GOLPH3, a Good Prognostic Indicator in Early-stage NSCLC Related to Tumor Angiogenesis

Background: Golgi phosphoprotein-3 (GOLPH3) is implicated in cancer development and progression. The aim of this study was to evaluate the prognostic significance of GOLPH3 protein and its association with tumor angiogenesis in patients with early-stage NSCLC. Materials and Methods: Immunohistochemistry was performed to determine GOLPH3 protein expression and allow assessment of intratumoral microvessel density (MVD) by counting CD-34 positive immunostained endothelial cells. Correlations of expression with MVD, clinicopathologic features and clinical prognosis were analyzed. Results: A notably higher level of GOLPH3 expression was found in early-stage NSCC tissues at the protein level. However, we do not find any correlation between GOLPH3 expression and clinicopathologic features ( p >0.05), although higher MVD was positively associated with GOLPH3 overexpression ( p <0.001). Expression of GOLPH3 was found to be an independent prognostic factor in early-stage NSCLC patients, those expressing high levels of GOLPH3 exhibiting a substantially lower 5-year overall survival than GOLPH3-negative patients (adjusted HR =1.899, 95% CI: 1.021-3.532, p =0.043). Conclusions: High expression of the GOLPH3 protein is common in early-stage NSCC, and is closely associated with tumor progression, increased tumor angiogenesis, and poor survival. We conclude a possibility of its use as a diagnostic and prognostic marker in early-stage NSCC patients.


Introduction
Lung cancer is the leading cause of death among the malignant tumors worldwide, and the incidence of lung cancer is increasing (Travis, 2011).Non-small-cell lung cancer (NSCLC) accounts for up to 80 % of all lung cancer cases (Brognard et al., 2001;Ren et al., 2004).Despite great advance in the treatment of cancers in recent years, the prognosis for patients with lung cancer remains poor, with 5-year survival rates less than 15% (Smith et al., 2010;Gao et al., 2011).Recurrence and the emergence of metastases are major causes of therapeutic failure in cancer patients, and 30-40% of stage I patients relapse after surgical resection (Fry et al., 1999).Moreover, prospective randomized data showed that adjuvant chemotherapy in stage IB has not achieved a significant survival benefit and even a detrimental effect was observed in stage IA (Crino et al., 1999).However, the selection criteria for use of adjuvant chemotherapy in patients with stage I NSCLC have not been established, only 4% of patients may actually benefit from such adjuvant therapy, while most patients will suffer undesired toxicity or potentially fatal side-effects, without gaining benefit in survival and RESEARCH ARTICLE GOLPH3, a Good Prognostic Indicator in Early-stage NSCLC Related to Tumor Angiogenesis Ming Lu, Yu Tian, Wei-Ming Yue, Lin Li, Shu-Hai Li, Lei Qi, Wen-Si Hu, Cun Gao, Li-Bo Si, Hui Tian* quality of life (Arriagada et al., 2004;Winton et al., 2005;Pignon et al., 2006).Therefore, there is an urgent need to identify novel biomarkers that will help select the patients with high chance of lung cancer recurrence and provide better prognosis and individualization treatment.
However, research studies have seldom studied the correlation between GOLPH3 expression and prognosis of Chinese patients with non-small cell lung cancer, specifically, the early-stage non-small cell lung cancer.Angiogenesis is an early event in carcinogenesis, and it plays an essential role in the processes of invasion and metastasis.Studies have shown that high MVD by CD34 is more closely related to poor survival than the other neoangiogenetic factors in stage IB-IIA NSCLC, because that these factors are more closely related to the metastatic process (Yano et al., 2000;Mineo et al., 2004).The correlation between GOLPH3 expression and prognosis suggested that GOLPH3 contribute to NSCLC invasion and metastasis by contributing to increased angiogenesis.To the best of our knowledge, this is the firstly made the study of relationship between GOLPH3 and cancer angiogenesis, and we believe that we have made some valuable progress.
Thus, to determine the role of GOLPH3 protein in the biologic behavior of early-stage NSCLC, we employed immunohistochemical method to examine GOLPH3 protein expression in clinical NSCLC samples, and we analyzed the relationships of GOLPH3 expression with variable clinicopathologic features and patient prognosis.Moreover, we investigated the possible angiogenic role by comparing GOLPH3 protein expression with intratumoral microvessel density (MVD) and assessed the independent prognostic factors affecting long-term survival.

