Supplementary Materials and Methods, Figures 1-8, Tables 1-3, Movie Legends 1-6 from Cancer Stem Cell Tumor Model Reveals Invasive Morphology and Increased Phenotypical Heterogeneity
<p>Supplementary tables S1-6. Supplementary table S1 Primers used for RT-qPCR analyses. Supplementary table S2 Clinical variables associated with differences in gene expression among 119 resected liver metastases. Supplementary table S3 613 genes whose expression was significantly upregulated and 481 genes whose expression was significantly (p<0.05) down-regulated in chemotherapy-treated tumors compared to chemo-naive tumors. Supplementary table S4 KEGG pathway finder analysis and Ingenuity Pathway Analysis of upregulated genes in chemo-treated compared to chemo-naive tumors. Supplementary table S5 P-values of expression levels of genes involved in mitochondrial dysfunction, ubiquinone synthesis and mitochondrial biogenesis upregulated in chemo-treated compared to chemo-naive tumors. Supplementary table S6 (additional information regarding the calculation listed below) ATP synthesis rates through glycolysis and OXPHOS.</p>
Abstract Background: Altered energy metabolism is one of the hallmarks of cancer. Tumor cells reprogram their energy metabolism to meet the demands of uncontrolled cell division. During tumorigenesis the vast majority of cancer cells become highly glycolytic (Warburg effect) accompanied by a decrease in oxidative metabolism. Chemotherapy is likely to affect the energy metabolism of tumor cells, but how specific drugs affect specific metabolic pathways is only beginning to be addressed. Methods: the effects of cytotoxic agents on energy metabolism were assessed by flow cytometric uptake of Mitotracker®, the ratio of mitochondrial and nuclear DNA on qRT-PCR, western blotting for protein levels of the different complexes of the respiratory chain and oxygen consumption rate by the Seahorse Extracellular Flux Analyzer. Cell death was analyzed by flow cytometric uptake of Propidium Iodide, Nicoletti assay and protein levels of caspases 3 and 8. Results: Gene expression analysis was performed on 119 resected liver metastases of colorectal tumors. Of all clinical variables tested, neoadjuvant chemotherapy was most prominently associated with changes in gene expression. Gene ontology and pathway analysis tools revealed that many of the chemotherapy-associated genes were involved in the regulation of oxidative phosphorylation (OxPhos). To test whether chemotherapy affects OxPhos patient derived colorectal spheroids were treated with the standard cytotoxic agents oxaliplatin and 5-fluorouracil. Chemotherapy strongly increases mitochondrial load, oxygen consumption rate and mitochondrial ATP synthesis. In line with these results chemo-treated tumor cells displayed a higher ratio of mitochondrial-to-nuclear DNA and expression of respiratory complex components was strongly increased following chemotherapy. Chemotherapy strongly induced expression of the histone deacetylase SIRT1, which has been implicated in mitochondrial biogenesis. Inhibition (by either nicotinamide, EX-527, Tenovin-6) or knockdown of SIRT1 prevented the chemotherapy-induced increase in oxidative phosphorylation. Moreover, SIRT1 knockdown greatly reduced tumor cell survival and clonogenic capacity following removal of chemotherapy. Conclusion: Chemotherapy induces an increase in oxidative phosphorylation via SIRT1 and this is required for tumor cell survival following drug removal. Since DNA repair requires ATP, mitochondrial biogenesis may be part of the tumor cell response to DNA-damaging agents. Post-chemotherapy targeting of SIRT1 (or OxPhos enzymes) may be an interesting novel approach to increase chemotherapy efficacy. Citation Format: Thomas T. Vellinga, Vincent C. de Boer, Tijana Borovski, Kari Trumpi, Szabolcs Fatrai, Onno Kranenburg, Inne H.M. Borel Rinkes, Jeroen Hagendoorn. Survival of colorectal cancer cells following chemotherapy relies on a SIRT1-dependent increase in oxidative phosphorylation. [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 3351. doi:10.1158/1538-7445.AM2014-3351
<div>Abstract<p>The recently developed concept of cancer stem cells (CSC) sheds new light on various aspects of tumor growth and progression. Here, we present a mathematical model of malignancies to investigate how a hierarchical organized cancer cell population affects the fundamental properties of solid malignancies. We establish that tumors modeled in a CSC context more faithfully resemble human malignancies and show invasive behavior, whereas tumors without a CSC hierarchy do not. These findings are corroborated by <i>in vitro</i> studies. In addition, we provide evidence that the CSC model is accompanied by highly altered evolutionary dynamics compared with the ones predicted to exist in a stochastic, nonhierarchical tumor model. Our main findings indicate that the CSC model allows for significantly higher tumor heterogeneity, which may affect therapy resistance. Moreover, we show that therapy which fails to target the CSC population is not only unsuccessful in curing the patient, but also promotes malignant features in the recurring tumor. These include rapid expansion, increased invasion, and enhanced heterogeneity. Cancer Res; 70(1); 46–56</p></div>
