LAPTM4B-35, a cancer-related gene, is associated with poor prognosis in TNM stages I-III gastric cancer patients

Abstract

Background: Lysosome-associated transmembrane protein 4β-35 (LAPTM4B-35), a member of the mammalian 4-tetratransmembrane spanning protein superfamily, has been reported to be overexpressed in several cancers. However the expression of LAPTM4B-35 and its role in the progression of gastric cancer (GC) remains unknown. The aim of this study was to investigate LAPTM4B-35 expression in GC, its potential relevance to clinicopathologic parameters and role of LAPTM4B-35 during gastric carcinogenesis.

Methods: In the present study, paraffin-embedded specimens with GC (n = 240, including 180 paired specimens) and 24 paired fresh frozen tissues were analyzed. qRT-PCR and immunohistochemistry (IHC) were used to analyze the expression of LAPTM4B-35 in GC. The effects of LAPTM4B-35 on GC cell proliferation, migration and invasion were determined by overexpression and knockdown assays.

Results: IHC showed that LAPTM4B-35 was expressed in 68.3% (123/180) of GC tissues, while in 16.1% (29/180) of their paired adjacent noncancerous gastric tissues (P = 0.000). LAPTM4B-35 mRNA levels in GC tissues were also significantly elevated when compared with their paired adjacent noncancerous tissues (P = 0.017). Overexpression of LAPTM4B-35 was significantly associated with degree of differentiation, depth of invasion, lymphovascular invasion and lymph node metastasis (P<0.05). Kaplan-Meier survival curves revealed that patients with LAPTM4B-35 expression had a significant decrease in overall survival (OS) in stages I-III GC patients (P = 0.006). Multivariate analysis showed high expression of LAPTM4B-35 was an independent prognostic factor for OS in stage I-III GC patients (P = 0.025).

Conclusion: These findings indicate that LAPTM4B-35 overexpression may be related to GC progression and poor prognosis, and thus may serve as a new prediction marker of prognosis in GC patients.

Fig 1. Expression analysis of LAPTM4B-35 in GC cell lines and GCs.

(A) mRNA (upper panel) and protein (lower panel) expression of LAPTM4B-35 in five gastric cancer cell lines. (B) relative mRNA expression of LAPTM4B-35 in gastric cancer and their adjacent noncancerous tissues.

Fig 2. Immunohistochemical staining with anti-LAPTM4B-35 protein in normal, noncancerous and carcinomas of the stomach.

(A) LAPTM4B-35 was not expressed in normal stomach mucosa. (B) LAPTM4B-35 was expressed in the dysplasia lesion. (C) LAPTM4B-35 negative staining in GC tissue. (E-H) LAPTM4B-35 positive staining in GC tissues. Original magnification: 100×.

Fig 3. Kaplan-Meier curves for overall survival of the GC patients.

A, Kaplan-Meier survival curves for OS in GC patients with LAPTM4B-35 positive and negative expression. (B, C, D) Kaplan-Meier curves for OS in subgroup of GC patients stratified by TNM stages I-III, IV and patients without lymphovascular invasion, respectively.

Fig 4. Effect of LAPTM4B-35 on cell proliferation.

(A) Expression of LAPTM4B-35 in BGC-823 and SGC-7901 after transfection with LAPTM4B expression vector and hCas9/gRNA were identified by Western-blot. BGC-823-AF shows BGC-823-overexpressing clone. (B) Growth curves determined by CCK-8 assay. Left panel: overexpression of LAPTM4B-35 promotes rapid increase in BGC-823 cell proliferation compared with wild-type and MOCK. Middle panel: knockdown of LAPTM4B-35 expression inhibits cell proliferation as compared with control and SGC-7901. Right panel: different cell proliferation state after incubation 3 days for BGC-823 and 2 days for SGC-7901. *P<0.05, BGC-823-AF vs MOCK, knockdown vs SGC-7901. For all data the mean and standard deviation represent the average of three independent experiments.

Fig 5. LAPTM4B-35 promotes gastric cancer cells migration.

(A) left upper panel: wound-healing assay of BGC-823-AF, MOCK and BGC-823; left lower panel: wound-healing assay of hCas9/gRNA, Control and SGC-7901. Photos were captured by an inverted phase-contrast microscope at 24h after wounding. Magnification = 100×. (B) Quantification of wound-healing rates. For all data the mean and standard deviation represent the average of three independent experiments.

Fig 6. LAPTM4B-35 promotes gastric cancer cells invasion.

(A), left upper panel: transwell assay of BGC-823-AF, MOCK and BGC-823 cells; left lower panel: transwell assay of hCas9/gRNA, Control and SGC-7901. Invasive cells were counted in randomly 5 selected microscopic fields. Magnification = 100×. (B) Quantification of migrating and invasive cells. For all data the mean and standard deviation represent the average of three independent experiments.