Materials
A total of 116 consecutive patients who were diagnosed with Stage Ⅰ NSCLC and treated with pulmonary lobectomy plus regional lymph node dissection from January 2000 through December 2001 at the Department of Thoracic Surgery, Qilu Hospital, were studied retrospectively.And they all had the clear pathological diagnosis without preoperatively radiotherapy and chemotherapy, and had no distant metastases.The data on their clinicopathologic features and follow-up were complete.And 57 cases were taken more than 5 cm from the tumor margin of normal lung tissues as a negative control.
The clinical characteristics of the patient are summarized in Table 1.For all patients, histological type and grade of cancer cell differentiation were reevaluated and determined by the classification system of the World Health Organization modified in 2004, and postsurgical pathological staging was determined based on the international staging system.Clinical follow-up data was available for a minimum of 5 years or until death.Informed consent was obtained from all patients and this study was approved by the Ethics Committee of Qilu Hospital (Shandong, China).

Immunohistochemistry
All specimens were collected during the surgery, fixed by 10% formalin and embedded in paraffin.The tissues were cut as 4 μm serial sections, and then deparaffinized using xylene and rehydrated through an ethanol series to water.High-temperature antigen retrieval was carried out in citrate buffer for 25 min in a microwave oven.Then the endogenous peroxidase enzyme activity was blocked using 3% H2O2 in methanol for 25 min at room temperature.The slides were then incubated with primary rabbit anti-GOLPH3 polyclonal antibody (Abcam) and rabbit anti-CD34 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4°C in a high humidity chamber, followed by incubation for 45 min at 37°C with biotinylated secondary antibodies and streptavidin-peroxidase complex.Finally, a 3, 30-diaminobenzidine solution was added, and the slides were counterstained with hematoxylin and mounted with neutral balsam.For negative controls, sections were incubated with PBS instead of the primary antibodies.

Evaluation of immunohistochemical staning
All sections were reviewed independently by three independent observers blinded to all clinical and pathologic information.Discordant cases were resolved by choosing the value consistent between two observers or the average of the scores.A reproducible semi-quantitative method that considered both staining intensity (0, negative; 1, weak; 2, moderate and 3, strong) and the percentage of positively stained cells (0, 0-5%; 1, 6-25%; 2, 26-50%; 3, 51-75%; 4, >76%) was adopted (Vermeulen PB et al., 2002) The staining index (SI) was calculated by multiplying the product of the staining intensity score and the proportion of positive tumor cells.For intratumoral microvessel density (MVD), microvessels were recorded by counting CD34 positively stained endothelial cells as descripted previously.The microvessel count (MVC) was determined independently by two pathologists in each case.Three representative areas of dense neovascularization were selected under low microscope power (×10 objective lens and ×10 ocular lens) and then vessels were counted at higher magnification (×20 objective lens and ×20 ocular lens).The average of the counts in three fields was recorded.Large vessels with thick, muscular walls were excluded, implementation of the calculation results are rounded.
The cutoff value for high and low expression was determined based on a heterogeneity value measured through log-rank statistical analysis with respect to overall survival (Dominik et al., 2005).

Statistical Analysis
The association between GOLPH3 expression and clinicopathological variables was plotted by the chi-square test.The correlation between intratumoral MVD and a patient's clinicopathologic factors and GOLPH3 protein immunoreactivity was analyzed by nonparametric test (Mann-Whitney U test or Kruskal-Wallis H test). Kaplan-Meier method was used to calculate the survival curves, and log-rank test was used to compare the difference between the survivals of patient subgroups.Multivariate Cox regression analysis was used to identify significant DOI:http://dx.doi.org/10.7314/APJCP.2014.15.14.5793 GOLPH3, a Good Prognostic Indicator in Early-stage NSCLC Related to Tumor Angiogenesis independent prognostic factors.Differences between groups were considered significant for P value<0.05.All statistical analyses were performed with SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA).

