Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

A MTA2-SATB2 chromatin complex restrains colonic plasticity toward small intestine by retaining HNF4A at colonic chromatin.

Plasticity among cell lineages is a fundamental, but poorly understood, property of regenerative tissues. In the gut tube, the small intestine absorbs nutrients, whereas the colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screening, we identify MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrains colonic plasticity. MTA2 loss in the adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-occupies DNA with HNF4A, an activating pan-intestinal transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic chromatin, and accumulation on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through an HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages, and their release unleashes plasticity.

SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

CRIF1 deficiency induces FOXP3LOW inflammatory non-suppressive regulatory T cells, thereby promoting antitumor immunity.

Recently identified human FOXP3lowCD45RA- inflammatory non-suppressive (INS) cells produce proinflammatory cytokines, exhibit reduced suppressiveness, and promote antitumor immunity unlike conventional regulatory T cells (Tregs). In spite of their implication in tumors, the mechanism for generation of FOXP3lowCD45RA- INS cells in vivo is unclear. We showed that the FOXP3lowCD45RA- cells in human tumors demonstrate attenuated expression of CRIF1, a vital mitochondrial regulator. Mice with CRIF1 deficiency in Tregs bore Foxp3lowINS-Tregs with mitochondrial dysfunction and metabolic reprograming. The enhanced glutaminolysis activated α-ketoglutarate-mTORC1 axis, which promoted proinflammatory cytokine expression by inducing EOMES and SATB1 expression. Moreover, chromatin openness of the regulatory regions of the Ifng and Il4 genes was increased, which facilitated EOMES/SATB1 binding. The increased α-ketoglutarate-derived 2-hydroxyglutarate down-regulated Foxp3 expression by methylating the Foxp3 gene regulatory regions. Furthermore, CRIF1 deficiency-induced Foxp3lowINS-Tregs suppressed tumor growth in an IFN-γ-dependent manner. Thus, CRIF1 deficiency-mediated mitochondrial dysfunction results in the induction of Foxp3lowINS-Tregs including FOXP3lowCD45RA- cells that promote antitumor immunity.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with colorectal cancer progression.

Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of ß-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

Triptolide inhibits esophageal squamous cell carcinoma progression by regulating the circNOX4/miR-153-3p/SATB1 signaling pathway.

BACKGROUND: To explore the role and mechanism of triptolide in regulating esophageal squamous cell carcinoma (ESCC) progression by mediating the circular RNA (circRNA)-related pathway. METHODS: The expression levels of circNOX4, miR-153-3p and special AT-rich sequence binding protein-1 (SATB1) were measured by qRT-PCR. Cell proliferation was confirmed by cell counting kit-8 assay and colony formation assay. Flow cytometry was employed to measure cell apoptosis and cell cycle process. Moreover, cell migration and invasion were detected using transwell assay. The protein levels of epithelial-mesenchymal transformation markers and SATB1 were determined by western blot analysis. Furthermore, dual-luciferase reporter assay and RIP assay were performed to confirm the interaction between miR-153-3p and circNOX4 or SATB1. Xenograft tumor models were built to verify the effects of triptolide and circNOX4 on ESCC tumor growth. RESULTS: CircNOX4 was highly expressed in ESCC tissues and cells, and its expression could be reduced by triptolide. Triptolide could inhibit ESCC proliferation, cell cycle process, migration, invasion, EMT process, and promote apoptosis, while these effects were reversed by circNOX4 overexpression. MiR-153-3p could be sponged by circNOX4, and the promotion effect of circNOX4 on the progression of triptolide-treated ESCC cells was abolished by miR-153-3p overexpression. SATB1 was a target of miR-153-3p. Also, SATB1 knockdown reversed the enhancing effect of miR-153-3p inhibitor on the progression of triptolide-treated ESCC cells. Triptolide reduced ESCC tumor growth by regulating the circNOX4/miR-153-3p/SATB1 axis. CONCLUSION: Triptolide could hinder ESCC progression, which was mainly achieved by regulating the circNOX4/miR-153-3p/SATB1 axis.

