Driver mutations such as EGFR and KRAS mutation, MET amplification, and ALK, RET, ROS1 translocations, play a key role in tumorigenesis and personalized therapy of lung adenocarcinoma (ADC). However, about 30% lung ADCs have not been found the known oncogenic driver mutations. Moreover, there was about 10-30% lung ADCs harbored KRAS mutation, which the clinical benefits of KRAS-targeting therapy have not been defined yet. So the exploration of molecular pathogenesis and potential personalized therapeutic targets in these ADCs is still one of the most important topics and should be further studied.
In the field of cancer research，high-throughputt gene expression profiling was applied in many aspects, including the screening of those tumor susceptible people, early diagnosis, the classification of molecular phenotype, targeted therapy, Monitoring and predicting tumor recurrence, therapeutic effect evaluation and prediction of prognosis. The global gene expression profiling was screened by high throughput genome scanning in EGFR mution ADCs, KRAS mutation ADCs and triple negative ADCs, which will provide experimental bases for searching new potential therapeutic targets.
Numerous studies have confirmed Polo-like kinase 1 (PLK1) is highly expressed in a broad spectrum of cancers and its overexpression often correlates with poor prognosis. PLK1 could play an ideal personalized therapeutic target of tumors. However, the study about PLK1 expression in lung ADC is very rare and whether or not PLK1 as a potential therapeutic target remains unknown so far. In the present study, the correlation with PLK1 overexprssion and the clinicopathological characteristics, molecular genetic alterations and patient prognosis was surveyed and the impact of PLK1 overexprssion on cell proliferation, cell cycle, apoptosis and tumor invasion was observed. The present study further demonstrated the clinic significance of PLK1 overexprssion and the value as a potentially personalized therapeutic target of lung ADC.
1. The global gene expression profiling was screened by Nimblegen DNA array based whole genome scanning in 7 lung ADC cases (2 cases of EGFR mution ADC, 2 cases of KRAS mutation ADC and 3 cases of triple negative ADC). The key cell signaling pathways and differentially Gene Ontology (GO) term-based genes were identified using clustering and discriminant analysis.
2. The expressions of PLK1, TTF1 and p53 protein in 266 patients with stage I-IV lung ADC were detected by immunohistochemistry (IHC). Mutations of EGFR 18-21 exons, KRAS 12/13 codons and EML4-ALK fusion were detected by directive gene sequencing，VENTANA immunohistochemical staining and fluorescence in situ hybridization (FISH), respectively. The correlations with PLK1, TTF1 and p53 expression and the clinicopathological characteristics, the mutation of EGFR and KRAS, and EML4-ALK fusion were further analyzed. Kaplan-Meier curves were constructed to analyze patient survival and Cox multivariate analyses were performed to identify independent factors indicating an unfavorable prognosis.
3. For the purpose of observing the influences of small molecular PLK1 inhibitor, Poloxin, on the biological properties of human lung ADC cell A549 (KRAS mutation, wild type p53), H1975 (EGFR mutation, p53 mutation) and H1563 (triple negative, wild type p53). MTT assay was performed to evaluate cell proliferation, and flow cytometry was performed to evaluate cell cycle and apoptosis, and transwell test was conducted to observe tumor cell invasion. Moreover, Real-time PCR and Westernblot were applied to analyze the expression of PLK1 mRNA and protein.
1. The significant difference in key cell signaling pathways was observed between lung ADCs and normal lung tissues using clustering and discriminant analysis, including fructose and mannose metabolism, HIF-1 signaling pathway, cell adhesion molecules, viral carcinogenesis, mucin type O-Glycan biosynthesis and other cell signaling pathways. There were 976 genes exhibiting significantly higher expression and 813 genes showing significantly lower expression using GO term-based genes analysis. Forty-five key genes involving the pathogenesis and evolution were identified, and some of which were crucial driver genes including EGFR, ERBB2, KRAS, MET, CDKN2A, ID1, RB1 and TP53. Some important genes involving lung ADC harbored EGFR mutation were identified such as EGFR, MYCL1, NKX2-1, SP-B/C, DUSP4, and KRAS, PLK1, CEACAM5, DDR1, LKB1 and TP53 involving lung ADC harbored KRAS mutation as well as AHNAK2, CYP24A1, EGFR, KRTAP5-8, MMP7 and PLK1 involving triple-negative lung ADC. Both lung ADC harbored KRAS mutation and triple-negative tumor showed significantly higher PLK1 expression.
2. PLK1 overexpression was exhibited in 62% (165/266) cases of lung ADC. There was positive correlation with PLK1 overexprssion and smoking history (P<0.05), metastesis/recurrence (P<0.01), and higher stage (stage III-IV) (P<0.05). According to different histological subtypes, PLK1 overexpression was more frequently found in colloid ADCs (87.5%), invasive mucinous ADCs (86.7%) and solid ADCs (81.1%), yet relatively uncommon in other ADC subtypes (P<0.01). PLK1 overexprssion was more common in mucin-product ADCs than in non mucin-product ADCs (P<0.05).
3. PLK1 overexpression significiantly correlate with KRAS mutation (P<0.01), p53 protein expression (P<0.01), other than TTF1 expression, EGFR mutation or EML4-ALK fusion (P>0.05). 79% (98/124) triple negative ADCs showed PLK1 overexpression. There was significant difference of PLK1 overexpression between triple negative and non-triple negative ADCs (P<0.01).
4. There was significant difference with respect to overall survival between lung ADCs with PLK1 overexpression and no or weak expression (P<0.01), as well as in the triple negative ADCs (P<0.05). Cox multivariate analysis indicated that stage was the most important determinant of overall survival (HR=2.874, P<0.001), and PLk1 overexpression was acted as an independent prognostic factor (HR=2.235, P<0.01).
5. Poloxin influenced significantly on the biological properties of human lung ADC cell lines with different molecular alterations. The effect was more obvious on A549 and H1563 as compared with H1975 cell line. The results showed that the ability of cell proliferation (P<0.01) and invasion (P<0.05) dramatically declined, the ratio of G2 cells (P<0.05) and the apoptotic index (P<0.05) significantly increased, and the level of PLK1 protein expression (P<0.01) sharply inhibited in vitro test.
1. There were amount of abnormally expressed genes in lung ADCs. The significant difference in gene expression profiling was confirmed among lung ADCs hanbored EGFR mutation, KRAS mutation and triple negative lung ADCs. These important founds laid a foundation on further study of pathogenesis and screening the potentially personalized therapeutic target of lung ADCs
2. Lung ADCs with PLK1 overexpression exhibited some definitely clinicopathological characteristics, such as smoking, ADCs with intra-/ extra-cellular mucin product, higher stage (stage III-IV), p53 expression, KRAS mutation and triple negative pattern. The cases of lung ADC with PLK1 overexpression presented unfavorable prognosis. PLK1 overexpression maybe plays an important role in pathogenesis and evolution of lung ADCs and acts as a predictor of patein’s strengthening treatment and follow-up.
4. In vitro tests of the study showed that Poloxin influenced significantly on the biological properties of human lung ADC cell lines, but sensitivity of cell lines with different molecular alterations was significant different. The results preliminarily indicated that PLK1 overexpression could act as a potentially personalized therapeutic target of lung ADCs hanbored KRAS mutation and triple negative lung ADCs.