TAGLN Expression Research Articles

SURG-03. GENE EXPRESSION CHANGES ASSOCIATED WITH RECURRENCE AFTER GROSS TOTAL RESECTION OF NEWLY DIAGNOSED WHO GRADE 1 MENINGIOMA

Abstract Patients who undergo gross total resection (GTR) of Central Nervous System World Health Organization (WHO) grade 1 meningioma constitute a “low risk” group, but some low risk meningiomas can recur despite reassuring clinical and histological features. In this study, gene expression in newly diagnosed WHO grade 1 meningiomas that had undergone GTR were evaluated for their association with recurrence. This was a retrospective, international, multi-center cohort study that included WHO grade 1 meningiomas that underwent GTR as first treatment. Normalized gene expression values from a previously validated 34-gene panel were evaluated for their association with recurrence. Kaplan-Meier, multivariable Cox proportional hazard analyses, and K-means clustering were performed to assess the association of genes of interest with recurrence and identify molecular subgroups among clinically and histologically low risk meningiomas. 442 patients with WHO grade 1 meningiomas that underwent GTR and had available gene expression profiling data were included in the study. Median follow-up was 5.0 years (interquartile range 2.6-7.7 years), local recurrence occurred in 36 patients (8.1%), 5-year local freedom from recurrence (LFFR) was 90.5%, and median time to recurrence of 2.9 years (range 0.5-10.7 years). Eleven genes were associated with local recurrence, including lower expression of ARID1B, ESR1, LINC02593, PGR, and TMEM30B and higher expression of CDK6, CDKN2C, CKS2, KIF20A, PGK1, and TAGLN. K-means clustering based on these 11 genes distinguished 2 molecular groups of clinically and histologically low risk meningiomas with significant differences in LFFR (HR 2.5, 95% CI 1.2-5.1, p=0.016). In conclusion, gene expression profiling may help to identify newly diagnosed WHO grade 1 meningiomas that have an elevated risk of recurrence despite GTR.

CAF-Associated Genes in Breast Cancer for Novel Therapeutic Strategies.

Breast cancer (BC) is the most common cancer in women, and therapeutic strategies for it are based on the molecular subtypes of luminal BC, HER2 BC, and triple-negative BC (TNBC) because each subtype harbors different unique genetic aberrations. Recently, features of the tumor microenvironment (TME), especially cancer-associated fibroblasts (CAFs), have been demonstrated to play a critical role in BC progression, and we would like to understand the molecular features of BC CAFs for novel therapeutic strategies. In a recent study, 115 CAF-associated genes (CAFGs) were identified in a public database of microdissection and microarray data (GSE35602) from 13 colorectal cancer (CRC) tumors. Using a public database (GSE10797) of 28 BC tumors, a similar analysis was performed. In BC, 59 genes from the 115 CAFGs identified in CRC (CRC CAFGs) were also closely associated with a CAFs marker, SPARC (R = 0.6 or beyond), and POSTN was of particular interest as one of the BC CAFGs with the highest expression levels and a close association with SPARC expression (R = 0.94) in the cancer stroma of BC tumors. In BC stroma, POSTN was followed in expression levels by DKK3, MMP2, PDPN, and ACTA2. Unexpectedly, FAP and VIM were not as highly associated with SPARC expression in the cancer stroma of BC tumors and exhibited low expression. These findings suggested that ACTA2 might be the most relevant conventional CAFs marker in BC, and ACTA2 was actually correlated in expression with many CRC CAFGs, such as SPARC. Surprisingly, the SE ratio values of the BC CAFGs were much lower (average SE = 3.8) than those of the CRC CAFGs (SE = 10 or beyond). We summarized the current understanding of BC CAFs from the literature. Finally, in triple-negative BC (TNBC) (n = 5), SPARC expression uniquely showed a close association with COL11A1 and TAGLN expression, representing a myofibroblast (myCAFs) marker in the cancer stroma of the BC tumors, suggesting that myCAFs may be molecularly characterized by TNBC in contrast to other BC phenotypes. In summary, CAFs could have unique molecular characteristics in BC, and such TME uniqueness could be therapeutically targeted in BC.

Gene Expression Changes Associated With Recurrence After Gross Total Resection of Newly Diagnosed World Health Organization Grade 1 Meningioma.

Patients who undergo gross total resection (GTR) of Central Nervous System World Health Organization (WHO) grade 1 meningioma constitute a "low-risk" group, but some low-risk meningiomas can recur despite reassuring clinical and histological features. In this study, gene expression values in newly diagnosed WHO grade 1 meningiomas that had undergone GTR were evaluated for their association with recurrence. This was a retrospective, international, multicenter cohort study that included WHO grade 1 meningiomas that underwent GTR, as first treatment, based on postoperative magnetic resonance imaging. Normalized gene expression values from a previously validated 34-gene panel were evaluated for their association with recurrence. Kaplan-Meier, multivariable Cox proportional hazard analyses, and K-means clustering were performed to assess the association of genes of interest with recurrence and identify molecular subgroups among clinically and histologically low-risk meningiomas. In total, 442 patients with WHO grade 1 meningiomas that underwent GTR and had available gene expression profiling data were included in the study. The median follow-up was 5.0 years (interquartile range 2.6-7.7 years), local recurrence occurred in 36 patients (8.1%), 5-year local freedom from recurrence was 90.5%, and median time to recurrence was 2.9 years (range 0.5-10.7 years). Eleven genes were associated with local recurrence, including lower expression of ARID1B, ESR1, LINC02593, PGR, and TMEM30B and higher expression of CDK6, CDKN2C, CKS2, KIF20A, PGK1, and TAGLN. Of these genes, PGK1 had the largest effect size. K-means clustering based on these 11 genes distinguished 2 molecular groups of clinically and histologically low-risk meningiomas with significant differences in local freedom from recurrence (hazard ratio 2.5, 95% CI 1.2-5.1, P = .016). Gene expression profiling may help to identify newly diagnosed WHO grade 1 meningiomas that have an elevated risk of recurrence despite GTR.

2‑D08 mediates notable anticancer effects through multiple cellular pathways in uterine leiomyosarcoma cells.

2',3',4'‑trihydroxyflavone (2‑D08), a SUMO E2 inhibitor, has several biological functions, including anticancer activity, but its effects on uterine leiomyosarcoma (Ut‑LMS) are unknown. The anticancer activity of 2‑D08 was explored in an in vitro model using SK‑LMS‑1 and SK‑UT‑1B cells (human Ut‑LMS cells). Treatment with 2‑D08 inhibited cell viability in a dose‑ and time‑dependent manner and significantly inhibited the colony‑forming ability of Ut‑LMS cells. In SK‑UT‑1B cells treated with 2‑D08, flow cytometric analysis revealed a slight increase in apoptotic rates, while cell cycle progression remained unaffected. Western blotting revealed elevated levels of RIP1, indicating induction of necrosis, but LC3B levels remained unchanged, suggesting no effect on autophagy. A lactate dehydrogenase (LDH) assay confirmed increased LDH release, further supporting the induction of apoptosis and necrosis by 2‑D08 in SK‑UT‑1B cells. 2‑D08‑induced production of reactive oxygen species and apoptosis progression were observed in SK‑LMS‑1 cells. Using Ki67 staining and bromodeoxyuridine assays, it was found that 2‑D08 suppressed proliferation in SK‑LMS‑1 cells, while treatment for 48 h led to cell‑cycle arrest. 2‑D08 upregulated p21 protein expression in SK‑LMS‑1 cells and promoted apoptosis through caspase‑3. Evaluation of α‑SM‑actin, calponin 1 and TAGLN expression indicated that 2‑D08 did not directly initiate smooth muscle phenotypic switching in SK‑LMS‑1 cells. Transcriptome analysis on 2‑D08‑treated SK‑LMS‑1 cells identified significant differences in gene expression and suggested that 2‑D08 modulates cell‑cycle‑ and apoptosis‑related pathways. The analysis identified several differentially expressed genes and significant enrichment for biological processes related to DNA replication and molecular functions associated with the apoptotic process. It was concluded that 2‑D08 exerts antitumor effects in Ut‑LMS cells by modulating multiple signaling pathways and that 2‑D08 may be a promising candidate for the treatment of human Ut‑LMS. The present study expanded and developed knowledge regarding Ut‑LMS management and indicated that 2‑D08 represents a notable finding in the exploration of fresh treatment options for such cancerous tumors.

