FellowPhD Tereza Goliaš
Project NameTargeting carbonic anhydrase IX and pyruvate dehydrogenase kinase 1 in hypoxic colorectal tumors
Host organisationBiomedical Research Center
Duration of the project01.06.2015 - 31.05.2018

Abstract
Glycolytic tumor cell metabolism fills the requirements for growth in the hypoxic environmental milieu. The ability to respond to changing environmental oxygen tension through the induction of carbonic anhydrase IX (CAIX) and pyruvate dehydrogenase kinase 1 (PDHK1), implicated in pH regulation and glucose metabolism, respectively, appears to be necessary for model tumor growth, and their high level of expression in human tumors predicts for poor patient outcome. These two hypoxia-regulated proteins therefore present promising new targets for the design of novel, non-standard chemotherapies that would negatively influence tumor cell proliferation and survival, and at the same time avoid normal tissue toxicity or increase efficacy of standard treatment regimens. This project examines molecular mechanisms governing regulation of these proteins in the hypoxic tumor microenvironment and attempts to target them in the background of colorectal cancer, and at the same time stratify patients who would benefit from such tumor microenvironment-directed therapy and improve their outcome.

Project Summary with Interim Results

Objective 1 of the fellowship is based on elucidating regulation of pyruvate dehydrogenase kinase 1 (PDHK1) activity in hypoxia, specifically the role of protein kinase A (PKA).  Objective 2 analyzes the connection between PDHK1 and carbonic anhydrase IX (CAIX) expression, and finally Objective 3 attempts to target PDHK1 and CAIX as combined modality treatment of hypoxic tumors.

Description of the work performed

Work performed in regards to Objective 1 included 2D-Western blots of normoxic and hypoxic cell lysates, where we wanted to see a differential shift in signal that would suggest covalent modifications of PDHK1.  We also immunoprecipitated overexpressed tagged PDHK1 from normoxic and hypoxic cells and sent to the Proteomics facility to be analyzed by mass spectrometry.  We further tested the effect of inhibitors and activators of PKA on PDHK1 activity.

For Objectives 2 and 3, we generated PDHK1 knockout cell lines by Cas9 genome editing, including the design and cloning of the targeting constructs.  We used these cell lines to prove that PDHK1 is the only PDHK (out of four) capable of phosphorylating Ser232 of the E1a subunit of pyruvate dehydrogenase (PDH).  We assessed the functional impact of the absence of this inhibitory phosphorylation on PDH by analyzing PDH activity, mitochondrial function (by SeaHorse Extracellular Flux Analyzer), growth of xenografted tumors, and clonogenic cell survival in conditions of limited glucose and glutamine.  We further tested the effects of CAIX inhibitor, pan-PDHK inhibitor and their combination on cell viability of control and PDHK1 knockout cells for Objective 3.

Description of the main results achieved

We saw a shift in hypoxic PDHK1 protein signal on 2D-Western blot pointing to a covalent modification of the protein in hypoxic conditions (where PDHK1 is activated), possibly phosphorylation.  After Br-cAMP treatment (PKA activator) of cells, we saw an increase in PDHK1 activity in normoxia.  Both of these results are promising in respect to the possible regulation of hypoxic PDHK1 activity by PKA.

A very important result is the proof that PDHK1 is the only kinase able to phosphorylate Ser232 of PDH E1a in vivo.  Its absence leads to a more active PDH even in hypoxia, which in turn stimulates oxidative metabolism under hypoxic conditions, reversing the Warburg effect.  Cells unable to phosphorylate Ser232 of PDH grow significantly slower than controls as xenografted tumors.

Treating cells with a CAIX inhibitor in combination with a pan-PDHK inhibitor had a synergic negative effect on cell viability.

Expected final results

Based on our main first-year results described above, we hope to show later on that PKA regulates PDHK1 activity, and that by targeting CAIX and PDHK1 together, we could show a significant negative effect on tumor cell proliferation and survival.