SFRP1 Research Articles

MiR-542-3p prevents ovariectomy-induced osteoporosis in rats via targeting SFRP1.

Secreted frizzled‐related protein‐1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR‐542‐3p and sFRP1 expression by RNA sequencing and qRT‐PCR. In addition, there was a significant negative correlation between miR‐542‐3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR‐542‐3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR‐542‐3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR‐542‐3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast‐induced mesenchymal stem cells (MSC), whereas the expression of miR‐542‐3p was significantly decreased. And miR‐542‐3p transfected MSCs showed a significant increase in osteoblast‐specific marker expression, indicating that miR‐542‐3p is necessary for MSC differentiation. Inhibition of miR‐542‐3p reduced bone formation, confirmed miR‐542‐3p play a role in bone formation in vivo. In general, these data suggest that miR‐542‐3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.

SFRP1 Promoter Methylation and Renal Carcinoma Risk: A Systematic Review and Meta-Analysis.

Epigenetic inactivation of tumor suppressor genes is an important molecular mechanism in the formation and development of human tumors. The purpose of our study was to evaluate the correlation between the methylation level of the secreted frizzled-related protein 1 (SFRP1) gene and the risk of renal cell carcinoma (RCC). The relevant literature was searched in detail in several electronic databases. The methodological heterogeneity was analyzed by meta-regression and subgroup analyses. The odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to summarize the dichotomous outcomes of our meta-analysis. The ten included articles contained 535 RCC samples and 475 normal controls. The results demonstrated that the methylation level of the SFRP1 promoter region was significantly correlated with an increased incidence of RCC (OR=13.72; 95% CI: 6.01-31.28; P=0.000). Furthermore, the eligible studies that had sufficient clinical data about the RCC cases were included in the analysis, and the results indicated that the frequency of SFRP1 promoter methylation was associated with a higher histological grade (P=0.000), tumor stage (P=0.033), tumor size (≥5 cm; P=0.029), and distant metastasis (P=0.047). Our results indicate that the methylation level of the SFRP1 promoter region is increased in patients with RCC compared to normal controls and might be involved in the occurrence and development of RCC. Additional well-designed studies are needed to further verify our conclusions.

Decreased expression of SFRP2 promotes development of the pituitary corticotroph adenoma by upregulating Wnt signaling.

Cushing's disease is primarily caused by pituitary adrenocorticotropin-secreting adenoma. However, its pathogenesis has remained obscure. In the present study, whole transcriptome analysis was performed by RNA sequencing (RNA-Seq) and expression of secreted frizzled-related protein 2 (SFRP2) was decreased in corticotroph tumors compared with normal pituitary glands. Furthermore, the RNA-Seq results were validated and the expression of SFRP2 in tumor tissues was analyzed by comparing another cohort of 23 patients with Cushing's disease and 3 normal human pituitary samples using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemistry staining. Clinically, there was an association between lower SFRP2 expression and aggressive adenoma characteristics, including larger size and invasiveness. Conversely, SFRP2 overexpression reduced the ability of AtT20 cells to proliferate and migrate, and reduced production of the adrenocorticotrophic hormone in vitro. Mechanistically, overexpressed SFRP2 reduced the level of β-catenin in the cytoplasm and nucleus, and decreased Wnt signaling activity in AtT20 cells. Therefore, SFRP2 appears to act as a tumor suppressor in Cushing's disease by regulating the activity of the Wnt signaling pathway.

The regulation of curcumin on cacinogenesis and modulation through Wnt/β‐catenin signaling in ovarian cancer cell line

