Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma

There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3’s regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation.

14-3-3γ is abundantly expressed, and overexpression of 14-3-3γ induces polyploidization of lung cancer cells [44]. Expression of 14-3-3γ is stimulated by IL-3, and 14-3-3γ overexpression promotes cell survival and growth by activating PI3K and MAPK signaling in hematopoietic cells [22]. These results suggest that 14-3-3γ is a crucial factor to modulate cell proliferation. Result from a proteomic study by two-dimensional difference gel electrophoresis and mass spectrometry reveals that 14-3-3γ is one of the potential biomarker of HCC [24]. A further study indicated that overexpression of 14-3-3γ is associated with extrahepatic metastasis and overall survival of HCC patients [16]. 14-3-3γ is thus considered a potential factor that contributes to HCC tumor progression by modulating cell proliferation and survival.
14-3-3β is implicated in the regulation of cell proliferation and migration. It was reported that overexpression of 14-3-3β induced cell proliferation, anchorage-independent growth and tumor growth of transformed cells via altering the activation of the MAPK signal pathway [17]. Moreover, increased 14-3-3β expression is associated with promoting cell proliferation in distinct types of cancer cells [45,46], whereas knockdown of 14-3-3β suppresses in vitro cell proliferation and in vivo tumor growth of rat hepatoma K2 cells [26]. 14-3-3β binds and forms a complex with 14-3-3β interactant 1 (FBI1) and transcriptionally suppresses the expression of MAPK phosphatase-1, thereby activating MAPK signaling to promote tumorigenicity and metastasis [47]. In addition, a further study focused on investigating the role of 14-3-3β on HCC tumor progression. Elevated 14-3-3β expression was significantly associated with higher incidence of metastasis and worse overall survival of HCC [17]. In addition, overexpression of 14-3-3β promotes HCC cell migration and proliferation, whereas treatment of the MEK-1 inhibitor abolished 14-3-3β-induced cell proliferation and migration [17]. These results suggest that 14-3-3β plays role as a promoting factor for cell proliferation of HCC.
4. Anti-Apoptotic Effects of 14-3-3 14-3-3 proteins protect cells from apoptosis by binding and retaining of phosphorylated Bad. Signaling from death insults leads to translocation of Bad and Bax to the mitochondrial membrane, where they form heterodimers with the anti-apoptotic Bcl-2 or Bcl-XL, thereby disrupting mitochondrial membrane potential and inducing permeability transition [48][49][50]. Through their high binding affinity for phosphorylated Bad, 14-3-3 proteins represent a major class of cytosolic proteins that play a physiological role in controlling apoptosis via the mitochondrial pathway. It has been reported that Akt phosphorylates Bad at Ser-136, ribosomal s6 kinase (RSK) at Ser-112 and protein kinase A (PKA) at Ser-155 [51][52][53][54][55]. Results from several studies suggest that phosphorylation at Ser-155 by PKA is a pre-requisite for Bad dissociation from Bcl-XL, and the dissociated Bad is subsequently phosphorylated at Ser-112 and/or Ser-136 to facilitate its binding to 14-3-3 proteins [56].
Another anti-apoptotic property of 14-3-3 proteins is attributed to their binding of phosphorylated Raf-1 and protection of Raf-1 kinase activity [40][41][42]. Raf-1 has been shown to be an important regulator of endothelial cell survival induced by vascular endothelial growth factor (VEGF) [59]. RSK is phosphorylated by ERK1/2, and RSK is involved in Raf-1/MEK-mediated cell survival signaling [60]. RSK has been shown to mediate cell survival by phosphorylating Bad at Ser-112 [60,61]. RSKs also phosphorylate transcriptional activators, such as cAMP response element-binding protein (CREB) [61]. Thus, binding of Raf-1 by 14-3-3 may play an important role in activating RSK, which, in turn, promotes survival by phosphorylating Bad and other transactivators.
Overexpression of 14-3-3ε correlates with the extrahepatic metastasis of HCC [15], implying that 14-3-3ε is potentially involved in regulating cancer cell EMT, migration and invasion. 14-3-3ε overexpression affects the expression of the EMT markers. It reduces E-cadherin levels and increases vimentin and N-cadherin levels [69], whereas knockdown of 14-3-3ε by siRNA inhibits EMT [69]. 14-3-3ε induces Snail and Zeb-1 expression, but has no significant effect on other E-cadherin repressors [69]. Although knockdown of either Snail or Zeb-1 by siRNAs suppresses HCC cell migration, 14-3-3ε-reduced E-cadherin expression was selectively abolished by the knockdown of Zeb-1, but not of Snail [69]. These results suggest that 14-3-3ε's regulation of EMT and cell migration is complicated and that multiple regulators or pathways are involved. 14-3-3ε is reversely correlated with E-cadherin in regards to the association of 14-3-3ε expression with clinicopathological parameters and E-cadherin levels in HCC tumors [69]. Notably, a combination of 14-3-3ε-positive and E-cadherin-negative expression is significantly associated with higher incidences of metastasis and poorer overall survival, whereas E-cadherin-positive expression has an attenuated prognostic outcome in 14-3-3ε-positive HCC patients [69]. These results support the notion that E-cadherin is a potential downstream factor regulated by 14-3-3ε expression in HCC. Thus, 14-3-3ε contributes to EMT regulation in HCC.
Partitioning defective 3 (Par-3) is one of the crucial factors involved in modulating cell polarity, consequently affecting EMT and migration. Par-3 participates in the polarity complex and regulates cell polarity through interacting with several GTP-bound regulators [71][72][73]. Par-3 expression is increased in HCC and is associated with distant metastasis and poor overall survival rates in HCC patients [74]. 14-3-3 (also known as Par-5 in nematodes and flies) interacts with Par-3 and controls cell polarity via a phosphorylation-dependent manner [75]. Furthermore, expression of Par-3 is significantly correlated with 14-3-3ε expression [74]. Thus, 14-3-3ε may synergize with Par-3 in controlling cell polarity, resulting in promoting HCC EMT and metastasis.

