Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine; however, their potential clinical application is hampered by the low efficiency of somatic cell reprogramming. ...Here, we show that the synergistic activity of synthetic modified mRNAs encoding reprogramming factors and miRNA-367/302s delivered as mature miRNA mimics greatly enhances the reprogramming of human primary fibroblasts into iPSCs. This synergistic activity is dependent upon an optimal RNA transfection regimen and culturing conditions tailored specifically to human primary fibroblasts. As a result, we can now generate up to 4,019 iPSC colonies from only 500 starting human primary neonatal fibroblasts and reprogram up to 90.7% of individually plated cells, producing multiple sister colonies. This methodology consistently generates clinically relevant, integration-free iPSCs from a variety of human patient's fibroblasts under feeder-free conditions and can be applicable for the clinical translation of iPSCs and studying the biology of reprogramming.
Carcinoma-associated fibroblasts (CAFs) are now widely appreciated for their contributions to tumor progression. However, the ability of CAFs to regulate anoikis, detachment-induced cell death, has ...yet to be investigated. Here, a new role for CAFs in blocking anoikis in multiple cell lines, facilitating luminal filling in three-dimensional cell culture, and promoting anchorage-independent growth is defined. In addition, a novel mechanism underlying anoikis inhibition is discovered. Importantly, it was demonstrated that CAFs secrete elevated quantities of insulin-like growth factor-binding proteins (IGFBPs) that are both necessary for CAF-mediated anoikis inhibition and sufficient to block anoikis in the absence of CAFs. Furthermore, these data reveal a unique antiapoptotic mechanism for IGFBPs: the stabilization of the antiapoptotic protein Mcl-1. In aggregate, these data delineate a novel role for CAFs in promoting cell survival during detachment and unveil an additional mechanism by which the tumor microenvironment contributes to cancer progression. These results also identify IGFBPs as potential targets for the development of novel chemotherapeutics designed to eliminate detached cancer cells.
The ability of CAF-secreted IGFBPs to block anoikis in breast cancer represents a novel target for the development of therapeutics aimed at specifically eliminating extracellular matrix-detached breast cancer cells.
Institutes of 1 Pharmacology and Toxicology and 2 Neuropathology, University of Göttingen, Göttingen; 3 Department of Biochemistry, Faculty of Chemistry, University of Kaiserslautern, Kaiserslautern, ...Germany; and 4 Fraunhofer-Institute of Toxicology and Experimental Medicine, Hannover, Germany
Submitted 8 December 2006
; accepted in final form 21 December 2007
The clarification of subcellular localization represents an important basis toward characterization of ATP-binding cassette (ABC) transporters and resolution of their roles in cellular physiology. Rat Abcb6 (rAbcb6) is a membrane-situated half-transporter belonging to the ABC protein superfamily. To investigate rAbcb6 subcellular distribution, the human colon adenocarcinoma line LoVo, which we found to be devoid of endogenous human ABCB6 mRNA, was employed for heterologous expression of rAbcb6 bearing a COOH-terminal epitope tag (rAbcb6-V5). Following subcellular fractionation, rAbcb6-V5 was observed as an N -glycosylated protein in fractions enriched with lysosomal/endosomal membrane proteins. Indirect immunofluorescence analyses of rAbcb6-V5 using antibodies against a rAbcb6-specific peptide or against the V5-tag revealed a punctate pattern that was colocalized with lysosome-associated membrane protein 1 (LAMP1), a marker of lysosomes/late endosomes. Substantial colocalization of tagged rAbcb6 with lysosomal/late endosomal marker was confirmed with living, unfixed LoVo cells coexpressing rAbcb6 fused to enhanced green fluorescent protein. Vesicular distribution in LoVo cells was consistent with localization of endogenous rAbcb6 expressed in rat primary hepatocyte cultures or in liver sections, as revealed by overlap of rat Lamp1 with rAbcb6 in double immunofluorescence analyses. Since several Abcb6-related half-transporters confer heavy metal tolerance, we investigated whether rAbcb6 expression in LoVo cells might affect sensitivity toward transition metal toxicity. Applying MTT viability assays, we found that expression of either rAbcb6-V5 or untagged rAbcb6 conferred tolerance toward copper, but not to cobalt or zinc. In summary, these results demonstrate that rAbcb6 is a glycosylated protein targeted to intracellular vesicular membranes and suggest involvement of rAbcb6 in transition metal homeostasis.
