcinoid H727 cells with up to 15 μmol/L of XN or carrier (DMSO), and analyzed the effect on cell growth by colony formation assay. The mechanism of growth inhibition was examined by flow cytometry and western analysis for the levels of pro-apoptotic and cell cycle regulatory proteins. To examine the anticancer activity of XN in vivo, subcutaneous BON carcinoid tumors developed in murine xenografts were subjected to intraperitoneal injections of XN (3 mg/kg bwt) or an appropriate volume of DMSO carrier every other day. Results: Treatment with xanthohumol significantly reduced the ability of carcinoid cells to form colonies in a dose-dependent manner. Flow cytometry and western analysis confirmed the observed decrease in cell viability, and showed the decrease was mediated through apoptosis. The carcinoid tumors in the mouse xenograft experiment showed a significant reduction in growth. Conclusions: XN treatment reduced cell viability, colony forming ability and increased cell death through apoptosis. In addition, administration of XN suppressed cancer cell growth in an in vivo xenograft mouse model. Our findings demonstrate for the first time the anti-proliferative effects of xanthohumol in carcinoid cell lines in vivo. Xanthohumol is already being used as a dietary supplement to encourage overall health and has shown few side effects. This fact combined with the potential anticarcinogenic activity found here warrants clinical investigation on patients with carcinoid disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 951. doi:1538-7445.AM2012-951

Abstract 2387: Fine mapping and characterization of a genetic determinant of susceptibility to estrogen-induced mammary cancer on chromosome 5 in the rat

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Although estrogens have long been implicated in the etiology of breast cancer, the mechanisms through which estrogens contribute to breast cancer development remain poorly defined. Furthermore many of the genes that influence the risk of developing breast cancer also remain to be determined. The ACI rat model of 17β-estradiol (E2)-induced mammary cancer is being used to elucidate the mechanisms through which estrogens contribute to breast cancer development and to identify novel genetic determinants of breast cancer susceptibility. Female ACI rats develop mammary cancer at an incidence nearing 100% when treated with physiological levels of E2. In contrast, both the Brown Norway (BN) and Copenhagen (COP) rat strains are resistant to E2-induced mammary cancer. A total of nine quantitative trait loci (QTL), designated Emca1 (Estrogen-induced mammary cancer) through Emca9, have been mapped in a series of intercrosses between ACI and COP rats (Emca1-2) or ACI and BN rats (Emca3-9). The purpose of the current study is to fine map the Emca1 and Emca8 genetic determinants of mammary cancer susceptibility, which were mapped to rat chromosome 5 (RNO5). ACI alleles at these QTL act to increase the number of mammary cancers per rat and to decrease the latency to the appearance of the first palpable mammary cancer. A series of congenic rat strains have been generated by introgressing defined segments of RNO5 from the resistant COP or BN rat strains onto the genetic background of the susceptible ACI strain. By defining the susceptibility of these congenic rat strains relative to that of the parental ACI strain, Emca8 has now been localized to an approximate 10 megabase (Mb) region of distal RNO5. Similar studies are underway to determine whether or not Emca1 maps to the same region of RNO5 as Emca8. As currently defined, Emca8 harbors approximately 200 known and predicted genes as well as a copy number variant (CNV) that differs between the ACI, COP and BN rat strains based on array comparative genomic hybridization (aCGH). To help identify the mechanisms through which Emca1 and Emca8 determine susceptibility to mammary cancer, we have defined gene expression profiles for normal mammary gland from E2 treated ACI, COP and BN rats and identified those genes that reside within Emca1 and Emca8 and are differentially expressed between ACI and COP or BN rats. Gene expression profiles were also defined for mammary cancers induced by E2 in ACI rats to identify those genes that reside within these QTL and are differentially expressed between normal and neoplastic mammary tissues. The mapping and expression data that define the genomic architecture of Emca1 and Emca8 will be presented and discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2387. doi:10.1158/1538-7445.AM2011-2387

