Protein phosphatase PHLPP induces cell apoptosis and exerts anticancer activity by inhibiting Survivin phosphorylation and nuclear export in gallbladder cancer

Abstract

Many factors regulate cancer cell apoptosis, among which Survivin has a strong anti-apoptotic effect and PHLPP is a tumor suppressor gene that can induce significant apoptosis. However, the relationship between PHLPP and Survivin in gallbladder carcinoma (GBC) has not been reported. This study found that PHLPP expression is decreased and Survivin expression is increased in GBC tissues and cell lines. Their expression levels showed an inverse relationship and were associated with poor prognosis of GBC patients. Loss of PHLPP can increase the level of phosphorylated Survivin and induce the nuclear export of Survivin, which thus inhibit cell apoptosis and promote cell proliferation in GBC cells. The process that PHLPP regulates Survivin phosphorylation and intracellular localization is involved in AKT activity. Re-overexpression of PHLPP in GBC cells can decrease AKT phosphorylation level. Reduced expression of PHLPP in GBC is associated with high expression of miR-495. Increasing PHLPP expression or inhibiting miR-495 expression can induce apoptosis and suppress tumor growth in GBC xenograft model in nude mice. The results revealed the role and mechanism of PHLPP and Survivin in GBC cells and proposed strategies for gene therapies targeting the miR-495 / PHLPP / AKT / Survivin regulatory pathway.

Keywords: Survivin; apoptosis; cell signaling; gallbladder carcinoma; protein phosphatase.

Figure 1. Expression of PHLPP and Survivin in GBC specimens and their relationships to prognosis in patients

(A) Clinical specimens of GBC and gallbladder tissues were subjected to dual-staining immunohistochemistry to detect the localization and expression of PHLPP and Survivin. The percentages of positive cells were counted for all of the slices within five high-power fields under microscope, and the positive cell percentages from <10% to <100% were defined as score 1 to 10, all cells that were negative were scored as zero. The expression scores were showed in area diagram. (B) The expression levels of PHLPP and Survivin showed an inverse relationship. (C) Different intracellular localization of Survivin in PHLPP-positive and –negative GBC cells, which were showed blue and red staining visualized by 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/tetranitroblue tetrazolium chloride (NBT) and 3-amino-9-ethylcarbazole (AEC), respectively, original magnification 400×. (D) The Kaplan-Meier analysis showed that both PHLPP and Survivin expression were closely associated with PFS in GBC patients, particularly the patients with PHLPP-negative and Survivin-positive had the worst prognosis in PFS.

Figure 2. PHLPP induces apoptosis and inhibits proliferation in GBC cells

(A) The EH-GB1 and GBC-SD cell lines were cultured, and 1 × 10⁶ cells at 24-well plates were infected with adenovirus Ad5-PHLPP (EH-GB1-PHLPP and GBC-SD-PHLPP), and then infected with adenovirus Ad5-shPHLPP (EH-GB1-shPHLPP and GBC-SD-shPHLPP), at a multiplicity of infection (MOI) of 100 pfu/cell. PHLPP expression was examined by Western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) The parental GBC cells and their sublines were stained with Annexin V / propidium iodide (PI), and cell apoptosis was measured with flow cytometer. The percentages of apoptotic cells, including the early and late apoptotic cells, were showed in histogram. **P < 0.01; ***P < 0.001. (C) The cell viability of GBC parental cells and cell sublines at 24 h, 48 h and 72 h was measured with the Cell Proliferation Kit in 96-well plates at 1 × 10⁴ cells/well. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. PHLPP inhibits Survivin phosphorylation and nuclear export

(A) Total protein, cytoplasmic protein and nuclear protein were extracted from 1 × 10⁶ cells of the parental EH-GB1 and GBC-SD cell lines and their cell sublines, and the total Survivin (t-Survivin) and phosphorylated Survivin (p-Survivin) were examined by Western blot, with GAPDH as the loading control. The densitometry analysis was performed to show the relative expression levels normalized with GAPDH density. *P < 0.05; **P < 0.01; ***P < 0.001. (B) The EH-GB1 and its cell sublines were cultured into Lab-Tek chambers at 1 × 10⁴ cells/well, fixed in 4% formaldehyde for 30 min, and incubated with the primary antibodies at working concentration 1:200 at 4°C overnight, and the TRITC-conjugated or FITC-conjugated secondary antibodies at working concentration 1:500 at room temperature for 1 h. The cells were counterstained with dihydrochloride (DAPI) for nuclear background staining and observed under laser-scanning confocal microscope.

