Increased PDK1 expression is associated with altered expression of genes involved in energy metabolism and ROS protection in NSCLC tumors.
We investigated PDK1 expression levels and correlated the data to a panel of genes involved in energy metabolism and ROS protection in two independent cohort datasets from lung tumors and normal tissue samples. Transcriptomic data was obtained from the lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) from the cancer genome atlas (TCGA LUAD & LUSC, RNA sequencing, n = 1016; adjacent normal, n = 110), and the GSE18842 microarray dataset with paired samples from normal tissue (n = 44) and lung adenocarcinoma and squamous-cell carcinoma (n = 46). In the TCGA data, the mutation status of EGFR is available, whereas in the GSE18842 data, paired data from normal- and cancer tissue from the same patient is available . The mRNA expression of PDK1 was significantly elevated in cancer tissue compared to normal tissue in both cohorts. In the TCGA EGFR wild-type samples (EGFRwt, n = 591), the upregulation was higher, compared to the group with EGFR mutations (EGFRmut, n = 76) (Fig. 1A), and in the GSE18842 cohort representing paired samples, the upregulation was prominent (Fig. 1B). To evaluate effects on glucose catabolism in tumors relative to normal tissue, we investigated expression levels of 22 related genes, both regulators and enzymes. The majority of these genes showed significant upregulation in cancer tissue compared to normal tissue (Fig. 1C). In particular, the expression of glucose transporter GLUT1 was increased, consistent with increased glucose uptake during tumor development.
Further, multiple glycolytic enzymes were increased, including PKM2 (only available data from the TCGA cohort), G6PD, PFKP, ALDOA, GAPDH, ENO1 and LDHA. For most genes, the findings were consistent in the two cohorts. These effects were generally similar in EGFR wild type (wt) compared EGFR mutated (mut) tumors; however, some genes displayed difference, such as the lactate importer, MCT1, which had lower expression in EGFRmut compared to EGFRwt tumors. Correlation analysis between PDK1 and the panel of genes involved in glucose metabolism revealed significant positive associations for a majority of genes (Rho values > + 0.3) (Fig. 1D). This indicates that PDK1 is co-regulated with the glycolytic machinery at the transcriptional level. Also here, the EGFRwt and EGFRmut groups showed similar results.
The EGFRmut group tended to display lower Rho values, which may be biologically relevant, though it could also result from the smaller sample size for this group. Interestingly, the oncogene c-MYC, known to interact with HIF1a to enhance glycolysis in cancer cells , was correlated to the expression of PDK1 in our data. However, only a trend was observed in EGFRmut and the GSE18842 cohort (Supplementary figure S1A). Also, NRAS has been shown to be a regulator of HIF1a and glycolysis , and this oncogene is also correlated to the expression of PDK1 in our data (Supplementary figure S1B). We found the majority of genes involved in gluconeogenesis and fatty acid oxidation (FAO) to be downregulated in tumor samples compared to normal tissue samples in both cohorts (Fig. 1E and 1F). Figure 1E shows data for selected genes encoding key enzymes and regulators of gluconeogenesis . The level of fructose bisphosphatase 1 (FBP1), a key regulator of gluconeogenesis activity was particularly decreased in tumors relative to normal tissue, and interestingly, as seen in Fig. 1D, FBP1 was strongly inversely correlated to the expression of PDK1. The correlation was more prominent in the EGFRwt group compared to the EGFRmut group, which may indicate that the metabolic changes during tumor development are different in the two groups. Genes involved in FAO was generally downregulated in NSCLC compared to normal tissue (Fig. 1F). ACOX4, CYP4A11, PPARG and PDK4 levels indicated that oxidation FAO was downregulated upon NSCLC development. Interestingly, PDK1 was inversely correlated to PDK4 (Supplementary figure S1C), suggesting distinct roles of the two enzyme family members.
Genes reflecting mitochondrial biogenesis and biomass displayed different types of responses (Fig. 1G). Interestingly, PPARGC1α (PGC1α), which is a key regulator of mitochondrial biogenesis, was strongly downregulated in the tumor samples. This was particularly evident in the EGFRmut group. In contrast, genes that are commonly used as markers of mitochondrial biomass, including TFAM, VDAC, TOM20 and ATPB, were mildly upregulated. Genes involved in mitophagy and autophagy also showed heterogeneous responses. PINK1 and PARK2, which are known modulators of mitophagy were significantly downregulated in the TCGA cohort. BNIP3 was upregulated, indicating an elevated level of apoptosis in cancer tissues. BNIP3 was also correlated to the level of PDK1 in both cohorts, and even to a higher degree in the EGFRmut group (Supplementary figure S1A). Altogether, we found no indication of loss of mitochondrial biomass, but the effects on regulators of mitochondrial biogenesis and dynamics may suggest that processes of mitochondrial quality control are not operating normally.