Ovarian cancer (OC) is the most lethal gynecologic malignancy and the top ten cancer of females in Taiwan and all over the world. Oncogenic activation of the Wnt signaling pathway is common in cancers, but mutation of beta‐catenin in ovarian cancer is rare. In addition to genetic events, epigenetic modification of secreted frizzled‐related protein (SFRP) family has been shown to be important in regulating Wnt signaling. The SFRPs act as antagonists of the Wnt ligands and inhibit the Wnt/β‐catenin signaling pathway. In the past research indicated the curcumin rich in curcuma longa has the capacity for anti‐ inflammatory, anti‐ oxidant and anti‐cancer effects as well as involve in the epigenetic regulation. It is helpful for inhibiting the tumor suppressor gene methylated and re‐expression. Therefore, the aim of this study is going to investigate the effects of curcumin on sFRP5 DNA methylation modification in ovarian cancer cell (SKOV3). Cell was treated with DMSO, 10 μM 5‐aza‐2′‐deoxycytidine (DAC), 5 μM DAC, 20 μM curcumin and respectively combined with 5 μM DAC in 96 hours, extracting the RNA and proteins for further analyze. The results of present study show that curcumin combined with 5 μM DAC may inhibit cancer cell colony formation and migration, the total DNMT activity, DNMT3a protein expression, also regulate tumor suppressor gene SFRP5 expression through Wnt/β‐catenin signaling pathway. As a result, it can attenuate or inhibit the ovarian cancer development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells with TGF-BETA, BMP2, GDF5 or combinations thereof does not result in a nucleus pulposus cell phenotype

Purpose: Regenerative approaches for intervertebral disc (IVD) degeneration, using multipotent progenitor cells, are thought to be a promising therapeutic approach for low back pain. Chondrogenic differentiation of mesenchymal stem cells (MSC) with GDF5 was shown to induce expression of nucleus pulposus cell phenotypic marker genes. However, the presence or absence of a fibrotic annulus fibrosus cell phenotype after GDF5 stimulation has not been evaluated. In addition, GDF5 has not been compared directly to the more commonly used BMP2 in MSC chondrogenesis and induction of an IVD-like phenotype. Methods: Bone-marrow derived MSC were obtained from 5 donors. Chondrogenic differentiation was performed in pellet cultures with TGF-beta, BMP2, GDF5 or combinations and evaluated by matrix deposition, histology and gene expression changes. IVD-like differentiation was evaluated by qPCR for validated NP (T, KRT19, CD24, CA12, FOXF1, PAX1) and AF (COL5A1, COL12A1, ADAMTS17, SFRP2) phenotypic marker genes. Down-stream signaling was evaluated by phospho-SMAD immunoblotting, target gene activation and the BRE-luciferase reporter assay. Results: Five MSC donors underwent TGF-beta-induced chondrogenic differentiation. After two weeks, the NP marker gene KRT19 was down regulated and the AF marker genes COL5A1, ADAMTS17 and SFRP2 were up regulated (Fig.1A). A detailed time course experiment revealed a rapid down regulation of KRT19/CA12 and up regulation of SFRP2 within 3 days. Up regulation of PAX1, COL5A1 and COL12A1 was observed from 5–7 days onwards. Subsequently, BMP2 (20 ng/ml) and GDF5 (isomolar concentration) with and without TGF-beta (10 ng/ml) were used to induce chondrogenesis. In combination with TGF-beta, only BMP2 enhanced sGAG deposition, which was reflected by increased ACAN expression (Fig.1B). BMP2 and GDF5 stimulation alone was not sufficient to induce chondrogenesis. The NP marker gene CA12 was induced by co-stimulation with GDF5 in comparison to TGF-beta alone. The AF marker gene SFRP2 was suppressed by co-stimulation with BMP2 when compared to TGF-beta alone. As GDF5 exerted little effects on chondrogenesis and gene expression, we evaluated downstream activation of SMAD1/5/8. pSMAD1/5/8C was highest in MSC stimulated with BMP2 followed by TGF-beta and GDF5. Dose-response studies revealed that GDF5 (up to 2 μg/ml) was unable to induce similar BRE-luciferase reporter activity as 20 ng/ml BMP2 (Fig.1C). Finally, MSC were differentiated with 1 μg/ml GDF5, TGF-beta or the combination. At this concentration GDF5 enhanced TGF-beta-induced chondrogenesis (sGAG and DNA content), but failed to induce differentiation alone (sGAG content, SOX9, COL2A1, ACAN). Neither NP or AF marker genes were differentially expressed between TGF-beta or TGF-beta and GDF5 stimulated MSC. Conclusions: Chondrogenic differentiation of bone-marrow derived MSC with TGF-beta, BMP2 or GDF5 failed to induce a specific NP cell phenotype based on 6 NP marker genes. In particular Brachyury/T and FOXF1 expression were difficult to detect in MSC prior to and after chondrogenic differentiation. The observed effects of GDF5 on MSC chondrogenesis and KRT19 down- and PAX1 up regulation by TGF-beta are in line with literature. BMP2 was more potent in inducing SMAD1/5/8 signaling and enhancing TGF-beta induced chondrogenic differentiation then GDF5 at equimolar concentrations. Nonetheless, BMP2 (20 ng/ml) enhanced sGAG content but not DNA content, while GDF5 (1 μg/ml) enhanced both sGAG and DNA content. Importantly, multiple AF marker genes (COL1A1, COL5A1, COL12A1, SFRP2) were consistently induced by TGF-beta-mediated chondrogenesis and only SFRP2 was suppressed by BMP2. Therefore, we conclude that differentiation of bone-marrow derived MSC with TGF-beta, or in combination with GDF5 or BMP2, induces a mixed chondro-fibroblast cell phenotype that does not resemble an intervertebral disc cell phenotype. Hence, we recommend the use of both positive and negative markers for the evaluation of NP-like differentiation of progenitor cells.

