In this study, we explored a new mechanism of DEPDC1-induced proliferation and migration of osteosarcoma cells. The expression of DEPDC1 in osteosarcoma was similar to that of EEF1A1 but was negatively correlated with FOXO3a. Therefore, we speculated that the complex formed by DEPDC1 and EEF1A1 enhanced the expression of EEF1A1 and ultimately led to the downregulation of FOXO3a, thus promoting the proliferation and migration of osteosarcoma cells in vitro and in vivo (Fig. 6).
DEPDC1 is a newly discovered tumor-related gene that has a highly conserved domain. Many studies have found that proteins with DEP domains can regulate many cellular functions, such as cell membrane anchoring, signal transduction, cell polarity establishment, and regulation of small molecule GTP enzyme activity . Recent studies have shown that DEPDC1 is overexpressed in bladder cancer, breast cancer, lung adenocarcinoma, and other malignant tumor types [19-21]. In addition, Harada et al. found that DEPDC1 mainly inhibited cell apoptosis through the NF–κB signalling pathway, and then promoted the progression of bladder cancer . Furthermore, DEPDC1 can also be used in the diagnosis and treatment of various tumors. Kretschmer et al. found that DEPDC1 and FOXM1 are significantly upregulated in ductal carcinoma in situ (DCIS), and thus can be used to identify early molecular markers of breast cancer . S-288310, a cancer peptide vaccine containing oncoantigens against DEPDC1, is well tolerated and can effectively prolong the survival time of patients with urothelial carcinoma of the bladder . However, it remains unclear whether DEPDC1 is the main mechanism of promoting the proliferation and migration of malignant tumors. In this study, we found that DEPDC1 was highly expressed in human osteosarcoma through the GEO database analysis and confirmed this result in osteosarcoma tissues and cells (Fig. 1). At the same time, through co-IP and RNA-seq experiments, we found that DEPDC1 inhibits the expression of FOXO3a in combination with EEF1A1, thereby promoting the proliferation and migration of osteosarcoma in vitro and in vivo (Fig. 2–5).
Eukaryotic translation elongation factor 1A (EEF1A) is an important molecule involved in the translation function in protein synthesis. EEF1A can be divided into two subtypes, EEF1A1 and EEF1A2. In humans, EEF1A is encoded by two genes on chromosomes 6 and 20, which are mainly involved in apoptosis, cell cycle regulation, protein degradation, and post-translation modifications [24-26]. EEF1 complex members are necessary in the eukaryotic elongation process. Recently, it has been found that EEF1A1 is related to cancer occurrence [13, 27]. Genetic changes in EEF1A1 were detected by The Cancer Genome Atlas (TCGA) to explore its potential impact on selected epigenetic modulators. However, the specific upstream and downstream regulatory molecules that bind to EEF1A1 were not accurately investigated, nor the clinical correlation between the expression of EEF1A1 and the prognosis of tumor patients . Furthermore, the role of EEF1A1 in the proliferation and metastasis of osteosarcoma has not been studied. In our study, we found that DEPDC1 directly binds to and promotes the expression of EEF1A1 in the nuclei of osteosarcoma cells, thus promoting the proliferation and migration of osteosarcoma cells in vitro and in vivo (Fig. 2 and 4).The 15–106, 107–180, and 407–527 fragments of DEPDC1 showed strong binding with EEF1A1 (Fig. 2E bottom). Furthermore the 15–106 domain of DEPDC1 is the DEP domain, named after Dishevelled, Egl-10, and Pleckstrin, in which this domain was first discovered. The function of this domain remains unclear, but is believed to be important for the membrane association of the signalling proteins in which it is present [29, 30]. In addition, this report for the first time revealed the relationship between the expression of DPEDC1/EEF1A1 and the clinical prognosis of osteosarcoma patients (Fig. 5). The expression of EEF1A1 in osteosarcoma is positively correlated with DEPDC1, and the high expression of both can reduce the survival time of osteosarcoma patients, which indicates that DEPDC1 and EEF1A1 are potential prognostic markers and therapeutic molecular targets of osteosarcoma.