SATB2 organizes the 3D genome architecture of cognition in cortical neurons.

The DNA-binding protein SATB2 is genetically linked to human intelligence. We studied its influence on the three-dimensional (3D) epigenome by mapping chromatin interactions and accessibility in control versus SATB2-deficient cortical neurons. We find that SATB2 affects the chromatin looping between enhancers and promoters of neuronal-activity-regulated genes, thus influencing their expression. It also alters A/B compartments, topologically associating domains, and frequently interacting regions. Genes linked to SATB2-dependent 3D genome changes are implicated in highly specialized neuronal functions and contribute to cognitive ability and risk for neuropsychiatric and neurodevelopmental disorders. Non-coding DNA regions with a SATB2-dependent structure are enriched for common variants associated with educational attainment, intelligence, and schizophrenia. Our data establish SATB2 as a cell-type-specific 3D genome modulator, which operates both independently and in cooperation with CCCTC-binding factor (CTCF) to set up the chromatin landscape of pyramidal neurons for cognitive processes.

Loss of SATB2 and CDX2 expression is associated with DNA mismatch repair protein deficiency and BRAF mutation in colorectal cancer.

The relationship between the expression of the SATB2 and CDX2 proteins and common molecular changes and clinical prognosis in colorectal cancer (CRC) still needs further clarification. We collected 1180 cases of CRC and explored the association between the expression of SATB2 and CDX2 and clinicopathological characteristics, molecular alterations, and overall survival of CRC using whole-slide immunohistochemistry. Our results showed that negative expression of SATB2 and CDX2 was more common in MMR-protein-deficient CRC than in MMR-protein-proficient CRC (15.8% vs. 6.0%, P = 0.001; 14.5% vs. 4.0%, P = 0.000, respectively). Negative expression of SATB2 and CDX2 was more common in BRAF-mutant CRC than in BRAF wild-type CRC (17.2% vs. 6.1%, P = 0.003; 13.8% vs. 4. 2%; P = 0.004, respectively). There was no relationship between SATB2 and/or CDX2 negative expression and KRAS, NRAS, and PIK3CA mutations. The lack of expression of SATB2 and CDX2 was associated with poor histopathological features of CRC. In multivariate analysis, negative expression of SATB2 (P = 0.030), negative expression of CDX2 (P = 0.043) and late clinical stage (P = 0.000) were associated with decreased overall survival of CRC. In conclusion, the lack of SATB2 and CDX2 expression in CRC was associated with MMR protein deficiency and BRAF mutation, but not with KRAS, NRAS and PIK3CA mutation. SATB2 and CDX2 are prognostic biomarkers in patients with CRC.

TOX, TWIST1, STAT4, and SATB1 protein expressions in early-stage mycosis fungoides.

BACKGROUND: Diagnosis of early mycosis fungoides (eMF) is challenging and often delayed as many of its clinical and histopathologic features may mimic various benign inflammatory dermatoses (BIDs). The products of the thymocyte selection-associated high mobility group box (TOX), twist family BHLH transcription factor 1 (TWIST1), signal transducer and activator of transcription 4 (STAT4), and special AT-rich sequence-binding protein 1 (SATB1) genes function as transcription factors and are involved in the pathogenesis of MF. OBJECTIVES: We aim to determine the diagnostic value of TOX, TWIST1, STAT4, and SATB1 protein expressions in eMF. METHODS: This non-randomized, controlled, prospective analytic study was conducted by performing immunohistochemistry staining with TOX, TWIST1, STAT4, and SATB1 polyclonal antibodies in lesional skin biopsies of eMF and BID patients. Nuclear staining of lymphocytes was compared between eMF and BIDs, and the capacity of these antibodies to predict eMF was determined. RESULTS: Immunostainings with anti-TWIST1 showed an increase in protein expression (p = 0.003) and showed a decrease with anti-SATB1 antibodies in eMF compared to BIDs (p = 0.005) while anti-TOX and anti-STAT4 antibodies did not exhibit significant differences (p = 0.384; p = 0.150). Receiver operating characteristic analysis showed that immunohistochemical evaluations of TWIST1 and SATB1 protein expressions can differentiate eMF (area under the curve [AUC]: 0.728, 95% confidence interval [CI]: 0.605-0.851, p = 0.002; AUC: 0.686, 95% CI: 0.565-0.807, p = 0.013). CONCLUSIONS: TWIST1 and SATB1 are potential diagnostic markers for the histologic diagnosis of eMF.

