Clinical Relevance And Underlying Mechanism Of Altered Expression Of Proto-oncogene BCL11A In Non-small Cell Lung Cancer

BackgroundLung cancer is the leading cause of cancer related death in China,including non small cell lung cancer(85%) and small cell lung cancer(15%) Despite much advance in the early diagnosis and standard treatment, the prognosis of NSCLC patients is still poor with 5-year survival less than 15%[25]. Only 15-25% of all NSCLC patients are candidates for surgery. Moreover, even in this selected population, approximately 50% of the patients will relapse after complete resection. Conventional multimodality therapies (surgery, radiation and chemotherapy) have reached a therapeutic ceiling in improving the five year overall survival rate of non-small cell lung cancer (NSCLC) patients. With the emerge of translational medicine in recent years,many basic researchs had worked from bench to beside,resulting in exploring many target therapy drugs and getting challenging effects. The most representative example in lung cancer translational research is about EGFR mutation and TKI respons[2]. For NSCLC patients with sensitive mutation, TKI gefitinib and erotinib can prlong the progress free survival of advanced stage patients.[3,4].Unfortunately,these therapeutic strategies unavoidably induce drug resistance and result in therapeutic failure, ultimately due to poor understanding of the disease and its resistance to the therapy.Non-small cell lung cancer is characterized by a variable clinical course and known for its unpredictable behavior. Molecular epidemiological studies have provided evidence that this disease arised through different molecular mechanisms including hereditarily somatic variation of genome DNA, abnormal gene expression in transcriptional level and transcript splice,dysregulation of microRNA,and procedurely dysfunction of protein expression. However, these molecular alterations were not enough to explain the heterogeneity of NSCLC[5,6].More and more molecular abnormality be found associated with specific tumor cell cycle,tumor differentiation,cell migration,apotosis and other biological process[7]. Clarity specific mechanism of gene expression regulation can be helpful for understanding tumor biological activity,and for clinical interference of abnormal target.Activation of proto-oncogene act as an important mechanism in tumorigensis including lung cancer.Proto-oncogene code many growth factor and receptor maintain normal growth and apotosis via accurate regulation network in normal condition[8].Several mechanisms including point mutation[9], gene fusion and chromosome translocation[10],gene amplication[11] can result in abnormal activation of proto-oncogene, acquire stronger capacity of proliferation and survival and promote tumorigenesis. In lung cancer, amplified oncogenes are present on 1p (NRAS, MYCL1, PAX7),3q(EIF4G1, TP73L, PIK3CA),5p (SKP2),7p (EGFR),8q (PTK2, MYC), 11q (CCND1,BIRC2, BIRC3) and 20q (E2F1). Important role of EGFR, k-ras mutation and EML4-ALK fusion gene in lung cancer carcinogensis haved been validated. Different from tumor surpressor, oncogene function dominantly, can malignantly transform cell only one allele activiated. However, these molecular alterations were not enough to explain the heterogeneity of NSCLC. The concept of epigenetics offers a partial explanation of the phenomena,such as methylation of p16 gene associated with squamous cell carcinoma and prognosis of early stage NSCLC patients[14,15].These heritable changes in gene expression that are not due to any alteration in the DNA sequence[14]。The best-known epigenetic alterations are methylation and histone deacetylation, a striking epigenetic modification called microRNA play a important role in gene regulation[16]. MicroRNAs constitute a large family of small, approximately 21-nucleotide-long, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans,plants and mammals. Lin4 is the first microRNA founded in nematode C. elegan, thousands of microRNAs have been identified in human, drosophila et(microrna.sanger.ac.uk).The mature microRNA inhibit protein translation via binding to specific region of mRNA by perfect or incomplete complementary. Several microRNA had been implicated in carcinogenesis including leukemia,lymphoma,lung cancer and breast cancer.Deregulated microRNAs can upregulate the oncogene expression and downregulate suppressor, result in dysfunction due to mutation or copy number variation.To explore the post-translated regulation mechanism of tumor tumorigenesis have being a research focus of tumor molecular genetics.Many microRNAs contributed to lung cancer initation and development, Takamizawa identified that let-7 was downregulated in NSCLC and correlated with poor pgognosis.Johnson found Ras family including K-Ras,H-Ras and N-Ras, were the target gene of let-7, they also identified that let-7 can repress tumor growth and associated with prognosis.MiR-128 can inhibit EGFR protein expression and LOH of mir-128 correlated with Gefitinib response.More and more microRNAs have been found associated with lung cancer carcinogenesis and prognosis,but the accurate mechanisms of downstream regulation remained unclear.To the summary, predicting and validating the target genes of microRNA, and establishing the stable relationship between microRNA and gene regulation are an important and promising work.PurposeThis study found a BCL11A gene which have different expression level between NSCLC and adjacent non-cancerous tissues by high mRNA array screening. Second,we used real-time PCR to validate the mRNA array result, and link the BCL11A mRNA level and clinicopathological factors to explore the role of BCL11A in NSCLC initation and development. Lastly, we investigated the mechanism of abnormal BCL11A expression turned to copy number and microRNA regulation.Experiment Design1.Surgical specimens were obtained with informed consent from 114 NSCLC patients (81 males and 33 females) who underwent potentially curative surgery at the Gangdong General Hospital (GGH) between 2003 and 2006. This study was approved by the Institutional Review Board (IRB) of Guangdong General Hospital. We found a ectopic BCL11A gene between NSCLC and adjacent non-cancerous tissues by mRNA array.2.To validate this different expression,we