PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2

Abstract

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.

Keywords: Akt; HCC; PEBP4; mTORC1; mTORC2.

Figure 1

The effect of PEBP4 knockdown on the mTORCs-mediated signaling pathways. (A) Western blot (WB) was performed using different HCC and immortalized normal hepatocytes as indicated. (B) Scan densitometric ratio of PEBP4 to actin. (C,D). MHCC97H cells were infected with sh-NC and different PEBP4 shRNA1-4. Cell extracts were blotted (C) and quantified by scan densitometry (D). (E) Extracts of MHCC97H cells with sh-PEBP4 and sh-NC were blotted with antibodies. (F,G) Relative scan densitometric units were determined by the ratio of phospho-proteins to their cognate total proteins or actin for (E). (H) MHCC97H cells with sh-PEBP4 or sh-NC were treated with or without IGF-1 (100 µg/mL) for 30 min and WB was performed using antibodies. (I,J) Relative scan densitometric units were determined for H. All graphs represent averages of three independent experiments (mean ± SEM). * p < 0.05, ** p < 0.01, ^## p < 0.01, ^&& p < 0.01, ^$$ p < 0.01.

Figure 2

Akt in mediating the effect of PEBP4 on mTORC1, but not mTORC2. (A) The MHCC97H cells containing sh-PEBP4 or sh-NC were infected with adenovirus encoding the active Akt1 mutant (Akt-S473D) or GFP as a control at different doses and Western blots carried out using antibodies, as indicated. (B,C) The immunoblotted bands were analyzed by densitometry, as in Figure 1. (D,G) The MHCC97L cells containing pCMV6-PEBP4 or pCMV6-NC were treated with or without MK-2206 (5 µM, 24 h), an Akt specific inhibitor, and extracted blotted with antibodies. (E,F,H,I) Quantification of bands in (D,G) was performed. Results were presented as mean ± SEM (n = 3). Significant differences were examined by student’s t-test. * p < 0.05, ** p < 0.01, ^## p < 0.01, ^&& p < 0.01, ^ϕϕ p < 0.01.

Figure 3

PEBP4 interacts Akt and mTOR. (A,B) Extracts of MHCC97H cells were immunoprecipated with anti-Akt antibodies (A) or anti-mTOR (B) antibodies and blotted with antibodies as indicated. (C,D) Extracts of MHCC97H/sh-PEBP4 and MHCC97H/sh-NC cells were immunoprecipitated with anti-Akt (C) or anti-mTOR antibodies (D), followed by WB. (E) HEK293T cells were transiently transfected with plasmid encoding PEBP4 aminoterminally tagged with myc epitope (N-myc-PEBP4) and the recombinant PEBP4 was immunoprecipitated by anti-myc antibody, followed by WB.

Figure 4

Knockdown of PEBP4 suppresses the growth of HCC. (A–D) MHCC97H cells containing sh-PEBP4 or sh-NC were examined by WB (A), cell proliferation (B), and colony formation (C,D). Colonies in triplicate were counted (D). (E) BALB/C nude mice (n = 5) were injected into right fossa axillaris with the cells as indicated in (A) and tumor volumes measured. (F,G) Mice were sacrificed after 20 days and tumors were weighed (G). Representative images of mice and isolated tumors (F) were presented. All quantitative measurements were presented as mean ± SEM and statistical significance was tested, ** p < 0.01.

Figure 5

Overexpression of PEBP4 promotes the growth of HCC. (A) MHCC97L cells were transfected with pCMV6-PEBP4 or pCMV6-NC (empty vector) and then examined by WB. (B) Cell proliferation. (C) Colony formation. Colonies in triplicate were counted (D). (E) BALB/C nude mice (n = 5) were injected into right fossa axillaris with the cells indicated in (A), and tumor volumes measured. (F,G) Mice were sacrificed after 28 days, and tumors were weighed (G). Representative images of mice (right fossa axillaris) and isolated tumors (F) were presented. Statistical analysis was performed as for Figure 4. * p < 0.05, ** p < 0.01.

Figure 6

The role of Akt in mediating the effect of PEBP4 on proliferation, migration, invasion, and EMT markers of HCC. (A–D) MHCC97H cells containing sh-PEBP4 or sh-NC were infected with adenovirus expressing Akt S473D or GFP. Cell proliferation (A), migration and invasion were assayed (B) and quantified (C,D). (E,F) EMT markers were examined by Western blot € and WB bands semi-quantified as opposed to actin (F). (G–L) MHCC97L cells transfected with pCMV6-PEBP4 or the empty vector pCMV6-NC were treated with MK2206 or DMSO, cell proliferation (G), cell migration, and invasion were examined (H) and quantified (I,J). EMT markers were examined by WB (K) and bands were quantified against actin (L). All experiments were repeated three times. Data were presented as mean ± SEM (n = 3), ** p < 0.01, ^## p < 0.01, ^ϕϕ p < 0.01.

Figure 7

The role of PEBP4 in regulation of metastasis of HCC. (A–C) MHCC97H/sh-NC or MHCC97H/sh-PEBP4 cells were injected into tail veins of nude mice (n = 5/group) and 10 weeks later, lungs were dissected and photographed. The representative images of lung appearance were shown (A). The metastatic nodules in the lung were counted and plotted (B). Representative hematoxylin and eosin (H&E) stained images of the lung were shown (magnification: ×40, ×100, ×400) (C). (D–F). MHCC97L/pCMV6-PEBP4 and MHCC97L/pCMV6-NC cells were injected, and lung metastasis was assessed similarly to (A–C). Arrows point to metastatic nodules. Data were presented as mean ± SEM (n = 5), ** p < 0.01.