Colony-forming Ability In Soft Agar Research Articles

Abstract 2102: Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model

Abstract Esophageal squamous cell carcinoma (ESCC) metastatic-initiating cells are heterogenous subpopulations that are hypothesized to promote distal metastasis. Pulmonary metastatic human-origin ESCC mice model was established in our laboratory, chronological flow cytometry collection of tumor cells isolated from metastatic mice lungs at different timepoints inoculated via intravenous injection was followed by 10X genomics multiplex single-cell transcriptomic sequencing (scRNA-seq). Bioinformatics analyses denoted a 7-signature panel previously introduced with preliminary data, follow-up validations of signature-enriched subpopulation in silico, in vitro, and in vivo were performed in this study. In silico bulk transcriptomic sequencing (RNA-seq) of single signature-enriched KYSE30 subpopulation followed by gene ontology (GO) enrichment analyses indicated significantly (FDR<0.05) enhanced biological processes involved in cell motility, cell migration and cell adhesion, compared to signature-low subpopulation. In vitro western blotting showed elevated mesenchymal markers expression, while diminished epithelial markers expression. In vitro real-time qPCR revealed single signature-enriched subpopulation has co-enrichment of other signature marker RNAs compared to control group. Further in vitro functional assays demonstrated signature-enriched subpopulation possesses strengthened spheroid formation ability (p<0.05) and soft agar colony formation ability (p<0.05) as compared to signature-low subpopulation. In addition to previous preliminary data on early survival ability in vivo, signature-enriched subpopulation was significantly more favorable in sustained cell survival and proliferation, and formation of larger size of metastatic lesions in mice lungs at 2 months post-intravenous injection as illustrated by flow cytometry (p<0.05) and immunohistochemistry (IHC) staining analyses (p<0.05) respectively as compared to signature-low subpopulation. To validate the protein expression of signature markers, 6-colour multiplex IHC staining was performed on KYSE30 cell line and metastatic mice lungs collected at different timepoints. Multiplex IHC staining exhibited that these signature markers were expressed in subpopulation of cells as well as expressed on inoculated tumor cells in metastatic mice lungs with timeline pattern. In summary, signature-enriched subpopulation manifested the metastatic-initiating properties in ESCC via various in silico, in vitro, and in vivo experimental analyses. Further multiplex staining in clinical tissue microarray would be performed and analyzed to examine feasibility of adopting these signature markers for facilitating clinical diagnosis and molecular targeting of metastatic ESCC. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Kwong Yiu Lam, Hongyu Zhou, Xinyuan Guan. Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2102.

Marinobazzanan, a Bazzanane-Type Sesquiterpenoid, Suppresses the Cell Motility and Tumorigenesis in Cancer Cells.

Marinobazzanan (1), a new bazzanane-type sesquiterpenoid, was isolated from a marine-derived fungus belonging to the genus Acremonium. The chemical structure of 1 was elucidated using NMR and mass spectroscopic data, while the relative configurations were established through the analysis of NOESY data. The absolute configurations of 1 were determined by the modified Mosher's method as well as vibrational circular dichroism (VCD) spectra calculation and it was determined as 6R, 7R, 9R, and 10R. It was found that compound 1 was not cytotoxic to human cancer cells, including A549 (lung cancer), AGS (gastric cancer), and Caco-2 (colorectal cancer) below the concentration of 25 μM. However, compound 1 was shown to significantly decrease cancer-cell migration and invasion and soft-agar colony-formation ability at concentrations ranging from 1 to 5 μM by downregulating the expression level of KITENIN and upregulating the expression level of KAI1. Compound 1 suppressed β-catenin-mediated TOPFLASH activity and its downstream targets in AGS, A549, and Caco-2 and slightly suppressed the Notch signal pathway in three cancer cells. Furthermore, 1 also reduced the number of metastatic nodules in an intraperitoneal xenograft mouse model.

Immunohistochemistry analysis of Pygo2 expression in central nervous system tumors.