Expression of several genes encoding antioxidant enzymes were upregulated in NSCLC compared to normal tissue (Fig. 1H). Three genes of the glutathione peroxidase (GPX) family were induced, particularly GPX2, and to a lesser extent for GPX7 and 8. GPX3 was significantly downregulated. The thioredoxins TXN and TXNRD1 were upregulated in the total TCGA cohort and in the EGFRwt group, however less in the EGFRmut group, suggesting differential regulation in cancer cells harboring EGFRwt compared to EGFRmut group. For the peroxidoxin family, 4 out of 6 genes were upregulated in the two cohorts. Further, the tumor mRNA expression of glutathione S-reductase (GSR) and superoxide dismutase 1 (SOD1) showed a trend of increase, whereas catalase (CAT) was significantly downregulated. The TCGA data showed mildly reduced expression of the mitochondrial SOD2 in the tumors. Interestingly, the mRNA level of multiple antioxidant genes correlated with PDK1 level (Fig. 1I). For this set of genes, PDK1 predominantly displayed significant or trending positive associations, such as for GPX2, GPX7 and TCNRD1. However, there were also negative associations, such as for GPX3 and TXBRD2. These data support the notion that regulation of PDK1 may be associated with contextual mechanisms of ROS defense.
DCA in combination with EGFR TKIs inhibit cell growth in NSCLC cell models.
Based on the finding that NSCLC tumors had upregulated PDK1, we investigated effects of the PDK inhibitor DCA on cells, either alone or in combination with EGFR TKI. We used HCC827 and HCC4006, two NSCLC cell lines which are sensitive to the first-generation EGFR TKI, erlotinib. We also use H1975 which is resistant to erlotinib, but sensitive to the third generation EGFR TKI rociletinib. Different dosage combinations of DCA and respective EGFR TKIs were tested. Periodic imaging microscopy during a treatment period of 14 days showed a time-dependent inhibition of proliferation and increased cell death in HCC827 cultures treated with both erlotinib and DCA, with apparently only dead cells remaining at the end of the treatment period (Fig. 2A). Assessment of proliferation was based on image analysis quantifying percent confluency (using the Incucyte software), and statistical analysis after 72 h showed a significant, but mild potentiated effect of the erlotinib + 20 mM DCA treatment compared to the agents alone (Fig. 2B). In cultures only treated with one of the agents, there were also reduced cell number, but the majority of these cells appeared intact and viable. A separate experiment was performed to test different dosage combinations of the agents measuring confluence and resazurin viability (Fig. 2C and Supplementary figure S2). The three different cell models displayed a similar pattern after 72 h treatment; there was an added antiproliferative effect of 20 or 25 mM DCA in combination with erlotinib/rociletinib. A concentration of 1 µM was required to give a significant effect of the EGFR TKIs, but for two of the cell types (HCC827 and H1975) the added effect of DCA was also evident at this concentration of the EGFR TKIs. These data demonstrate that DCA in NSCLC cell inhibits cell proliferation, and further suggest that the added effect in combination with EGFR TKIs may serve to counteract development of cellular EGFR TKI resistance.
Acute effects of DCA on NSCLC cell metabolism.
Treatment with DCA is expected to activate mitochondrial pyruvate oxidation, by releasing PDK-mediated inhibition of the PDH enzyme complex. To investigate how this acutely affects mitochondrial respiration and glycolysis in cultured NSCLC cells, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Addition of DCA, alone or in combination with EGFR TKI, gave a small acute increase in OCR in HCC827 cells, but not in the two other cell types (H1975 and HCC4006) (Fig. 3A and 3B, and Supplementary Fig. 3A-D). The minor effect of DCA on OCR may suggest that an internal switch in fueling pathways for oxidative metabolism occurs without major effect on the respiratory rate under these conditions. In contrast, ECAR acutely decreased upon DCA addition in all three cell lines. It was confirmed that the addition of the DCA solution (pH 7.4) did not change the pH of the medium itself, and therefore the most plausible explanation is that DCA caused decreased cellular lactate secretion (Fig. 3C and 3D). The associated decrease in ECAR supports a metabolic shift with enhanced propensity for mitochondrial oxidation at the expense of lactate production. Of note, these effects of DCA were generally unaffected by the presence of EGFR TKI. Next, to confirm if DCA treatment caused increased mitochondrial oxidation of pyruvate, as well as its precursors lactate and glucose, we incubated cells with 14C-labeled versions of these substrates, and measured production of 14CO2. Figure 3E positions in which pathway steps the detected 14CO2 is produced, depending on 14C-labeled substrates used in the central energy pathway. The data clearly demonstrated increased pyruvate oxidation after treatment with DCA in all three cell lines (Fig. 3F). DCA caused significantly increased rate of lactate oxidation in HCC4006 and H1975, whereas it was decreased in HCC827 (Fig. 3G). All cell lines exhibited significant or trending decrease in glucose oxidation upon DCA treatment (Fig. 3H). In support of decreased cellular lactate production, we found mRNA levels of LDHA to be decreased in the three cell lines after treatment with 20 mM DCA for 48 h. Further, the mRNA level of PGC1α was strongly upregulated in DCA treated cells, supporting metabolic adaptations involving mitochondria (Fig. 3I). These functional and regulatory data confirm that DCA promotes mitochondrial pyruvate oxidation in NSCLC cells, and thereby antagonizes several metabolic signatures of cancer cell proliferation and therapy resistance. Noteworthy, these changes in cell metabolism had minor effects on mitochondrial respiratory rate.
