The level of TCP1 increases with successive generations of xenograft tumors and is associated with the tumorigenicity of HL-60 cells in vivo
Previous studies have indicated that successive implantation of HL-60 cells in nude mice could increase tumor formation in vivo. However, the underlying mechanisms are still unclear. For this purpose, we established four generations of HL-60 cell lines according to previous descriptions 35. As expected, the four generations of HL-60 cell lines exhibited increasing subcutaneous tumor formation ability with tumor formation rates of 30% (3/10) for HL-60-G1, 50% (5/10) for HL-60-G2, 80% (8/10) for HL-60-G3 and 100% (10/10) for HL-60-G4 cells (Fig. 1A). To address the possible molecular changes during the acquisition of tumorigenicity, five cell lysates were prepared and subjected to 2D-DIGE combined with mass spectrometry for proteomic analysis (Fig. 1B). The results of 2D-DIGE showed up to 2153 matched protein spots on the gels. Among these, 33 spots were significantly upregulated in the highly tumorigenic cells, whereas 54 spots were downregulated (Supplementary Fig. 1A). One differentially expressed protein, master No. 71, was identified as TCP1 via MALDI-TOF/TOF MS analysis (Supplementary Fig. 1B). To further verify TCP1 expression in tumor nodules, we performed IHC staining to detect TCP1 protein in tumor tissues formed by different generations of tumor cells. As shown by IHC analysis, the expression of TCP1 increased with increasing generations (Fig. 1C), which suggested that TCP1 may be closely related to the formation of malignant tumors. We then tested this hypothesis in tissue samples with AML and a variety of leukemia cells. RT-qPCR and WB showed that the expression of TCP1 in the initial untreated and recurrent marrow samples of AML was significantly higher than that in AML bone marrow samples in the normal and remission stages (Fig. 1D, E). The expression level of TCP1 in various malignant hematological cells was also higher than that in normal bone marrow specimens (Fig. 1F). The results indicate that TCP1 is highly expressed in leukemia cells and is related to the tumorigenicity of HL-60 cells in vivo.
TCP1 is highly expressed in many solid tumors and is related to poor outcomes in patients with PDAC and HCC
Given the upregulated expression of TCP1 in AML patients and leukemic cell lines (Fig. 1) and HCC17, we speculated that TCP1 might be upregulated in various malignant solid tumors. To explore the clinical significance of TCP1, we examined the expression of TCP1 in PDAC, HCC, lung cancer, lymphoma, gastric cancer, and thyroid cancer. The average level of TCP1 in tumor tissues was higher than that in adjacent tissues in all types of cancer examined (Fig. 2A), indicating that TCP1 is markedly increased in many solid tumors. Subsequently, we focused on the expression of TCP1 in pancreatic cancer and liver cancer by IHC and immunofluorescence (IF). The results showed that the expression of TCP1 in poorly differentiated PDAC and HCC17 was higher than that in well-differentiated and moderately differentiated tumors (Fig. 2B, C, F), and the overall survival (OS) time of patients with high levels of TCP1 was significantly shorter than that of patients with low levels of TCP1 (Fig. 2D). Pearson correlation analysis showed that the TCP1 level was negatively correlated with recurrence time in patients with PDAC and HCC (Fig. 2E, G), suggesting that TCP1 is associated with poor prognosis of malignant tumors.
Suppression of TCP1 inhibits the proliferation, migration, and invasion in PDAC and HCC cells
To investigate the effect of TCP1 on the phenotype of PDAC and HCC cells, we conducted functional experiments to evaluate the influence of TCP1 knockdown on cell proliferation, migration, and invasion. First, BxPC-3, PANC-1, SMMC-7721, and Huh-7 cells were infected with lentivirus containing shRNA targeting TCP1. TCP1 protein expression was significantly decreased in all shTCP1 cell lines (Fig. 3A). MTT and colony formation assay results showed that shTCP1 significantly inhibited cell growth and colony formation (Fig. 3B, C). Next, we examined the effect of shTCP1 on cell migration by Transwell assays. shTCP1 significantly inhibited the migration and invasion in four cells types (Fig. 3D). Thus, TCP1 knockdown successfully attenuated the proliferation, migration, and invasion of various cancer cell models in vitro.
Suppression of TCP1 expression inhibits tumor growth and metastatic spread in animal models
To explore the effect of altered TCP1 expression on the processes of tumor proliferation, we established a xenograft model in nude mice using PANC-1, SMMC-7721, and Huh-7 cancer cell lines. Consistent with the in vitro results, xenograft tumors with downregulated TCP1 expression grew more slowly than the control xenografts (Fig. 4A). Compared with the controls, TCP1 suppression showed significant growth inhibition as tumor volume decreased and tumor weight was lost in all three cell lines (Fig. 4B, C). To further investigate the role of TCP1 in PDAC and HCC metastasis, we injected SMMC-7721 and BxPC-3 cells labeled with luciferase into the mice by tail veins to track their metastasis. The metastatic ability of pancreatic cancer and liver cancer cells in vivo was detected by a living imaging system. As illustrated, knocking down TCP1 significantly reduced metastatic spread in both SMMC-7721 and BxPC-3 cells (Fig. 4D, E). Furthermore, the OS rates of mice treated with shTCP1 cancer cells were significantly longer than those of the control mice (Fig. 4F). In conclusion, these results suggest that knockdown of TCP1 could significantly decrease tumor burden in vivo.