Pygo2 as a Wnt signaling pathway component has been detected in multiple cancer types. In this study, we identified Pygo2 expression features by immunohistochemistry in 73 central nervous system tumor specimens, in comparison with 14 normal brain tissues and surrounding non-tumorous tissues of tumor. Our study indicated that 59% of the patient tumor specimens exhibited positive Pygo2-staining and increases intensity with the grade of malignancy, especially for WHO grade III and IV gliomas, was observed high level expression, compared with normal brain tissues. Five out of nine WHO grade III anaplastic astrocytomas and seven out of nine WHO grade IV glioblastomas showed Pygo2-positive staining. The analysis of Pygo2 gene expression by quantitative real-time PCR of additional ten fresh patient samples yielded similar results. Further studies performed with stable cell lines in vitro demonstrated that Pygo2 render cells higher proliferation rate, migration and anchorage-independent colony-forming ability in soft agar. Taken together, our studies suggest an important role of Pygo2 in brain tumor progression.

Histone Methyltransferase Setd7 Regulates Nrf2 Signaling Pathway by Phenethyl Isothiocyanate and Ursolic Acid in Human Prostate Cancer Cells.

This study aims to investigate the role of the epigenetic regulator SET domain-containing lysine methyltransferase 7 (Setd7) in regulating the antioxidant Nrf2 pathway in prostate cancer (PCa) cells and examines the effects of two phytochemicals, phenethyl isothiocyanate (PEITC) and ursolic acid (UA). Lentivirus-mediated shRNA knockdown of Setd7 in LNCaP and PC-3 cells decreases the expression of downstream Nrf2 targets, such as NAD(P)H: quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase theta 2 (Gstt2). Downregulation of Setd7 decreases soft agar colony formation ability of PCa cells. Knockdown of Setd7 increases reactive oxygen species (ROS) generation. Furthermore, Setd7 knockdown attenuates Nqo1 and Gstt2 expression in response to H2 O2 challenge, whereas increased DNA damage is observed in Setd7 knockdown cells in comet assay. Interestingly, Setd7 expression could be induced by the dietary phytochemicals PEITC and UA. Chromatin immunoprecipitation (ChIP) assays show that Setd7 knockdown decreased H3K4me1 enrichment in the Nrf2 and Gstt2 promoter regions, while PEITC and UA treatments elevated the enrichment. Taken together, these results indicate that Setd7 knockdown decreases Nrf2 and Nrf2-target genes expression and that PEITC and UA induce Setd7 expression, which activates the Nrf2/antioxidant response element (ARE) signaling pathway and protects DNA from oxidative damage.

Evaluation of tumorigenic potential of CeO2 and Fe2O3 engineered nanoparticles by a human cell in vitro screening model

With rapid development of novel nanotechnologies that incorporate engineered nanomaterials (ENMs) into manufactured products, long-term, low dose ENM exposures in occupational settings is forecasted to occur with potential adverse outcomes to human health. Few ENM human health risk assessment efforts have evaluated tumorigenic potential of ENMs. Two widely used nano-scaled metal oxides (NMOs), cerium oxide (nCeO2) and ferric oxide (nFe2O3) were screened in the current study using a sub-chronic exposure to human primary small airway epithelial cells (pSAECs). Multi-walled carbon nanotubes (MWCNT), a known ENM tumor promoter, was used as a positive control. Advanced dosimetry modeling was employed to ascertain delivered vs. administered dose in all experimental conditions. Cells were continuously exposed in vitro to deposited doses of 0.18μg/cm2 or 0.06μg/cm2 of each NMO or MWCNT, respectively, over 6 and 10weeks, while saline- and dispersant-only exposed cells served as passage controls. Cells were evaluated for changes in several cancer hallmarks, as evidence for neoplastic transformation. At 10weeks, nFe2O3- and MWCNT-exposed cells displayed a neoplastic-like transformation phenotype with significant increased proliferation, invasion, and soft agar colony formation ability compared to controls. nCeO2-exposed cells showed increased proliferative capacity only. Isolated nFe2O3 and MWCNT clones from soft agar colonies retained their respective neoplastic-like phenotypes. Interestingly, nFe2O3-exposed cells, but not MWCNT cells, exhibited immortalization and retention of the neoplastic phenotype after repeated passaging (12–30 passages) and after cryofreeze and thawing. High content screening and protein expression analyses in acute exposure ENM studies vs. immortalized nFe2O3 cells, and isolated ENM clones, suggested that long-term exposure to the tested ENMs resulted in iron homeostasis disruption, an increased labile ferrous iron pool, and subsequent reactive oxygen species generation, a well-established tumorigenesis promotor. In conclusion, sub-chronic exposure to human pSAECs with a cancer hallmark screening battery identified nFe2O3 as possessing neoplastic-like transformation ability, thus suggesting that further tumorigenic assessment is needed.

