Role For Argonaute Research Articles

Abstract 2538: Role of Argonaute 2 in regulation of immune microenvironment in pancreatic cancer

Abstract KRAS mutations have been observed in nearly 95% of pancreatic ductal adenocarcinoma (PDAC), however targeting KRAS remains a major therapeutic challenge. Previous studies from our group have discovered a novel interaction between KRAS and Argonaute 2 (AGO2) and have uncovered a significant role of this interaction in regulating KRAS signaling. Knockout of AGO2 in genetically engineered mouse models of KRAS driven pancreatic and lung cancer dramatically impacted tumor progression. Intriguingly, in pancreatic cancer loss of AGO2 expression resulted in early pancreatic intraepithelial lesions (PanINs) that failed to progress to PDAC. We observed increased senescence in these AGO2 knockout lesions that abrogated PDAC progression. Further, there was pronounced infiltration of immune cells in pancreata lacking AGO2 in comparison to wild type. We particularly observed a 20-fold increase in natural killer (NK) cells population in pancreata lacking AGO2. This instigated us to evaluate potential role of AGO2 in regulating immune microenvironment in pancreatic cancer. Gene set enrichment analysis of AGO2 knockout SW1990 PDAC cells revealed significant upregulation of inflammatory and interferon response pathways. We observed increased expression of several signaling proteins implicated in immune activation pathways. Further characterization of these knockout cells indicated upregulation of MHC proteins that consequently added to mechanistic insights. Additionally, we are also exploring syngeneic models of pancreatic cancer and we will present findings from our ongoing studies to evaluate the impact of AGO2 knockout on immune axis. Overall, our findings suggest potential involvement of AGO2 in regulating immune microenvironment in pancreatic cancer. Citation Format: Jennifer Hon, Carson Kenum, Pushpinder S. Bawa, Vijaya L. Dommeti, Anastasia A. Sahu, Miriam Gandham, Chi-Chiang Li, Zainab I. Taher, Sylvia Zelenka-Wang, Jean C. Tien, Sunita Shankar, Seema Chugh, Arul M. Chinnaiyan. Role of Argonaute 2 in regulation of immune microenvironment in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2538.

Roles of the Core Components of the Mammalian miRISC in Chromatin Biology.

The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have shed light on the role of AGO and TNRC6 in mammalian chromatin biology and on the implications these novel mechanisms may have in our understanding of regulating gene expression.

Abstract 2578: An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis

Abstract The RAS gene family is among the most commonly mutated genes in cancer, and targeting the potential binding partners of mutated RAS may present a promising alternative therapeutic strategy. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of KRAS through its Switch II domain. Further, we demonstrated a role for AGO2 through the development of a KRASG12D driven mouse model of pancreatic cancer. Specifically, our findings revealed that AGO2 ablation drove early precursor pancreatic intraepithelial (PanIN) lesions to enter oncogene-induced senescence in lieu of progressing to late stage PanINs and pancreatic ductal adenocarcinoma. These studies suggested that AGO2 plays a key role in the development of mutant KRAS driven cancers. In order to assess the role of AGO2 in other mutant RAS driven cancers, we performed co-IP of purified AGO2 and purified NRAS/HRAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2, regardless of GDP or GTP nucleotide loading. Using a panel of cell lines (including T24 cells (HRASG12V) and Mel-Juso cells (NRASQ61L)), we observed that AGO2 interacted with both wild-type (WT) and mutant forms of HRAS and NRAS. Additionally, using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupted the interaction between WT RAS and AGO2. However, the mutant NRAS-, HRAS-, and KRAS-AGO2 interactions were resistant to EGFR regulation. Considering the strong role of AGO2 in mutant KRAS driven cancer, we next asked if loss of AGO2 could induce a similar inhibition of RAS driven disease in human cell line models. In a panel of mutant NRAS and HRAS driven cell lines, AGO2 knockdown led to a substantial decrease in cell proliferation; however, WT RAS expressing cells were unperturbed following loss of AGO2. Similar to the loss of AGO2 in the KRASG12D driven mouse model, knockdown of AGO2 led to increased beta-galactosidase staining in a panel of oncogenic NRAS/HRAS driven cells. Furthermore, knockdown of AGO2 was associated with an induction of multiple senescence pathways, including increased p53, p21, and p16 expression. These results suggest that decreased AGO2 expression is sufficient to induce senescence in mutant but not WT RAS driven cells. In these cell line models, AGO2 loss reduced not only the mutant RAS isoform expression but also the WT RAS isoforms. Finally, knockdown of AGO2 led to an inhibition of T24 and Mel-Juso cell migration and metastasis in a zebrafish xenograft model. Taken together, these results suggest that 1) AGO2 interactions with mutant HRAS and NRAS play a key role in mutant RAS driven oncogenesis and 2) both mutant HRAS and NRAS depend on AGO2 to overcome senescence. Citation Format: Ronald F. Siebenaler, Jessica Waninger, Sunita Shankar, Seema Chugh, Shih-Chun Chu, Jean Tien, Vijaya L. Dommeti, Malay Mody, Anudeeta Gautam, Carson Kenum, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2578.