Figure 4. PHLPP inhibits Survivin phosphorylation through AKT signaling pathway

(A) The expressions of total AKT (t-AKT) and phosphorylated AKT (p-AKT) were examined in 1 × 10⁶ cells of the parental EH-GB1 and GBC-SD cell lines and their cell sublines by Western blot, with GAPDH as the loading control. The densitometry analysis was performed to show the relative expression levels normalized with GAPDH density. ***P < 0.001. (B) The EH-GB1 cells were transfected with AKT shRNA vector (shAKT) and mock control shRNA vector (shMC), and the expressions of t-AKT, PHLPP and p-Survivin were examined by Western blot, with GAPDH as the loading control. The densitometry analysis was performed to show the relative expression levels normalized with GAPDH density. ***P < 0.001.

Figure 5. PHLPP was regulated by miR-495 in GBC cells

(A) The expression of miR-495 in 30 cases of GBC primary tumor tissues, 10 cases of GBC liver metastatic tissues and 10 cases of normal gallbladder mucosa tissues was examined by qRT-PCR. *P < 0.05; **P < 0.01. (B) The EH-GB1 and GBC-SD cells were transfected with miR-495 mimic and miR-495 inhibitor, with miNC as the negative control. After confirming miR-495 expression levels, the PHLPP expression was detected by Western blot, with GAPDH as the loading control. The densitometry analysis was performed to show the relative expression levels normalized with GAPDH density. ***P < 0.001. (C) The regulation of miR-495 on PHLPP expression was measured by luciferase reporter assay. EH-GB1 and GBC-SD cells at 1 × 10⁶ cells/well in 24-well plates were transfected with miR-495 mimic or miR-495 inhibitor at 20 μg/well and cultured for 24 h. The wells were co-transfected with 20 ng pRL-TK together with 200 ng pGL3-miR495Luc, pGL3-miR495Ctrl, or pGL3-Control. The luciferase activity was measured 48 h later. The positive control vector pGL3-Control was used to normalize the relative activities of pGL3-miR495Luc and pGL3-miR495Ctrl. ***P < 0.001. (D) The aforementioned cells transfected with miR-495 mimic or miR-495 inhibitor were measured their viability at 48 h and 72 h after transfection. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Effect of PHLPP or miR-495 on mouse GBC xenograft model

(A) Nude mice were implanted with 5 × 10⁶ cells / mouse of EH-GB1 cells, and divided into five groups after forming xenograft tumors 12 days later. Each mouse received a total dose of 1 × 10⁹ pfu adenovirus, or 50 μg miR-495 mimic and miR-495 inhibitor. The tumor diameters were measured weekly and the tumor volumes were calculated. *P < 0.05; **P < 0.01; ***P<0.001 compared with the blank control group. (B) The xenograft tumors were collected and weighed. *P < 0.05; **P < 0.01; ***P < 0.001. (C) The xenograft tumors were fixed in 10% neutral buffered formalin for 6 h, paraffin-embedded, sectioned and prepared for detecting the expressions of PHLPP, p-AKT and p-Survivin by immunohistochemistry, and the cell apoptosis rates were detected by TUNEL assay. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7. The schematic diagram of miR-495/PHLPP/AKT/Survivin regulatory pathway potentially existed in GBC

The phosphorylation of AKT is a key step for regulating cell proliferation and apoptosis. The extracellular cytokines and survival factors activate AKT signaling, which promotes the phosphorylation of Survivin and finally results in apoptosis inhibition and cell proliferation. However, PHLPP, that is under the regulation of miR-495, may dephosphorylate AKT and inactivate AKT signaling pathway, and also possibly dephosphorylate Survivin and promotes Survivin transportation into nuclei, then inactivates the function of Survivin, finally results in an increase of cell apoptosis and decrease of cell proliferation.