The Forkhead transcription factors (FOXO), also named Forkhead-like protein (FKHR), is a family transcription factors that was identified in 2000. There are four types in mammals, FOXO1, FOXO3a, FOXO4, and FOXO6, which are distributed on different chromosomes . The common feature of this family is the conserved DNA domain, namely, the Forkhead box. This protein family regulates apoptosis, cell cycle, cell proliferation, DNA damage repair, and cancer development, and inhibits tumor cell proliferation . FOXO3a is among the most widely studied members of the Forkhead family. It is located on human chromosome 6q21 and is expressed in gastrointestinal, liver, ovary, prostate, and breast tissue as well as others [32, 33]. FOXO3a dysfunction leads to uncontrolled cell proliferation and DNA damage accumulation, resulting in tumorigenic effects [34, 35]. The main mechanism regulating FOXO3a activity and its target genes is the control of the nuclear–cytoplasmic shuttling of FOXO3a. The phosphorylation of FOXO3a leads to its translocation from the nucleus to cytoplasm, followed by binding to 14-3-3 protein in the cytoplasm, and then FOXO3a is degraded in a ubiquitin-/proteasome-dependent manner [36, 37]. The balance of nuclear–cytoplasmic shuttling is crucial for maintaining the function of FOXO3a. The loss of this balance leads to the occurrence and development of various diseases including cancer [38, 39]. Studies have shown that the Wnt–β-catenin and PI3K–AKT–FOXO3a pathways have a central role in cancer. AKT phosphorylates FOXO3a, promoting its translocation from the cell nucleus to the cytoplasm. When this effect was reversed by PI3K and AKT inhibitors, the accumulation of FOXO3a in the nucleus increased, which promoted the apoptosis of colon cancer cells and inhibited their metastasis . Additionally, Hu et al. found that nuclear exclusion of FOXO3a by AKT contributed to cell survival. They also observed that Iκβ kinase (IKK) can promote FOXO3a phosphorylation, inhibiting the expression of FOXO3a, and finally causing FOXO3a protein hydrolysis through the ubiquitin-dependent proteasome pathway. The expression and accumulation of FOXO3a in the nucleus is reduced by IKKβ, which promotes the proliferation of breast cancer cells and is related to the low survival rate of breast cancer . However, there have been no further studies on whether oncogenes are involved in the transcription and expression of FOXO3a, affecting the balance of nuclear–cytoplasmic shuttling and promoting the occurrence and development of tumors. Lei et al. found that DEPDC1 overexpression facilitated cell proliferation and tumor growth through increasing the expression of FOXM1 in TNBC cells . FOXM1 is negatively regulated by FOXO3a, supports cell survival, drug resistance, colony formation and proliferation in vitro, and promotes tumor development in vivo . At the same time, the FOXO3-FOXM1 axis is a key cancer drug target and a modulator of cancer drug resistance . But so far, there is no report to explore the relationship between DEPDC1 and FOXO3a and their interaction mechanism. In this study, we found that FOXO3 had the best correlation with DEPDC1 expression in human osteosarcoma cells by RNA-seq (Fig. 3A, B and Additional file 4: Supplementary Fig. S2). Later, we found that DEPDC1 forms a complex with EEF1A1. Upregulation of DEPDC1 promoted the accumulation of EEF1A1 in the nuclei of osteosarcoma cells and then inhibited the expression of FOXO3a, restricting its distribution to the cytoplasm, while the expression of phosphorylated FOXO3a increased (Fig. 2–4). When DEPDC1 was downregulated, the opposite result was obtained (Fig. 2–4). Furthermore, the expression of FOXO3a was negatively correlated with DEPDC1 in human osteosarcoma and lowexpression of FOXO3a shortened the survival time of patients with osteosarcoma (Fig. 5). Therefore, we observed that inhibition of FOXO3a expression could significantly promote tumor proliferation and migration while reducing the survival time of tumor patients, which is consistent with many previous reports. Wasim et al. found that EEF1A1 can activate AKT dependent cell migration and tumor proliferation [44, 45]. In addition, AKT phosphorylates FOXO3a, promoting tumor proliferation and metastasis .
Cyclin D1 is an important regulator of the cell cycle and has a vital role in tumor development. In the cell cycle, cyclin D1 levels are regulated by many factors and change periodically. Cyclin D1 regulates cell proliferation from G1 phase to S phase. Overexpression of cyclin D1 shortens the time from G1 to S phase and accelerates the transformation of the cell cycle, leading to uncontrolled cell proliferation and migration and finally tumor occurrence . In addition, cyclin D1 is the critical downstream molecule of the FOXO3a signalling pathway, and is negatively correlated with FOXO3a expression (Fig. 3C, D). Tudzarova et al. found that FOXO3a activates the ARF–Hdm2–p53–p21 pathway, and p53 in turn activates expression of the Wnt/β-catenin signalling antagonist DKK3, leading to cyclin D1 downregulation . Zheng et al. also found that FOXO transcription factors repress cyclinD1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses cyclin D1expression . Lin et al. found that FLOT1 knockout inhibited the proliferation and tumorigenicity of breast cancer cells by upregulating FOXO3a and then downregulating cyclin D1 . Meanwhile, the overexpression of EEF1A1 regulates G1-phase progression to promote HCC proliferation through the STAT1-cyclin D1 pathway . STAT1 has been proved to have the same effect as AKT, which can phosphorylate FOXO3a . However, it has not been reported whether the complex formed by DEPDC1 and EEF1A1 in human osteosarcoma weakens the inhibitory effects of FOXO3a on cyclin D1 to ultimately affect tumor progression.
Combined with this study and many previous studies, we first found that the proliferation and migration of osteosarcoma cells are affected through the DEPDC1–EEF1A1–FOXO3a–cyclin D1 signalling pathway in vitro and in vivo. Therefore, we speculated that in human osteosarcoma cells, high expression of DEPDC1 permits it to form a complex with EEF1A1 and promote the expression of EEF1A1, thus inhibiting the synthesis of FOXO3a. At the same time, FOXO3a translocates from the nucleus and is phosphorylated, which eventually decreases the inhibitory effect of FOXO3a on cyclin D1 and promotes the growth of human osteosarcoma. However, there are many limitations in this study, such as how FOXO3a is degraded after leaving the nucleus and how it affects the expression of downstream cyclin D1 in osteosarcoma, thus inhibiting the progression of osteosarcoma. Further studies are required to elucidate this. In conclusion, our study suggests that the DEPDC1–EEF1A1–FOXO3a–cyclin D1 pathway may be a promising target for the prevention and treatment of human osteosarcoma.