Abstract A20: An essential role for Argonaute 2 in mouse models of KRAS driven cancers

Abstract In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing that is required for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed the genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D driven pancreatic cancer. Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in survival of KRASG12D mutant mice deficient in AGO2 expression. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis; however rather than progressing to PDAC, these lesions undergo profound oncogene-induced senescence (OIS). Since PanIN development requires EGFR and is not AGO2 dependent, we probed the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS (using co-IP and PLA analyses). In wild-type RAS expressing cells, grown in media containing serum, RAS-AGO2 co-localization was limited to the intracellular regions of the cells, which dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular basal levels. Using phosphorylation-deficient AGO2 mutants, we demonstrate that the disruption of wild type-RAS-AGO2 interaction is due to AGO2Y393 phosphorylation, a target of EGFR. Interestingly, the mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild-type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. Taken together, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. In the lung cancer mouse model, we also observed a similar dependence of AGO2 in KRAS-driven lung adenocarcinoma. Along with related abstracts detailing the mechanisms of OIS mediated by AGO2 (Ronald Siebenaler) and evidence of direct interaction between oncogenic KRAS and AGO2 with an affinity of 200nM (Jessica Waninger), we present our latest studies related to the KRAS-AGO2 interaction. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Xiao-Ming Wang, Kristin M. Juckette, Alice Xu, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Lisha Wang, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS driven cancers [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A20.

Inferring targeting modes of Argonaute-loaded tRNA fragments

ABSTRACT Transfer RNA fragments (tRFs) are an emerging class of small RNA molecules derived from mature or precursor tRNAs. They are found across a wide range of organisms and tissues, in small RNA fraction or loaded to Argonaute in numbers comparable to microRNAs. Their functions and mechanisms of action are largely unknown, and results obtained on individual tRFs are often hard to generalize. Here we predicted binding mechanisms and specific target interaction sites of 26 human Argonaute-loaded tRFs of different types using large-scale meta-analyses of available experimental data. Strikingly, our findings matched all interaction sites detected in a recent experimental screen, confirming the validity of our computational approach. Such sites are primarily located on the 5ʹ end of tRFs and often involve additional binding along the tRF length, similar to microRNAs. Indicative of multiple layers of regulation, diverse regulatory non-coding RNAs comprised a third of the tRF targets, with the rest being protein-coding transcripts. In the latter, coding sequence and 3ʹ UTRs were the likely primary target regions, although we observed interactions of tRFs with 5ʹ UTRs. Another novel phenomenon we report, a large number of putative interactions between tRFs and introns, is compatible with the roles of Argonaute in the nucleus. Further, observed tRF-intron binding modes suggest a mechanism of interaction of tRFs with Argonaute-dependent introns, and we predict here >20 candidate introns of this type. Taken together, these results present tRFs as regulatory molecules with a rich functional spectrum.