Piezo1 mediated calcium-calcineurin signaling pathway regulates gastrointestinal smooth muscle cell phenotype in a stiffness dependent manner

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 6880: Immune infiltration in inflammatory and myofibroblastic cancer-associated fibroblast high microenvironments of colorectal cancer

Abstract Background: Cancer-associated fibroblasts are major modulators of the tumor microenvironment (TME) and play a large role in the immune system’s response to tumors. The direct effect that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) have on immune infiltration is not well studied in colorectal cancer (CRC). Here we investigate the effects that myCAFs and iCAFs have on immune infiltration in human CRCs. Methods: The Cancer Genome Atlas (TCGA) Colorectal Adenocarcinoma PanCancer Atlas dataset was accessed via cBioPortal. Cancers were separated into myCAF prominent (MP) by high gene expression (Z-score > 1) of ACTA2 and TAGLN and iCAF prominent (IP) by expression of PDPN and ICAM1. Gene Set Enrichment Analysis (GSEA) was performed on differential expression results comparing these groups. Additionally, bulk RNA sequencing results were analyzed for immune cell subsets using Cibersortx. Statistical analysis was completed in R. Results: Of 592 cancer samples, 24 were MP and 9 IP. There was no significant difference in age (MP median age 63, IP median age 70, q = 0.6) nor sex (MP 17 female/7 male; IP 4 female/5 male, q=0.9). MP have 1/24 profiled patients with MSIsensor score >=3.5 and 2/24 patients with MSI MANTIS score > 0.4; IP have 3/9 profiled patients with MSIsensor score >=3.5 and 3/9 patients with MSI MANTIS score >0.4. IP cancers were enriched for gene sets associated with enhanced immune cell infiltration (Inflammatory Response, Interferon Gamma Response, Allograft rejection, TNFA signaling via NFKB, IL6 JAK STAT3 signaling, Interferon Alpha Response, Complement, IL2 STAT5 signaling and Apoptosis, all q< 0.001). MP cancers were enriched in the KRAS signaling Down gene set (q=.009). IP cancers had a higher proportion of infiltrating immune cells than MP cancers by CibersortX analysis (iCAF median = 2.829, myCAF median= 2.284, p < 0.01). IP cancers had higher CD4 memory cells (p <0.001, iCAF median = 0.455, myCAF mean = 0.318), NK resting cells (p<0.001, 0.228, 0.103), M1-like macrophages (p <0.01, 0.1977,0.107), and neutrophils (p<0.01, 0.0298, 0). MP cancers had a higher number of NK activated cells (p = 0.04, iCAF median = 0, myCAF median = 0). M1-like macrophage gene markers were higher expressed in IP cancers compared to MP cancers, though not significantly (NOS2 log2FC = 0.66, q = 0.652; IL1A 3.25, 0.0375; IL23A 1.34, 0.151; IL26 1.31, 0.32). Additionally, IP cancers tended to express markers for activated CD8+ T cells, though only IFNG was significant (GZMB, log2FC = 0.97, q = 0.337; IFNG, 2.16, q<0.001; PRF1, 1.12, q=0.198). Conclusion: These analyses indicate that iCAFs play the major role in modulating the immune infiltration of cancers. These data highlight the need for further research into the mechanisms by which different CAF subtypes alter the immune microenvironment. Citation Format: Yousef Gadalla, Katherine A. Johnson, Sharanya Nath, Cheri A. Pasch, Dustin A. Deming. Immune infiltration in inflammatory and myofibroblastic cancer-associated fibroblast high microenvironments of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6880.

