The clinical association of programmed cell death protein 4 (PDCD4) with solid tumors and its prognostic significance: a meta-analysis

Background Programmed cell death protein 4 (PDCD4) is a novel tumor suppressor protein involved in programmed cell death. Its association with cancer progression has been observed in multiple tumor models, but evidence supporting its association with solid tumors in humans remains controversial. This study aimed to determine the clinical significance and prognostic value of PDCD4 in solid tumors. Methods A systematic literature review was performed to retrieve publications with available clinical information and survival data. The eligibility of the selected articles was based on the criteria of the Dutch Cochrane Centre proposed by the Meta-analysis Of Observational Studies in Epidemiology group. Pooled odds ratios (ORs), hazard ratios (HRs), and 95% confidence intervals (CIs) for survival analysis were calculated. Publication bias was examined by Begg’s and Egger’s tests. Results Clinical data of 2227 cancer patients with solid tumors from 23 studies were evaluated. PDCD4 expression was significantly associated with the differentiation status of head and neck cancer (OR 4.25, 95% CI 1.87–9.66) and digestive system cancer (OR 2.87, 95% CI 1.84–4.48). Down-regulation of PDCD4 was significantly associated with short overall survival of patients with head and neck (HR: 3.44, 95% CI 2.38–4.98), breast (HR: 1.86, 95% CI 1.36–2.54), digestive system (HR: 2.12, 95% CI 1.75–2.56), and urinary system cancers (HR: 3.16, 95% CI 1.06–9.41). Conclusions The current evidence suggests that PDCD4 down-regulation is involved in the progression of several types of solid tumor and is a potential marker for solid tumor prognoses. Its clinical usefulness should be confirmed by large-scale prospective studies.


Background
Programmed cell death protein 4 (PDCD4) is a novel tumor suppressor protein involved in programmed cell death. The PDCD4 gene is located on chromosome 10q24, and its allelic loss/gain is frequently reported in human cancers. Up-regulation of PDCD4 is observed after the initiation of apoptosis, suggesting that loss of, or reduced, PDCD4 expression could contribute to the anti-apoptotic property of cancer cells [1]. In mouse epidermal JB6 cells, which are resistant to anchoragedependent cell death and neoplastic transformation, high levels of PDCD4 expression could be induced in response to the presence of tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate [2] and tumor necrosis factor-alpha [3]. Therefore, it has been suggested that PDCD4 is a potent tumor suppressor gene. PDCD4 could inhibit neoplastic transformation through inhibition of adaptor protein-1 (AP-1) activation [3]. Structurally, PDCD4 could interact with RNA helicase eukaryotic translation initiation factor 4A (eIF4A), inhibiting its helicase activity and affecting protein translation [4,5]. In addition, PDCD4 could inhibit nuclear factor kappaB (NF-κB)-dependent transcription and related pathways [6].
Loss of important tumor suppressors with critical functions during the transformation process is a hallmark of cancer. Identifying key tumor suppressor proteins is important for the sub-classification of tumors at different stages with different behaviors. Moreover, the elucidation of the pathways associated with tumor suppressors could help identify predictive markers for prognostic use and provide novel insights into cancer treatment. Accumulated results in preclinical studies indicate that PDCD4 is a novel tumor suppressor gene with anti-neoplastic properties [7][8][9]. Nevertheless, some studies have suggested the conflicting conclusion that PDCD4 does not exert a tumor-suppressing effect in certain malignancies, such as non-small cell lung cancer [10,11]. To explore whether PDCD4 consistently acts as a tumor suppressor and positive prognostic marker for solid tumors, we conducted an updated meta-analysis to evaluate the clinical significance and prognostic value of PDCD4 in human cancers.

Literature search
A systematic literature search through PubMed, EMBASE, and MEDLINE was performed using the following main keywords: "PDCD4" and "cancer" or "carcinoma" or "tumor" or "malignancy. " All studies that examined the expression status of PDCD4 were recruited regardless of the detection methods used. The last search was performed on January 12th, 2016.

Study selection
Two reviewers (JZHL and WG) manually screened and selected the eligible studies independently. Studies that were not reported in English or Chinese, review articles, studies that had used cell lines and animal models without any data on human tissue samples, and studies without sufficient data for calculation were excluded from the analysis.

Methodology quality assessment
The studies on prognosis were evaluated using the criteria of the Dutch Cochrane Centre proposed by Metaanalysis Of Observational Studies in Epidemiology (MOOSE) group [12]. The following inclusion criteria were used: (1) trial dealt with PDCD4; (2) clear definition of study design; (3) clear definition of outcome assessment, including overall survival (OS), disease-specific survival (DSS), disease-free survival (DFS), and relapsefree survival (RFS); (4) clear definition of cut-off score of PDCD4 expression or high/low evaluation; and (5) a follow-up period of at least 12 months.

Data extraction
The selected publications were accessed by two reviewers (JZHL and WG). The following details were retrieved from the selected papers: (1) general information, including the first author, publication year, case populations, cancer types, sample types, and test methods; (2) the case number in each of the sub-groups with different PDCD4 expression levels and diverse clinicopathologic properties; and (3) the results of survival analysis, including hazard ratios (HRs) and 95% confidence intervals (CIs).

Statistical analysis
Pooled odd ratios (ORs), HRs, and 95% CIs were calculated for the evaluation of the clinicopathologic association of PDCD4 and its prognostic value in solid tumors. To check whether there was homogeneity among the individual ORs/HRs for the selection of the optimal effects model analysis, a heterogeneity test with the inconsistency index (I 2 ) statistic and Q statistic (P value) was performed. Substantial heterogeneity was indicated when I 2 ≥ 50% and P < 0.05, and a random effects model was adopted; a fixed effects model was appropriate when I 2 < 50% and P > 0.05. After a suitable model had been chosen, Forest plots with pooled OR/HR and 95% CIs were then retrieved from the individual HRs and 95% CIs. Pooled OR/HR > 1 suggested that the worst prognoses were more likely to occur in the patients with no, or low levels of, PDCD4 expression than those with high levels of PDCD4 expression. Statistically significant differences between groups with diverse PDCD4 expression levels was determined if the 95% CI did not overlap 1. In addition to the calculation of overall OR/HR and 95% CI, subgroup analysis was performed with respect to the case population (Asian/European/North American), cancer type (brain tumor/head and neck cancer/breast cancer/digestive system cancer/respiratory system tumor/ gynecologic tumor/urinary system cancer), and sample type (protein/RNA).
Publication bias was assessed by the Begg's and Egger's tests [13]. P < 0.05 represented a statistically significant publication bias. All analyses were performed with Stata software version 12.0 (Stata Corporation, College Station, TX, USA).

Study characteristics and qualitative assessment
According to the selection criteria, 493 articles were found in the initial screening. After removing 445 irrelevant articles, 33 articles were selected for further evaluation. According to the critical checklist of the Dutch Cochrane Centre, 23 articles fulfilled all of the quality assessment criteria. These 23 articles, involving a total of 2227 solid tumor cases, were included in the meta-analysis. Figure 1 shows the selection process. The characteristics of the included studies are shown in Table 1.

Associations of PDCD4 down-regulation with the clinicopathologic parameters of cancer patients
The associations between PDCD4 expression and the clinicopathologic features of patients with solid tumors are shown in Table 2. Four studies of digestive system cancers [7,[14][15][16] and one study of urinary system cancers [17] examined associations between tumor size and PDCD4 expression level. All of the studies were carried out on Asian patients using antigen-based methods, and the combined OR indicated no significant association. Low PDCD4 expression level was associated with advanced T category of urinary system cancers (OR 4.87, 95% CI 1.69-14.00) [17] and head and neck cancers (OR 2.15, 95% CI 1. 10-4.19) [18]. However, PDCD4 expression level was not associated with the T category of digestive system cancers (OR 0.98, 95% CI 0.46-2.08) [15,19]. There was no obvious evidence for an association between PDCD4 expression and the N category of head and neck cancers [20,21], respiratory system cancers [22], and digestive system cancers [7,15,19,23]. Low PDCD4 level was associated with advanced M category of urinary system cancers (OR 4.87, 95% CI 1.69-14.00) [17] and advanced clinical stage of head and neck cancers (OR 2.30, 95% CI 1.44-3.69) [18,20,21,24].