Development of resistance to EGFR TKI is associated with EMT and altered metabolic signature. To model the development of erlotinib resistance in NSCLC cells, we treated HCC827 and HCC4006 cells with 1 µM erlotinib for more than 4 weeks, and established sublines that survived (Supplementary figure S4A). The resistant sublines are referred to as HCC827/BERL and HCC4006/BERL. In addition, we used an established H1975 cell line, with a subline (H1975/COR1-1) resistant to the third generation EGFR TKI rociletinib (CO-1686), provided by Clovis oncology [85, 86]. The H1975 cell line is known to be resistant towards erlotinib as they have a T790M mutation in EGFR exon 20, but sensitive towards rociletinib. The resistance phenotype of the three cell models was confirmed by drug response experiments measuring confluence after 72 h treatment with their corresponding EGFR TKI (Fig. 4A-4C). We further aimed to study the clonogenic potential upon treating cells with rociletinib and erlotinib. As expected, clonogenic potential was absent in the HCC827 and HCC4006 EGFR TKI sensitive cell lines treated with either erlotinib or rociletinib, while H1975 cells were resistant against erlotinib but sensitive to rociletinib (Fig. 4D). All resistant cell lines were resistant to both rociletinib and erlotinib (Fig. 4D). By measuring mRNA (Fig. 4E-G) and using microscopy and immunocytochemistry, typical features of EMT were documented in EGFR TKI resistant cells, including mesenchymal morphology, low E-cadherin levels and high vimentin levels (Fig. 4H - J). The effects on E-cadherin and vimentin protein levels were supported by western blot analysis, and c-MET mRNA was reduced (Supplementary figure S4 B-C). This supports previous findings for the H1975 model [85, 86]. The mRNA expression of PDH subunits and the PDKs were evaluated in the resistant compared to the parental cells (Fig. 4K). EGFR TKI resistance was not associated with consistent effects on PDHA and PDHB mRNA levels. PDK1, PDK2 and PDK4 was upregulated in both HCC827/BERL and HCC4006/BERL compared to the parental cells. In H1975/COR1-1 cells compared to the parental, PDK2 was upregulated, whereas PDK3 and PDK4 were downregulated. Upregulation of PDK1 protein expression in EGFR TKI resistant cells was confirmed in the HCC827 and HCC4006 cell models, and this was consistent with increased phosphorylation of PDH E1-alpha subunit (Fig. 4M). When measuring mRNA of a selection of genes associated with glucose metabolism, we found strong upregulation of one or more MCT and GLUT mRNA in accordance with the patient samples in Fig. 1. MCT1 and/or MCT2 was upregulated in the resistant cell lines compared to their respective parental cells (Fig. 4L). Furthermore, MPC1 tended to be downregulated and MPC2 upregulated in erlotinib resistant cell lines compared to sensitive control. Interestingly, PGC1α (PPARC1) expression greatly increased upon drug resistance. The mRNA level of FBP1 was severely downregulated in the resistant cells compared to parental control. Furthermore, FBP1 protein could only be detected in the parental cells of the HCC4006 cell line, and not in the HCC827/PAR cell line (Fig. 4L). Expression of SOD2 protein and mRNA was increased in the erlotinib resistant cells (Fig. 4M and 4L). Minor effects were observed regarding mRNA and protein expression of LDHA upon drug resistance (Fig. 4M and 4L).
Mitochondrial respiration and glycolysis in EGFR TKI resistant NSCLC cells.
