Regulation of Protein Arginine Methyl Transferase 5 by Novel Serine 15 Phosphorylation in Colorectal Cancer

Indiana University-Purdue University Indianapolis (IUPUI)The overexpression of protein arginine methyltransferase 5 (PRMT5) is strongly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Previously, we demonstrated that PRMT5 overexpression could substantially augment activation of NF-κB via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect on the transcriptional competence of p65. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. We report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A-PRMT5), in either HEK293 cells or HT29, DLD1 and HCT116 CRC cells attenuated NF-κB activation compared to wild type (WT)-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, we found that overexpression of S15A-PRMT5 mutant attenuated the expression of a subset of NF-κB target genes through decreased p65 occupancy at their respective promoters. Importantly, the S15A-PRMT5 mutant also reduced IL-1β-induced methyltransferase activity of PRMT5 as well as its ability to form a complex with p65. Finally, we observed that the S15A-PRMT5 mutant diminished the growth, migratory and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, our findings provide strong evidence that novel phosphorylation of PRMT5 at S15 is critical to its regulation of NF-κB and plays an essential role in promoting the cancer-associated functions exerted by the PRMT5/NF-κB axis. Therefore, development of inhibitors to block phosphorylation of PRMT5 at S15 could become a potential novel therapeutic approach to treat CRC.2020-10-1

Epigenetic Biomarkers and Their Therapeutic Applications in Colorectal Cancer

Colorectal cancer (CRC) is one of the most aggressive cancers worldwide and is known to develop through a stepwise process involving the accumulation of several genetic and epigenetic alterations. Furthermore, numerous studies have highlighted the significant role that certain epigenetic enzymes play in CRC pathogenesis, particularly those that govern chromatin components in the promoter regions of tumor suppressors and oncogenes. Here, we delineate the relationship between CRC-associated epigenetic marks, their modifying enzymes, and the classification of CRC into distinct molecular pathways or subtypes. Moreover, we discuss some of the most prominent methyltransferases, demethylases, acetyltransferases, and deacetylases, which have been targeted for preclinical and clinical CRC treatment. Notably, inhibitors against these epigenetic enzymes are a promising new class of anticancer drugs, with several obtaining Food and Drug Administration (FDA) approval for the treatment of blood and solid tumors. By highlighting the epigenetic molecular pathways leading to CRC development as well as providing an update on current CRC epigenetic therapies, this chapter sheds fresh insight into new and emerging avenues for future therapeutics

Modulating the modulators: regulation of protein arginine methyltransferases by post-translational modifications

The therapeutic potential of targeting protein arginine methyltransferases (PRMTs) is inextricably linked to their key roles in various cellular functions, including splicing, proliferation, cell cycle regulation, differentiation, and DNA damage signaling. Unsurprisingly, the development of inhibitors against these enzymes has become a rapidly expanding research area. However, effective targeting of PRMTs requires a deeper understanding of the mechanistic details behind their regulation at multiple levels, involving those mechanisms that alter their activity, interactions, and localization. Recently, post-translational modifications (PTMs) of PRMTs have emerged as another crucial aspect of this regulation. Here, we review the regulatory role of PTMs in the activity and function of PRMTs, with emphasis on the contribution of these PTMs to pathological states, such as cancer

Phosphorylation of NF-κB in Cancer

The proinflammatory transcription factor nuclear factor-κB (NF-κB) has emerged as a central player in inflammatory responses and tumor development since its discovery three decades ago. In general, aberrant NF-κB activity plays a critical role in tumorigenesis and acquired resistance to chemotherapy. This aberrant NF-κB activity frequently involves several post-translational modifications of NF-κB, including phosphorylation. In this chapter, we will specifically cover the phosphorylation sites reported on the p65 subunit of NF-κB and their relationship to cancer. Importantly, phosphorylation is catalyzed by different kinases using adenosine triphosphate (ATP) as the phosphorus donor. These kinases are frequently hyperactive in cancers and thus may serve as potential therapeutic targets to treat different cancers

Role of post-translational modification of the Y box binding protein 1 in human cancers

Y box binding protein-1 (YBX1) belongs to a DNA- and RNA-binding family of transcription factors, containing the highly conserved cold shock domain (CSD). YBX1 is involved in a number of cellular functions including transcription, translation, DNA damage repair etc., and it is upregulated during times of environmental stress. YBX1 is localized in both the cytoplasm and the nucleus. There, its nuclear translocation is observed in a number of cancers and is associated with poor prognosis and disease progression. Additionally, YBX1 expression is upregulated in a variety of cancers, pointing towards its role as a potential oncogene. Under certain circumstances, YBX1 also promotes the expression of multidrug resistance 1 (MDR1) gene, which is involved in the development of drug resistance. Thus, it is critical to understand the mechanism of YBX1 regulation and its downstream effects on promoting cancer development. A number of recent studies have highlighted the mechanisms of YBX1 regulation. Mass spectrometric analyses have reported several post-translational modifications that possibly play an important role in modulating YBX1 function. Phosphorylation is the most widely occurring post-translational modification in YBX1. In vivo analyses of sites like S102 and more recently, S165 illustrate the relationship of post-translational regulation of YBX1 in promoting cell proliferation and tumor growth. This review provides a comprehensive and up-to-date account of post-translational modifications identified in YBX1. This knowledge is a key in allowing us to better understand the mechanism of YBX1 regulation, which will aid in development of novel therapeutic strategies to target YBX1 in many types of cancer in the future

Role of Novel Serine 316 Phosphorylation of the p65 Subunit of NF-κB in Differential Gene Regulation

Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers

Methylation of NF-κB and its Role in Gene Regulation

The nuclear factor κB (NF-κB) is one of the most pivotal transcription factors in mammalian cells. In many pathologies NF-κB is activated abnormally. This contributes to the development of various disorders such as cancer, acute kidney injury, lung disease, chronic inflammatory diseases, cardiovascular disease, and diabetes. This book chapter focuses on how methylation of NF-κB regulates its target genes differentially. The knowledge from this chapter will provide scientific strategies for innovative therapeutic intervention of NF-κB in a wide range of diseases

Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer

Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF- B in colon cancer

poster abstractY-box binding protein 1 (YBX1) is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor B (NF-B), has never been previously reported. Here, we show that overexpression of wild type YBX1 (wtYBX1) activates NF-B, suggesting that YBX1 is a potential NF-B activator. Furthermore, using mass spectrometry analysis, we identified novel phosphorylation of serine 165 (S165) on YBX1. Overexpression of the S165A-YBX1 mutant in either 293 cells or colon cancer HT29 cells showed dramatically reduced NF-B activating ability as compared to that of wtYBX1, confirming that S165 phosphorylation is critical for the activation of NF-B by YBX1. We further show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-B-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared to wtYBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-B activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer