Melania E. Mercado-Pimentel

Multiple Transforming Growth Factor-β Isoforms and Receptors Function during Epithelial-Mesenchymal Cell Transformation in the Embryonic Heart

Epithelial-mesenchymal cell transformation (EMT) is a critical process during development of the heart valves. Transition of endothelial cells into mesenchymal cells in the atrioventricular (AV) canal and the outflow tract regions of the heart form the cardiac cushions that eventually form the heart valves. Collagen gel invasion assay has aided in the identification of molecules that regulate EMT. Among those, transforming growth factor-β (TGF-β) ligands and receptors demonstrate a critical role during EMT. In the chick, TGF-β ligands and some receptors have specific functions during EMT. TGF-β2 mediates endothelial cell-cell activation and separation, and TGF-β3 mediates cell invasion into the extracellular matrix. Receptors involved in the EMT process include TGF-β receptor type II (TBRII), TBRIII, endoglin and the TBRI receptors, ALK2 and ALK5. In contrast, in the mouse model, TGF-β2 is the only ligand involved in EMT. The TGF-β2 null mouse has either increased EMT or a mesenchymal cell proliferation after EMT. However, functional studies of TGF-β1 in vivo and in vitro showed that TGF-β1 functions in the EMT of the mouse AV canal. Latent TGF-β-binding protein (LTBP-1) and endoglin have a role in the EMT process. Therefore, TGF-βs mediate cardiac EMT in both embryonic species. Further studies will reveal the identification of ligand and receptor-specific activities.

TGFβ‐mediated RhoA expression is necessary for epithelial‐mesenchymal transition in the embryonic chick heart

Endothelia in the atrioventricular canal (AVC) of the embryonic heart undergo an epithelial‐mesenchymal transition (EMT) and migrate into the underlying extracellular matrix. We explore here whether RhoA mediates this EMT. RhoA was detected in all cells of the chick heart during the stages studied. Expression was elevated when EMT was actively occurring. Explants treated with C3 exoenzyme in collagen gel cultures showed a significant decrease in mesenchymal cell numbers. siRNA was used to inhibit RhoA mRNA, and both activated endothelial and mesenchymal cells decreased significantly with treatment. Loss of RhoA produced a reduction of RhoB, cyclin‐b2, and β‐catenin messages showing that these genes are regulated downstream of RhoA. In contrast, runx‐2 was not reduced. Inhibition of TGFβ3 or TGFβ2 activity caused a large reduction of RhoA message. These data place RhoA in TGFβ regulated pathways for both endothelial activation and mesenchymal invasion and demonstrate a functional requirement during EMT. Developmental Dynamics 235:1589–1598, 2006.

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.

The induction of S100p expression by the Prostaglandin E2 (PGE2)/EP4 receptor signaling pathway in colon cancer cells

Background: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated. Methodology/Prinicipal Findings: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility. Conclusions/Significance: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.

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.

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