To determine if development of EGFR TKI resistance affects mitochondrial function in NSCLC cells, we measured respiratory rates following sequential additions of specific modulators. For two of the models (HCC827, H1975) the resistant cells had significantly lower rates of mitochondrial respiration compared to the respective parental cells (Fig. 5A-E, Supplementary figure S5). This effect was found both regarding basal respiratory activity, and uncoupled maximal respiratory capacity. Further, as the leak respiration upon oligomycin addition was normal or low, the integrity of the inner mitochondrial membrane is intact. In the HCC4006 model, there was an increase in basal respiration, whereas maximal respiratory capacity was reduced, in the resistant cells compared to the sensitive control cells. Characterization of the glycolytic function in EGFR TKI resistant cells was performed in a separate experiment (Fig. 5F-5L). Noteworthy, endogenous ECAR reflects glycolytic activity supported by cellular glucose stores, and other cellular processes influencing extracellular acidification, and these contributes only to a minor part compared to activity induced by addition of extracellular glucose (Supplementary Figure S5F) . Resistant cells of the HCC827 and HCC4006 models had lower basal ECAR after adding glucose, and maximal ECAR in presence oligomycin, compared to the parental cells (Fig. 5F-I). HCC827/BERL had similar ECAR levels compared to the parental cells. The glucose induced ECAR varied between the three cell lines; however, the spare glycolytic capacity was slightly reduced in resistant cell lines. To sum-up, the resistant cells of the HCC827 and H1975 models, exhibited low rates of mitochondrial respiration, while glycolysis was similar to the parental cells. In contrast, the resistant HCC4006/BERL cells had relatively normal rates of mitochondrial respiration, but low glycolytic rate, compared to the parental cells. To further investigate the metabolic changes upon drug resistance, we measured oxidation rates of pyruvate, lactate and glucose (Fig. 5J-L). The HCC827/BERL cells had significantly lower oxidation of all the three substrates, compared to the parental cells. H1975/COR1-1 only showed reduced glucose oxidation, and HCC4006/BERL had increased pyruvate and lactate oxidation. In summary, it is interesting to see that there was generally an inverse relationship between effects of resistance on basal respiration and glycolysis ECA rates for the three models. The rate of oxygen consumption is further reflected in the substrate oxidation measurements, where HCC827/BERL is oxidizing less pyruvate, lactate and glucose, whereas the HCC4006/BERL which has an increased oxygen consumption rate upon development is oxidizing more of the three substrates compared to parental cells. The changes in substrate oxidation for the H1975/COR1-1 cells are however more modest compared to parental cells, also reflecting the general rate of oxidition, confirming the consistency of metabolic alterations for the three NSCLC cell models.
DCA inhibits cell proliferation in NSCLC cells resistant to EGFR TKI therapy.
Based on our previous findings, we investigated how DCA treatment influences metabolism in the resistant clones. In combination with 1 µM EGFR TKI, DCA demonstrated a dose-dependent effect in the EGFR TKI resistant cells (Fig. 6A). An experiment to evaluate colony formation capacity of the EGFR TKI resistant cells showed that upon treating cells with 10 mM DCA, the clonogenic potential was decreased. When combining 10 mM DCA with 1 µM EGFR TKI, the clonogenic potential was even lower in HCC4006/BERL, and at both 20 mM DCA, and 1 µM EGFR TKI combined with 20 mM, DCA the clonogenic potential was totally absent for all subtypes (Fig. 6B). Upon investigating the metabolic effects of DCA (Fig. 6C-D and Supplementary figure S6), the HCC827/BERL cells showed a significant increase in OCR acutely after addition of DCA. H1975/COR1-1 displayed a minor increase in OCR after DCA addition, whereas no effect was seen in HCC4006/BERL cells. However, exposure to DCA reduced ECAR in all the three resistant cell lines (Fig. 6D and Supplementary figure S6).
Furthermore, the three resistant cell lines showed increased pyruvate and lactate oxidation after treatment with 20 mM DCA for 24 h, accompanied by a trending or significant decrease in glucose oxidation (Fig. 6E-G). These data suggest that even though the three cell lines show different metabolic phenotypes, all increase pyruvate and lactate oxidation upon DCA treatment. DCA also reduced their glycolytic rate, and reduced cell viability upon treatment.
Radiation increases the effect of DCA and EGFR TKI in NSCLC cells.
Our findings so far suggest that DCA causes a metabolic shift in NSCLC cells, and that this may increase the sensitivity to EGFR TKI treatment. One possible theory is that enforced activation of the pyruvate oxidation axis results in metabolic stress, including increased ROS production. Thus, we investigated if the effects of DCA and erlotinib could be enhanced in combination with ionizing radiation. NSCLC cells were treated with ionizing radiation, in clinically relevant doses, minutes before drugs were added, and the proliferation monitored by periodic microscopy. Furthermore, 8 Gy radiation added to the effect of DCA and erlotinib, both in parental cells and resistant cells (Fig. 7A-B). The proliferation curves showed that the inhibited proliferation rate was more pronounced in cells sensitive to EGFR TKI (Fig. 7C). At 72 h post treatment (Fig. 7D), the cell growth was reduced significantly in the combined DCA and erlotinib treated cells compared to control. However not statistically significant, there was a trend suggesting that ionizing radiation added to the therapeutic effect of DCA and EGFR.