Abstract 1589: Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is primarily characterized by a desmoplastic reaction, mainly consisting of cancer-associated fibroblasts (CAFs). In order to investigate the heterogeneity of CAFs, we initially conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using pancreas tissue from 12-week-old KPC (K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre) PDAC mouse models. We identified three clusters enriched with myofibroblast CAF (myCAF) gene markers and one cluster enriched with inflammatory CAF (iCAF) gene markers. Next, we performed single-cell RNA sequencing (scRNA-seq) on pancreas tissues from KPC mice. scRNA-seq revealed four distinct fibroblast clusters, each exhibiting characteristic gene expression profiles of normal fibroblasts, iCAF, myCAF and antigen-presenting CAF (apCAF). These findings suggest that CAFs in the myCAF cluster identified in scATAC-seq may have potential to differentiate into either myCAFs or other CAF subtypes based on certain cues. Next, we focus on the expression of myCAF marker genes, particularly two commonly used markers, Tagln and Acta2. Among myCAFs, two subtypes were identified: Tagln-positive and Acta2-positive CAFs, and Tagln-negative and Acta2-positive CAFs. To confirm our findings, we conducted Immunohistochemical analysis (IHC) on KPC pancreas samples. We observed CAFs that were positive for both Talng and αSMA exclusively in close proximity to tumor cells. Fibroblast expressing only αSMA mostly located in normal area. Tagln is known as an actin-binding protein and plays an important role in activated fibroblasts. Orthotopic PDAC mouse models using Tagln homozygous knockout mice showed a significant reduction in tumor weight compared to the wild-type mice (P = 0.0076). Indeed, we found that high TAGLN expression in PDAC samples was associated with poor survival, as demonstrated by the analysis of the TCGA dataset. These data suggest that myCAFs expressing TAGLN contribute to the promotion of PDAC growth. Targeting stromal TAGLN might be a potential therapeutic strategy to counteract PDAC progression. Citation Format: Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Maruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1589.

Involvement of TGFBI-TAGLN axis in cancer stem cell property of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.

Identification of distinct vascular mural cell populations during zebrafish embryonic development.

Mural cells are an essential perivascular cell population that associate with blood vessels and contribute to vascular stabilization and tone. In the embryonic zebrafish vasculature, pdgfrb and tagln are commonly used as markers for identifying pericytes and vascular smooth muscle cells. However, the overlapping and distinct expression patterns of these markers in tandem have not been fully described. Here, we used the Tg(pdgfrb:Gal4FF; UAS:RFP) and Tg(tagln:NLS-EGFP) transgenic lines to identify single- and double-positive perivascular cell populations on the cranial, axial, and intersegmental vessels between 1 and 5 days postfertilization. From this comparative analysis, we discovered two novel regions of tagln-positive cell populations that have the potential to function as mural cell precursors. Specifically, we found that the hypochord-a reportedly transient structure-contributes to tagln-positive cells along the dorsal aorta. We also identified a unique mural cell progenitor population that resides along the midline between the neural tube and notochord and contributes to intersegmental vessel mural cell coverage. Together, our findings highlight the variability and versatility of tracking both pdgfrb and tagln expression in mural cells of the developing zebrafish embryo and reveal unexpected embryonic cell populations that express pdgfrb and tagln.

Identifying genetic loci that are associated with changes in gene expression in PTSD in a South African cohort.

The molecular mechanisms underlying posttraumatic stress disorder (PTSD) are yet to be fully elucidated, especially in underrepresented population groups. Expression quantitative trait loci (eQTLs) are DNA sequence variants that influence gene expression, in a local (cis-) or distal (trans-) manner, and subsequently impact cellular, tissue, and system physiology. This study aims to identify genetic loci associated with gene expression changes in a South African PTSD cohort. Genome-wide genotype and RNA-sequencing data were obtained from 32 trauma-exposed controls and 35 PTSD cases of mixed-ancestry, as part of the SHARED ROOTS project. The first approach utilised 108 937 single-nucleotide polymorphisms (SNPs) (MAF > 10%) and 11 312 genes with Matrix eQTL to map potential eQTLs, while controlling for covariates as appropriate. The second analysis was focused on 5638 SNPs related to a previously calculated PTSD polygenic risk score for this cohort. SNP-gene pairs were considered eQTLs if they surpassed Bonferroni correction and had a false discovery rate <0.05. We did not identify eQTLs that significantly influenced gene expression in a PTSD-dependent manner. However, several known cis-eQTLs, independent of PTSD diagnosis, were observed. rs8521 (C > T) was associated with TAGLN and SIDT2 expression, and rs11085906 (C > T) was associated with ZNF333 expression. This exploratory study provides insight into the molecular mechanisms associated with PTSD in a non-European, admixed sample population. This study was limited by the cross-sectional design and insufficient statistical power. Overall, this study should encourage further multi-omics approaches towards investigating PTSD in diverse populations.

