CYR61/CCN1: A novel mediator of redox response in corneal stromal cells of keratoconus.

Keratoconus (KC) is a progressive, multifactorial and ectatic corneal disorder that characterized by steepening thinning of the cornea. It was previously demonstrated that oxidative stress has a strong link with KC progression. However, the molecular mechanism underlying oxidative stress response in KC remains unclear. Hence, the present study analyzed the heterogeneity of response of corneal stromal cells (CSCs) to oxidative stress in order to further illustrate how oxidative shape the pathophysiology of KC. Single-cell transcriptomics analysis revealed that CSCs demonstrated significant higher oxidative stress score in the KC group compared to the Ctrl group. The expression of oxidative markers verified by experiments illustrated elevated oxidative stress levels and insufficient antioxidant levels in CSCs of KC. In further single-cell transcriptomics analysis, we identified CYR61 to distinguish different subgroups of CSCs responding to oxidative stress. The cornea stroma cells in KC could be differentiated into CYR61high cells and CYR61low cells. Of note, the CYR61high cells showed lower score in collagen production process and higher score in collagen catabolic process. Further experiments illustrated that CYR61 was elevated in KC and associated with collagen production.

Cysteine-rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high-fat diet fed mice and HepG2 cells.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is strongly associated with insulin resistance development. Hepatic lipid accumulation and inflammation are considered the main drivers of hepatic insulin resistance in MASLD. Cysteine-rich 61 (Cyr61 also called CCN1), a novel secretory matricellular protein, is implicated in liver inflammation, and its role in MASLD is not clearly understood. Therefore, we investigated the role of Cyr61 in hepatic insulin resistance and lipid metabolism as major factors in MASLD pathogenesis. In high-fat diet (HFD)-fed C57BL/6J mice, Cyr61 was downregulated or upregulated via viral transduction. Measurements of glucose homeostasis, histological assessment of liver tissues, and gene expression and signaling pathways of lipogenesis, fatty acid oxidation, and inflammation were performed using liver samples from these mice. Cyr61 levels in HepG2 cells were reduced using RNAi-mediated gene knockdown. Inflammation and insulin resistance were evaluated using real-time polymerase chain reaction and western blotting. HFD/AAV-shCyr61 mice exhibited enhanced glucose tolerance via the protein kinase B pathway, reduced hepatic inflammation, decreased lipogenesis, and increased fatty acid oxidation. Notably, HFD/AAV-shCyr61 mice showed elevated protein expression of sirtuin 6 and phosphorylated-AMP-activated protein kinase. In vitro experiments demonstrated that inhibition of Cyr61 downregulated pro-inflammatory cytokines such as interleukin-1 beta, IL-6, and tumor necrosis factor-alpha via the nuclear factor kappa B/c-Jun N-terminal kinase pathway, and alleviated insulin resistance. Cyr61 affected hepatic inflammation, lipid metabolism, and insulin resistance. Inhibition of Cyr61 reduced inflammation, recovered insulin resistance, and altered lipid metabolism in vivo and in vitro. Therefore, Cyr61 is a potential therapeutic target in MASLD.

CYR61 is Involved in Neonatal Hypoxic-ischemic Brain Damage Via Modulating Astrocyte-mediated Neuroinflammation.

The activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in astrocytes has been found in the hypoxic-ischemic brain damage (HIBD) model. Cysteine rich angiogenic inducer 61 (CYR61) is secreted by reactive astrocytes. However, the effects of CYR61 on HIBD and its related mechanisms remain unclear. This study sought to explore the role of CYR61 in the activation of astrocytes and the NLRP3 inflammasome in neonatal HIBD. HIBD models were established in 7-day Sprague-Dawley rat pups. Neurobehavioral evaluation and 2,3,5-triphenyl-tetrazolium chloride staining were performed. In addition, rat primary astrocytes were used to establish the cell model of HIBD in vitro by oxygen-glucose deprivation/reperfusion (OGD/R). Then, CYR61-overexpression and sh-CYR61 viruses mediated by lentivirus were transduced into ODG/R-treated primary astrocytes. The expressions of related genes were evaluated using real-time quantitative PCR, western blot, immunofluorescence staining, and Enzyme-linked immunosorbent assay. The results showed that hypoxia-ischemia induced short-term neurological deficits, neuronal damage, and cerebral infarction in neonatal rats. In vivo, the expressions of CYR61, NLRP3, and glial fibrillary acidic protein (GFAP) were up-regulated in the HIBD model. In vitro, CYR61 exhibited high expression. CYR61 overexpression increased the expressions of GFAP and C3, whereas decreased S100A10 expression. CYR61 overexpression increased the expression of NLRP3, ASC, caspase-1 p20 and IL-1ß. CYR61 overexpression activated NF-κB by promoting the phosphorylation of IκBα and p65. Thus, CYR61 is involved in neonatal HIBD progress, which may be related to the activation of astrocytes, the NLRP3 inflammasome, and the NF-κB signaling pathway.

CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.

Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.

Inhibitory Effects and Mechanisms of the Novel Shikonin Derivative DMAKO-20 on Melanoma Metastasis and Invasion.

