PGAM5 expression and macrophage signatures in non-small cell lung cancer associated with chronic obstructive pulmonary disease (COPD)

NSCLC presents with locally advanced or metastatic disease in 80% of cases [1, 2], which accounts for its dismal prognosis [3]. Although smoking is by far the most common risk factor for NSCLC, other risk factors include pulmonary fibrosis, tuberculosis, radiation therapy and COPD [4]. Up to 70% of lung cancer smokers have pre-existing COPD prior to cancer diagnosis [5, 6]. COPD patients are at an increased risk of developing lung cancer, irrespective of their smoking history [7–9]. Smokers with airflow obstruction have a five-fold increased risk of lung cancer compared to those with normal lung function [5]. As well as airflow obstruction, emphysema diagnosed on computed tomography (CT) is also another independent risk factor for lung cancer [10, 11]. The presence of COPD and/or emphysema are also predictors of reduced survival of NSCLC patients or increased risk of recurrence following surgery [12–15].

Oxidative stress is a well-recognised driver of carcinogenesis and is present in smokers and COPD patients, with the greatest degree of oxidative stress present in the airways of patients with COPD [16]. Reactive oxygen species produced in mitochondria from oxidative phosphorylation represent the main cellular source of oxidative stress, and increased production of mitochondrial ROS seen in airway epithelial cells of patients with COPD is associated with mitochondrial dysfunction [16]. Under increased oxidative stress, metabolically-active cells may undergo an increase in mitochondrial biogenesis and also in the degradation of damaged mitochondria within autolysosomes through the process of mitophagy. An inability to increase biogenesis and/ or induce mitophagy may lead to the accumulation of dysfunctional mitochondria and subsequently, increased oxidative stress [17].

The prognosis of lung cancer is also dependent on the immune inflammatory response within the tumour stroma [18]. One of the key components of the stroma is the tumour-associated macrophage which has traditionally been divided into the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. The presence of M1 macrophages within the tumour islets of NSCLC may confer a survival advantage while the presence of M2 macrophages is a poor prognostic factor [19]. The effector functions of macrophages in the M1 and M2 states are dependent on changes in mitochondrial metabolism [20].

We therefore reasoned that mitochondrial dysfunction could be a driving mechanism underlying the increased propensity of COPD patients to develop cancer and the poor prognosis of NSCLC patients with COPD. In the current study, we demonstrated enhanced expression of the mitophagy-inducing protein PGAM5 in malignant epithelial cells, as well as in the alveolar macrophages from normal lung and adjacent to cancer and that their expression was related to the survival of non-small cell lung cancer patients. We also showed that the expression of PGAM5 was related to specific macrophage phenotypes. Our data suggests that the expression of PGAM5 in lung cancer is associated with specific macrophage phenotypes and patient survival.

COPD patients are at increased risk of NSCLC while patients with established NSCLC have a worse outcome if they have concomitant COPD [5–9, 12–15]. Oxidative stress is an initiator of carcinogenesis [35] and the prognosis of NSCLC is partly dependent on the metabolic state of its tumour-associated macrophages [36]. Both the increased oxidative stress in COPD and the metabolic state of macrophages are dependent on the state of the mitochondrion [16, 20]. We therefore reasoned that there would be an altered expression of selective mitochondrial-related genes within tumour tissue and that this would be correlated with a change in patient survival. Fourteen mitochondrial-related genes showed a ≥ 2 fold difference in expression between NSCLC and normal lung tissue and their expression was correlated with a reduction in patient survival. Although dysfunction in mitophagy has previously been demonstrated in COPD and NSCLC, there have been no published studies of the mitophagy-related protein, PGAM5, in the pathogenesis of COPD and NSCLC.

PGAM5 functions in multiple cell death pathways [37] and regulates mitophagic protection against cell necroptosis [38]. PGAM5 catalyses the dephosphorylation of FUNDC1 which enhances its interaction with microtubule-associated protein 1A/1B-light chain 3, leading to mitophagy [39]. PGAM5 also promotes inflammasome activation in macrophages [40]. We report for the first time the expression of PGAM5 in lung tissue from COPD and NSCLC patients. There was 2.5 fold increase in PGAM5 mRNA expression in cancer tissue compared to background/ normal lung tissue. Using immunohistochemistry, the tumour cells but not the normal tissue expressed PGAM5. This confirmed that both mRNA and protein expression of PGAM5 are higher in cancer tissue than in normal tissue, although it is difficult to quantify the degree of increase in protein expression. PGAM5 was consistently expressed in the malignant epithelial cells of lung cancer patients, with no difference in the level of expression across the 3 groups of patients, even if adenocarcinoma or squamous cell carcinoma cases were considered separately across the groups. The level of expression of PGAM5 in lung cancer itself cannot therefore explain why patients with COPD and NSCLC have the worse outcome. However, PGAM5 expression in carcinomas from smokers with moderate emphysema showed lower expression compared to those from smokers with mild or no emphysema. Although the significance of this finding may be limited by the small number of patients in the moderate emphysema group, this may reflect reduced mitophagy in this group of patients, compared to those with mild or no emphysema. This also points to the need of subclassifying patients according to emphysema severity in future studies. PGAM5 is also expressed in alveolar macrophages at the edge of the cancer.