Circulating Wnt inhibitory factor 1 levels are associated with development of cardiovascular disease

Background and aimsWnt signaling is involved in atherosclerotic plaque formation directly and indirectly by modulating cardiovascular risk factors. We investigated whether circulating concentrations of Wnt inhibitors are associated with cardiovascular events in subjects with intermediate cardiovascular risk. Methods904 non-diabetic subjects participating in the SAPHIR study were assessed. In the SAPHIR study, middle-aged women without overt atherosclerotic disease at study entry were followed up for 10 years. 88 patients of our study cohort developed cardiovascular disease at follow-up (CVD group). Subjects of the CVD group were 1:2 case-control matched for age, sex, BMI and smoking behavior with subjects without overt cardiovascular disease after a 10 year-follow-up (control group). 18 patients of the CVD group and 19 subjects of the control group were retrospectively excluded due to fulfilling exclusion criteria. Baseline circulating sclerostin, dickkopf (DKK)-1, secreted frizzled-related protein (SFRP)-1 and Wnt inhibitory factor (WIF)-1 levels were assessed by ELISA. ResultsBaseline systemic SFRP-1 and WIF-1 levels were significantly higher in patients with cardiovascular events (n = 70) when compared to healthy controls (n = 157) while DKK-1 and sclerostin levels were similar in both groups. Logistic regression analysis revealed WIF-1 as a significant predictor of future cardiovascular events. ConclusionsOur data suggest that increased SFRP-1 and WIF-1 levels precede the development of symptomatic atherosclerotic disease. Assessment of systemic WIF-1 levels, which turned out to be independently associated with CVD, might help to early identify patients at intermediate cardiovascular risk.

GSK3-activated STAT5 regulates expression of SFRPs to modulate adipogenesis.

Adipogenesis is a tightly regulated cellular process and is closely associated with obesity and its associated metabolic disorders, such as diabetes. Multiple transcription factors and signaling pathways are involved in the regulation of adipogenesis. Here, we report that glycogen synthase kinase (GSK3), which was reported to play an important role in many cellular processes, is essential to adipocyte differentiation at early and terminal differentiation phases. Mechanistically, GSK3 modulates adipogenesis through regulation of both canonical Wnt pathways, which involve Wnt/β-catenin signaling, and noncanonical Wnt pathways, which include JNK and Ras-related C3 botulinum toxin substrate signaling. GSK3-regulated adipogenesis is also mediated by secreted frizzled-related proteins (SFRPs), especially SFRP1, the canonical Wnt antagonist. The obesity-induced increase of Sfrp1 expression can be reversed by the GSK3 inhibitor. GSK3-regulated expression of Sfrp is mediated by signal transducer and activator of transcription 5 (STAT5). We demonstrated that GSK3 activates STAT5 through regulation of its phosphorylation to bind to the promoter of Sfrp genes and the peroxisome proliferator-activated receptor γ gene to stimulate their expression, which could ultimately lead to a modulated adipogenic process. Our findings identify a GSK3/STAT5/SFRP/Wnt regulatory axis of adipogenesis and shed light on the molecular mechanism of adipogenesis by suggesting that different pathways and adipogenic regulators coordinately modulate adipocyte differentiation.-Wang L., Wang, Y., Meng, Y., Zhang, C., Di, L. GSK3-activated STAT5 regulates expression of SFRPs to modulate adipogenesis.

