Special AT-rich Sequence-binding Protein 1 Protein Research Articles

Upregulation of ubiquitin carboxy‑terminal hydrolase 47 (USP47) in papillary thyroid carcinoma exvivo and reduction of tumor cell malignant behaviors after USP47 knockdown by stabilizing SATB1 expression invitro.

Aberrant ubiquitination contributes to cancer development, including thyroid carcinoma. The present study assessed the expression of ubiquitin carboxy-terminal hydrolase 47 (USP47) and underlying molecular events in the development of papillary thyroid carcinoma (PTC). The effects of USP47 on PTC cell invasion and migration were analyzed by Transwell assays, while. the effects of USP47 and SATB1on PTC cell gene expression and changes in tumor cell metabolism were assayed by reverse transcription-quantitative PCR, western bolt, or ELISA, respectively. The expression of USP47 mRNA and protein was upregulated in PTC tissue and associated with the PTC tumor size. Knockdown of USP47 expression in PTC cell lines (TPC-1 and K1), decreased the cell proliferation mobility and invasion capacities, whereas USP47 overexpression in these cell lines showed an inverse effect and promoted cell glycolysis and glutamine metabolism. Moreover, expression of special AT-rich sequence-binding protein-1 (SATB1) was high in PTC tissue and was associated with USP47 expression. SATB1 expression promoted tumor cell glycolysis and glutamine metabolism, while USP47 protein bound to and deubiquitinated SATB1 to increase its intracellular levels, thus promoting glycolysis and glutamine metabolism. USP47 promotion of PTC development may be due to its stabilization of SATB1 protein, suggesting that targeting the USP47/SATB1 signaling axis may serve as a therapeutic intervention for PTC.

Elevated Expression of SATB1 Predicts Unfavorable Clinical Outcomes in Colon Adenocarcinoma.

Special AT-rich sequence-binding protein 1 (SATB1) belongs to the chromatin-remodeling protein which regulates different genes expression. High expression of SATB1 was found to be associated with the development of certain carcinomas. However, the functions of SATB1 in colon adenocarcinoma (CAC) remains unclear yet. Our study aims to investigate the potential role of SATB1 in CAC and whether it is associated with the unfavorable symptoms of CAC patients. The expression pattern of SATB1 was measured in CAC samples and adjacent noncancerous samples through quantitative real-time polymerase chain reaction and immunohistochemistry staining. We performed univariate and multivariate analyses to evaluate the clinical role of SATB1 in enrolled patients. The Kaplan-Meier analyses and log-rank tests were carried out to assess the clinicopathologic characteristics. The effect of SATB1 in human colon cancer cells was examined through cellular experiments. The expression level of SATB1 in CAC tissues was significantly elevated compared with adjacent control tissues. High expression of SATB1 in tumor tissue was found to be associated with lymph node metastasis and advanced TNM stage. Higher SATB1 level in CAC patients indicated a worse 5-year survival time. Moreover, high SATB1 was defined as an independent poor prognostic factor. Cellular experiments showed that inhibition of the SATB1 protein level in human colon cells could suppress the migration and invasion capabilities. Our findings revealed that high expression of SATB1 was significantly correlated with the poor clinical features and prognosis of CAC patients. It indicated that SATB1 might serve as a potential prognostic predictor and novel drug target for CAC treatment.

Small Interfering RNA-Mediated Knockdown of SATB1 Increases Cell Proliferation through NF-κb Activation in Acute Myeloid Leukemia