Expression of GOLPH3 in Early-Stage NSCLC and its relation to clinicopathologic profiles
Immunohistochemical staining for GOLPH3 was performed in 116 tumors and 57 normal lung tissues.As shown in Figure . 1 A and B, immunohistochemical analysis showed that the GOLPH3 protein was immunostained in the cancer cell cytoplasm.The correlation of GOLPH3 overexpression with clinicopathologic features was analyzed.Table 1 summarized the association of GOLPH3 expression with various clinicopathological features of early-stage NSCLC tissues.However, we observed no statistically significant correlation between GOLPH3 protein expression and any clinicopathological features of NSCLC tissues (p> 0.05, Table 1).The mean value of GOLPH3 overexpression in 116 early-stage NSCLC tissues was 58.62 %, significantly higher than normal lung tissues (42.11%, p= 0.041; Table 2).

Correlation between MVD and clinicopathologic factors of Early-Stage NSCLC
Intratumoral MVD was quantified by counting CD34positive endothelial cells in cancer tissues (Figure .1D), and the staining intensity of MVD ranged broadly from 12 to 79 microvessels/ 200×magnification field (Median: 47.69/HPF).Meanwhile, we found that MVD was significantly correlated with invasion depth (p=0.031,Mann-Whitney U test), while no significant correlations were observed between MVD and other clinicopathologic factors (p>0.05,Table 1).

Correlation of GOLPH3 Protein with MVD
Statistical analysis demonstrated that there was significantly more MVD in tumors with GOLPH3 protein high expression than that in those with GOLPH3 protein low expression (p<0.001,Mann-Whitney U test; Figure .2).

Univariate and multivariate survival analysis
In order to investigate the prognostic implications of    WhitneyUtest demonstrated that tumors with GOLPH3 protein high expression showed significantly higher intratumoral MVD than tumors with GOLPH3 protein low expression (P< 0.01)

5796
GOLPH3 expression in overall survival and progressionfree survival of early-stage NSCLC, the detailed clinical information of all 116 NSCLC patients in high GOLPH3 expression and low GOLPH32 expression groups was reviewed.At the time of this analysis, with a median follows up of 52.00 months (range 15-82 months), 53 patients (45.69%) were alive and 44 patients (37.93%) remained free of disease.As determined by the log-rank test, patients with positive GOLPH3 expression showed a more unfavorable prognosis than those with no over expression (p=0.001, Figure 3A).Meanwhile, the patients with high GOLPH3 expression have a high rate of tumor recurrence (p=0.001, Figure 3B).Moreover, the univariate analysis revealed that the high MVD predicted poorer overall and progression-free survival of NSCLC patients (p =0.019 and p = 0.014, resp., Table 3, Figure 3C and D).Furthermore, the multivariate analyses identified GOLPH3 protein overexpression and high MVD as independent prognostic factors for progression-free survival (p= 0.038 and p= 0.044, resp., Table 3).However, only the GOLPH3 protein overexpression retained its significance as an independent prognosticator for distasteful overall as well as progression-free survival (p= 0.043, Table 3).We further analyzed the prognostic significance of GOLPH3 protein in selective patient subgroups stratified according to the invasion depth of NSCLC.Univariate analysis demonstrated that the overall 5-year survival and progression-free 5-year survival rate of patients with GOLPH3 protein high expression was significantly lower than that of the remaining patients among T2 (p= 0.001 and P=0.002, respectively; Figure . 4).However, this trend was not found in patients with T1 invasion depth (p=0.290 and p=0.183, respectively; Figure .4).

