Association of Irisin/FNDC5 with ERRα and PGC-1α Expression in NSCLC

Katarzyna Nowińska, Karolina Jabłońska, Urszula Ciesielska, Aleksandra Piotrowska, Katarzyna Haczkiewicz-Leśniak, Konrad Pawełczyk, Marzenna Podhorska-Okołów, Piotr Dzięgiel, Katarzyna Nowińska, Karolina Jabłońska, Urszula Ciesielska, Aleksandra Piotrowska, Katarzyna Haczkiewicz-Leśniak, Konrad Pawełczyk, Marzenna Podhorska-Okołów, Piotr Dzięgiel

Abstract

The rapid growth and division of cancer cells are associated with mitochondrial biogenesis or switching to glycolysis. ERRα, PGC-1α and irisin/FNDC5 are some of the proteins that can influence these processes. The aim of this study was to determine the correlation of these proteins in non-small cell lung cancer (NSCLC) and to investigate their association with clinicopathological parameters. Immunohistochemistry reactions were performed on tissue microarrays (860 NSCLC, 140 non-malignant lung tissue). The normal fibroblast cell line (IMR-90) and lung cancer cell lines (NCI-H1703 and NCI-H522) were used as co-cultures. The mRNA levels of FNDC5 and ESRRA (encoding ERRα) were assessed in IMR-90 cells after co-culture with lung cancer cells. We observed a decreased level of ERRα with an increase in tumor size (T), stages of the disease, and lymph node metastases (N). In the adenocarcinoma (AC) subtype, patients with a higher ERRα expression had significantly longer overall survival. A moderate positive correlation was observed between FNDC5 mRNA and ESRRA mRNA in NSCLCs. The expression of FNDC5 mRNA in IMR-90 cells increased after 24 h, and ESRRA gene expression increased after 48 h of co-culture. The ERRα receptor with PGC-1α participates in the control of FNDC5/irisin expression. Normal fibroblasts revealed an upregulation of the FNDC5 and ESRRA genes under the influence of lung cancer cells.

Keywords: ERRα; ESRRA; FNDC5; NSCLC; PGC-1α; irisin; non-small cell lung cancer.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Immunohistochemical reactions (IHC) indicating lack of irisin (A) and ERRα (B) expression. Weak positive IHC reaction for PGC-1α (C) in non-malignant lung tissue (NMLT). Positive cytoplasmic IHC reaction indicating irisin expression in skeletal muscle (positive control—D). Irisin expression in NSCLC cancer cells and stromal cells (grade of malignancy G1—G, G2—J, G3—M). Nuclear expression of ERRα in kidney (positive control—E). ERRα expression in NSCLC cancer cells (G1—H, G2—K, G3—N). Positive cytoplasmic expression of PGC-1α in prostate (positive control—F). PGC-1α in NSCLC cancer cells and stromal cells (G1—I, G2—L, G3—O). Magnification ×200. Arrows indicate a positive reaction.
Figure 2
Figure 2
Comparison of irisin expression levels detected by immunohistochemistry (IHC) in NSCLC (n = 860) cells (A,C,E,G) and in stromal cells (B,D,F,H) according to the tumor size (A,B), malignancy grade (C,D), lymph node status (E,F), and tumor stage (G,H), * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001. Kaplan–Meier survival curves show the prognostic impact of irisin expression levels in cancer cells (I) and stromal cells (J) on overall survival (OS) in patients with NSCLC. Patients were grouped according to the median value of expression levels.
Figure 3
Figure 3
Comparison of ERRα receptor expression levels detected by immunohistochemistry (IHC) in non-small cell lung cancer NSCLC (n = 860, A,C,E,G) and adenocarcinoma subtype—AC (n = 344, B,D,F,H) according to the tumor size (A,B), the grade of malignancy (C,D), the lymph node status (E,F), and the tumor stage (G,H), * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001.
Figure 4
Figure 4
Kaplan–Meier survival curves show the prognostic impact of ERRα expression levels on overall survival (OS) in patients with NSCLC (A), AC (D), SCC (G). Kaplan–Meier survival curves show the prognostic impact of PGC-1α expression levels in cancer cells on overall survival (OS) in patients with NSCLC (B), AC (E), SCC (H). Kaplan–Meier survival curves show the prognostic impact of PGC-1α expression levels in stromal cells on overall survival (OS) in patients with NSCLC (C), AC (F), SCC (I). Patients were grouped according to the median value of expression levels.
Figure 5
Figure 5
Comparison of PGC-1α expression levels detected by immunohistochemistry (IHC) in NSCLC (n = 860) cells (A,C,E,G) and in stromal cells (B,D,F,H) according to the tumor size (A,B), malignancy grade (C,D), lymph node status (E,F), and tumor stage (G,H). * p ≤ 0.05, ** p ≤ 0.005.
Figure 6
Figure 6
Correlations of ERRα receptor expression levels with diagnostic markers were strong positive—Ki-67 (A), moderate positive—EGFR (B), moderate positive—p63 (C) and weak positive—PD-L1 (D) in NSCLC (n = 860).
Figure 7
Figure 7
Correlations of PGC-1α expression levels in stromal cells with diagnostic markers were moderate positive—Ki-67 (A), weak positive—EGFR (B), weak positive—p63 (C) and weak positive—PD-L1 (D) in NSCLC (n = 860).
Figure 8
Figure 8
Correlations between irisin expressed in cancer cells with PGC-1α expressed in cancer cells (A) and stromal cells with PGC-1α (stromal cells) (B), and ERRα receptor (C). Correlations between ERRα and PGC-1α in NSCLC (n = 860) cancer cells (D).
Figure 9
Figure 9
Comparison between control (n = 16) and NSCLCs (n = 56) of FNDC5 mRNA (A) and ESRRA mRNA (B) expression levels. The moderate positive correlation between mRNA FNDC5 and mRNA ESRRA expression levels in NSCLC patients (C), * p ≤ 0.05, ** p ≤ 0.005.
Figure 10
Figure 10
Comparison of the expression level of FNDC5 mRNA after co-culture in IMR-90 cells in the empty insert (control) (A,B) and the insert with lung cancer cells [NCI-H1703 (C,D) and NCI-H522 (E,F)], * p ≤ 0.05, ** p ≤ 0.005, *** p < 0.001.
Figure 11
Figure 11
Comparison of the expression level of ESRRA mRNA after co-culture in IMR-90 cells in the empty insert (control) (A,B) and the insert with lung cancer cells [NCI-H1703 (C,D) and NCI-H522 (E,F)], * p ≤ 0.05, ** p ≤ 0.005.
Figure 12
Figure 12
Positive immunogold reaction (black dots—indicated by arrows) point to irisin/FNDC5 expression in the cell cytoplasm in NCI-H522 cells—magnification on the right (A), in NCI-H1703 cell mitochondria-M membrane—magnification on the right (B), in rough endoplasmic reticulum-RER and in cytoplasmic extensions of A549 cell (C), N-nucleus, magnification ×25,000.

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