PER1 Serves as a Tumor Suppressor in Breast Cancer by Regulating MEK5/ERK5 Signaling Pathway

Abstract

Background: Breast cancer (BC) is one of the most frequently diagnosed malignant tumors worldwide. Period circadian protein homolog 1 (PER1) is a primary component of the biorhythm molecular oscillation system. The objective of this study was to elucidate the association between PER1 and clinical BC outcomes and determine the potential effect of PER1 on BC tumor development.

Methods: Immunohistochemical staining for PER1 was performed on 30 normal breast tissue and 172 BC samples. Those BC cases were categorized into two groups to analyze the prognostic significance of PER1 expression. The expression of key proteins in the MEK/ERK pathway (ERK1/2, ERK5, P38, JNK1/2/3) and their phosphorylation levels (p-ERK1/2, p-ERK5, p-P38, and p-JNK1/2/3) were elucidated by western blot test. XMD17-109, a specific ERK5 inhibitor, was used to treat BT-549 and MCF-7 BC cells with knockdown of PER1.

Results: Increased PER1 expression was identified in 26 and 80 normal breast and BC tissues, respectively, whereas low expression was detected in 4 normal and 92 BC tissues. Although no differences were observed in the estrogen receptor (ER), menstrual cycle, TNM, progesterone receptor (PR), and HER-2 stages, age, and tumor size between the two cohorts, both the rate of axillary lymph node metastasis (P<0.05) and vascular tumor thrombosis (P<0.05) were enhanced in the low cohort. Furthermore, the low-PER1 group had the worst overall survival (HR: 0.44, 95% CI: 0.20-0.96, P=0.035) and relapse-free survival (HR: 0.29, 95% CI: 0.13-0.67, P=0.002). PER1 overexpression reduced phosphorylation levels of ERK5 in Lenti-blast-PER1-MDA-MB-231 BC cells (P<0.05), while PER1 silencing had the opposite effect on the pGenesil-1-PER1-MCF-7 cells (P<0.05). Colony formation, 5-ethynyl-2'-deoxyuridine, and Transwell cell migration and invasion assays revealed that XMD17-109 antagonized the enhancement of cell proliferate, migration, and invasion by PER1 knockdown (P<0.05).

Conclusion: PER1 plays an anti-tumor role by regulating the MEK5/ERK5 pathway in BC.

Keywords: MEK5/ERK5; PER1; breast cancer; circadian clock gene; tumor suppressor.

Figure 1

Representative pictures of positive and negative expression of PER1 protein in BC tissues (100× and 400×). (A) Positive expression. (B) Negative expression.

Figure 2

PER1 affects BC patient’s prognosis. (A) RFS and OS of 172 BC patients based on the ten-year follow‑up data. (B) RFS and OS in lymph node metastasis subgroup. (C) RFS and OS in vascular tumor thrombus subgroup.

Figure 3

The key protein expression of the MAPK pathway in the breast cancer cell with low and high PER1 expression. *P<0.05 vs pGenesil-1-KB group; ^#P<0.05 vs Lenti-blast-KB group.

Figure 4

Effects of ERK5 inhibitor XMD17-109 on MEK5/ERK5 pathway activity. *P<0.05 vs pGenesil-1-KB group; ^#P<0.05 vs pGenesil-1-PER1 group.

Figure 5

XMD17-109 reverses the enhancement of the proliferative ability in BC cells with low expression of PER1 (100×). (A) Colony Formation Assay. (B) EdU Assay. *P<0.05 vs pGenesil-1-KB group; ^#P<0.05 vs pGenesil-1-PER1 group.

Figure 6

XMD17-109 reverses the increased migration and invasion ability in BC cells with low expression of PER1 (100×). (A) Transwell tumor cell migration assay. (B) Transwell tumor cell invasion assay. *P<0.05 vs pGenesil-1-KB group; ^#P<0.05 vs pGenesil-1-PER1 group.