Clinical outcome and expression of mutant P53, P16, and Smad4 in lung adenocarcinoma: a prospective study

Background Whole-exome sequencing has shown that lung adenocarcinoma (LAC) can be driven by mutant genes, including TP53, P16, and Smad4. The aim of this study was to clarify protein alterations of P53, P16, and Smad4 and to explore their correlations between the protein alterations and clinical outcome. Methods We investigated associations among P53 mutant (P53Mut) expression, and P16 and Smad4 loss-of-expression, with clinical outcome in 120 LAC patients who underwent curative resection, using immunohistochemical (IHC) methods. Results Of the 120 patients, 76 (63.3%) expressed P53Mut protein, whereas 54 (45.0%) loss of P16 expressed and 75 (62.5%) loss of Smad4 expressed. P53Mut expression was associated with tumor size (P = 0.041) and pathological stage (P = 0.025). Loss of P16 expression was associated with lymph node metastasis (P = 0.001) and pathological stage (P < 0.001). Loss of Smad4 expression was associated with tumor size (P = 0.033), lymph node metastasis (P = 0.014), pathological stage (P = 0.017), and tumor differentiation (P = 0.022). Kaplan-Meier survival analysis showed that tumor size (P = 0.031), lymph node metastasis (P < 0.001), pathological stage (P < 0.001), P53Mut protein expression (P = 0.038), and loss of p16 or Smad4 expression (P < 0.001) were significantly associated with shorter overall survival(OS), whereas multivariate analysis indicated that lymph node metastasis (P = 0.014) and loss of p16 or Smad4 expression (P < 0.001) were independent prognostic factors. Analysis of protein combinations showed patients with more alterations had poorer survival (P < 0.001). Spearman correlation analysis showed that loss of Smad4 expression inversely correlated with expression of P53Mut (r = −0.196, P = 0.032) and positively with lost P16 expression (r =0.182, P = 0.047). Conclusions The findings indicate that IHC status of P53Mut, P16, and Smad4 may predict patient outcomes in LAC.


Background
Lung cancer is the leading cause of cancer death worldwide [1], and the proportion of lung cancer patients with lung adenocarcinoma (LAC) is reportedly increasing [2]. Survival rates for LAC have improved dramatically in the last decade, owing to identification of driver mutations in LAC [3]. For example, EGFR tyrosine kinase inhibitor have been approved for treatment of LACs that carry EGFR gene mutations, which has greatly improved the prognosis of such patients [4,5]. Discovery of genetic biomarkers for cancers is expected to rapidly expand [6]. Identified driver gene alteration for LAC currently includes mutations of EGFR, KRAS, PIK3CA, BRAF, STK11, DDR2, TP53, Smad4, P16, RET, and ALK, among others [7][8][9][10][11]. As driver genes are found, their relationships to each other and to patients' prognoses must be verified.
Genetic alterations of P53, P16, and Smad4 have been found in pancreatic cancer, and appear to be strongly associated with its malignant behavior [12][13][14]. In our previous study with a genetically engineered mouse model, we found P53 Mut 's potentially malignant gain-of-function was promoted by inactivating the inhibitory actions of transforming growth factor β (TGF-β), caused by downregulation of smad4, which in turn was synergistically caused by P53 Mut and deficient P16/P19. Although these three genes have been studied individually in LAC, little is known about how they interact, or their combined effect on prognosis. Here, we investigated mutant P53, P16, and Smad4 in LAC by immunohistochemical (IHC) staining and correlated these mutations with clinicopathological features and patients' OS.

Patients and tissue samples
This study included 120 patients with LAC who underwent surgical resection between January 2007 and March 2009 at the Nanjing Medical University-Affiliated Cancer Hospital, Nanjing, China. All patients had complete medical records and complete follow-up data. The last followup date was March 2014. Patients who died of causes other than LAC before this date were excluded. Their clinicopathological data were collected from medical records and follow-up data were obtained through telephone interviews or by consulting the police population information system. These patients' mean age was 59.4 years (range: 35 to 85 years), including 58 men and 62 women. Before their surgeries, all patients underwent CT scans or B-ultrasonic examinations to exclude locoregional or widespread metastases. All patients underwent radical resections; no patients received radiotherapy or chemotherapy before surgery. This study was approved by the Ethics Committee of Nanjing Medical University.

