Browsing by Author "Wei, Han"

Now showing 1 - 10 of 13
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    A complex signature network that controls the upregulation of PRMT5 in colorectal cancer
    (Elsevier, 2022-03) Wei, Han; Hartley, Antja-Voy; Motolani, Aishat; Jiang, Guanglong; Safa, Ahmad; Prabhu, Lakshmi; Liu, Yunlong; Lu, Tao; Pharmacology and Toxicology, School of Medicine
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    Adapting AlphaLISA high throughput screen to discover a novel small-molecule inhibitor targeting protein arginine methyltransferase 5 in pancreatic and colorectal cancers
    (Impact Journals, 2017-05-23) Prabhu, Lakshmi; Wei, Han; Chen, Lan; Demir, Özlem; Sandusky, George; Sun, Emily; Wang, John; Mo, Jessica; Zeng, Lifan; Fishel, Melissa; Safa, Ahmad; Amaro, Rommie; Korc, Murray; Zhang, Zhong-Yin; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An in silico structure prediction suggested that PR5-LL-CM01 inhibits PRMT5 by binding with its active pocket. Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF- B in colon cancer
    (Office of the Vice Chancellor for Research, 2015-04-17) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao
    Y-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.
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    Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer.
    (Impact Journals, 2015-10-06) Prabhu, Lakshmi; Mundade, Rasika; Wang, Benlian; Wei, Han; Hartley, Antja-Voy; Martin, Matthew; McElyea, Kyle; Temm, Constance J.; Sandusky, George; Liu, Yunlong; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Y-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 κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of
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    Development of AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases
    (Royal Society of Chemistry, 2017-11-21) Prabhu, Lakshmi; Chen, Lan; Wei, Han; Demir, Özlem; Safa, Ahmad; Zeng, Lifan; Amaro, Rommie E.; O’Neil, Bert H.; Zhang, Zhongyin; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.
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    KDM2A Deficiency in the Liver Promotes Abnormal Liver Function and Potential Liver Damage
    (MDPI, 2023-09-27) Martin, Matthew; Motolani, Aishat; Kim, Hyeong-Geug; Collins, Amy M.; Alipourgivi, Faranak; Jin, Jiamin; Wei, Han; Wood, Barry A.; Ma, Yao-Ying; Dong, X. Charlie; Mirmira, Raghavendra G.; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    Dysregulation of metabolic functions in the liver impacts the development of diabetes and metabolic disorders. Normal liver function can be compromised by increased inflammation via the activation of signaling such as nuclear factor (NF)-κB signaling. Notably, we have previously identified lysine demethylase 2A (KDM2A)—as a critical negative regulator of NF-κB. However, there are no studies demonstrating the effect of KDM2A on liver function. Here, we established a novel liver-specific Kdm2a knockout mouse model to evaluate KDM2A’s role in liver functions. An inducible hepatic deletion of Kdm2a, Alb-Cre-Kdm2afl/fl (Kdm2a KO), was generated by crossing the Kdm2a floxed mice (Kdm2afl/fl) we established with commercial albumin-Cre transgenic mice (B6.Cg-Tg(Alb-cre)21Mgn/J). We show that under a normal diet, Kdm2a KO mice exhibited increased serum alanine aminotransferase (ALT) activity, L-type triglycerides (TG) levels, and liver glycogen levels vs. WT (Kdm2afl/fl) animals. These changes were further enhanced in Kdm2a liver KO mice in high-fat diet (HFD) conditions. We also observed a significant increase in NF-κB target gene expression in Kdm2a liver KO mice under HFD conditions. Similarly, the KO mice exhibited increased immune cell infiltration. Collectively, these data suggest liver-specific KDM2A deficiency may enhance inflammation in the liver, potentially through NF-κB activation, and lead to liver dysfunction. Our study also suggests that the established Kdm2afl/fl mouse model may serve as a powerful tool for studying liver-related metabolic diseases.
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    Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer
    (American Society for Biochemistry and Molecular Biology, 2017-02-24) Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao; Medical and Molecular Genetics, School of Medicine
    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.