Abstract 15554: Smooth Muscle Cells Specific Gene Inactivation of Lysyl Oxidase Reduces Atherosclerosis Burden and Plaque Calcification in Hyperlipidemic Mice

Aim: Dissect the mechanisms by which LOX increases atherogenesis and plaque calcification in hyperlipidemic mice. Methods: LOX conditional knockout [LOX f/f Myh11-CreER T2 ApoE -/- ] and control littermate [LOX wt/wt Myh11-CreER T2 ApoE -/- ] mice were injected with tamoxifen before high fat diet (HFD) feeding for atherosclerosis and calcification assessment and molecular analyses. Results: scRNA sequence showed full Lox gene inactivation in 72.5% of the aortic SMCs without any significant Loxl compensatory regulation. Gene deletion in SMCs decreased global arterial LOX deposition by ~3-fold in relation to control mice. Surprisingly, the loss of vascular LOX neither promoted aneurysm nor affected the aorta's stiffness. However, it reduced atherosclerosis burden (WT: 23±1 vs. KO: 13±2 %, p ≤0.01) and calcium deposition (11.8±3 versus 5±0.4%, p≤ 0.05) within the plaque after 16 weeks of high fat diet (HFD). Using bulk RNA sequencing, we further demonstrated that Lox gene inactivation leads to a marked decrease in Frizzled Related Protein-3 (FRZB) gene expression (127-fold and p≤0.0001), a biphasic modulator of the proatherogenic Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. The compromised FRZB /β-catenin/TCF4 signaling axis reduced the downstream production of Bone Morphogenetic Protein 2 (BMP2), which is responsible for the osteogenic transition of SMCs. Further scRNAseq analysis of atherosclerotic aortas from conditional KO and control mice also showed a reduction of BMP2 in SMCs. Interestingly, genetic inactivation of LOX increased the number of contractile SMCs (KO: 40 vs. WT: 7 %), characterized by the high expression of ACTA2 and TAGLN, in the aorta of HFD fed mice when compared to the control group. BAPN, an irreversible inhibitor of LOX activity, was not able to reduce BMP2 expression and protein levels nor reduce calcium deposition in vitro, suggesting that the pro-calcification effect of LOX is not activity dependent. Conclusions: Vascular gene inactivation of Lox protects mice against atherosclerosis by reducing β-catenin/TCF4/BMP2 proatherogenic signaling.

Molecular Mechanism of Mouse Uterine Smooth Muscle Regulation on Embryo Implantation.

Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.

Gene-Expression Profiling of Mucinous Ovarian Tumors and Comparison with Upper and Lower Gastrointestinal Tumors Identifies Markers Associated with Adverse Outcomes.

Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04-7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04-1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01-1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Low expression of IGFBP4 and TAGLN accelerate the poor overall survival of osteosarcoma

Osteosarcoma is a common malignant bone tumor characterized by the production of osteoid stroma by the tumor. However, effect of IGFBP4 and TAGLN on the survival of osteosarcoma is unclear. The GEO database was used to identify the differentially expressed genes (DEGs) between control samples and osteosarcoma. Genes for biological process (BP), cellular composition (CC), and molecular function (MF) were examined using DAVID, Metascape, and GSEA. GSE14359 and GSE36001 were downloaded in the GEO database. GEO2R was used to find DEGs between control samples and osteosarcoma. The cytoHubb also found the hub genes of IGFBP4 and TAGLN. The Kaplan–Meier method was used to analyze overall survival. A total of 134 patients with osteosarcoma were enrolled in this study. The RNA levels of IGFBP4 and TAGLN were evaluated by RT-qPCR. The correlation between IGFBP4 and TAGLN expression and their associations with clinical indicators were analyzed using Spearman's rho test and Pearson's Chi-squared test. Univariate and multivariate Cox regression analyses were used to determine the potential prognostic factors. And the animal model was used to verify the role of hub genes on the osteosarcoma by the RT-qPCR and immunofluorescence. Support Vector Machine (SVM) was performed to construct the correlation among the expression of IGFBP4, TAGLN, and osteosarcoma. Through bioinformatics, IGFBP4 and TAGLN were identified as the hub genes of osteosarcoma. And osteosarcoma patients with high expression levels of IGFBP4 (HR = 0.56, P = 0.013) and TAGLN (HR = 0.52, P = 0.012) had better overall survival times than those with low expression levels. The results showed that pathologic grade (P = 0.017), tumor metastasis (P < 0.001), and enneking stage (P < 0.001) were significantly correlated with IGFBP4. Also, pathologic grade (P = 0.002), tumor metastasis (P < 0.001), and enneking stage (P < 0.001) were significantly related to the TAGLN. Spearman’s correlation coefficient displayed that IGFBP4 were significantly correlated with the tumor metastasis (ρ = − 0.843, P < 0.001), enneking stage (ρ = − 0.500, P < 0.001), and TAGLN (ρ = 0.821, P < 0.001). IGFBP4 (HR = 0.252, 95% CI 0.122–0.517, P < 0.001) and TAGLN (HR = 0.155, 95% CI 0.089–0.269, P < 0.001) were significantly associated with overall survival. Based on the qPCR and immunofluorescence, IGFBP4 and TAGLN were down-regulated in the osteosarcoma tissue than the control group. And the SVM presented that there exists strong relationship among the expression of IGFBP4, TAGLN, and osteosarcoma. IGFBP4 and TAGLN may be attractive molecular targets for osteosarcoma, opening a new avenue for research into the disease.