Background: Special AT-rich sequence-binding protein 1(SATB1) is a chromatin-remodeling protein that has been shown to regulate gene expressions and aberrant cell growth in different types of cancer. Up-regulation of SATB1 is linked with progression and metastasis of tumors. It is also reported that SATB1 is involved in the differentiation of hematopoietic lineages. However, less is known about the association between SATB1 and hematology malignancy. We previously reported that SATB1 expressions were decreased in T cell leukemia/lymphoma (T-ALL), and knockdown of SATB1 significantly enhanced invasiveness of Jurkat cell through the activation of Wnt/β-catenin signaling pathway in vitro. The contrary roles of SATB1 in solid organ tumors and hematology malignancy may provide hints to study the function of SATB1. Here, we investigated the role of SATB1 in acute myeloid leukemia (AML).Methods: To characterize SATB1 mRNA and protein expression in AML, we performed qRT-PCR and Western blot on peripheral blood mononuclear cells (PBMCs) from 52 newly diagnosed AML patients, as well as AML cell lines NB4, HEL, THP-1 and HL-60. Stable HL-60 cell lines with knockdown of SATB1 by shRNAs sequences (HL-60 SATB1-shRNA1 and HL-60 SATB1-shRNA2) were established. CCK-8 cell proliferation assay and flow cytometric analysis were performed to evaluate the cell proliferation and cell cycle. The invasiveness of HL-60 SATB1-shRNAs and HL-60 CTR cells was analyzed and compared by transwell assay. In order to study the role of SATB1 in vivo, murine model was established using HL-60 SATB1-shRNAs treated nude mice and tumorigenicity was comopared by tumor size. After demonstrating the association between SATB1 knockdown and tumorigenicity in vitro and in vivo, we next investigated NF-κB, AKT and MAPK signaling pathways by western blot.Results:l. SATB1 expression was significantly lower in AML patients than normal controls, which is similar to what we found in T-ALL patients. SATB1 expression was different before and after treatment, especially after complete remission (CR) of AML. Increased SATB1 expression was associated with better clinical outcome. We further investigated the roles of SATB1 on AML cells by shRNAs mediated knockdown of SATB1. Knockdown of SATB1 promoted the proliferation of HL-60 SATB1 shRNA1 cells and HL-60 SATB1 shRNA2 cells compared to the control. However, the transwell assay did not show increased invasiveness in SATB1 depleted HL-60 cells. The in vivo experiment showed that tumor size and weight in SATB1 knockdown mice were significantly increased compared to the control mice. In the mechanism study, we investigated NF-κB, AKT and MAPK signaling pathways by western blot. We found that the expression of NF-κB p65 in HL60 SATB1-shRNAs cells was increased, which suggested that SATB1 regulated AML cell growth via the activation of NF-κB signaling pathway.Conclusions: Impairment of SATB1 is associated with AML. SATB1 expressions were decreased in AML. Knockdown of SATB1 promoted cell proliferation and tumorigenicity of AML via the activation of NF-κB signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

SATB1 Expression of Colorectal Adenomatous Polyps is Higher than that of Colorectal Carcinomas.

"Special AT-rich sequence-binding protein-1" (SATB1) is a global genome organizer and is found to have effects on carcinogenesis and progression of various malignancies including colorectal carcinoma (CRC). We aimed to investigate the expression of SATB1 in CRC and colorectal adenomatous polyps (CAP), the correlation between clinicopathologic parameters, and overall survival. We examined 227 CRCs and 129 CAPs. SATB1 protein expression was evaluated by immunohistochemistry. We found higher SATB1 expression in adenomatous epithelium than in CRC tissues (55.0% vs. 42.7%, respectively) (P<0.05). None of the adjacent normal colorectal mucosa stained positive in CRC cases, and only one of the adjacent normal mucosa of the CAP cases was positive. SATB1 expression of left-sided CRC was higher than that of right-sided CRC (46.3% vs. 28.6%, respectively) (P<0.05), and SATB1 expression of conventional adenocarcinomas was higher than that of mucinous carcinomas (45.5% vs. 6.3%, respectively) (P<0.05). SATB1 expression was higher in CAPs consisting of high-grade dysplasia than in polyps with low-grade dysplasia (77.8% vs. 51.4%) (P<0.05). SATB1 expression did not correlate with patients' overall survival. In conclusion, due to the higher expression of SATB1 in CAP than in CRC, we think SATB1 may have a role in the early stages of carcinogenesis of CRCs. This is the first study investigating SATB1 expression in CAPs. Besides this is the first report that shows different SATB1 expressions in conventional colorectal adenocarcinoma and mucinous carcinoma, and also in right-sided and left-sided CRC. Our results, with supporting new studies, can provide SATB1 as a possible candidate for targeted therapy for CRC patients.

