Targeting undruggable phosphatase overcomes trastuzumab resistance by inhibiting multi-oncogenic kinases.

AIMS: Resistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. METHODS: Four public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. RESULTS: PTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed "undruggable," was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. CONCLUSIONS: Antibody-conjugated saRNA represents an innovative approach for targeting "undruggable" PTPs.

HuR facilitates miR-93-5p-induced activation of MAP3K2 translation via MAP3K2 3'UTR ARE2 in hepatocellular carcinoma.

MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3' untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3'UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3'UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3'UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.

saKLK1-374 is more difficult to induce KLK1 expression in normal prostate cell lines than that in prostate cancer cell lines: Rethinking the universality of RNA activation.

RNA activation, as a method of regulating gene expression at the transcriptional level, is far less widely used than RNA interference because of the insufficient understanding of the mechanism and the unstable success rate. It is necessary to analyze the failure cases of RNA activation to promote the application of RNA activation. When we validated the saRNAs designed to induce KLK1 expression, we found that saKLK1-374 can upregulate KLK1 expression in prostate tumor cell lines, but failed in normal prostate cell lines. To determine whether the RNA activation of normal cells is difficult only when the target gene is KLK1, we tested p21WAF1/CIP1 as the target gene in RNA activation experiments of normal and cancer prostate cells. Next, to determine whether the above phenomenon exists in other tissues, we used normal and cancerous bladder cells to perform RNA activation experiments with KLK1 and p21WAF1/CIP1 as targets. We have also extended the time from transfection to detection to evaluate whether a longer incubation time can make saRNA upregulate the target genes in normal cells. Fluorescently labeled dsRNA was transfected to evaluate the transfection efficiency, and the expression of Ago2 and IPO8 necessary for RNA activation was also detected. The p21WAF1/CIP1 could be significantly upregulated by saRNA in prostate cancer cells, but not in normal prostate cells. The expression of KLK1 in bladder-derived cell lines was extremely low and could not be induced by saRNA. The p21WAF1/CIP1 was upregulated by saRNA to a higher extent in bladder cancer cells but to a lower extent in normal bladder cells. Prolonging incubation time could not make saRNA induce the expression of target genes in normal cells. Compared with tumor cells used in this study, normal cells had lower transfection efficiency or lower expression of Ago2 and IPO8. Although it has been currently found that normal cell lines in the prostate and bladder might be more difficult to be successfully induced target gene expression by exogenous saRNA than tumor cells due to low transfection efficiency or Ago2 and IPO8 expression, it is not certain that this phenomenon occurs in other types of tissue. However, researchers still need to pay attention to the transfection efficiency and/or the expression levels of Ago2 and IPO8 when conducting RNA activation experiments in normal cells.

Preparation of a miR-155-activating nucleic acid nanoflower to study the molecular mechanism of miR-155 in inflammation.

At present, the molecular mechanisms underlying inflammation remain unclear. In recent years, research on inflammation has focused on stimulating cell inflammation by using exogenous pro-inflammatory substances such as lipopolysaccharide (LPS) or inflammatory factors. To investigate the molecular mechanism of inflammation from a new perspective, we designed a nucleic acid nanoflowers (NFs) complex to directly activate inflammatory genes to study the inflammatory response without the need for external microbial factors to trigger an inflammatory response. An RNAa-type target gene-activated NFs was designed. Human umbilical vein endothelial cells (HUVECs) were transfected with NFs carrying small activating RNA (saRNAs) to directly co-activate microRNA (miR)-155 and SHIP1 genes. After RNA activation (RNAa)-type NFs were transferred into HUVECs, the expression of miR-155 and pro-inflammatory and cancer-related factors increased, anti-inflammatory factors were reduced, cell proliferation increased, and cell migration was promoted. IL-1ß protein levels were decreased and SHIP1 expression was downregulated. When miR-155 and its target SHIP1 were both activated, the expression of both was unaltered, maintaining cell homeostasis. This points towards miR-155 overexpression can trigger inflammation, and that miR-155 and its target genes act as a molecular switch role in the development of inflammation.

Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.

