PAPAS promotes differentiation of mammary epithelial cells and suppresses breast carcinogenesis

Highlights

• •The lncRNA PAPAS represses rRNA gene activity in mammary epithelial cells (MECs)
• •Upregulation of PAPAS is required for lactogenic differentiation of MECs
• •Breast cancer cells curb R-loop-dependent PAPAS synthesis
• •Reconstitution of PAPAS in breast cancer suppresses tumor growth and metastasis

Summary

Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoiding breast cancer development. Here, we show that lactogenic differentiation of murine MECs requires silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and lung invasion and activates many interferon-regulated genes previously linked to metastasis suppression. Mechanistically, PAPAS transcription depends on R-loop formation at the 3′ end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.

Introduction

Cell proliferation depends on ribosome biogenesis, which is initiated and driven by synthesis of ribosomal RNA (rRNA) in the nucleolus. Given the high energy demand of ribosome production,¹ transcription of rRNA genes (rDNA) into a precursor transcript (pre-rRNA) is tightly regulated in response to developmental and environmental cues.^2,3 Epigenetic silencing of rDNA upon cellular stress and differentiation is mediated by the long non-coding RNA (lncRNA) PAPAS (promoter and pre-rRNA antisense), an rDNA antisense transcript generated by RNA polymerase II (RNA Pol II).^4,5,6,7 In quiescent fibroblasts and terminally differentiated cells, PAPAS guides the histone methyltransferase Suv4-20h2 (also known as KMT5c) to rDNA, thereby triggering trimethylation of histone H4 at lysine 20 (H4K20me3) and chromatin compaction.^4,5 This PAPAS-mediated epigenetic silencing differs from H4K20me3 deposition at constitutive heterochromatin, where Suv4-20h2 recruitment depends on H3K9me3 and heterochromatin protein 1 (HP1).⁸ In accord with terminal differentiation being disrupted during cancer pathogenesis,⁹ loss of H4K20me3 is common in human cancers, and PAPAS is progressively downregulated in a model of skin carcinogenesis.^5,10

Female breast cancer is the fifth leading cause of cancer mortality worldwide and was the most frequently diagnosed cancer in 2020.¹¹ Most breast cancers are adenocarcinomas originating from the mammary epithelium, which undergoes extensive remodeling throughout puberty, pregnancy, lactation, and weaning.¹² The plasticity and hormone responsiveness of mammary epithelial cells (MECs) have been linked to cancer susceptibility.¹³ Despite the molecular heterogeneity of breast cancers, loss of H4K20me3 is a common epigenetic aberration, while H3K9me3 is not affected, and Suv4-20h2 levels are even higher than in healthy breast tissues.^14,15,16,17 Thus, we hypothesize that diminished H4K20me3 levels in mammary tumors result, at least in part, from perturbation of PAPAS-mediated Suv4-20h2 recruitment to rDNA. Given that PAPAS silences rDNA in myogenic, adipogenic, and enterocytic differentiation,⁴ we posit that the lncRNA exerts this function also upon lactogenic differentiation of MECs, thereby restricting neoplastic growth.

In this study, we investigate the role of PAPAS in normal development and carcinogenesis of the mammary epithelium and provide mechanistic insights into the downregulation of PAPAS in breast cancer cells. Our findings establish that PAPAS exerts a dual function as a driver of cellular differentiation and as a tumor suppressor. Importantly, reconstitution of PAPAS expression in breast cancer cells restricts neoplastic growth in immunocompromised as well as immunocompetent hosts and diminishes metastasis to the lungs. PAPAS downregulation in human breast cancer samples and cell lines correlates with tumor aggressiveness, emphasizing the therapeutic relevance of our findings.

Results

PAPAS represses rDNA transcription upon lactogenic differentiation

The murine MEC line HC11 can be differentiated by exposure to a mixture of lactogenic hormones comprising dexamethasone, insulin, and prolactin (DIP).^18,19 In the presence of DIP, post-confluent HC11 cells adopt a polarized phenotype and secrete a milk-like fluid toward the bottom of the monolayer, thereby forming dome-like structures^20,21 (Figure 1A). Accordingly, elevated expression of the Csn2 and Wap genes, encoding the milk proteins β-casein and whey acidic protein, respectively, serves as a marker for differentiation efficiency^18,19,20,21 (Figures 1B, 1C, and S1A). In line with lactogenic differentiation of HC11 cells causing cell cycle arrest at the G0/G1 stage, DIP-treated cells exhibited a strong reduction in the messenger RNA (mRNA) coding for the mitotic kinase Nek2²¹ (Figure S1B). To test if rDNA activity was affected in the differentiated state, we monitored pre-rRNA levels as well as RNA Pol I occupancy at the promoter and gene body and found a clear decrease by DIP treatment (Figures 1D, 1E, S1C, and S1D). In contrast, PAPAS was upregulated about 10-fold and coincided with enhanced H4K20me3 abundance at rDNA (Figures 1F, 1G, and S1E). Of note, PAPAS-mediated rDNA silencing did not reduce the levels of mature rRNA (Figure S1F), which is highly abundant and long lived, and thus facilitates robust RNA concentration homeostasis in cells.²²