High Expression Of Yes-associated Protein Promotes Tumor Development In Non-small Cell Lung Cancer (NSCLC)

BackgroundWith increased industrialization and population aging, lung cancer has been identified as the tumour with the highest morbidity and mortality among all kinds of malignant tumors. During the past decades, there has been a great development in the treatment for lung cancer, and even the mortality rate of lung cancer in male patients has declined slightly. However, the five-year survival rate of non-small cell lung cancer (NSCLC) has remained less than15%, and most patients died in the first year after diagnosis. The treatment of lung cancer has reached a bottleneck stage. So it is the issue must be solved concerning how to early diagnose and how to find the main mechanism of inducible resistance during chemotherapy for lung cancer.Hippo pathway is a newly discovered cell signal transduction pathway in recent years. It contains4components:Wts (also known as lats)、Hpo、Sav (also known as shar-pei)、Mats。It can downregulate the expression of Drosophila inhibitor of apoptosis protein1(DIAP1)、bantam、Cyclin E (CycE) to regulate the balance of cell proliferation and apoptosis to inhibit the cell overgrowth, and to regulate the characterization of contact inhibiton bewteen cells. Thus, Hippo pathway can regulate the volume and size of organs.Yes-associated protein (YAP) was first discovered and named in1994, it is a multifunctional intra-cellular junction protein and transcriptional co-activator. YAP plays a signal transduction and gene transcription regulatory role in normal cells. Recent studies have shown that YAP is the target molecules in Hippo pathway. The abnormal regulation of Hippo pathway can lead to tumors, and YAP is the key factor of this pathway.Recent researches have shown that YAP was over-expressed in the liver cancer, gastric cancer, ovarian cancer, prostate cancer, colon cancer and esophagus cancer tissues, indicating that YAP may be involved in tumor development and progression as carcinogenic factors. Studies have found that to modulate the cell density through the Hippo pathway can inhibit the expression of transcriptional co-activator YAP. However, the suppressor of Lats tumor protein kinase phosphorylation can lead to inactivation of YAP oncoprotein, and inactivation of the YAP phosphorylation in vivo will diminish the role of growth-promoting. On the contrary, YAP overexpression can cause excessive cell proliferation by overcoming the contact inhibition between cells. They also found that YAP expression was also increased in human liver cancer, gastric cancer, ovarian cancer, prostate cancer, colon cancer and esophagus cancer; and further showed that YAP regulated the cell proliferation by the Hippo pathway which may play an important role in tumors development. Someone else have found that YAP showed high expression in liver cancer and was related to the serum AFP expression and the degree of tumor differentiation. YAP was an independent prognostic indicators of related overall survival and disease-free survival rate in liver cancer patients. And YAP could enhance the transformed phenotype of ovarian cancer cell lines and can confer the resistance of ovarian cancer to chemotherapy drugs. Further studies indicated that high expression of YAP correlates with poor patient prognosis.Previous studies showed that the cellular response to chemotherapy is critically dependent on functional p73, and that c-Jun promotes cell death by stabilizing p73following cisplatin treatment. Danovi et al identify that YAP acted as a novel and critical downstream effector of c-Jun-mediated apoptosis following cisplatin treatment. They found that YAP contributed to cell death by stabilising p73protein by competing with the E3ubiquitin-protein ligase Itch for p73binging. These conclusions are similar to the previous reports.In summary, YAP was found overexpressed in a variety of tumor tissues and it was related to the differentiation, metastasis, chemotherapy sensitivity, prognosis etc, which suggested that YAP may be an important oncogene and it may play a key role in tumorigenesis and tumor development. But, a few reports suggested that YAP in breast cancer act as a tumor suppressor. Our study aimed to observe the expression of YAP in non-small cell lung cancer and to explore its possible role in tumor development and progression.Objective 1. To detect the expression of YAP in non-small cell lung cancer with