Abstract 94: Role of Argonaute 2 in oncogene induced senescence in a pancreatic cancer mouse model

Abstract Pancreatic cancer is a highly devastating malignancy with a very poor survival rate of 7%. Mutations in KRAS have been identified in more than 90% of PDAC patients. Previous work from our lab has shown that KRAS directly interacts with Argonaute 2 (AGO2) to promote cellular transformation. To study the involvement of AGO2 in KRASG12D-driven cancers, AGO2 expression was ablated in the KrasG12D/+; p48 Cre model (KC) model of pancreatic cancer. AGO2 floxed mice (AGO2loxP/loxP) were crossed with KC mice, resulting in oncogenic KRAS expression along with knockout of AGO2 in pancreatic acinar cells. Survival and disease progression were compared between wild-type (AGO2+/+; KRASG12D; p48Cre), heterozygous (AGO2fl/+; KRASG12D; p48Cre), and homozygous (AGO2fl/fl; KRASG12D; p48Cre) experimental mice groups. Homozygous knockout of AGO2 in KC mice resulted in significantly increased survival as compared to wild type and heterozygous mice. Pancreatic ductal adenocarcinoma (PDAC) and metastases were restricted to the wild-type and heterozygous mice. Pancreas from AGO2fl/fl; KRASG12D; p48Cre mice develop only early pancreatic intraepithelial lesions (PanINs), which fail to progress to PDAC. Senescence-associated β-galactosidase staining showed strong and significant increase in senescence in PanIN lesions mice lacking AGO2 expression as compared to AGO2+/+; KRASG12D; p48Cre mice. This suggests that AGO2 prevents oncogene induced senescence (OIS) as a result of KRASG12D expression and allows PanIN to PDAC progression. To gain mechanistic insights of OIS due to AGO2 loss, we evaluated markers for OIS including p16, p53, p21, gamma γH2AX, and RAS-associated signaling (pERK and pAkt). Analysis of PanIN lesions lacking AGO2 showed increased p16 levels and high levels of phospho-ERK, compared to PDAC from pancreas with AGO2 expression. In order to extend these observations in cell line models, we performed AGO2 knockdown in T24 cells harboring HRASG12V. Surprisingly, cells with low AGO2 levels underwent OIS, which was similar to the pancreatic mouse model and was accompanied with increased phospho-ERK signaling and p16 expression. Further studies are underway to determine the contribution of the RAS-AGO2 interaction in the development of OIS. Additionally, we are using CRISPR/Cas9 technology to screen pancreatic cancer cell lines with AGO2 knockout for their dependence on AGO2 and their ability to undergo OIS in the absence of AGO2 expression. We will present findings from our ongoing studies involving the role of AGO2 loss in the KPC (KRASG12D; p53fl/+; Cre) model, wherein OIS will be assessed in the absence of p53, a canonical inducer of cellular senescence. Citation Format: Seema Chugh, Jean C. Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Z. Wang, Sanjana Eyunni, Kristin M. Juckette, Lisha Wang, Sunita Shankar, Arul M. Chinnaiyan. Role of Argonaute 2 in oncogene induced senescence in a pancreatic cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 94.

Abstract 957: An essential role for Argonaute 2 in mouse models of KRAS-driven cancers