New Insights into Juxtaglomerular Cells via Single‐Cell RNA‐Sequencing

The regulation of renin release from juxtaglomerular (JG) cells at the systemic or local level has been extensively investigated and well understood. It includes three primary mechanisms: intrarenal baroreceptor, salt feedback via macula densa, and β1‐adrenergic receptor activation. However, further understanding of the control of renin synthesis and secretion in JG cells at the cellular or molecular level is limited. Additionally, JG cells are generally considered to be derived from the smooth muscle (SM) cells in the afferent arterioles via a reversible metaplastic transformation. The number of JG cells could be significantly increased under specific conditions, which is also known as the process of JG cells recruitment. Nevertheless, the similarities and differences at the molecular level between JG cells and SM cells as well as the intracellular mechanisms underlying the JG cells recruitment have not been fully clarified. An emerging technique, the single‐cell RNA sequencing, provides a new tool to precisely investigate the molecular characterization of JG cells. In the present study, we isolated and sequenced a total of 9815 cells from a single‐cell stock derived from the renal cortex of C57BL/6J mice (~20 mg cortical tissue per mouse, n=5). Using stringent quality controls, we further analyzed 7557 cells. Unbiased clustering analysis demonstrated 16 distinct cell clusters in the t‐distributed stochastic neighbor embedding (tSNE) map, and each cell cluster was defined based on the expression level of the representative cell type‐specific marker genes. In particular, a cluster consisting of 284 cells was identified as smooth muscle (SM) cells by the high and distinct expression of both Acta2 (α‐smooth muscle actin) and Tagln (Transgelin). Moreover, on the basis of the expression level of the marker gene Ren1 (Renin), 34 JG cells were further distinguished from this cluster of SM cells. We profiled the whole‐transcriptome of these JG cells and correlated it with the transcriptional level of Ren1. The top 5 genes that were most positively correlated with the Ren1 expression were Nr2f1 (R=0.79), Akr1b7 (R=0.68), Smim15 (R=0.64), Gng11 (R=0.60) and Sdc1 (R=0.59). We also compared the whole‐transcriptome between these JG cells and SM cells. The top 5 genes with the largest difference in the normalized transcript count were Ren1 (+4.28), Akr1b7 (+1.53), Sfrp2 (+1.01), Fam46a (+0.88), and Sdc1 (+0.75). Thus, aldo‐keto reductase 1B7 (Akr1b7) and syndecan‐1 (Sdc1) could be two potential candidates that may participate in the control of renin release from JG cells. However, previous studies using the Akr1b7‐deficient mice have shown that aldo‐keto reductase 1B7 is not of major relevance for the modulation of renin production and secretion. Therefore, we speculate that syndecan‐1 (Sdc1), a transmembrane heparan sulfate proteoglycan, may play a role in the regulation of renin release as well as JG cells recruitment, especially through the mechanism of intrarenal baroreceptor. This hypothesis will be tested with the Sdc1‐deficient mouse model in our future studies.

Fibroblast-Secreted Phosphoprotein 1 Mediates Extracellular Matrix Deposition and Inhibits Smooth Muscle Cell Contractility in Marfan Syndrome Aortic Aneurysm.

