Role of RNA binding proteins of the Drosophila behavior and human splicing (DBHS) family in health and cancer.

RNA-binding proteins (RBPs) play crucial roles in the functions and homoeostasis of various tissues by regulating multiple events of RNA processing including RNA splicing, intracellular RNA transport, and mRNA translation. The Drosophila behavior and human splicing (DBHS) family proteins including PSF/SFPQ, NONO, and PSPC1 are ubiquitously expressed RBPs that contribute to the physiology of several tissues. In mammals, DBHS proteins have been reported to contribute to neurological diseases and play crucial roles in cancers, such as prostate, breast, and liver cancers, by regulating cancer-specific gene expression. Notably, in recent years, multiple small molecules targeting DBHS family proteins have been developed for application as cancer therapeutics. This review provides a recent overview of the functions of DBHS family in physiology and pathophysiology, and discusses the application of DBHS family proteins as promising diagnostic and therapeutic targets for cancers.

Roles of Noncoding RNAs in Regulation of Mitochondrial Electron Transport Chain and Oxidative Phosphorylation.

The mitochondrial electron transport chain (ETC) plays an essential role in energy production by inducing oxidative phosphorylation (OXPHOS) to drive numerous biochemical processes in eukaryotic cells. Disorders of ETC and OXPHOS systems are associated with mitochondria- and metabolism-related diseases, including cancers; thus, a comprehensive understanding of the regulatory mechanisms of ETC and OXPHOS systems is required. Recent studies have indicated that noncoding RNAs (ncRNAs) play key roles in mitochondrial functions; in particular, some ncRNAs have been shown to modulate ETC and OXPHOS systems. In this review, we introduce the emerging roles of ncRNAs, including microRNAs (miRNAs), transfer-RNA-derived fragments (tRFs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs), in the mitochondrial ETC and OXPHOS regulation.

A FRET-based respirasome assembly screen identifies spleen tyrosine kinase as a target to improve muscle mitochondrial respiration and exercise performance in mice.

Aerobic muscle activities predominantly depend on fuel energy supply by mitochondrial respiration, thus, mitochondrial activity enhancement may become a therapeutic intervention for muscle disturbances. The assembly of mitochondrial respiratory complexes into higher-order "supercomplex" structures has been proposed to be an efficient biological process for energy synthesis, although there is controversy in its physiological relevance. We here established Förster resonance energy transfer (FRET) phenomenon-based live imaging of mitochondrial respiratory complexes I and IV interactions using murine myoblastic cells, whose signals represent in vivo supercomplex assembly of complexes I, III, and IV, or respirasomes. The live FRET signals were well correlated with supercomplex assembly observed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and oxygen consumption rates. FRET-based live cell screen defined that the inhibition of spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase that belongs to the SYK/ zeta-chain-associated protein kinase 70 (ZAP-70) family, leads to an increase in supercomplex assembly in murine myoblastic cells. In parallel, SYK inhibition enhanced mitochondrial respiration in the cells. Notably, SYK inhibitor administration enhances exercise performance in mice. Overall, this study proves the feasibility of FRET-based respirasome assembly assay, which recapitulates in vivo mitochondrial respiration activities.

Estrogen-Inducible LncRNA BNAT1 Functions as a Modulator for Estrogen Receptor Signaling in Endocrine-Resistant Breast Cancer Cells.

Recent advances in RNA studies have revealed that functional long noncoding RNAs (lncRNAs) contribute to the biology of cancers. In breast cancer, estrogen receptor α (ERα) is an essential transcription factor that primarily promotes the growth of luminal-type cancer, although only a small number of lncRNAs are identified as direct ERα targets and modulators for ERα signaling. In this study, we performed RNA-sequencing for ER-positive breast cancer cells and identified a novel estrogen-inducible antisense RNA in the COL18A1 promoter region, named breast cancer natural antisense transcript 1 (BNAT1). In clinicopathological study, BNAT1 may have clinical relevance as a potential diagnostic factor for prognoses of ER-positive breast cancer patients based on an in situ hybridization study for breast cancer specimens. siRNA-mediated BNAT1 silencing significantly inhibited the in vitro and in vivo growth of tamoxifen-resistant ER-positive breast cancer cells. Notably, BNAT1 silencing repressed cell cycle progression whereas it promoted apoptosis. Microarray analysis revealed that BNAT1 silencing in estrogen-sensitive breast cancer cells repressed estrogen signaling. We showed that BNAT1 knockdown decreased ERα expression and repressed ERα transactivation. RNA immunoprecipitation showed that BNAT1 physically binds to ERα protein. In summary, BNAT1 would play a critical role in the biology of ER-positive breast cancer by modulating ERα-dependent transcription regulation. We consider that BNAT1 could be a potential molecular target for diagnostic and therapeutic options targeting luminal-type and endocrine-resistant breast cancer.