HOXC6 promotes gastric cancer cell invasion by upregulating the expression of MMP9.

Previous studies have demonstrated that the homoeboxC6 (HOXC6) gene is highly expressed in gastric cancer tissues and is associated with the depth of tumor invasion, and is associated with poor prognosis of gastric cancer patients expressing HOXC6. The present study investigated the effect and underlying mechanism of HOXC6 on the proliferation and metastasis of gastric cancer cells invitro. Reverse transcription‑quantitative polymerase chain (PCR) reaction was used to investigate the expression levels of HOXC6 in different gastric cancer cell lines and the effect of different levels of expression on the proliferation of gastric cancer cells was determined by cell growth curve and plate colony formation. The effect of HOXC6 on the anchorage‑independent proliferation of gastric cancer cells was determined by soft agar colony formation assay while the Transwell invasion assay was used to investigate the effect of different levels of HOXC6 expression on the invasive and metastatic abilities of gastric cancer cells. Semi‑quantitative PCR was used to detect the effect of different levels of HOXC6 expression on the expression of matrix metalloproteinase (MMP)2 and MMP9 in gastric cancer cells. Immunoblotting was used to assess MMP9 signaling in the gastric cancer cells. The HOXC6 gene is highly expressed in the majority of the gastric cancer cell lines. Overexpression of HOXC6 promoted gastric cancer cell proliferation and colony formation ability while HOXC6 downregulation inhibited cell proliferation and clone forming ability. HOXC6 overexpression also enhanced the soft agar colony formation ability of gastric cancer cells while HOXC6 downregulation decreased the colony formation ability. Upregulated HOXC6 increased the migration and invasion abilities of gastric cancer cells while interfering with HOXC6 expression inhibited the migration and invasion of the gastric cancer cells. The expression of MMP9 was enhanced with an upregulation of HOXC6 expression while HOXC6 downregulation lowered MMP9 gene expression levels. Increased expression of HOXC6 in gastric cancer cell lines significantly activated extracellular signal‑regulated kinase signaling and upregulated MMP9. The HOXC6 gene promotes the proliferation of gastric cancer cells while upregulation of MMP9 promotes migration and invasion of gastric cancer cells.

Abstract 3918: Synergistic targeting of fibroblast growth factor receptor and AKT pathways significantly inhibits prostate cancer progression