immunohistochemical staining.2. To detect the expression of YAP in non-small cell lung cancer with Western blot.3. To detect the expression of YAP in non-small cell lung cancer with RT-PCR.4. To observe the expression of YAP in different pathological types, different stages, and different differentiation to suggest the possible role of YAP in the tumorigenesis and tumor development.Methods1.40patients with primary lung cancer and received the surgical treatment were selected from January2009to January2010, in thoracic surgery department of Shandong Provincial Hospital. None of these patients received chemotherapy or radiotherapy.2.250mmm3tumour tissue samples and adjacent tissues samples (the distance to the tumour’s visible edge>2cm) were taken respectively and immediately placed in-80℃refrigerator for spare.3. Among40patients,28were males,12were females and patients aged from40to75years old, with a median age of57years old.4. According to the2002international staging system by Union for International Cancer Control (UICC), there were17patients with stage Ⅰ (4cases with la,13cases with Ib),12with stage Ⅱ (1case with Ⅱa,11cases with Ⅱb),11with stage Ⅲ (8cases with Ⅲa,3cases with Ⅲb).5. According to pathological types, there were18squamous cell carcinoma,20adenocarcinoma,1squamous adenocarcinoma and1large cell carcinoma.6. According to lymph node metastasis, there were18cases with No,12cases,10cases with N2.7. According to the differentiation,4cases were well differentiated,29cases were moderately differentiated and7cases were poorly differentiated.8. All procedures of this study complied with the ethics standards established by Shandong Provincial Hospital Commission responsible for the human experiment and obtained its approval, informed consent was signed.9. To detect the expression of YAP in non-small cell lung cancer with immunohistochemical staining.9.1Specimens taken from patients were fixed in4%formalin for24hours, after gradiently dehydrated and paraffin-embedded, pathological section was done with4um slice thickness.9.2Put the sections in3%H2O2at room temperature for20minutes to remove endogenous peroxidase; washed3times for5minutes each time by distilled water.9.3In0.01M citrate buffer (PH6.0), antigen retrieval was performed in the microwave for10minutes; after natural cooling, washed three times using PBS (PH7.4) for5minutes each time.9.4Drop20ul YAP first mouse anti-human antibody diluted with PBS (1:200, Shanghai Rui Qi Biotechnology Co., Ltd.) in each slice, drop20ul PBS in the negative controlled sections.9.54℃incubation in a humidified chamber overnight; washed three times for5minutes each time using PBS (PH7.4).9.6In each slice drop20ul HRP-conjugated goat anti-mouse secondary antibody diluted with PBS (1:200dilution, Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd.); PBS washed3times for2minutes each time.9.7Drop diaminobenzidine (DAB) chromogenic reagent (Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd.), control the time under microscope; wash with PBS, nuclei stained with hematoxylin; gradient alcohol dehydration, vitrification by dimethylbenzene; mounted with neutral gum.9.8In Each slice select5×400visual fields, image analysis system Leica QwinV3was applied to calculate the average gray value of positive area in each field.10. To detect the expression of YAP in non-small cell lung cancer with Western blot10.1SDS lysis buffer (1.74mg/ml (lOmmol/L) PMSF (phenylmethylsulfonyl fluoride):PMSF0.174g, isopropanol100ml, dissolved and stored in a1.5ml microcentrifuge tube, save under-20℃) was used to extract the total protein from the fresh NSCLC tissues:).10.2The30ug of total protein for each sample was fractionated by10%SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (GE Healthcare).10.3YAP protein was detected with monoclonal mouse anti-human YAP (1:500dilution; material Technology Co., Ltd. Shanghai Ruiqi Biotechnology Co., Ltd.).