Abstract In 2016, we identified a direct interaction between RAS and Argonaute 2 (AGO2), a key mediator of RNA-mediated gene silencing, that is essential for KRAS-driven oncogenesis using pancreatic and lung cancer cell line models. Recently, we employed a genetically engineered mouse model of pancreatic cancer to define the effects of conditional loss of AGO2 in KRASG12D-driven pancreatic cancer (KC model). Genetic ablation of AGO2 did not interfere with development of the normal pancreas or KRASG12D-driven early precursor pancreatic intraepithelial neoplasia (PanIN) lesions. However, AGO2 loss prevents progression from early to late PanIN lesions, development of pancreatic ductal adenocarcinoma (PDAC), and metastatic progression. This results in a dramatic increase in the survival of KRASG12D mutant mice deficient in AGO2 expression. Mechanistically, lack of PanIN to PDAC progression was due to oncogene-induced senescence (OIS) through activation of EGFR-wild type RAS-phosphoERK signaling in the absence of AGO2. Using validated pan-RAS and AGO2 antibodies for immunofluorescence (IF) and proximity ligation assay (PLA), we observed increased RAS and AGO2 co-localization at the plasma membrane in mouse and human pancreatic tissues associated with PDAC progression. While AGO2 ablation permits PanIN initiation driven by the EGFR-RAS axis, these lesions undergo OIS rather than progressing to PDAC. Further, we used co-IP and PLA analyses to probe the effects of EGF stimulation in cell lines expressing wild-type and mutant forms of KRAS. In wild-type RAS expressing cells, RAS-AGO2 co-localization and interaction were limited to the intracellular regions of the cells, and dramatically increased and shifted to the plasma membrane under conditions of stress (serum starvation). Interestingly, EGF stimulation disrupted this membrane RAS-AGO2 interaction and restored it to intracellular levels. Using phosphorylation-deficient AGO2 mutants, we further demonstrate that the disruption of wild-type RAS-AGO2 interaction is due to EGFR-mediated AGO2Y393 phosphorylation. Interestingly, mutant KRAS-AGO2 interaction is not subject to EGFR activation, suggesting that although both the wild type and mutant RAS bind AGO2, they are differentially regulated through growth factor receptor activation. We will discuss our ongoing studies evaluating the effects of AGO2 ablation in the KRASG12Ddriven lung cancer mouse model and PDAC progression with p53 loss (KPC model). Our recent in vivo work supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR and wild-type RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression. Citation Format: Sunita Shankar, Jean Ching-Yi Tien, Ronald F. Siebenaler, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Kristin M. Juckette, Alice Xu, Xiao-Ming Wang, Seema Chugh, Malay Mody, Sanjana Eyunni, Andrew Goodrum, Grace Tsaloff, Yuping Zhang, Ingrid J. Apel, Javed Siddiqui, Richard D. Smith, Heather A. Carlson, John Tesmer, Xuhong Cao, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in mouse models of KRAS-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 957.

The expanding roles of Argonautes: RNA interference, splicing and beyond.

Argonaute (AGO) protein family is highly conserved in eukaryotes and prokaryotes, reflecting its evolutionarily indispensible role in maintaining normal life cycle of the organisms. Small RNA-guided, AGO-dependent RNA interference (RNAi) is a well-studied pathway for gene expression regulation, which can be performed at transcriptional, posttranscriptional or translational level. In addition to RNAi, growing pieces of evidence point to a novel role of AGOs in pre-mRNA (messenger RNA precursor) splicing in animals. Many noncoding RNAs (ncRNAs) share common structural features with protein-coding genes, indicating that these ncRNAs might be subject to AGO-mediated splicing. Finally, we provide a comprehensive view that RNAi, transcription and RNA splicing are highly interactive processes, all of which involve several key factors such as AGOs. In this regard, the AGO proteins contribute to orchestrate an exquisite gene regulatory network in vivo. However, more research efforts are needed to reach a thorough understanding of the AGO activities.