PF462 SPECIAL AT‐RICH SEQUENCE BINDING PROTEIN 1 (SATB1) IS MARKEDLY EXPRESSED IN THE DARK ZONE GERMINAL CENTER B CELLS ON MURINE SPLEEN

Background:Special AT‐rich Sequence Binding Protein 1 (SATB1) is known as a protein that binds to the AT‐rich region near the enhancer of various genes. SATB1 is expressed predominantly in the thymus, and controls T lineage‐specific gene transcription by organizing chromatin configurations in the cell nucleus. In addition, we previously reported that SATB1 plays important roles in the early differentiation of hematopoietic stem cells (HSCs) (Immunity, 2013; Cell Reports, 2018). Indeed, exogenous induction of SATB1 in HSCs enhances both T and B lymphopoietic potentials, whereas SATB1 deficiency skews HSC differentiation toward myeloid lineage at the expense of lymphocyte production. Furthermore, SATB1 also associates with the enhancer of immunoglobulin heavy chain gene. Thus, SATB1 plays a pivotal role in various aspects of lymphopoiesis, but its significance in the B lineage development has yet to be determined.Aims:We performed this experiment to know the role of SATB1 in the B lineage development.Methods:We used SATB1/Tomato reporter mice to monitor the precise SATB1 expression during B cell maturation in vivo. We also generated B cell‐specific SATB1 conditional knock out mice using Cre‐loxP‐mediated inactivation of SATB1. We crossed SATB1‐flox mice with mice expressing Cre recombinase under the control of the Mb1 gene promotor.Results:At first, we analyzed the B lineage development of SATB1/Tomato reporter mice. In bone marrow (BM), while markedly increasing with early lymphoid differentiation, SATB1/Tomato expression decreased in pro‐B and pre‐B stages and almost diminished in surface IgM+ immature B cells. However, SATB1/Tomato expression was revived after immature B cells migrated to the spleen. The expression of SATB1/Tomato increased with the maturation from T1 to T3 transitional splenic B cells. The up‐regulation of SATB1/Tomato in developing B cells positively correlated with IgD expression. Indeed, while transcripts for SATB1 gene were barely detected in the splenic IgM+IgD− stage, they were actively induced in IgM+IgD+ cells and became abundant in IgM−IgD+ B cells. When we incubated murine splenic B cells with an anti‐IgM antibody in vitro, SATB1/Tomato+ IgD+ population was significantly induced. Incubation of murine splenic B cells with anti‐IgM and anti‐CD40 also induced the same population. These results suggest that B cell receptor signaling induces SATB1 expression along with the transition of IgM to IgD. The acquisition of antigens by naive B cells triggers the formation of germinal centers (GCs). The mature GC is divided into two compartments, called the light zone (LZ) and the dark zone (DZ). The naïve B cells located in LZ receive the positive selection of higher‐affinity B cells by CD4+ helper T cells and migrate to DZ by expressing the DZ‐homing receptor CXCR4. The B cells in DZ are highly proliferative. The analysis of SATB1/Tomato reporter mice showed that SATB1+ IgD+ splenic B cells had a significantly higher expression of CXCR4 than the SATB1− IgD+ cells. In addition, CD83, the activation marker of LZ B cells, decreased in IgD+ B cells with increased expression of SATB1. The above results imply that the SATB1 protein might be involved in the modulation of Ig heavy chain and the formation of DZ in GC during splenic B cell maturation. To test this hypothesis, we analyzed Mb1‐Cre:SATB1 Flox/Flox mice. The histological analysis showed that the follicles of the spleen in Mb1‐Cre: SATB1 Flox/Flox mice are enlarged compared to those in control SATB1 Flox/Flox mice.Summary/Conclusion:With these results, we conclude that SATB1 plays some role in the formation of GCs.

