Biology subjects

Shen, S.

Publications and source records attributed to Shen, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

CD46 targeted 212Pb alpha particle radioimmunotherapy for prostate cancer treatment

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 20 Ci 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (10 Ci 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment. SignificanceThis study reports a novel CD46 targeted 212Pb alpha particle radioimmunotherapy, 212Pb-TCMC-YS5, that is well tolerated and shows potent anti-tumor activity (tumor growth inhibition and increase of animal survival) in vivo in three prostate cancer small animal models, i.e., a subcutaneous and an intraprostate orthotopic mCRPC cell line-derived xenograft models, and a prostate cancer patient-derived xenograft model. Given that YS5 is a clinical stage human antibody, this YS5-based 212Pb alpha particle therapy has potential of translation to the clinic for treatment of mCRPC patients.

bioengineering↗

bioRxiv2022

Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. TRIAGE-Cluster integrates patterns of repressive chromatin deposited across diverse cell types with weighted density estimation to determine cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method that evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

bioinformatics↗

bioRxiv2022

An integrated cell barcoding and computational analysis pipeline for scalable analysis of differentiation at single-cell resolution

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.

genomics↗

bioRxiv2022

Highly synchronized cortical circuit dynamics mediate spontaneous pain

Cortical neural dynamics mediate information processing for the cerebral cortex, implicated in fundamental biological processes, such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes cortical network into an aberrant state, and if so, whether it can be brought back to a normal operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with robust spontaneous pain-like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos-expressing neurons, and selective activation of GABAergic interneurons, significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More importantly, restoring a normal range of neural dynamics, through attenuating pain-induced S1 synchronization, alleviated pain-like behavior. These results suggest spontaneous pain pushes S1 regional network into a synchronized state, whereas reversal of this synchronization alleviates pain.

neuroscience↗

bioRxiv2022

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

molecular biology↗

bioRxiv2022

Molecular formula discovery via bottom-up MS/MS interrogation

A substantial fraction of metabolic features remains undetermined in mass spectrometry (MS)-based metabolomics. Here we present bottom-up tandem MS (MS/MS) interrogation to illuminate the unidentified features via accurate molecular formula annotation. Our approach prioritizes MS/MS-explainable formula candidates, implements machine-learned ranking, and offers false discovery rate estimation. Compared to the existing MS1-initiated formula annotation, our approach shrinks the formula candidate space by 42.8% on average. The superior annotation accuracy of our bottom-up interrogation was demonstrated on reference MS/MS libraries and real metabolomics datasets. Applied on 155,321 annotated recurrent unidentified spectra (ARUS), our approach confidently annotated >5,000 novel molecular formulae unarchived in chemical databases. Beyond the level of individual metabolic features, we combined bottom-up MS/MS interrogation with global peak annotation. This approach reveals peak interrelationships, allowing the systematic annotation of 37 fatty acid amide molecules in human fecal data, among other applications. All bioinformatics pipelines are available in a standalone software, BUDDY (https://github.com/HuanLab/BUDDY/).

bioinformatics↗

bioRxiv2022

Arginine methylation of Puf4 drives diverse protein functions

The evolutionarily conserved Pumilio domain-containing RNA binding proteins (RBPs) are involved in many steps of post-transcriptional gene regulation, including RNA stability, polyadenylation, deadenylation, and translation. RBPs are post-translationally modified by methylation of arginine/glycine-rich domains, though the consequences of these modifications are not well known. We determined the arginine methylation and phosphorylation landscape of the Pumilio domain-containing RBP Puf4 from the basidiomycete fungus Cryptococcus neoformans. We found that methyl-deficient Puf4 mutants do not complement critical PUF4 deletion phenotypes, such as resistances to endoplasmic reticulum stress and antifungals, and cell wall remodeling. Methyl-deficient mutants also exhibit unique RNA and protein interactions. Lastly, we identified intra-protein cross talk between post-translationally modified methylated and phosphorylated residues. Overall, we show that post-translational modifications, particularly arginine methylation, of Puf4 regulate the functions of this RBP.

molecular biology↗

bioRxiv2022

Distinct Single-cell Immune Ecosystems Distinguish True and De Novo HBV-related Hepatocellular Carcinoma Recurrences