Abstract The fibroblast growth factor (FGF) and AKT pathways are important stimuli to prostate cancer (PCa) development and progression. FGF and AKT pathways are becoming increasingly attractive as targets for PCa therapeutic intervention. Dual-targeting of FGF receptor (FGFR) and AKT signaling might be highly efficacious in the treatment of PCa. In this study, we evaluated the combined treatment effect of FGFR inhibitor AZD4547 and AKT inhibitor AZD5363 on PCa progression in vitro. Proliferation assay shows that AZD4547 weakly inhibits PCa cell proliferation, while AZD5363 demonstrates strong proliferation inhibition effect. The combined treatment of AZD4547 and AZD5363 further inhibits cell proliferation compared to AZD4547 monotreatment. Both AZD4547 and AZD5363 inhibit cell anchorage-independent growth and invasion, and combined treatment synergistically decrease cell soft agar colony formation ability and invasiveness. Molecular mechanisms of the dual targeting of FGFR and AKT were explored by immunoprecipitation (IP) and western blot (WB). IP on 22RV1 cells shows that FGFR4 phosporylation was inhibited by AZD4547 but not AZD5363, while was strongly decreased in the combined treatment. WB demonstrats that in VCaP and LNCaP cells PLCγ phosphorylation was significantly inhibited by AZD4547, slightly decreases by AZD5363, and strongly inhibited by combined treatment. FGFR2α phosphorylation was remarkably inhibited by AZD4547 in VCaP and 22RV1 cells, slightly decreased by AZD5363 in VCaP cells, but was strongly increased by AZD5363 in 22RV1 cells. Combined treatment of both cell lines shows remarkable inhibition of FRS2α phosphorylation. Erk1/2 and MEK phosphorylation was dramatically decreases by AZD4547, while was not inhibited, or to some extent, increased by AZD5363. Notably, the combined treatment synergistically inhibited MAPK signaling in PCa cells. AKT phosphorylation was dramatically enhanced by AZD5363 as an inactive hyperphosphorylated form. It was inhibited by AZD4547, but was still highly enhanced in combined treatment because that the stimulation effect of AZD5363 was too strong to be attenuated. Both AZD4547 and AZD5363 suppress STAT3 phosphorylation in LNCaP and 22RV1 cells, and the combined treatment shows synergistic inhibition of STAT3 activation. STAT1 phosphorylation was inhibited by AZD5363 and combined treatment but not by AZD4547 alone. WB also demonstrate that AZD5363 strongly inhibit phosphorylation of AKT substrate and downstream proteins PRAS40, GSK3β and S6, which are not, or slightly decreased by AZD4547, but are remarkably inhibited by the combined treatment. This study provides a preclinical proof of concept that combination of a FGFR inhibitor with an AKT inhibitor has profound potential to treat prostate cancer. Citation Format: Shu Feng, Michael Ittmann. Synergistic targeting of fibroblast growth factor receptor and AKT pathways significantly inhibits prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2015-3918

Abstract 2478: Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis

Abstract Background: Tid1, a DnaJ cochaperone protein mainly located in mitochondria, functions as a tumor suppressor in different types of solid tumors. Recently, we first demonstrate that Tid1 is negatively associated with cell migration, tumor status, and survival prognosis in head and neck squamous cell carcinoma (HNSCC) (J Pathol 2009; 219:347-55). As a cochaperone protein like Tid1, it should be instrumental to find the Tid1 client/interacting proteins, consequently, to understand the physiological function of those Tid1 client/interacting proteins as therapeutic targets for future HNSCC treatment. Methods: First, we applied affinity chromatography and systemic proteomics analysis to identify galectin-7 as one of client/interacting proteins of Tid1. Subsequently, using the metastatic and/or non-metastatic cells determined tumorigenic properties of HNSCC with overexpression and/or knockdown of galectin-7 and/or Tid1. The nuclear and cytosolic subfractions were isolated to determine the role of Tid1 of galectin-7 nuclearization. We also attempted to correlate the expression of Tid1 and galectin-7 with tumor stage of HNSCC patient tissues by immunohistochemical assay. Finally, site-directed mutagenesis was applied to identify the novel N-link glycosylated sites of Tid1, by which to regulate the interaction and stability of galectin-7. Results: We found metastatic cells expressed lower level of Tid1 and higher level of galectin-7 than non-metastatic cells. For in vitro tumorigenic analyses, we observed that overexpression of galectin-7 resulted in elevated migration, invasion and soft agar colony formation abilities in HNSCC. Inversely, co-overexpression of Tid1 reduced the expression of galectin-7, impaired the galectin-7 mediated migration, invasion and soft agar colony formation ability of HNSCC, and retarded nuclearization of galectin-7. Moreover, knockdown of galectin-7 reduced migration, invasion and soft agar colony formation ability of HNSCC metastatic cells. Further, we found that galectin-7 ubiquitination as revealed by antiubiquitin immunoblot analysis in immunoprecipitates. Finally, the negative relationship was between Tid1 and Galectin-7 expression in HNSCC clinical specimen. We found out that Tid1 interacts with galectin-7 through N-linked glycosylation. Tid1 mutants of N-link glycosylated sites restored the level and the tumorigenic function of galectin-7. Conclusion: Our studies first pointed out that galectin-7 plays pivotal roles in HNSCC tumorigenesis. Notably, we identified galectin-7, which is the client/interacting protein of Tid1. We hypothesized that Tid1/ Hsp70 may regulate galectin-7 stability by ubiquitinylation. The molecular mechanism by which Tid1 negatively modulates the galectin-7 warrants further investigation. Citation Format: Yu-Syuan Chen. Glycosylation of tid1 regulates galectin-7 ubiquitination and localization to suppress head and neck cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2014-2478

