April 29, 2021
Supplementary Components1. distinguishing FBP1 from previously-identified tumour suppressors ([[[(((expression correlates significantly with advanced tumour stage and worse patient prognosis (Fig. 1cCd), whereas expression does not (Extended Data Fig. 3dCe), suggesting that FBP1 may harbour novel, nonenzymatic function(s). Open in a separate window Figure 1 Integrative analyses reveal that FBP1 is ubiquitously inhibited and exhibits tumour-suppressive functions in ccRCCa, Metabolic gene set analysis of RNAseq data provided by the TCGA ccRCC project (http://cancergenome.nih.gov). 480 ccRCC tumour and 69 adjacent normal tissues were included. 2,752 genes encoding all known human metabolic enzymes and transporters were classified according to KEGG (http://www.genome.jp/kegg/). Generated metabolic gene sets were ranked based on their median fold expression changes in ccRCC tumour vs. normal tissue, and plotted as median median absolute deviation. b, Immunohistochemistry staining of a representative kidney tissue microarray with FBP1 antibody. T: ccRCC tumours; N: adjacent normal kidney. c, Normalized RNASeq reads of in 69 normal kidneys and 480 ccRCC tumours grouped into Stage ICIV by TCGA. d, Kaplan-Meier survival curve of 429 ccRCC patients enrolled in the TCGA database. Patients were equally divided into two groups (top and bottom 50% expression) based on expression levels in their tumours. e, Growth of 786-O ccRCC cells in low serum medium (1% FBS), with or without ectopic FBP1 expression. f, Xenograft tumour growth of 786-O cells with or without ectopic FBP1 expression. End-point tumour weights were measured and plotted. g, Growth of human HK-2 proximal renal tubule cells with or without FBP1 inhibition in 1% serum medium. Values represent means.d. (four Mouse monoclonal to 4E-BP1 technical replicates, from two independent experiments). *p 0.01. To investigate functional roles for FBP1 in ccRCC progression, we ectopically expressed FBP1 in 786-O ccRCC tumour cells to levels observed in HK-2 proximal tubule cells (Extended Data Fig. 4a). FBP1 expression significantly inhibited 2D culture (Fig. 1e), anchorage-independent (Extended Data Fig. 4b), and xenograft tumour growth (Fig. 1f and Extended Data Fig. 4c). Similarly, enforced FBP1 expression inhibited growth of RCC10 and 769-P ccRCC cells (Extended Data Fig. 4dCe), AZM475271 and A549 lung cancer cells preferentially under hypoxia (Extended Data Fig. 4f and 4g). These total outcomes confirmed that FBP1 can suppress ccRCC AZM475271 and various other tumour cell development, an impact pronounced when in conjunction with HIF activation significantly. In HK-2 cells, FBP1 depletion, however, not G6Computer ablation or ectopic PFKL appearance, was sufficient to market HK-2 cell development (Fig. expanded and 1g Data Fig. 4hCj). Since FBP1 may be the rate-limiting enzyme in gluconeogenesis10, we manipulated FBP1 appearance in renal cells and assessed glucose metabolism. FBP1 inhibition elevated blood sugar lactate and uptake secretion in HK-2 cells cultured in 10 mM blood sugar, (Fig. 2a), an impact augmented by reducing glucose levels to at least one 1 mM (Prolonged Data Fig. 5aCb). To assess glycolytic flux, we performed isotopomer distribution evaluation using [1, 2-13C] blood sugar as the tracer, which creates glycolytic and TCA intermediates formulated with two 13C atoms (M2 species), as well as corresponding M1 species from the pentose phosphate pathway (PPP; Extended Data Fig. 5c). We observed elevated M2 enrichment of four AZM475271 TCA intermediates (malate, aspartate, glutamate, and citrate) in FBP1-depleted HK-2 cells (Fig. 2bCc). In contrast, G6PC inhibition failed to promote glucose-lactate turnover (data not shown), suggesting that FBP1, but not G6PC, is a critical regulator of glucose metabolism in renal cells. Consistent with this result, ectopic FBP1 expression in a expression and HIF activity was recapitulated in primary ccRCC tumours (Fig. 3b). In contrast, expression did not correlate with HIF activity in ccRCC cells or tumour tissues (Extended Data Fig. 6gCh). Interestingly, FBP1 also inhibited HIF activity in A549 lung cancer cells cultured at 0.5% O2 (Fig. 3c), demonstrating that this effect is not specific to renal cells. Moreover, FBP1 expression reduced canonical HIF target ([[promoters, but not in the non-hypoxia responsive (expression) based on expression levels, and their relative HIF activities were quantified and plotted as described in Methods. c, HIF reporter activity in hypoxic RCC4 and A549 cells (0.5% O2) with or without ectopic FBP1 expression. d, qRT-PCR analysis of HIF target genes in RCC10 cells expressing vector or FBP1. e, ChIP assays evaluating the chromatin binding of FBP1 to HREs in the promoter, or to a non-hypoxia responsive region of the locus. RNA Polymerase II antibody was used as a positive control. f, Immunofluorescent staining of.