IHC analyses
Specimens of primary LAC from 120 patients were cut into 5-μm tissue sections and deparaffinized by routine methods. The slides were steamed for 20 min in sodium citrate buffer. After cooling for 5 min, the slides were IHC stained for P53 Mut , P16 and Smad4. At least five different distinct regions of the primary tumor were IHC-labeled for each case to evaluate for potential heterogeneity. IHC labeling was carried out using P53 Mut mouse monoclonal antibody (clone SC126 diluted 1:100, Santa Cruz Biotechnology, Dallas, USA), CDKN2A/P16 rabbit monoclonal antibody (clone SC468 diluted 1:100, Santa Cruz Biotechnology), and Smad4/Dpc4 mouse monoclonal antibody (clone SC-7966, diluted 1:100, Santa Cruz Biotechnology) as reported [15]. Labeling was detected by adding biotinylated secondary antibodies. Positive controls were taken from sections known to be positive from pancreatic carcinoma specimens. For the negative controls, 1% PBS was used in place of primary antibodies. Results were evaluated independently by two experienced pathologists. P53 Mut was considered positive when ≥10% of tumor cell nuclei showed strong staining with a dark brown color. P16 and Smad4 were considered positive when ≥ 20% of tumor cell cytoplasm and nuclei showed staining with a brown color (Figure 1).

Statistical analysis
Statistical analysis of group differences was performed using χ 2 tests. The 1-, 3-, and 5-year survival rates were estimated using life tables; OS was estimated using the Kaplan-Meier method, and the differences were assessed by the log-rank test. Cox proportional hazards models were generated for multivariate analysis. Correlation analysis used the Spearman test. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software (version 17.0, SPSS).

Clinicopathological features and OS
Univariate analysis results were based on log-rank tests of clinicopathological characteristics in relation to OS. Tumor size (P = 0.031), lymph node metastasis (P < 0.001), and pathological stage (P < 0.001) were significantly associated with shorter OS (Table 2).

Protein alterations and OS
Loss of P16 and Smad4 IHC labeling was associated with a significantly shorter OS (P < 0.001). There were significant differences in positive labeling of P53 Mut with regard to OS (P = 0.038). Next, based on the number of altered proteins, we classified the patients into eight groups: (n = 10); and P53mut+/P16−/Smad4− (n = 28). Kaplan-Meier survival analysis showed that the P53mut−/ P16+/Smad4+ group had the longest OS and the P53 mut+/P16−/Smad4-group had the shortest OS (P < 0.001). The higher number of altered proteins robustly reflected major differences in survival outcome. The results showed patients with more protein alterations had poorer survival rates (Table 2, Figure 2).

Multivariate analyses of factors affecting OS
Multivariate models using Cox proportional hazards analysis were conducted with the parameters that were    (Table 3).

