Supplementary Table from Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers

Supplementary Data from Oncohistone Mutations Occur at Functional Sites of Regulatory ADP-Ribosylation

Abstract Long noncoding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression of lncRNA152 (lnc152; a.k.a. DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown of lnc152 promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression of lnc152 inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice, lnc152 inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated that lnc152 downregulates genes controlling angiogenesis. Using pull down assays followed by LC/MS-MS, we identified RBM47, a known tumor suppressor in breast cancer, as a lnc152-interacting protein. The effects of lnc152 in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate that lnc152 is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC. Implications: This study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC.

Miscarriage is a common complication of pregnancy for which there are few clinical interventions. Deficiency in endometrial stromal cell decidualization is considered a major contributing factor to pregnancy loss; however, our understanding of the underlying mechanisms of decidual deficiency are incomplete. ADP ribosylation by PARP-1 and PARP-2 has been linked to physiological processes essential to successful pregnancy outcomes. Here, we report that the catalytic inhibition or genetic ablation of PARP-1 and PARP-2 in the uterus lead to pregnancy loss in mice. Notably, the absence of PARP1 and PARP-2 resulted in increased p53 signaling and an increased population of senescent decidual cells. Molecular and histological analysis revealed that embryo attachment and the removal of the luminal epithelium are not altered in uterine Parp1, Parp2 knockout mice, but subsequent decidualization failure results in pregnancy loss. These findings provide evidence for a previously unknown function of PARP-1 and PARP-2 in mediating decidualization for successful pregnancy establishment.

During gestation, the female reproductive tract must maintain pregnancy while concurrently preparing for parturition. Here, we explore the transitions in gene expression and protein turnover (fractional synthesis rates [FSR]) by which the cervix implements a transition from rigid to compliant. Shifts in gene transcription to achieve immune tolerance and alter epithelial cell programs begin in early pregnancy. Subsequently, in mid-to-late pregnancy transcriptional programs emerge that promote structural reorganization of the extracellular matrix (ECM). Stable isotope labeling revealed a striking slowdown of overall FSRs across the proteome on gestation day 6 that reverses in mid-to-late pregnancy. An exception was soluble fibrillar collagens and proteins of collagen assembly, which exhibit high turnover in nonpregnant cervix compared with other tissues and FSRs that continue throughout pregnancy. This finding provides a mechanism to explain how cross-linked collagen is replaced by newly synthesized, less cross-linked collagens, which allows increased tissue compliance during parturition. The rapid transition requires a reservoir of newly synthesized, less cross-linked collagens, which is assured by the high FSR of soluble collagens in the cervix. These findings suggest a previously unrecognized form of "metabolic flexibility" for ECM in the cervix that underlies rapid transformation in compliance to allow parturition.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.

Abstract Estrogens, such as 17β- estradiol (E2), act through estrogen receptor alpha (ERα), a ligand-regulated transcription factor that binds across the genome to promote enhancer formation and regulate gene expression. ERα is expressed in approximately 70% of breast cancers, where it regulates the transcription of genes involved in mitogenic and inflammatory pathways. We are exploring the acetylation of ERα on lysines (K) 266 and 268, modifications that we previously showed enhance the DNA binding and transcriptional activities of ERα in biochemical and cell-based assays. ERα acetylation is catalyzed by the lysine acetyltransferases p300 and CBP in an E2- and steroid receptor coregulator (SRC)-dependent manner. Acetylation-dependent activation of ERα has potential implications in breast cancers associated with enhanced coregulator interactions, such as SRC-3/Amplified in Breast (AIB1) gene amplifications and gain-of-function ERα mutations in endocrine resistant metastatic tumors, such as Y537S and D538G. Notably, the constitutively-activated Y537S mutant exhibits E2-independent K266/268 acetylation and further enhanced acetylation with E2 stimulation when expressed in breast cancer cells. Enhanced ERα acetylation may promote the gain-of-function ERα activity in this context. Our hypothesis is that acetylation of ERα alters its function by increasing E2-responsive gene transcription and signaling in breast cancers. We are using the ER-positive MCF-7 breast cancer cell line with a knockdown/re-expression strategy with biochemical mimics of acetylated (K266/268Q) or unacetylated (K266/268R) ERα. Genomic ERα binding profiles using chromatin immunoprecipitation-sequencing (ChIP-seq) has defined both overlapping and unique sets of transcriptional targets and recruitment kinetics for the ERα mutants. Collectively, our results suggest that the transcriptional activities of the ERα acetylation mutants share a common altered mechanism of recruitment to chromatin with potential implications on enhancer activation. Our current efforts are focused on investigating the role of ERα acetylation on chromatin accessibility, enhancer activity, and target gene transcription.

Abstract Estrogens, such as 17β-estradiol (E2), act through estrogen receptor alpha (ERα), a ligand-regulated transcription factor that binds across the genome to promote enhancer formation and regulate gene expression. ERα is expressed in approximately 70% of breast cancers, where it regulates the transcription of genes involved in mitogenic and inflammatory pathways. We are exploring the the acetylation of ERα on lysines 266 and 268, a modification that enhances the DNA binding and transcriptional activities of ERα. ERα acetylation is catalyzed by the lysine acetyltransferases p300 and CBP in an E2- and steroid receptor coregulator (SRC)-dependent manner. Acetylation-dependent activation of ERα has potential implications in breast cancers associated with enhanced coregulator interactions, such as SRC-3/Amplified in Breast (AIB1) gene amplifications and gain-of-function ERα mutations in endocrine resistant metastatic tumors, such as Y537S and D538G. Increased association of ERα with SRCs and p300/CBP leading to enhanced ERα acetylation may promote gain-of-function ERα activity. Our hypothesis is that acetylation of ERα alters its function by increasing E2-responsive gene transcription and signaling in breast cancers. Our current efforts are focused on investigating the role of ERα acetylation on chromatin binding and accessibility, enhancer activity, and target gene transcription. We are using the ER-positive MCF-7 breast cancer cell line with a knockdown/re-expression strategy with biochemical mimics of acetylated (K266/268Q) or unacetylated (K266/268R) ERα. Genomic ERα binding profiles using chromatin immunoprecipitation-sequencing (ChIP-seq) has defined overlapping, but unique, sets of transcriptional targets and recruitment kinetics for the ERα mutants. Current efforts are focused on defining global transcriptional responses associated with the unique binding activities using precision run-on sequencing (PRO-seq). Ultimately, our goal is to define how acetylation affects the binding of ERα to chromatin and its effect on the transcriptional activity of E2-target genes in breast cancers in the context of gain-of-function ERα mutations and coregulator amplifications. Supported in part by a postdoctoral fellowship from the Lalor Foundation to YMV, and grants from the NIDDK, NICHD, and CPRIT to WLK, as well as support from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment. Citation Format: Vasquez YM, Setlem R, Murakami S, Kraus WL. Role of estrogen receptor alpha acetylation in estrogen-dependent gene regulation in breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-16.