Benign bronchial and alveolar epithelial cells do not express PGAM5 while pre-neoplastic epithelium such as squamous dysplasia and carcinoma in situ express PGAM5. The association of PGAM5 expression with worse prognosis in squamous cell carcinoma and its expression in the sequential transformation of squamous dysplasia into malignant squamous cells suggests a pathological role for PGAM5 in lung squamous cell carcinoma. The mechanisms by which PGAM5 may contribute to the malignant transformation of the epithelium is uncertain. It is possible that this may be related to the action of PGAM5 on cell death pathways, leading to uncontrolled cellular proliferation secondary to its overexpression.

PGAM5 expression was only detected within alveolar macrophages in non-cancerous lung tissue. The level of expression in alveolar macrophages in non-cancerous tissue was higher in smokers than non-smokers, with a trend towards highest expression in COPD or emphysematous patients. The phenotype of alveolar macrophages is altered in COPD [41] and the increased oxidative stress, resulting from excess free radicals in cigarette smoke, leads to mitochondrial damage and increased turnover, including mitophagy. This may be reflected by the increased expression of PGAM5 in alveolar macrophages. Increased mitochondrial turnover may also lead to a change in the metabolic phenotype of the alveolar macrophages which is known to be related to their immunomodulatory phenotypes [20]. PGAM5 in the alveolar macrophages may also lead to the activation of the inflammasome, which is important in triggering the inflammatory response in COPD [40].

In GSE 72194, PGAM5 expression within tumours was correlated with 9 out of 49 macrophage signatures (‘modules 7, 9, 10, 11, 28, 36, 39, 42, 49’) obtained from a study of macrophage molecular phenotypes by Xue et al. [28]. In GSE 31210, PGAM5 expression was negatively correlated with the macrophage ‘module 36’ (similar to signature of monocytes activated by tumour necrosis factor + Pam3CSK4 + prostaglandin E2) expression in lung cancer, which itself is inversely associated with mortality. This may partly explain our original observation that PGAM5 expression is associated with a worse outcome in NSCLC. We speculate that products from macrophages stimulated by PGAM5 may affect epithelial cell function and may account, at least in part, for the increased risk of lung cancer in patients with COPD. Further work is needed to elucidate the relevant mechanisms.

The findings of this study are limited by the modest number of cases we have analysed with immunohistochemistry, findings that would need to be confirmed in a larger study. Another limitation relates to the potential differences in tumour stroma content between the different tumour samples within the datasets used. To quantify RNA expression within the tumour cells and not stroma, laser capture microdissection of tumour cells would have to be performed. Although such data may be available from individual studies with small number of samples, large datasets mostly include data from studies in which laser capture tissue dissection was not carried out. However, our data on PGAM5 expression has been validated by immunohistochemistry with the demonstration of PGAM5 specifically in tumour cells and not in stromal cells. Variations of RNA expression due to intratumoural heterogeneity have been reduced by the relatively large number of tumour samples [151 squamous cell carcinomas and 140 adenocarcinomas] examined at an early stage of cancer (Stage 1).

Also, in our study, it was not possible to determine if there were macrophage signatures specific to COPD-associated cancer in comparison to non-COPD associated cancer. We propose that the tumour-associated macrophages in COPD-associated lung cancer are different from those in non-COPD associated cancer. This would account for the worse prognosis of the former, as we have shown that specific macrophage phenotypes are associated with prognosis.

In summary, the expression of specific mitochondrial-related proteins and macrophage signatures are associated with the outcome of NSCLC. The expression of PGAM5 in lung cancer was correlated with specific macrophage phenotypes, one of which was associated with lung cancer mortality. PGAM5 is expressed in pre-malignant and malignant epithelial cells, but not in benign epithelium and may therefore play a role in the malignant transformation of airway epithelial cells.