LIMR Cancer: Investigation of accentuated mitochondrial ROS and NLRP3 inflammasome activation in cystic fibrosis and colorectal carcinoma.

Summary

Cystic fibrosis (CF) is associated with an accentuated inflammatory response to bacterial lipopolysaccharides (LPS), increased resting energy expenditure and a higher than expected prevalence of gastrointestinal and other malignancies. Our group, as well as others, have demonstrated a link between CF (bi-allelic mutations to the Cystic Fibrosis Transmembrane Conductance Regular gene [CFTR] gene) and an increased risk of colorectal cancer (CRC), which also presents at a younger age. The incidence of a single CFTR gene mutation in the CRC population is also higher than expected, suggesting an association between CFTR function and CRC risk.

We have previously demonstrated that CF cells (monocytes and bronchial airway epithelial cells) express features of autoinflammation, with an exaggerated NLRP3 inflammasome response and elevated mitochondrial reactive oxygen species (mROS). This hyper inflammatory response results in lung and other organ damage and drives systemic inflammation.

We have also demonstrated (submitted) increased mitochondrial respiration in CF cell lines expressing CFTR F508del/F508del (which is the most common CF causing mutation) with respect to basal, maximal, spare respiratory capacities and ATP production, as well as increased mitochondrial ROS generated via forward electron transport. Calcium blockers, BAPTA-AM and thapsigargin normalised mitochondrial metabolism, suggesting a calcium-mediated mechanism. Calcium flux experiments show that CFTR modulators, which are used in clinical practice and target stabilisation of the CFTR protein, decrease intracellular calcium flux in CF cells to the same extent as BAPTA-AM, highlighting the importance of calcium and chloride in metabolism in CF.

The first part of the project will investigate the role of calcium, mitochondrial OCR, mROS and NLRP3 expression in cells with single CF mutations (carriers). This is very important as 1:25 of the UK population are carriers of the CFTR gene and it is unknown if this predisposes to accentuated inflammation, especially as a carrier status predisposes individuals to increased respiratory disease as well as colorectal cancer.

The second part of the project will focus on NLRP3 and other inflammasomes, mROS and calcium flux in both healthy and CF colonic cells as well as colorectal cell cultures. Specific repurposed drugs will be used to assess effectiveness at normalising CF related cellular aberrations in vitro. This will include the highly effective CFTR modulators (ivacaftor/elexacaftor/tezacaftor) which have revolutions the clinical outcome in up to 90% of patients with CF. This work will help to better understand the potential pro oncogenic milieu of CF colonic cells and whether drugs such as CFTR modulators can down regulate some of these aberrant cellular processes.

This research is highly relevant to cancer more generally as there is growing literature supporting the role of the CFTR gene as a cancer suppressor gene in a wide spectrum of cancers. Gut inflammation is also elevated in CF, with increased levels of faecal calprotectin. While the aetiology is multifactorial, the inflammatory process is likely to be mediated through mROS and inflammasome activation. Elevated concentration of intracellular Calcium, ROS and NLRP3 have been associated with the promotion of cancer and tumour cell proliferation.

The previous projects have received a young investigator award as well as best immunology and basic science poster awards at sequential European Cystic Fibrosis Society Meetings. Therefore, we are well placed to deliver this innovative work.

Techniques associated with this project:

• Cell cultures
• Cytokine assays
• Glycolytic and mitochondrial assays (Extracellular Flux Analyser) Seahorse XFe96
• Ion flux assays
• Caspase assays

References

1. The risk of colorectal cancer in individuals with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: An English population-based study. Birch RJ, Peckham D, Wood HM, Quirke P, Konstant-Hambling R, Brownlee K, Cosgriff R, Consortium GER, Burr N, Downing A.J Cyst Fibros. 2023 May;22(3):499-504.

1. Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis. Jarosz-Griffiths HH, Scambler T, Wong CH, Lara-Reyna S, Holbrook J, Martinon F, Savic S, Whitaker P, Etherington C, Spoletini G, Clifton I, Mehta A, McDermott MF, Peckham D.Elife. 2020 Mar 2;9:e54556. doi: 10.7554/eLife.54556.PMID: 32118580

1. ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis. Scambler T, Jarosz-Griffiths HH, Lara-Reyna S, Pathak S, Wong C, Holbrook J, Martinon F, Savic S, Peckham D, McDermott MF.Elife. 2019 Sep 18;8:e49248. doi: 10.7554/eLife.49248.

This project is part of the International PhD Academy: Medical Research

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