PSPC1 is a potential prognostic marker for hormone-dependent breast cancer patients and modulates RNA processing of ESR1 and SCFD2.

Breast cancer is the most common cancer type among women worldwide. The majority of breast cancer expresses estrogen receptor (ER) and endocrine therapy is a standard treatment of ER-positive breast cancer. However, development of the therapy resistance is still a major challenge and thus new therapeutic approaches are needed. Here we show that an RNA-binding protein, PSPC1, play a crucial role in ER-positive breast cancer growth through post-transcriptional gene regulation. We showed that siRNA-mediated PSPC1 silencing suppressed the proliferation of ER-positive breast cancer cells. Strong immunoreactivity (IR) of PSPC1 was correlated with poor prognosis for ER-positive breast cancer patients. Using immunoprecipitation, RNA-immunoprecipitation (RIP) and quantitative PCR (qPCR) experiments, we showed that PSPC1 interacted with PSF and was involved in post-transcriptional regulation of PSF target genes, ESR1 and SCFD2. Strong SCFD2 IR was correlated with poor prognosis for ER-positive breast cancer patients and combinations of PSPC1, PSF, and SCFD2 IRs were potent prognostic factors. Moreover, we identified DDIAS and MYBL1 as SCFD2 downstream target genes using microarray analysis, and finally showed that SCFD2 silencing suppressed tamoxifen-resistant breast tumor growth in vivo. These results indicated that PSPC1 and SCFD2 axis could be a promising target in the clinical management of the disease.

Emerging Roles of COX7RP and Mitochondrial Oxidative Phosphorylation in Breast Cancer.

Metabolic alterations are critical events in cancers, which often contribute to tumor pathophysiology. While aerobic glycolysis is a known characteristic of cancer-related metabolism, recent studies have shed light on mitochondria-related metabolic pathways in cancer biology, including oxidative phosphorylation (OXPHOS), amino acid and lipid metabolism, nucleic acid metabolism, and redox regulation. Breast cancer is the most common cancer in women; thus, elucidation of breast cancer-related metabolic alteration will help to develop cancer drugs for many patients. We here aim to define the contribution of mitochondrial metabolism to breast cancer biology. The relevance of OXPHOS in breast cancer has been recently defined by the discovery of COX7RP, which promotes mitochondrial respiratory supercomplex assembly and glutamine metabolism: the latter is also shown to promote nucleic acid and fatty acid biosynthesis as well as ROS defense regulation. In this context, the estrogen-related receptor (ERR) family nuclear receptors and collaborating coactivators peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) are essential transcriptional regulators for both energy production and cancer-related metabolism. Summarizing recent findings of mitochondrial metabolism in breast cancer, this review will aim to provide a clue for the development of alternative clinical management by modulating the activities of responsible molecules involved in disease-specific metabolic alterations.

TRIM39 is a poor prognostic factor for patients with estrogen receptor-positive breast cancer and promotes cell cycle progression.