Long non-coding RNA LINC00491 accelerates head and neck squamous cell carcinoma progression through regulating miR-508-3p/SATB1 axis and activating Wnt signaling pathway.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy of the head and neck epidermis. Accumulating long non-coding RNAs (lncRNAs) have been proven to be involved in the occurrence and development of HNSCC. LncRNA long intergenic non-protein coding RNA 491 (LINC00491) has been confirmed to regulate the progression of some cancers. In our study, we aimed to explore the potential biological function of LINC00491 and expound the regulatory mechanism by which LINC00491 affects the progression of HNSCC. RT-qPCR was utilized to analyze the expression of LINC00491 in HNSCC cell lines and the normal cell line. Functionally, we carried out a series of assays to measure cell proliferation, apoptosis, migration and invasion, such as EdU assay, colony formation, wound healing and western blot assays. Also, mechanism assays including RNA pull down and RIP were also implemented to investigate the interaction of LINC00491 and RNAs. As a result, we discovered that LINC00491 was highly expressed in HNSCC cells. In addition, LINC00491 depletion suppressed cell proliferation, migration and EMT process. Furthermore, we discovered that LINC00491 could bind to miR-508-3p. MiR-508-3p overexpression can restrain HNSCC cell growth. Importantly, miR-508-3p can target SATB homeobox 1 (SATB1) in HNSCC cells. Further, Wnt signaling pathway was proved to be activated by LINC00491 through SATB1 in HNSCC cells. In a word, LINC00491 accelerated HNSCC progression through regulating miR-508-3p/SATB1 axis and activating Wnt signaling pathway.

A dual function for the chromatin organizer Special A-T rich Binding Protein 1 in B-lineage cells.

SATB1 (Special A-T rich Binding protein 1) is a cell type-specific factor that regulates the genetic network in developing T cells and neurons. In T cells, SATB1 is required for lineage commitment, VDJ recombination, development and maturation. Considering that its expression varies during B-cell differentiation, the involvement of SATB1 needs to be clarified in this lineage. Using a KO mouse model in which SATB1 was deleted from the pro-B-cell stage, we examined the consequences of SATB1 deletion in naive and activated B-cell subsets. Our model indicates first, unlike its essential function in T cells, that SATB1 is dispensable for B-cell development and the establishment of a broad IgH repertoire. Second, we show that SATB1 exhibits an ambivalent function in mature B cells, acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells, respectively. Third, our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response, in which this factor limits somatic hypermutation of Ig genes.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

The role of transcription factors in shaping regulatory T cell identity.

Forkhead box protein 3-expressing (FOXP3+) regulatory T cells (Treg cells) suppress conventional T cells and are essential for immunological tolerance. FOXP3, the master transcription factor of Treg cells, controls the expression of multiples genes to guide Treg cell differentiation and function. However, only a small fraction (<10%) of Treg cell-associated genes are directly bound by FOXP3, and FOXP3 alone is insufficient to fully specify the Treg cell programme, indicating a role for other accessory transcription factors operating upstream, downstream and/or concurrently with FOXP3 to direct Treg cell specification and specialized functions. Indeed, the heterogeneity of Treg cells can be at least partially attributed to differential expression of transcription factors that fine-tune their trafficking, survival and functional properties, some of which are niche-specific. In this Review, we discuss the emerging roles of accessory transcription factors in controlling Treg cell identity. We specifically focus on members of the basic helix-loop-helix family (AHR), basic leucine zipper family (BACH2, NFIL3 and BATF), CUT homeobox family (SATB1), zinc-finger domain family (BLIMP1, Ikaros and BCL-11B) and interferon regulatory factor family (IRF4), as well as lineage-defining transcription factors (T-bet, GATA3, RORγt and BCL-6). Understanding the imprinting of Treg cell identity and specialized function will be key to unravelling basic mechanisms of autoimmunity and identifying novel targets for drug development.

Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s.

The genome organizer, special AT-rich binding protein-1 (SATB1), functions to globally regulate gene networks during primary T cell development and plays a pivotal role in lineage specification in CD4+ helper-, CD8+ cytotoxic-, and FOXP3+ regulatory-T cell subsets. However, it remains unclear how Satb1 gene expression is controlled, particularly in effector T cell function. Here, by using a novel reporter mouse strain expressing SATB1-Venus and genome editing, we have identified a cis-regulatory enhancer, essential for maintaining Satb1 expression specifically in TH2 cells. This enhancer is occupied by STAT6 and interacts with Satb1 promoters through chromatin looping in TH2 cells. Reduction of Satb1 expression, by the lack of this enhancer, resulted in elevated IL-5 expression in TH2 cells. In addition, we found that Satb1 is induced in activated group 2 innate lymphoid cells (ILC2s) through this enhancer. Collectively, these results provide novel insights into how Satb1 expression is regulated in TH2 cells and ILC2s during type 2 immune responses.

Increased GS-II lectin binding and SATB2 downregulation are biological features for sessile serrated lesions and microvesicular hyperplastic polyps.

Sessile serrated lesions (SSLs) and microvesicular hyperplastic polyps (MVHPs) are colorectal lesions displaying gastric differentiation. Griffonia simplicifolia-II (GS-II) is a lectin specific to terminal α/ßGlcNAc residues. Here, we assessed GS-II binding and performed immunostaining for HIK1083 (specific to terminal αGlcNAc residues), MUC5AC, MUC6, and special AT-rich sequence binding protein 2 (SATB2) in SSLs, MVHPs, and tubular adenomas (TAs). We observed MUC5AC positivity in 28 of 30 SSLs, but in only three of 23 TAs. Moreover, 24 of 30 SSLs were MUC6-positive, while none of the 23 TAs were MUC6-positive. None of the 30 SSLs or 23 TAs showed HIK1083 positivity. All 30 SSLs and 26 MVHPs were GS-II-positive, while only seven of 23 were in TAs. GS-II staining was mainly distributed in the Golgi region, but SSLs and MVHPs showed goblet cell distribution, in 20 of 30 and 19 of 26 cases, respectively. All SSLs, MVHPs, and TAs were SATB2-positive, but 21 of 30 SSLs and 12 of 26 MVHPs showed decreased staining intensity relative to adjacent mucosa, a decrease seen in only two of 23 in TAs. These results indicate overall that increased terminal ßGlcNAc and decreased SATB2 expression are characteristics of SSLs and MVHPs.

SATB1 Chromatin Loops Regulate Megakaryocyte/Erythroid Progenitor Expansion by Facilitating HSP70 and GATA1 Induction.

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

SATB1 regulates 3D genome architecture in T cells by constraining chromatin interactions surrounding CTCF-binding sites.

Special AT-rich sequence binding protein 1 (SATB1) has long been proposed to act as a global chromatin loop organizer in T cells. However, the exact functions of SATB1 in spatial genome organization remain elusive. Here we show that the depletion of SATB1 in human and murine T cells leads to transcriptional dysregulation for genes involved in T cell activation, as well as alterations of 3D genome architecture at multiple levels, including compartments, topologically associating domains, and loops. Importantly, SATB1 extensively colocalizes with CTCF throughout the genome. Depletion of SATB1 leads to increased chromatin contacts among and across the SATB1/CTCF co-occupied sites, thereby affecting the transcription of critical regulators of T cell activation. The loss of SATB1 does not affect CTCF occupancy but significantly reduces the retention of CTCF in the nuclear matrix. Collectively, our data show that SATB1 contributes to 3D genome organization by constraining chromatin topology surrounding CTCF-binding sites.

