Increased STIP1 and Hsp90 correlate with progression and prognosis of lung adenocarcinoma

Background: Stress-inducible phosphoprotein 1 (STIP1) and heat shock protein 90 (Hsp90) have been found to be correlated with malignant tumors. The aim of this investigation was to study the relationship between their expressions and lung adenocarcinoma (LAC). Methods: The expressions of STIP1 and Hsp90 in LAC cells and tissues were tested by immunohistochemistry and western blot; the correlation between their expressions and clinicopathological parameters of LAC was analyzed by survival analysis and multiple regression analysis. Results: Expressions of STIP1 and Hsp90 were higher in A549 cells and LAC tissues than that in 16 human bronchial epithelial cells (16HBE cells) (P < 0.05) and adjacent normal lung tissues (P < 0.05). The expression of STIP1 and Hsp90 in LAC showed a strong positive correlation (P < 0.05) and significantly correlated with lymph node metastasis (P < 0.05), advanced clinical stage (P < 0.05) and shorter survival (P < 0.05) of LAC. Conclusions: Increased expressions of STIP1 and Hsp90 were closely related to malignant biological behavior of LAC, suggesting that they could be used as potential biomarkers and prognostic indicators for LAC.


Introduction
Lung cancer is one of the most malignant tumors with the fastest growth in morbidity and mortality and the greatest threat to population health and life [1]. In the past 20 years, the incidence of lung cancer has been reported to increase significantly in China [2] and the incidence of lung adenocarcinoma (LAC) has exceeded that of lung squamous cell carcinoma (LSCC) [2]. Clinical studies show that LAC is closely related to specific carcinogenic driver genes such as epidermal growth factor receptor (EGFR), anaplasticlymphoma kinase (ALK), mesenchymal to epithelial transition factor (MET), rearranged during transfection (RET), and proto-oncogene tyrosine-protein kinase ROS (ROS1). Structural changes (mutation, overexpression or rearrangement) of these specific oncogenes can trigger and promote the development of LAC cells [3][4][5]. So far, it has been known that lung cancer patients associated with driving genetic mutations have reached 62% of the total number of lung cancers [6,7]. In any case, it is believed that there are more unknown driving genes associated with the development of LAC that have not been discovered [8].
Although the human genome map has been drawn, the function of the protein encoded by the gene is extremely complicated, so the study of protein function has become the focus of the post-genome era [9]. We previously screened a differential protein stress-inducible phosphoprotein 1 (STIP1) based on isobaric tags for relative and absolute quantification (iTRAQ) in combination with two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) and found that its expression was significantly higher in LAC cells than in 16 human bronchial epithelial cells (16HBE cells). The STIP1 gene is located in the human chromosome 11q13 coding region and consists of 14 exons with a protein molecular weight of 63 kDa, which is located near many cancer-related genes [10]. It is reported that up-regulated STIP1 promotes the growth, colony formation and migration of liver cancer cells [11] and high STIP1 expression is correlated with advanced T stage of colorectal cancer [12]. Heat shock protein 90 (Hsp90) plays an important role in protein folding, subunit assembly, and regulation of cell growth and survival [13,14] and has been found to be highly expressed in some solid tumors, which is closely related to tumor progression and prognosis [15,16]. The purpose of this study was to quantify the difference in expressions of STIP1 and Hsp90 in LAC cells and tissues, and to verify their associations with LAC clinical parameters.

Material And Methods
Cell culture

Western blot
The cultured A549 cells were lysed with RIPA lysate to extract total cellular protein. The BCA method was used to determine the protein concentration of each sample. After the boiling water bath denaturation treatment, an equal amount of total protein was subjected to polyacrylamide gel electrophoresis. After electrophoresis, the total protein was transferred to a PVDF membrane. After the PVDF membrane was blocked with a 10% skim milk powder solution, the corresponding primary antibody was added and incubated at 4°C overnight. The PVDF membrane was rinsed and then incubated with a secondary antibody at 37 °C for 45 min. The PVDF membrane was illuminated by ECL method and was exposed in a dark room with X-ray film.
Tissue microarray (TMA) construction The tissue sections were observed and labeled under light microscope. Tissue array instrument was used to locate and drill a hole with a diameter of 1mm on the recipient wax block. A tissue column with a diameter of 1 mm was drilled from the tumor tissue and inserted into the hole of the recipient wax block. The prepared tissue chip wax block was incubated in a 37 °C incubator for 1 h. After cooling at room temperature, it was cut into 4 μm thick tissue sections and placed in an oven at 60 °C overnight. The sections were subjected to HE staining to see if the samples were accurate, and the qualified sections were kept at -4 °C for use.

Immunohistochemistry (IHC)
The expressions of STIP1 (dilution concentration of mouse anti-human STIP1 polyclonal antibody, 1:500; Abcam) and Hsp90 (dilution concentration of mouse anti-human Hsp90 polyclonal antibody, 1:300; Boster) in tissues were detected by immunohistochemical staining. A positive expression of STIP1 in thyroid cancer was used as a positive control for STIP1 and a positive expression of Hsp90 in breast cancer was used as a positive control for Hsp90. PBS buffer instead of primary antibody was used as a negative control.
The expressions of STIP1 and Hsp90 were stained as tan and brownish yellow, which were distributed in the cytoplasm of LAC cells. The semi-quantification analysis of IHC were performed by the following criteria: 0 for tumor cells without staining; medium staining was 1 point; more than 75% of tumor cells were stained for 2 points; more than 1 point is considered a positive expression [17].
Statistical analysis SPSS 20.0 software was used for statistical analysis. The chi-square test and Fisher's exact test were used for comparison of counting data between groups. The one-way ANOVA was used to compare the measurement data. Correlation analysis between the enumeration data was performed by the cruskal-wallis rank sum test. Kaplan-meier method was used to analyze the survival data. Bilateral test was taken and P < 0.05 was considered statistically significant.

