Correlation between expression of XB130, AKT and prognosis in non-small cell lung cancer patients

Objective To examine XB130 and AKT expression levels in the pathological tissues of non-small cell lung cancer (NSCLC) followed by analysis of their relationship with other common prognostic indicators.Methods A retrospective analysis of the clinical and pathological characteristics of 120 NSCLC subjects from August 1, 2014 to August 1, 2015 was conducted. The expression of XB130 and AKT was detected by immunohistochemistry. Correlation analysis of XB130 with various other factors was analyzed by the Chi-squared test. Relationship between XB130, AKT and patient overall survival (OS), as well as progression-free survival (PFS) was examined using Cox analysis.Results The XB130 expression was significantly correlated to tumor differentiation (P = 0.004), T staging (P = 0.045), lymphatic metastasis (P = 0.028), and AKT expression (P = 0.043). Univariate analysis of the Cox proportional hazard model revealed that high XB130 expression, lymphatic metastasis, T staging, and high AKT expression were adverse prognostic factors for NSCLC (P < 0.05). Besides, the multivariate analysis demonstrated that high XB130 expression, lymph node metastasis, and high AKT expression were the independent prognostic factors (P < 0.05).Conclusion XB130, AKT, and lymph node metastasis are independent risk factors for NSCLC prognosis. XB130 might affect patient prognosis through AKT. XB130 marker the and a candidate the treatment with

the multivariate analysis demonstrated that high XB130 expression, lymph node metastasis, and high AKT expression were the independent prognostic factors (P < 0.05).Conclusion XB130, AKT, and lymph node metastasis are independent risk factors for NSCLC prognosis. XB130 might affect patient prognosis through AKT.
XB130 may be a prognostic marker for the survival and a candidate for the treatment of NSCLC, with potential therapeutic prospects.

Background
Lung cancer is one major malignancy in the world [1,2], whose morbidity and mortality rates in China in 2015 were 733.3‰ and 610.2‰, respectively. More than 3 85% of lung cancers are NSCLC [3]. Although early NSCLC can be cured by surgery, about 70% of subjects show metastasis when diagnosed [3]. Moreover, drug resistance has emerged even in the presence of advanced lung cancer treatment, such as molecularly targeted therapy and immunotherapy (reference: Strategies for the treatment of non-small-cell lung cancer after drug resistance to EGFR-TKI). The prognosis of advanced NSCLC patients is poor, showing one-year average survival and lower than 20% 5-year survival rate [4][5][6][7]. For lung cancer patients, the currently proven carcinogens include smoking, air pollution, environmental factors, ionizing radiation, ras, myc gene family, etc. Immunohistochemical staining proceduresThe negative control group was also immunohistochemically stained. The procedure was the same as the experimental group, and only the primary antibody was substituted by the PBS buffer. Then the counting process was performed by two pathologists, and the tissue sections were counted separately by the double-blind method. When the results of the grouping were inconsistent, the third pathologist recounted and grouped. According to the result determination method, all the slices were grouped, and the obtained data was used for statistical analysis.

