LACTB suppresses carcinogenesis in lung cancer and regulates the EMT pathway

Abstract

Lung cancer causes thousands of deaths worldwide every year, and present therapeutics show little benefit for advanced-stage patients. Researchers do not know why and how lung cancer begins. Lactamase β (LACTB) is a tumor-suppressor in some cancers. However, its role in lung cancer is unknown. By analyzing the TCGA database and Kaplan-Meier Plotter database, LACTB was found to be downregulated in lung cancer tissues but the methylation level was increased. Patients with high LACTB expression exhibited improved survival. Then, in vitro assays demonstrated that LACTB overexpression inhibited cell migration and invasion, and induced apoptosis in H1299 and H1975 cells. Knockdown of LACTB caused the reverse effects. Moreover, a much higher apoptotic rate and more potent inhibitory effects on H1299 and H1975 cells were obtained when LACTB was combined with docetaxel. In addition, members of the epithelial-mesenchymal transition (EMT) signaling pathway were assessed using western blot analysis. The expression of E-cadherin was decreased while levels of N-cadherin and vimentin were increased after knockdown of LACTB in lung cancer cells. By contrast, overexpression of LACTB increased the level of E-cadherin but decreased N-cadherin and vimentin. Therefore, LACTB is a tumor suppressor in lung cancer that inhibits cell migration and invasion and induces cell apoptosis. Meanwhile, LACTB was found to strengthen the anticancer role of docetaxel and to suppress the EMT pathway in lung cancer.

Keywords: LACTB; docetaxel; epithelial-mesenchymal transition; lung cancer.

Figure 1

LACTB is clinically associated with lung cancer. (A) LACTB was found to be significantly decreased in lung cancer tissues compared to that noted in normal tissues based on TCGA database. (B) The methylation level of LACTB in lung cancer tissues was increased significantly compared to normal tissues based on the TCGA database. (C) The survival probability of lung cancer patients with high LACTB expression was much higher than the patients with low LACTB expression. ^*P<0.05, statistical difference compared to the normal tissues. LACTB, lactamase β; TCGA, The Cancer Genome Atlas.

Figure 2

LACTB negatively regulates the cell survival in lung cancer. (A) LACTB was found to be overexpressed in H1299 cells by transfection with a plasmid carrying LACTB. In the control group (Ctrl), empty vector pCDH was transfected into cells. (B) LACTB was overexpressed in H1975 cells by transfection with a plasmid carrying LACTB. (C) LACTB was knocked down in H1299 cells by shRNA targeting LACTB. (D) LACTB was knocked down in H1975 cells by shRNA targeting LACTB. (E) Overexpression of LACTB inhibited cell survival in H1299 and H1975 cells. (F) Knockdown of LACTB promoted cell survival in H1299 and H1975 cells. ^*P<0.05, statistical difference compared with the Ctrl or shNC group. LACTB, lactamase β.

Figure 3

Overexpression of LACTB suppresses lung cancer cell migration in a wound-healing assay. (A) Cell migration behavior was determined by a wound-healing assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared with the Ctrl group. LACTB, lactamase β.

Figure 4

Knockdown of LACTB promotes lung cancer cell migration in a wound-healing assay. (A) Cell migration behavior was determined by a wound-healing assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared to the shNC group. LACTB, lactamase β.

Figure 5

Overexpression of LACTB suppresses lung cancer cell migration in a Transwell assay. (A) Cell migration behavior was determined by a Transwell assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared to the Ctrl group. LACTB, lactamase β.

Figure 6

Knockdown of LACTB promotes lung cancer cell migration in a Transwell assay. (A) Cell migration behavior was determined by a Transwell assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared to the shNC group. LACTB, lactamase β.

Figure 7

Overexpression of LACTB suppresses cell invasion in lung cancer. (A) Cell invasion behavior was determined by a Transwell assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared with the Ctrl group. LACTB, lactamase β.

Figure 8

Knockdown of LACTB promotes cell invasion in lung cancer. (A) Cell migration behavior was determined by a Transwell assay in H1299 and H1975 cells. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared to the shNC group. LACTB, lactamase β.

Figure 9

LACTB promotes the cell killing effect of docetaxel on lung cells. (A) Combination of LACTB with docetaxel further reduced the survival rate of H1299 cells. (B) Combination of LACTB with docetaxel further reduced the survival rate of H1975 cells. (C) Knockdown of LACTB partially reversed the growth inhibition of H1299 cells by docetaxel. (D) Knockdown of LACTB partially reversed the growth inhibition of H1975 cells by docetaxel. ^*P<0.05, statistical difference. LACTB, lactamase β

Figure 10

Overexpression of LACTB enhances the cell apoptotic rate by docetaxel in H1975 cells. (A) Cell apoptosis rate in H1975 cells was detected by FACS analysis. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared with the Ctrl group. LACTB, lactamase β.

Figure 11

Knockdown of LACTB reduces the cell apoptotic rate by docetaxel in H1975 cells. (A) Cell apoptosis rate in H1975 cells was detected by FACS analysis. (B) The statistical analysis of data in (A). ^*P<0.05, statistical difference compared to the shNC group. LACTB, lactamase β.

Figure 12

LACTB regulates the EMT signaling pathway. (A) Overexpression of LACTB promoted E-cadherin expression and reduced the level of Snail, N-cadherin, vimentin, MMP2 and MMP9 in H1975 cells. By contrast, knockdown of LACTB increased the level of Snail, N-cadherin, vimentin, MMP2 and MMP9 while reducing E-cadherin in H1975 cells. (B) Gray value analysis of the protein bands (on the left) shown in in A. (C) Gray value analysis of the protein bands (on the right) in (A). ^*P<0.05, statistical difference compared to the Ctrl or shNC group. LACTB, lactamase β; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase.