Cyr61 Protein Expression Research Articles

Therapeutic Effect of 1,3‐Thiazin‐6‐One for the Treatment of Brain Cancer Through Increased Accumulation in the Brain Glioblastoma Cells

AbstractIn the present study a library of five (2‐(amino)‐1,3‐thiazin‐6‐one) compounds was synthesized and investigated as against glioblastoma cells in vitro and in vivo in the mice model. The results revealed that all of the five 1,3‐thiazin‐6‐one compounds (4a, 4b, 4c, 4d and 4e) exhibited cytotoxicity against U87MG and 9 L brain cancer cell lines. Moreover, it was found that 9 L cells showed slightly higher sensitivity towards the compounds 4a, 4b, 4c, 4d and 4e compared to U87MG cells. It was observed that the compounds 4a, 4b, 4c, 4d and 4e showed a time‐dependent increase in uptake efficiency by U87MG and 9 L cells. Furthermore, the data revealed that uptake of the compounds and therefore internalization was maximum during initial 1 h of the treatment. Among the synthesized compounds, compound 4c containing trifluoromethyl moiety showed higher uptake efficiency compared to the compound 4a, 4b, 4d and 4e. Treatment of U87MG and 9 L cell tumor spheroids with compound 4c significantly (P<0.05) inhibited the tumor growth compared to the control spheroids. In vivo data revealed that treatment with compound 4c led to a significant (P<0.05) decrease in glioblastoma growth in mice in dose‐dependent manner. Growth of glioblastoma in mice was almost completely inhibited after 28 days of treatment with 2 mg/kg dose of compound 4c. Pharmacokinetic studies showed that compound 4c remained in circulation for longer duration in mice and its terminal half‐life was found to be 6.5 h. Treatment of U87MG and 9 L cells with compound 4c led to a prominent decrease in expression of CYR61 protein. In conclusion, findings of the present study suggest that compound 4c acts as a potential therapeutic agent for the treatment of advanced brain cancer through activation of Hippo pathway. Therefore, more studies need to be performed to investigate the detailed mechanism underlying the inhibitory effect of compound 4c against the glioblastoma.

Disruption of the Hippo pathway promotes the proliferation of childhood acute lymphoblastic leukemia cells, inhibits apoptosis and chemosensitivity

ABSTRACT Background Despite advancements in chemotherapy and stem cell transplantation, the recurrence and chemoresistance of childhood acute lymphoblastic leukemia (cALL) remain a significant challenge, thus indicating the need for novel therapeutic targets. Research design and methods The protein levels of YAP1, p-YAP1, TAZ, and Cyr61 of cALL patients and healthy volunteers were measured by western blot analysis. Then the leukemic cell line SUP-B15 was transfected with sh-YAP1 and pcDNA3.1-YAP1 to knockdown or overexpress YAP1. The viability, chemosensitivity, apoptosis, migration, and invasion of SUP-B15 cells were determined by MTT, flow cytometry, and Transwell assay. Results The cALL patients had higher YAP1, TAZ, and Cyr61 protein expression and lower p-YAP1 protein expression in bone marrow tissues compared with healthy volunteers (p < 0.01). In SUP-B15 cells, YAP1 knockdown upregulated p-YAP1 protein expression (p < 0.01) and downregulated TAZ and Cyr61 protein expression (p < 0.01). In addition, knocking down YAP1 significantly inhibited cell viability, migration, and invasion, and induced apoptosis (p < 0.01). YAP1 knockdown also reduced the IC50 value following treatment with vincristine, daunorubicin, cyclophosphamide, and dexamethasone (p < 0.05). Conclusions Disruption of the Hippo pathway attenuates the development of cALL by promoting cell proliferation while suppressing apoptosis and drug sensitivity.

Effects of umbilical cord mesenchymal stem cells on expression of CYR61, FSH, and AMH in mice with premature ovarian failure.

This study aimed to investigate the effects of umbilical cord mesenchymal stem cells on the expression of CYR61, FSH and AMH in mice with premature ovarian failure. For this purpose, thirty SPF female SD mice were selected as the research object, 10 of which were control group, namely group α, and 20 mice with premature ovarian failure model were established by cyclophosphamide. The mice were divided into the model group, namely the β group and the umbilical cord mesenchymal stem cell transplantation group (γ group), with 10 mice in each group. ELSA method was used to determine the levels of follicle-stimulating hormone (FSH), Luteinizing hormone (LH), estradiol (Estradiol) in serum. The changes of E2, Antimullerian hormone (AMH) and cysteine-rich protein 61 in ovarian tissues were determined by the protein imprinting method. Connective tissue growth factor (CTGF) and caspase-3 protein expression. Results showed that in fertility rate, γ group > α group > β group, the difference was statistically significant (P<0.05), in litter size, α group > γ group > β group, the difference was statistically significant (P<0.05). The levels of serum E2 and AMH in α group > γ group > β group, and the levels of serum FSH and LH in β group > γ group > α group were statistically significant (P<0.05). The growth follicles were α group > γ group > β group, and the atresia follicles were β group > γ group > α group, and there was a statistical difference among all groups (P<0.05). There was no difference in luteal number among the three groups (P>0.05). In terms of CYR61 and CTGF protein expression, α group > γ group > β group, and in terms of caspase-3, β group > γ group > α group had statistical significance (P<0.05). It is concluded that intervention with umbilical cord mesenchymal stem cells can significantly improve the expression levels of CYR61 and AMH, reduce the level of FSH, promote cell survival, improve the reproductive quality of mice, and restore the physiological function of the ovary. It is feasible to treat premature ovarian failure with umbilical cord mesenchymal stem cells.

Effects of umbilical cord mesenchymal stem cells on expression of CYR61, FSH and AMH in mice with premature ovarian failure.

The aim of this study was to objective to investigate the effects of umbilical cord mesenchymal stem cells on the expression of CYR61, FSH and AMH in mice with premature ovarian failure. For this purpose, thirty SPF female SD mice were selected as the research object, 10 of which were control group, namely group α, and 20 mice with premature ovarian failure model were established by cyclophosphamide. The mice were divided into model group, namely β group and the umbilical cord mesenchymal stem cell transplantation group (γ group), with 10 mice in each group. ELSA method was used to determine the levels of follicle-stimulating hormone (FSH), Luteinizing hormone (LH), estradiol (Estradiol) in serum. The changes of E2, Antimullerian hormone (AMH) and cysteine-rich protein 61 in ovarian tissues were determined by the protein imprinting method. Connective tissue growth factor (CTGF) and caspase-3 protein expression. Results showed that in fertility rate, γ group > α group > β group, the difference was statistically significant (P<0.05), in litter size, α group > γ group > β group, the difference was statistically significant (P<0.05). The levels of serum E2 and AMH in α group > γ group > β group, and the levels of serum FSH and LH in β group > γ group > α group were statistically significant (P<0.05). The growth follicles were α group > γ group > β group, and the atresia follicles were β group > γ group > α group, and there was a statistical difference among all groups (P<0.05). There was no difference in luteal number among the three groups (P>0.05). In terms of CYR61 and CTGF protein expression, α group > γ group > β group, and in terms of caspase-3, β group > γ group > α group had statistical significance (P<0.05). It is concluded that intervention with umbilical cord mesenchymal stem cells can significantly improve the expression levels of CYR61 and AMH, reduce the level of FSH, promote cell survival, improve the reproductive quality of mice, and restore the physiological function of the ovary. It is feasible to treat premature ovarian failure with umbilical cord mesenchymal stem cells.

Expression of Shh, Gli1, and Cyr61 in Gastric Cancer Predicts Overall Survival of Patients: A Retrospective Study.

This study aimed to evaluate the expression levels of Shh, Gli1, and Cyr61 proteins in gastric cancer tissues and analyze the relationship between these three proteins and the clinicopathological factors and prognosis of patients. This was a retrospective study. Four hundred gastric cancer tissue specimens from patients who underwent radical gastrectomy in Zhangye People's Hospital affiliated to Hexi University between February 2013 and February 2021 underwent immunohistochemical analysis. The positive expression rates of Shh, Gli1, and Cyr61 in gastric cancer tissues were 55.5%, 56.5%, and 64.5%, respectively. The expressions of Shh, Gli1, and Cyr61 in gastric cancer tissues were significantly correlated with tumor size, depth of invasion, and degree of differentiation (P < .05). The expression of Shh protein was positively correlated with the expression of Gli1 protein (P < .01), and the expression of Gli1 protein was positively correlated with the expression of Cyr61 protein (P < .01). Univariate and multivariate analyses showed that the expression of Shh, Gli1, and Cyr61 could predict the prognosis of patients (P < .05). Receiver operating characteristic curve analysis combined with TNM staging could better predict the three-year overall survival of patients (P < .05). Shh, Gli1, and Cyr61 proteins are significantly expressed in gastric cancer tissues and are risk factors for the prognosis of patients with gastric cancer.

Expression of scrum CYR61 mRNA and protein in patients with tibial fracture and its relationship with prognosis

Objective: To explore the relationship between the expression of serum CYR61 mRNA and protein and prognosis in patients with tibial fracture. Methods: Eighty-six patients with tibiofibular fractures (fracture group) admitted to Xi'an Central Hospital from July 2016 to July 2018 and 84 healthy controls (control group) who underwent physical examination in our hospital were selected as the study subjects. The imaging healing score (RUST) and prognosis were divided into two subgroups, 74 in the normal healing group and 10 in the abnormal healing group. The serum CYR61 mRNA and protein expression levels were compared and the relationship between serum CYR61 mRNA and protein expression levels and fracture prognosis was analyzed. Result: The relative expression levels of CYR61 mRNA and protein in the normal healing group were (2.35±0.49) and (0.33±0.10), respectively (2.0210.29) and (0.23±0.07) in the abnormal healing group and 1.8810.37 in the control group.), (0.26±0.06), the difference between the three groups was statistically significant (P&lt;0.05), and the serum CYR61 mRNA and CYR61 protein in the normal healing group were significantly higher than the control group (P&lt;0.05), and the serum CYR61 mRNA in the abnormal healing group. CYR61 protein was significantly lower than the normal healing group (P&lt;0.05). The RUST of the normal healing group was (11.3511.51), which was higher than that of the abnormal healing group (7.1710.95), and the difference was statistically significant (P&lt;0.05). Serum CYR61 mRNA and protein expression levels were significantly positively correlated with prognosis (P&lt;0.05). ROC curve analysis showed that the AUC of serum CYR61 mRNA and protein were 0.735 and 0.778, respectively, and the Cut-off values were 2.363 and 0.338, respectively. The sensitivities were 60.80% and 54.10%, respectively, and the specificities were 80.00% and 90.00%, respectively. They were 0.408 and 0.441, respectively, thus indicating that serum CYR61 mRNA and protein expression levels have predictive value for prognosis. Conclusion: The higher the mRNA and protein expression levels of serum CYR61 in early stage, the better the prognosis of fracture healing in patients with tibiofibular fractures. To a certain extent, serum CYR61 can be used as a reference for predicting the prognosis of tibiofibular fractures.

Cyr61 Promotes Inflammation of a Gouty Arthritis Model in Rats.

Background Cyr61 is considered a novel proinflammatory factor. Gouty arthritis (GA) is a self-limited inflammatory reaction caused by monosodium urate (MSU) crystals. In this study, we assessed the role of Cyr61 in the inflammatory process of GA. Methods We investigated the expression of Cyr61 in MSU-induced rat gout models and MSU-stimulated rat fibroblast-like synovial (FLS) cells. After inhibiting the expression of Cyr61, levels of IL-1β, TNF-α, and IL-6 were detected by ELISA, qPCR, western blot, and immunohistochemical methods. We probed the downstream NF-κB signaling pathway using the NF-κB inhibitor PDTC, and levels of NF-κB and p-NF-κB were detected by western blot and qPCR. Results Our results demonstrate that Cyr61 plays a potent role in the formation of local inflammation in vitro and in vivo. Cyr61 was highly expressed in synovial tissues of gout models, and the expression of Cyr61 protein was also significantly increased in MSU-stimulated FLS cells. Cyr61 promoted MSU-induced acute inflammation via the NF-κB signaling pathway. Conclusions Our study has revealed that Cyr61 is an important regulatory factor for the initiation of inflammation in GA. The high expression of Cyr61 protein can induce synovial cells to produce many inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, partly in an NF-κB-dependent manner. Thus, inhibition of Cyr61 could be a new target and strategy for the prevention and treatment of GA.

