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Antisense RNAs are ubiquitous in human cells, yet their role is largely unexplored. Here we profiled antisense RNAs in the MDA-MB-231 breast cancer cell line and its highly lung metastatic derivative. We identified one antisense RNA that drives cancer progression by upregulating the redox enzyme NADPH quinone dehydrogenase 1 (NQO1), and named it NQO1-AS. Knockdown of either NQO1 or NQO1-AS reduced lung colonization in a mouse model, and investigation into the role of NQO1 indicated that it is broadly protective against oxidative damage and ferroptosis. Breast cancer cells in the lung are dependent on this pathway, and this dependence can be exploited therapeutically by inducing ferroptosis while inhibiting NQO1. Together, our findings establish a role for NQO1-AS in the progression of breast cancer by regulating its sense mRNA post-transcriptionally. Because breast cancer predominantly affects females, the disease models used in this study are of female origin and the results are primarily applicable to females.

RBMS1 putative regulons, RBMS1 target list, RBMS1 80-gene signature list

Supplementary Figure 4

Supplementary Methods

The description of benchmarking PRADA

The schematics of the data flow used for analysis

Identifying master regulators that drive pathologic gene expression is a key challenge in precision oncology. Here, we have developed an analytic framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a posttranscriptional regulator of RNA stability by directly binding its target mRNAs. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients.Significance:By applying a new analytic approach to transcriptomic data from clinical samples and models of colon cancer progression, we have identified RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.See related commentary by Carter, p. 1261.This article is highlighted in the In This Issue feature, p. 1241

RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3’UTR of the RORC transcript.In vivoDMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identifytransfactors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

GI toxicity is a common dose-limiting adverse effect of platin chemotherapy treatment. Up to 50% of cancer survivors continue to experience symptoms of chronic constipation or diarrhea induced by their chemotherapy for many years after their treatment. This drug toxicity is largely attributed to damage to enteric neurons that innervate the GI tract and control GI motility. The mechanisms responsible for platin-induced enteric neurotoxicity and potential preventative strategies have remained unknown. Here, we use human pluripotent stem cell derived enteric neurons to establish a new model system capable of uncovering the mechanism of platin-induced enteric neuropathy. Utilizing this scalable system, we performed a high throughput screen and identified drug candidates and pathways involved in the disease. Our analyses revealed that excitotoxicity through muscarinic cholinergic signaling is a key driver of platin-induced enteric neuropathy. Using single nuclei transcriptomics and functional assays, we discovered that this disease mechanism leads to increased susceptibility of specific neuronal subtypes, including inhibitory nitrergic neurons, to platins. Histological assessment of the enteric nervous system in platin-treated patients confirmed the selective loss of nitrergic neurons. Finally, we demonstrated that pharmacological and genetic inhibition of muscarinic cholinergic signaling is sufficient to rescue enteric neurons from platin excitotoxicityin vitroand can prevent platin-induced constipation and degeneration of nitrergic neurons in mice. These studies define the mechanisms of platin-induced enteric neuropathy and serve as a framework for uncovering cell type-specific manifestations of cellular stress underlying numerous intractable peripheral neuropathies.

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

Antisense RNAs are ubiquitous in human cells, yet the role that they play in healthy and diseased states remains largely unexplored. Here, we developed a computational framework to catalog and profile antisense RNAs and applied it to poorly and highly metastatic breast cancer cell lines. We identified one antisense RNA that plays a functional role in driving breast cancer progression by upregulating the redox enzyme NQO1, and hence named NQO1-antisense RNA or NQO1-AS. This upregulation occurs via a stabilizing interaction between NQO1-AS and its complementary region in the 3’UTR of NQO1 mRNA. By increasing expression of NQO1 protein, breast cancer cells are able to tolerate higher levels of oxidative stress, enabling them to colonize the lung. During this process the cancer cells become dependent on NQO1 to protect them from ferroptosis. We have shown that this dependence can be exploited therapeutically in xenograft models of metastasis. Together, our findings establish a previously unknown role for NQO1-AS in the progression of breast cancer by serving as a post-transcriptional regulator of RNA processing and decay for its sense mRNA.

