Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression

Abstract

Background and Objectives: The Connexin43 (Cx43) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Keywords: Cx43 gene mutation; lung cancer.

Figure 1

Immunohistochemical analysis of Cx43 protein expression using a monoclonal Cx43 anti-human antibody on paraffin sections of lung tumor specimens. (A) Negative control showing no Cx43 immunostaining (×200). (B) Positive control showing Cx43 protein expressed in the membrane of non-tumor lung tissue (arrow) (×400). (C) Nuclear localization of Cx43 in tumor cells (arrow) (patient 888C) (×400). (D) Cx43 expression in the cytoplasm of tumor cells (arrow) (patient 245D) (×400).

Figure 2

Growth curves of CL-3 lung cancer cells expressing wild-type or mutant Cx43. Cells were transfected with wild-type Cx43, vector control, or mutant Cx43 constructs (Cx43-18,19 and Cx43-57). Cell numbers were counted daily for 7 days. Data points represent the mean of three independent experiments + standard error of the mean.

Figure 3

Kaplan–Meier survival curves for NSCLC patients stratified by Cx43 protein localization. Three groups are represented: patients with Cx43 membrane expression and negative immunostaining (black line), patients with Cx43 positive staining in the cytoplasm (dotted line), and patients with Cx43 positive staining in the nucleus (grey line). The x-axis represents days after surgery, while the y-axis shows the survival percentage.