SCC-S2 is overexpressed in colon cancers and regulates cell proliferation

SCC-S2 was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of SCC-S2 and its biological roles in colon cancers have not been reported. The aim of this study is to investigate the expression and clinical significance of SCC-S2 in colon cancers and explore its biological function in colon cancer cells. We analyzed the expression pattern of SCC-S2 in 92 colon cancer tissues by immunohistochemistry and found that SCC-S2 was overexpressed in 45 (48.9 %) of 92 colon cancer specimens. There was a significant association between SCC-S2 overexpression and TNM stage (p = 0.0387), lymph node metastasis (p = 0.0219), and proliferation index (p = 0.0279). siRNA knockdown of SCC-S2 expression in CACO2 and HCT116 cells decrease cell proliferation, colony formation, and soft agar colony formation ability. Furthermore, western blot analysis showed that SCC-S2 depletion decreased cyclin D1 and phospho-Rb levels. In conclusion, we demonstrated that SCC-S2 is overexpressed in colon cancers and contributes to malignant cell growth by cyclin D1 and phospho-Rb modulation, which makes SCC-S2 a candidate therapeutic target in colon cancer.

Potential for the embryonic morphogen Nodal as a prognostic and predictive biomarker in breast cancer

IntroductionThe re-emergence of the tumour growth factor-beta (TGF-beta)-related embryonic morphogen Nodal has recently been reported in several different human cancers. In this study, we examined the expression of Nodal in a series of benign and malignant human breast tissues to determine the clinical significance of this expression and whether Nodal could represent a potential therapeutic target in breast cancer.MethodsTissue sections from 431 therapeutically naive patients diagnosed with benign or malignant breast disease were stained for Nodal by immunohistochemistry and analysed in a blinded manner. The degree of Nodal staining was subsequently correlated with available clinical data, such as diagnoses and disease stage. These tissue findings were further explored in breast cancer cell lines MDA-MB-231 and MDA-MB-468 treated with a Nodal blocking antibody to determine biological effects for target validation.ResultsA variable degree of Nodal staining was detected in all samples. The intensity of Nodal staining was significantly greater in undifferentiated, advanced stage, invasive breast cancer compared with benign breast disease or early stage breast cancer. Treatment of human breast cancer cells in vitro with Nodal blocking antibody significantly reduced proliferation and colony-forming ability in soft agar, concomitant with increased apoptosis.ConclusionsThese data suggest a potential role for Nodal as a biomarker for disease progression and a promising target for anti-Nodal therapy in breast cancer.

Abstract 2961: Reactivation of EWS-FLI1 suppressed FOXO1 expression as a novel therapeutic strategy for Ewing's sarcoma

Abstract The EWS-FLI1 chimeric protein, characterizing Ewing's sarcoma (ESFT), is prototypic for an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of recognition motifs for forkhead box (FOX) proteins in EWS-FLI1 repressed promoters. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibiting FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, FOXO1 is excluded from the nucleus as a consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, augmented by chemical inhibition of PI3K, or by mutation of CDK2 or AKT phosphorylation sites. Furthermore, ChIP experiments revealed that EWS-FLI1 directly binds the promoter of FOXO1 in vitro suggesting a multi-layered regulation of FOXO1 expression by transcriptional repression and post-translational modification. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability. A significant overlap between EWS-FLI1 repressed and FOXO1 activated genes was observed. Treatment of ESFT cell lines with Methyl-Seleninic-Acid (MSA) led to re-activation of endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner, and induced massive cell death, which we found to be at least partially FOXO1-dependent. Taken together, these data confirm our hypothesis that a repressive sub-signature of EWS-FLI1 regulated genes is due to suppression of FOXO1. FOXO1 re-activation by small molecules may constitute a novel therapeutic strategy in the treatment of ESFT. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. 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 2961. doi:1538-7445.AM2012-2961

