Research of The Molecular Mechanism of NARF in Promoting Breast Cancer Metastasis

Introduction

Breast cancer is the leading cause of cancer death for women globally, and the vast majority of breast cancer–related deaths are due to metastasis. Advanced breast cancers are characterized by intratumoral hypoxia, which is associated with metastasis, treatment failure, and patient mortality.

Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1). HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis.

Increased expression of nuclear prelamin A recognition factor (NARF), a relatively uncharacterized protein previously found to be associated with the nuclear lamina, has also been identified as a mitochondrial protein associated with abnormal iron deposition in multiple sclerosis brains.

However, no studies have reported a role for NARF in cancer or transcriptional regulation. The study of the molecular mechanism of hypoxia-induced BCSC specification and promotion of breast cancer metastasis is important for the treatment and drug development of breast cancer.

Methods

MDA-MB-231 and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium. To investigate whether NARF expression correlated with a HIF signature in breast cancers and whether the NARF gene HIF-1 binding site is present within a functional HRE, ChIP-qPCR assays, RT-qPCR and immunoblotting assays were performed, respectively. To further investigate in depth the correlation between NARF and BCSC phenotype and the role of NARF in cancer or transcriptional regulation, lentiviral transduction, luciferase reporter gene assay, mammosphere assay, aldehyde dehydrogenase assay, animal studies, immunofluorescence assay, immunohistochemistry and bioinformatics and statistics were performed.

Results

ChIPsequencing and related test data suggest that NARF is a hypoxia-inducible gene. Hypoxia induces NARF expression in a HIF-1–dependent manner, demonstrating that NARF is a direct HIF-1 target gene. By comparing NARF gene expression with 20 genetic BCSC markers in 1218 breast cancer patient samples from the TCGA database, it was determined that NARF expression significantly correlated with the expression of BCSC phenotype-related genes and that NARF contributed to hypoxia-induced BCSC enrichment.

To further investigate the role of NARF in BCSC enrichment, we generated MDA-MB-231 and MCF-7 subclones by stable transduction with a lentiviral vector encoding one of five different shRNAs targeting NARF or NTC vectors, and performed immunoblotting assays as well as animal experiments. The results suggest that exposure of breast cancer cells to hypoxia enriches the BCSC population and that knockdown (KD) of NARF eliminates this effect. Furthermore, NARF KD impairs tumor initiation and lung metastasis.

In order to investigate the molecular mechanism of this effect in depth, a detailed analysis of the regulation of NARF gene expression in a variety of breast cancer cell lines was performed. The results show that NARF protein is recruited to OCT4 binding sites at the NANOG, SOX2, and KLF4 genes in hypoxic breast cancer cells. NARF, in turn, recruits KDM6A, which erases repressive H3K27me3 marks, enabling OCT4-mediated transcription of the NANOG, SOX2, and KLF4 genes, which is required for specification of BCSCs that are essential for tumor initiation and metastasis.

Fig. 1 HIF-1–dependent NARF expression promotes OCT4-mediated breast cancer stem cell specification.

Summary

This study identifies NARF as a HIF-1 target gene and demonstrates a critical role for NARF in BCSC specification. This study shows that exposure of breast cancer cells to hypoxia induces NARF mRNA and protein expression as a result of direct binding of HIF-1 to the NARF gene promoter. Exposure of breast cancer cells to hypoxia enriches the BCSC population, and knockdown (KD) of NARF abrogates this effect. Consistent with these findings, NARF KD inhibited tumor initiation and the metastatic potential of breast cancer cells in an orthotopic mouse model. Investigation of the molecular mechanisms underlying these effects revealed that NARF recruits histone lysine demethylase 6A (KDM6A) to OCT4 binding sites, thereby licensing OCT4-mediated transcriptional activation of the NANOG, SOX2, and KLF4 genes.

Reference:

Yang, Y.; et al. NARF is a hypoxia-induced coactivator for OCT4-mediated breast cancer stem cell specification. Science Advances, 2022, 8(49): eabo5000.

* For research use only. Not intended for any clinical use.

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Research of The Molecular Mechanism of NARF in Promoting Breast Cancer Metastasis

Introduction

Methods

Results

Summary

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