LoVo; rat hepatocytes; lysosomes; endosomes; glycosylation; copper tolerance
Address for reprint requests and other correspondence: K. I. Hirsch-Ernst, Institute of Pharmacology and Toxicology, Univ. of Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany (e-mail: khirsche{at}med.uni-goettingen.de )
Abstract
The tumor microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated ...fibroblasts (CAFs) in the microenvironment are a critical player in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the importance of CAFs in promoting tumor progression and the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in other non-tumorigenic cell lines and in breast cancer cell lines. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space.
In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. However, we discovered that the degradation of Mcl-1 was strongly inhibited by CAF-conditioned media suggesting that Mcl-1 was not properly targeted to the proteasome. Furthermore, using mass spectrometric analyses, we have identified proteins of the IGFBP family that are differentially secreted by CAFs in a fashion that regulates anoikis induction. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis.
Citation Format: Kelsey Weigel, Ana Jakimenko, Brooke Conti, William Kaliney, Matthew M. Champion, Zachary T. Schafer. The regulation of anoikis by carcinoma-associated fibroblasts in 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 1634. doi:10.1158/1538-7445.AM2013-1634
Abstract
The initial step of the metastatic cascade in most carcinomas occurs when tumor cells break through the basement membrane and invade locally into the surrounding microenvironment. This ...microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated fibroblasts (CAFs) in the microenvironment are a critical player in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the importance of CAFs in promoting tumor progression and the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in two additional non-tumorigenic cell lines: BPH-1 and HMEC. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis.
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 2083. doi:1538-7445.AM2012-2083
Abstract
The synthetic retinoid fenretinide, N-(4-hydroxyphenyl) retinamide (4-HPR), is selectively cytotoxic to cancer cells via increases in dihydroceramide levels and generation of reactive oxygen ...species and has clinical activity against recurrent neuroblastoma (J Clin Oncol 27: 15s abstr 10009, 2009). Metabolism of ceramides into various sphingolipid species, including sphingosine, and phosphorylation of dihydrosphingosine and sphingosine by sphingosine kinase 1 (SPHK1) are anti-apoptotic and thought to counter cytotoxicity mediated by ceramides. Multistep selection for resistance to 4-HPR was performed with a 4-HPR sensitive neuroblastoma cell line, SMS-KCNR (IC99=4.6 uM), resulting in the 4-HPR resistant line, KCNR-FR (IC99=19.9 uM). We generated a TaqMan low-density array (TLDA) that measures expression of 42 genes involved in ceramide synthesis and metabolism by quantitative RT-PCR. Genes overexpressed in KCNR-FR relative to SMS-KCNR included ceramide desaturase 2 (14.1-fold), sphingosine-1-phosphate receptors S1PR2, 3, and 4 (2.4, 1.6, and 1.3-fold, respectively), and SPHK1 (5.2-fold). These data point toward 4-HPR resistance being mediated by sphingosine-1-phosphate or dihydrosphingosine-1-phosphate, both of which are generated via phosphorylation of sphingosine by SPHK1. SPHK1-specific RNAi knockdown decreased SPHK1 mRNA expression (36% ± 0.007 of scrambled RNAi, p<0.05) and significantly increased apoptosis (35.8 ± 1.7%; p<0.05) in KCNR-FR treated with 4-HPR relative to scrambled RNAi controls (8.8 ± 6.5%; p<0.05). Safingol (L-threo-dihydrosphingosine), a putative sphingosine kinase inhibitor in phase I trials, partially reversed KCNR-FR resistance to 4-HPR (Combination Index = 0.56). Thus, selection for resistance to 4-HPR in neuroblastoma was associated with increased expression of multiple components of the anti-apoptotic sphingosine kinase signaling pathway, and such resistance can be partially overcome by targeting sphingosine kinase.
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 5262.
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