UHRF1 knockdown induces cell cycle arrest and apoptosis in breast cancer cells through the ZBTB16/ANXA7/Cyclin B1 axis

Abstract

Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) is involved in tumorigenicity through DNA methylation in various cancers, including breast cancer. This study aims to investigate the regulatory mechanisms of UHRF1 in breast cancer progression. Herein, we show that UHRF1 is upregulated in breast cancer tissues and cell lines as measured by western blot analysis and immunohistochemistry. Breast cancer cells are transfected with a UHRF1 overexpression plasmid (pcDNA-UHRF1) or short hairpin RNA targeting UHRF1 (sh-UHRF1), followed by detection of cell proliferation, invasion, apoptosis, and cell cycle. UHRF1 overexpression promotes proliferation and invasion and attenuates cell cycle arrest and apoptosis in breast cancer cells, while UHRF1 knockdown shows the opposite effect. Moreover, methylation-specific PCR and ChIP assays indicate that UHRF1 inhibits zinc finger and BTB domain containing 16 (ZBTB16) expression by promoting ZBTB16 promoter methylation via the recruitment of DNA methyltransferase 1 (DNMT1). Then, a co-IP assay is used to verify the interaction between ZBTB16 and the annexin A7 (ANXA7) protein. ZBTB16 promotes ANXA7 expression and subsequently inhibits Cyclin B1 expression. Rescue experiments reveal that ZBTB16 knockdown reverses the inhibitory effects of UHRF1 knockdown on breast cancer cell malignancies and that ANXA7 knockdown abolishes the inhibitory effects of ZBTB16 overexpression on breast cancer cell malignancies. Additionally, UHRF1 knockdown significantly inhibits xenograft tumor growth in vivo. In conclusion, UHRF1 knockdown inhibits proliferation and invasion, induces cell cycle arrest and apoptosis in breast cancer cells via the ZBTB16/ANXA7/Cyclin B1 axis, and reduces xenograft tumor growth in vivo.

1. Introduction

Breast cancer is the most common cancer in women worldwide and is the second leading cause of cancer-related death among women [1]. Currently, clinical therapeutic strategies include surgical resection, chemotherapy, adjuvant radiotherapy, and targeted biotherapy [2]. Although significant improvements have rapidly been made in the management of breast cancer, the outcomes remain seriously unsatisfactory owing to the increasing incidence rate and tumor recurrence and metastasis [3]. Thus, there is an urgent need to clarify the molecular mechanism of breast cancer progression and formulate more effective clinical treatment strategies.

Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1), also known as ICBP90 in humans and Np95 in mice, has been identified as a multidomain protein. It has been reported that UHRF1 is upregulated in multiple cancers, and its dysregulation is associated with proliferation, cell cycle arrest and apoptosis in cancer cells. For instance, UHRF1 is elevated in cutaneous squamous cell carcinoma (cSCC), and knockdown of UHRF1 attenuates cSCC cell proliferation, migration, and invasion, leading to G2/M cell cycle arrest and increased apoptosis [4]. UHRF1 expression is upregulated in patients with hepatocellular carcinoma (HCC), while knockdown of UHRF1 inhibits proliferation and metastasis and induces G2/M cell cycle arrest in HCC cells [5]. UHRF1 has also been identified as an important epigenetic factor that regulates DNA methylation. The SET and RING-associated (SRA) domain of UHRF1 has been proven to be required for the maintenance of DNA methyltransferase 1 (DNMT1)-mediated DNA methylation [6]. Accumulating evidence indicates that UHRF1 is involved in tumorigenicity by promoting DNA promoter methylation and subsequently silencing tumor suppressor genes. It was found that UHRF1 induces DNMT1-mediated promoter methylation and subsequently downregulates the expressions of tumor suppressor genes, thus inhibiting cell cycle arrest and apoptosis in cervical cancer [7]. UHRF1 facilitates gastric cancer proliferation and growth in vitro and in vivo by mediating hypermethylation of several tumor suppressor genes [8]. Notably, it was previously reported that UHRF1 promotes the proliferation, invasion and migration of breast cancer cells [9]. Moreover, short hairpin RNA (shRNA) lentiviral system-mediated UHRF1 knockdown inhibits breast cancer cell proliferation [10], indicating that UHRF1 may serve as a promising therapeutic target for breast cancer. However, the regulatory mechanisms of UHRF1 in breast cancer progression have not been fully characterized.

Zinc finger and BTB domain containing 16 (ZBTB16), also known as promyelocytic leukemia zinc finger (PLZF) or zinc finger protein 145 (ZFP145), is a member of the zinc finger and BTB/POZ domain-containing family of proteins (ZBTBs). Recent studies have shown that ZBTB16 participates in various major biological processes, such as hematopoiesis, spermatogenesis, stem cell maintenance, tumor suppression and immune regulation [11,12]. Downregulation of ZBTB16 has been detected in various cancers and malignant cell lines. ZBTB16 is significantly downregulated in HCC [13].