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    PRMT5-mediated methylation of YBX1 regulates NF-κB activity in colorectal cancer
    (Nature Publishing Group, 2020-09-28) Hartley, Antja-Voy; Wang, Benlian; Mundade, Rasika; Jiang, Guanglong; Sun, Mengyao; Wei, Han; Sun, Steven; Liu, Yunlong; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    The multifunctional protein Y-box binding protein 1 (YBX1), is a critical regulator of transcription and translation, and is widely recognized as an oncogenic driver in several solid tumors, including colorectal cancer (CRC). However, very little is known about the upstream or downstream factors that underlie YBX1′s regulation and involvement in CRC. Previously, we demonstrated that YBX1 overexpression correlated with potent activation of nuclear factor κB (NF-κB), a well-known transcription factor believed to be crucial in CRC progression. Here, we report a novel interaction between NF-κB, YBX1 and protein arginine methyltransferase 5 (PRMT5). Our findings reveal for the first time that PRMT5 catalyzes methylation of YBX1 at arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-κB activation and its downstream target gene expression. Importantly, when WT-YBX1 is overexpressed, this methylation exists under basal (unstimulated) conditions and is further augmented upon interleukin-1β (IL-1β) stimulation. Mechanistically, co-immunoprecipitation studies reveal that the R205 to alanine (A) mutant of YBX1 (YBX1-R205A) interacted less well with the p65 subunit of NF-κB and attenuated the DNA binding ability of p65. Importantly, overexpression of YBX1-R205A significantly reduced cell growth, migration and anchorage-independent growth of CRC cells. Collectively, our findings shed important light on the regulation of a novel PRMT5/YBX1/NF-κB axis through PRMT5-mediated YBX1-R205 methylation. Given the fact that PRMT5, YBX1 and NF-κB are all among top crucial factors in cancer progression, pharmacological disruption of this pivotal axis could serve as the basis for new therapeutics for CRC and other PRMT5/YBX1/NF-κB-associated cancers.
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    Regulation of a PRMT5/NF-κB Axis by Phosphorylation of PRMT5 at Serine 15 in Colorectal Cancer
    (MDPI, 2020-05-23) Hartley, Antja-Voy; Wang, Benlian; Jiang, Guanglong; Wei, Han; Sun, Mengyao; Prabhu, Lakshmi; Martin, Matthew; Safa, Ahmad; Sun, Steven; Liu, Yunlong; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    patients. Importantly, our previous work demonstrated that PRMT5 overexpression could substantially augment activation of the nuclear factor kappa B (NF-κB) via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. Here, we report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Interestingly, we identified for the first time that the oncogenic kinase, PKCι could catalyze this phosphorylation event. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A), in either HEK293 cells or CRC cells HT29, DLD1, and HCT116 attenuated NF-κB transactivation compared to WT-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, the S15A mutant when compared to WT-PRMT5, could downregulate a subset of IL-1β-inducible NF-κB-target genes which correlated with attenuated promoter occupancy of p65 at its target genes. Additionally, the S15A mutant reduced IL-1β-induced methyltransferase activity of PRMT5 and disrupted the interaction of PRMT5 with p65. Furthermore, our data indicate that blockade of PKCι-regulated PRMT5-mediated activation of NF-κB was likely through phosphorylation of PRMT5 at S15. Finally, inhibition of PKCι or overexpression of the S15A mutant attenuated the growth, migratory, and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, we have identified a novel PKCι/PRMT5/NF-κB signaling axis, suggesting that pharmacological disruption of this pivotal axis could serve as the basis for new anti-cancer therapeutics.
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    Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond
    (Landes Bioscience, 2014) Mundade, Rasika; Ozer, Hatice Gulcin; Wei, Han; Prabhu, Lakshmi; Lu, Tao; Department of Pharmacology and Toxicology, IU School of Medicine
    Many biologically significant processes, such as cell differentiation and cell cycle progression, gene transcription and DNA replication, chromosome stability and epigenetic silencing etc. depend on the crucial interactions between cellular proteins and DNA. Chromatin immunoprecipitation (ChIP) is an important experimental technique for studying interactions between specific proteins and DNA in the cell and determining their localization on a specific genomic locus. In recent years, the combination of ChIP with second generation DNA-sequencing technology (ChIP-seq) allows precise genomic functional assay. This review addresses the important applications of ChIP-seq with an emphasis on its role in genome-wide mapping of transcription factor binding sites, the revelation of underlying molecular mechanisms of differential gene regulation that are governed by specific transcription factors, and the identification of epigenetic marks. Furthermore, we also describe the ChIP-seq data analysis workflow and a perspective for the exciting potential advancement of ChIP-seq technology in the future.