PMC

(a) 2-deoxyglucose uptake is diminished on transfection of DERL3 in HCT-116 cells. Data shown represent the mean±s.d. P-value obtained by Student’s t-test (**P<0.01). (b) Glucose-dependent growth is observed in empty vector-transfected HCT-116 cells, but not in DERL3-transfected cells in low- and high-glucose content media. P-values are those associated with Student’s t-test (*P<0.05). (c) Lactate production is depleted on transfection of DERL3 in HCT-116 cells in low- and high-glucose content media. Data shown represent the mean±s.d. P-values are those associated with Student’s t-test (**P<0.01). (d) Oxygen consumption rate is higher in HCT-116 DERL3-transfected cells (**P<0.01) and is also associated with a greater production of ATP (**P<0.01) (e) and ROS at the mitochondrial (f) and total (g) levels (*P<0.05 in both cases). Data shown represent mean±s.e.m. of biological duplicates. All P-values were according to Student’s t-test.

(a) Methylation-specific PCR analyses for DERL3 in primary human colorectal tumours. The presence of a band under the U or M lanes indicates unmethylated or methylated sequences, respectively. Normal colon (NC) and in vitro methylated DNA (IVD) are shown as positive controls for the unmethylated and methylated sequences, respectively. (b) Illustrative immunohistochemical examples demonstrate that SLC2A1 overexpression in human primary colorectal tumours is associated with DERL3 promoter CpG island hypermethylation. (c) Methylation-specific PCR analyses for DERL3 in benign colorectal adenomas. (d) The presence of DERL3 hypermethylation in primary colorectal tumours is significantly associated with shorter relapse-free survival in two cohorts of patients (discovery and validation groups). (e) Percentage of hypermethylated DERL3 samples in cancer cell lines, primary tumours from our study and data available from The Cancer Genome Atlas (TCGA) consortium.

(a) MTT assays for cell viability of DERL3 and empty vector-transfected HCT-116 on the use of four drugs that target cancer metabolism. Empty vector HCT-116 cells (DERL3-deficient) were significantly more sensitive to the PKM2 inhibitor shikonin. Data shown represent mean±s.d. (b) Shikonin significantly reduces tumour growth in the empty vector HCT-116-derived tumours generated in nude mice, but does not affect the growth rate of tumours derived from HCT-116 DERL3-transfected cells. Ten mice were used for each condition. Data shown represent mean±s.d. (c) DERL3 promoter CpG island hypermethylation is significantly associated with enhanced sensitivity to shikonin in the Sanger panel of colorectal cancer cell lines. (d) The presence of DERL3 promoter CpG island hypermethylation is significantly associated with enhanced sensitivity to the AKT inhibitor VIII in the Sanger set of colorectal cancer cell lines. The box plots display the distribution of IC₅₀ values with the central solid line representing the median and the limits of the box, the upper and lower quartiles. The whiskers represent the minimum and maximum values excluding outliers (<1.5 × the interquartile range). (e) Empty vector HCT-116 cells were significantly more sensitive to the antiproliferative effect of the AKT inhibitor VIII than were DERL3-transfected cells.

(a) Mass spectrometry spectra obtained by the SILAC approach in empty vector (red asterisk)- and DERL3 (blue asterisk)-transfected cells: upper column, recovery of DERL3 protein expression in HCT-116-transfected cells confirmed by multiple SILAC light signals originated by two tryptic peptides; middle column, strong downregulation of signals from two SLC2A1 peptides (VTILELFR and TFDEIASGFR with log2 fold changes of 4.22 and 3.56, respectively) in DERL3-transfected cells; lower column, signals from peptides of a non-DERL3 differentially regulated protein (ACTB) show light:heavy signal patterns close to the expected 1:1 proportion. (b) Western blot, (c) cell cytometry and (d) immunofluorescence show downregulation of the SLC2A1 protein in DERL3 stably transfected HCT-116 cells in comparison with empty vector-transfected cells. (e) Semiquantitative and quantitative reverse transcription-PCR shows that DERL3-dependent downregulation of SLC2A1 occurred at the protein level and was not associated with a difference in SLC2A1 mRNA levels. Data shown represent mean±s.e.m. of biological triplicates. (f) Treatment of DERL3-FLAG and pIRES2-eGFP HCT-116-transfected cells with cycloheximide in a time-course experiment followed by western blotting shows that SLC2A1 protein degradation occurs only in the presence of DERL3.

(a) Bisulphite genomic sequencing of DERL3 promoter CpG Island. CpG dinucleotides are represented as short vertical lines and the transcriptional start site (TSS) is represented as a long black arrow. The locations of the bisulphite genomic sequencing primers are indicated by black arrows. At least eight single clones are shown for each sample. Presence of a methylated or unmethylated cytosine is indicated by a black or a white square, respectively. (b) Methylation-specific PCR (MSP) analyses. The locations of the MSP primers are indicated by white arrows. The presence of a band under the U or M lanes indicates unmethylated or methylated sequences, respectively. Normal lymphocyte/colon (NC and NL) and in vitro methylated DNA (IVD) are shown as positive controls for the unmethylated and methylated sequences, respectively. Expression levels of the DERL3 transcript were determined by semiquantitative PCR (c) and real-time reverse transcription-PCR (d). (e) The expression of DERL3 RNA transcript was restored in the HCT-116, HCT-15, COLO-205 and SW48 cells by treatment with the demethylating drug 5-aza-2-deoxycytidine (AZA). Genetic disruption of the two major DNA methyltransferases DNMT1 and DNMT3B (DKO cells) also restored DERL3 expression in HCT-116 cells. Data shown represent mean±s.e.m. of biological triplicates. (f) Protein levels of DERL3 were recovered after AZA treatment in HCT-116 cells and in DKO cells.

(a) Cell proliferation differences between DERL3-expressing clones and the corresponding controls determined by the MTT assay and monitored for 7 days; P-values for exact binomial test (**P<0.01). (b) Colony formation assay. The number of colonies formed 2 weeks after seeding 1,000 cells on 35 mm plates and maintained in selection media were quantified and plotted; P-values for Student's t-tests (***P<0.005). HCT-116 cells transfected with DERL3 or empty vector were injected into the flanks of severe combined immunodeficiency (SCID) mice. Tumour volume was measured for 30 days (c) and tumour weight determined at the end of that period (d). P-values are those associated with Mann–Whitney U-tests (**P<0.01). (e) Orthotopic growth study implanting equal tumour pieces from the subcutaneous model in the colon tract. Tumour weight was measured at 30 days. P-values are those associated with Mann–Whitney U-tests (***P<0.005). (f) Illustrative examples of differential peritoneal colonization and hematoxylin and eosin stain (H&E) staining of colonized liver and lungs are shown. (g) Illustrative surgery samples and H&E staining of colonized liver and lung after spleen injection are shown.