CSPG4P12 polymorphism served as a susceptibility marker for esophageal cancer in Chinese population.

BACKGROUND: Chondroitin sulfate proteoglycan 4 pseudogene 12 (CSPG4P12) has been implicated in the pathogenesis of various cancers. This study aimed to evaluate the association of the CSPG4P12 polymorphism with esophageal squamous cell carcinoma (ESCA) risk and to explore the biological impact of CSPG4P12 expression on ESCA cell behavior. METHODS: A case-control study was conducted involving 480 ESCA patients and 480 healthy controls to assess the association between the rs8040855 polymorphism and ESCA risk. The CSPG4P12 rs8040855 genotype was identified using the TaqMan-MGB probe method. Logistic regression model was used to evaluate the association of CSPG4P12 SNP with the risk of ESCA by calculating the odds ratios (OR) and 95% confidence intervals (95%CI ). The effects of CSPG4P12 overexpression on cell proliferation, migration, and invasion were examined in ESCA cell lines. Co-expressed genes were identified via the CBioportal database, with pathway enrichment analyzed using SangerBox. The binding score of CSPG4P12 to P53 was calculated using RNA protein interaction prediction (RPISeq). Additionally, Western Blot analysis was performed to investigate the impact of CSPG4P12 overexpression on the P53/PI3K/AKT signaling pathway. RESULTS: The presence of at least one rs8040855 G allele was associated with a reduced susceptibility to ESCA compared to the CC genotype (OR = 0.51, 95%CI = 0.28-0.93, P = 0.03). Stratification analysis revealed that the CSPG4P12 rs8040855 C allele significantly decreased the risk of ESCA among younger individuals (≤ 57 years) and non-drinkers (OR = 0.31, 95%CI = 0.12-0.77, P = 0.01; OR = 0.42, 95%CI=0.20-0.87, P = 0.02, respectively). CSPG4P12 expression was found to be downregulated in ESCA tissues compared to adjacent normal tissues. Overexpression of CSPG4P12 in ESCA cells inhibited their proliferation, migration, and invasion capabilities. Furthermore, Western Blot analysis indicated that CSPG4P12 overexpression led to a reduction in PI3K and p-AKT protein expression levels. P53 silencing rescues the inhibitory effect of CSPG4P12 on p-AKT. CONCLUSION: The CSPG4P12 rs8040855 variant is associated with reduced ESCA risk and the overexpression of CSPG4P12 inhibited the migration and invasion of ESCA cells by P53/PI3K/AKT pathway. These findings suggest that CSPG4P12 may serve as a novel biomarker for ESCA susceptibility and a potential target for therapeutic intervention.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma.

BACKGROUND: As a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear. METHODS: Gene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC. RESULTS: Our findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression. CONCLUSIONS: Our findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Transcriptome Sequencing Analysis of lncRNA and mRNA Expression Profiles in Bone Nonunion.

Background: Bone nonunion is a serious complication of fracture. This study explored the differentially expressed lncRNAs (DELs) and mRNAs (DEGs) and identified potential lncRNA-mRNA interactions in bone nonunion. Methods: We extracted total RNA from three bone nonunion and three bone union patient tissue samples. RNA sequencing was performed to detect DELs and DEGs between bone nonunion and union tissue samples. The lncRNAs and genes with absolute log2-fold change (log2FC) > 1 and adjusted p value < 0.05 were further chosen for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. lncRNA and targeted mRNA interaction networks were constructed. Results: We observed 179 DELs and 415 DEGs between the bone nonunion and union tissue samples. GO analysis indicated that DELs and DEGs were mainly enriched in the chondroitin sulfate proteoglycan biosynthetic process. DELs and DEGs were enriched in "ECM-receptor interaction" and "Staphylococcus aureus infection" KEGG pathways. Several potential lncRNA-mRNA interactions were also predicted. Conclusions: This study identified bone nonunion-associated lncRNAs and mRNAs using deep sequencing that may be useful as potential biomarkers for bone nonunion.

A comprehensive atlas of Aggrecan, Versican, Neurocan and Phosphacan expression across time in wildtype retina and in retinal degeneration.

As photoreceptor cells die during retinal degeneration, the surrounding microenvironment undergoes significant changes that are increasingly recognized to play a prominent role in determining the efficacy of therapeutic interventions. Chondroitin Sulphate Proteoglycans (CSPGs) are a major component of the extracellular matrix that have been shown to inhibit neuronal regrowth and regeneration in the brain and spinal cord, but comparatively little is known about their expression in retinal degeneration. Here we provide a comprehensive atlas of the expression patterns of four individual CSPGs in three models of inherited retinal degeneration and wildtype mice. In wildtype mice, Aggrecan presented a biphasic expression, while Neurocan and Phosphacan expression declined dramatically with time and Versican expression remained broadly constant. In degeneration, Aggrecan expression increased markedly in Aipl1-/- and Pde6brd1/rd1, while Versican showed regional increases in the periphery of Rho-/- mice. Conversely, Neurocan and Phosphacan broadly decrease with time in all models. Our data reveal significant heterogeneity in the expression of individual CSPGs. Moreover, there are striking differences in the expression patterns of specific CSPGs in the diseased retina, compared with those reported following injury elsewhere in the CNS. Better understanding of the distinct distributions of individual CSPGs will contribute to creating more permissive microenvironments for neuro-regeneration and repair.

Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Novel regulatory roles of small leucine-rich proteoglycans in remodeling of the uterine cervix in pregnancy.

The cervix undergoes rapid and dramatic shifts in collagen and elastic fiber structure to achieve its disparate physiological roles of competence during pregnancy and compliance during birth. An understanding of the structure-function relationships of collagen and elastic fibers to maintain extracellular matrix (ECM) homeostasis requires an understanding of the mechanisms executed by non-structural ECM molecules. Small-leucine rich proteoglycans (SLRPs) play key functions in biology by affecting collagen fibrillogenesis and regulating enzyme and growth factor bioactivities. In the current study, we evaluated collagen and elastic fiber structure-function relationships in mouse cervices using mice with genetic ablation of decorin and/or biglycan genes as representative of Class I SLRPs, and lumican gene representative of Class II SLRP. We identified structural defects in collagen fibril and elastic fiber organization in nonpregnant mice lacking decorin, or biglycan or lumican with variable resolution of defects noted during pregnancy. The severity of collagen and elastic fiber defects was greater in nonpregnant mice lacking both decorin and biglycan and defects were maintained throughout pregnancy. Loss of biglycan alone reduced tissue extensibility in nonpregnant mice while loss of both decorin and biglycan manifested in decreased rupture stretch in late pregnancy. Collagen cross-link density was similar in the Class I SLRP null mice as compared to wild-type nonpregnant and pregnant controls. A broader range in collagen fibril diameter along with an increase in mean fibril spacing was observed in the mutant mice compared to wild-type controls. Collectively, these findings uncover functional redundancy and hierarchical roles of Class I and Class II SLRPs as key regulators of cervical ECM remodeling in pregnancy. These results expand our understating of the critical role SLRPs play to maintain ECM homeostasis in the cervix.

Glutaredoxin 2 promotes SP-1-dependent CSPG4 transcription and migration of wound healing NG2 glia and glioma cells: Enzymatic Taoism.

Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.

Maternal Smc3 protects the integrity of the zygotic genome through DNA replication and mitosis.

Aneuploidy is frequently observed in oocytes and early embryos, begging the question of how genome integrity is monitored and preserved during this crucial period. SMC3 is a subunit of the cohesin complex that supports genome integrity, but its role in maintaining the genome during this window of mammalian development is unknown. We discovered that, although depletion of Smc3 following meiotic S phase in mouse oocytes allowed accurate meiotic chromosome segregation, adult females were infertile. We provide evidence that DNA lesions accumulated following S phase in SMC3-deficient zygotes, followed by mitosis with lagging chromosomes, elongated spindles, micronuclei, and arrest at the two-cell stage. Remarkably, although centromeric cohesion was defective, the dosage of SMC3 was sufficient to enable embryogenesis in juvenile mutant females. Our findings suggest that, despite previous reports of aneuploidy in early embryos, chromosome missegregation in zygotes halts embryogenesis at the two-cell stage. Smc3 is a maternal gene with essential functions in the repair of spontaneous damage associated with DNA replication and subsequent chromosome segregation in zygotes, making cohesin a key protector of the zygotic genome.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

A Chinese Case of Cornelia de Lange Syndrome Caused by a Pathogenic Variant in SMC3 and a Literature Review.

Purpose: Cornelia de Lange syndrome (CdLS) is a rare congenital developmental disorder, and cases caused by variants in SMC3 are infrequent. This article describes a case of CdLS related to a pathogenic variant in SMC3 and performs a literature review. Methods: We collected clinical data and biological samples from a 12-year-old boy with "short stature for 11 years". Gene variants in the proband were detected by whole-exome sequencing, and the variants in his parents were verified by Sanger sequencing. All SMC3-related CdLS patients from the PubMed and Web of Science databases were collected and summarized using the available data. Results: A pathogenic variant in SMC3 in the proband, c.1942A>G, was identified. Neither of his parents carried the same variant. Twenty-eight patients were diagnosed with CdLS with variants in SMC3, including the cases in this study and those reported in the literature, where half of the variant types were missense, followed by 32% (9/28) with a deletion and 11% (3/28) with a duplication. All patients showed symptoms of verbal development delay and intellectual disability to different degrees, and 90% patients had long eyelashes while 89% patients had arched eyebrows. Conclusion: This study summarized different gene variants in SMC3 and the frequencies of the various clinical manifestations according to the reported literature. For CdLS caused by SMC3 variants, short stature and facial dysmorphic features are the two most important clinical clues. Definite diagnosis of this rare disease may be challenging clinically; thus, it is significant to use molecular diagnosis.