|3-actin protein served as an internal reference.10.4After DAB coloring TotallabTL120gel analysis system was used to analyze the integrated optical density (IOD) of various samples to obtain the relatively calculated amount of YAP protein.11. To detect the expression of YAP in non-small cell lung cancer with RT-PCR.11.1RNAiso PLUS kit (TaKaRa Company, China) was applied to extract total RNA from each sample (about50mg).11.2PrimeScript1st Strand cDNA Synthesis Kit (TaKaRa Company, China) was applied for reverse transcription.11.3Premix Taq (?) Version2.0kit was used for PCR amplification. The system capacity was20ul. Reaction condition:. Before cycle started, the sample was preheated at94℃for3min. Each cycle was consisted of30cycles of30s at94℃,30s at64℃, and60s at72℃; It is30cycles totally,72℃,60s. β-actin served as an internal reference.11.4The YAP and P-actin primer sequences were: YAP forword:5’-AACTCGGCTTCAGCCATGA-3’ YAP reverse:5’-GCTACGCAGGGCTAACTCCTGT-3’ β-actin forword:5’-ATAGCACAGCCTGGATAGCAACGTAC-3’ β-actin reverse:5’-CACCTTCTACAATGA GCTGCGTGTG-3’11.5Amplified products were electrophoresed on a2%agarose gels. The relative expression levels were calculated by Totallab TL120gel analysis system.12. Statistical analysis Statistical analysis was performed using Statview4.5(SAS Institute, NC, USA). All values are expressed as the mean±standard error (SE) unless otherwise stated. The Chi-square-test was used to make the correlated analysis between YAP expression and the clinical and pathological factors of patients. Other data analysis used t-test. The significance was set at P<0.05。Results1. The expression of YAP in non-small cell lung cancer with immunohistochemical staining:The results showed that YAP was mainly expressed in the nucleus with only a small part in the cytoplasm.40cases of NSCLC tumors showed70.0%YAP-positive (28/40), of which32.5%(13/40) strongly positive,37.5%(15/40) weakly positive. The normal lung tissue is only7.5%(3/40) positive. The YAP expression was mainly in alveolar type Ⅱ epithelial cells.2. The expression of YAP in non-small cell lung cancer with Western blot:Western blotting results have also confirmed the result described above. In40cases of NSCLC cancer, the expression of YAP protein was significantly elevated in28cases. The elevated YAP expression in adenocarcinoma was more significant. This result indicated that YAP expression was significantly increased in NSCLC and was related with the increased gene transcription levels.3. The expression of YAP in non-small cell lung cancer with RT-PCR:The YAP expression in NSCLC tumor tissues was compared with the normal lung tissue in mRNA level. The results showed that the YAP mRNA in tumor tissues were significantly higher than the normal lung tissue, and this expression was elevated significantly in adenocarcinoma4. YAP expression related to clinical and pathologic factors in NSCLC:Further analysis of YAP immunohistochemistry results, and clinical and pathologic factors in NSCLC patients showed that the intensity of YAP expression correlated with tumor T stage, TNM stage and the degree of lymph node metastasis. In lymph node metastasis samples, the YAP expression was significantly higher than those without lymph node metastasis (P=0.0134). In late T stage, late TNM stage, YAP expression was also increased compared with early stage,(P=0.0117, P=0.0385), indicating that YAP was involved in the progression of NSCLC; There is no correlation between YAP expression and patient age, gender or tumor differentiation. The positive rate in adenocarcinoma patients was higher than squamous cell carcinoma patients. This results were similar to mRNA and Western Blot results (P=0.0415).Conclusions1. Our study showed that YAP in NSCLC cancer tissues was significantly higher than the pericancerous tissues in mRNA level; we also found that the YAP expression was elevated more significantly in adenocarcinoma.2. It indicated that YAP expression was significantly increased in NSCLC and was related with the increased level of gene transcription.3. YAP expression was mainly assembled in the nucleus, only a small part of the expression in the cytoplasm. Only a few YAP expression was found in the normal lung tissue alveolar type II epithelial cells.4. The levels of YAP expression had no relation with age, gender, tumor size and tumor differentiation, but positively correlated with tumor T stage, TNM stage, lymph node metastasis.5. The result of immunohistochemistry experiment also showed that YAP expression was significantly higher in NSCLC tumor tissues than the pericancerous tissues, and the higher expression was found more significantly in adenocarcinoma tissues. The above further proved that YAP may play a key role in the tumorigenesis and the development of NSCLC, the expression of YAP may become an early marker in the development of NSCLC.6. The detection of the YAP expression using immunohistochemistry technology may become an important molecular biology indicator of the tumorigenesis of NSCLC in clinical practice.