Role of PHDs in the initiation and progression of colorectal cancer

Abstract

Colorectal cancer (CRC) is the 4th most common cancer in the world. It is also the third most common contributor to cancer-related deaths worldwide. The average life-time risk of CRC is 3-5% and this is known to be influenced by lifestyle factors (diet, alcohol consumption, exercise) as well as genetic factors. One well-known genetic contributor is a gene called APC (Adenomatous Polyposis Coli). APC is mutated in around 80% of CRCs and this leads to the upregulation of the Wnt signalling pathway. In this project, I was interested in the role of APC in early CRC cancer development and I aimed to explore new research avenues in this context. For this, the manipulation of the hypoxia signalling pathway was of particular interest to me. The term “hypoxia” describes a state of decreased oxygen concentration inside cells. While cells can commonly encounter hypoxic states, a tightly regulated transcriptional response ensures quick adaptation and ultimately the restoration of normal oxygen levels. This is under the control of a transcription factor called HIFα (Hypoxia inducible factor), which itself is controlled by oxygen-sensing enzymes called prolyl hydroxylases (PHDs). PHDs use molecular oxygen to hydroxylate HIFα and tag it for proteasomal degradation. When oxygen is scarce, PHD activity is inhibited and HIFα will accumulate instead. Interestingly, in many cancers, hypoxic states and HIFα accumulation are not resolved and this has been associated with poor prognosis and resistance to chemotherapy and radiotherapy. However, in this project I found that the chemical inhibition of PHDs by a drug called Molidustat can induce a severe cell-death phenotype of CRC cells in vitro. Importantly, this death phenotype only occurred in Apcfl/fl murine intestinal organoid cultures, while WT control organoids remained largely unaffected. Making use of clonogenicity assays, I could further observe that this drug treatment directly affected the organoids’ Lgr5+ stem cell population. Using a previously published method called Cellular Thermal Shift Assay (CETSA), I was able to confirm binding of Molidustat to its putative target PHD2 in live cells in vitro. However, extending this target-engagement method to the whole proteome, I was also able to identify other potential players involved in this cell death phenotype. Glutathione s-transferase-π-1 (GSTP1), an enzyme involved in cellular detoxification and ROS scavenging, proved particularly interesting in this regard. I used chemical proteomics and biochemical enzymatic activity assays to further validate the potential inhibition of GSTP1 by Molidustat. I hypothesised that the inhibition of PHD2 (and upregulation of HIF) would sensitise cells to perturbations in ROS. In line with this, I could see a significant increase of H2O2-dependent ROS in cells after treatment with Molidustat, which was ablated by the addition of NAC (N-Acetyl Cysteine). My findings thus provide potential insights into the mechanisms involved in the CRC cell death following Molidustat treatment. While further mechanistic experiments are needed, I am hoping that I might have uncovered a therapeutically useful synthetic lethality between PHD2 and the ROS-detoxification system in CRC.