Identification of Lysosome-Associated Protein Transmembrane-4 as a Novel Therapeutic Target for Osteosarcoma Treatment

Abstract

Objective: The aim of the study is to evaluate the expression of lysosome-associated protein transmembrane-4 (LAPTM4B) in human osteosarcoma tissue samples collected in our hospital, and to explore the possible correlations between the clinical pathological features of osteosarcoma patients and LAPTM4B expression.

Methods: Immunohistochemical (IHC) assays were performed to detect the expression levels of LAPTM4B in 62 tissue samples of osteosarcoma tissues and corresponding non-tumor tissues. According to LAPTM4B staining intensity in tumor tissues, osteosarcoma patients were classified into LAPTM4B high expression and low expression groups. In addition, the potential correlations between LAPTM4B expression levels and clinical pathological features were evaluated. In addition, we detected the effects of LAPTM4B on the proliferation and invasion of esteosarcoma cells through colony formation assay and transwell assay, respectively. We further explored the potential effects of LAPTM4B on tumor growth and metastasis using in vivo animal model.

Results: We revealed that LAPTM4B was highly expressed in human osteosarcoma tissues. We determined the significance between LAPTM4B and clinical features, including the tumor size (P = 0.004*) and the clinical stage (P = 0.035*) of osteosarcoma patients. Our results further demonstrated that ablation of LAPTM4B obviously blocked the proliferation and invasion of osteosarcoma cells in vitro and restrained tumor growth and metastasis in mice.

Conclusion: We investigated the potential involvement of LAPTM4B in osteosarcoma progression and confirmed LAPTM4B as a novel therapeutic target for osteosarcoma.

Keywords: Invasion; LAPTM4B; Osteosarcoma; Proliferation; Therapeutic target.

Figure 1

LAPTM4B was obviously highly expressed in osteosarcoma tissues from patients. (A) Immunohistochemistry (IHC) revealed LAPTM4B expression levels in osteosarcoma tissues and the representative images are exhibited (100× and 400× magnification, respectively). (B) IHC assays exhibited LAPTM4B expression levels in corresponding adjacent tissues and the representative images are shown (100× and 400× magnification, respectively).

Figure 2

The expression levels of LAPTM4B were obviously decreased in both MG‐63 and U‐2 OS cells after the transfection of LAPTM4B‐targeted shRNA plasmids. (A) The results of quantitative PCR assays showed the obviously reduced expression levels of LAPTM4B in its short hairpin RNA (shRNA) plasmid‐transfected MG‐63 and U‐2 OS cells, respectively. (B) Immunoblot assays revealed the efficient decrease of LAPTM4B expression levels after the transfection of its shRNA plasmids in both MG‐63 and U‐2 OS cells. Results are presented as mean ± SD, *P < 0.05.

Figure 3

LAPTM4B regulates or affects and invasion of osteosarcoma cells in vitro. (A). Colony formation assays were performed using MG‐63 and U‐2 OS cells transfected with control or LAPTM4B short hairpin RNA (shRNA) plasmids, and colony numbers were manually counted (×1). (B). Transwell assays were conducted using both MG‐63 and U‐2 OS cells transfected with control or LAPTM4B shRNA plasmids, and the invasion degree was quantified by the numbers of stained cells (×200). Results are presented as mean ± SD, *P < 0.05.

Figure 4

LAPTM4B contributes to tumor growth and metastasis of osteosarcoma cells in mice. (A) MG‐63 cells infected with control or LAPTM4B short hairpin RNA (shRNA) lentivirus were subcutaneously implanted into nude mice. After 2 weeks, tumors were isolated and photographed, and the volume of tumors was measured every week (n = 4 in each group). The tumor growth curves were calculated and analyzed according to the average volume of five tumors in between LAPTM4B knockdown and control groups. (B) MG‐63 cells infected with control or LAPTM4B shRNA lentivirus were sequentially implanted into the caudal vein of nude mice. Eight weeks later, tumors were isolated from mice and the metastasis volume was calculated in each group (n = 5 for each group). (C). Western blot assays were performed and exhibited the expression levels of LAPTM4B in control or knockdown group tumors isolated from mice. Results are presented as mean ± SD, *P < 0.05.