Expression of cysteine-rich protein 61 protein in prostate carcinoma and their clinical significance

Objective To investigate the expression and clinical significance of Cysteine-rich protein 61 (Cyr61) protein in prostate cancer. Methods We selected 68 cases of the prostate cancer tissues and 60 cases of benign prostatic hyperplasia tissues in our hospital. Immunohistochemistry was used to detect the expression of Cyr61 protein in prostate cancer tissues and benign prostatic hyperplasia tissues, and then the difference of expression between the two groups and its relationship with pathological parameters of prostate cancer were analyzed. Results The expression of Cyr61 protein in prostate cancer tissues was significantly higher than that in benign prostatic hyperplasia tissues (85.3% vs. 8.3%, χ2=75.536, P 0.05), TNM staging (χ2=0.342, P>0.05), regional lymph node metastasis (χ2=0.176, P>0.05) and distant metastasis (χ2=0.673, P>0.05), but was significantly correlated with preoperative total prostate specific antigen (tPSA) level (χ2=8.492, P<0.05), differentiation degree (χ2=13.028, P<0.05) and Gleason scores (χ2=4.032, P<0.05). Conclusion The expression of Cyr61 protein in prostate cancer tissues were significantly up-regulated, which helps to assess the progress of prostate cancer. Key words: Cysteine-rich protein 61 protein; Prostate carcinoma; Expression

Angiotensin Ⅱ induces apoptosis of HEK293T cells by up-regulating Cyr61 expression

To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism. Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10-7 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting. The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells (P &lt; 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group (P &lt; 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% vs (3.87 ± 0.83)%, P &lt; 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment (P &lt; 0.05). The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.

Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

BackgroundOvarian cancer is considered one of the lethal cancers responsible for high mortality and morbidity across the world. The prognosis and the survival rate of ovarian cancer is far from decent. Cysteine-rich 61 (Cyr61) also known as CCN1, is a member of CCN-family of growth factors, reported to be significantly overexpressed in several malignancies which include, but are not limited to, ovarian cancer. Recent studies have revealed that women with type 2 diabetes mellitus have an elevated risk of ovarian cancer. Hence, administration of an antidiabetic drug with anticancer effects such as metformin may act as an effective therapeutic regime against ovarian cancer.Material/MethodsCell viability and apoptosis were examined by MTT and Annexin V/PI double staining respectively. Cell migration was determined by Boyden Chamber assay. Transient knockdown of Cyr61 in ovarian cancer cells was achieved by transecting the cells with siRNA for Cyr61using Lipofectamine 2000.ResultsOur results indicated that treatment of ovarian cancer cells with metformin caused significant downregulation of Cyr61 protein expression levels ultimately favoring apoptosis. Transient knockdown of Cyr61 resulted in the inhibition of cell proliferation and migration. This was also associated with the concomitant downregulation of pAkt and pmTOR confirming the role of Cyr61 as an upstream modulator of Akt signaling. Conversely the extracellular supplementation of recombinant Cyr61 attenuates the cytotoxic properties of metformin in ovarian cancer cells.ConclusionsTaken together, we concluded that metformin exhibits anticancer effects and Cyr61 acts as a direct target for metformin in ovarian cancer cells.

Huaier Augmented the Chemotherapeutic Sensitivity of Oxaliplatin via Downregulation of YAP in Hepatocellular Carcinoma.

For unresectable Hepatocellular carcinoma (HCC), chemotherapy is still an important treatment strategy. Oxaliplatin (Oxa) is an effective treatment of HCC after sorafenib treatment failure. However, the intrinsic or acquired resistance of Oxa affected the chemotherapeutic sensitivity. By analyzing the data of GEO Database, we found that Oxa aberrantly increased the expression of Cysteine-rich61 (Cyr61) in HCC cell lines. Subsequently, in Bel-7404 and SMMC-7721 cells after treated with Oxa, it was verified that the expression of Cyr61 and Yes-associated protein (YAP) was increased. Moreover, we found that blockade of YAP promoted Oxa-induced cell apoptosis for the first time. Meanwhile, our previous study demonstrated that Huaier (HE) inhibited the expression of YAP. Further study found that combination treatment of Oxa and HE had a significantly synergistic anti-cancer effect and significantly inhibited the expression of YAP and apoptosis related proteins. Taken together, we have observed that overexpression of YAP significantly reduced the chemotherapeutic sensitivity of Oxa in HCC for the first time. Combination treatment of Oxa and HE solved this problem.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0–20 μM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Clinical relevance of Cyr61 expression in patients with hormone-dependent breast cancer.