Discussion
In this meta-analysis, we examined the association between PDCD4 expression and the clinicopathologic parameters of cancers from different anatomical sites, including the brain, head and neck, breast, and digestive, gynecologic, and urinary systems. Our results indicated Epithelial differentiation was a determining factor in the prognoses of head and neck cancers. Poorly differentiated cancers were highly proliferative compared with their highly differentiated counterparts. In addition, differentiation status was important in maintaining a tumorigenic and treatment-resistant cancer stem cell subpopulation in head and neck cancers [36]. Hence, cancer treatment with differentiation inducers (such as retinoic acid) could inhibit cancer cell proliferation and is known as differentiation cancer therapy [37,38]. In head and neck cancers, it had been demonstrated that valproic acid or all-trans retinoic acid could inhibit the growth of head and neck squamous cell carcinomas by inducing terminal differentiation [39]. Furthermore, differentiation therapy could help in suppressing and eradicating the cancer stem cell population in head and neck squamous cell carcinomas [36,40]. The association between PDCD4 expression and cancer cell differentiation was clearly demonstrated in peripheral blood cancers. PDCD4 expression was induced in NB4 and HL-60 acute myelocytic leukemia (AML) cell lines, primary human promyelocytic leukemia (AML-M3) cells, and CD34 + hematopoietic progenitor cells in the presence of all-trans retinoic acid. Differentiation induction could be prevented if PDCD4 was silenced using small interfering RNA (siRNA) [41]. In solid tumors, PDCD4 expression was inhibited by the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway. During adipogenic differentiation of adipose tissue-derived mesenchymal stem cells, PDCD4 expression was reduced and AKT phosphorylation was increased in a time-dependent manner [42]. Because of the association between PDCD4 and cancer differentiation, PDCD4 restoration could be a novel approach for differentiation cancer therapy, resulting in effective suppression of solid tumor development and further improving the prognosis [43].
In urinary system cancer, PDCD4 suppression was associated with the metastatic status of the patients [17]. PDCD4-knockout mice developed spontaneous lymphomas with systematic dissemination and frequent liver/renal metastasis [44]. Preclinical studies have indicated that PDCD4 could control key genes involved in cancer migration and metastasis [25,45,46]. The expression of urokinase plasminogen activator surface receptor (u-PAR), which mediates plasmin-mediated extracellular matrix degradation, was shown to be controlled by PDCD4 [45]. In PDCD4-knockdown cancer cells, epithelial cadherin 1 (E-cadherin) promoter activity was inhibited. In contrast, in colorectal cancer cells overexpressing PDCD4, E-cadherin protein level was increased accordingly [25]. In ACHN and 786-O renal cancer cells, PDCD4 regulated AKT phosphorylation, leading to the migration or invasion of cancer cells via the up-regulation of the mammalian target of rapamycin complex 1 (mTORC1) [46]. Metastasis of cancer cells is a main cause of death in laryngeal carcinoma patients. Moreover, molecular factors involved in metastasis, especially in the epithelial-mesenchymal transition (EMT), could be a possible mechanism of cancer cell resistance. Because low PDCD4 expression was significantly associated with metastasis in urinary system cancers, PDCD4 could be a novel target for improving the prognosis of this malignancy.
Several limitations were observed in this meta-analysis. A small sample size was observed in one cancer group. Moreover, multiple cut-off criteria, discrepancies among diverse tumor properties, and the therapy received also affected the results. In most of the studies included in prognostic assessment, all clinical stages were represented in the respective cancer cases. However, the study reported by Dou et al. [32], only focused on advanced stage rectal cancer, and the low PDCD4 expression within these patients was not significantly associated with the 5-year OS or DFS indicated by the 95% CI overlapping 1. Furthermore, the subcellular localization of the PDCD4 protein affects cancer behavior. During tumor progression, PDCD4 protein translocation from the nucleus to the cytoplasm was observed in cancer cells [47,48]. Accumulation of cytoplasmic PDCD4 protein is reported in both normal and cancer cell lines [49]. Almost all of the studies included in our meta-analysis measured the total PDCD4 protein level in the tissue samples rather than the separate nuclear or cytoplasmic PDCD4 protein level. In the study conducted by Nagao et al. [15], both nuclear and cytoplasmic PDCD4 protein pools were examined in pancreatic cancer patients. Different ORs and 95% CIs were obtained for the assessment of clinical stage and OS. A similar situation was observed in the study by Kakimoto et al. [50] that examined the association between PDCD4 expression and histological differentiation. These findings suggest that the prognostic value of PDCD4 in human malignancies should be studied by further stratified analysis, including the determination of precise cellular localization of PDCD4.