RBM3 staining
Five high-magnification fields were randomly selected, and 100 cells were counted.
The depth of coloration and the number of positive cells were determined. Staining intensity scores: colorless for 0, light yellow for 1, brown for 2, and tan for 3.
Positive cell percentage scoring criteria: 25% of positive staining cells were low expression, and >25% were high expression.
AKT stainingFive high-magnification fields were randomly selected, and 200 cells were counted. The staining intensity of cancer cells and the proportion of stained cells were comprehensively scored. The film was read by a pathologist in a blinded way. Once the cytoplasm and/or nucleus were stained, it was judged to be a positive cell. Staining intensity: unresponsive cells for 0, pale yellow for 1, brown yellow for 2, and sepia for 3. The stained cell percentage indicated: < 10% was 0, < 20% was 1 point, < 50% was 2 points, and ≥ 50% was 3 points. Staining intensity multiplied by stained cell percentage was used for evaluating the AKT level. The integral 0 to 2 and 3 to 9 were classified into negative and positive expression, respectively.
Statistical methodSPSS 21.0 software was used to perform the statistical analysis.
Correlation comparisons were conducted with the chi-square test. Cox regression analysis was applied for the survival analysis. P < 0.05 was statistically significant. expression is also high, with their expression levels significantly correlated.
XB130, AKT, and lymph node metastasis are independent prognostic factors affecting PFS and OSWe then performed the univariate analysis of Cox proportional hazard model ( Table 2). Overall survival (OS) and progression-free survival (PFS) in subjects having high XB130 level were significantly less than those in subjects having low XB130 level (P = 0.000), indicating that patients having high XB130 expression were more likely to have worse survival and prognosis and high XB130 expression is one of the adverse factors affecting the prognosis of NSCLC.
Significant differences in OS and PFS were discovered between subjects with and without lymphatic metastasis (P = 0.000). Subjects having lymphatic metastasis had worse prognosis than subjects without lymphatic metastasis, indicating that lymphatic metastasis affects the prognosis of NSCLC. Besides, OS (P = 0.002) and PFS (P = 0.003) were significantly different between subjects having low AKT level and those having high AKT level. Subjects with low AKT level had a lower risk, indicating that high expression of AKT is one of the factors affecting the prognosis of NSCLC. Taken together, high XB130 expression, lymphatic metastasis, and high AKT expression were adverse risk factors for NSCLC prognosis (P < 0.05).We then conducted the multivariate analysis of Cox proportional hazard model (Table 3).
Patients having high XB130 or AKT expression had a worse prognosis (P < 0.05), 7 indicating that high XB130 level and high AKT level were independent risk factors for the prognosis of NSCLC, which is not affected by age, gender, lymph node metastasis, T staging, smoking or not, and the degree of differentiation. When comparing to the patients with non-lymph node metastasis, those with lymphatic metastasis had a worse prognosis (P = 0.000), indicating that lymphatic metastasis is an independent prognostic factor, not affected by age, gender, smoking or differentiation. It was worth noting that the univariate analysis of Cox proportional hazard model demonstrated that T staging affected the prognosis of NSCLC, but multivariate analysis showed that T staging did not affect the NSCLC prognosis. The possible reason is that T staging has a lower impact on prognosis than lymph node metastasis, high XB130 expression, and high AKT expression, and the prognostic effect of T-stage is interfered by the confounders.

Discussion
Lung cancer is very malignant due to high morbidity and mortality. More proliferation, survival, invasion [9,13,14] but also in signal transduction [15].
Controlled by Rac and cytoskeleton [13], XB130 activates c-Src and PI3K/AKT pathway [14], which in turn regulates cytoskeleton [13]. These signaling pathways are critical for the lung cancer progression [16,17]. As an adapter protein, XB130 is crucial for signal transduction, cancer cell proliferation, survival, and tumorigenesis. level is significantly related to cell proliferation, angiogenesis, and poor prognosis in a variety of tumor patients [8][9][10][11][12]. However, there have been few studies about the clinical value of XB130 in cancers including NSCLC. To study the relationship between XB130 expression and prognosis of gastric cancer, 411 patients were selected as subjects [12]. They found that both mRNA and protein of XB130 were detected in normal gastric tissues. The OS was significantly shorter in subjects having high XB130 compared with those having low XB130. The XB130 expression predicts the sensitivity of tumors to several clinical drugs. 5-fluorouracil (5-FU), cisplatin, and irinotecan inhibited the activity of wild-type and XB130-knockout gastric cancer cells in a concentration-dependent manner, with cisplatin, irinotecan and 5-FU being more sensitive to XB130-knockout and wild-type cells, respectively.
In 5-FU treatment, the survival rate was higher in subjects having high XB130 level when comparing to those having low level, suggesting that the reduction in XB130 protein level is a prognostic factor for gastric cancer patients with short survival time, high recurrence rate, and chemotherapy response.

Ethics approval and consent to participate
Our study was approved by Qingdao Municipal Hospital (approval no. QSL2014015067). All patients provided written informed consent prior to enrollment in the study.

Consent for publication
Written informed consent was obtained from all participants.

Availability of data and materials
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they had no competing interests.

Funding
None.

Authors' contributions
Conception and design: LC.
Analysis and interpretation of data: XYZ.
Drafting the article: ZLH Revising it critically for important intellectual content: KG,YL,CYG,TLZ.   Figure 1 Immunohistochemical staining of XB130 and AKT in lung cancer tissues.