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

CYR61—An angiogenic biomarker to early predict the impaired healing in diaphyseal tibial fractures

SummaryBackgroundAngiogenesis is a prerequisite for fracture repair, whereas insufficient blood supply is likely to result in impaired healing. In the present study, we aimed to determine the correlation of simple tibial fracture healing outcome with serial estimation of CYR61 expressions in the early phase of healing.MethodsIn total, 107 adult fractured patients and 97 healthy controls were analysed. Peripheral blood samples were taken from controls (at once) and fractured patients at 4th, 7th, 10th, 15th, 20th and 28th days of post-fracture follow-ups to quantify the CYR61 mRNA and protein expression by qRT-PCR and Western blotting assay, respectively. Clinic-radiological follow-up was done at 6th, 10th, 16th, 20th, and 24th weeks of post-fracture follow-ups using RUST scores to analyse the fracture healing progression and their final outcomes.ResultsBy considering controls as Group I (n = 97), as per the clinico-radiological status at 24th week, fracture patients were divided into two groups: Group II (normal healing, n = 91) and Group III (impaired healing, n = 16). Both CYR61 mRNA and protein expressions were lower (baseline) in Group I than in Groups II and III; however, a significant difference was observed only with the Group II. In both groups, expressions of CYR61 mRNA as well as protein gradually upregulated from the baseline to a peak and then declined. Both, the CYR61 mRNA as well as protein expressions were significantly higher at all follow-ups in Group II than in Group III. Mean RUST scores between Group II and Group III showed a significant statistical difference at each follow-up. Significant correlation was found between the CYR61 expressions and the RUST score (fracture healing progression).ConclusionWe conclude that CYR61 expression provides an early prediction of the healing outcomes of simple diaphyseal tibial fractures.The translational potential of this articleSuch an approach would benefit not only the patients' wellbeing but also the entire health care system in terms of the cost implications associated with long lasting treatment interventions and hospitalisation. However, the authors recommend further multicentric study with a large sample size to increase the validity, reliability, and generalisability of our observation and inferences.

Correlation between Cyr61 expression and clinicopathologic parameters in adenomyosis

Adenomyosis, a benign invasion of endometrium, is closely related to endometriosis. Cysteine-rich 61 (Cyr61), a protein present in all endometrial tissues and menstrual effluents, is known to be associated with endometriosis. However, its relation to adenomyosis has not been determined thus far. Therefore, here, we aimed to investigate the expression of Cyr61 protein in adenomyosis and determine the correlation between Cyr61 expression and clinicopathologic parameters in patients with adenomyosis. One hundred and twenty patients with histologically diagnosed adenomyosis, who underwent hysterectomy for non-endometrial disease were enrolled in this study. Patients were interviewed using a standard questionnaire consisting of sociodemographic characteristics and reproduction history. The severity of dysmenorrhea and menorrhagia was evaluated using the visual analogue scale (VAS) and pictorial blood-loss assessment chart (PBAC). Samples of serum, endometrial tissue, and peritoneal fluid were collected, and Cyr61 mRNA levels were determined by RT-PCR. The Cyr61 protein levels in endometrial and ectopic lesions were determined by immunohistochemistry and those in serum and peritoneal fluid, by ELISA. We found that expression of Cyr61 was higher in the ectopic endometrium than in the eutopic endometrium. Cyr61 expression in the endometrium was correlated with age, number of natural labors, PBAC score, VAS score, uterine volume, adenomyosis type, and concurrent endometriosis. The Cyr61 protein level in the ascites was higher than that in serum, and no correlation existed between them. Our results suggest that the expression of Cyr61 may be indirectly related to the degree of dysmenorrhea and Cyr61 may be involved in the pathogenesis of adenomyosis.

