Supplementary Figure 6 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 3 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure Legends, Supplementary Tables 1-2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 1 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 4 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Lasting immunosuppressive effect on lung metastases following the suppressive regimen of bortezomib.

Calreticulin expression on bortezomib-treated tumor cells.

Supplementary Figure 5 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Increase in the number of lung metastases following the suppressive regimen of bortezomib.

Legends for Supplementary Figures S1-S3.

AbstractCancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c+CD8+ dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8+ T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2−/− mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy. Cancer Res; 75(24); 5260–72. ©2015 AACR.

Abstract Many tumors have been demonstrated to contain a subpopulation of cancer stem cells (CSC) whose epigenetic modifications convey resistance to conventional therapies. Furthermore, tumor expression of genes associated with pluripotent embryonic cells, such as Oct-4, Sox-2 and Nanog, often correlates with poor prognosis. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) would be a possible alternative to cell surface phenotype for identification of CSC populations. Therefore we evaluated Nanog expression in 4T1.2 murine breast carcinoma cells, as a biomarker for CSC sub-populations. Introduction of a Nanog promoter reporter that drives destabilized GFP into 4T1.2 cells resulted in a stable expression of GFP in about 1% of the cells. These GFP+ cell could be easily isolated by FACS cell sorting. On subsequent in vitro cell culture of GFP+ populations, up to 80% of the cells became GFP- over a period of 4 weeks. This would be consistent with a subsequent differentiation of these cells. By contrast GFP- populations remained 100% GFP-. In vitro CSC surrogate assays show that Nanog-GFP+ cells produced more spheroids in soft agar and under non-adherent growth conditions. More importantly, in vivo cell transfer studies demonstrated that Nanog-GFP+ cells were more efficient at generating experimental lung metastases when compared to Nanog-GFP-cells. Interestingly Nanog-GFP+ cells were more resistant to the chemotherapeutic drugs paclitaxel and bortezomib. However targeting the extrinsic apoptosis pathway with a combination of bortezomib and agonist antibodies to the TRAIL death receptor DR5 was equally effective against both Nanog-GFP+ and Nanog-GFP- cells. These findings suggest that Nanog promoter activity is a robust marker of highly metastatic 4T1.2 CSC subpopulations, and suggests that eradication of such cells may be required for improved anti-cancer therapies. Funded by NCI Contract HHSN261200800001E Citation Format: Alan D. Brooks, Rachel L. de Kluyver, Jimmy K. Stauffer, Marcella Kaddoura, Thomas J. Sayers. Using Nanog expression to identify breast cancer stem cell populations. [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 2656. doi:10.1158/1538-7445.AM2013-2656

Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.

Abstract Functional heterogeneity of transformed cells exists within a single tumor mass and between distinct tumor nodules. Many tumors have also been demonstrated to contain a subpopulation of tumor initiating cells (TICs) whose epigenetic modifications convey resistance to conventional therapies. It has been hypothesized that TICs represent the terminal product of epithelial-mesenchymal transition which is linked to metastatic potential. Therefore we examined the metastatic potential of TICs in the 4T1 murine breast carcinoma model, and evaluated Nanog expression as a biomarker for highly metastatic 4T1 sub-populations. Traditionally TICs have been identified and isolated through expression of selected cell surface markers and functional assays, however cell surface phenotype alone fails to identify CSC in all tumors from the same organ or in all patients. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) could be superior to cell surface phenotype for identification of TICs. Thus we adapted a commercial promoter reporter system where the Nanog promoter drives copepod GFP which has been destabilized to better reflect rapid changes in transcriptional activity. I ntroduction of a Nanog promoter reporter construct into 4T1.2 cells resulted in stable expression of GFP in about 0.5% of the cells. In vivo tumor transfer studies confirmed that Nanog promoter activity (4T1.2-GFPpos cells) was associated with a significantly enhanced ability to form experimental lung metastases. In addition, in vitro TIC surrogate assays showed that 4T1.2 GFPpos cells produced increased numbers of spheroids in both soft agar and under non-adherent growth conditions. Finally Nanog-GFPpos cells were more resistant to killing by Paclitaxel than Nanog-GFPneg cells, but were more sensitive to Salinomycin. Together these findings suggest that Nanog promoter activity is a robust marker of metastatic 4T1.2 TICs. We have previously observed that administration of the proteasome inhibitor Bortezomib (Bzb) has moderate anti-4T1.2 tumor activity in vivo therefore we examined the sensitivity of the various 4T1.2 populations to Bzb treatment in vitro. 4T1.2 Nanog-GFPpos cells were resistant to the cytostatic effects of Bzb while 4T1.2 Nanog-GFPneg cells were sensitive. The molecular basis of this difference is under investigation, but is not due to differences in accumulation of ubiquitinated proteins. Taken together, these findings show that Nanog promoter activity is a marker of highly metastatic TICs, and suggest that eradication of TICs may be required for curative anti-cancer therapies. 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 3349. doi:10.1158/1538-7445.AM2011-3349