Abstract 3029: An in vivo role for Argonaute 2 in KRAS driven pancreatic cancer

Abstract Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we recently identified an interaction between RAS and Argonaute 2 (AGO2), the core component of the RNA silencing machinery exerting translational control of mRNA transcripts. RAS and AGO2 co-sediment and co-localize in intracellular endomembrane bound organelles. The AGO2 N-terminal domain directly binds the KRAS Switch II region, irrespective of GDP/GTP bound to RAS. Functionally, we observed that AGO2 was required for maximal oncogenic KRAS levels and AGO2 knock-down attenuates cell proliferation in mutant KRAS-dependent cells. Intriguingly, AGO2 mediated microRNA processing (RISC activity) was attenuated in cancer cells expressing mutant KRAS compared to those expressing wild type RAS. Yet, these initial investigations using cell line models do not reveal a clear role for AGO2 in KRAS driven oncogenesis. Therefore, we probed the role of AGO2 in the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model driven by oncogenic KRAS. Towards this end, we generated LSL-KrasG12D; Pft1a Cre; AGO2flox/flox (KCA) mice. Preliminary analysis (8-10 weeks of age) suggests that compared to LSL-KRASG12D (KC) mice, homozygous loss of AGO2 results in increased KRAS driven Acinar to Ductal Metaplasia (ADM) phenotype considered as precursors of PanIN (pancreatic intraepithelial neoplasia) and PDAC. This mirrors the phenotype of loss of Dicer in the same model further reinforcing a role for microRNAs in restraining KRAS oncogenic programs. Yet unlike Dicer, loss of AGO2 alone did not cause any gross or histological changes in the development of the mouse pancreata. In an effort to understand the underlying mechanisms that are regulated by AGO2, we also studied the activated levels of a variety of signaling molecules in pancreata obtained from different genotypes. Primary analysis indicates that wild type RAS levels are elevated during AGO2 loss and has a bearing on PDAC initiation. Using both Western blot and Immunohistochemical analyses, we demonstrate a previously unknown and critical role for AGO2 in KRAS-MAPK signaling pathway. Citation Format: Sunita Shankar, Jean Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Ingrid A. Apel, Jiaqi Shi, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An in vivo role for Argonaute 2 in KRAS driven pancreatic 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 3029. doi:10.1158/1538-7445.AM2017-3029

Abstract 393: Pancreatic Beta Cell Export of miR-375 to High-Density Lipoproteins is Inversely Associated With Insulin Secretion

microRNAs (miRNAs) are critical regulators of glucose metabolism and contribute to the pathogenesis of Type 2 Diabetes (T2D). Recently, we reported that high-density lipoproteins (HDL) transport and deliver functional miRNAs to recipient cells. Here, we report that miR-375 is decreased on HDL in two models of chronic hyperglycemia -- T2D human subjects and Zucker Diabetic Fatty (ZDF) rats. Since miR-375 expression in the islets is 10X greater than in other organs, we tested whether pancreatic beta cells have the ability to export miR-375 to HDL through in vitro export assays, incubating HDL with INS1 beta cells or primary human islets. Indeed, we found miR-375 to be readily exported to HDL from INS1 cells and primary islets in vitro . To determine if cholesterol transporters contribute to HDL-miR-375 export from beta cells, Abca1, Abcg1 and Scarb1 (SR-BI) were inhibited using siRNAs; however, we found that knockdown of each of these transporters failed to affect the beta cell’s ability to export miR-375 to HDL. Nonetheless, enhancing insulin secretion with tolbutamide resulted in the suppression of HDL-miR-375 export, suggesting that miRNA export and insulin secretion are inversely regulated. To determine the roles of Argonaute (Ago) family proteins in HDL-miRNA export, INS1 cells were transfected with siRNAs against Eif2c1-4 to knockdown Ago1-4. We found HDL-miR-375 export to be suppressed when Ago1, but not Ago2-4, were inhibited, suggesting that miRNA export is downstream of miRNA processing by Ago1. We are currently investigating the relationship between HDL-miR-375 export, insulin secretion, and miRNA processing in pancreatic beta cells to elucidate the mechanism(s) controlling HDL-miR-375 export. Collectively, results suggest that a large fraction of HDL-miRNAs originate from pancreatic beta cells and HDL-miRNAs are exported independent of cholesterol transporters.