Aberrant expression of the sFRP and WIF1 genes in invasive non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) are the most common pituitary tumors and mainly invade the sphenoid, cavernous sinus or dura mate. Aberrant regulation of the Wnt signaling pathway plays an important role in tumorigenesis. This study was designed to investigate the relationships between secreted frizzled-related proteins (sFRPs), WIF1 genes and the invasion of NFPAs by tissue microassays (TMAs) of samples from 163 patients. Significantly weaker staining of WIF1 and sFRP4 were detected in the invasive group compared with the non-invasive group by TMAs (p = 0.002, p < 0.001). Univariate analysis showed a significant correlation between tumor invasion and low expression of WIF1 and sFRP4 (p = 0.002, p < 0.001). A similar trend was observed when analyzing the mRNA and protein levels through RT-PCR and western blot experiments. Methylation of the WIF1 promoter was significantly increased in invasive NFPAs compared with the noninvasive group (p = 0.004). The average progression free survival time in the high WIF1 group was longer than that in the low WIF1 group (p = 0.025). Furthermore, RT-PCR measured the levels of 11 miRNAs targeting WIF1 according to the Targetscan database and PubMed. The levels of miRNA-137, miRNA-374a-5p and miRNA-374b-5p in the invasive group were 0.037-fold, 0.577-fold and 0.44-fold that of the noninvasive group (p = 0.003, p = 0.049 and p = 0.047). Overexpression of miRNA-137 could inhibit the proliferation and invasion of GH3 cells through cell viability and Transwell experiments (p < 0.05). Furthermore, the WIF1 level was upregulated after overexpression of miRNA-137 compared with miRNA-137-NC (control miRNA) in GH3 cells. Our data suggest that WIF1 may be potential biomarker for the aggressiveness of NFPAs. miRNA-137 plays an important role in the Wnt signaling pathway by affecting promoter methylation of WIF1.

74P - Promoter hypermethylation of Wnt pathway inhibitor SFRP1 gene and its expression levels in human astrocytomas

74P - Promoter hypermethylation of Wnt pathway inhibitor SFRP1 gene and its expression levels in human astrocytomas

TARGETED DELIVERY OF SOLUBLE FIZZLED RELATED PROTEIN 1 ATTENUATES TRANSVERSE AORTIC CONSTRICTION INDUCED HYPERTROPHIC HEART FAILURE

Wnt signaling pathway activation facilitates the progression of hypertrophic heart failure. Soluble fizzled related protein 1 (SFRP1) is the negative regulator of Wnt signaling pathway. This study was aimed to investigate the effects and safety profile of targeted sfrp1 gene delivery in hypertrophic

Secreted frizzled-related protein 1 regulates the progression of neuropathic pain in mice following spinal nerve ligation.

Previous studies have shown that the Wnt/β-catenin signaling pathway plays an important role in modulating neuropathic pain after sciatic nerve injury. In this study, we explored the role of secreted frizzled-related protein 1 (SFRP1), a Wnt antagonist, in neuropathic pain using a mouse model following spinal nerve ligation (SNL). We found SNL-induced SFRP1 downregulation in the spinal cord. Further, overexpression of SFRP1 via spinal injection into the spinal cord attenuated SNL-induced allodynia, hyperalgesia, and neuroinflammation. Consistently, in vitro assays also showed decreased expression of SFRP1 in spinal cord astrocytes after exposure to lipopolysaccharide (LPS). Overexpression of SFRP1 significantly alleviated the secretion of LPS-induced proinflammatory factors in spinal cord astrocytes. Furthermore, spinal injection of LPS-treated astrocytes induced allodynia and hyperalgesia, which were reversed by the overexpression of SFRP1 in these cells. Additionally, SNL increased Wnt3a and β-catenin levels and also induced an increase in nuclear expression of β-catenin; these effects were all attenuated by SFRP1. Finally, we found that downregulation of SFRP1, mainly through DNA methylation, is involved in the pathogenesis of neuropathic pain. Taken together, these results suggested that the SFRP1/Wnt3a/β-catenin signaling pathway might be a suitable therapeutic target for neuropathic pain.

Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids.

Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2'-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.

Secreted frizzled-related protein 2 (SFRP2) expression promotes lesion proliferation via canonical WNT signaling and indicates lesion borders in extraovarian endometriosis.

What is the role of SFRP2 in endometriosis? SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis, regulating endometriosis lesion growth and indicating endometriosis lesion borders together with CTNNB1 (also known as beta catenin). Endometriosis is a common, chronic disease that affects women of reproductive age, causing pain and infertility, and has significant economic impact on national health systems. Despite extensive research, the pathogenesis of endometriosis is poorly understood, and targeted medical treatments are lacking. WNT signaling is dysregulated in various human diseases, but its role in extraovarian endometriosis has not been fully elucidated. We evaluated the significance of WNT signaling, and especially secreted frizzled-related protein 2 (SFRP2), in extraovarian endometriosis, including peritoneal and deep lesions. The study design was based on a cohort of clinical samples collected by laparoscopy or curettage and questionnaire data from healthy controls and endometriosis patients. Global gene expression analysis in human endometrium (n = 104) and endometriosis (n = 177) specimens from 47 healthy controls and 103 endometriosis patients was followed by bioinformatics and supportive qPCR analyses. Immunohistochemistry, Western blotting, primary cell culture and siRNA knockdown approaches were used to validate the findings. Among the 220 WNT signaling and CTNNB1 target genes analysed, 184 genes showed differential expression in extraovarian endometriosis (P < 0.05) compared with endometrium tissue, including SFRP2 and CTNNB1. Menstrual cycle-dependent regulation of WNT genes observed in the endometrium was lost in endometriosis lesions, as shown by hierarchical clustering. Immunohistochemical analysis indicated that SFRP2 and CTNNB1 are novel endometriosis lesion border markers, complementing immunostaining for the known marker CD10 (also known as MME). SFRP2 and CTNNB1 localized similarly in both the epithelium and stroma of extraovarian endometriosis tissue, and interestingly, both also indicated an additional distant lesion border, suggesting that WNT signaling is altered in the endometriosis stroma beyond the primary border indicated by the known marker CD10. SFRP2 expression was positively associated with pain symptoms experienced by patients (P < 0.05), and functional loss of SFRP2 in extraovarian endometriosis primary cell cultures resulted in decreased cell proliferation (P < 0.05) associated with reduced CTNNB1 protein expression (P = 0.05). SFRP2 and CTNNB1 improved extraovarian endometriosis lesion border detection in a relatively small cohort (n = 20), although larger studies with different endometriosis subtypes in variable cycle phases and under hormonal medication are required. The highly expressed SFRP2 and CTNNB1 improve endometriosis lesion border detection, which can have clinical implications for better visualization of endometriosis lesions over CD10. Furthermore, SFRP2 acts as a canonical WNT/CTNNB1 signaling agonist in endometriosis and positively regulates endometriosis lesion growth, suggesting that the WNT pathway may be an important therapeutic target for endometriosis. This study was funded by the Academy of Finland and by Tekes: Finnish Funding Agency for Innovation. The authors have no conflict of interest to declare.

In-Vitro Effects of Secreted Frizzled-Related Protein 1 (SFRP1) On Human Corneal Epithelial Cells