Abstract Obesity is a risk factor for development of certain cancers but the basis for this risk is unclear. In this study, we developed a novel mouse model that demonstrates directly how lipogenic phenotypes commonly associated with diet-induced metabolic syndromes can influence hepatic cancer development. Activated AKT and β-catenin (AKT/CAT) genes were hydrodynamically codelivered using the Sleeping Beauty transposon to initiate liver tumorigenesis. AKT/CAT and MET/CAT combination induced microscopic tumor foci by 4 weeks, whereas no tumorigenesis resulted from delivery of AKT, MET, or CAT alone. Primary AKT/CAT tumor cells were steatotic (fatty) hepatocellular adenomas which progressed to hepatocellular carcinomas (HCC) upon in vivo passage, whereas primary MET/CAT tumors emerged directly as frank HCC. Conversion of AKT/CAT tumor cells to frank HCC during passage was associated with induction of the human HCC marker α-fetoprotein and the stem cell marker CD133. Using hierarchical clustering and gene set enrichment analysis, we compared the primary murine AKT/CAT and MET/CAT tumors to a panel of 53 human HCCs and determined that these two mouse models could be stratified as distinct subtypes associated in humans with poor clinical prognosis. The chief molecular networks identified in primary and passaged AKT/CAT tumors were steatosis and lipid metabolic pathways, respectively. Our findings show how coactivation of the AKT and CAT pathways in hepatocytes can efficiently model development of a lipogenic tumor phenotype. Furthermore, we believe that our approach could speed the dissection of microenvironmental factors responsible for driving steatotic-neoplastic transformation to frank carcinoma, through genetic modification of existing immunodefined transgenic models. Cancer Res; 71(7); 2718–27. ©2011 AACR.

Commentary to: Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis Pamela M. Holland, Robert Miller, Jon Jones, Heather Douangpanya, Julia Piasecki, Martine Roudier and William C. Dougall

The immune system surveys the skin for keratinocytes (KCs) infected by viruses or with acquired genetic damage. The mechanism by which T cells mediate KC elimination is however undefined. In this study we show that antigen-specific CD8 T cells can eliminate antigen-bearing KCs in vivo and inhibit their clonogenic potential in vitro, independently of the effector molecules perforin and Fas-ligand (Fas-L). In contrast, IFN-gamma receptor expression on KCs and T cells producing IFN-gamma are each necessary and sufficient for in vitro inhibition of KC clonogenic potential. Thus, antigen-specific cytotoxic T lymphocytes (CTLs) may mediate destruction of epithelium expressing non-self antigen by eliminating KCs with potential for self-renewal through an IFN-gamma-dependent mechanism.

Recently we reported that gene codon composition determines differentiation-dependent expression of the PV L1 genes in mouse primary keratinocytes (KCs) in vitro and in vivo (Zhao et al. 2005, Mol. Cell Biol. 25:8643-8655). Here, we investigated whether generalized substitution of isoencoding codons affects the duration of expression of PV L1 genes in mouse and human KCs in day 1 culture transiently transfected with native (Nat) and codon modified (Mod) L1 genes. Following transient transfection, KC continuously transcribed both Nat and Mod PV L1 genes for at least 12 days, with the levels of L1 mRNAs from the Mod L1 genes significantly higher than those from the Nat L1 genes. However, continuous L1 protein expression at day 9 post-transfection was observed for both mouse and human KCs transfected with the Nat L1 genes only. Further, aa-tRNAs prepared from D8 KC cultures enhanced translation of two PV Nat L1 DNAs in RRL lysate and PV Nat L1 mRNAs in D0 cell-free lysate, whereas aa-tRNAs from D0 KCs enhanced translation of PV Mod L1 mRNAs in D8 cell-free lysate. It appears that aa-tRNAs in less-differentiated and differentiated KCs differentially match the PV Nat and Mod L1 mRNAs to regulate their translations in vitro.