Expression of special AT rich sequence binding protein-1 and proliferation cell nuclear antigen gene in transitional cell carcinomas of bladder and clinical significances

Objective To investigate the significance of the expression of special AT rich sequence binding protein-1 (SATB1) and proliferation cell nuclear antigen (Ki-67) gene in transitional cell carcinoma of bladder (BTCC). Methods The expression of SATB1 and Ki-67 was detected by immunohistochemistry in 60 cases of bladder carcinoma tissues and 30 cases of normal bladder tissues respectively. Then their relationship with pathological grade, clinical stage were analyzed. The correlation between SATB1 and Ki-67 was also evaluated. Results The positive rate of SATB1 protein in bladder carcinoma was 85.0% (51/60), significantly higher than that in the normal bladder tissues (P=0.000). The positive rate of Ki-67 protein in human bladder carcinoma was 80.0% (48/60), obviously higher than that in the normal bladder tissue (P=0.000). The amounts of SATBl and Ki-67 were positively correlated with the pathological grades (P=0.001, 0.002), clinical stages (P=0.003, 0.007)and lymphatic metastasis (P=0.001, 0.002) of BTCC. There was positive correlation between SATB1 and Ki-67 gene expression in bladder carcinoma tissue (r=0.601, P=0.002). Conclusion Positive expression of SATB1 and Ki-67 gene may play an important role in the development of BTCC. SATB1 and Ki-67 gene may serve as an important molecular biological indicator in diagnosing and predicating the biological behaviors of BTCC. Key words: Bladder carcinoma; Special AT rich sequence binding protein-1; Proliferation cell nuclear antigen

Silencing SATB1 overcomes temozolomide resistance by downregulating MGMT expression and upregulating SLC22A18 expression in human glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system and has a very poor prognosis. Currently, patients were treated by resection followed by radiotherapy plus concurrent temozolomide (TMZ) chemotherapy. However, many patients are resistant to TMZ-induced DNA damage because of upregulated expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). In this study, upregulation of SATB1 and MGMT, and downregulation of SLC22A18 resulted in acquisition of TMZ resistance in GBM U87 cells. Inactivation of special AT-rich sequence-binding protein 1 (SATB1) using short hairpin RNA (shRNA) downregulated MGMT expression and upregulated solute carrier family 22 member 18 (SLC22A18) expression in GBM cells. This suggested SATB1-mediated posttranscriptional regulation of the MGMT and SLC22A18 protein levels. Immunohistochemical analysis of malignant glioma specimens demonstrated a significant positive correlation between the levels of MGMT and SATB1, and a negative correlation between the levels of SLC22A18 and SATB1. Importantly, in recurrent, compared with the primary, lesions in 15 paired identical tumors, the SATB1 and MGMT protein levels were increased and the SLC22A18 levels were decreased. Finally, in TMZ-resistant GBM, SATB1 knockdown enhanced TMZ efficacy. Consequently, SATB1 inhibition might be a promising strategy combined with TMZ chemotherapy to treat TMZ-resistant GBM.

Inhibition of prostate cancer DU145 cell growth with small interfering RNA targeting the SATB1 gene.

Prostate cancer is a common visceral cancer of men worldwide. It is important to develop a more effective treatment for prostate cancer to overcome the treatment resistance that occurs with recurrence. RNA interference has been demonstrated to be a powerful tool for gene knockdown and has potential as a cancer treatment. It has been previously demonstrated that staining of special AT-rich sequence-binding protein 1 (SATB1) was stronger in prostatic carcinoma with metastasis compared with prostatic carcinoma without metastasis. In the present study, SATB1 small interfering (si)RNA was transfected into prostate cancer DU145 cells and normal human lung fibroblast cells, and cell proliferation was investigated using a Cell Counting kit-8. Three siRNA were transfected into cells using siPORT Lipid Transfection agent, and blank control and negative control groups were established. The cells were harvested and SATB1 mRNA and protein expression was determined by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. DU145 cell adhesion, migration and invasion capabilities were determined using cell adhesion, Transwell and Transwell with Matrigel assays, respectively. Silencing SATB1 significantly inhibited DU145 cell growth, adhesion, migration and invasive capability in vitro, indicating that a SATB1-targeting siRNA was successfully engineered. The results of the present study suggest that SATB1 siRNA may be a potential agent for treating human prostate cancer.

The interactions between SATB1 and F-actin are important for mechanisms of active cell death.

The direct involvement of nuclear actin filaments in gene transcription and remodeling of chromatin is still debatable. However, nuclear localization of F-actin and its interactions with other nuclear matrix proteins have been reported. The aim of the study was to estimate the interactions between nuclear F-actin and one of the matrix proteins, special AT-rich sequence-binding protein 1 (SATB1), during active cell death induced in vitro by geldanamycin (GA). The expression of SATB1 was modified by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against SATB1 or expression plasmid with cloned cDNA of SATB1. The amount and localization of F-actin were altered by changes of cofilin-1 (CFL1) expression in MCF-7 cells. The association between SATB1 and F-actin during GA-induced cell death was analyzed using confocal and transmission electron microscopy. Our studies revealed the colocalization between nuclear F-actin and SATB1 protein, during GA-induced death of breast cancer MCF-7 cells. The colocalization was enhanced in cells with overexpressed SATB1 and cofilin-1. At the ultrastructural level the SATB1 and F-actin complexes were seen at the border of condensed and decondensed chromatin. The presence of SATB1/F-actin molecular complexes was confirmed by magnetic separation of F-actin and interacting proteins. We suggest that the molecular interactions between SATB1 and F-actin are necessary for active cell death to occur.

Divergent expression patterns of SATB1 mRNA and SATB1 protein in colorectal cancer and normal tissues.

Special AT-rich sequence-binding protein 1 (SATB1) is a ‘genome organizer,’ and it has been proposed as a factor that affects the development and progression of various human neoplasms, including colorectal cancer (CRC). This study aimed to compare SATB1 expression in a group of CRC patients and healthy subjects at the mRNA and protein levels. We collected paired tumor tissue and unchanged mucosa of the large intestine from 102 CRC patients as well as 53 biopsies of normal colon mucosa obtained from healthy patients during screening colonoscopy. Tissue samples were quantified for SATB1 mRNA by quantitative PCR, while SATB1 protein expression was determined by Western blotting and immunohistochemistry. SATB1 mRNA level in tumor tissues was over twofolds lower than in samples of corresponding unchanged tissues and fourfolds lower than in biopsies of healthy colon mucosa. Western blotting analysis revealed that SATB1 protein content in tumor and unchanged tissues of CRC patients was over sixfold and fivefolds higher than in biopsies of healthy colon mucosa, respectively. Immunohistochemical staining demonstrated higher nuclear and cytoplasmic SATB1 reactivity in the tumor tissue compared to unchanged mucosa of CRC patients. Despite these differences, SATB1 mRNA, protein, and immunoreactivity levels did not correlate with patients’ clinicopathological data and their overall survival, but the latter analysis was limited by a relatively short period of follow-up. In conclusion, we suggest that some as yet unidentified posttranscriptional mechanisms that regulate SATB1 expression may be altered in the CRC tissue.

Expression and significance of SATB1 in the development of breast cancer.

Special AT-rich sequence binding protein 1 (SATB1) is a recently discovered gene regulator that can promote the growth and metastasis of breast cancer. However, its expression in different stages of breast cancer development have not been examined. We explored the role of SATB1 in the development of breast cancer by detecting SATB1 expression levels in different stages of breast cancer. SATB1 expression was determined using an immunohistochemical streptavidin peroxidase method; the relationship between clinicopathological features of breast cancer and SATB1 expression was analyzed using the X(2) test. Positive rates of SATB1 protein in normal breast tissue, normal breast ductal hyperplasia tissue, precancerous lesions of breast cancer, non-invasive cancer, early invasive carcinoma, and invasive breast cancer tissue were, respectively, 6.25 (2/32), 6.4 (3/47), 20.4 (10/49), 45.0 (9/20), 52.9 (9/17), and 76.6% (72/94). SATB1 in the latter 3 groups was significantly higher than in the first 3 groups (P < 0.05). The positive rate of SATB1 protein in invasive non-special types of breast cancer (88.5%, 54/61) was significantly higher than in the special type of invasive breast cancer (54.5%, 18/33) and early invasive breast cancer (52.9%, 9/17) (P < 0.05). SATB1 protein expression in breast cancer with lymph node metastasis was generally increased, and the difference was statistically significant (P < 0.05). SATB1 protein expression showed an increasing trend in different stages of breast cancer development. Overexpression indicated poor prognosis.

Decreased SATB1 Expression in ALT and Promoted Invasion of Jurkat Cell in Vitro

BACKGROUND: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types cancer. However, their expression patterns and function values for adult T-cell leukemia (ALT) are still largely unknown.METHODS: Expression of SATB1 in normal and ALT peripheral blood leucocytes (n=50 for normal, n=30 for ALT) were evaluated by RT-qPCR in mRNA level. Expression of protein SATB1 in leukemia cell lines were evaluated by Weston Blot.SATB1-shRNA was constructed in psuper-retro-puro retrovirus vector. Jurkat-SATB1-sh and control stable cell lines were established after virus infection and puromycin selection. Cellular proliferation and invasion of Jurkat-ctr and SATB1 knockdown Jurkat cells were evaluated by CCK-8 kit and transwell matrigel invasion assay, respectively.RESULTS: SATB1 mRNA expression was decreased in ALT (p<0.001) compared with normal peripheral blood leucocytes cells. SATB1 protein expression in Jurkat cell was higher than other leukemia cell lines. SATB1-shRNA was constructed successfully. Weston Blot show Jurkat-SATB1-sh and control stable cell lines were established successfully. Knockdown of SATB1 expression significantly enhanced invasion of Jurkat cell in vitro but no effect in proliferationCONCLUSIONS: Decreased expression of SATB1 in ALT was associated with cell invasion. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5199: Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer

Abstract Recently, genome organizer protein special AT-rich sequence-binding protein 1(SATB1) has been reported to promote a metastatic phenotype and correlate with poor prognosis in breast cancer. The mechanism of SATB1 elevation in breast cancer remains elusive. This study was to investigate whether microRNAs (miRNAs) play a role in modulating SATB1 expression in ductual breast cancer by using archival formalin-fixed and paraffin-embedded (FFPE) clinical samples. Firstly, four canonical target prediction databases, including TargetScan, PicTar, miRanda and miRDB, were utilized to predict the interaction between miRNA and the 3′-UTR of SATB1 mRNA. The results of the prediction software programs were then integrated by displaying Venn diagram. Secondly, the Qiagen miRNeasy FFPE kit was used to isolate total RNA from 93 specimens with primary invasive ductual breast cancer. Expression of candidate miRNA transcriptional levels was measured using TaqMan MicroRNA Assays with miRNA-specific stem loop primers by relative real-time quantitative RT-PCR. The relative levels of miRNA expression were calculated from the relevant signals by normalization with the signal for RNU6B miRNA expression in each sample. Meanwhile, SATB1 protein expression levels were assessed by immunohistochemistry(IHC). Finally, correlations between the expression levels of these miRNAs and SATB1 were analyzed. Overall 22 miRNAs, 24 miRNAs, 76 miRNAs and 75 miRNAs were found by TargetScan, PicTar, miRanda and miRDB, to target SATB1 respectively, whereas only miR-7, miR-21, miR-23a, miR-23b, miR-34a and miR-155 were found by all four programs to be predicted to regulate SATB1. Quantitative RT-PCR detection analysis showed that expression levels of miR-34a were much higher in SATB1-negative than in SATB1-positive tumors (p&amp;lt;0.001). whereas lower miR-7 expression showed a strong trend toward an association with elevated SATB1 expression (P = 0.056). We then extended the analysis of miR-34a expression in breast cancer tissues to include all scores for SATB1 protein expression, assessed semiquantitatively by IHC. miR-34a expression levels were gradually decreased as SATB1 protein scores increased. Moreove, our quantitative RT-PCR results showed no differences in miR-21, miR-23a, miR-23b, and miR-155 expression between SATB1-negative and SATB1-positive tumors. We thus conclude that SATB1 maybe a novel target of miR-34a in human breast cancer. Downregulation of miR-34a promotes human breast cancer to progress through reduced repression of SATB1. Restoration of miR-34a may prove therapeutic in breast cancer patients in which aberrant SATB1 expression plays a role. Note: This abstract was not presented at the meeting. Citation Format: Weiming Duan, Zixing Chen, Li Yao, Min Tao, Kai Chen, Wei Liu, Jiannong Cen. Distinct expression pattern of microRNAs bioinformatically predicted to target SATB1 in human breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2014-5199

Influence of special AT-rich sequence binding protein 1 silencing on the invasion of glioma U251 cells

Objective To study the effect of special AT-rich sequence binding protein 1 (SATB1) short hairpin RNA (shRNA) on the invasion of human glioma cells,and explore its mechanism.Methods The recombinant plasmid of small hairpin RNA targeting SATB1 gene was constructed,and transfected into glioma U251 cells by electroporation.Untransfected group,control-shRNA-green fluorescent protein (GFP) group and SATB1-shRNA group were set up in the experiment.The expression of SATB1 mRNA and protein in U251 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively.The expression of some functional proteins was also studied by Western blotting.Enzyme-linked immunosorbent assay (ELISA) was used to examine the changes of concentration of ectocytic matrix metalloproteinases 2 (MMP-2) and MMP-9.The invasion ability of U251 cells in the 3 groups was evaluated by scarification and Transwell assays.Results At 48 h after transfection,the concentration of extracellular MMP-2 in the SATB1-shRNA group [(69.4 ± 3.5) μg/L] was significantly lower than in the control group [(152.5 ± 2.6) μg/L] and the control-shRNA-GFP group [(150.5 ± 5.2) μg/L] (P ＜ 0.05 for both).The concentration of extracellular MMP-2 in the SATB1-shRNA group [(40.6 ±2.4) μμg/L] was significantly lower than in the control group [(86.7 ± 6.7) μg/L] and the control-shRNA-GFP group [(89.8 ± 10.5) μg/L] (P ＜ 0.05).As compared with the untransfected group and control-shRNA-GFP group,the SATB1-shRNA group showed significantly lower expression level of MMP-2 and MMP-9 (P ＜ 0.05).Meanwhile tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and SLC22A18 in the SATB1-shRNA group was significantly up-regulated.ELISA also indicated obvious changes of concentration of ectocytic MMP-2 and MMP-9.The scarification and Transwell assays revealed that the invasion ability in the SATB1-shRNA group was significantly decreased as compared with that in the rest two groups (P ＜ 0.05).Conclusion SATB1-targeted RNA intereference could inhibit the expressions of SATB1 and decrease the invasion ability of U251 cells in vitro. Key words: Glioma; Special AT-rich sequence binding protein 1 ; Invasion

Fluorosed mouse ameloblasts have increased SATB1 retention and Gαq activity.

Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.

Overexpression of SATB1 is associated with biologic behavior in human renal cell carcinoma.

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of SATB1 in RCC remains unclear. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of SATB1 was significantly associated with depth of invasion (P<0.001), lymph node status (P = 0.001) and TNM stage (P = 0.009). SATB1 knockdown inhibited the proliferation, migration and invasion of 786-O cells, whereas SATB1 overexpression promoted the growth and aggressive phenotype of ACHN cells in vitro. Furthermore, SATB1 expression was positively correlated with ZEB2 expression (P = 0.013), and inversely linked to levels of SATB2 and E-cadherin (P = 0.005 and P<0.001, respectively) in ccRCC tissues. Our data provide a basis for the concept that overexpression of SATB1 may play a critical role in the acquisition of an aggressive phenotype for RCC cells through EMT, providing new insights into the significance of SATB1 in invasion and metastasis of ccRCC, which may contribute to fully elucidating the exact mechanism of development and progression of RCC.

