1. Introduction

Liver cancer rankis as the sixth most frequentcommonly diagnosed cancer and the third leading cause of cancetumor-associated death^[1]. Ambong primaryut 85% of liver tumors, approximately 85% of cancer cases correspond to hepatocellular carcinoma (HCC)^[1][2], an aggressive tumalignancyor with high incidence and mortality rates^[3][4][5]. SiUnce the majority of HCC cases are detected at advanced stages^[3][4]fortunately, only a slight percentage of patients are eligible for curative threrapeutical approacheatments^[2][3].^[4] Despite the latest advances in diagnostic and therapeutic strategies,nd the prognosis of HCC remains very poor, with an overall 5-year survival rate lower than 18%^[6]. Thus, therere is an imperative need to identify novefore, finding new functional biomarkers able to optimize early diagnosis, guide treatment application ancould improve HCC patients’ survival outcomes.

The forkhead box (FOX) family is organized into 19 different sub-families subgroup (FOXO) of transcription factors that share a highly conserved DNA-binding domain named forkhead box or winged-helix domain. In mammals, the O subgroup (FOXO) is is composed of FOXO1, FOXO3, FOXO4 and FOXO6^[7][8][9]. Specifically, FOXO3 has shown to playexert physiological and pathological rolefunctions by controlling the transcription of key target genes involved in multiple cellular processes^[9][10][11]. Nonetheless, conthere are opposing radictory reports about the role of FOXO3 expression in cancer, finding that FOXO3 upregulation can act either as a tumor-suppressive or an oncogenic mechanism, depending on the tumor typ are found in literature or specific circumstances^[7][10].

Hepatocytes usuallyIn exhibit high FOXO3 levels^[7], reganrd this transcriptiono HCC, factor seems to be implicated in the pathogenesis of liver disease^[12]. Regarding liver canertain articer, several studies sustain that abnormal FOXO3 overexpression could representconstitute an unfavorable hallmark in HCC, being linked to more aggressive phenotypes and a worse long-term prognosis^{[12][13][14][15][16][17]}. ConvOtherselywise, other investigationstudies defend the correlassociation of low levels of FOXO3 expression with poorer HCC patient outcomes^[18][19][20]. Thus, ex

Wisting HCC articles analyzing the relationship of FOXO3 levels with survival parameters and clinicopathological factors show controversial results, being necessary to clarify th the aim of clarifying the role played by FOXO3 in HCC.

Theon HCC preasent research constitutes the first systematic review with meta-analysis aimed to explore t well as of investigating the potential correlation between value of FOXO3 expression and HCC pathogenesis, clinicopathological features and patient outcomes. Thus, we sought to contribute to a deeper understanding of HCC and provide a novel molecule with diagnostic and prognostic value, enabling better diagnosis and patient surveillance.

2. Materials and Methods

♦ 2.1. Study Objectives

The aim of thias a new biomarker in HCC, a recent systematic review with meta-analysis (PROSPERO regis investigration was tocode: CRD42021237321) evaluated the dicorrelagnostic and prognostic value tion of FOXO3 expression in patients with HCC, focusing on the association of FOXO3 expression with tumor development and pathogenesis, survival parameters, as well as with several tumor and patient characteristics.

T and clinicopathological factors. It needs to be mentioned that the csturrent meta-analysis was performed followingdy followed the PRISMA guidelines^[21]., Moreover, the study protocol was registered in PROSPERO, being assigned the CRD42021237321 registration code.

♦ 2.2. Literature Search Strategy

An exhaudetermining the quality of the selected investigationstive literature search was performed in the databases Cochrane, Embwith the Newcastle-Ottawa scale (NOS) criteriase^[22], PubMed, Scopus and Web of Science (WOS), establishing 30th April 2021 as the final inclusion date. The eligible studies were identified by performing the statitiscal analysis using the search strategies indicated in Table 1.

Table 1. Spreviously reported mearcth strateodology for each database^[23][24].