Introduction: Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. Results: Using the multi-region TRACERx lung cancer study, we uncovered that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR driven lung cancer and identified subclonal enrichment of APOBEC mutational signatures. To model APOBEC mutagenesis in lung cancer, several novel EGFR mutant mouse models containing a human APOBEC3B transgene were generated. Using these models, it was uncovered that APOBEC3B expression is detrimental at tumor initiation when expressed continuously in a p53 wildtype background. This detrimental effect is likely due to elevated chromosomal instability, which was observed to increase significantly with APOBEC3B expression in an EGFR mutant TP53 deficient mouse model. Induction of subclonal expression of APOBEC3B in an EGFR mutant mouse model with tyrosine kinase inhibitor (TKI) therapy resulted in a significant increase in resistant tumor development. Significant downregulation of the base excision repair gene uracil-DNA glycosylase (UNG) was also observed in APOBEC3B expressing mice, which paralleled findings in patient tumors and cell lines treated with TKI therapy. Finally, a mouse mutational signature was identified in APOBEC3B expressing cell lines, reinforcing the idea that APOBEC driven mutagenesis contributes to TKI resistance. Conclusion: This study demonstrates a unique principle by which targeted therapy induces changes within tumors ideal for APOBEC driven tumor evolution, fueling therapy resistance. Citation Format: Manasi Mayekar, Deborah Caswell, Natalie Vokes, Emily K. Law, Wei Wu, William Hill, Eva Gronroos, Andrew Rowan, Maise Al Bakir, Clare Weeden, Caroline E. McCoach, Collin M. Blakely, Nuri Alpay Temiz, Ai Nagano, Daniel L. Kerr, Julia K. Rotow, Oriol Pich, Franziska Haderk, Michelle Dietzen, Carlos Martinez Ruiz, Bruna Almeida, Lauren Cech, Beatrice Gini, Joanna Przewrocka, Chris Moore, Miguel Murillo, Bjorn Bakker, Brandon Rule, Cameron Durfee, Shigeki Nanj, Lisa Tan, Lindsay K. Larson, Prokopios P. Argyris, William L. Brown, Johnny Yu, Carlos Gomez, Philippe Gui, Rachel I. Vogel, Elizabeth A. Yu, Nicholas J. Thomas, Subramanian Venkatesan, Sebastijan Hobor, Su Kit Chew, Nicholas McGranahan, Nnennaya Kanu, Eliezer M. Van Allen, Julian Downward, Reuben S. Harris, Trever Bivona, Charles Swanton. Targeted cancer therapy induces APOBEC fueling the evolution of drug resistance [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 2197.

Introductory paragraphThe clinical success of targeted cancer therapy is limited by drug resistance that renders cancers lethal in patients1-4. Human tumours can evolve therapy resistance by acquiringde novogenetic alterations and increased heterogeneity via mechanisms that remain incompletely understood1. Here, through parallel analysis of human clinical samples, tumour xenograft and cell line models and murine model systems, we uncover an unanticipated mechanism of therapy-induced adaptation that fuels the evolution of drug resistance. Targeted therapy directed against EGFR and ALK oncoproteins in lung cancer induced adaptations favoring apolipoprotein B mRNA-editing enzyme, catalytic polypeptide (APOBEC)-mediated genome mutagenesis. In human oncogenicEGFR-driven andALK-driven lung cancers and preclinical models, EGFR or ALK inhibitor treatment induced the expression and DNA mutagenic activity ofAPOBEC3Bvia therapy-mediated activation of NF-κB signaling. Moreover, targeted therapy also mediated downregulation of certain DNA repair enzymes such as UNG2, which normally counteracts APOBEC-catalyzed DNA deamination events. In mutantEGFR-driven lung cancer mouse models, APOBEC3B was detrimental to tumour initiation and yet advantageous to tumour progression during EGFR targeted therapy, consistent with TRACERx data demonstrating subclonal enrichment of APOBEC-mediated mutagenesis. This study reveals how cancers adapt and drive genetic diversity in response to targeted therapy and identifies APOBEC deaminases as future targets for eliciting more durable clinical benefit to targeted cancer therapy.

Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.

Broad dysregulation of gene expression control is a hallmark of cancer progression. Identifying the underlying master regulators that drive pathological gene expression is a key challenge in precision oncology. Here, we have developed a network analytical framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a post-transcriptional regulator of RNA stability by directly binding and stabilizing ~80 target mRNAs. Measurements in more than 180 clinical samples as well as survival analyses in publicly available datasets, have shown that RBMS1 silencing and the subsequent downregulation of its targets are strongly associated with disease progression and poor survival in colon cancer patients. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients.SignificanceBy applying a new analytical approach to transcriptomic data from clinical samples and models of colon cancer progression, we have uncovered RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, Par4-deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial mt-Nd2, and Snora75, a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

Here we performed a systematic search to identify breast-cancer-specific small noncoding RNAs, which we have collectively termed orphan noncoding RNAs (oncRNAs). We subsequently discovered that one of these oncRNAs, which originates from the 3' end of TERC, acts as a regulator of gene expression and is a robust promoter of breast cancer metastasis. This oncRNA, which we have named T3p, exerts its prometastatic effects by acting as an inhibitor of RISC complex activity and increasing the expression of the prometastatic genes NUPR1 and PANX2. Furthermore, we have shown that oncRNAs are present in cancer-cell-derived extracellular vesicles, raising the possibility that these circulating oncRNAs may also have a role in non-cell autonomous disease pathogenesis. Additionally, these circulating oncRNAs present a novel avenue for cancer fingerprinting using liquid biopsies.

