Oncogenic role of PDK4 in human colon cancer cells

Abstract

Background: Cancer cells maintain high rates of glycolysis. Pyruvate dehydrogenase kinases (PDK) contribute to this phenomenon, which favours apoptosis resistance and cellular transformation. We previously reported upregulation of PDK4 in normal mucosa of colorectal cancer (CRC) patients compared with controls and in preneoplastic intestine of our mouse model. Decreased methylation of four consecutive PDK4 CpGs was observed in normal mucosa of patients. Although other members of the PDK family have been investigated for transformation potential, PDK4 has not been extensively studied.

Methods: PDK4 methylation in blood of CRC patients and controls was evaluated by pyrosequencing. PDK4 expression in human colon carcinoma cells was down-regulated by RNAi. Cellular migration and invasion, apoptosis and qRT-PCR of key genes were assessed.

Results: Pyrosequencing revealed decreased methylation of the same four consecutive CpGs in the blood of patients compared with controls. Cellular migration and invasion were reduced and apoptosis was increased following transient or stable inhibition of PDK4. Expression of vimentin, HIF-1 and VEGFA was reduced.

Conclusions: These studies demonstrate the involvement of PDK4 in transformation. Methylation assessment of PDK4 in the blood may be useful for non-invasive CRC detection. PDK4 should be considered as a target for development of anticancer strategies and therapies.

Figure 1

DNA methylation of PDK4 in peripheral blood.(A) A cohort of 80 individuals was analysed for four CpGs individually and for average methylation of all four CpGs. Controls (40 individuals), white bars; CRC patients (40 subjects), black bars. Values are means±s.e.m. (B) Data for a second cohort (29 controls and 18 patients) are presented as in A. *P<0.05, **P<0.01 and ***P<0.005; independent t-tests.

Figure 2

PDK4 siRNA knockdown decreases migration, invasion and resistance to apoptosis in LoVo cells.At least three similar experiments were performed and representative results are shown. Data are expressed as means±s.e.m. (A) GAPDH or PDK4 expression in mock-transfected cells or cells transfected with scrambled siRNA or specific siRNA against GAPDH (positive control) or PDK4. Expression is presented in arbitrary units with TUBULIN for normalisation. Bars represent means of triplicates. (B) Effect of scrambled siRNA- or PDK4 siRNA transfection on migration and invasion. Bars represent the mean of stained cells in 15 fields. Average number of cells migrated/invaded was significantly lower with PDK4 siRNA than scrambled siRNA. *P<0.001 and **P<0.0005 (compared with mock- or scrambled siRNA-treated cells, independent t-test). (C) Vimentin was evaluated by Western blotting. DCA (positive control) was used at 50 mM. (D) Cleavage of PARP, assessed by western blotting. PDK4 siRNA transfection resulted in PARP cleavage. DCA (50 mM) or etoposide (20 μM) were used as positive controls. siRNA, small interfering RNA.

Figure 3

PDK4 siRNA knockdown decreases migration, invasion and resistance to apoptosis in DLD1 cells.Representative results from two experiments are shown. Data are expressed as means±s.e.m. (A) GAPDH or PDK4 expression after mock- or scrambled siRNA (white bars) or after specific siRNAs (black bars) against GAPDH (positive control) or PDK4. Expression is presented in arbitrary units with TUBULIN for normalisation. Bars represent mean of triplicates. (B) Effect of scrambled siRNA- or PDK4 siRNA transfection on migration/invasion. Bars represent mean of stained cells in 15 fields. Number of cells migrated/invaded was significantly lower in PDK4 siRNA transfectants than in scrambled siRNA. *P<0.01 and **P<0.001 (different from mock- or scrambled siRNA-treated cells, independent t-test). (C) Vimentin was evaluated by western blotting. (D) Cleavage of PARP, assessed by western blotting. PDK4 siRNAs transfection resulted in PARP cleavage. DCA (50 mM) or etoposide (20 μM) were used as positive controls. siRNA, small interfering RNA.

Figure 4

PDK4 shRNA knockdown in LoVo cells decreases migration, invasion and expression of HIF1A and VEGFA genes.(A) PDK4 knockdown by shRNA was effective in the two transductants, assessed by qRT-PCR with TUBULIN for normalisation. (B) Average number of cells migrated or invaded was significantly lower for cells expressing PDK4 shRNA. Three experiments were performed and representative results are shown. (C) Significantly decreased expression of HIF1A was observed compared with scrambled shRNA. (D) Similar decreases were observed for VEGFA. Results are expressed as means±s.e.m. Bars represent mean of triplicates for expression assays and mean of stained cells in 15 fields for migration/invasion assessments. Asterisks denote significant differences compared with scrambled shRNA. *P<0.01, **P<5 × 10⁻⁴, ***P<1 × 10⁻⁴ and ****P<5 × 10⁻⁵ (independent t-tests).