Overexpression of PIN1 Enhances Cancer Growth and Aggressiveness with Cyclin D1 Induction in EBV-Associated Nasopharyngeal Carcinoma

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1's potential role as a therapeutic target.

Methods: The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated.

Results: We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo.

Conclusions: This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients.

Fig 1. Overexpression of PIN1 in EBV-associated NPC.

(A) IHC staining was used to illustrate the overexpression of PIN1 in representative NPC primary tumors (NPC1-NPC4). All of the cases were EBV-positive undifferentiated carcinomas. NPC1, NPC3 and NPC4 are from the patients with stage 3 disease. NPC2 is from a patient with stage 2 NPC. Strong PIN signals were found in the tumor cells, which are indicated by yellow “_*”. The adjacent normal epithelium served as the control in which weak PIN1 signals were observed. The red arrows indicate normal nasopharyngeal epithelium. (B) Expression of PIN1 in NPC tumor lines, xenografts and immortalized normal NP cells, detected by qRT-PCR (upper panel) and Western blot (lower panel). For qRT-PCR, the PIN1 transcription in NP460 was used as a reference. The relative expression of PIN1 transcripts was indicated as a fold difference over the reference. β-actin was used for loading normalization. Similarly, ACTIN was used as an internal loading control in the Western blot analysis. The weak PIN1 expression in NP69 and NP460 suggested that the downregulation of PIN1 involved post-translational regulation.

Fig 2

PIN1 suppression inhibits cell growth, DNA synthesis, colony formation ability and cyclin D1 expression in NPC cells (A) qRT-PCR and (B) Western blot were used in the downregulation of PIN1 transcription and protein expression in PIN1 siRNAs (siPIN1 544 and siPIN1 545)-treated NPC cells and C666-1, respectively. In these PIN1 knockdown cells, reduced cyclin D1 expression was observed. ACTIN was used as the loading control in the Western blot analysis. (C) WST-1 assay revealed growth inhibition in the NPC cells transfected with siPin1 544 and siPin1 545. (D) Colony formation ability was significantly suppressed in PIN1-silenced C666-1 cells. Representative photos of colonies formed by siRNA-treated and control cells are shown. Statistical significance was determined by Student t-test, where a P-value of less than 0.05 was considered significant (*P < 0.05, **P < 0.01). (E) A BrdU assay was used to reveal the significant inhibition of DNA synthesis in the PIN knockdown NPC cells.

Fig 3. PIN1 inhibitor Juglone suppresses tumor cell growth and induces caspase-3 activity.

(A) HK-1 (left panel) and C666-1 (right panel) were treated with the PIN1 inhibitor Juglone for 24 hours to determine the drug dose sensitivity. The IC50 values of the C666-1 and HK1 cells were 6 μM and 10 μM, respectively. (B) The expression of cyclin D1 was suppressed by Juglone in a dose-dependent manner, but no significant changes in β-catenin expression were detected. The expression of cyclin D1 and β-catenin in the Juglone-treated C-666 xenograft model was examined by Western blot. Reduced expression of cyclin D1 was observed in the tumor that had received Juglone treatment. PIN1 inhibition resulted in suppression of cyclin D1 both in vitro and in vivo, and there was a modest reduction in the β-catenin level in the in vivo model. (C) The C666-1 cells exhibited significantly higher caspase-3 activity after Juglone treatment, compared with their untreated or DMSO-treated counterparts. This indicates that PIN1 inhibition can activate the caspase apoptotic pathway in NPC cells. Statistical significance was determined by Student t-test, P-value of less than 0.05 was considered significant (*P < 0.05, **P < 0.01).

Fig 4. PIN1 inhibitor Juglone suppresses tumor growth in vivo.

(A) Using in vivo imaging, the tumor growth of luciferase-tagged C666-1 was revealed after treatment with different dosages of Juglone (ROI counts per million photons per second). The luciferase signal was increased in the control untreated tumors during the Juglone treatment. In the mice treated with Juglone in different doses (0.5, 1 and 1.5 mg/kg), consistently lower luciferase signals were observed during the 8-day treatment periods. (B) Significant inhibition of tumor growth was shown in the mice treated with 0.5, 1 and 1.5 mg/kg Juglone. Four nude mice were used in each study group. Statistical significance was determined by Student t-test, where a P-value of less than 0.05 was considered significant (*P < 0.05, **P < 0.01).

Fig 5. PIN1 overexpression enhances anchorage-independent growth in nasopharyngeal epithelial cells.

(A) The overexpression of PIN1 was detected in NP69 cells transfected with Pin1-expressing lenti-viral vectors (Pin1 1# and Pin1 2#) by qPCR (left panel) and Western blot (middle panel). Representative photos of PIN1-transfected cells by bright field (right upper panel) and fluorescence field (right lower panel) are shown. The transfection efficiency was monitored by GFP expression. (B) Enhanced anchorage-independent growth on soft agar in NP69 cells with PIN1-overexpression, compared with control cells (left upper panel, colonies in 6-well plates; left lower panel, colonies under microscope; right panel, average data in histogram). (C-D) No significant effect of PIN1 overexpression on cell growth and colony formation ability was detected in the immortalized nasopharyngeal epithelial cells NP69, by WST-1 and colony formation assay, respectively. Statistical significance was determined by Student t-test, where a P-value of less than 0.05 was considered significant (*P < 0.05).

Fig 6. Overexpression of PIN1 activates the MAPK/JNK pathway.

(A) Using the Cancer 10-Pathway Reporter Luciferase assay, significant activation of the MAPK/JNK signaling pathway was observed in two stably PIN1-overxpressing NP69 cell lines (NP69lenti-PIN1 1# and NP69lenti-PIN1 2#). For these PIN1-expressing cells, the activity was also moderately activated in the NOTCH, P53/DNA damage and NF-κB pathways. (B) Western blot analysis demonstrated the upregulation of c-Jun, phosphorylated c-Jun (Ser63 and Ser73) and cyclin D1 in the stable PIN1-transfected NP69 cells. Statistical significance was determined by Student t-test, where a P-value of less than 0.05 was considered significant (*P < 0.05).