Abstract 4142: The effects of SBHA on Notch 1 expression and platinum sensitivity in ovarian cancer cells

Objective: Despite advances in radical surgical debulking and intraperitoneal chemotherapy, ovarian cancer remains the most deadly gynecologic malignancy. The Notch signaling pathway plays an important role in cellular differentiation, proliferation, and apoptosis. We have previously found Notch 1 intracellular domain (NICD) to be highly expressed in the most platinum resistant ovarian cancer cell lines. Recent evidence has shown that histone deacetylase inhibitors (HDACi9s) decrease platinum resistance in oral squamous cell cancers. In addition, the HDACi suberoyl bis-hydroxamic acid (SBHA) has been found to modify Notch 1 expression in other cancers. We hypothesized that SBHA would reduce ovarian cancer cell proliferation through inhibition of the Notch 1 signaling pathway and help sensitize ovarian cancer cells to platinum. Methods: Following treatment with SBHA, NICD expression in the ovarian carcinoma cell lines OVCAR3, SKOV3, and CaOV3 was analyzed by Western blot. The effects of SBHA and cisplatin on ovarian cancer cell growth were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay. Results: NICD expression by Western blot was consistently reduced in all 3 cell lines following treatment with SBHA. Dose-dependent growth inhibition of ovarian cancer cells was demonstrated with SBHA treatment. Furthermore, the addition of SBHA to cisplatin resulted in additional reduction of NICD and further growth suppression in SKOV3, the most platinum resistant cell line tested. Conclusions: SBHA treatment of ovarian cancer cells led to decreased NICD expression and dose dependent growth reduction in ovarian cancer cells. In addition, we found that SBHA in combination with platinum further reduced NICD expression and growth in SKOV3 cells. These findings provide a compelling argument for further study into the role that Notch 1 signaling may be playing in platinum resistance and ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4142.

Abstract 4147: Xanthohumol decreases Notch1 expression and cell growth in human epithelial ovarian cancer cell lines

Epithelial ovarian cancer is the most lethal of all gynecologic malignancies in the United States with the majority of cases detected with advanced disease. Therefore, it is critical that novel therapies be developed that can effectively treat ovarian cancer. Notch1 is a transmembrane receptor that upon activation is cleaved by gamma-secretase resulting in Notch1 intracellular domain (NICD) translocation to the nucleus where it heterodimerizes with the transcription factor CBF-1 converting CBF-1 from a transcriptional repressor to a transcriptional activator via recruitment of coactivators. Deregulation of the Notch pathway has been observed in cancers such as T cell lymphoblastic leukemia, pancreatic, and breast. In addition, it has been shown that NICD is expressed in ovarian cancer cell lines and ovarian tumors. Xanthohumol, a prenylated chalcone derived from hops used in brewing beer, has been shown to exhibit anti-cancer properties. Since xanthohumol has been shown to inhibit cancer cell growth and invasion, we hypothesized that the Notch1 signaling pathway is suppressed by xanthohumol leading to decreased cell growth. OVCAR3 and SKOV3 are human epithelial ovarian cancer cell lines that express high levels of NICD. These cells were treated with increasing doses of xanthohumol, and cell growth was measured by 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay on days 0, 2, 4, and 6. In addition, protein lysates were isolated on days 2 and 4 of treatment, detected by western blot using a NICD primary antibody, and protein expression was evaluated by densitometry. These treatments resulted in decreased cell growth when measured by MTT assay. In addition, when analyzed by western blot, there was a decrease in NICD protein expression. In conclusion, xanthohumol was a potent inhibitor of ovarian cancer cell growth, and our results suggest that xanthohumol is affecting the Notch pathway. Therefore, it is essential that further investigation into the role that xanthohumol has on the Notch pathway be pursued. These outcomes could prompt the use of xanthohumol as a potential chemotherapeutic agent in ovarian cancer patients who do not respond long term to standard chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4147.