ABSTRACTPurpose: Limbal corneal epithelial cells (LCECs) are responsible for corneal epithelial cell regeneration. However, corneal central epithelial cells (CCECs) are also suggested to display potential for self-renewal. Additionally, a better understanding of molecules that regulate corneal epithelial cell regeneration is important for studying conditions affecting the cornea, for example, keratoconus. Given our previous findings of reduced levels of secreted frizzled-related protein 1 (SFRP1) in tears from keratoconus patients compared to controls, we investigated the effects of SFRP1 on the proliferation and survival of cultured central and limbal human corneal epithelial cells.Material and methods: Limbal and central corneal explants were established from postmortem human corneas, and cultured in CnT-PR, an epithelial-specific tissue culture media. Subcultured cells from explants were immunostained for the cytokeratins CK3, 12, 19, and the proliferative/oligopotent markers Ki67 and p63. BrdU flow cytometry, Alamar Blue and LDH assays were used to assess effects of SFRP1 treatment on central and LCECs.Results: Primary limbal and central corneal epithelial cells were successfully cultured in vitro to confluence (P6 and P4, respectively). They all expressed varying levels of cytokeratins CK3, CK12 and CK19, and Ki67 and p63. Additionally, they showed significantly increased metabolic activity after SFRP1 treatment (p < 0.05), with a maximum response at 1 μg/mL of SPRF1. No difference in proliferation was detected in SFRP1 treated LCECs; however, a reduction in cell death was noted (p < 0.05).Conclusion: Similar to the LCECs, primary human CCECs can be cultured in vitro, and expressed epithelial markers. SFRP1 demonstrated an improvement on the metabolic activity of both CCECs and LCECs, which in LCECs could be resulted from reduced cell death. This may have implications in degenerative corneal disorders, such as keratoconus.

Chemoprevention of colorectal cancer by black raspberry anthocyanins involved the modulation of gut microbiota and SFRP2 demethylation.

Freeze-dried black raspberry (BRB) powder is considered as a potential cancer chemopreventive agent. In this study, we fed azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated C57BL/6J mice with a diet containing BRB anthocyanins for 12 weeks, and this led to a reduction in colon carcinogenesis. These animals had consistently lower tumor multiplicity compared with AOM/DSS-treated mice not receiving BRB anthocyanins. In AOM/DSS-treated mice, the number of pathogenic bacteria, including Desulfovibrio sp. and Enterococcus spp., was increased significantly, whereas probiotics such as Eubacterium rectale, Faecalibacterium prausnitzii and Lactobacillus were dramatically decreased, but BRB anthocyanins supplement could reverse this imbalance in gut microbiota. BRB anthocyanins also caused the demethylation of the SFRP2 gene promoter, resulting in increased expression of SFRP2, both at the mRNA and protein levels. Furthermore, the expression levels of DNMT31 and DNMT3B, as well as of p-STAT3 were downregulated by BRB anthocyanins in these animals. Taken together, these results suggested that BRB anthocyanins could modulate the composition of gut commensal microbiota, and changes in inflammation and the methylation status of the SFRP2 gene may play a central role in the chemoprevention of CRC.

Abstract PR05: Familial cancer patient specific iPSCs based study of a potential oncogenic factor, sFRP2, in osteosarcoma

Abstract Osteosarcoma (OS), despite being a serious pediatric cancer with the lowest survival rate, is not well studied due to its rare occurrence. For this reason, studying OS in a patient-specific manner is more challenging. In an attempt to establish a method to study OS in a patient-specific manner, we previously generated Li-Fraumeni syndrome (LFS---a germline P53 mutation disease) patient specific iPSCs, and demonstrated that the LFS-iPSC derived mesenchymal stem cells (MSCs) and osteoblasts (OBs) can recapitulate tumorigenic phenotypes in vitro and in vivo (Lee, Kim, et al., Cell 2015). Taking advantage of this new platform, we focused on finding potential therapeutic targets. We found SFRP2 (secreted frizzled related protein 2) to be highly expressed in LFS iPSC-derived MSCs and OBs by global RNA-seq gene expression analysis. Using global transcript profiling of ectopic overexpression (OE) of sFRP2 in wild-type MSCs, we discovered that sFRP2 regulates osteoblast differentiation, cell adhesion, cell proliferation, and apoptosis-related gene sets. Further functional assays revealed that sFRP2 OE contributes to the generation of abnormal osteoblast cells, which are the cause of OS, by suppressing osteogenic alkaline phosphatase activity, mineralization, and osteogenic BMP and WNT signaling. Furthermore, ectopic OE of sFRP2 is associated with LFS mediated tumorigenesis. sFRP2 OE in MSCs and OBs facilitates cell proliferation through cell cycle alteration and increases anchorage independency. In in ovo and in vivo xenograft assays (subcutaneous and intratibial injections), the tumor size is significantly increased in sFRP2 OE group and decreased in knockdown sFRP2 group. Interestingly, we also found that sFRP2 OE augments angiogenesis in vitro and in vivo, suggesting sFRP2 has a role as a paracrine factor. In the future, we will analyze sFRP2 expression levels in human OS tissue samples to determine whether sFRP2 OE has clinical relevance. Conclusively, this study demonstrates that P53 mutation-mediated osteosarcoma exerts oncogenicity through sFRP2 overexpression and that targeting of sFRP2 is a potential therapeutic strategy for OS treatment. This abstract is also being presented as Poster A22. Citation Format: Huen Suk Kim, Ye X. Yuan, Jeffrey M. Bernitz, Andreia Gomes, Christopher Schaniel, Dung-Fang Lee, Ihor R. Lemischka. Familial cancer patient specific iPSCs based study of a potential oncogenic factor, sFRP2, in osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR05.