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Identification of a germline CSPG4 variation in a family with neurofibromatosis type 1-like phenotype.

Neurofibromatosis type 1 (NF1), an autosomal dominant and multisystem disorder, is generally considered to be caused by NF1 inactivation. However, there are also numerous studies showing that Neurofibromatosis type 1-like phenotype can be caused by the abnormalities in the other genes. Through targeted parallel sequencing, whole-exome sequencing, de novo genomic sequencing, and RNA isoform sequencing, we identified a germline V2097M variation in CSPG4 gene probably increased susceptibility to a NF1-like phenotype family. Besides, a series of in vitro functional studies revealed that this variant promoted cell proliferation by activating the MAPK/ERK signaling pathway via hindering ectodomain cleavage of CSPG4. Our data demonstrate that a germline variation in the CSPG4 gene might be a high risk to cause NF1-like phenotype. To our knowledge, this is the first report of mutations in the CSPG4 gene in human diseases.

Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

Transcription-mediated supercoiling regulates genome folding and loop formation.

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.

Integration of transcriptomics and metabolomics reveals anlotinib-induced cytotoxicity in colon cancer cells.

BACKGROUND: Mounting evidences suggested that anlotinib exhibits effective anti-tumor activity in various cancer types, such as lung cancer, glioblastoma and medullary thyroid cancer. However, its function in colon cancer remains to be further revealed. METHODS: Colon cancer cells (HCT-116) were treated with or without anlotinib. Transcript and metabolite data were generated through RNA sequencing and liquid chromatography-tandem mass spectrometry, respectively. The integrated analysis transcriptomics and metabolomics was conducted using R programs and online tools, including ClusterProfiler R program, GSEA, Prognoscan and Cytoscape. RESULTS: We found that differentially expressed genes (DEGs) were mainly involved in metabolic pathways and ribosome pathway. Structural maintenance of chromosome 3 (SMC3), Topoisomerase II alpha (TOP2A) and Glycogen phosphorylase B (PYGB) are the most significant DEGs which bring poor clinical prognosis in colon cancer. The analysis of metabolomics presented that most of the differentially accumulated metabolites (DAMs) were amino acids, such as L-glutamine, DL-serine and aspartic acid. The joint analysis of DEGs and DAMs showed that they were mainly involved in protein digestion and absorption, ABC transporters, central carbon metabolism, choline metabolism and Gap junction. Anlotinib affected protein synthesis and energy supporting of colon cancer cells by regulating amino acid metabolism. CONCLUSIONS: Anlotinib has a significant effect on colon cancer in both transcriptome and metabolome. Our research will provide possible targets for colon cancer treatment using anlotinib.

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth­factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF­mutant CSPG4­positive or ­negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient­derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4­positive cells as compared to pre­therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug­resistance in melanoma cells.

NG2/CSPG4, CD146/MCAM and VAP1/AOC3 are regulated by myocardin-related transcription factors in smooth muscle cells.

The present work addressed the hypothesis that NG2/CSPG4, CD146/MCAM, and VAP1/AOC3 are target genes of myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MKL1, MRTF-B/MKL2) and serum response factor (SRF). Using a bioinformatics approach, we found that CSPG4, MCAM, and AOC3 correlate with MYOCD, MRTF-A/MKL1, and SRF across human tissues. No other transcription factor correlated as strongly with these transcripts as SRF. Overexpression of MRTFs increased both mRNA and protein levels of CSPG4, MCAM, and AOC3 in cultured human smooth muscle cells (SMCs). Imaging confirmed increased staining for CSPG4, MCAM, and AOC3 in MRTF-A/MKL1-transduced cells. MRTFs exert their effects through SRF, and the MCAM and AOC3 gene loci contained binding sites for SRF. SRF silencing reduced the transcript levels of these genes, and time-courses of induction paralleled the direct target ACTA2. MRTF-A/MKL1 increased the activity of promoter reporters for MCAM and AOC3, and transcriptional activation further depended on the chromatin remodeling enzyme KDM3A. CSPG4, MCAM, and AOC3 responded to the MRTF-SRF inhibitor CCG-1423, to actin dynamics, and to ternary complex factors. Coincidental detection of these proteins should reflect MRTF-SRF activity, and beyond SMCs, we observed co-expression of CD146/MCAM, NG2/CSPG4, and VAP1/AOC3 in pericytes and endothelial cells in the human brain. This work identifies highly responsive vascular target genes of MRTF-SRF signaling that are regulated via a mechanism involving KDM3A.