Revealing differential tumor immune microenvironment (TIME) characteristics between true versus de novo hepatocellular carcinoma (HCC) recurrence could help optimal development and use of immunotherapies. Here, we studied the TIME of recurrent HBV-related HCCs by 5and VDJ single-cell and bulk RNA-sequencing, flow cytometry, and multiplexed immunofluorescence. Analyses of mutational profiles, evolutionary trajectories, and clonal architecture using whole-exome sequencing identified de novo versus true recurrences, some of which occurred before clinical diagnosis. The TIME of truly recurrent HCCs was characterized by an increased abundance in KLRB1+CD8+ T cells with memory phenotype and low cytotoxicity. In contrast, we found an enrichment in cytotoxic and exhausted CD8+ T cells in the TIME of de novo recurrent HCCs. Transcriptomic and interaction analyses showed an upregulated GDF15 expression level on HCC cells in proximity to dendritic cells, which may have dampened antigen presentation and inhibited anti-tumor immunity in the TIME of truly recurrent lesions. In contrast, we found that myeloid cells crosstalk with T cells mediated T cell exhaustion and immunosuppression in the TIME of de novo recurrent HCC. In conclusion, our results support genomic diagnosis and immune profiling for guiding immunotherapy implementation based on the type of HCC recurrence and TIME. HighlightsO_LITruly recurrent lesions are seeded before primary tumor diagnosis, and that de novo cancer can occur earlier than the clinically used 2-year limit. C_LIO_LIScRNA-seq unravels distinct immune ecosystems in true versus de novo HCC recurrences, highlighting the need for different immunotherapy strategies for two types of HCC recurrence. C_LIO_LICD8+ T cells in de novo recurrence displayed cytotoxic and exhausted phenotypes while those in truly recurrent lesions showed a memory phenotype with weak cytotoxicity. C_LIO_LIHCC cells expressing the inhibitory molecule GDF15 were in the proximity of DCs only in truly recurrent lesions. C_LIO_LIHigh GDF15 expression level was associated with truly recurrent HCC and worse prognosis. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/494526v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1b76bf0org.highwire.dtl.DTLVardef@10f889aorg.highwire.dtl.DTLVardef@d16b52org.highwire.dtl.DTLVardef@dceb7a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

HOPX governs a molecular and physiological switch between cardiomyocyte progenitor and maturation gene programs

This study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

developmental biology↗

bioRxiv2022

Molecular basis of broad neutralization against SARS-CoV-2 variants including Omicron by a human antibody

Omicron, a newly emerging SARS-CoV-2 variant, carried a large number of mutations in the spike protein leading to an unprecedented evasion from many neutralizing antibodies (nAbs). Here, we performed a head-to-head comparison of Omicron with other existing highly evasive variants in terms of their reduced sensitivities to antibodies, and found that Omicron variant is significantly more evasive than Beta and Mu variants. Of note, some key mutations occur in the conserved epitopes identified previously, especially in the binding sites of Class 4 nAbs, contributing to the increased Ab evasion. We also reported a broadly nAb (bnAb), VacW-209, which effectively neutralized all tested SARS-CoV-2 variants and even SARS-CoV. Finally, we determined six cryo-electron microscopy structures of VacW-209 complexed with the spike ectodomains of wild-type, Delta, Mu, C.1.2, Omicron, and SARS-CoV, and revealed the molecular basis of the broadly neutralizing activities of VacW-209 against SARS-CoV-2 variants. Overall, Omicron has once again raised the alarm over virus variation with significantly compromised neutralization. BnAbs targeting more conserved epitopes among variants will continue to play a key role in pandemic control and prevention. One sentence summaryStructural and functional analyses reveal that a human antibody named VacW-209 confers broad neutralization against SARS-CoV-2 variants including Omicron by recognizing a highly conserved epitope.

immunology↗

bioRxiv2022

The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5{Delta} mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5{Delta} mutant showed that Rmt5 is critical for the homeostasis of eIF2 and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5{Delta} clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5{Delta} phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA-binding proteins. SignificanceThe fungal pathogen Cryptococcus neoformans is a huge threat for people living with immune deficits, especially HIV/AIDS. Its virulence potential is dependent on virulence factors, stress adaptation, and thermotolerance. Post-transcriptional gene regulation is important for these pathogenic processes, but the mechanisms that govern post-transcriptional regulator function are unexplored. Protein arginine methylation is a major modification of post-transcriptional regulators that has not been investigated in pathogenic fungi. Here we investigated the role of arginine methylation by arginine methyltransferases on the biology and virulence of C. neoformans. Phenotypic characterization of deletion mutants revealed pleiotropic functions for RMTs in this pathogen. Further investigation of the Rmt5 interactome using proximity-dependent biotinylation revealed interactions with RNA binding proteins and translation factors, thereby impacting virulence-associated processes.

microbiology↗

bioRxiv2021

scGAD: single-cell gene associating domain scores for exploratory analysis of scHi-C data

