MicroRNA regulation of hypoxia-induced tumorigenicity and metastatic potential of colon tumor-initiating cells

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The initiaton and progression of colorectal cancer (CRC), which is the second most common cause of cancer mortality in Western countries, are driven by a subpopulation of highly tumorigenic cells, known as cancer stem cells or tumor-initiating cells (TICs). These self-renewing TICs are, to a large extent, responsible for therapy resistance, cancer recurrence, and metastasis formation. TICs are known to extensively interact with their microenvironment and can be influenced by various extrinsic factors, such as inflammatory signaling or tumor hypoxia. Previous expression profiling studies have shown that microRNAs (miRNAs) are involved in the regulation of CRC inititation and metastatic progression. Moreover, specifc miRNAs have been identified as potential mediators of the cellular response to hypoxia. On the other hand, the molecular mechanisms that link hypoxia, miRNA expression, colon TIC regulation, and CRC progression, remain poorly understood. Thus, the main objectives of this work were to analyze the effects of hypoxia on the miRNA expression of colon TICs and to identify miRNAs that regulate metastasis initiation. In a first phase, we generated and thoroughly characterized different stable TIC-enriched spheroid cultures (SCs), both from CRC cell lines and from primary patient material. Each established SC was thereby shown to display key TIC properties, including substantial plasticity, in vitro and in vivo self-renewal capacity and, most importantly, extensive tumorigenic potential. Moreover, the individual SCs displayed increased chemoresistance capacity, compared to adherent counterpart cultures. Taken together, we could demonstrate that the spheroid system is a suitable model to study colon TICs, thereby laying the methodological foundation for the following subparts of this project. In a second step, we studied the influence of hypoxia on the miRNA expression profile of our established SCs. MiR-210-3p was thereby identified as the miRNA with the strongest response to hypoxia. Importantly, both hypoxic culture conditions and stable overexpression of miR-210 were shown to promote in vitro and in vivo self-renewal capacity of our colon TIC-enriched cultures. Moreover, by promoting lactate production and by repressing mitochondrial respiration, miR-210 was found to trigger the metabolic reprogramming of colon TICs towards a glycolytic and aggressive phenotype. Finally, we studied the role of miRNAs in the context of TIC-driven metastasis formation. By comparing primary tumor- and lymph node metastasis-derived SCs, we were able to identify the miR-371~373 cluster as an important regulator of tumorigenic and metastatic potential. Stable overexpression of the entire miR-371~373 cluster, followed by gene and protein expression analysis, enabled us to uncover the transforming growth factor beta receptor II (TGF-βRII) and the inhibitor of DNA binding 1 (Id1) as miR-371~373 cluster-responsive proteins. Most importantly, different sphere, tumor, and metastasis formation assays revealed that the miR-371~373/TGF-βRII/Id1 signaling axis regulates the self-renewal capacity and metastatic colonization potential of colon TICs. Taken together, our findings emphasize the strong plasticity of colon TICs and clearly illustrate that miRNAs can act as potent modulators of essential TIC properties. Accordingly, we could show that miR-210 and the miR-371~373 cluster are involved in metabolic reprogramming of TICs and in the regulation of metastasis formation, respectively. Altogether, our study contributes to a better understanding of the molecular mechanisms that drive TIC-induced tumor progression and may provide indications for interesting miRNA biomarker candidates and target molecules for future TIC-specific therapies.