COPB2 promotes hepatocellular carcinoma progression through regulation of YAP1 nuclear translocation

Abstract

Objectives: Although Yes-associated protein 1 (YAP1) is an important oncogene in hepatocellular carcinoma (HCC) progression, its nuclear localization prevents it from being considered a potential therapeutic target. Recently, studies have reported that coatomer protein complex subunit beta 2 (COPB2) also plays a critical role in HCC development; however its mechanism of action is unclear. This study aimed to investigate the role of COPB2 and YAP1 in the progression of HCC and to elucidate the underlying mechanisms.

Methods: COPB2 and YAP1 expression in HCC tissues were first analyzed by database searches and immunohistochemistry. Nomogram and artificial neural network models were established based on COPB2 and YAP1 expression. Cell proliferation was detected by cell counting kit-8 and clone formation assay, while cell migration and invasion were assessed using Transwell assays. Finally, the potential mechanisms underlying COPB2 regulation of YAP1 nuclear translocation were explored by immunofluorescence assay and Western blot.

Results: COPB2 combined with YAP1 expression was associated with overall postoperative survival in HCC patients and was an independent prognostic factor. High expression of both COPB2 and YAP1 in patients may reduce the efficacy of postoperative transarterial chemoembolization therapy. In vitro experiments revealed that COPB2 affected the sensitivity of HCC cells to Cisplatin (DDP) by regulating YAP1 nuclear translocation.

Conclusions: Our findings suggest that COPB2/YAP1 affects the drug sensitivity of HCC cells to DDP and that targeting COPB2/YAP1 may be a promising strategy for the precision treatment of HCC.

Keywords: Cisplatin (DDP); Coatomer protein complex subunit beta 2 (COPB2); Hepatocellular carcinoma (HCC) prognosis; Yes-associated protein 1 (YAP1).

Figure 1. Expression of coatomer protein complex subunit beta 2 (COPB2) and Yes-associated protein 1 (YAP1) in hepatocellular carcinoma (HCC) tissues in the database. (A) In patient 2280, COPB2 expression was low in the cytoplasm, and YAP1 expression was low in the nucleus. (B) In patient 2279, COPB2 was highly expressed in the cytoplasm, while YAP1 was highly expressed in the nucleus.

Figure 2. Correlation of coatomer protein complex subunit beta 2 (COPB2) and Yes-associated protein 1 (YAP1) expression in hepatocellular carcinoma tissues in tissue microarrays. (A) In patient A, COPB2 expression was low in the cytoplasm, and YAP1 expression was low in the nucleus. (B) In patient B, COPB2 was highly expressed in the cytoplasm, and YAP1 was highly expressed in the nucleus.

Figure 3. Kaplan-Meier survival analysis between coatomer protein complex subunit beta 2/Yes-associated protein 1 (COPB2/YAP1) expression and overall survival (OS) of hepatocellular carcinoma patients after surgery. (A) Patients with higher COPB2 expression have a shorter OS. (B) Patients with higher YAP1 expression have a shorter OS. (C) Patients with increased expression of both COPB2 and YAP1 have the shortest OS.

Figure 4. Nomogram predicting the probability of survival at 3 and 5 years. (A) Nomogram of hepatocellular carcinoma patients after surgery based on coatomer protein complex subunit beta 2 and Yes-associated protein 1 expression. (B–C) Good calibration for predicting survival at 3 and 5 years.

Figure 5. An ANN model related to COPB2 and YAP1 expression. (A) Schematic representation of an artificial neural network (ANN) for predicting overall survival after surgery in patients with hepatocellular carcinoma. (B) The importance of the variables in the ANN model.

Figure 6. Kaplan-Meier survival analysis between coatomer protein complex subunit beta 2/Yes-associated protein 1 (COPB2/YAP1) expression and overall survival (OS) of hepatocellular carcinoma patients receiving transarterial chemoembolization after surgery. (A) Patients with higher COPB2 expression have a shorter OS. (B) Patients with higher YAP1 expression have a shorter OS. (C) Patients with increased expression of both COPB2 and YAP1 have the shortest OS.

Figure 7. Coatomer protein complex subunit beta 2 (COPB2) mediates the drug sensitivity of hepatocellular carcinoma cells to DDP (5 μg/mL) through the regulation of Yes-associated protein 1 (YAP1). (A) Huh7 and SK-hep1 were transfected with si-RNA to knock down COPB2 and plasmids to overexpress YAP1, and Western blot was used to detect the efficiency. (B) Cell proliferation was detected by CCK-8 assay at different time points. (C) Representative picture and quantification of colony formation. (D–E) Representative picture and quantification of the Transwell assay. All data are displayed as mean ± standard deviation (SD). **p < 0.01, ***p < 0.001.

Figure 7. Coatomer protein complex subunit beta 2 (COPB2) mediates the drug sensitivity of hepatocellular carcinoma cells to DDP (5 μg/mL) through the regulation of Yes-associated protein 1 (YAP1). (A) Huh7 and SK-hep1 were transfected with si-RNA to knock down COPB2 and plasmids to overexpress YAP1, and Western blot was used to detect the efficiency. (B) Cell proliferation was detected by CCK-8 assay at different time points. (C) Representative picture and quantification of colony formation. (D–E) Representative picture and quantification of the Transwell assay. All data are displayed as mean ± standard deviation (SD). **p < 0.01, ***p < 0.001.

Figure 8. Knockdown of coatomer protein complex subunit beta 2 (COPB2) promotes the exit of Yes-associated protein 1 (YAP1) from the nucleus and affects its stability. (A) Representative images of immunofluorescent staining for YAP1 distribution. (B) Representative pictures of Western blotting analysis of pLATS1 and pYAP1 in Huh7 and SK-hep1 cells transfected with si-NC and si-COPB2. All data are displayed as mean ± standard deviation (SD). **p < 0.01.