Abstract 2099: Tid1 regulates galectin-7 mediated tumorigenesis of oral squamous cell carcinoma

Abstract Background: We previously identified Tid1, a DnaJ cochaperon protein, functions as a tumour suppressor in oral squamous cell carcinoma (OSCC) tumourigenesis (J Pathol 2009;219:347-55). To further understand the molecular mechanisms mediated by Tid1 on oral tumorigenesis, we applied affinity chromatography and systemic proteomics analysis to identify galectin-7, which specifically interacted with Tid1. Others have shown that Galectin-7 plays a number of important roles in tumor development. However, the role of Galectin-7 in OSCC tumourigenesis remains unclear. Herein, we elucidated the mechanism mediated by Tid1 on regulating galectin-7 mediated cellular signaling and tumorigenesis in OSCC. Methods: First, through HA-tag affinity chromatography and mass spectrometry we identified substrate proteins that specifically interact with Tid1. Secondly, co-immunoprecipitation analysis and confocal microscope was performed to confirm the protein-protein interaction between galectin-7 and Tid1. In vitro tumorigenic properties of OSCC overexpressing galectin-7 were determined. Subsequently, the tumorigenic properties of OSCC after co-overexpression of galectin-7 and Tid1 were investigated. Finally, immunoprecipitation analysis was performed to recognize the Tid1 induced deregulation of galectin-7 by enhancing ubiquitination. Results: We found out that Tid1 interacted with galectin-7. Furthermore, overexpression galectin-7 expression resulted in up-regulation migration, invasion and soft agar colony formation abilities in OSCC. Importantly, co-overexpression of Tid1 decreased the galectin-7 expression and impaired galectin-7-mediated migration, invasion and soft agar colony formation ability of OSCC. Finally, we found galectin-7 ubiquitination as revealed by antiubiquitin immunoblot analysis in immunoprecipitates. Conclusion: Our studies first pointed out that galectin-7 played pivotal roles in OSCC tumorigenesis. Notably, we identified galectin-7 that specifically interacted with Tid1. We hypothesized that Tid1/ Hsp70 may regulate galectin-7 stability by ubiquitinylation. The molecular mechanism by which Tid1 negatively modulates the galectin-7 warrants further investigation. 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 2099. doi:10.1158/1538-7445.AM2011-2099

Abstract 2392: Primary cultures established from estrogen receptor negative breast tumors: Method and characterization

Abstract Approximately 35% of breast cancers lack expression of estrogen receptor (ER) protein. ER-negative (ER-) breast cancer carries a worse prognosis than ER positive (ER+) breast cancer, but more importantly, it precludes use of less toxic endocrine therapies. Our objective was to establish primary cultures from dissociation of ER- breast tumors in order to provide an alternative cellular model that can be used as a valuable tool for both in vitro and in vivo studies of ER- breast cancer. A total of eight ER- tumors were successfully dissociated into primary cultures: four from ER-/PR-/H2N- and four from ER-/PR-/H2N+ primary breast tumors with a 100% success rate. The epithelial-enriched cell pellet was placed in culture and for experiments carried for a maximum of 30 passages. These cells will be hereafter referred to as “dissociated tumor” (DT) cultures. Cells were grown in 2D culture and their in vitro morphology, proliferation rates, mammosphere and soft agar colony formation ability, and CD44/CD24 surface marker expression (tumor initiating cell (TIC) content) are determined. In addition we have performed gene expression profiling and established their tumorigenic potential in NOD/SCID female mice. Each culture exhibited its own relatively unique morphology; two of the eight cultures grew mainly as suspensions while the remaining six DT cultures grew with a higher percentage of attached, mesenchymal phenotype cells. Proliferation rates ranged from 38 to 60 hours, and while all of the DT cultures had the ability to form mammospheres, five of the eight DTs (DT13, DT16, DT22, DT25 and DT28) formed colonies in soft agar. Analysis of CD44 and CD24 surface markers expression showed that while all DT cultures were CD44 positive, expression of CD24 varied among DTs. Of the eight DT cultures, we found five of them to have a high (>85%) CD44+/CD24-/lo cell content, one the CD44+/CD24high phenotype and two of eight have populations with increasing levels of CD24 expression ranging from -/lo to medium. This CD44+/CD24-/lo phenotype is stable when examined over several passages. Microarray analysis comparing the DT cultures to cancer cell lines showed that they clustered with each other and with several breast cancer cell lines of known ER- status and EGFR or Her2 overexpression status. In agreement with the soft agar assay, DT16, DT22, DT25 and DT28 had the ability to form tumors when injected into the mammary fat pad of female NOD/SCID mice. In summary, our study describes primary cultures of dissociated ER negative breast cancer cells that provide an alternative, primary cell based model that allows both in vitro as well as in vivo experimental approaches. The use of these types of cellular models may lead to a better understanding of ER- breast cancer biology as well as being a valuable tool for screening potential therapeutic options for ER- breast cancer. 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 2392. doi:10.1158/1538-7445.AM2011-2392