Results
Expression levels of STIP1 and Hsp90 in A549 cells are higher than that in 16HBE cells A Western blot method was used to determine the expression levels of STIP1 and Hsp90 in A549 and 16HBE cells, we found that the expressions of STIP1 and Hsp90 in A549 cells were higher than that in 16HBE cells ( Figure 1A). Quantitative analysis suggested that the expression levels of STIP1 (0.88 ± 0.17) and Hsp90 (0.79 ± 0.27) in the A549 cells were higher than that in 16HBE (0.46 ± 0.13 for STIP1; 0.52 ± 0.19 for Hsp90, P< 0.001) ( Figure 1B).

Multiple regression analysis of clinical parameters
and overall survival of patients with LAC As shown in Table 6, a total of 8 parameters were included in the analysis, including

Discussion
Lung cancer is one of the major diseases that cause cancer-related death. Among them, LAC is the most common pathological type, and it has gradually increased in recent years [1]. Studies have shown that some gene mutations and protein expression abnormalities are closely related to the occurrence and development of LAC, and some molecular targeted drugs have been used in clinical practice [3,18,19]. STIP1 is a is an adaptor protein that combines heat shock protein 70 and heat shock protein 90 to modulate biological effects such as transcription, translation, and protein folding, and promotes the growth of tumor cells [12,20,21]. According to a recent meta-analysis, the high expression of STIP1 was related to lymph node metastasis, advanced TNM stage and shorter survival of patients compared with that of tumor tissues with low STIP1 expression, which was expected to be a molecular marker of malignancy or provide new ideas for the development of targeted drugs [22]. However, the malignant tumors included in the meta-analysis have only ovarian cancer, liver cancer, gastric cancer, thyroid cancer, and colon cancer. This also reflects the current lack of research on the expression pattern of STIP1 in LAC. In our study, we investigated the association and prognostic significance of STIP1 and Hsp90 with LAC clinical features by methods of molecular biology and biostatistics.
We first used the Western blot method to determine the expression patterns of STIP1 and Hsp90 in A549 and 16HBE cells and found that the expressions of STIP1 and Hsp90 in A549 cells were higher than that in 16HBE cells. This finding further validated our previous findings in proteomics, namely STIP1 expression was significantly higher in LAC cells than in 16HBE cells. And some studies have observed that the expression of STIP1 is increased in other tumor cells and tissues, and suggests that STIP1 expression plays a role in the occurrence, invasion and metastasis of malignant tumors [23, 24]. Next, we used the method of IHC to detect expression patterns in STIP1 and Hsp90 in LAC tissues and adjacent normal lung tissues. Similarly, we found that the expressions of STIP1 and Hsp90 in LAC tissues were higher than that in adjacent normal lung tissues. These results from our study suggest that STIP1 and Hsp90 may play an important role in the occurrence and development of LAC, and the synergistic increase of the two may be risk factor for LAC.
Hsp90 has been found to be highly expressed in the serum and tissues of lung cancer patients and up-regulation of Hsp90 is related to the occurrence, development and outcome of lung cancer [25]. We further analyzed the association between the expression of STIP1 and Hsp90 and various clinical parameters of LAC and found that the expression of STIP1 was associated with poor differentiation, lymph node metastasis and advanced stage of LAC and that the expression of Hsp90 was related to lymph node metastasis and advanced stage of LAC. Lymph node metastasis and poor differentiation are the malignant biological characteristics of tumors and are often used to determine the treatment of tumors and to predict the prognosis of tumors [26][27][28]. Previous studies suggest that STIP1 is elevated in patients with lymph node metastasis and advanced clinical stages, and indicate that STIP1 expression may serve as a potentially valuable biomarker for cancer [11,12,[20][21][22]24]. As a chaperone, Hsp90 can activate signals of some cancer- Based on the phenomenon that STIP1 and Hsp90 have increased synchronously in LAC, we further analyzed the correlation between the two in LAC tissues and found that they had a significant positive correlation, indicating that there was an intrinsic link between their expressions in LAC. STIP1 has been shown to act as an adaptor which can guide Hsp90 to Hsp70 in cytoplasm to form a customer protein complex and ultimately regulate its molecular chaperone activity, which play complex roles in RNA splicing, signal transcription, protein folding, signal transduction and cell cycle regulation [10,31].
Overexpression of STIP1 in cancer cells is associated with regulation of the JAK2-STAT3 signaling pathway. The interaction of STIP1 and HSP90 promotes maturation of the JAK2 protein and forms a scaffold complex that transduces JAK2-STAT3 signaling. However, inhibiting the expression of STIP1 can block the interaction between STIP1-Hsp90 and showed an antitumor effect [23]. From our study, we deduce that STIP1 and Hsp90 have synergistic effects in the occurrence and development of LAC, and the common increase in both may play a role in the progress of LAC. During our follow-up, 64 out of 73 patients were followed up with complete survival data. We found that patients with expressions of STIP1 and Hsp90 had a shorter survival than those without expressions, which means that

Competing interests
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Consent for publication
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Ethics approval and consent to participate  TNM staging: T = primary tumor; N = regional lymph nodes; M = distant metastasis; ★ , The 8th edition of the TNM classification for non-small lung cancer.