Loss of special AT‐rich sequence‐binding protein 1 (SATB1) predicts poor survival in patients with colorectal cancer

Special AT-rich sequence-binding protein 1 (SATB1) is a cell type-specific matrix attachment region binding protein, functioning as a global genome organizer. This study aims to investigate the expression pattern and the prognostic value of SATB1 in colorectal cancer. Prospectively collected data were obtained and tissue microarrays were constructed from a cohort of 352 patients. SATB1 protein expression was evaluated by immunohistochemistry and scored by two independent investigators. SATB1 expression was predominantly nuclear in both normal and cancer tissues. Loss of SATB1 nuclear expression was seen in 22% of colorectal cancers compared to 1.5% of adjacent normal colorectal tissue, and was associated with worse overall survival (P=0.02) independent of age and stage of disease (HR 2.48 with 95% CI 1.31-4.70). Loss of SATB1 expression was more evident in younger patients (P=0.03), tumours with mucinous or signet ring histology (P=0.0001) and poor differentiation (P=0.005). SATB1 expression was associated with a survival advantage in patients with Dukes C tumours who received adjuvant chemotherapy. Loss of SATB1 nuclear expression correlates with poor survival and a less favourable response to adjuvant chemotherapy in colorectal cancer. The value of SATB1 in individualized colorectal cancer therapy warrants further evaluation.

Expression of SATB1 protein in the ductal breast carcinoma tissue microarrays — preliminary study

Special AT-rich sequence-binding protein 1 (SATB1) is a nuclear matrix protein which interacts with specific regions of DNA, ensuring its proper organization and function in the cell. The expression of SATB1 was primarily found in thymocytes, but its increased levels were observed in various types of cancers. However, the knowledge of the function and application possibilities of this protein is still limited. The aim of this study was to investigate the expression of SATB1 protein using immunohistochemistry and tissue microarray (TMA) technique and determine its possible relationship with the proliferative marker Ki-67, estrogen a (ER) and progesterone (PR) receptors as well as grade of histological malignancy (G). The study was performed on material of 48 archival invasive ductal breast cancers (IDC). The TMAs were prepared with the use of 0.6 mm diameter punches. Immunohistochemical reactions were carried out using antibodies against Ki-67, ER, PR and SATB1 proteins. The intensity of the nuclear reaction was evaluated using a light microscope and computer-assisted image analysis. Expression of Ki-67 and SATB1 protein was observed in 89.58% and 31.25% of cancer cases, respectively. 62.5% of tumors were classified as ER-positive, and 47.92% as PR-positive. Statistical analysis showed a moderate positive correlation between Ki-67 and SATB1 expression (r = 0.291, p = 0.045 independently on the receptor status, and r = 0.392, p = 0.032 in ER-negative tumors). The expression of the Ki-67 antigen increased with higher grade of histological malignancy (G). The results suggest that SATB1 protein may play an indirect role in the cell proliferation and should be evaluated in relation to the other markers. Further studies concerning determination of its role in cancer progression and metastasis, in terms of application as therapeutic target and prognostic marker, are recommended.

Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl2-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8%). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.

Phosphorylated SATB1 is associated with the progression and prognosis of glioma

Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene regulator, and high expression of SATB1 is associated with progression and poor prognosis in several malignancies. Here, we examine the expression pattern of SATB1 in glioma. Microarray analysis of 127 clinical samples showed that SATB1 mRNA was expressed at lower levels in highly malignant glioblastoma multiforme (GBM) than in low-grade glioma and normal brain tissue. This result was further confirmed by real-time RT-PCR in the clinical samples, three GBM cell lines, primary SU3 glioma cells and tumor cells harvested by laser-capture microdissection. Consistent with the mRNA levels, SATB1 protein expression was downregulated in high-grade glioma, as shown by western blotting. However, phospho-SATB1 levels showed an opposite pattern, with a significant increase in these tumors. Immunohistochemical analysis of phospho-SATB1 expression in tissue microarrays with tumors from 122 glioma cases showed that phospho-SATB1 expression was significantly associated with high histological grade and poor survival by Kaplan–Meier analysis. In vitro transfection analysis showed that phospho-SATB1 DNA binding has a key role in regulating the proliferation and invasion of glioma cells. The effect of SATB1 in glioma cell is mainly histone deacetylase (HDAC1)-dependent. We conclude that phospho-SATB1, but not SATB1 mRNA expression, is associated with the progression and prognosis of glioma. By interaction with HDAC1, phospho-SATB1 contributes to the invasive and proliferative phenotype of GBM cells.