Abstract A05: An in vivo model reveals a role for Argonaute 2 in oncogenic KRAS driven pancreatic cancer initiation

Abstract Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we recently identified an interaction between RAS and Argonaute 2 (AGO2), the core component of the RNA silencing machinery exerting translational control of mRNA transcripts. RAS and AGO2 co-sediment and co-localize in intracellular endomembrane bound organelles. The AGO2 N-terminal domain directly binds the KRAS Switch II region, irrespective of GDP/GTP bound to RAS. Functionally, we observed that AGO2 was required for maximal oncogenic KRAS levels and AGO2 knock-down attenuates cell proliferation in mutant KRAS-dependent cells. Intriguingly, AGO2 mediated microRNA processing (RISC activity) was attenuated in cancer cells expressing mutant KRAS compared to those expressing wild type RAS. Yet, these initial investigations using cell line models do not reveal a clear role for AGO2 in KRAS driven oncogenesis. Therefore, we probed the role of AGO2 in the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model driven by oncogenic KRAS. Towards this end, we generated LSL-KrasG12D; Pft1a Cre; AGO2flox/flox (KCA) mice. Preliminary analysis (8-10 weeks of age) suggests that compared to LSL-KRASG12D (KC) mice, homozygous loss of AGO2 results in increased KRAS driven Acinar to Ductal Metaplasia (ADM) phenotype considered as precursors of PanIN (pancreatic intraepithelial neoplasia) and PDAC. This mirrors the phenotype of loss of Dicer, another microRNA processing enzyme, in the same model further reinforcing a role for microRNAs in restraining KRAS oncogenic programs. Yet unlike Dicer, loss of AGO2 alone did not cause any gross or histological changes in the development of the mouse pancreata. In an effort to understand the underlying mechanisms that are regulated by AGO2, we also studied the activated levels of a variety of signaling molecules in pancreata obtained from different genotypes. Using both Western blot and Immunohistochemical analyses, we demonstrate a previously unknown and critical role for AGO2 in KRAS-MAPK signaling pathway. Together our data from the in vivo mouse model suggests that AGO2 is an important regulator of KRAS signaling and oncogenic KRAS alters the translational machinery through its interaction with AGO2. Citation Format: Sunita Shankar, Jean Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Arul M. Chinnaiyan. An in vivo model reveals a role for Argonaute 2 in oncogenic KRAS driven pancreatic cancer initiation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A05.

Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion

Successful embryo implantation requires synchronous development and communication between the blastocyst and the endometrium, however the mechanisms of communication in humans are virtually unknown. Recent studies have revealed that microRNAs (miRs) are present in bodily fluids and secreted by cells in culture. We have identified that human blastocysts differentially secrete miRs in a pattern associated with their implantation outcome. miR-661 was the most highly expressed miR in blastocyst culture media (BCM) from blastocysts that failed to implant (non-implanted) compared to blastocysts that implanted (implanted). Our results indicate a possible role for Argonaute 1 in the transport of miR-661 in non-implanted BCM and taken up by primary human endometrial epithelial cells (HEECs). miR-661 uptake by HEEC reduced trophoblast cell line spheroid attachment to HEEC via PVRL1. Our results suggest that human blastocysts alter the endometrial epithelial adhesion, the initiating event of implantation, via the secretion of miR, abnormalities in which result in implantation failure.

