Evaluation of AXIN1 and AXIN2 as targets of tankyrase inhibition in hepatocellular carcinoma cell lines

AbstractAXIN1 mutations are observed in 8–10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing β-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear β-catenin accumulation nor clearly enhanced expression of β-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced β-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control β-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to β-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the β-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of β-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on β-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced β-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing β-catenin signaling.

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Comprehensive analysis of β-catenin target genes in colorectal carcinoma cell lines with deregulated Wnt/β-catenin signaling

BMC Genomics ◽

10.1186/1471-2164-15-74 ◽

2014 ◽

Vol 15 (1) ◽

pp. 74 ◽

Cited By ~ 115

Author(s):

Andreas Herbst ◽

Vindi Jurinovic ◽

Stefan Krebs ◽

Susanne E Thieme ◽

Helmut Blum ◽

...

Keyword(s):

Colorectal Carcinoma ◽

Cell Lines ◽

Target Genes ◽

Carcinoma Cell ◽

Comprehensive Analysis ◽

Carcinoma Cell Lines

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Interactions of the Mdm2/p53 and Proteasome Pathways: Implications for Combination Strategies to Enhance the Anti-Myeloma Activity of Bortezomib

Blood ◽

10.1182/blood.v112.11.2651.2651 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2651-2651

Author(s):

Melissa G. Ooi ◽

Patrick J. Hayden ◽

Douglas W. McMillin ◽

Joseph M. Negri ◽

Jake Delmore ◽

...

Keyword(s):

Cell Lines ◽

Target Genes ◽

Therapeutic Option ◽

Single Agent ◽

Binding Pocket ◽

Missense Mutations ◽

Apoptotic Pathway ◽

Drug Classes ◽

The Future

Abstract The “guardian of the genome” p53 is inactivated in most human cancers via missense mutations in its DNA binding core domain or via overexpression of the human homolog of Mdm2 (Hdm2), an E3 ubiquitin ligase that binds and ubiquitinates p53 thereby leading to its degradation via the ubiquitin/proteasome pathway. Direct inhibition of Mdm2 function could potentially stabilize p53 and activate the p53 apoptotic pathway, which could be a useful non-genotoxic approach for the treatment of cancer. Nutlin-3, a cis-imidazoline small molecule with affinity for the p53-binding pocket of Mdm2 and capable of disrupting the p53-Mdm2 interaction, can activate p53 and induce apoptosis in vitro in many malignancies, including multiple myeloma (MM) cells. We hypothesized that suppression of Mdm2-mediated p53 ubiquitination may synergize with accumulation of p53 triggered by bortezomib. To address this question, we evaluated the response of MM cells vs. select models of epithelial cancers to bortezomib and its combination with nutlin-3. We observed that breast (MDA-MB-231-luc); prostate (DU145); thyroid (SW579, FRO, WRO, TT) and colon (ARO/HT-29) cancer cell lines exhibited synergistic apoptotic response to the combination of sublethal concentrations of bortezomib plus nutlin-3. This synergistic killing effect was associated with synergistic increase in the expression of p53, p21, Mdm2, Bax, Noxa, PUMA and the cleavage of caspase-3, caspase-9 and PARP. In contrast, MM cell lines (MM-1S, MM-1R, NCI-H929, KMS-11) exhibited mostly an additive effect when treated with the combination of bortezomib plus nutlin-3. To further probe the biological relevance of these findings in the context of the bone microenvironment in MM, we compared the transcriptional profile of p53 target genes as well as the response to nutlin-3 in MM cells cultured alone vs. in the presence of bone marrow stromal cells (BMSCs). We observed that co-culture with BMSCs attenuates the response of MM cells to single-agent nutlin-3 and is associated, in p53 wild-type cells, with increased amplitude of the transcriptional signature of genes suppressed by activated p53, suggesting that BMSCs suppress p53 activity in MM cells. Overall, these results suggest a complex correlation between the Mdm2/p53 and proteasome pathways: many p53 mutant epithelial cancer models can become more bortezomib-responsive by Mdm2 inhibition, while MM cells, with higher baseline responsiveness to bortezomib and nutlin-3, typically do not exhibit significant increases in their response to the combination of these 2 drug classes compared to the single-agent treatments. These observations suggest that concurrent Mdm2 inhibition may extend the spectrum of bortezomib applications to tumor types with currently limited single-agent response to proteasome inhibition. In MM, p53 mutations have been historically considered to be present in only late stage disease (e.g. plasma cell leukemia/extramedullary MM), but their prevalence may increase in the future as more patients with advanced MM survive longer thanks to recently introduced drug classes (thalidomide, bortezomib, lenalidomide). In those MM patients who harbor p53 pathway lesions and have developed bortezomib-resistance/refractoriness, combinations of bortezomib with Mdm2 inhibitors may also represent in the future an intriguing potential therapeutic option that merits further preclinical and clinical evaluation.

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Plasma protein regulation by thyroid hormone

Journal of Endocrinology ◽

10.1677/joe.0.1790367 ◽

2003 ◽

Vol 179 (3) ◽

pp. 367-377 ◽

Cited By ~ 36

Author(s):

KH Lin ◽

HY Lee ◽

CH Shih ◽

CC Yen ◽

SL Chen ◽

...

Keyword(s):

Thyroid Hormone ◽

Plasma Protein ◽

Hormone Receptors ◽

Target Genes ◽

Transcriptional Level ◽

Carcinoma Cell Lines ◽

Iron Binding Protein ◽

Dose Dependent ◽

Made In

Thyroid hormones (THs) regulate growth, development, differentiation and metabolic processes by interacting and activating thyroid hormone receptors (TRs). Although much progress has been made in our understanding of the transcriptional regulation of many TR target genes, little is known of the regulation of plasma protein gene expression by TRs. To investigate the role of TRs in plasma protein expression we used human hepatocellular carcinoma cell lines and carried out cDNA microarray analysis. Our results indicate that several plasma proteins including transferrin, prothrombin, angiotensinogen, haptoglobin, alpha-2-HS-glycoprotein alpha and beta chain, complement, lipoproteins and fibrinogen are up-regulated by THs. Furthermore, clusterin, alpha-2-macroglobulin precursor, prothymosin alpha and alpha-fetoprotein were found to be down-regulated by THs.Transferrin, an iron-binding protein expressed in all mammals, and mainly synthesized in the liver, was investigated further. Immunoblot and Northern blot analyses revealed that exposure of HepG2-TRalpha1 sub-lines and HepG2-Neo cells to tri-iodothyronine (T(3)) induced time- and dose-dependent increases in the abundance of transferrin mRNA and protein, with the extent of these effects correlating with the level of expression of TRalpha1. Nuclear run-on experiments indicate that this induction is functioning at the transcriptional level. Moreover, cyclohexamide treatment did not eliminate the induction of transferrin by TH. Thus, our results suggest that the induction of transferrin by TH is direct and may in fact be mediated by an as yet unidentified response element in the promoter region.

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