Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Development of a Small Molecule Downmodulator for the Transcription Factor Brachyury.

Brachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed "undruggable". Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA-approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure-based drug design approach, aided by mass-spectrometry and X-ray crystallography, to develop DHC-156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase-inhibition from this novel scaffold while demonstrating that DHC-156 induces the post-translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Integrating Clinical Cancer and PTM Proteomics Data Identifies a Mechanism of ACK1 Kinase Activation.

Beyond the most common oncogenes activated by mutation (mut-drivers), there likely exists a variety of low-frequency mut-drivers, each of which is a possible frontier for targeted therapy. To identify new and understudied mut-drivers, we developed a machine learning (ML) model that integrates curated clinical cancer data and posttranslational modification (PTM) proteomics databases. We applied the approach to 62,746 patient cancers spanning 84 cancer types and predicted 3,964 oncogenic mutations across 1,148 genes, many of which disrupt PTMs of known and unknown function. The list of putative mut-drivers includes established drivers and others with poorly understood roles in cancer. This ML model is available as a web application. As a case study, we focused the approach on nonreceptor tyrosine kinases (NRTK) and found a recurrent mutation in activated CDC42 kinase-1 (ACK1) that disrupts the Mig6 homology region (MHR) and ubiquitin-association (UBA) domains on the ACK1 C-terminus. By studying these domains in cultured cells, we found that disruption of the MHR domain helps activate the kinase while disruption of the UBA increases kinase stability by blocking its lysosomal degradation. This ACK1 mutation is analogous to lymphoma-associated mutations in its sister kinase, TNK1, which also disrupt a C-terminal inhibitory motif and UBA domain. This study establishes a mut-driver discovery tool for the research community and identifies a mechanism of ACK1 hyperactivation shared among ACK family kinases. IMPLICATIONS: This research identifies a potentially targetable activating mutation in ACK1 and other possible oncogenic mutations, including PTM-disrupting mutations, for further study.

Methylated ctDNA Quantification: Noninvasive Approach to Monitoring Hepatocellular Carcinoma Burden.

BACKGROUND: Noninvasive, precision monitoring of hepatocellular carcinoma (HCC) treatment efficacy would greatly facilitate personalized therapy and improve patient outcomes. We hypothesize that quantifying methylated circulating tumor DNA (ctDNA) can be used to effectively monitor HCC burden without the need for biopsy. STUDY DESIGN: Blood samples were collected from 25 patients, 21 with HCC and 4 with benign liver masses, at various timepoints throughout the course of treatment at a high-volume academic medical center. Quantification of methylated ctDNA molecules assessed CpG sites on more than 550 preselected cancer-specific amplicons. The tumor methylation score (TMS) was calculated by measuring the difference between the amount of methylation in the plasma and buffy coat with a normal cutoff value of 120 or less. RESULTS: Among 10 patients with surgical HCC (5 surgical resections and 5 liver transplants), TMS revealed a statistically significant, rapid postoperative decline in 9. One patient who had a persistently elevated TMS on postoperative day 1 was subsequently found to have had metastatic disease. Patients in the negative control cohort all had normal-range pre- and postoperative TMS. Preoperative TMS correlated moderately with tumor burden on pathology (Spearman r = 0.54) of surgical specimens. From 11 subjects undergoing systemic therapy or Y90 radioembolization, analysis of 16 time periods demonstrated that the change in TMS (ΔTMS) was better associated with tumor progression than the change in Δalpha-fetoprotein (area under the curve 0.800 and 0.783, respectively). A composite score combining ΔTMS and Δalpha-fetoprotein further improved performance for detecting tumor progression with an area under the curve of 0.892. CONCLUSIONS: These findings indicate that ctDNA methylation scores can effectively evaluate changes in tumor burden without the need for tumor biopsy.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Clinical impact of myosteatosis measured by magnetic resonance imaging on long-term outcomes of hepatocellular carcinoma after radical hepatectomy.

AIMS: A variety of factors have been reported to affect long-term outcomes after radical resection of hepatocellular carcinoma (HCC). However, the indicators remain controversial. The purpose of this study was to evaluate the relationship between myosteatosis of the multifidus muscle and long-term outcomes after radical surgery for HCC. METHODS: We retrospectively analyzed clinicopathological data for 187 patients with HCC who underwent radical surgery at Tokushima University between January 2009 and December 2020 and measured the density of fat in the multifidus muscle at L3 on their preoperative magnetic resonance images (MRI). Associations of myosteatosis and clinicopathological factors with long-term outcomes were evaluated. RESULTS: The patients were divided into a myosteatosis-negative group (n = 122) and a myosteatosis-positive group (n = 65). The cancer-specific survival rate after hepatectomy was significantly worse in the myosteatosis-positive group than in the myosteatosis-negative group (p = 0.03). Univariate analysis identified multiple tumors, stage III/IV disease, an alfa-fetoprotein level ≥ 10 ng/ml, PIVKA-II ≥ 400 AU/ml, vp(+) status, and myosteatosis to be prognostic factors for cancer-specific survival. Multivariate analysis revealed multiple tumors, an alfa-fetoprotein level ≥ 10 ng/ml, and myosteatosis to be independent prognostic factors. CONCLUSIONS: Myosteatosis measured by MRI is a simple and useful predictor of the long-term outcome after radical surgery for HCC.

Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1 ubiquitin-associated (UBA) domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. No experimentally determined molecular structure of this unusual UBA domain is available. We fused the UBA domain to the 1TEL variant of the translocation ETS leukemia protein sterile alpha motif (TELSAM) crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and crystallize at protein concentrations as low as 0.2 mg/mL. Our studies support a mechanism of 1TEL fusion crystallization and show that 1TEL fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Eomes restricts Brachyury functions at the onset of mouse gastrulation.

Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs) Eomesodermin (Eomes) and Brachyury sharing partially redundant functions. Gross differences in mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene regulation by Eomes and Brachyury remain poorly understood. Here, we combine mouse embryonic and stem-cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale, binding of both TFs overlaps at promoters of target genes but shows specificity for distal enhancer regions that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs the specification of anterior mesoderm (AM) and DE by Eomes and caudal mesoderm by Brachyury. Remarkably, EOMES antagonizes BRACHYURY gene regulatory functions in coexpressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives.

Transcriptomic evidence for Brachyury expression in the caudal tip region of adult Ptychodera flava (Hemichordata).

Most metazoans have a single copy of the T-box transcription factor gene Brachyury. This gene is expressed in cells of the blastopore of late blastulae and the archenteron invagination region of gastrulae. It appears to be crucial for gastrulation and mesoderm differentiation of embryos. Although this expression pattern is shared by most deuterostomes, Brachyury expression has not been reported in adult stages. Here we show that Brachyury of an indirect developer, the hemichordate acorn worm Ptychodera flava, is expressed not only in embryonic cells, but also in cells of the caudal tip (anus) region of adults. This spatially restricted expression, shown by whole-mount in situ hybridization, was confirmed by Iso-Seq RNA sequencing and single-cell RNA-seq (scRNA-seq) analysis. Iso-Seq analysis showed that gene expression occurs only in the caudal region of adults, but not in anterior regions, including the stomochord. scRNA-seq analysis showed a cluster that contained Brachyury-expressing cells comprising epidermis- and mesoderm-related cells, but which is unlikely to be associated with the nervous system or muscle. Although further investigation is required to examine the roles of Brachyury in adults, this study provides important clues for extending studies on Brachyury expression involved in development of the most posterior region of deuterostomes.

The evolutionary history of Brachyury genes in Hydrozoa involves duplications, divergence, and neofunctionalization.

Brachyury, a member of T-box gene family, is widely known for its major role in mesoderm specification in bilaterians. It is also present in non-bilaterian metazoans, such as cnidarians, where it acts as a component of an axial patterning system. In this study, we present a phylogenetic analysis of Brachyury genes within phylum Cnidaria, investigate differential expression and address a functional framework of Brachyury paralogs in hydrozoan Dynamena pumila. Our analysis indicates two duplication events of Brachyury within the cnidarian lineage. The first duplication likely appeared in the medusozoan ancestor, resulting in two copies in medusozoans, while the second duplication arose in the hydrozoan ancestor, resulting in three copies in hydrozoans. Brachyury1 and 2 display a conservative expression pattern marking the oral pole of the body axis in D. pumila. On the contrary, Brachyury3 expression was detected in scattered presumably nerve cells of the D. pumila larva. Pharmacological modulations indicated that Brachyury3 is not under regulation of cWnt signaling in contrast to the other two Brachyury genes. Divergence in expression patterns and regulation suggest neofunctionalization of Brachyury3 in hydrozoans.

FHOD1 is upregulated in glioma cells and attenuates ferroptosis of glioma cells by targeting HSPB1 signaling.