Fibrillin 1 (Fbn1) mutation causes Marfan syndrome (MFS) with thoracic aortic aneurysm (TAA) as the main complication. The mechanisms for extracellular matrix (ECM) homeostasis disruption in MFS TAA are unclear. Here, we found ECM-related gene secreted phosphoprotein 1 (Spp1) increased in Fbn1C1041G/+ mice using transcriptome sequencing and a distinct fibroblast subcluster with Spp1 as the strongest marker was identified with analysis of the MFS mouse aortic single-cell sequencing dataset. Immunostaining confirmed elevated Spp1 in adventitial fibroblasts, and Spp1 might regulate fibroblast and smooth muscle cell (SMC) communication primarily through Itga8/Itgb1. Then, we observed Spp1 reduced contractile genes Acta2 and Tagln expression in SMCs and increased collagen expression in fibroblasts, which might contribute to TAA development. Finally, we also found elevated SPP1 plasma level was associated with an increased risk of TAA in patients. Therefore, SPP1 may serve as a biomarker and therapeutic target for TAA.

Polydatin Glycosides Improve Monocrotaline-Induced Pulmonary Hypertension Injury by Inhibiting Endothelial-To-Mesenchymal Transition.

Objective: To study the effect of polydatin on the injury of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). Methods: SD rats were induced to develop PAH injury by a single subcutaneous injection of MCT (60 mg/kg). From the second day, rats in the administration group were orally given sildenafil (20 mg/kg) and polydatin (30 or 60 mg/kg) for 3 weeks. At the end of the experiment, right ventricular hypertrophy (RVH) index of SD rats was calculated, pathological damage was assessed by HE staining, transcription levels of target genes were detected by RT-PCR and Elisa, and expression levels of Endothelial-to-mesenchymal transition (EndMT) related proteins were detected by immunohistochemistry (IHC) and immunofluorescence (IF). Finally, molecular docking analysis was used to verify the interaction of polydatin on the main targets. Results: Polydatin could significantly restore the body function, reduce MCT-induced PAH injury, reduce serum biochemical indices; polydatin could effectively inhibit EndMT process by decreasing the expression of N-cadherin, β-catenin and vimentin; polydatin could down-regulate TAGLN expression and increase PECAM1 expression to reduce pulmonary vascular remodeling. The interaction between polydatin and EndMT target was confirmed by molecular docking operation. Conclusion: Pharmacological experiments combined with Combining molecular docking was first used to clarify that polydatin can reduce the pulmonary endothelial dysfunction and pulmonary vascular remodeling induced by MCT by inhibiting EndMT. The results of the study provide new ideas for the further treatment of PAH injury.

The canonical smooth muscle cell marker TAGLN is present in endothelial cells and is involved in angiogenesis.

Elongation of vascular endothelial cells (ECs) is an important process in angiogenesis; however, the molecular mechanisms remain unknown. The actin-crosslinking protein TAGLN (transgelin, also known as SM22 or SM22α) is abundantly expressed in smooth muscle cells (SMCs) and is widely used as a canonical marker for this cell type. In the course of studies using mouse embryonic stem cells (ESCs) carrying an Tagln promoter-driven fluorescence marker, we noticed activation of the Tagln promoter during EC elongation. Tagln promoter activation co-occurred with EC elongation in response to vascular endothelial growth factor A (VEGF-A). Inhibition of phosphoinositide 3-kinase (PI3K)-Akt signaling and mTORC1 also induced EC elongation and Tagln promoter activation. Human umbilical vein endothelial cells (HUVECs) elongated, activated the TAGLN promoter and increased TAGLN transcripts in an angiogenesis model. Genetic disruption of TAGLN augmented angiogenic behaviors of HUVECs, as did the disruption of TAGLN2 and TAGLN3 genes. Tagln expression was found in ECs in mouse embryos. Our results identify TAGLN as a putative regulator of angiogenesis whose expression is activated in elongating ECs. This finding provides insight into the cytoskeletal regulation of EC elongation and an improved understanding of the molecular mechanisms underlying the regulation of angiogenesis.

