FOXO3 in human hepatocellular carcinomarole on tumor development, prognosis and autophagy-related sorafenib resistance acquisition

Abstract

Liver cancer constitutes one of the main malignancies contributing to world’s cancer burden and associated deaths, ranking the sixth in terms of incidence and the third in terms of mortality. Hepatocellular carcinoma or hepatocarcinoma (HCC) represents the main hepatic tumor subtype, accounting for ~80% of liver cancer cases, and unfortunately, HCC patient outcomes are very disappointing. The poor prognosis of HCC is mostly derived from two major reasons. First, there is great difficulty and inaccuracy in detecting the disease at early stages, which leads to the frequent diagnosis of HCC cases at advanced phases, when curative treatments are not available. Furthermore, in addition to the failed surveillance programs and late HCC diagnosis, the efficacy of palliative therapies for patients with advanced HCC is limited, which is largely due to the early development of chemoresistance by HCC cells. Therefore, in order to ameliorate the clinical success of HCC patients, it is imperative to find functional biomarkers that can improve early diagnosis and prognostic evaluation of HCC, as well as to discover novel therapeutic targets that allow the achievement of more effective therapeutic approaches. Sorafenib has been the only approved treatment for advanced HCC for a decade, becoming the mainstay therapy since 2007. Indeed, although additional first-line treatments have recently been introduced, sorafenib still constitutes one of the major antitumor drugs employed in the clinical setting of advanced HCC. Nonetheless, although sorafenib has been shown to significantly extend patients’ survival, its long-term efficacy is restricted, which is tightly related to the development of drug resistance during the first months of administration. In light of these discouraging results, there is an ongoing search for the mechanisms underlying the acquisition of sorafenib resistance in HCC, as targeting these cytoprotective mechanisms may help prevent or overcome such chemoresistance and, thus, enhance advanced HCC patient survival. Among the cellular strategies responsible for sorafenib resistance acquisition, the catabolic process of macroautophagy, hereinafter referred to as “autophagy”, has been suggested to be involved. This self-digestive, recycling and housekeeping process enables cells to remove harmful intracellular components within autophagolysosomes. For this reason, autophagy has been shown to display a dual-faced nature in cancer, either avoiding HCC origination and progression, or contrarily triggering the survival of HCC cells. Consequently, autophagy activation has been reported to counteract or promote sorafenib resistance depending on the specific cellular context, being necessary to fully understand the molecular regulatory mechanisms of autophagy driving the failure or success of sorafenib therapy. The transcription factor forkhead box O3 (FOXO3), expressed by hepatocytes, mediates the regulation of key processes such as cell proliferation, apoptosis, response against stress or autophagy. FOXO3 action is important for maintaining cellular homeostasis, but it has also been associated with pathological conditions. In fact, dysregulation of FOXO3 expression and activity has been found in several cancer types, including HCC. Nevertheless, the precise involvement of FOXO3 in HCC development and progression has not been widely addressed. Furthermore, altered FOXO3 expression and activity have also been linked to the loss of sorafenib sensitivity by HCC cells. This effect has been partially attributed to the potential modulatory action exerted by FOXO3 on the autophagy pathway. However, little research has investigated the interplay between FOXO3, autophagy and sorafenib resistance acquisition in HCC, finding only two reports whose conclusions are controversial. For these reasons, the main objective of the current PhD Thesis was to analyze the potential diagnostic and prognostic value of FOXO3 expression in patients with HCC, as well as to unravel the role played by autophagy and its modulation by FOXO3 on the acquisition of sorafenib resistance by HCC cells. On the one hand, in order to study the diagnostic and prognostic significance of FOXO3 in HCC, we performed a systematic review with meta-analysis. Specifically, the aim of this investigation was to analyze the correlation of FOXO3 expression with tumor development, survival outcomes and clinicopathological features of HCC patients. For this, we systematically searched for eligible articles including this information in Embase, PubMed, Scopus, Web of Science and Cochrane Library. Meanwhile, with the purpose of investigating the controversial crosstalk between FOXO3, autophagy and sorafenib resistance acquisition in HCC, we performed an experimental research using two sorafenib-resistant human HCC cell lines, HepG2S1 and HepG2S3, derived from the parental non-resistant HepG2 line. Additionally, we checked the possible correlation between FOXO3 and autophagy-related markers in HCC patient samples employing the UALCAN database. A total of five studies comprising 1,059 HCC cases were included in our meta-analysis, observing that approximately half of HCC patient samples displayed elevated expression of FOXO3. Pooled results from the comparison of FOXO3 expression between HCC samples and healthy liver tissues revealed that FOXO3 overexpression significantly correlates with the existence of HCC. These results indicate that FOXO3 levels may be upregulated during the development and establishment of HCC. Thus, this feature could be useful for diagnosing HCC. Likewise, overall results from the meta-analysis found that enhanced expression of FOXO3 is associated with worse survival outcomes of HCC patients, and subgroup analysis also detected a significant relationship between FOXO3 high levels and an increased risk of invasion. Therefore, elevated expression of FOXO3 seems to be linked to poor HCC prognosis, potentially representing an unfavorable clinical feature. Altogether, although additional large-scale studies focusing on the clinical value of FOXO3 expression in HCC are needed, results from our systematic review with meta-analysis show that FOXO3 could be a potential diagnostic and prognostic biomarker for HCC monitoring. On the other hand, in vitro results from our experimental research indicated that sorafenib-resistant HepG2S1 and HepG2S3 HCC cell lines are autophagy-competent, finding that both cell lines display an enhanced basal autophagy flux compared to non-resistant parental HepG2 cells. Besides, we proved that such increased autophagy flux exerts a protective action against sorafenib antitumor effects, since pharmacological inhibition of the autophagic process significantly raised sorafenib-resistant HepG2S1 and HepG2S3 cell death. Meanwhile, we verified that the expression of autophagy-related markers positively correlates with FOXO3 in human HCC samples, also detecting FOXO3 upregulation in both chemoresistant HCC cell lines. Interestingly, FOXO3 knockdown significantly suppressed the increased autophagic rate and prompted HepG2S1 and HepG2S3 cell death, thereby suggesting that FOXO3 upregulation may be implicated in the activation of pro-survival autophagy in sorafenib-resistant HCC cells. This finding was confirmed by treating our resistant cells with regorafenib, one of the main second-line agents employed in the clinical setting of advanced HCC after failure of first-line therapy with sorafenib. Here, we uncovered for the first time that regorafenib effectively inhibits FOXO3 and the related cytoprotective autophagy in sorafenib-resistant HCC cells. Hence, these results support that aberrant FOXO3 upregulation and subsequent activation of pro-survival autophagy contribute to the loss of sorafenib sensitivity by HCC cells. Collectively, although further research should be accomplished, overall findings of the current PhD Thesis highlight that FOXO3 could constitute an interesting diagnostic and prognostic biomarker, as well as a promising therapeutic target, in HCC. Enhanced FOXO3 expression appears to be related to HCC development and progression. Therefore, FOXO3 evaluation could assist in early-stage HCC diagnosis and prognostic prediction in the clinical practice. In addition, since FOXO3 upregulation seems to be involved in sorafenib resistance acquisition through the induction of autophagy, targeting pro-survival FOXO3/autophagy axis could yield better outcomes in advanced HCC patients.