TCP1 inhibition accelerates the degradation of c-Myc via the ubiquitin-proteasome pathway
Our in vitro and in vivo results showed that TCP1 had a strong effect on tumor cell proliferation. To better understand the molecular mechanism of TCP1-mediated tumor progression, we examined whether TCP1 inhibition affects the core signaling networks that are known to play an important role in HCC and PDAC, including PI3K/AKT/mTOR, MAPK signaling pathways and the key genes of cell proliferation. According to the results of the WB (Supplementary Fig. 2), we assessed the influence of TCP1 on the expression of the tumor proliferation-related protein c-Myc in TCP1 knockdown cell models. The results showed that the protein level of c-Myc but not the mRNA level was significantly decreased (Fig. 5A, B). Therefore, we speculated that TCP1 could affect the stability of the c-Myc protein. Subsequently, we used the protein synthesis inhibitor cycloheximide (CHX) to measure the degradation of the c-Myc protein. We found that the degradation of c-Myc was faster in cancer cells with TCP1 knockdown than in control cells, implying that TCP1 may regulate the protein stability of c-Myc (Fig. 5C). Then, MG132 was added to inhibit the activity of the 26S proteasome. The degradation of c-Myc protein was significantly inhibited in cancer cell models treated with both CHX and MG132 compared with those treated with CHX alone (Fig. 5C). Moreover, we observed that the degradation rate of c-Myc was considerably accelerated in the shTCP1 group compared with the scramble group in all types of cancer examined (Fig. 5D). Successively, using ubiquitination assays, we found that TCP1 knock-down increased the ubiquitination level of c-Myc (Fig. 5E). Thus, the above results indicated that suppression of TCP1 expression promotes the ubiquitination of c-Myc in cells, which eventually accelerates the degradation of the c-Myc protein.
TCP1 regulates the stability of c-Myc through AKT/GSK-3β and ERK signaling pathways
Previous studies have pointed out that the phosphorylation of Thr58 decreases the stability of c-Myc and promotes its degradation via the ubiquitin-proteasome pathway; the phosphorylation of Ser62, on the other hand, stabilizes c-Myc28,29. It has been reported that the phosphorylation of c-Myc is regulated mainly by the AKT/GSK-3β and ERK pathways22. To determine whether TCP1 regulates c-Myc protein stability through its phosphorylation, we manipulated TCP1 expression levels in above four cell lines and determined changes in the phosphorylation of c-Myc. While the total protein level of c-Myc and the ratio of p-c-Myc Ser62 to p-c-Myc Thr58 were decreased in TCP1 knockdown cells, TCP1 overexpression showed the opposite effect, providing evidence that TCP1 regulates the stability of c-Myc mainly by altering the ratio of p-c-Myc Ser62 to p-c-Myc Thr58 (Fig. 6A-D). Then, we assessed the levels of key proteins of the AKT/GSK-3β and ERK signaling pathways. The phosphorylation of AKT Ser473, GSK-3β Ser9, and ERK was significantly reduced with TCP1 knock-down but increased with TCP1 overexpression (Fig. 6A, C). However, the total protein levels of AKT, GSK-3β, and ERK were not changed in either TCP1 knock-down or overexpression cells (Fig. 6A, C). These results suggest that TCP1 regulates the AKT/GSK-3β and ERK signaling pathways. After knocking down TCP1 and inhibiting GSK-3β activity with AR-A014418 (AR), the protein level of c-Myc was restored, which demonstrated that TCP1 regulates the stability of c-Myc by modulating the AKT/GSK-3β signaling axis (Fig. 6E, F). Next, we used different concentrations of the AKT-specific inhibitor AKT inhibitor VIII (AKT-iVIII) to inhibit AKT activity in the four cell lines, we found that the TCP1 protein levels were not changed (Fig. 6G), which indicates that the TCP1 protein is an upstream regulator of the AKT/GSK-3β pathway. These results revealed that TCP1 regulates the stability of the c-Myc protein through the AKT/GSK-3β and ERK signaling pathways, elucidating the mechanism of TCP1 in pancreatic cancer and liver cancer.
Overexpression of TCP1 promotes the expression of c-Myc by regulating the AKT/GSK-3β and ERK signaling pathways, thus promoting the occurrence of primary liver cancer
To gain global insight into the mechanism of TCP1 in hepatocarcinogenesis, WT and TCP1-KI mice were used to construct the primary HCC model induced by DEN. The number of tumor nodules (the red arrows), the ratio of liver weight, and tumor volume significantly increased in TCP1-KI group compared with WT group (Fig. 7A, B), indicating that TCP1 promotes the occurrence and development of primary HCC. Histological analysis showed that the liver tissues of the TCP1-KI group had no complete liver lobules (Fig. 7C). The nuclei of the hepatocytes were enlarged, and the vacuolation was seriously disordered compared with that in WT group (Fig. 7C). TCP1-KI group showed obvious pathological features of hepatoma. In addition, IHC showed that the expression of c-Myc and Ki67 was significantly increased in the TCP1-KI group (Fig. 7C). Furthermore, the influence of TCP1 overexpression on the AKT/GSK-3β and ERK pathways was noted by WB. The overexpression of TCP1 could activate the AKT/GSK-3β and ERK pathways to promote the expression of c-Myc (Fig. 7D), which further confirmed our hypothesis in vivo.
Here, we discovered an important role for TCP1 in promoting cancer cell proliferation and metastasis through c-Myc. TCP1 increased the phosphorylation of ERK, leading to the phosphorylation of c-Myc Ser62, which could promote cell proliferation. Moreover, the level of phosphorylated c-Myc Thr58 was significantly decreased after TCP1 upregulated the phosphorylation of AKT/GSK-3β, which led to the inhibition of c-Myc degradation (Fig. 8). Taken together, our findings reveal a novel mechanism of TCP1 and indicate that TCP1 may be a valuable molecular marker for cancer diagnosis and prognosis evaluation.