WOS, Web of Science

♦ 2.3. Inclusion and Exclusion CriteriaMain Findings

The articles satisfying the following criteria were selected: (1) studies involving patients diagnosed with HCC; (2) determination of FOXO3 expression at translational or transcriptional level in tumor tissue; (3) relationship of FOXO3 levels with tumor presence/absence, survival data or clinicopathological features reported.

Studies complying with the following criteria were excluded: (1) studies exclusively accomplished with pre-clinical models; (2) reviews, book chapters, meeting communications and similar articles; (3) studies without mandatory data directly provided or in which it cannot be estimated; (4) articles with no available full-text in English.

♦ 2.41. Data Extraction and Quality AssessFOXO3 and HCC development

ArticPooles screening, as well as data extraction and quality assessmentd results from each included study, was independently performed by four researchers. Discrepancies were solved by discussion and final consensus.

Tthe comparison of FOXO3 expression between tumor tissues and he quality of selected articles was determined using the Newcastle–Ottawa scale (NOS), which scores studies from 0 to 9^[22]. thy normal liver samples proved that elevated levels of FOXOnly3 high-quality studies (NOS score ≥ 5) were included in the quantitative synthesis. The bsignificantly correlate with HCC paseline ctharacteristics of selected studieogenesis are shown in (OR, 15.98; 95% CI, 1.96–130.02; p = 0.01) (Figure 1a, Table 2.1).

TablFigure 21. Baseline characteristics of selected articles.

healthy liver tissues.

Aside from Zighou et al.^[15], which did not inform about the xpatients’ origin, all reported patients came from Asia, mainly from China (73.05%)^[12][19][20] ession and the rest from Korea (26.95%)^[16]. Although Ahn et al.^[16] and Zhou et al.^[15] did not proevide gender data, the reported population was predominantly male (76.53%). Regarding risk factors, within the five papers included, three evaluated hepatitis B infection^[16][19][20], and onral clinicopatholy one assessed hepatitis C and alcoholic condition^[16]. Likgical fewise, two researches^[19][20] also validated the puresence of cirrhosis i in HCC patients. In summary, considering the total number of patients for whom the corresponding information is known, 73.28% of patients suffered from hepatitis B, 31.35% from hepatitis C, 32.64% from cirrhosis, 34.05% from alcoholism and 26.49% from hepatitis B, C and alcoholism simultaneously.

♦ 3.2. Association of FOXO3 Expression with HCC Pathogenesis

Two out of the five studies included in this meta-analysis compared FOXO3 levels between HCC samples and normal liver tissues. Pooled results showed that enhanced FOXO3 expression is significantly related to HCC development (OR, 15.98; 95% CI, 1.96–130.02; p = 0.01), finding an elevated heterogeneity (I² = 60.30%, Q-test p = 0.11) (Figure 2a, Table 3).

Figure 2. Forest plots of the studies assessing the relationship between FOXO3 overexpression and (a) tumor pathogenesis or (b) OS in HCC patients.

AFP, alpha-fetoprotein; CI, confidence interval; HRBV, hazard ratio; OS, overall survivalepatitis B virus; REML, Restricted Maximum Likelihood; TNM, tumor-node-metastasis.

Table 31. AnEvalysisuation of the association of enhanced FOXO3 high expression with tumor developmentlevels with HCC pathogenesis, survival and clinicopathological factoreatures.

Assessment of the correlation of FOXO3 high expression with (a) tumor pathogenesis and (b) overall survival (OS) in HCC patients.