14-3-3 Proteins Promote HCC Cell Migration
Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase associated with and activated by integrins. FAK stimulates downstream signaling to regulate cell adhesion, migration and apoptosis [79][80][81][82]. FAK is overexpressed in HCC and is a potential prognostic factor predicting worse overall survival rates and higher incidences of metastasis [83]. It was reported that expression of FAK is dually regulated by p53 and NFκB [84,85]. Activated NFκB bound to the binding site on the FAK promoter region induces FAK expression and enhances cell migration [84,85]. Expression of FAK is tightly correlated with 14-3-3ε, and the positive expression of either 14-3-3ε or FAK is associated with the tumor size and extrahepatic metastasis of HCC [86]. 14-3-3ε induces FAK expression and promoter activity. Transiently-forced expression of 14-3-3ε induces nuclear translocation of NFκB, as well as enhancing the binding capacity of NFκB to the FAK promoter [86]. FAK is a crucial factor regulating cell migration, and 14-3-3ε promotes cell migration via the activation of the NFκB/FAK pathway in HCC.
14-3-3β has been implicated in modulating cell proliferation, migration and tumor growth in HCC [34]. Expression of 14-3-3β in HCC tumors is elevated, and its expression is significantly associated with distant metastasis, whereas only rare incidences of metastasis were found in 14-3-3β-negative HCC patients [34]. These results suggest that 14-3-3β plays an important role in promoting HCC tumor migration, invasion and metastasis. Forced expression of 14-3-3β increases HCC cell migration, proliferation, anchorage-independent growth and in vivo tumor growth [34]. These effects are abolished by siRNA knockdown of 14-3-3β or treatment with pharmacological inhibitors of MEK-1/2 [34]. It has been shown that 14-3-3β binds and maintains the activity of Raf-1, thereby enhancing Raf/MEK/ERK signaling [87,88]. These studies imply that 14-3-3β is a potential effector promoting HCC progression via the MAPK signal pathway.
Intriguingly, 14-3-3σ was reported to contribute to cell invasion and metastasis in selective subtypes of breast cancer. Although the expression of 14-3-3σ is silenced in most types of breast cancer, 14-3-3σ stabilizes a soluble complex of actin and intermediate filaments to enhance cell invasion of the more aggressive and malignant breast cancers [89]. 14-3-3σ expression is significantly correlated with poor clinical outcomes of basal-like subtype breast cancer. This was seen by analyzing the association of 14-3-3σ with breast cancer clinicopathological characteristics [89]. These results suggest that 14-3-3σ is involved in cancer cell aggressiveness and tumor metastasis.

Conclusions
Selective 14-3-3 isoforms are overexpressed and are potential prognostic markers of HCC. 14-3-3 isoforms contribute to HCC EMT and migration/invasion and are associated with a higher risk of extrahepatic metastasis ( Table 1). Overexpression of 14-3-3 proteins is thus a potential effector and can serve as a diagnostic marker for the more malignant types of HCC. 14-3-3 isoforms form homo-dimers or hetero-dimers that interact with their ligand proteins through Ser/Thr phosphorylation. As 14-3-3β, 14-3-3ε, 14-3-3σ and 14-3-3ζ isoforms are overexpressed in HCC, they may form different types of dimmers that interact with common or distinct partner proteins, thereby regulating joint or specific signaling and downstream targets. This may explain the specificity of select 14-3-3 isoform expression in different tissues or malignancies. In summary, 14-3-3ε and 14-3-3ζ contribute to HCC EMT by suppressing E-cadherin via Zeb-1 and αB-crystallin/Slug regulation, respectively. Additionally, 14-3-3β, 14-3-3γ and 14-3-3σ promote cell migration and proliferation of HCC mediated by activating Raf/MEK/ERK, PI3K/Akt signaling and increasing β-catenin/HSF-1α/HSP70 expression. The signaling pathways of JNK and p38/MAPK are involved in modulating 14-3-3ζ-associated chemosensitivity of HCC cells. Thus, 14-3-3 isoforms regulate cell proliferation, EMT and cell migration of HCC by synergistic networks to promote HCC tumor progression ( Figure 1). Finally, expression of 14-3-3ζ is associated with sorafenib resistance, and 14-3-3σ is considered a potential target for drug resistance. In conclusion, expression of particular 14-3-3 isoforms is a potential prognostic marker for clinical outcomes of HCC. Targeting selective 14-3-3 isoforms and related effectors can be beneficial in developing therapeutic strategies for HCC. We therefore conclude that 14-3-3 proteins are promising prognostic markers and therapeutic targets of HCC.

Conflicts of Interest
The authors declare no conflict of interest.