BACKGROUND: As a new type of regulatory cell death, ferroptosis has been proven to be involved in cancer pathogenesis and therapeutic response. However, the detailed roles of ferroptosis or ferroptosis-associated genes in glioma remain to be clarified. METHODS: Here, we performed the TMT/iTRAQ-Based Quantitative Proteomic Approach to identify the differentially expressed proteins between glioma specimens and adjacent tissues. Kaplan-Meier survival was used to estimate the survival values. We also explored the regulatory roles of abnormally expressed formin homology 2 domain-containing protein 1 (FHOD1) in glioma ferroptosis sensitivity. RESULTS: In our study, FHOD1 was identified to be the most significantly upregulated protein in glioma tissues. Multiple glioma datasets revealed that the glioma patients with low FHOD1 expression displayed favorable survival time. Functional analysis proved that the knockdown of FHOD1 inhibited cell growth and improved the cellular sensitivity to ferroptosis in glioma cells T98G and U251. Mechanically, we found the up-regulation and hypomethylation of HSPB1, a negative regulator of ferroptosis, in glioma tissues. FHOD1 knockdown could enhance the ferroptosis sensitivity of glioma cells via up-regulating the methylated heat-shock protein B (HSPB1). Overexpression of HSPB1 significantly reversed FHOD1 knockdown-mediated ferroptosis. CONCLUSIONS: In summary, this study demonstrated that the FHOD1-HSPB1 axis exerts marked regulatory effects on ferroptosis, and might affect the prognosis and therapeutic response in glioma.

The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions.

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called "14-3-3 site-finder". We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.

The Occurrence of Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Its Predictive Factors in Patients with Oral Squamous Cell Carcinoma.

OBJECTIVES: To determine the occurrence of Activated Leukocyte Cell Adhesion Molecule (ALCAM) and its predictive factors in patients with oral squamous cell carcinoma (OSCC). METHODS: This cross sectional study was concocted on 102 patients with OSCC referred to Imam Khomeini Hospital of Tehran during 1997-2015. The data collection tool a checklist consisted of demographic and pathologic (lymph node involvement, differentiation, tumor size and tumor location) characteristics which extracted from patients' medical records. To evaluate ALCAM, a new sample of tumor tissue was prepared from archive. Finally, the multivariable logistic regression model was used to determine the predictive factors of ALCAM by STATA14. RESULTS: the number (%) of men and women were 70 (68.6) and 32 (31.4%), respectively. The mean age (S.D) of participants was 61.7 (15.6) years. Of the total samples, 32 (38.2), 19 (18.6), 36 (35.3) and 8 (7.8%) samples were related to the tongue, oral mucosa, skin and lips, respectively. More than half of the tumors had good differentiation and lymph node involvement and 74.5% were ≥20 mm. Also, 79.41% of the samples were positive for the overall incidence of ALCAM. The most important predictors of the overall incidence of ALCAM were tumor size (OR: 3.46, 95% CI: 1.71 - 7.01) and tumor location (OR: 3, 95% CI: 1.03 - 8.72). Similarly, for incidence of cytoplasmic ALCAM were age (OR: 2.56, 95% CI: 1.38 - 4.76) and location of the tumor (OR: 3.23, 95% CI: 1.08 - 9.64). However, the only predictor of membranous ALCAM incidence was lymph node involvement (OR: 0.36, 95% CI: 0.19 - 0.66). CONCLUSION: The results of our study suggest preliminary evidence for the potential clinical application of ALCAM as a prognostic biomarker for OSCC which may be the basis for future clinical application, however further studies are recommended.

The noncatalytic regions of the tyrosine kinase Tnk1 are important for activity and substrate specificity.

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an in vitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, in vitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.

Uterine Sarcoma With FGFR1-TACC1 Gene Fusion: A Case Report and Review of the Literature.

With the growing availability of RNA sequencing technology in the pathology laboratory, new gene fusion-associated malignancies are increasingly being characterized. In this article, we describe the second ever reported case of a uterine sarcoma harboring a FGFR1-TACC1 gene fusion. The patient, a 53-yr-old perimenopausal woman, was found to have a 6 cm mass spanning the lower uterine segment and endocervix. Histologically, this was a spindle cell neoplasm with coagulative necrosis, moderate cytologic atypia, and increased mitotic activity. By immunohistochemistry, the neoplastic cells coexpressed CD34 and S100, and lacked smooth muscle marker expression. RNA sequencing revealed the presence of a FGFR1-TACC1 gene fusion. This report provides further evidence to suggest that FGFR1-TACC1 may be a recurrent fusion in a subset of uterine sarcomas. RNA sequencing using a panel that includes FGFR-TACC family fusions should be considered for uterine sarcomas that do not fit conventional diagnostic criteria, particularly as tumors with these fusions may be amenable to targeted therapy.

CircMED12L Protects Against Hydrogen Peroxide-induced Apoptotic and Oxidative Injury in Human Lens Epithelial Cells by miR-34a-5p/ALCAM axis.