SummaryQuantitative tools are needed to leverage the unprecedented resolution of single-cell high-throughput chromatin conformation (scHi-C) data and to integrate it with other single-cell data modalities. We present single-cell gene associating domain (scGAD) scores as a dimension reduction and exploratory analysis tool for scHi-C data. scGAD enables summarization at the gene level while accounting for inherent gene-level genomic biases. Low-dimensional projections with scGAD capture clustering of cells based on their 3D structures. scGAD enables identifying genes with significant chromatin interactions within and between cell types. We further show that scGAD facilitates the integration of scHi-C data with other single-cell data modalities by enabling its projection onto reference low-dimensional embeddings. AvailabilityscGAD is part of the BandNorm R package at https://sshen82.github.io/BandNorm/articles/scGAD-tutorial.html. Contactkeles@stat.wisc.edu, yzheng23@fredhutch.org Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2021

Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection

BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.

molecular biology↗

bioRxiv2021

Reporting amyloid beta levels via bioluminescence imaging with amyloid reservoirs inAlzheimer's disease models

Bioluminescence imaging has changed daily practice in preclinical research of cancers and other diseases in the last decades; however, it has been rarely applied in preclinical research of Alzheimers disease (AD). In this report, we demonstrated that bioluminescence imaging could be used to report the levels of amyloid beta (A{beta}) species in vivo. We hypothesized that AkaLumine, a newly discovered substrate for luciferase, could bind to A{beta} aggregates and plaques. We further speculated that the A{beta} species have the reservoir capacity to sequester and release AkaLumine to control the bioluminescence intensity, which could be used to report the levels of A{beta}s. Our hypotheses have been validated via in vitro solution tests, mimic studies with brain tissues and mice, two-photon imaging with AD mice, and in vivo bioluminescence imaging using transgenic AD mice that were virally transduced with aka Luciferase (AkaLuc), a new luciferase that generates bioluminescence in the near infrared window. As expected, compared to the control group, we observed that the A{beta} group showed lower bioluminescence intensity due to AkaLumine sequestering at early time points, while higher intensity due to AkaLumine releasing at later time points. Lastly, we demonstrated that this method could be used to monitor AD progression and therapeutic effectiveness of avagacestat, a well-studied gamma-secretase inhibitor. Importantly, a good correlation (R2 = 0.81) was established between in vivo bioluminescence signals and A{beta} burdens of the tested AD mice. We believe that our approach can be easily implemented into daily imaging experiments and has tremendous potential to change daily practice of preclinical AD research.

neuroscience↗

bioRxiv2021

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in mRNA stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (P. yoelii) and human (P. falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knock-out of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in rodent and human malaria parasites. Restoration of the nsun2 gene in the knock-out parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites. SignificanceModifications of RNA including methylations of cytosine (m5C) and adenosine (m6A) have important roles in RNA metabolism, cellular responses to stress, and biological processes of differentiation and development. Here, we report on the profiles of m5C mRNA modifications in malaria parasites that infect rodents (Plasmodium yoelii) and humans (Plasmodium falciparum). These parasites have genes that encode homologs of human and plant NSUN2 methyltransferases (m5C "writers"). We show that one of these homologs, termed PyNSUN2, stabilizes mRNA transcripts in P. yoelii and mediates m5C-associated development of the parasite sexual stages (gametocytes). Further research on m5C and other epitranscriptomic modifications may yield new insights into molecular pathways of gametocyte development and mosquito infectivity that can be exploited to interrupt malaria transmission.

microbiology↗

bioRxiv2021

Comprehensive Analysis of Spatial Architecture in Primary Liver Cancer

Heterogeneity is the major challenge for cancer prevention and therapy. Here, we firstly constructed high-resolution spatial transcriptomes of primary liver cancers (PLCs) containing 84,823 spots within 21 tissues from 7 patients. The sequential comparison of spatial tumor microenvironment (TME) characteristics from non-tumor to leading-edge to tumor regions revealed that the tumor capsule potentially affects intratumor spatial cluster continuity, transcriptome diversity and immune cell infiltration. Locally, we found that the bidirectional ligand-receptor interactions at the 100 m wide cluster-cluster boundary contribute to maintaining intratumor architecture. Our study provides novel insights for diverse tumor ecosystem of PLCs and has potential benefits for cancer intervention.

cancer biology↗

bioRxiv2021

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

biochemistry↗

bioRxiv2021

Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression ofPAK3 via miRNA regulation

Over 85% of lung cancer patients harbor overt or subclinical metastases at diagnosis, and therefore most patients die of progressive metastatic disease despite aggressive local and systemic therapies. Somatic mutations in the Smad4 gene have been found in non-small-cell lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generated a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and/or Smad4 fl/fl LOF mutations. The Smad4fl/fl; p53 fl/fl; KrasG12D (SPK) mutant mice manifested a much higher incidence of tumor metastases than the p53 fl/fl; KrasG12D (PK) mice. Molecularly, PAK3 was identified as a novel downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice was achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components suggests clinical use of Smad4 LOF as a potential marker for prognosis in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

cancer biology↗