Discussion
The molecular basis of lung cancer is complex and heterogeneous. Over the last decades, identification of driver mutations in LAC has led to the development of targeted agents, several of which are in clinical trials and are already approved for clinical use [6]. Recent whole-exome sequencing studies of numerous human cancers have conclusively shown TP53 to be the most frequently mutated gene in human cancers [15,16]. The P53 protein and its downstream pathways are important in preventing tumor formation, but TP53 mutation is common in cancers. Moreover, unlike other tumor-suppressor genes that only lose their tumorsuppressor functions, the P53 Mut gene may endow its mutant protein with new activities that actively promote tumor progression and increased resistance to anticancer treatments [17]. Because P53 Mut proteins have longer half-life than wild-type P53, which can accumulate in the nucleus, we only can detect P53 Mut proteins by IHC. Although Ding et al. found that 45% of LAC patients had TP53 mutations [18], the clinical implications of mutant P53 in LAC may still be conflicting. P53 alterations are reported to predict poor survival in patients with non-small cell lung cancer (NSCLC) [19][20][21][22]. However, Ahn et al. reported that P53 Mut protein expression did not correlate with OS in NSCLC [23]. In our study, the P53 Mut frequency was 63.3%, which was higher than that previously reported. The results of univariate analyses showed that higher P53 Mut IHC expression predicted shorter OS. However, the multivariate analysis indicated that higher P53 Mut expression did not independently predict poorer OS. Furthermore, mice that express P53 Mut reportedly have a more aggressive and metastatic tumor profile than that of mice with null or wild-type P53 [24,25]. Conversely, Jackson et al. reported that P53 Mut protein in lung showed no detectable gain-of-function activity [26]. Although the present study found no relationship between P53 Mut and lymph node metastasis, P53 Mut expression was linked to tumor size and pathological stage. The role of TP53 mutations as a prognostic marker in NSCLC were reported conflicting. This may be due to the molecular heterogeneity and differing functional effects specific to various TP53 genotypes, methodological issues related to the assessment of mutation status, and design issues related to small sample size and nonhomogeneous groups of patients. Furthermore, the context in which these mutations occur, the initiating events and other secondary molecular alterations, may matter as well. Hence, a larger sample size and more complete experiment methods will be required in the future to obtain reliable and consistent results.
P16 is an important tumor-suppressor gene that has been found to affect cell-cycle by inactivating the cyclindependent kinase inhibitor [27][28][29]. P16 alterations in NSCLC were mainly homozygous deletions, promoter hypermethylation and point mutations [30]. The relationship between P16 expression and lung cancer is still unclear. Although some studies reported that P16 expression increased in NSCLC [31,32], another found the P16 gene to be a commonly inactivated tumor-suppressor gene in NSCLC, and altered P16 and P53 genes to be frequently found in the same tumors [30]. In this study, loss of P16 was linked to lymph node metastasis and pathological stage, which accords with the study that found complete P16 inactivation in advanced NSCLC [30].
Smad4 is a tumor-suppressor gene with a key role in the TGF-β signaling pathway [33]. Because of its mediatory role in the growth-inhibitory effects of TGF-β in normal cells and its loss in some tumors, Smad4 is considered a tumor-suppressor gene [34]. Alterations of Smad4 gene were reported in pancreatic, colorectal, gastric, esophageal, and breast tumors; its loss is associated with tumorigenesis and progression [35][36][37][38][39]. However, the role of Smad4 in LAC is unclear. NSCLC reportedly features low Smad4 expression, which is closely correlated with lymph node metastasis but not with histological type or differentiation [40]. Our study found the loss of Smad4 was key to LAC occurrence and development; our IHC results showed a 62.5% loss rate for Smad4 in patients with LAC. Negative Smad4 labeling was associated with tumor size, lymph node metastasis, differentiation, and pathological stage, and patients with Smad4 negative specimens had worse OS. Thus, reduced Smad4 expression in LAC may predict poor prognosis.
We also found that patients with more protein alterations had worse OS. Possibly, accumulated protein alterations greatly influence LAC development; this would also indicate that combinations of protein alterations are more accurate predictors for patient outcome than single alterations.
We used Spearman correlation analysis to investigate the relationship among smad4, P53 Mut , and P16. Although expressions of Smad4 and P53 Mut were inversely correlated, Smad4 expression was positively correlated with P16 expression. A previous study revealed that in SMMC-7221 hepatocellular carcinoma cells, TGF-β inhibited proliferation by upregulating P16 expression and increased apoptosis by activating caspase 3 in a Smad4-dependent manner [41]. Another study showed that low Smad4 expression is related to the high p53 expression in breast  tumors [42]. As these results are similar to ours, we speculated that LAC could have a similar mechanism. In another study with results that accorded with ours, knocked-down P53 (using siRNA) reportedly increased Smad4 activity and promoted apoptosis in MCF-7 breast cancer cells [39]. Montserrat et al. found that P16 was a commonly inactivated tumor-suppressor gene in NSCLC and that P16 alterations and P53 mutations were frequently found in the same tumor [30]. However, in this paper, we found no correlation between P16 expression and TP53 expression.

Conclusions
In conclusion, alterations of the P53, P16, and Smad4 proteins were strongly associated with LAC malignancy of LAC. Their IHC assessment at the time of diagnosis may provide a new prognostic method, assisting in deciding optimal treatment strategies for patients with LAC.