CSI, cumulative survival; DFS, disease-free survival; F, female; FOXO3, forkhead box O3; onfidence interval; HR, hazard ratio; IHC, immunohistochemistry; M, male; NR, not reported; OS, overall survival. * Tumor samples included for survival analysis. ** From UALCAN database. ¹ Final scores were calculated by multiplying the score obtained for percentage of positive cells (0, ≤5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, >75%) by the score registered for staining intensity (0, no signal; 1, weak; 2, moderate; 3, strong). ² Final scores were calculated by multiplying the score obtained for percentage of positively stained tumor cells (0, 0–10%; 1, 11–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%) by the score registered for staining intensity (0, negative; 1, weak; 2, intermediate; 3, strong). ³ Fina; REML, Restricted Maximum Likel scores were calculated by multiplying the score obtained for percentage of positive cells stained (0, no staining; 1, 1 ≤ 1 < 25%; 2, 25% ≤ 2 < 50%; 3, 50% ≤ 3 < 75%; 4, ≥75%) by the score registered for staining intensity (0, negative; 1, weak; 2, moderate; 3, strong)hood.

♦ 2.52. Statistical AnalysisFOXO3 and OS

MetBa-analysis synthesis was conducted using the STATA software version 16 (Stata Corporation, College Station, TX, USA). Tsed on the results of the rtole of FOXO3 expression on HCC was examined in two steps. In the first step, we tal of the included articles, pooled the overall survival (OS) data by hazard ratio (HR) and 95% confidence interval (CI) to unravel the correlation between data demonstrated that FOXO3 and HCC patients’ life expectancy. OS was established as the time from the intervention date until the day of decease or the last follow-up visit. Parmar method^[23] washigh levels significantly correlate with employed to estimate these data when no explicit information was reported in the primary wer OS ratestudy.  (HRs and respective , 1.79; 95% CIs were, 1.11–2.89; combinedp throughout= the studies0.02) (Figure 1b, Table 1).

♦ 2.3. FOXO3 and Clinicopathological Features

In ithe second step, the power of the association of FOXO3 overexpression with tumor existence or ially, although all available data on clinicopathological features was estimated by odds ratio (OR) with 95% CI. It needs to be mentioned that specific thresholds or cut-off values were established to calculate the possible coactors were pooled and analyzed, the investigators did not observe any correlation betweenof enhanced FOXO3 high levels and certainexpression with different clinicopathological factors:s such as alpha-fetoprotein (AFP), 50 ng/ml; tumor-node-metastasis (TNM) staging, I-II/III-IV; tumor size, 5 cm. Combined HR > 1 and OR > 1 denoted an upper risk of poor prognosis and a higher incidence of the tested feature when high expression of FOXO3, respectively, considering significant when p < 0.05.

Heterogene levels, cirrhosis, gender, hepatity was evaluated by chi-square-based Q-test and I² stats B vistic. The Restricted Maximum Likelihood (REML) method was used as the random-effect model in cases where heterogeneity was detected (Q-test p-value < 0.10 and/or I² ≥ 50%). Otherwus (HBV) infection, invasion, metastasise, the fixed-effects model with Inverse Variance (IV) method was employed^[24]. To examine umor-node-methe heterogeneity sources, we performed subgroup analyses based on sample size, patients’ origin, NOS score or follow-up time.

Publicstasis (TNM) statgion bias was explored through the evaluation of funnel plot asymmetry and Egger’s test. When Egger’s p-value < 0.05 and fng, tumor nodunnel plot was asymmetric, significant publication bias existed. In this case, trim-and-fill method was used to estimate a corrected effect arity and tumor size after(Figure 2, Table 1). adjustment, Howhich helped determine whether the publication bias substantially affected the robustness of the pooled results.

3. Results

♦ 3.1. Study Characteristics

A total of 441 appliever, certain parameters sucable records were identified through a database search, but 268 studies were duplicates, and after scanning titles and abstracts, another additional 56 non-original articles were excluded. The full-text of 117 articles was checked for eligibility, finding 69 articles without patient samples, one without English full-text, 10 without HCC patients and 32 without FOXO3-related tumor pathogenesis, survival or clinicopathological features evaluation as invasion showed an elevated heterogeneity among data. Therefore, these 112 papers were also removed from the study. Finally, five articlesubgroup analysis was^{[12][15][16][19][20]} were assessed for quality and data extraction. All these studies reached the quality threshold according to NOS score (Table 2) anubsequently conducted in ord were included for quantitative meta-analysis (Figure 1).

Fi to investigure 1. PRISMA flow diagram of study selection.e FOXO3, forkhead box O3; HCC, hepatocellular carcinoma; WOS, Web of Science.

As indicathed in Table 2, t he articles included in the present meta-analysis were published from 2009 to 2020, and involved a total of 1059 HCC cases. Zhou et al.^[15] did erogeneity causes anot directly report sample collection procedure or patient information. The rest of the samples were obtained by surgical resection, and the enrolled patients did not receive any pre- or post-operative treatment potentially find a significant correlation. The

Figure 2. Anumber of tumor samples across studies ranged from 91 to 365, and 537 olysis of the totals (50.71%) exhibited FOXO3 overexpression. All included studies^{[12][15][16][19][20]} provided dassociata relating FOXO3 expression with OS, four articles^{[12][16][19][20]}  on betwith clinicopathological features and two investigations^[12][16] also compared en FOXO3 levels between tumoral and

AFP, alpha-fetoprotein; CI, confidence interval; FEM, fixed-effects model; FOXO3, forkhead box O3; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; OR, odds ratio; OS, overall survival; REM, random-effects model; TNM, tumor-node-metastasis.

♦ 32.34. Correlation between FOXO3 Expression and OSSubgroup Analysis for OS and Invasion 

FuIn orthermore, we assessed the prognostic value of FOXO3 expression using the total of the enrolled articles. Pooled results showed a significant correlation between high FOXO3 expression and OS (HR, 1.79; 95% CI, 1.11–2.89; p = 0.02), and a great der to find the heterogeneity sources for every heterogeneity across studies was detected (I² =us 91.66%, Q-test p = 0.00) (Figure 2b, Table 3).

♦ 3.4. Correlation of FOXO3 Expression with Clinicopathological Features

We pooled the avarilable data from the selected five papers to determine the hypothetical correlation between high FOXO3 expression and several clinicopathological characteristics. However, enhanced FOXO3 expression was not statistically correlated with the evaluated features, which included: AFP, cirrhosis, gender , hepatitis B virus (HBV) infection, invasion, metastasis, TNM staging, tumor nodularity and tumor sizable analyzed, subgroup analysis was carried out for parameters involving more (Figure 3, Table 3).

Moreover, heterogeneity tests revealed that heterogeneity was substantial in the following cases: AFP (I² = 86.14%, Q-test p = 0.01), cirrhosis (I² = 87.81%, Q-test p = 0.00), invasion (I² = 80.92%, Q-test p = 0.00) and TNM staging (I² = 95.72%, Q-test p = 0.00). Otherwise, assumable heterogeneity was found in gender, HBV infection, metastasis, tumor nodularity and tumor size (Figure 3, Table 3).

Figure 3. Forest plots of the studies assessing the relationship between FOXO3 overexpression and specific clinicopathological features in HCC patients. AFP, alpha-fetoprotein; CI, confidence interval; HBV, hepatitis B virus; REML, Restricted Maximum Likelihood; TNM, tumor-node-metastasis.

♦ 3.5. Subgroup Analysis

San two original stubgroup analysdis for heterogeneous parameters was performed aes according to sample size, NOS score, patients’ origin or follow-up time, in order to evaluate the possible heterogeneity sourcese. Subgroups composed of only one report were not considered.