RNA activation (RNAa) is an uncharacterized mechanism of transcriptional activation mediated by small RNAs, such as microRNAs (miRNAs). A critical issue in RNAa research is that it is difficult to distinguish between changes in gene expression caused indirectly by post-transcriptional regulation and direct induction of gene expression by RNAa. Therefore, in this study, we seek to identify a key factor involved in RNAa, using the induction of ZMYND10 by miR-34a as a system to evaluate RNAa. We identify the positive transcription elongation factors CDK9 and DDX21, which form a complex with nuclear AGO and TNRC6A, as important transcriptional activators of RNAa. In addition, we find that inhibition of DDX21 suppresses RNAa by miR-34a and other miRNAs without inhibiting post-transcriptional regulation. Our findings reveal a strong connection between RNAa and release of paused Pol II, facilitating RNAa research by making it possible to separately analyze post-transcriptional regulation and RNAa.

Let-7c increases BACE2 expression by RNAa and decreases Aß production.

MicroRNAs (miRNAs) are highly conserved, non-coding transcripts that regulate gene expression in various ways. Evidence suggests that miRNAs may be a contributory factor in neurodegeneration, including Alzheimer's disease (AD), Parkinson's disease (PD), and triplet repeat disorders. In order to further understand the potential roles of miRNAs in the pathogenesis of AD, we analyzed Down syndrome (DS), a special model of AD, by using a TaqMan microRNA array and found that miRNA let-7c was up-regulated in both DS and AD. ELISA assay showed that let-7c reduced the expression level of Aß significantly. Real-time quantitative-polymerase chain reaction (RT-qPCR) was conducted to reveal that the expression level of let-7c increased dramatically in DS cells, patients with DS and mice with AD compared with normal ones respectively. Additionally, western blotting illustrated that let-7c suppressed the expression of Aß by inducing BACE2 to cut C99 and increase the content of C83/80. BACE2 expression was inhibited by let-7c and luciferase reporter gene assay revealed that let-7c increased the activity of wild-type BACE2 promoter but not 3'UTR. Furthermore, promoter analysis of BACE2 confirmed that let-7c could bind to BACE2 in the sequence between -1368 and -1347. In addition, immunoblotting assay demonstrated that let-7c induced BACE2 expression by RNAa. To the best of our knowledge, our study revealed for the first time that let-7c up-regulated BACE2 expression and decreased Aß production.

Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells.

The loss of inner ear hair cells leads to irreversible acoustic injury in mammals, and regeneration of inner ear hair cells to restore hearing loss is challenging. ATOH1 is a key gene in the development and regeneration of hair cells. Small activating RNAs (saRNAs) can target a gene to specifically upregulate its expression. This study aimed to explore whether small activating RNAs could induce the differentiation of human adipose-derived mesenchymal stem cells into hair cell-like cells with a combination of growth factors in vitro and thus provide a new strategy for hair cell regeneration and the treatment of sensorineural hearing loss. Fifteen small activating RNAs targeting the human ATOH1 gene were designed and screened in 293 T and human adipose-derived mesenchymal stem cells, and 3 of these candidates were found to be capable of effectively and stably activating ATOH1 gene expression. The selected small activating RNAs were then transfected into hair cell progenitor cells, and hair cell markers were examined 10 days after transfection. After transfection of the selected small activating RNAs, the expression of the characteristic markers of inner ear hair cells, POU class 4 homeobox 3 (POU4F3) and myosin VIIA (MYO7A), was detected. Human adipose-derived mesenchymal stem cells have the potential to differentiate into human hair cell progenitor cells. In vitro, small activating RNAs were able to induce the differentiation of hair cell progenitor cells into hair cell-like cells. Therefore, RNA activation technology has the potential to provide a new strategy for the regeneration of hair cells.

RNA Activation-A Novel Approach to Therapeutically Upregulate Gene Transcription.

RNA activation (RNAa) is a mechanism whereby RNA oligos complementary to genomic sequences around the promoter region of genes increase the transcription output of their target gene. Small activating RNA (saRNA) mediate RNAa through interaction with protein co-factors to facilitate RNA polymerase II activity and nucleosome remodeling. As saRNA are small, versatile and safe, they represent a new class of therapeutics that can rescue the downregulation of critical genes in disease settings. This review highlights our current understanding of saRNA biology and describes various examples of how saRNA are successfully used to treat various oncological, neurological and monogenic diseases. MTL-CEBPA, a first-in-class compound that reverses CEBPA downregulation in oncogenic processes using CEBPA-51 saRNA has entered clinical trial for the treatment of hepatocellular carcinoma (HCC). Preclinical models demonstrate that MTL-CEBPA reverses the immunosuppressive effects of myeloid cells and allows for the synergistic enhancement of other anticancer drugs. Encouraging results led to the initiation of a clinical trial combining MTL-CEBPA with a PD-1 inhibitor for treatment of solid tumors.

Antitumor Activity of Small Activating RNAs Induced PAWR Gene Activation in Human Bladder Cancer Cells.

Small double-stranded RNAs (dsRNAs) have been proved to effectively up-regulate the expression of particular genes by targeting their promoters. These small dsRNAs were also termed small activating RNAs (saRNAs). We previously reported that several small double-stranded RNAs (dsRNAs) targeting the PRKC apoptosis WT1 regulator (PAWR) promoter can up-regulate PAWR gene expression effectively in human cancer cells. The present study was conducted to evaluate the antitumor potential of PAWR gene induction by these saRNAs in bladder cancer. Promisingly, we found that up-regulation of PAWR by saRNA inhibited the growth of bladder cancer cells by inducing cell apoptosis and cell cycle arrest which was related to inhibition of anti­apoptotic protein Bcl-2 and inactivation of the NF-κB and Akt pathways. The activation of the caspase cascade and the regulation of cell cycle related proteins also supported the efficacy of the treatment. Moreover, our study also showed that these saRNAs cooperated with cisplatin in the inhibition of bladder cancer cells. Overall, these data suggest that activation of PAWR by saRNA may have a therapeutic benefit for bladder cancer.

Targeted p21 activation by a new double stranded RNA suppresses human prostate cancer cells growth and metastasis.

We have previously demonstrated that miR-1236-3p and its sequence homology dsRNA, dsRNA-245 (which is completely complementary to the p21 promoter) had potential ability to upregulate p21 expression by targeting specific promoter sequence and inhibited bladder cancer (BCa). However, we still know little about the effect of miR-1236-3p on prostate cancer and which dsRNA has an inhibitory effect on prostate cancer (PCa)? Here, we confirmed that miR-1236-3p was decreased in PCa cells and tissues. MiR-1236-3p inhibited PCa cells growth and metastasis by activating p21. Furthermore, we demonstrated that dsP21-245 could inhibit PCa cells growth and metastasis by activating p21 expression. Microarray experiments displayed that miR-1236-3p could affect AKT signaling pathway. We demonstrated that miR-1236-3p significantly suppressed the AKT pathway by inhibiting TLR2 expression while activating p21 expression in PCa cells; this influence was independent of p21 activation. In summary, our results provided evidence that both endogenous and exogenous small RNAs might function to induce p21 expression by interacting with the same promoter region, therefore impeding PCa development. Additionally, our results indicated that miRNA activation could activate the expression of some unknown genes as well as cell signaling pathways. This indicated the need for the further study of clinical applications of RNA activation.

Upregulation of FHIT gene expression in endometrial carcinoma by RNA activation.

Endometrial carcinoma is the most common malignant tumors of the reproductive system, and fragile histidine triad (FHIT) plays an important role in multiple tumors. The purpose of this study was to investigate the expression of FHIT gene in endometrial carcinoma, and its effect on proliferation, invasion, and metastasis after upregulation. In vitro, the endometrial carcinoma cell lines were cultured. The FHIT-saRNA expression vector was constructed. The endometrial carcinoma cell line that upregulated the expression of FHIT was established, and whether the saRNA had a direct targeting regulation on the FHIT was verified. A difference of expression of FHIT in normal endometrial and endometrial carcinoma was detected. We detected the proliferation of endometrial carcinoma cell lines before and after activating FHIT. The endometrial carcinoma cell lines were compared with the corresponding transiently transfected cell lines in their capabilities of cell migration and invasion. The results showed that the expression of FHIT in endometrial carcinoma was significantly decreased or even deficient compared with normal endometrium. Upregulating the expression of FHIT is related to inhibiting the proliferation, invasion and metastasis of endometrial carcinoma. The possible mechanism is related to the regulation of cell cycle regulation, and plays a role in inhibiting tumor proliferation. The research on molecular mechanism in the development and progression of endometrial carcinoma has important theoretical significance for improving the diagnosis, treatment and prognosis of clinical tumors.