Bioinformatics-based identification of miR-542-5p as a predictive biomarker in breast cancer therapy

BackgroundTamoxifen is the first-line hormone therapy for estrogen receptor alpha positive (ERα+) breast cancer. However, about 40% of patients with ERα + breast cancer who receive tamoxifen therapy eventually develop resistance resulting in a poor prognosis. The aim of this study was to mine available data sets in the Gene Expression Omnibus (GEO) database, including in vitro (cell lines) and in vivo (tissue samples), and to identify all miRNAs associated with tamoxifen resistance (TamR) in breast cancer. Secondly, this study aimed to predict the key gene regulatory networks of newly found TamR-related miRNAs and evaluate the potential role of the miRNAs and targets as potential prognosis biomarkers for breast cancer patients.ResultMicroarray data sets from two different studies were used from the GEO database: 1. GSE66607: miRNA of MCF-7 TamR cells; 2. GSE37405: TamR tissues. Differentially expressed microRNAs (miRNAs) were identified in both data sets and 5 differentially expressed miRNAs were found to overlap between the two data sets. Profiles of GSE37405 and data from the Kaplan-Meier Plotter Database (KMPD) along with Gene Expression Profiling Interactive Analysis (GEPIA) were used to reveal the relationship between these 5 miRNAs and overall survival. The results showed that has-miR-542-5p was the only miRNA associated with overall survival of ERα + breast cancer patients who received adjuvant tamoxifen. Targets of has-miR-542-5p were predicted by miRanda and TargetScan, and the mRNA expression of the three 3 target gene, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Beta (YWHAB), Lymphocyte Antigen 9 (LY9), and Secreted Frizzled Related Protein 1 (SFRP1) were associated with overall survival in 2 different databases. Copy-number alterations (CNAs) of SFRP1 confer survival disadvantage to breast cancer patients and alter the mRNA expression of SFRP1 in cBioPortal database.ConclusionThis study indicates that miRNA has-miR-542-5p is associated with TamR and can predict prognosis of breast cancer patients. Furthermore, has-miR-542-5p may be acting through a mechanism involving the target genes YWHAB, LY9, and SFRP1. Overall, has-miR-542-5p is a predictive biomarker and potential target for therapy of breast cancer patients.

Secreted Frizzled Related Protein 2 Modulates Epithelial–Mesenchymal Transition and Stemness via Wnt/β-Catenin Signaling in Choriocarcinoma

Background/Aims: Choriocarcinoma (CC) is a highly aggressive gestational trophoblastic neoplasia; however, the underlying molecular mechanisms of its invasiveness and metastasis remain poorly understood. Human secreted frizzled-related protein 2 (SFRP2) could function as a tumor promoter or suppressor in different tumors, yet the role it plays in CC’s invasion and metastasis is thoroughly unclear. The current study was aimed to explore the function and underlying mechanism of SFRP2 in CC. Methods: The expression of SFRP2 in CC tissues was examined via immunohistochemistry. The methylation level and expression of SFRP2 in CC cell lines, JEG-3 and JAR were examined via bisulfite sequencing PCR (BSP), western blotting and quantitative RT-PCR. The biological role of increasing expressed SFRP2 through its promoter demethylation with 5-Aza-2’-deoxycytidine (5-Aza) was examined by a series of in vitro functional studies. Furthermore, lentivirus transfection technology was adopted to investigate the biological roles of SFRP2 knockdown in JEG-3 and JAR cells in vitro and in vivo. Moreover, its downstream signaling pathway was investigated. Results: SFRP2 was downregulated in CC tissues, and its expression was inversely related to its promoter hypermethylation frequency in JEG-3 and JAR cells. Increased SFRP2 through its promoter demethylation inhibited cell migration, invasion and colony formation in JEG-3 and JAR cells, whereas decreased SFRP2 reversed the epithelial-mesenchymal transition (EMT) process and stemness in JEG-3 and JAR cells both in vitro and vivo. Mechanistically, SFRP2 regulated the EMT and stemness of CC cell lines via canonical Wnt/β-catenin signaling, validated by the usage of a Wnt activator and inhibitor. Conclusion: The current study indicates that downregulated SFRP2 has potent tumor-promotive effects in CC through the modulation of cancer stemness and the EMT phenotype via activation of Wnt/β-catenin signaling in vitro and in vivo.