Discussion
The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cell-signaling pathways commonly deregulated in human cancers.This has led to the prediction that mTOR inhibitors may be useful in oncology, and derivatives of one such molecule, rapamycin (from which mTOR derives its name), are currently in clinical development.And it has been reported that overexpression of GOLPH3 promotes cell transformation   doi.org/10.7314/APJCP.2014.15.14.5793 GOLPH3, a Good Prognostic Indicator in Early-stage NSCLC Related to Tumor Angiogenesis via enhancing the activity of the serine/threonine kinase mTOR.Recent studies have found that the development of a variety of solid tumors abnormally activation is related with mTOR signal transduction pathways in breast cancer, glioma and so on, so the GOLPH3 has become an important target for cancer therapy (Guertin et al., 2007;Scott et al., 2009).However, few studies have investigated the expression and significance of GOLPH3 in earlystage NSCC, especially for the prognosis of early-stage NSCLC.In the present study, we focused on early-stage NSCLC cancer patients to determine the correlation of GOLPH3 expression with clinicopathological parameters and survival.
Furthermore, we found that high GOLPH3 expression and high MVD were strongly associated with decreased overall survival and progression-free survival in earlystage NSCLC patients.In multivariable analysis, GOLPH3 expression and MVD retained its independence factors in predicting progression-free survival for NSCLC patients, suggesting that GOLPH3 protein and MVD may be potential prognostic factors for the relapse of early-stage NSCLC patients.However, in multivariate analysis, only GOLPH3 expression could independently and significantly predict overall 5-year survival.These findings provide evidence that GOLPH3 could be regarded as a biomarker for predicting the outcome of early-stage NSCLC patients.
In this study, our results showed that there was no significant correlation between GOLPH3 expression and the clinicopathological features of early-stage NSCLC.However, high MVD was significantly associated with T status in early-stage NSCLC, but not associated with other clinicopathological features.To a certain extent, T stage is a critical process of tumor development, and the difference in the correlation between MVD and clinicopathological features may reflect that the microvessel density is an important aspect of tumor development.And angiogenesis plays a vital role in tumor initiation, progression and metastasis, and it has been considered one of the hallmarks of cancer (Butler et al., 2010;Hanahan et al., 2011).We studied the relationship between the expression of GOLPH and MVD.Notably, our results showed that the overexpression of GOLPH3 significantly associated with increased angiogenic activity measured as intratumoral MVD, suggesting that GOLPH3 plays crucial role in NSCLC tumorigenesis by the induction or/and promotion of tumor angiogenesis.Furthermore, the patients with positive GOLPH3 expression showed a more unfavorable prognosis than those with no over-expression in T2 invasion depth of NSCLC, not in T1 stage.These findings suggest that the carcinogenicity of GOLPH3 protein may be an independent carcinogenic factor, working along with the tumor progression gradually.Although there is no definitive finding on the regulatory mechanism of angiogenesis by GOLPH3, our results highlight the potential role of GOLPH3 in tumor angiogenesis.
Taken together, our data support the assumption that GOLPH3 protein over-expression is common in earlystage NSCLC and significantly correlated with tumor angiogenesis.Moreover, it is an independent prognostic factor for early-stage NSCLC patients.

Figure 1 .
Figure 1.Expression Pattern of GOLPH3 in lung cancer tissues.(a) High GOLPH3 expression in lung adenocarcinoma.(b): High GOLPH3 expression in lung squamous cell carcinoma tissues.(c): Negative GOLPH3 expression in NSCLC tissues.(d): Intratumoral microvessels were stained as brown by the anti-CD34 monoclonal antibody in lung cancer tissues (magnification×400).

Figure 3 .
Figure 3. Kaplan-Meier Curves of Disease-Free and Overall Survival Stratified According to the Status of GOLPH3 Protein Expression and MVD Figure 4. Kaplan-Meier Survival Curves of Patients Stratified According to Invasion Depth.

Table 1 . Correlations of Clinicopathologic Variables of Early-stage NSCLC with GOLPH3 and MVD
*P a Chi-square test; P b Nonparametric test (Mann-Whitney U test or Kruskal-Wallis H test)