RNA activation in insects: The targeted activation of endogenous and exogenous genes.

RNA activation (RNAa) is a newly emerging area of research in which dsRNA targeting promoter regions can induce the expression of the target gene. Although still in its infancy, it is already having significant impacts in several research areas in particular as cancer therapeutics. So far, the scope of RNAa has been limited to mammals and Caenorhabditis elegans with no indication of its prevalence in insects. In this study, we aimed to demonstrate the presence of RNAa in the insect dengue vector Aedes aegypti. Furthermore, we looked to uncover some details surrounding the involvement of host factors in order to present this as a new technique for insect research. The outcomes of this study provide new opportunities to further research into arthropod-borne diseases and insect biology in the same way as RNA interference.

Developing small activating RNA as a therapeutic: current challenges and promises.

RNA activation (RNAa) allows specific gene upregulation mediated by a small activating RNA (saRNA). Harnessing this process would help in developing novel therapeutics for undruggable diseases. Since its discovery in mid 2000s, improvements of saRNA design, synthetic chemistry and understanding of the biology have matured the way to apply RNAa. Indeed, MiNA therapeutics Ltd has conducted the first RNAa clinical trial for advanced hepatocellular carcinoma patients with promising outcomes. However, to fully realize the RNAa potential better saRNA delivery strategies are needed to target other diseases. Currently, saRNA can be delivered in vivo by lipid nanoparticles, dendrimers, lipid and polymer hybrids and aptamers. Further developing these delivery technologies and novel application of RNAa will prove to be invaluable for new treatment development.

Small RNA-induced INTS6 gene up-regulation suppresses castration-resistant prostate cancer cells by regulating ß-catenin signaling.

Small RNAs play an important role in gene regulatory networks. The gene suppressive effect of small RNAs was previously the dominant focus of studies, but during the recent decade, small RNA-induced gene activation has been reported and has become a notable gene manipulation technique. In this study, a putative tumor suppressor, INTS6, was activated by introducing a promoter-targeted small RNA (dsRNA-915) into castration-resistant prostate cancer (CRPC) cells. Unique dynamics associated with the gene upregulation phenomenon was observed. Following gene activation, cell proliferation and motility were suppressed in vitro. Downregulation of Wnt/ß-catenin signaling was observed during the activation period, and the impairment of ß-catenin degradation reversed the tumor suppressor effects of INTS6. These results suggest the potential application of small activating RNAs in targeted gene therapy for CRPC.

Current Advances in Small Activating RNAs for Gene Therapy: Principles, Applications and Challenges.

Small activating RNAs (saRNAs) are small double-stranded RNAs that could mediate the target-specific gene expression by targeting selected sequences in gene promoters at both the transcriptional and epigenetic levels. This phenomenon of gene manipulation is known as RNA activation (RNAa), which opens up a new pathway for RNA-based gene therapeutics in contrast to RNA interference. Although the exact molecular mechanism of RNAa mediated by saRNAs still remains foggy, some studies have provided the possible ones to explain it. Furthermore, mounting evidence exhibit that saRNAs not only provide a new approach to study gene function and manipulate transcriptional activity, but also promise a great potential for clinical therapy against various diseases, especially cancer. Cancer-associated genes could be up-regulated by saRNAs to modulate cell cycle and proliferation, induce cell senescence and apoptosis, inhibit cancer cell invasion and migration, and reverse chemotherapy resistance. Herein, we summarize the known mechanisms of saRNAs on up-regulating specific gene expression and focus on the potential applications of saRNAs in gene therapy. In addition, some concerns about mechanisms and challenges for delivery of saRNAs are involved in this review. The precise mechanisms of saRNAs need to be further illustrated and some novel delivery systems for saRNAs are expected to be developed for clinical applications.

