This study demonstrated that GOLPH3 was significantly over-expressed in human HCC tumor tissues. In addition, high expression of GOLPH3 was closely correlated with the serum AFP level, a widely used serum marker of HCC. In vitro, we found that the down-regulation of GOLPH3 resulted in decreasing cell proliferation and increasing cell apoptosis. Moreover, diminished GOLPH3 could inhibit tumorigenic properties of HCC cells in vivo. Furthermore, mTOR signaling pathway was involved in the pathological mechanism of GOLPH3 in HCC.
Hepatocellular carcinoma (HCC) is a cancer with a high mortality rate due to the fact that diagnosis usually occurs at an advanced stage. So it remains a serious threat to human health. Over the past decades, no significant improvements have been achieved regarding the early diagnosis and treatment of HCC. The understanding of the molecular oncogenesis underlying HCC is still lacking. Many studies have showed that GOLPH3 is an oncogene, which is involved in tumorigenesis and correlated with poor prognosis. Furthermore, some previous studies report that GOLPH3 overexpression is associated with poor clinical outcome in HCC [
25,
26]. In this study, we also discovered that GOLPH3 was high expressed in the HCC tumor tissues (Fig.
1). It is reported that HCC patients with AFP level ≤ 20 ng/ml may benefit the most from hepatectomy, but patients with AFP level > 20 ng/ml need comprehensive therapy, surgical resection and close follow-up examinations [
27]. So we put the 20 ng/ml AFP as a standard. And the results showed that the up-regulation of GOLPH3 was strongly correlated with high AFP level (Table
1). What’s more, that low expression of GOLPH3 promoted HCC cell apoptosis and inhibited tumorigenesis was found by functional studies in vitro and in vivo (Fig.
2 and Fig.
3).
The mammalian target of rapamycin (mTOR), a phosphatidylinositol 3 kinase (PI3K)-related serine/theronine kinase, plays a central role in regulating cell growth, proliferation and survival, in part by regulating translation initiation. mTOR functions in two structural and functional distinct protein complexes: mTORC1, which also contains two positive regulatory subunits, Raptor and mLST8, and two negative regulators, PRAS40 and DEPTOR; and mTORC2, which also contains Rictor, mSin1 and Protor, and also mLST8 and DEPTOR [
28‐
32]. As a part of mTOR complexes, mTORC1 directly activates S6 K1 (p70 ribosomal protein S6 kinase), phosphorylated at Thr389 [
33,
34], the major rapamycin-sensitive site. S6 K1 clearly plays an important role in the regulation of cell growth, cell cycle progression and cell proliferation [
31]. While mTORC2 was found to play a critical role in phosphorylating Akt (Ser473), which is one of the most important survival kinases, involved in regulating a wide array of cellular processes, including metabolism, growth, proliferation and apoptosis [
35‐
37]. In the HCC tissue specimens, immunochemistry revealed that the protein expression of p-mTOR (Ser2448), p-Akt (Ser473), and p-S6 K1 (Thr389) in the HCC tumor tissues were significantly higher than those in the paired normal groups (Fig.
4a). These results were verified in HCC cell lines (Fig.
4b). What’s more, by knocking down the GOLPH3 expression in HCC cells, the expression of p-mTOR (Ser2448), p-Akt (Ser473) and p-S6 K1 (Thr389) were remarkably decreased in the HCC cell lines and in the xenograft tumor model (Fig.
5a and Fig.
5b). These results indicated that GOLPH3 could be involved in HCC tumorigenesis via activating both mTORC1 and mTORC2. For further study, we also noted that knockdown GOLPH3 in the HCC cell lines diminished expression of Raptor rather than Rictor (see Additional file
1: Figure S1A). Research has suggested that Raptor can regulate mTOR activity and functions as a scaffold for recruiting mTORC1 substrates [
38]. Perhaps GOLPH3 could regulate Raptor to activate mTOR signaling cascade in HCC. Studies have demonstrated that mTOR controls NF-κB activity by stimulating IKK [
39] and GOLPH3 could sustain the activation of the NF-κB signaling pathway in HCC [
26]. Thus, the interplay among GOLPH3, mTOR and NF-κB in the progression of HCC is likely to be complex and needs to be further investigated. What’s more, research has shown that DNA-PK, a multimeric complex consisting of a catalytic subunit, DNA-PKcs, and the regulatory subunits, Ku70 and Ku86, plays a critical role in the DNA damage response, particularly in the recognition and repair of double stranded breaks [
40]. DNA-PK could phosphorylate GOLPH3 and play an important role in DNA-PK/GOLPH3/MYO18A pathway to regulate cell survival following DNA damage [
41]. In addition, Hemmings and colleagues observed that DNA-PK could phosphorylate AKT on Ser473 [
42]. And in our study, we discovered that when knocking down the GOLPH3 expression in HCC cells, the expression of DNA-PKcs and Ku70 was decreased (Additional file
1: Figure S1B). Perhaps this was because GOLPH3 and DNA-PK could via regulating the phosphorylation of AKT influence each other in Golgi’s function during DNA damage response. The more exact mechanism between DNA-PK/GOLPH3/MYO18A pathway and GOLPH3/mTOR pathway to regulate function of GOLPH3 in HCC progression will be explored in our further studies. And we think it is likely that there exist one or more proteins involved in GOLPH3-associated xsignaling pathway in HCC which may have therapeutic utility.