In rRegard to OS, a correlation was shown with high FOXO3 expression when subgroups were based on sample size n > 100 (HR, 2.13; 95% CI, 1.37–3.33; p = 0.00) and n > 200/300 (HR, 2.44; 95% CI, 1.21–4.94; p = 0.01), as well as on follow-up moning OS parameths (≤120/240) (HR, 1.96; 95% CI, 1.08–3.55; p = 0.03). However, heterogeneity still continued to be high in wall of these previous subgroups: sample size n > 100 (I² = 74.73% and Q-tes successt p = 0.01), n > 200/300 (I² = 81.20% and Q-test p = 0.02) and foullow-up months (≤120/240) (I² = 93.12% and Q-test p = 0.00) (Figure 4a, Table 4). Othey rwise, FOXO3 overexpression was significantly associated to OS solved when NOS ≤ 6 or follow-up > 60 months (HR, 1.58; 95% CI, 1.24–2.02; p = 0.00), and heterogeneity was successfully solved, as shown by I² = 15.43% and Q-test p = 0.28), (Figure 4a, Table 4).

Figure 4. Falsorest plots after subgroup analysis of the studies assessing the finding a significant correlationship between high FOXO3 overexpression and (a)levels and poor OS or (b) invasion in HCC patients. CI, confidence interval; HR, hazard ratio; NOS, Newcastle–Ottawa scale; OS, overall survival; REML, Restricted Maximum Likelihood.

 (Figure 3a, Table 4.2). SubgMoroup analysis.

PSublication bias evaluation of the potential correlation of FOXO3 overexpression with group analysis for (a) tumor pathogenesis, (b) OS and (cb) the invassessed clinicopathological features by funnel plots asymmetrysion. AFP, alpha-fetoprotein; CI, confidence interval; HBV, hepatitis B virus; HR, hazard ratio; IV, Inverse VariancNOS, Newcastle–Ottawa scale; OS, overall survival; REML, Restricted Maximum Likelihood.

Table 2. Subgroup analysis.

CI, confidence interval; FEM, fixed-effects model; FOXO3, forkhead box O3; HR, hazard ratio; NOS, Newcastle–Ottawa scale; OR, odds ratio; OS, overall survival; REM, random-effects model.

Additionallyeover, heterogeneity in invasion was resolved based onin the subgroup NOS = 6 and Korean provenance (I² = 0.00% and Q-test p = 0.39), obshowerving also a strong associfor the first time a significant correlation with high levels of FOXO3 (OR, 2.95; 95% CI, 1.67–5.21; p = 0.00). CurAdditiousnally, the elimination of Chen et al.^[19]  also led to an assumable heterogeneity (I²=29.99% and Q-test p = 0.23) and a significant relation with FOXO3 overexpression (OR, 2.13; 95% CI, 1.44–3.16; p = 0.00) (Figure 4b, Table 4).

The rest of the suassociation bgroups exhibited an elevated heterogeneity and did not follow a correlation with tween FOXO3 expression (Figure 4, Table 4). Howeover, overall, subgroup analysis helped disclose that sample size, follow-up time, NOS score and patients’ origin contribute, at least in part, to the reported heterogeneity.

♦ 3.6. Publication Bias

Althouxpression and high an Egger’s test could not be accomplished for the comparison of HCC tumor vs. non-tumor samples (Table 5), funnel plot analysis likely showed a slight asymmetry (Figure 5a). In regard to OS, asymmetry was oprobserved (Figure 5b) and a significant result was obtained in the Egger’s test (p = 0.00) (Table 5), which denoted pubilication bias. Hence, the trim-and-fill method was performed, but no “missing” studies were imputed, and the global effect size remained unchanged (Table 5). Cy of invasionversely, publication bias was not detected for all the assessed clinicopathological characteristics (Figure 5c3b, Table 52).

Figure 53.

CI, confidence interval; TNFEM, tumor-node-metastasisfixed-effects model; FOXO3, forkhead box O3; HR, hazard ratio; NOS, Newcastle–Ottawa scale; OR, odds ratio; OS, overall survival; REM, random-effects model.

Table 5. Evaluation of publication bias.

AFP, alpha-fetoprotein; CI, confidence interval; FEM, fixed-effects model; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HR, hazard ratio; OR, odds ratio; OS, overall survival; REM, random-effects model; TNM, tumor-node-metastasis. * Convergence not achieved during tau2 estimation.