RNA activation technique and its applications in cancer research.

RNA activation (RNAa) is a mechanism of gene activation mediated by small activating RNAs. The activation of gene expression by small activating RNA has excellent targeting specificity and flexibility, with a persistent and strong effect. Studies have shown that the RNAa technique has broad prospects for application in the research on tumor pathogenesis and the treatment of tumors. This paper reviews the literature on RNAa with regard to the course of discovery, the mechanisms and characteristics of action, and the current status and prospects of application.

S-phase kinase-associated protein 2 impairs the inhibitory effects of miR-1236-3p on bladder tumors.

We have previously demonstrated that miR-1236-3p has the robust ability to up-regulate p21 expression by targeting the p21 promoter, thus inhibiting bladder cancer progression. Microarray experiments displayed that miR-1236-3p significantly increased the expression of the oncogenic F-box protein S-phase kinase-associate protein 2 (Skp2) while activating p21 expression in bladder cancer cells. Here, we confirmed that Skp2 was over-expressed following transfection with miR-1236-3p. Further, we demonstrated that miR-1236-3p and its sequence homology dsRNA, dsRNA-245 (which is completely complementary to the p21 promoter), both are able to potently induce p21 expression. We found that dsRNA-245 did not induce changes in Skp2 expression, while miR-1236-3p could increase Skp2 expression; this influence was independent of p21 activation. Moreover, transfection of miR-1236-3p or dsRNA-245 into bladder cancer cells significantly inhibited cell proliferation and clonegenesis and induced cell cycle arrest mainly by regulating p21 expression. However, the growth inhibition caused by dsRNA-245 was more effective than that caused by miR-1236-3p. This difference in effect size is mainly related to the miR-1236-3p-induced expression of Skp2. In summary, our results provided evidence that both endogenous and exogenous small RNAs might function to induce p21 expression by interacting with the same promoter region, therefore impeding bladder cancer cell growth. Additionally, our results indicated that microRNA activation can activate the expression of some tumor suppressor genes as well as some oncogenes. This indicated the need for the further study of clinical applications of RNA activation.

saRNAdb: Resource of Small Activating RNAs for Up-regulating the Gene Expression.

RNA activation (RNAa) is the process of enhancing selective gene expression at transcriptional level using double-stranded RNAs, targeting gene promoter. These RNA molecules are usually 21 nucleotides long and termed as small activating RNAs (saRNAs). They are involved in gene regulation, epigenetics, gain-of-function studies and have potential therapeutic applications for various diseases especially cancer. RNAa is opposite to RNA interference in functionality; however, both processes share some protein machinery. There are many RNA interference centered online resources but no one for saRNAs; therefore, we developed "saRNAdb" database (http://bioinfo.imtech.res.in/manojk/sarna/). It contains 2150 manually curated saRNA entries with detailed information about their nucleotide sequences, activities, corresponding target gene, promoter and other experimental data. Besides, saRNA-promoter binding location, predicted saRNA features, tools (off-target, map) and RNAa-related proteins with their interacting partners are provided. saRNAdb is expected to assist in RNA research especially for nucleic acid-based therapeutics development.

miRNA activation is an endogenous gene expression pathway.

Transfection of small non-coding RNAs (sncRNAs) molecules has become a routine technique widely used for silencing gene expression by triggering post-transcriptional and transcriptional RNA interference (RNAi) pathways. Moreover, in the past decade, small activating (saRNA) sequences targeting promoter regions were also reported, thereby a RNA-based gene activation (RNAa) mechanism has been proposed. In this regard, Turner and colleagues recently discovered an endogenous microRNA (miRNA) which binds its promoter in order to upregulate its own expression. Interestingly, several miRNA-induced RNA activation (miRNAa) phenomena have since then been identified. My objective here is to introduce the reader into the emergent miRNAa research field, as well as bring together important discoveries about this unexplored transcriptional activation pathway.