Abstract 552: Aberrant expression of CYR61 in bladder cancer and its preoperative diagnostic potential in muscle invasive bladder cancer

Abstract Cysteine-rich 61 (CYR61) is involved in development and progression in different types of human cancer. Nonetheless, the role of CYR61 in bladder cancer has not been reported. In this study, we explored CYR61 expression in tumor tissues of urothelial carcinoma of bladder and evaluated its diagnostic potential for the muscle invasive bladder cancer in preoperative urine. The global gene expression analysis was applied with the tumor tissues of 16 non-muscle invasive bladder cancers (NMIBCs) and 14 muscle invasive bladder cancers (MIBCs) using microarrays (the Agilent). After bioinformatic analysis, 10 genes that their expression signatures showed significant differences between the invasive and non-invasive tumors were selected, and then verified using RT-qPCR in the aforementioned 30 bladder cancer samples. Seven out of the10 genes was further validated in an independent cohort of 54 bladder cancers (32 NMIBCs and 22 MIBCs). Among the 4 genes that sustained significantly associated with tumor invasiveness through the validation, CYR61 was investigated in protein level by immunohistochemistry (IHC) staining in 115 formalin-fixed paraffin-embedded tumor tissues (59 NMIBCs and 56 MIBCs); followed by enzyme-linked immune sorbent assay (ELISA) applied in preoperative urine sample of 104 patients with bladder cancer (21 NMIBCs and 83 MIBCs). The mRNA transcription of CYR61 was significantly up-regulated in tumor tissues of MIBCs compared to those NMIBCs (fold change&amp;gt;2.0 and P value &amp;lt; 0.001) in both the discovery phase (microarray study) and the validation phase. The IHC staining further confirmed that expression of CYR61 protein was significantly associated with MIBCs. An intensive expression of CYR61 was observed in 64.9% of MIBCs, but in only 17.2% of NMIBCs (p = 0.001). On the other hand, we did not find any association between CYR61 expression and other analyzed clinicopathologic parameters, such as tumor size, tumor grade, tumor recurrence, patient's age, gender, drinking or smoking. CYR61 protein levels in the urine collected from the preoperative patients were determined by ELISA and measured as the ratio of urinary creatinine. We found an average 2.5 fold CYR61 urinary content in the MIBCs than that in the NMIBCs (10.48±9.36 ng/mg VS 4.12±2.26 ng/mg, p&amp;lt;0.001). Urinary CYR61 had 87.0% sensitivity and 61.7% specificity to predicate MIBCs (AUC 0.815, 95% CI 0.722-0.908, p&amp;lt;0.001). In conclusion, aberrant over-expression of CYR61 was associated with muscle invasion of bladder urothelial carcinoma in both mRNA and protein levels. The abundance of CYR61 protein in urine sample could be a potential biomarker for preoperatively differentiating MIBCs or NMIBCs, while its merits still need further investigation in large cohorts of the bladder cancer. Citation Format: Huang Chen, Jianzhong Shou, Yu Liu, Wei Zheng, Hao Xi, Jianhui Ma, Yan ning Gao. Aberrant expression of CYR61 in bladder cancer and its preoperative diagnostic potential in muscle invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 552. doi:10.1158/1538-7445.AM2015-552

Combined treatment with troglitazone and lovastatin inhibited epidermal growth factor-induced migration through the downregulation of cysteine-rich protein 61 in human anaplastic thyroid cancer cells.

Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

Abstract 18954: Novel Role of De Novo Matricellular Protein Cyr61 in Bridging Lysophosphatidic Acid and Integrin Pathways Leading to Smooth Muscle Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid, markedly induces smooth muscle cell (SMC) migration, which is one of the important processes involved in atherosclerotic lesion development. In order to search for key molecules that control LPA-induced SMC migration, we performed gene microarray analysis and attempted to identify LPA-induced extracellular molecules responsible for LPA-triggered cell migration. We observed that LPA selectively induced the expression of the matricellular protein Cyr61, a multifunctional matrix-related protein, which has been implicated in cell angiogenesis, proliferation, adhesion and migration. We hypothesize that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induces temporal and spatial expression of Cyr61 protein in SMCs. LPA-induced de novo Cyr61 proteins promptly accumulate in the Golgi apparatus and then translocate to the extracellular matrix. Cyr61 specific siRNAs diminishes LPA-induced cell migration, indicating the novel regulatory role of the induced matricellular protein Cyr61 in LPA-induced cell migration. Using primary SMCs derived from LPA receptor 1 (LPA1) knockout and LPA2 knockout mice, our data reveal that LPA1 is required for LPA-induced Cyr61 expression in SMCs and cell migration. Furthermore, LPA induces profound activation of focal adhesion kinase (FAK) in SMCs in later time points, coinciding with the accumulation of Cyr61in the extracellular matrix. FAK is an intracellular kinase important in regulating cell migration. Our data demonstrated that knockdown of de novo Cyr61 expression blocked LPA-induced cellular FAK activation and cell migration. Integrin alpha6 and beta1 have been reported to interact with Cyr61 in SMCs. We found that knockdown of the expression of integrin alpha6 or beta1 prevented LPA-induced FAK activation and cell migration. Taken together, these data reveal that a novel LPA/Cyr61 pathway controls cell migration and that de novo Cyr61 in extracellular matrix bridges LPA and integrin signaling, which in turn, activates FAK leading to cell migration. Understanding this novel pathway will provide new insights into mechanisms underlying the development of cell migration related disorders including atherosclerosis and restenosis.

Knockdown of cysteine-rich 61 inhibits proliferation, migration, and invasiveness of prostate carcinoma PC-3 cells

The overexpression of wild-type cysteine-rich 61 (Cyr61) is associated with the aggressiveness of cancer and poor prognosis in different human cancers. The aim of this study was to examine the expression of Cyr61 protein in prostate adenocarcinomas and to investigate the effects of inhibiting Cyr61 expression using small interfering RNA on the proliferation, migration, invasiveness, and sensitivity of prostate carcinoma PC-3 cells to TNF-related apoptosis-inducing ligand. Cell proliferation was measured by XTT assay. Colony formation assay, wound healing assay, and Matrigel invasion assay were also examined. Immunohistochemical staining of prostate tumor tissues showed that prostate carcinomas had significantly increased Cyr61 level compared with benign glands adjacent to carcinoma. siRNA-based knockdown of Cyr61 resulted in decreased cellular proliferation, clonogenicity, migration, and invasion. At the same time, Cyr61 silencing effectively reduced the levels of phosphorylated Akt and integrin-β3. Cyr61-specific siRNA combined with TRAIL increased the apoptosis of PC-3 cells and the cleavage of apoptosis hallmarkers such as PARP and caspase-3. Taken together, our data provide evidence that Cyr61 modulates integrin-β3 level as well as PI3-kinase/Akt activity through which Cyr61 may mediate tumorigenesis, and suggest the potential importance of Cyr61 targeting on enhancing the therapeutic efficacy of prostate cancer.

Abstract 67: The Novel Role of the Matricellular Protein Cyr61 in the Extracellular Matrix Bridging LPA and Integrin Signaling Pathways Leading to Smooth Muscle Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid, markedly induces smooth muscle cell (SMC) migration. We identified a novel function of the de novo synthesized matricellular protein Cyr61, which bridges LPA and integrin signaling pathways leading to cell migration. LPA transiently and markedly induced Cyr61 mRNA expression and the temporal and spatial expression of Cyr61 proteins in SMCs. The de novo synthesized Cyr61 proteins promptly accumulate in the Golgi apparatus and then translocate to the extracellular matrix. Using primary SMCs from LPA receptor knockout mice, we identified that LPA1 receptor is required for LPA-induced Cyr61 expression. Neutralization or knockdown of Cyr61 protein expression blocked LPA-induced cell migration, indicating the novel regulatory role of the induced matricellular protein Cyr61 in LPA-induced cell migration. LPA induces the activation of intracellular focal adhesion kinase (FAK) in SMCs. Interestingly, 1) knockdown of Cyr61 blocked LPA-induced cellular FAK activation and cell migration, and 2) knockdown of the expression of integrins alpha6, beta1 and beta3 prevented LPA or Cyr61-induced FAK activation and cell migration. These data reveal for the first time that a novel LPA/Cyr61 pathway controls cell migration and that the matricellular protein Cyr61 bridges LPA signaling and integrin signaling, which, in turn, activates FAK leading to cell migration.