Abstract 5338: A repressive EWS-FLI1 subsignature is the result of FOXO1 suppression and nuclear exclusion

Abstract The EWS-FLI1 chimeric protein, characteristic of Ewing′s sarcoma (ESFT), constitutes the prototype of an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of ETS binding motifs in EWS-FLI1 activated target promoters, while EWS-FLI1 repressed promoters showed most prominently enrichment of recognition motifs for forkhead box (FOX) proteins. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibition of FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, ectopic FOXO1 was found excluded from the nucleus as consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, a directly EWS-FLI1 activated kinase, augmented by chemical inhibition of PI3K, or by mutation of CDK2 phosphorylation sites. These results suggest an important role for CDK2 in FOXO1 repression by EWS-FLI1. To test the contribution of FOXO1 to the EWS-FLI1 repressive signature on the ESFT transcriptome, the overlap between genes activated by EWS-FLI1 knockdown and genes activated by ectopically expressed, nuclear directed FOXO1 was determined and compared to the effects of EWS-FLI1 knockdown under concomitant FOXO1 silencing. A significant overlap between FOXO1 activated and EWS-FLI1 repressed genes was identified (P<10e-20, hypergeometric test). Taken together, these data confirm our hypothesis that a repressive subsignature of EWS-FLI1 regulated genes is due to suppression of FOXO1. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability suggesting that FOXO1 plays an important role in ESFT oncogenesis. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. 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 5338. doi:10.1158/1538-7445.AM2011-5338

Identification and Characterization of a Novel Melanoma Tumor Suppressor Gene on Human Chromosome 6q21

By characterizing a complex chromosome rearrangement involving 6q and 17p in melanoma cell line UACC-930, we isolated a candidate tumor suppressor gene at 6q21, named prenyl diphosphate synthase subunit 2 (PDSS2), which was interrupted by an inversion breakpoint. The purpose of this study was to determine the tumor-suppressive potential of PDSS2 in the development of melanoma. To isolate the rearranged 6q in UACC-930 cells, a bacterial artificial chromosome clone (RP1-67A8) covering the breakpoint at 6q21 was digested with HindIII and each DNA fragment was used as the probe for the breakpoint in Southern blotting. The HindIII fragment probe covering the breakpoint was then used to screen an EcoRI-digested DNA library generated from UACC-930. To characterize the tumor-suppressive potential of PDSS2, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was shown by both in vitro and in vivo assays. The differential expression of PDSS2 in benign nevi and primary melanoma samples was also studied. Down-regulation of PDSS2 was observed in 59 of 87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7 of 66, 10.6%; P < 0.001). In addition, an overexpression of the PDSS2 in UACC-903 cells could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. Our results support the proposal that PDSS2 is a novel tumor suppressor gene that plays an important role in the development of malignant melanoma.

The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1‐dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1‐dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose‐ and time‐dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony‐forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway‐associated diseases. J. Cell. Biochem. 106: 682–692, 2009. © 2009 Wiley‐Liss, Inc.

Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects

Bcr-Abl acquires its transforming ability through its upregulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. In chronic myelogenous leukemia (CML) cells, Bcr-Abl phosphorylates Bcr on tyrosine residues reducing its kinase activity. Overexpression of BCR in BCR-ABL+ cells produces a phosphoserine form of Bcr, which inhibits the oncogenic effects of BCR-ABL. To investigate the inhibitory effects of Bcr on Bcr-Abl, we expressed BCR/GFP in TonB210 cells, which contain a tetracycline-inducible BCR-ABL. In nude mice injected with cell clones of TonB210/BCR/GFP, tumor formation was delayed, and tumors were 50% smaller compared with the TonB210/GFP. In addition, TonB210/ BCR/GFP cells had little colony-forming ability in soft agar compared with TonB210/GFP cells. In contrast, a point mutant of BCR (Y360F), which disrupts its kinase activity, not only blocked Bcr's inhibitory effects but also enhanced the oncogenic effects of Bcr-Abl in a solid tumor model and in soft agar colony assays. Similar effects were observed with a second BCR kinase domain mutant, S354A. These results indicate that the inhibitory function of Bcr directed toward Bcr-Abl requires its kinase function.

Phosphorylated retinoid X receptor α loses its heterodimeric activity with retinoic acid receptor β

A malfunction in retinoid X receptor (RXR) alpha due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRalpha loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRalpha affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRalpha and retinoic acid receptor beta was impaired when 293T cells were transfected with phosphomimic mutant RXRalpha (T82D/S260D), whereas this interaction was activated at a level similar to wild-type RXRalpha-transfected cells when the cells were transfected with an unphosphorylated mutant RXRalpha (T82A/S260A). Treating the T82A/S260A-transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D-transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D-transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony-forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRalpha abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor beta, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRalpha phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth.

Insulin‐like growth factor binding protein‐related protein 1 (IGFBP‐rP1) has potential tumour‐suppressive activity in human lung cancer

The expression of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is decreased in various tumours, but the role of IGFBP-rP1 in lung cancer is not yet clear. In this study, IGFBP-rP1 expression in lung cancer cell lines was evaluated and reduced expression of IGFBP-rP1 was found. In tissue microarrays containing 138 primary tumours and 20 normal lung tissues analysed by immunohistochemistry, 58 tumours (42%) exhibited no expression of IGFBP-rP1, while all 20 normal lung tissues showed high expression. In squamous cell lung cancer, low expression of IGFBP-rP1 was significantly linked to high-grade tumours. Treatment with 5-aza-2'-deoxycytidine restored the expression of IGFBP-rP1 in three of four lung cancer cell lines. Sequencing of PCR products of sodium bisulphite-treated genomic DNA from the three lung cancer cell lines revealed a heterogeneous methylation pattern in the region of exon 1 and intron 1. Stable transfection of IGFBP-rP1 full-length cDNA into the H2170 lung cancer cell line led to increased expression of IGFBP-rP1 protein. IGFBP-rP1-positive transfectants exhibited remarkably reduced colony-forming ability in soft agar, suppression of tumour growth rate in nude mice, and increased apoptotic cell number as well as activated caspase-3 expression level. The data suggest that IGFBP-rP1 is a tumour suppressor inactivated by DNA methylation in human lung cancer.

Correlation of malignant phenotypes of human tumour cell lines with augmented expression of Hsp72 protein measured by laser scanning cytometry

Augmented expression of members of the heat shock protein 70 (Hsp70) family are frequently observed in various human cancers. In this study, we examined applicability of laser scanning cytometer (LSC) to evaluate the level of Hsp72, which is the member constitutively expressed and significantly induced after heat shock, in human tumour cell lines. The relative nuclear content of Hsp72 measured by LSC correlated well with the relative intracellular content determined by Western blotting (R = 0.906). Furthermore, there was a close relationship between the relative nuclear content of Hsp72 measured by LSC and the colony-forming ability in soft agar, one of the malignant characteristics of tumour cells (R = 0.880). These results indicate that LSC measurement is useful for predicting the degree of malignancy of cancer cells, as it is reliable, faster than Western blotting and more objective and quantitative than visual measurements.