Expresión de EDNRB y CDX2 posibles biomarcadores en progresión al cáncer cervical

De acuerdo a la historia natural del cáncer del cuello uterino, en donde las lesiones preneoplásicas de bajo y alto grado pueden presentar fenómenos de regresión o progresión, existe gran interés en la búsqueda de biomarcadores que permita predecir la evolución de las lesiones preneoplásicas del cérvix hacia la progresión o regresión de la enfermedad. Estos biomarcadores pudieran ser de origen genético, o epigenético que alteren la expresión de los genes y que pudieran estar asociados con la carcinogénesis en diferentes tipos de tejido humano. El objetivo del estudio fue analizar la expresión del mARN de los genes SFRP1, PTPRN, CDO1, EDNRB, CDX2, EPB41L3 y HAND1 en muestras negativas para lesiones intraepiteliales cervicales (n=9), muestras con lesiones intraepiteliales de bajo grado (n=10) y alto grado (n=11). Se realizó análisis de expresión de los genes mencionados mediante qRT-PCR y el análisis de los datos se realizó mediante la prueba no paramétrica de ANOVA. La diferencia estadística se determinó en valores p&lt; 0,05. Para los genes EDNRB y CDX2 se observó disminución 66,7% en las muestras sin alteraciones histológicas cervicales, comparado con una disminución en la expresión del 50% en muestras con LIEBG y para el grupo de LIEAG del 36,4% para el gen EDNRB y del 27,3% para el gen CDX2 dando una diferencia estadísticamente significativa p= 0,02. Sugiriendo que EDNRB y CDX2 podrían ser útiles como posibles biomarcadores en la carcinogénesis cervical.

Role and alterations of DNA methylation during the aging and cancer

Besides the genetic research, increasing number of scientific studies focus on epigenetic phenomena - such as DNA methylation - regulating the expression of genes behind the phenotype, thus can be related to the pathomechanism of several diseases. In this review, we aim to summarize the current knowledge about the evolutionary appearance and functional diversity of DNA methylation as one of the epigenetic mechanisms and to demonstrate its role in aging and cancerous diseases. DNA methylation is also characteristic/also appear to prokaryotes, eukaryotes and viruses. In prokaryotes and viruses, it provides defence mechanisms against extragenous DNA. DNA methylation in prokaryotes plays a significant role in the regulation of transcription, the initiation of replication and in Dam-directed mismatch repair. In viruses, it participates not only in defence mechanisms, but in the assembly of capsids as well which is necessary for spreading. In eukaryotes, DNA methylation is involved in recombination, replication, X chromosome inactivation, transposon control, regulation of chromatin structure and transcription, and it also contributes to the imprinting phenomenon. Besides the above-mentioned aspects, DNA methylation also has an evolutionary role as it can change DNA mutation rate. Global hypomethylation appearing during aging and in cancerous diseases can lead to genetic instablility and spontaneous mutations through its role in the regulation of transposable elements. Local hypermethylated alterations such as hypermethylation of SFRP1, SFRP2, DKK1 and APC gene promoters can cause protein expression changes, thus contribute to development of cancer phenotype. DNA methylation alterations during aging in cancerous diseases support the importance of epigenetic research focusing on disease diagnostics and prognostics. Orv Hetil. 2018; 159(1): 3-15.