Overexpressed LAPTM4B-35 is a risk factor for cancer recurrence and poor prognosis in non-small-cell lung cancer

Background The expression levels and clinical significances of Lysosomal-associated protein transmembrane-4β-35 (LAPTM4B-35) protein are unknown in the non-small-cell lung cancer (NSCLC). This study aimed to explore the expression and prognostic value of LAPTM4B-35 in NSCLC patients. Methods The clinicopathological and survival data of 107 NSCLC patients who received radical surgery from 2007 and 2011 were reviewed. The LAPTM4B-35 expression of the paired tumors and adjacent normal specimens were detected, and the association between LAPTM4B-35 and clinical variables was explored. Kaplan-Meier analysis and Cox regression (Proportional hazard model) were performed to investigate the prognostic significance for NSCLC. Results LAPTM4B-35 was over expressed in NSCLC tissues. The elevated LAPTM4B-35 expression was associated with cancer recurrence (P = 0.031). The 5-year median OS and PFS were significantly worse in the LAPTM4B-35 overexpressed group. Multivariate Cox analysis showed that LAPTM4B-35 over-expression was an independent factor for OS and PFS in NSCLC(P = 0.018, P = 0.026, respectively). Conclusions The overexpressed LAPTM4B-35 was an independent prognostic biomarker for NSCLC, which could predict cancer recurrence and poor over survival. And that may be applied as potential target for NSCLC treatment.

www.impactjournals.com/oncotarget results Patient clinicopathological characteristics Table 1 summarized the clinicopathological characteristics of all the NSLCL patients. Among these patients, the median age in this study was 65 years (range, 28 to 81 years), and 85 (79.4%) patients were male and 22(20.6%) patients were female. 34(31.8%) patients were SCC and 73(68.2%) were non-SCC. 48 lymph node negative patients received postoperative adjuvant chemotherapy alone, while 55 lymph node metastasis patients had chemo-radiotherapy treatment.

lAPtM4b-35 was overexpressed in nsclc cancer tissues
The mRNA level of LAPTM4B-35 was detected by qRT-PCR, and as the Figure 1A indicated, the LAPTM4B-35 mRNA was overexpressed in tumor tissues. Consistently, our Western Blotting data showed the LAPTM4B-35 protein expression was also significantly elevated in cancer tissues ( Figure 1B).

lAPtM4b-35 expression and clinicopathological variables
We further compared the clinicopathological parameters based on the LAPTM4B-35 protein level. As the Table 1 showed, the elevated LAPTM4B-35 protein expression was significantly correlated with histology (p

dIscussIon
In the present study, we first explored the LAPTM4B-35 expression in NSCLC, and then investigated the association of LAPTM4B-35 protein expression with clinicopathological factors and prognosis. Our study found that the LAPTM4B-35 protein was expressed in NSCLC, and the elevated   [1]. NSCLC accounted for about 85% of all lung carcinomas, and it included non-SCC and SCC [6,16]. Systematic clinical studies and basic research on NSCLC had improved the prognosis of the NSCLC, however, the long-term outcome of the NSCLC remained poor [17][18][19][20]. It is significant to identify the new biomarkers to improve the prognosis of NSCLC.
LAPTM4B was originally identified in hepatocellular carcinoma. And further studies found LAPTM4B-35 was overexpressed in several kinds of solid tumors [21,22]. What was more, LAPTM4B-35 played critical role in tumorigenesis and tumor metastasis [23,24]. Recently, Qiao and her colleagues reported that the LAPTM4B-35 expression was up regulated in SCLC patients [13]. Unfortunately, there was not too much information about the expression and clinical significance of LAPTM4B-35 in NSCLC. Therefore, we detected the LAPTM4B-35 expression, collected clinicopathological and survival data, and explored the association between the LAPTM4B-35 protein expression and clinicopathological factors. Consistently, our study found that the LAPTM4B-35 was overexpressed in NSCLC compared to the normal tissues ( Figure 1). We further investigated the associations of elevated LAPTM4B-35 protein expression and clinicopathological characters in NSCLC. As the table 1 showed, the higher LAPTM4B-35 protein expression correlated with aggressive features (including poor histopathologic differentiation, lymph node metastasis and advanced clinical stage.) and tumor recurrence .
The potential limitations including: the relative small size population limited the degree of evidence. Accordingly, larger and muti-center clinical study needs to be conducted to validate our report. Besides, our results and the previous studies all found the LAPTM4B-35 was associated with tumorigenesis, progression and aggressive clinicopathological features, and the potential molecular mechanisms for these processes need to be elucidate.

conclusIons
Our study identified that the LAPTM4B-35 was elevated in NSCLC, the elevated LAPTM4B-35 expression was associated with aggressive clinicopathological features and poor prognosis, suggesting that LAPTM4B-35 protein could be applied in predicting patient's prognosis.

MAterIAls And MetHods
Patients who were confirmed NSCLC histologically from 2007 to 2011 were enrolled in this study. The inclusion criteria included: (a) received radical surgery; (b) had the complete clinical and follow-up informations. The exclusion criteria included: (a) distant metastasis was found before or during the operation; (b) patients who received treatment prior to radical surgery. Base on the above criteria, total of 107 patients were enrolled in this study. Written informed consents were obtained from all participants according to the Helsinki Declaration, and this study protocol was approved by the Ethics Committee of our hospital, Tianjin, China.
The clinical information such as age, gender, tumor size, histology, TNM stage, clinical stage and survival data were collected. The follow-up data was obtained from outpatient visit or telephone follow-up. The OS was defined as time from the day of diagnosis to the day of last visit or death. The median follow-up time was 64.5 months.

Western blot
Western blotting was conducted as previously described [14,15]. In brief, tumor tissues were lysed with NP40 lysis buffer. Equal amounts of proteins were separated by SDS-PAGE, then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% nonfat milk, and incubated with GAPDH and anti-LAPTM4B-35 polyclonal antibody (dilution, 1µg/ml; Abcam, Cambridge, UK.) at 4 o C overnight, and then probed with secondary antibody at room temperature for 1 hour. Signals were detected using enhanced chemiluminescence detection system (Pierce, Rockford, IL, USA). The intensity of hybridization signals was quantified using Image analysis program. The protein levels were normalized to those of GAPDH. The LAPTM4B-35 protein levels were divided into two groups based on the median expression level.

statistical analysis
Continuous variables were described using mean ±standard deviation; the categorical variables were analyzed by a chi-squared test. The Kaplan-Meier method and the Multivariate analyses were conducted to identify significant independent factors for the prognosis. The statistical analyses were performed using SPSS version 18.0 (SPSS, Chicago, IL, United States). Significance was defined as p-Values (two sides) < 0.05.