43. Discussion

Primary liver cancer, mainly represented by HCC^[2][6][25], remains a global unsolved health challenge^[1][2]. Asymptomatic presentation at early stages, deficient diagnostic mtethodologychniques and post-therapy recurrence are common features of this HCC, a lethal primary liver tumor with a remarkably poordisappointing prognosis^{[2][6][15][16][19][25]}. MoreoEver, aln though increasing efforts are being put into HCC biomarker discovery^{[24][25][26][27]}, effective molecules that assist tumorble to improve HCC detection and predict therapy response are still lacking.

FOXO3, Mean important member belongwhile, ing to the evolutionary conserved FOXO sub-family^[7][8][10], is a c has beentral transcription factor that governs downstream targets involved in key cellular processes^[10]. Isuggest has been reported that theFOXO3 deregulation of FOXO3 expression is icould be involved in cancer emergence^[10][12][16] and progression^[12][16][19]. FOXO3, which is mainly expressed by liver cells^[7], seems to be connected to the development of hepatic disease^[12], bbut the exact linkage between FOXO3 expression and primary liver cancer has not been uncoverlarified yet.

Therefore, we performed the pcurresent systematic review with meta-analysis tnt study was carried out to accurately determine the relassociationtionship of FOXO3 overehigh expression with tumor development, survival outcomrate and clinicopathological factoreatures, examining the potential usefulness of this factor as a diagnostic and prognostic biomarker for HCC monitoring.

A total of fThive high-quality studies, enrolling 1059 HCC cases, were selected for conducting thiss systematic review with meta-analysis. Approximately half of the cases included in this investigation showed high FOXO3 expression, and most patients came from Asia, mainly from China, mainly accomplished with Chinese population, which is not surprising as this geographical area accounts for nearly 75% of HCC incidence worldwide^[6] andsince most new HCC cases usually abcout 50% of new HCC patients are Chineseme from China^[28].

The, currdent meta-analysis revealetected a significant correlation between FOXO3 high expression and HCC pathogenesis. Interestingly, Lu et al.^[29]  evidenced that FOXO3 is highlyenhanced FOXO3 expressed and overactivated in HCC patients, which waion and activity is associated with strong liver damage and overexpression of HCC-related genes, suggesting that FOXO3 overexpression is involved in tumorigenesis promotion. Additional reports from pre-clinical studies also indicated that FOXO3 upregulation is related to HCC oncogenicity via the overexpression of the long noncoding RNAs (lncRNAs) PRR34 antisense RNA 1 (PRR34-AS1)^[17] and LOC554202^[30], or by the circular RNA circFBXO11/miR-605 axis^[13]. Contrary to all the above-mentiwioned results, the article by se, Wu et al.^[31]  described that reduction in FOXO3 nuclear translocation and activity could be involved in sepiapterin reductase-mediated HCC progression. Considering all the previous evidence, which mostly defends a tumor-promoting action of the positive regulation of FOXO3, our metaThus, this meta-analysis certainly supports the findings reported by the majority of studies and underscores the role of FOXO3 overexpression on fostering HCC development. Hence, the suggests that upregulation of FOXO3 may constitute a suitable diagnostic factor able to complement classic techniques.

MeanwFurthile, primary pooled results showed ermore, a significant association between FOXO3 overexpression and shorter OS of poor survival outcomes was registered in HCC patients. Chen et al.^[32] emphas, indicatizedng that FOXO3 expression is related to shorter survival time and cancer progressiocould constitute a negative prognostic factor in this tumor. Similar reports were shown in invasive ductal breast carcinoma^[32], which was also observed in glioblastoma human samples^[33]. Likewise, high levels of this factor were significantly related to a shorter disease-free survival (DFS) time and an increased Ki-67 proliferation index in tripleand triple-negative breast cancer (TNBC) patientsamples^[34]. Otherwise, Zhao et al.^[35]  observed that FOXO3 transactivity is impaired in HCC due to FOXO3 dodownregulation, which ccould be linked with the enhancement of cell proliferation promoted by thyroid hormone receptor-interacting protein 6 (TRIP6). However, correlation between TRIP6 and FOXO3 expression in HCC individuals and its impact on survival rate were not assessed^[35]. Therefore,

Addithere is no conclusive statement about the impact of FOXO3 on HCC prognosis based on such a study.