detect BCL11A expression both in mRNA and protein levels, and link BCL11A level with patient's clinicopathological factors and prognosis.3.To investigate the mechanism of BCL11A deregulation in NSCLC,we performed aCGH to evaluate BCL11A gene copy number variance.4.A non-coding RNA called microRNA can regulate gene expression post-transcriptionally and contribute lung cancer tumorigenesis. This study analysized the different microRNA expression of 37 paired NSCLC and adjacent non-cancerous tissues via Agilent human microRNA expression array. We found several microRNAs which are downregulated in NSCLC, on the other hand, we search potential microRNAs that regulated BCL11A protein and conduct intersection set.5. Exploring the fuction of these microRNAs and validate the relationship between microRNAs and BCL11A gene.Result1.BCL11A gene was upregulated in NSCLC based on Affymetrix GeneChip(?) platform,the average folds were 3.9.2.To validate the up-regulated BCL11A expression in NSCLC, Taqman(?) real time qRT-PCR assays were used to detect the same set of total RNA samples used in Affymetrix GeneChip(?) tests. Again, BCL11A mRNA levels were found significantly different between 114 NSCLC tissues and 42 adjacent normal tissues, with up- regulation in cancer tissues (P=0.004). In the 42 pairs of cancer versus non-tumor adjacent tissues, BCL11A expression at mRNA level was also up-regulated in cancer tissues(P=0.024). BCL11A mRNA have a minus relationship with disease stage (P=0.040) and lymph node status(P=0.013). Patients with an early disease stage and no lymph node involvement have a higher BCL11A mRNA expression. Furthermore, high BCL11A mRNA levels correlate with better overall survival and longer disease free survival.Cox analysis result demonstrated that BCL11A mRNA were an independent prognosis factor of desease free survival for early stage NSCLC patients.3.Expression of BCL11A at the protein level was investigated in 113 NSCLC tissues and 25 non-cancerous adjacent lung tissues by immunohistochemistry. BCL11A protein staining was not found in the 25 adjacent normal lung tissues.80 out of 113 NSCLC patients had positive staining for BCL11A protein. The positive expression rates in adenocarcinoma, squamous carcinoma and large cell carcinoma were 63.2% (43/68),85.7%(31/36) and 66.7%(6/9) respectively. Interestingly, BCL11A localized predominantly in nucleus of squamous cell carcinoma, however, in adenocarcinoma, the immunohistochemical staining was detectable predominantly in cytoplasm of cancer cells. Subsquently,we linked BCL11A protein level with clinicopathological factors and found that BCL11A protein levels BCL11A mRNA have a minus relationship with disease stage (P=0.029) and lymph node status(P=0.029). Patients with an early disease stage and no lymph node involvement have a higher BCL11A protein expression. These results coincide with previous mRNA results.Similarily, high BCL11A protein levels correlated with better overall survival and longer disease free survival. Patients with higer BCL11A protein expression had longer overall survival(P=0.051),escepically for early stage patients(P=0.028). Cox analysis result demonstrated that BCL11A protein levels were an independent prognosis factor of desease free survival for NSCLC patients [P=0.002, HR 0.396,95%(CI) 0.219-0.715]. Moreover, we found that BCL11A protein levels can not only predict disease free survival but also predict overall survival in early stage NSCLC patients,however, there were not correlation between BCL11A protein and prognosis in advanced stage patients.5.To investigate the mechanism of BCL11A deregulation in NSCLC,we performed aCGH to evaluate BCL11A gene copy number variance. The result indicated that BCL11A gene ampilified frequently in squamous cell carcinoma,but not in adenocarcinoma or large cell carcinoma,the normalized intensity of probes in squamous,adenocarcinoma and large cell carcinoma were 0.345,0.026 and 0.230 respectively. We also associated BCL11A gene CNV with BCL11A mRNA level, the result suggested BCL11A mRNA level had a positive correlation with BCLl 1A CNV in squamous cell carcinoma (rs=0.346, P=0.041).These data suggest that BCL11A gene overexpression in squamous cell carcinoma may result from genomic CNV, but the mechanism for adenocarcinoma and large cell carcinoma remain unclear.6. Meanwhile, this study compared microRNAs profiles between 37 NSCLC tissue and adjacent non-cancerous tissues by Agilent microRNA array. The result demonstrated many microRNAs including let-7 family,mir-30 cluster,mir-34 family and mir-1 were downregulated in NSCLC. Subsequently, we validated this result by Taqman real-time PCR successfully. We predicted the microRNA clusters which can potentially regulate BCL11A gene translation,and conduct intersection with microRNA clusters downregulated in NSCLC, several microRNAs can meet the requirement including miR-l,miR-30a,miR-103 and miR-217. Among these microRNAs, miR-1 and miR-30a were selected to further analysis, the result demonstrated that miR-30a but not miR-1 can suppress BCL11A gene translation post-transcriptionally.Primary Conclusion1. BCL11A is upregulated both in mRNA and protein levels,indicating that proto-oncogene BCL11A is activiated in NSCLC.2.BCL11A expressed exclusively in NSCLC tissues but not in adjacent non-cancerous tissues,suggesting that BCL11A might be a special tumor marker.3.The deregulation of BCL11A in NSCLC have a significant histological difference,BCL11A levels in squamous carcinoma are much higher than other types. 4.BCL11A associasted with disease stage and lymph node stage both in mRNA and protein levels.5.Patients with high BCL11A expression both in mRNA or protein levels have a better prognosis BCL11A mRNA is a independent prognosis for disease free survival, but BCL11A Protein can not only predict disease free survival but also predict overall survival.Which suggested that BCL11A can be a better prognosis factor than BCL11AmRNA.6.MicroRNAs differently expressed between NSCLC and adjacent non-cancerous tissues, many microRNAs are downregulated in NSCLC tissues.7.BCL11A overexpression in squamous cell carcinoma mostly result from BCL11A gene copy number gain,however, hsa-miR-30a inactivation might be another mechanism for activation of proto-oncogene BCL11A.