Objective— Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. Approach and Results— DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX −/− ). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX −/− mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gα s -linked G-protein–coupled receptor pathway in human platelets. Conclusions— This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit–linked G-protein–coupled receptor–dependent manner.

3523 Background: CRC progression accompanies dysregulations in pathways of gene expression control. Regulatory pathways that govern RNA decay have emerged as key mechanisms coopted by cancer cells. Here, we have described a novel regulatory program that acts as a suppressor of metastasis in CRC. Methods: We have developed a computational approach called PRADA that identifies master regulators of aberrant mRNA stability. By applying this tool to a compendium of gene expression data collected from patient samples and colon cancer cell lines, we have identified a novel regulatory program involved in CRC metastasis. We have used xenograft models and genomic technologies to functionally dissect this pathway. We have also performed multivariate analysis in public datasets, as well as qPCR-based measurements in tumor samples to establish its clinical relevance. Results: PRADA identified the RNA-binding protein RBMS1 as a key factor in CRC metastasis. RBMS1, which is silenced in highly metastatic cells, binds and stabilizes a large regulon of mRNAs. Silencing RBMS1 in established lines resulted in increased liver colonization in xenograft models and its over-expression reduced metastasis. We also identified the set of genes that are directly bound and regulated by RBMS1 and function downstream. Loss of RBMS1 and its regulon provide a signature predictive of RFS in localized CRC (n = 574, HR = 0.48, p = 0.038) even when controlled for known prognostic factors in a multivariate analysis (Table). We also observed a 4-fold reduction (P

Due to an increasing diversity of bacterial pathogens known to be transmitted by hard ticks (Acari: Ixodidae) in North America, a comprehensive assay is needed to detect and differentiate among these numerous tick-borne pathogens. We describe an expanded protocol using a combination of multiplex polymerase chain reaction and reverse line blot hybridization to detect a greater diversity of infectious agents than were previously detectable. Ten novel oligonucleotide probes, either individually or in concert, enabled or enhanced identification of six Borrelia species, three Rickettsia species, and one Ehrlichia species. Simultaneous detection of these numerous tick-borne pathogens can advance surveillance efforts and improve accuracy of detection and, thus, reporting.

Unlike its conventional counterpart, a new vector basis function based meshless method that preserves the divergence property of electromagnetic fields is proposed for solving transient electromagnetic problems. Its divergence properties are investigated through numerical experiment; it is found that artificial charges generated by the proposed method are not present while they are in the conventional meshless method in source free regions.

The Third Asia Pacific Conference on Evidence-Based Medicine was successfully concluded on the 28th November 2004 at the Chinese University of Hong Kong. Professor Han Qi De, Vice Chairman of the Standing Committee of the People's National Congress of China, Professor Huang Jie Fu, Vice Minister of the Ministry of Health of China, Dr Chow York, the Secretary for Health, Welfare and Food of the Hong Kong Special Administrative Region of China, and Professor Lawrence Lau, Vice Chancellor of the Chinese University of Hong Kong expressed congratulations on the opening of the Conference. Dr Muir Gray CBE, Founding Director of Oxford Institute of Health Sciences and Professor Paul Glasziou, Director of the Oxford Centre for EBM delivered the keynote speeches. Over 300 delegates from 28 countries and regions worldwide participated in the conference. One hundred and seven delegates gave presentations. Over 250 delegates attended the two training programmes on EBM and systematic reviews. The conference received enthusiastic participation and contribution from all the delegates during the entire course of the programmes. The conference concluded that knowledge will be the major enemy of disease in the 21st century and all clinical and health care decisions should be based on the best evidence available. Evidence from scientific research in human subjects constitutes the knowledge that can be used to guide clinical practice directly and inform health care decisions. In the past few decades, science has advanced greatly, but there was collective ignorance about the scientific evidence on the effects of health care. Today, there is still a gap between current scientific knowledge and its applications: between what we know and what is actually being done. Good knowledge of health care is like clean drinking water to a person. Generating, summarizing and disseminating evidence from scientific research must be organized collectively for utilization in the way we supply drinking water. Local commitments as well as international efforts are essential for successfully translating knowledge into actions to continually improve the quality and efficiency of health care. During the conference, representatives from 12 countries and regions in the Asia Pacific also discussed and formed the ‘Asia Pacific EBM Network’ to strengthen further collaborations and facilitate the development of evidence-based medicine in this region. Professor Jin Ling Tang, Director of the Chinese Cochrane Centre Hong Kong Branch was elected the chairman of the network. In addition, the Chinese book Evidence-Based Decision Making in Healthcare by Dr Muir Gray CBE and Professor Jin Ling Tang and published by Peking University Medical Press was for the first time introduced to readers during the conference.