Charlton Cooper

Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Students t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression.

Intermittent Hypoxia Induces Proteasome-Dependent Down-Regulation of Estrogen Receptor α in Human Breast Carcinoma

Purpose: Hypoxia may influence gene expression to promote malignancy, and acute hypoxia has been shown to transiently repress estrogen receptor (ER)-α expression in breast cell lines. However, the effect of intermittent hypoxia, which is likely more prevalent in breast cancers, remains to be determined. Experimental Design: ER-α expression was assessed by Western blot and immunohistochemistry in a selected cohort of 51 ER-α–positive breast carcinomas, in relation to markers of hypoxia. The effect of acute and intermittent hypoxia on ER-α expression was also determined in MCF7 and ZR-75 breast cell lines, together with the role of proteasome function with the proteasome inhibitor bortezomib. Results: Regional loss of ER-α expression occurs in breast tumors and is consistently present in hypoxic regions defined by the proximity of necrosis and induction of hypoxia-induced genes carbonic anhydrase IX (CA-IX) and glucose transporter 1 (Glut-1), in both in situ (n = 29; P < 0.0001) and invasive (n = 20; P = 0.0001) carcinomas. In MCF7 and ZR-75 cells, ER-α is transiently down-regulated by acute hypoxia and rapidly restored by reoxygenation. However, intermittent, acute hypoxia can cause a similar down-regulation of ER-α that is not attributable to decreased mRNA and persists in MCF7 cells despite reoxygenation for up to 14 days. This effect occurs with no change in cell viability but a corresponding reduction in growth response to estradiol. However, ER-α expression can be restored by bortezomib. Conclusions: Intermittent hypoxia can cause persistent changes in proteasome function that may contribute to reduced ER-α expression in breast tumors and consequently to diminished response and development of resistance to endocrine therapy.

The protein encoded by the functional steroid receptor RNA activator is a new modulator of ER alpha transcriptional activity.

The steroid receptor RNA activator gene (SRA1) encodes for a functional RNA (SRA) as well as a protein (SRAP). While several groups reported on SRA‐RNA mechanism of action, SRAP exact function remains to be elucidated, mainly due to a lack of studies investigating the function of the protein independently of its RNA counterpart. Using two independent models to examine its specific functions, SRAP was found to enhance estrogen receptor alpha activity in a ligand and response‐element dependent manner. Our data therefore suggest that both transcript and protein products of the SRA1 gene co‐modulate the transcriptional activity of steroid receptors.

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.

Steroid Receptor RNA Activator Protein (SRAP): a potential new prognostic marker for estrogen receptor-positive/node-negative/younger breast cancer patients

IntroductionThe steroid receptor RNA activator is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, Steroid Receptor RNA Activator Protein (SRAP), whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters.MethodsFollowing antibody validation, SRAP expression was assessed by tissue-microarray (TMA) analysis of 372 breast tumors. Clinical follow-up and parameters such as steroid receptor and node status were available for all the corresponding cases. Immunohistochemical scores were independently determined by three investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests.ResultsSRAP levels were significantly (Mann-Whitney rank sum test, P < 0.05) higher in estrogen receptor-alpha positive (ER+, n = 271), in progesterone receptor positive (PR+, n = 257) and in older patients (age > 64 years, n = 182). When considering ER+ tumors, PR+ tumors, or younger patients (≤ 64 years), cases with high SRAP expression had a significantly (Mantel-Cox test, P < 0.05) worse breast cancer specific survival (BCSS) than those with low SRAP levels. SRAP also appeared as a very powerful indicator of poor prognostic for BCSS in the subset of ER+, node negative and young breast cancer patients (Cox regression analysis, n = 60, BCSS Hazard Ratio = 8.61, P < 0.006).ConclusionsOur data suggest that SRAP levels might provide additional information on potential risk of recurrence and negative outcome in a specific set of patients with otherwise good prognosis when considering only estrogen receptor and nodal status.

The steroid receptor RNA activator protein is recruited to promoter regions and acts as a transcriptional repressor

MINT‐7761068: SRAP (uniprotkb:Q9HD15) physically interacts (MI:0915) with HDAC2 (uniprotkb:Q92769) by anti bait coimmunoprecipitation (MI:0006)

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1.

Objective: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). Methods: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. Results: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. Conclusion: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process.

The steroid receptor RNA activator protein (SRAP) controls cancer cell migration/motility

Our results highlight for the first time a link existing between SRA1 gene expression and cell motility.

Steroid Receptor RNA Activator Protein (SRAP): A Potential New Prognostic Marker for Estrogen Receptor-Positive/Node-Negative/Younger Breast Cancer Patients.

Purpose: The steroid receptor RNA activator (SRA) is a functional RNA suspected to participate in the mechanisms underlying breast tumor progression. This RNA is also able to encode for a protein, SRAP, whose exact function remains to be determined. Our aim was to assess, in a large breast cancer cohort, whether levels of this protein could be associated with outcome or established clinical parameters. Experimental Design: Following antibody validation, we have assessed SRAP expression by tissue-microarray (TMA) analysis of 372 tumors with known steroid receptor and node status. Clinical follow-up was available for all the corresponding patients. Immunohistochemical scores were independently determined by two investigators and averaged. Statistical analyses were performed using standard univariate and multivariate tests. Results: SRAP levels were significantly (Mann-Whitney rank sum test, P Conclusion: Our data suggest that SRAP might be a new predictor of breast cancer specific survival in younger breast cancer patients with ER+/node negative tumors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2017.

The roles of SRA1 gene in breast cancer

The Steroid receptor RNA activator (SRA) gene has been implicated in estrogen receptor signaling pathw ay. First identified as a RNA coregulator, SRA had been shown to increase steroid receptor activity. SRA RNA exp ression is altered during breast tumorigenesis and its molecul ar role in underscoring these events has been sugge sted. The subsequent identification of molecules capable of b inding SRA, including RNA helicase p68, SRA stem-loop interacting RNA binding protein (SLIRP), and steroi dogenic factor 1 (SF1) indicates SRA function is no t exclusively limited to modulate steroid receptor activity. A re cent genome-wide expression analysis by depleting SRA in cancer cells has further expanded our understanding of a b roader biological role played by SRA. In addition, several RNA isoforms have been found to encode an endogenous protein (SRAP), which is well conserved among Chordata. Interestingly, SRAP also modulates steroid receptor activity and functions as a co-regulator in estrog en receptor signaling. The recent observation that a higher exp ression of SRAP protein is associated with poorer s urvival in breast cancer patients treated with tamoxifen, highlights the potential relevance of this protein in cancer. Together, the SRA1 gene encodes both functional RNA and protein (SRAP) products, making it a unique member amongst the growing population of steroid receptor co-regulators.