Consideringonally, it has FOXO3 as a potential biomarker in HCC constitutes a novel approach. As a consequence, there is still little research in the literature that analyzes the possible connection between een found that high FOXO3 levels and survival outcome, being all this evidence included in the current systematic review with meta-analysis. Even though our study proved the potential of FOXO3 as a negative prognostic factor in HCC, additional large-scale investigations should be performed in this cancer type to confirm such an encouraging resultmay trigger HCC invasiveness.

Regarding the evaluated clDinicopathological features, subgroup analysis showed that invasion statistically correlates with high levels of FOXO3 in articles with a medium–high NOS score or those harboring Korean patients, suggesting that elevated FOXO3 expression may trigger HCC invasiveness. Additionally, it has been reported thafferent articles also determined that FOXO3 expression accentuates invasiveness and tumor expansion in glioblastoma^[33], pancreatic cancer^[36] and HeLa and melanoma MDA-MB-435 cells^[37], being also correlated to perineural invasion in TNBC samples^[34].

 Contrariwise, FOXO3 oppositely impacted the invasive capabilities of breast tumors, depending on the estrogen receptor α (ERα) status^[38]. BMoresidesover, Yang et al.^[39]  demonstrated the potential of bortezomib to suppressinhibit cell migration and invasion via FOXO3 by upregulation ng FOXO3 in cholangiocarcinoma and HCC in vitro models. However, these results were not tested in vivo nor in HCC patients. Moreover, it needs to be mentioned that boand bortezomib is not currently approved for HCC treatment, due to it not showing enough efficacy against this tumor, and it does not re does not represent a major chemotherapeutic drug within the HCC field. It is know

On that FOXO3 displays a dual role in cancer, promoting malignant phenotypes or inhibiting cancer progression depending on the tumor type or specific tumor-related circumstances. This fact likely drives contrasting findings among different reports. Therefore, although our meta-analysis revealed for the first time a significant correlationother hand, although no correlation was found between enhanced FOXO3 levels and the risk of invasion, further studies are required to unequivocally unravel the impact of FOXO3 on the modulation of HCC invasion capability.

Iany other evaluated clin regard to clinicopathological factors evaluated other than invasion, there was not a significant association between FOXO3 overexpression and AFP eature, Chen et alevels,.^[40] cirrhosis, gender, HBV infection, metastasis, TNM staging, tumor nodularity or tumor size. However, despite no correlation observed between ndicated that FOXO3 levels and HBV infection, Chen et acoul.^[40] proved that FOXO3 participates in the HBV-mediated HCC tumorigenesis. GWiven the absence of additional HCC investigations, novel th regard to studies analyzing the relationship of HBV infection or other clinicopathological factors with FOXO3 expression will help unravel the potential of this central transcription factor to predict HCC-associated features and reinforce its usefulness as a novel biomarker.

With resccompect to the possible association between FOXO3 expression and the aforementioned clinicopathological parametersished in other tumors, an investigation conducted with nasopharyngeal carcinoma samples observed that low FOXO3 expression correlates with advanced clinical stages and higher T stages, apart from s well as with lymph node metastasis and distant metastasis^[41]. Reduced FOXO3 levels in colorectal cancer^[42], esophageal squamous cell carcinoma (ESCC)^[43] and pancreatic ductal adenocarcinoma samples^[44]  have been also correlassociated with more advanced disease. Meanwhile, deDeregulation of FOXO3 levels has shown to differentially influence lymph node metastasis in invasive ductal carcinoma^[32], TNBC^[34] and bladder carcinoma^[45], finding that the interplay β-catenin-FOXO3 can also behave as a a metastasis promoter in colon cancer^[46]. MorBeoversides, FOXO3 downregulation in ESCC patients accounted for lymph node metastasis^[43], and its low expression correlated with a larger tumor size in gastric adenocarcinoma^[47].