Tripartite motif (TRIM) family proteins are involved in various biological processes and the pathophysiology of cancers. However, the roles of TRIM39, a TRIM family member, in breast cancer is not well-understood. Here, we performed immunohistochemical study of TRIM39 protein in clinical estrogen receptor-positive (ER+ ) breast cancer tissues from 108 patients. TRIM39 immunoreactivity (IR) was positively correlated with advanced stage (p < 0.001), large invasive tumor size (p = 0.012), and positive lymph node status (p = 0.002). Positive TRIM39 IR was significantly correlated with short disease-free survival (DFS) (p = 0.001). Multivariate analysis revealed that the TRIM39 status is an independent prognostic factor in DFS (p = 0.049). Microarray analysis of MCF-7 breast cancer cells treated with siRNA revealed that TRIM39 knockdown downregulated the cell cycle- and cell division-related genes, including MLLT11, CDCA3, CDC25C, BIRC5, and ANP32E. Consistently, TRIM39 knockdown significantly suppressed proliferation and cell cycle transition to S phase in MCF-7 and 4-hydroxytamoxifen-resistant (OHTR) breast cancer cells. These results suggest that TRIM39 promotes ER+ breast cancer growth by promoting cell cycle progression.

OCT1 Is a Poor Prognostic Factor for Breast Cancer Patients and Promotes Cell Proliferation via Inducing NCAPH.

Octamer transcription factor 1 (OCT1) is a transcriptional factor reported to be a poor prognostic factor in various cancers. However, the clinical value of OCT1 in breast cancer is not fully understood. In the present study, an immunohistochemical study of OCT1 protein was performed using estrogen receptor (ER)-positive breast cancer tissues from 108 patients. Positive OCT1 immunoreactivity (IR) was associated with the shorter disease-free survival (DFS) of patients (p = 0.019). Knockdown of OCT1 inhibited cell proliferation in MCF-7 breast cancer cells as well as its derivative long-term estrogen-deprived (LTED) cells. On the other hand, the overexpression of OCT1 promoted cell proliferation in MCF-7 cells. Using microarray analysis, we identified the non-structural maintenance of chromosomes condensin I complex subunit H (NCAPH) as a novel OCT1-taget gene in MCF-7 cells. Immunohistochemical analysis showed that NCAPH IR was significantly positively associated with OCT1 IR (p < 0.001) and that positive NCAPH IR was significantly related to the poor DFS rate of patients (p = 0.041). The knockdown of NCAPH inhibited cell proliferation in MCF-7 and LTED cells. These results demonstrate that OCT1 and its target gene NCAPH are poor prognostic factors and potential therapeutic targets for patients with ER-positive breast cancer.

Long Intergenic Noncoding RNA OIN1 Promotes Ovarian Cancer Growth by Modulating Apoptosis-Related Gene Expression.

Patients with advanced ovarian cancer usually exhibit high mortality rates, thus more efficient therapeutic strategies are expected to be developed. Recent transcriptomic studies revealed that long intergenic noncoding RNAs (lincRNAs) can be a new class of molecular targets for cancer management, because lincRNAs likely exert tissue-specific activities compared with protein-coding genes or other noncoding RNAs. We here show that an unannotated lincRNA originated from chromosome 10q21 and designated as ovarian cancer long intergenic noncoding RNA 1 (OIN1), is often overexpressed in ovarian cancer tissues compared with normal ovaries as analyzed by RNA sequencing. OIN1 silencing by specific siRNAs significantly exerted proliferation inhibition and enhanced apoptosis in ovarian cancer cells. Notably, RNA sequencing showed that OIN1 expression was negatively correlated with the expression of apoptosis-related genes ras association domain family member 5 (RASSF5) and adenosine A1 receptor (ADORA1), which were upregulated by OIN1 knockdown in ovarian cancer cells. OIN1-specifc siRNA injection was effective to suppress in vivo tumor growth of ovarian cancer cells inoculated in immunodeficient mice. Taken together, OIN1 could function as a tumor-promoting lincRNA in ovarian cancer through modulating apoptosis and will be a potential molecular target for ovarian cancer management.

Mechanisms of Apoptosis-Related Long Non-coding RNAs in Ovarian Cancer.

Ovarian cancer is a health-threatening malignancy of ovary in female reproductive systems and one of the most common gynecological malignancies worldwide. Due to rare early symptoms, ovarian cancers are often diagnosed at advanced stages and exhibit poor prognosis. Thus, efforts have been paid to develop alternative diagnostic and therapeutic strategies for the disease. Recent studies have presented that some long non-coding RNAs (lncRNAs) play roles in apoptosis of ovarian cancer cells through various mechanisms involved in the regulation of transcription factors, histone modification complexes, miRNAs, and protein stability. Because evasion of apoptosis in cancer cells facilitates to promote tumor progression and therapy resistance, apoptosis regulatory mechanisms of lncRNAs may be promising new targets in ovarian cancer. In this review, we introduce the recent findings in regard to the molecular mechanisms of apoptosis-related lncRNAs in ovarian cancer cells.