PURPOSE: Cataract is the leading cause of visual impairment and reversible blindness. Despite advances in surgical removal of cataracts, cataract continues to be a leading public-health issue due to the complications after surgery. Circular RNAs (circRNAs) have been showed to be implicated in the pathophysiology of age-related cataract (ARC). Herein, this work elucidated the role and mechanism of circMED12L in the process of ARC. METHODS: Human lens epithelial cells (HLECs) were exposed to hydrogen peroxide (H2O2) in experimental groups. Levels of genes and proteins were measured by qRT-PCR and western blotting. Cell growth was evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The oxidative stress was assessed by detecting the activity of malondialdehyde, catalase, and superoxide dismutase. The interaction between miR-34a-5p and circMED12L or ALCAM (activated leukocyte cell adhesion molecule) was validated using dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: CircMED12L expression was lower in the lens epithelium of ARC patients and H2O2-induced HLECs compared with the normal individuals and untreated cells. Functionally, forced expression of circMED12L could alleviate H2O2-induced viability inhibition, as well as apoptotic and oxidative injury in HLECs. Mechanistically, circMED12L/miR-34a-5p/ALCAM constituted a feedback loop in HLECs. MiR-34a-5p was increased, while ALCAM was decreased in ARC patients and H2O2-induced HLECs. High expression of miR-34a-5p reversed the protective effects of circMED12L on HLECs under H2O2 treatment. Besides, inhibition of miR-34a-5p could repress H2O2-induced apoptotic and oxidative injury in HLECs, which were abolished by subsequent ALCAM knockdown. CONCLUSION: Overexpression of circMED12L could protect against H2O2-induced apoptosis and oxidative stress in HLECs by miR-34a-5p/ALCAM axis.

FGFR1/MAPK-directed brachyury activation drives PD-L1-mediated immune evasion to promote lung cancer progression.

Immune checkpoint inhibitors provide promising benefits for patients with cancer. However, efficacy has been encumbered by high resistance rates. It is critical to understand the basic mechanisms of tumor-mediated resistance to this treatment modality. Previous studies have found that the transcription factor brachyury is highly expressed in lung cancer. Here, we show that brachyury activation induces the upregulation of PD-L1 leading to inactivation of T cell proliferation in vitro and inhibited infiltration of CD8+ and CD3+ T cells into tumor in an immunocompetent mouse model. We further demonstrate that FGFR1/MAPK activation regulates brachyury and PD-L1 expressions and promotes immunosuppression. Blocking FGFR1/MAPK suppresses brachyury and PD-L1 expressions, revives immune activity, and reverses the resistance to anti-PD-1 treatment to produce a durable therapeutic response. We also find that lung cancer patients with high activation of the FGFR1-MAPK-brachyury-PD-L1 signature and low expression of CD8A, CD3D, or PDCD1 have worse survival outcomes. These findings elucidate a novel mechanism of immune escape from immune checkpoint therapy and provide an opportunity to enhance its therapeutic efficacy in the treatment of a subset of FGFR1/MAPK/brachyury/PD-L1-driven lung cancer.

Proteome profiling of enzalutamide-resistant cell lines and serum analysis identified ALCAM as marker of resistance in castration-resistant prostate cancer.

Enzalutamide (ENZA) is a frequently used therapy in metastatic castration-resistant prostate cancer (mCRPC). Baseline or acquired resistance to ENZA have been observed, but the molecular mechanisms of resistance are poorly understood. We aimed to identify proteins involved in ENZA resistance and to find therapy-predictive serum markers. We performed comparative proteome analyses on ENZA-sensitive parental (LAPC4, DuCaP) and -resistant prostate cancer cell lines (LAPC4-ENZA, DuCaP-ENZA) using liquid chromatography tandem mass spectrometry (LC-MS/MS). The top four most promising candidate markers were selected using bioinformatic approaches. Serum concentrations of selected markers (ALCAM, AGR2, NDRG1, IDH1) were measured in pretreatment samples of 72 ENZA-treated mCRPC patients using ELISA. In addition, ALCAM serum levels were measured in 101 Abiraterone (ABI) and 100 Docetaxel (DOC)-treated mCRPC patients' baseline samples. Results were correlated with clinical and follow-up data. The functional role of ALCAM in ENZA resistance was assessed in vitro using siRNA. Our proteome analyses revealed 731 significantly differentially abundant proteins between ENZA-sensitive and -resistant cells and our filtering methods identified four biomarker candidates. Serum analyses of these proteins revealed only ALCAM to be associated with poor patient survival. Furthermore, higher baseline ALCAM levels were associated with poor survival in ABI- but not in DOC-treated patients. In LAPC4-ENZA resistant cells, ALCAM silencing by siRNA knockdown resulted in significantly enhanced ENZA sensitivity. Our analyses revealed that ALCAM serum levels may help to identify ENZA- and ABI-resistant patients and may thereby help to optimize future clinical decision-making. Our functional analyses suggest the possible involvement of ALCAM in ENZA resistance.