Abstract W P221: Argonaute 2 Mediates Sildenafil-Promoted Axonal Outgrowth

Molecular mechanisms are not fully known for the inhibitory effect of chondroitin sulfate proteoglycans (CSPGs) on axonal outgrowth. The present study investigated the effect of CSPGs on miRNA expression and the role of Argonaute 2 (Ago2) in mediating Sildenafil-induced axonal outgrowth under CSPG conditions. Primary rat embryonic cortical neurons were cultured in a microfluidic chamber which separates somata and axons. Selective application of CSPGs within the axonal compartment markedly inhibited axonal outgrowth (567±15 μm vs 688 ± 21 μm in control, n=5, p<0.05). Axonal application of Sildenafil, a PDE5 inhibitor, completely reversed the inhibitory effect of CSPG on axonal outgrowth (671±17 μm, n=5). Analysis of miRNA PCR array showed that axonal application of CSPGs robustly changed miRNA profiles not only in axons but also in somata, indicating that axonal signals regulate soma miRNAs. Sildenafil inversely altered miRNA up- and down-regulated by CSPGs, including miR-29c, miR-9a, miR-20b, miR-298 and miR-34a. Attenuation of Ago2, a regulator of miRNA activity, in neurons by siRNA abolished Sildenafil-increased axonal outgrowth under CSPG conditions, suggesting that miRNAs are required for Sildenafil-enhanced axonal outgrowth. One of putative targets of miR-29c is integrin β1. Western blot analysis showed that CSPGs and Sildenafil reduced and increased, respectively, axonal integrin β1 and its downstream protein, focal adhesion kinase, suggesting that miR-29c regulates these protein levels. To examine cause-effect of integrin β1, we applied a neutralizing antibody against integrin β1 into the axonal compartment and found that the antibody attenuated the Sildenafil-reversed inhibitor effect of CSPGs on axonal outgrowth (523±34 μm vs 671±17 μm in Sildenafil, n=5, p<0.01). In conclusion, the present study indicates that axonal signals induced by CSPGs and Sildenafil change miRNA profiles in the entire neuron and that Sildenafil abolishes the CSPG inhibitory effect on axons via Ago2.

On plant stem cells and animal stem cells

A comparison of plant and animal stem cells can highlight core aspects of stem-cell biology. In both kingdoms, stem cells are defined by their clonogenic properties and are maintained by intercellular signals. The signaling molecules are different in plants and animals stem cell niches, but the roles of argonaute and polycomb group proteins suggest that there are some molecular similarities.

The varied roles of nuclear argonaute-small RNA complexes and avenues for therapy.

Argonautes are highly conserved proteins found in almost all eukaryotes and some bacteria and archaea. In humans, there are eight argonaute proteins evenly distributed across two clades, the Ago clade (AGO1-4) and the Piwi clade (PIWIL1-4). The function of Ago proteins is best characterized by their role in RNA interference (RNAi) and cytoplasmic post-transcriptional gene silencing (PTGS) – which involves the loading of siRNA or miRNA into argonaute to direct silencing of genes at the posttranscriptional or translational level. However, nuclear-localized, as opposed to cytoplasmic, argonaute-small RNA complexes may also orchestrate the mechanistically very different process of transcriptional gene silencing, which results in prevention of transcription from a gene locus by the formation of silent chromatin domains. More recently, the role of argonaute in other aspects of epigenetic regulation of chromatin, alternative splicing and DNA repair is emerging. This review focuses on the activity of nuclear-localized short RNA-argonaute complexes in a mammalian setting and discusses recent in vivo studies employing nuclear-directed sRNA for therapeutic interventions. These studies heed the potential development of RNA-based drugs which induce epigenetic changes in the cell.

Autoregulation of microRNA biogenesis by let-7 and Argonaute

SUMMARYMicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways1. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65 nucleotide (nt) precursors that are then cleaved by Dicer, resulting in the mature 22 nt forms2,3. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base-pairing to recognize sequences in mRNA transcripts, leading to translational repression and destabilization of the target mRNAs4,5. Here we show that the miRNA complex also targets and regulates non-coding RNAs (ncRNAs) that serve as substrates for the miRNA processing pathway. We found that the C. elegans Argonaute, ALG-1, binds to a specific site at the 3′ end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA via a conserved complementary site in its own primary transcript, thus creating a positive feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding mRNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of auto-regulation of let-7 biogenesis sets a new paradigm for controlling miRNA expression.

A Role for ARGONAUTE in Apomixis

Apomixis is a form of asexual reproduction in angiosperms that results in the production of viable seed from maternal tissues of the ovule in the absence of meiosis and fertilization (reviewed in [Bicknell and Koltunow, 2004][1]). Apomixis occurs in a number of wild plant species, but in none of the