Vitamin K-Dependent γ-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

Long Non-coding RNAs Involved in Metabolic Alterations in Breast and Prostate Cancers.

Breast and prostate cancers are the most prevalent cancers in females and males, respectively. These cancers exhibit sex hormone dependence and thus, hormonal therapies are used to treat these cancers. However, acquired resistance to hormone therapies is a major clinical problem. In addition, certain portions of these cancers initially exhibit hormone-independence due to the absence of sex hormone receptors. Therefore, precise and profound understanding of the cancer pathophysiology is required to develop novel clinical strategies against breast and prostate cancers. Metabolic reprogramming is currently recognized as one of the hallmarks of cancer, as exemplified by the alteration of glucose metabolism, oxidative phosphorylation, and lipid metabolism. Dysregulation of metabolic enzymes and their regulators such as kinases, transcription factors, and other signaling molecules contributes to metabolic alteration in cancer. Moreover, accumulating lines of evidence reveal that long non-coding RNAs (lncRNAs) regulate cancer development and progression by modulating metabolism. Understanding the mechanism and function of lncRNAs associated with cancer-specific metabolic alteration will therefore provide new knowledge for cancer diagnosis and treatment. This review provides an overview of recent studies regarding the role of lncRNAs in metabolism in breast and prostate cancers, with a focus on both sex hormone-dependent and -independent pathways.

Long Noncoding RNAs Involved in the Endocrine Therapy Resistance of Breast Cancer.

Long noncoding RNAs (lncRNAs) are defined as RNAs longer than 200 nucleotides that do not encode proteins. Recent studies have demonstrated that numerous lncRNAs are expressed in humans and play key roles in the development of various types of cancers. Intriguingly, some lncRNAs have been demonstrated to be involved in endocrine therapy resistance for breast cancer through their own mechanisms, suggesting that lncRNAs could be promising new biomarkers and therapeutic targets of breast cancer. Here, we summarize the functions and mechanisms of lncRNAs related to the endocrine therapy resistance of breast cancer.

Roles of Splicing Factors in Hormone-Related Cancer Progression.

Splicing of mRNA precursor (pre-mRNA) is a mechanism to generate multiple mRNA isoforms from a single pre-mRNA, and it plays an essential role in a variety of biological phenomena and diseases such as cancers. Previous studies have demonstrated that cancer-specific splicing events are involved in various aspects of cancers such as proliferation, migration and response to hormones, suggesting that splicing-targeting therapy can be promising as a new strategy for cancer treatment. In this review, we focus on the splicing regulation by RNA-binding proteins including Drosophila behavior/human splicing (DBHS) family proteins, serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in hormone-related cancers, such as breast and prostate cancers.

Exportin-5 mediates nuclear export of SRP RNA in vertebrates.

The signal recognition particle is a ribonucleoprotein complex that is essential for the translocation of nascent proteins into the endoplasmic reticulum. It has been shown that the RNA component (SRP RNA) is exported from the nucleus by CRM1 in the budding yeast. However, how SRP RNA is exported in higher species has been elusive. Here, we show that SRP RNA does not use the CRM1 pathway in Xenopus oocytes. Instead, SRP RNA uses the same export pathway as pre-miRNA and tRNA as showed by cross-competition experiments. Consistently, the recombinant Exportin-5 protein specifically stimulated export of SRP RNA as well as of pre-miRNA and tRNA, whereas an antibody raised against Exportin-5 specifically inhibited export of the same RNA species. Moreover, biotinylated SRP RNA can pull down Exportin-5 but not CRM1 from HeLa cell nuclear extracts in a RanGTP-dependent manner. These results, taken together, strongly suggest that the principal export receptor for SRP RNA in vertebrates is Exportin-5 unlike in the budding yeast.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells.

Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.