The tumor immune microenvironment shapes response to pembrolizumab in microsatellite instability-high endometrial cancer

Objectives: Despite FDA approval of pembrolizumab in microsatellite instability-high (MSI-H) / mismatch repair deficient solid tumors, approximately half of patients with MSI-H endometrial cancer are treatment-refractory. Our unpublished analysis of MSI-H endometrial cancer samples from the Cancer Genome Atlas (TCGA) suggests the possibility of immunologically ‘hot’ and ‘cold’ tumor microenvironments (TME); these differences may explain variable treatment responses. We sought to evaluate MSI-H endometrial tumor samples to examine differences in the TME and identify transcriptomic signatures associated with response/resistance to pembrolizumab. Methods: Archival tumor samples from MSI-H endometrial cancer patients treated at the University of Texas MD Anderson Cancer Center were obtained under an IRB-approved protocol. Tissue samples originating from patients who did not receive pembrolizumab treatment (‘untreated cohort’; n=11) were submitted for RNA sequencing analysis (RNA-seq) to evaluate TCGA-derived signatures in an independent sample set. Pre-treatment archival tumor samples (‘treated cohort’; n=23) from patients who were later treated with pembrolizumab were collected and submitted for RNA-seq to identify predictive immuno-genomic signatures associated with treatment response. Results: In the untreated cohort, there were observable differences in the transcriptomic profiles of the samples. Four samples were immunologically ‘hot’ as evidenced by an abundance of pro-inflammatory immune cell infiltrate (CD8+ T-cells, B-cells, monocytes, and dendritic cells). Three samples had a paucity of pro-inflammatory immune cell infiltrate (‘cold’) while 4 samples had intermediate amounts (‘warm’). In the treated cohort, there were 14 responders, 7 non-responders, and 2 patients with unknown response. The 14 responders consisted of samples with ‘hot’ (5/5; 100%), ‘cold’ (6/8; 75%), and ‘warm’ TMEs (3/8; 37.5%) while the 7 non-responders consisted of only ‘cold’ (2/8; 25%) and ‘warm’ (5/8; 62.5%) TME samples. We observed an enrichment of fibroblasts and endothelial cell transcriptomic signatures in the samples of the non-responders compared to responders (p=0.018). In particular, there was a significantly higher gene expression of PAMR1, HHIP, and MMRN1 in the non-responders. Subdividing samples into complete response, partial response, and no response to pembrolizumab, we observed a trend of increasing enrichment of fibroblast and endothelial cell transcriptomic signatures as response decreased. Specifically, there was increasing expression of TAGLN (fibroblast gene) and other endothelial cell genes (EMCN, KDR, MMRN1, MYCT1, PEAR1, PTPRB, and TEK). Conclusions: The TME composition appears to be heterogeneous among MSI-H endometrial cancer patients. Increased presence of fibroblasts and endothelial cells in the TME may contribute to innate resistance to pembrolizumab. Treatment aimed toward the reduction of these cellular subpopulations may improve sensitivity to PD-1 inhibitors. Future studies are needed to validate these findings.

M6A demethylase ALKBH5 suppresses proliferation and migration of enteric neural crest cells by regulating TAGLN in Hirschsprung's disease

ObjectivesThis study aims to investigate the role of demethylase ALKBH5 mediated demethylation of TAGLN mRNA in the occurrence of Hirschsprung's disease (HSCR), and to clarify how ALKBH5 reduces the m6A level of TAGLN mRNA and inhibits its degradation, thereby inhibiting the proliferation and migration of neural crest cells, and potentially contributing to the occurrence of HSCR. Material and methodsQuantitative real-time PCR (qRT-PCR) and Western-Blot (WB) were conducted to test the expression level of ALKBH5 and TAGLN genes. Cell function assays were adopted to detect cell phenotypes. The qRT-PCR and methylated RNA immunoprecipitation (MeRIP-qPCR) were used to test the regulation of TAGLN by ALKBH5. Results1. Compared with control intestinal tissue, the expression level of TAGLN and ALKBH5 in the aganglionic intestinal tissue of HSCR is increased.2. The MeRIP-PCR and dualluciferase report confirmed that ALKBH5 could bind to m6A sites of TAGLN mRNA and reduce the m6A level of TAGLN mRNA.3. In vitro cell experiments confirmed that overexpression of ALKBH5 can inhibit the degradation of TAGLN mRNA, increase the expression of TAGLN, thereby inhibiting cell proliferation and migration.4. A zebrafish model of ALKBH5 overexpression was constructed. Studies have shown that ALKBH5 could inhibit the proliferation and migration of zebrafish enteric neurons. ConclusionsALKBH5 could demethylate TAGLN mRNA and up-regulate TAGLN expression, leading to the inhibition of proliferation and migration of enteric neural crest cells and contributing to the occurrence of HSCR.