RESUMO
Cell division, growth, and differentiation are energetically costly and dependent processes. In adult stem cell-based epithelia, cellular identity seems to be coupled with a cell's metabolic profile and vice versa. It is thus tempting to speculate that resident stem cells have a distinct metabolism, different from more committed progenitors and differentiated cells. Although investigated for many stem cell types in vitro, in vivo data of niche-residing stem cell metabolism is scarce. In adult epithelial tissues, stem cells, progenitor cells, and their progeny have very distinct functions and characteristics. In our study, we hypothesized and tested whether stem and progenitor cell types might have a distinctive metabolic profile in the intestinal lineage. Here, taking advantage of the genetically accessible adult Drosophila melanogaster intestine and the availability of ex vivo single cell sequencing data, we tested that hypothesis and investigated the metabolism of the intestinal lineage from stem cell (ISC) to differentiated epithelial cell in their native context under homeostatic conditions. Our initial in silico analysis of single cell RNAseq data and functional experiments identify the microRNA miR-277 as a posttranscriptional regulator of fatty acid ß-oxidation (FAO) in the intestinal lineage. Low levels of miR-277 are detected in ISC and progressively rising miR-277 levels are found in progenitors during their growth and differentiation. Supporting this, miR-277-regulated fatty acid ß-oxidation enzymes progressively declined from ISC towards more differentiated cells in our pseudotime single-cell RNAseq analysis and in functional assays on RNA and protein level. In addition, in silico clustering of single-cell RNAseq data based on metabolic genes validates that stem cells and progenitors belong to two independent clusters with well-defined metabolic characteristics. Furthermore, studying FAO genes in silico indicates that two populations of ISC exist that can be categorized in mitotically active and quiescent ISC, of which the latter relies on FAO genes. In line with an FAO dependency of ISC, forced expression of miR-277 phenocopies RNAi knockdown of FAO genes by reducing ISC size and subsequently resulting in stem cell death. We also investigated miR-277 effects on ISC in a benign and our newly developed CRISPR-Cas9-based colorectal cancer model and found effects on ISC survival, which as a consequence affects tumor growth, further underlining the importance of FAO in a pathological context. Taken together, our study provides new insights into the basal metabolic requirements of intestinal stem cell on ß-oxidation of fatty acids evolutionarily implemented by a sole microRNA. Gaining knowledge about the metabolic differences and dependencies affecting the survival of two central and cancer-relevant cell populations in the fly and human intestine might reveal starting points for targeted combinatorial therapy in the hope for better treatment of colorectal cancer in the future.
RESUMO
Although B-Raf(V600E) is the most common somatic mutation in papillary thyroid carcinoma (PTC), how it induces tumor aggressiveness is not fully understood. Using gene set enrichment analysis and in vitro and in vivo functional studies, we identified and validated a B-Raf(V600E) gene set signature associated with tumor progression in PTCs. An independent cohort of B-Raf(V600E)-positive PTCs showed significantly higher expression levels of many extracellular matrix genes compared with controls. We performed extensive in vitro and in vivo validations on thrombospondin-1 (TSP-1), because it has been previously shown to be important in the regulation of tumor angiogenesis and metastasis and is present in abundance in tumor stroma. Knockdown of B-Raf(V600E) resulted in TSP-1 down-regulation and a reduction of adhesion and migration/invasion of human thyroid cancer cells. Knockdown of TSP-1 resulted in a similar phenotype. B-Raf(V600E) cells in which either B-Raf(V600E) or TSP-1 were knocked down were implanted orthotopically into the thyroids of immunocompromised mice, resulting in significant reduction in tumor size and fewer pulmonary metastases from the primary carcinoma as compared with the control cells. Treatment of orthotopic thyroid tumors, initiated 1 week after tumor cell implantation with PLX4720, an orally available selective inhibitor of B-Raf(V600E), caused a significant tumor growth delay and decreased distant metastases, without evidence of toxicity. In conclusion, B-Raf(V600E) plays an important role in PTC progression through genes (i.e., TSP-1) important in tumor invasion and metastasis. Testing of a patient's thyroid cancer for B-Raf(V600E) will yield important information about potential tumor aggressiveness and also allow for future use of targeted therapies with selective B-Raf(V600E) inhibitors, such as PLX4720.