ColAlectively, all the available evidence concerning FOXO3 regulation and the associated cancer featuretogether, these reports highlights the double-edged action played by this crucial transcription factor, finding that deregulation of FOXO3 expression and activity may definitely determine tumor promotion or suppression depending on the cancer type, cellular context or genomic profile.

IFOXO3, fin summary, this study is the first comprehensive, detailed and systematic meta-analysis evaluating and demonstrating the diagnostic and prognostic value of FOXO3 in HCC, thus covering an unexplored research pathway. It is worth mentioning that all published and available clinical evidence on the associatiing that deregulation of FOXO3 with HCC development, survival parameters and clinicopathological features was checked and, if appropriated, included in this work. Moreover, our meta-analysis contains important and informative analysis, such as heterogeneity assessment, subgroup analysis and publication bias evaluation. Therefore, although further studies are needed to corroborate our findings, this novel systematic review with meta-analysis constitutes the first addressing the clinical significance of FOXO3 as a biomarker in HCC, thereby setting the basis for future investigations and providing a new potential molecule that could successfully assist HCC detection and prognostic evaluation.

Howeveexpression and activity may lead to cancer, there are still some limitations in the current study that need to be considered. The number of articles included both in qualitative and quantitative synthesis was low, and the evaluation of the diagnostic and clinicopathological significance of FOXO3 in HCC involved even fewer reports, due to these data being missing in some of the studies. Thus, the volume of the research was relatively small, and more investigations are required to verify and complete the results obtained by this innovative meta-analysis.

On the other hand, although only one promotion or suppaper was excluded during the assessment of eligible articles because the full-text was not written in English, exclusion of studies published in languages other than English probably accounts for publication bias, discarding investigations with relevant results. Despite one of the five included articles not reporting the patients’ ethnicity, the rest of the studies were carried out with Asian patient samples. There is a lack of investigations performed with people from other geographical regions, which leads to a disbalance in terms of patients’ origin, likely contributing to bias apparition. Additionally, not every included report evaluated HCC-associated risk factors, such as HBV infection, thereby missing relevant information that could condition global results. In regard to studies evaluating FOXO3 levels by immunohistochemistry (IHC), they established very similar but not completely identical criteria for the definition of “high” FOXO3 expression, which could cause mild heterogeneity. HRs from two out of the five included studies had to be estimated from survival curves, as this information was not directly reported, which could slightly contribute to variability apparition among articles. Furthermore, only one of the selected articles provided DFS information, an interesting prognosis-related variable, but this fact prevented determining its potential association with FOXO3 expressionession depending on the cancer type, cellular context or genomic profile.

54. Conclusions

In conclusion, our systematic review with meta-analysis has been the first to evaluate the potential of the transcription factor FOXO3 as a novel and functional biomarker in cancer and, more precisely, in HCC. This novel study demonstrated that an increased his study proved for the first time that an enhanced FOXO3 expression of FOXO3 may could be an unfavorable clinical factor with diagnostic and prognostic valusignificance in HCC, being related toassociated with tumor development, poor OS and a high probabilityhigh risk of invasion. Thuserefore, the evaluation of FOXO3 levels could constitutes a promising strategyapproach to optimize and complement HCC detection and, specifically, to guide patient surveillance and make an accurate prognosis. However, these findings need to be confirmed by additional high-quality, well-designed and large-scale investigations. 

Graphical Abstract. FOXO3, Forkhead box O3; HCC, hepatocellular carcinoma; OS, overall survival.

This entry is an adaptation from doi.org/10.3390/cancers13215349