Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

Abstract

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Insight into the Role and Regulation of Gap Junction Genes in Lung Cancer and Identification of Nuclear Cx43 as a Putative Biomarker of Poor Prognosis

“…Moreover, mRNA expression is typically derived from all cells present in the tumour sample (e.g., in the lung, there would be a mix of pneumocytes, bronchial epithelium, endothelial cells, macrophages and other cell types). Indeed, a recent study showed that cancer-associated fibroblasts can form Cx43-mediated gap junctions with NSCLC cells to support their malignant progression [64]. The ratios of these cell types in a tumour change.…”

Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches

“…Interestingly, it has been shown that CAFs are able to stimulate cancer cell proliferation and progression through multiple mechanisms. For instance, in a lung cancer model, CAFs underwent increased aerobic glycolysis and promoted the epithelial mesenchymal transition, migration and invasion of non-small-cell lung carcinoma (NSCLC) cells, in contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation, thereby an activation of the PIK3/Akt and MAPK/ERK pathways occurred (123). Furthermore, colorectal cancer cells induce oxidative stress in microenvironment fibroblast, which then undergo metabolic changes, including increased expression of glycolytic enzymes, reduced TCA cycle enzymes and autophagy proteins such as microtubule-associated protein 1A/1B-light chain 3 (LC3), Bcl-2 interacting protein 3(BNIP3), and p62 (124).…”

Peer Review #1 of "Identification of a six-gene metabolic signature predicting overall survival for patients with lung adenocarcinoma (v0.1)"

“…The abnormal expression of PKM promoted cancer growth, invasion, and metastasis by governing aerobic glycolysis (Prakasam et al, 2017;Zahra et al, 2020) and induced cancer treatment resistance (Calabretta et al, 2016). Furthermore, PKM is overexpressed in non-small cell lung cancer (NSCLC) and involved in the development and prognosis of NSCLC (Luo et al, 2018). TPI1 is a crucial enzyme in carbohydrate metabolism, catalyzing the interconversion of dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate during glycolysis and gluconeogenesis.…”