Expression of SATB2 in primary cutaneous sarcomatoid neoplasms: a potential diagnostic pitfall.

SATB2 can be used as an immunohistochemical marker for osteoblastic differentiation. The differential diagnosis of a cutaneous sarcomatoid neoplasm sometimes includes osteosarcoma when the tumour concomitantly involves the skin, soft tissue, and bone, or when there is a past medical history of osteosarcoma. As the utility of SATB2 immunohistochemistry in these scenarios was unclear, we aimed to determine the frequency and the pattern of SATB2 expression in a variety of cutaneous sarcomatoid neoplasms. SATB2 expression by immunohistochemistry was evaluated by intensity (0-3) and extent (0-100%) of staining to generate an h-score for each case. Expression levels were classified into high-positive (h-score ≥100), low-positive (20-99), and negative (<20) groups. Positive SATB2 expression was observed in 18/23 (78%) atypical fibroxanthomas (AFX), 10/19 (53%) pleomorphic dermal sarcomas, 9/20 (45%) cutaneous sarcomatoid squamous cell carcinomas, 14/39 (36%) sarcomatoid melanomas, 2/13 (15%) poorly differentiated cutaneous angiosarcomas, 10/17 (59%) high-grade cutaneous leiomyosarcomas, and 7/8 (88%) osteosarcoma controls. With the exception of AFX, all cutaneous neoplasms showed significantly lower average h-scores than osteosarcoma. AFX gave the highest average h-score (71) and percentage of high-positive cases (48%) among all examined cutaneous neoplasms. Only two (1.5%) of all cutaneous cases showed strong intensity of staining. Common SATB2 expression in various cutaneous sarcomatoid neoplasms poses a potential diagnostic pitfall when the differential diagnosis includes osteosarcoma. Requirement of strong staining and a high-positive h-score improves the specificity of SATB2 in differentiating these tumours from osteosarcoma.

SAFB2 Inhibits the Progression of Breast Cancer by Suppressing the Wnt/ß-Catenin Signaling Pathway via NFAT5.

Aberrant scaffold attachment factor-B2 (SAFB2) expression is associated with several malignant tumors. In this study, we investigated how SAFB2 worked in the process of breast cancer as well as the underlying mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis were used to investigate the expression of SAFB2 and nuclear factor of activated T cells 5 (NFAT5). Cellular proliferative ability was detected with cell counting kit 8 (CCK8), colony formation and 5-Ethynyl-2'-deoxyuridine (EdU) staining assays. Cell apoptosis was measured via flow cytometry and western blotting analysis. Wound healing, transwell assays, and western blotting analysis were executed to estimate cell migration and invasion. The relationship between SAFB2 and NFAT5 was verified by RNA immunoprecipitation (RIP) assay and NFAT5 mRNA stability was examined with actinomycin (Act) D assay. Western blotting analysis also tested the expression of Wnt/ß-catenin signaling-associated proteins. As a result, SAFB2 was downregulated in breast cancer cell lines, while NFAT5 was highly expressed in most breast cancer cell lines. Overexpression of SAFB2 suppressed the proliferation, migration, and invasion while exacerbated the apoptosis of breast cancer cells. SAFB2 interacted with NFAT5 mRNA and declined the stability of NFAT5 mRNA. Overexpression of NFAT5 counteracted anti-proliferative, anti-metastatic and pro-apoptotic effects of SAFB2 in breast cancer cells. Mechanistically, SAFB2 overexpression inhibited the Wnt/ß-catenin signaling pathway, while this effect was partially eliminated by NFAT5. Collectively, SAFB2 hindered breast cancer development and inactivated Wnt/ß-catenin signaling via regulation of NFAT5, suggesting that SAFB2 might be a promising therapeutic target for breast cancer.