Benjamin C. Onyeagucha

S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer.

Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1.

How microRNAs influence both hereditary and inflammatory-mediated colon cancers

MicroRNAs have emerged as important post-translational regulators of gene expression and are involved in several physiological and pathological states including the pathogenesis of human colon cancers. In regards to tumor development, microRNAs can act as oncogenes or tumor suppressors. Two hereditary predispositions (i.e., Lynch syndrome and familial adenomatous polyposis) contribute to the development of colon cancer. In addition, individuals who suffer from inflammatory bowel diseases such as Crohns disease or ulcerative colitis have a higher risk of developing colon cancer. Here, we discuss the occurrence of the deregulated expression of microRNAs in colon cancer that arise as a result of hereditary predisposition and inflammatory bowel disease.

The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells

S100P signaling through the receptor for advanced glycation end‐products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA‐21 (miR‐21). We show that exogenous S100P up‐regulates miR‐21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR‐21. Furthermore, blockage of RAGE with anti‐RAGE antibody suppresses S100P induction of miR‐21. In addition, we found that S100P induction of miR‐21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c‐Fos, and AP‐1 family members, at the miR‐21 gene promoter.

MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.The radiation and chemotherapy used for treating medulloblastoma patients cause debilitating side effects. Here, the authors show that miR-584 acts as a therapeutic adjuvant as it sensitizes medulloblastoma to radiation and chemotherapy by targeting HDAC1 or eIF4E3 to enhance spindle defects and DNA damage.

Abstract 2336: Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3’UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn’t have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation. Citation Format: Benjamin Chidi Onyeagucha, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Nourhan Abdelfattah, Santosh Timilsina, Rosa M. Guzman, Carla Zeballos, Vijay Eedunuri, Sanjay Bansal, Hima Bansal, Tabrez A. Mohammad, Yidong Chen, Manjeet K. Rao. Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2017-2336

Abstract 1806: AP-1 transcriptionally regulates expression of miR-155 in colon cancer cells.

Accumulating evidence indicates elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these overwhelming evidences, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Both S100P and miR-155 expressions are up-regulated in colon tumor specimens. Ectopic S100P expression leads to elevation of miR-155 level. Conversely, knockdown of S100P results in a decrease in miR-155 levels. Exogenous S100P induces miR-155 expression, but blockage of the RAGE receptor with anti-RAGE antibody suppresses the induction of miR-155 by exogenous S100P. Attenuation Blockage of AP-1 activation by S100P, through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppresses miR-155 induction by exogenous S100P. Exogenous S100P treatment stimulates the enrichment of c-Fos, an AP-1 family member at the miR-155 promoter site. Finally, functional study shows that miR-155 knockdown decreases colon cancer cell cell cell growtholon formation and motilityotility in S100P stably transfected cells and parental cells. Taken together, these data demonstrate that the expression of miR-155 is regulated by S100P is dependent on RAGE activation and stimulation of AP-1. Furthermore, the results show that miR-155 is a downstream target of S100P/RAGE signaling and a critical player in S100P functions in colon cancer cells. Citation Format: Benjamin C. Onyeagucha, Melania Mercado-Pimentel, Erik Flemington, Mark A. Nelson. AP-1 transcriptionally regulates expression of miR-155 in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2013-1806

Abstract 2398: S100P/RAGE signaling activates AP1 and NF-kB in miR-21/RECK regulation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The receptor for advanced glycation end-products (RAGE) plays a role in different pathological diseases including cancer. Several ligands activate RAGE, among them are AGEs (advance glycation end-products), HMGB1 (amphoterin), amyloid-α peptide, and the S100 Ca2+ binding family. Activation of RAGE by S100P stimulates many cell processes in cancer mediated by AP1, NF-kB and ERK1/2. Recently, these transcription factors were shown to induce expression of the oncogene, miR-21. Our data show that S100P over-expression in LS174T and SW480 colon cancer cells induces miR-21 expression. It is well known that S100P has an intracellular and an extracellular function when it interacts with Ezrin and RAGE respectively. To decipher if the extracellular function of S100P mediated by its interaction with RAGE induces miR-21, we treated SW480 normally expressing RAGE and not S100P with exogenous human recombinant (hr)-S100P. These results show that S100P/RAGE signaling induces miR-21 expression. To determine if the induction of miR-21 by S100P/RAGE signaling is mediated by AP1 and NF-kB, we performed luciferase studies with wild type, and mutated AP1 and NF-kB pri-miR-21 promoter constructs in cells expressing only the RAGE receptor. These data show that S100P/RAGE signaling mediates miR-21 induction by the activation of AP1 and NF-kB. Additionally, LS174T cells expressing RAGE and S100P rendered similar results when they ectopically over-express S100P. However, ectopic S100P expression in SW480 cells induced miR-21 independent of AP1 and NF-kB. These results suggest that S100P has another mechanism of regulating miR-21. Our previous data indicated that over-expression of S100P down regulates the reversion-inducing cysteine-rich protein with Kazal motifs (RECK). RECK is an anti metastatic gene, inhibitor of metalloproteinases and a target of miR-21. Our data shows that RAGE expressing cells treated with exogenous hr-S100P down regulate RECK expression, suggesting that miR-21 induction by S100P/RAGE signaling represses RECK. To determine if there is a correlation in expression levels of miR-21 with RECK, RAGE, and S100P, we used the combined method of in situ hybridization (ISH) and immunohistochemical (IHC) techniques on human colorectal cancer tissues. We found that there are three groups of cells in the malignant epithelium as well as in the surrounding tissue. One group of cells expresses high levels of miR-21, another group expresses high levels of RECK while the third group expresses both. Together, these data show that S100P/RAGE regulates miR-21/RECK expression mediated by AP1 and NF-kB and suggest that in cancer this signaling pathway remodels the extracellular matrix by the activation of metalloproteinases inducing epithelial mesenchymal transition to allow cell migration/invasion in colon cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2398. doi:1538-7445.AM2012-2398

Abstract 2300: MicroRNA-101 (miR-101) posttranscriptionally regulates the expression of EP4 receptor in colon cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The predominant product of cyclooxygenase (COX-2) in the colon, prostaglandin (PG) E2 promotes carcinogenesis. Expression of the PGE2 receptor EP4 is upregulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs. A bioinformatics search revealed a conserved target site for miR-101 within the EP4 receptor-3′ UTR. In both colorectal cancer cell lines and human specimens, we observed an inverse correlation between miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3′-UTR. In contrast, a mutant EP4 receptor-3′-UTR was unaffected. Ectopic expression miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2300. doi:1538-7445.AM2012-2300