BACKGROUND: Cutaneous melanoma is a malignant tumor with an increasing incidence, prone to recurrence and metastasis. This study aims to explore the effects and mechanisms of the novel shikonin derivative 5,8-dimethyl alkannin oxime derivative (DMAKO-20) on the metastasis and invasion of melanoma cells. METHODS: The inhibitory effects of DMAKO-20 on the melanoma cell line A375 were investigated through Cell Counting Kit-8 (CCK-8), Transwell and angiogenesis experiments. Network pharmacology and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to explore potential sites and pathways involved in this process. Additionally, quantitative polymerase chain reaction (qPCR) and Western blot experiments were conducted before and after drug treatment to verify the expression trends of related pathways and proteins. RESULTS: DMAKO-20 demonstrated selective inhibition of proliferation, invasion and migration of melanoma cells at low concentrations. The WNT pathway appears to be implicated in this process, as DMAKO-20 effectively attenuates its activation, consequently reducing matrix metalloproteinase 9 (MMP9) and Cellular Communication Network Factor 1 (CCN1)/cysteine-rich angiogenic inducer 61 (CYR61) levels. Such modulation inhibits melanoma dissemination and invasion into other tissues. CONCLUSION: DMAKO-20 exhibits the capability to suppress metastasis and invasion of melanoma cells, suggesting its potential for clinical application as an adjuvant therapy against melanoma.

Chemotherapy-initiated cysteine-rich protein 61 decreases acute B-lymphoblastic leukemia chemosensitivity.

PURPOSE: Chemoresistance is a major challenge for acute lymphoblastic leukemia (ALL) treatment. Cysteine-rich protein 61 (Cyr61) plays an important role in drug resistance modulation of tumor cells, and Cyr61 levels are increased in the bone marrow of patients with ALL and contribute to ALL cell survival. However, the effect of Cyr61 on B cell acute lymphoblastic leukemia (B-ALL) cell chemosensitivity and the regulatory mechanisms underlying Cyr61 production in bone marrow remain unknown. METHODS: Nalm-6 and Reh human B-ALL cell lines were used in this study. Cyr61 levels were assessed using quantitative real-time PCR (qRT-PCR), western blot analysis, and enzyme-linked immunosorbent assay. The effect of Cyr61 on B-ALL cell chemosensitivity to daunorubicin (DNR) was evaluated using cell viability and flow cytometry analyses. The regulatory mechanisms of Cyr61 production in bone marrow were examined using qRT-PCR and western blot analysis. RESULTS: Cyr61 knockdown and overexpression increased and decreased the chemosensitivity of B-ALL cells to DNR, respectively. Cyr61 attenuated chemotherapeutic drug-induced apoptosis by upregulating B cell lymphoma-2. Notably, DNR induced DNA damage response and increased Cyr61 secretion in B-ALL cells through the ataxia telangiectasia mutated (ATM)-dependent nuclear factor kappa B pathway. CONCLUSION: DNR induces Cyr61 production in B-ALL cells, and increased Cyr61 levels reduce the chemosensitivity of B-ALL cells. Consequently, targeting Cyr61 or related ATM signaling pathway may present a promising treatment strategy to enhance the chemosensitivity of patients with B-ALL.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Targeting cell-type-specific, choroid-peripheral immune signaling to treat age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness featuring pathogenic neovascularization of the choroidal vasculature (CNV). Although systemic immunity plays a role in AMD, the ocular signals that recruit and activate immune cells remain poorly defined. Using single-cell RNA sequencing, we prospectively profile peripheral blood mononuclear cells from 65 individuals including AMD and controls, which we integrate with existing choroid data. We generate a network of choroid-peripheral immune interactions dysregulated in AMD, including known AMD-relevant gene vascular endothelial growth factor (VEGF) receptor 2. Additionally, we find CYR61 is upregulated in choroidal veins and may signal to circulating monocytes. In mice, we validate that CYR61 is abundant in endothelial cells within CNV lesions neighboring monocyte-derived macrophages. Mechanistically, CYR61 activates macrophage anti-angiogenic gene expression, and ocular Cyr61 knockdown increases murine CNV size, indicating CYR61 inhibits CNV. This study highlights the potential of multi-tissue human datasets to identify disease-relevant and potentially therapeutically modifiable targets.

Inhibition of Increased Invasiveness of Breast Cancer Cells With Acquired Tamoxifen Resistance by Suppression of CYR61.

BACKGROUND/AIM: Hormone sensitivity-targeted therapy with selective estrogen receptor modulators (SERMs), such as 4-hydroxytamoxifen (4-OHT), is the mainstay of treatment for breast cancers (BCs) that express estrogen receptor α (ERα). However, development of resistance limits this therapy approach. The question arises whether changes associated with 4-OHT resistance could be exploited therapeutically. MATERIALS AND METHODS: First, 4-OHT-resistant sublines of ERα-positive breast carcinoma cell lines MCF-7 and T47D were generated. Viability was assessed by the Alamar Blue assay. Cell invasion was quantified in modified Boyden chambers with Matrigel. Changes in expression of CYR61, S100A4, and ERα were examined by RT-qPCR. Expression of CYR61 was suppressed by transient gene silencing using siRNA. Successful suppression was verified by western blot. Efficacy of 4-OHT treatment was analyzed by quantification of viability using Alamar Blue assay. Correlation of CYR61 levels in patients with luminal A BC to distant metastases-free survival was determined by Kaplan-Meier analysis. RESULTS: ERα-positive MCF-7 and T47D BC cells exhibit an extremely weak invasion rate. Acquired tamoxifen resistance significantly increased the invasive behavior of both tamoxifen-resistant MCF-7-TR and T47D-TR sublines. In addition, expression of CYR61 and S100A4 showed significantly increased levels, whereas expression of ERα was decreased. Suppression of CYR61 expression resulted in a significant decreased invasion rate. In addition, expression of S100A4 was reduced, whereas expression of ERα was increased. Furthermore, suppression of CYR61 resulted in re-sensitization to 4-OHT. High CYR61 levels in patients with luminal A BC resulted in reduced distant metastases-free survival. CONCLUSION: The prometastatic factor CYR61 appears to play an important role in the increased invasiveness of tamoxifen-resistant ERα-positive BC cells. Its suppression leads to a lower invasion rate. Given the few therapeutic options available for tamoxifen-resistant BC, therapy that reduces CYR61 may improve its treatability in future.

Is the Hippo Pathway Effector Yes-Associated Protein a Potential Key Player of Dairy Cattle Cystic Ovarian Disease Pathogenesis?

Cystic ovarian disease (COD) in dairy cattle is characterized by preovulatory follicles that become cysts, fail to ovulate and persist in the ovary; consequently, interfering with normal ovarian cyclicity. The intraovarian key players that orchestrate the alterations occurring in the preovulatory follicle and that culminate with cyst formation and persistence, however, remain uncertain. Interestingly, the Hippo pathway effector yes-associated protein (YAP) has been described in humans and mice as a key player of anovulatory cystic disorders. To start elucidating if YAP deregulation in ovarian follicle cells can be also involved in the pathogenesis of COD, we have generated a series of novel results using spontaneously occurring cystic follicles in cattle. We found that mRNA and protein levels of YAP are significantly higher in granulosa (GCs) and theca cells (TCs) isolated from cystic follicles (follicular structures of at least 20 mm in diameter) in comparison to respective cell types isolated from non-cystic large follicles (≥12 mm). In addition, immunohistochemistry and Western blot analyses used to determine YAP phosphorylation pattern suggest that YAP transcriptional activity is augmented is cystic GCs. These results were confirmed by a significant increase in the mRNA levels encoding for the classic YAP-TEAD transcriptional target genes CTGF, BIRC5 and ANKRD1 in GCs from follicle cysts in comparison to non-cystic large follicles. Taken together, these results provide considerable insight of a completely novel signaling pathway that seems to play an important role in ovarian cystic disease pathogenesis in dairy cattle.

Corrigendum: Serum CYR61 is associated with airway inflammation and is a potential biomarker for severity in chronic obstructive pulmonary disease.

[This corrects the article DOI: 10.3389/fmed.2021.781596.].

Transformer 2ß regulates the alternative splicing of cell cycle regulatory genes to promote the malignant phenotype of ovarian cancer.

Late-stage ovarian cancer (OC) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is unclear. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. In this study, we explored the molecular functions of a canonical RBP, Transformer 2ß homolog (TRA2B), in cancer cells. TRA2B knockdown in HeLa cells and subsequent whole-transcriptome RNA sequencing (RNA-seq) analysis revealed the TRA2B-regulated alternative splicing (AS) profile. We disrupted TRA2B expression in epithelial OC cells and performed a series of experiments to confirm the resulting effects on OC cell proliferation, apoptosis and invasion. TRA2B-regulated AS was tightly associated with the mitotic cell cycle, apoptosis and several cancer pathways. Moreover, the expression of hundreds of genes was regulated by TRA2B, and these genes were enriched in the functions of cell proliferation, cell adhesion and angiogenesis, which are related to the malignant phenotype of OC. By integrating the alternatively spliced and differentially expressed genes, we found that AS events and gene expression were regulated independently. We then explored and validated the oncogenic functions of TRA2B by knocking down its expression in OC cells. The high TRA2B expression was associated with poor prognosis in patients with OC. In ovarian tissue, TRA2B expression showed a gradual increasing trend with increasing malignancy. We demonstrated the important roles of TRA2B in ovarian neoplasia and aggressive OC behaviors and identified the underlying molecular mechanisms, facilitating the targeted treatment of OC.

CircUSP48 promotes malignant behavior by regulating CYR61 via miR-365 in osteosarcoma.

Even though circular RNAs (circRNAs), a class of non-coding endogenous RNA, play a crucial role in the progression of osteosarcoma (OS), the specific function of hsa_circ_0000028 (circUSP48) remains unclear. This study aims to elucidate the mechanism by which circUSP48 regulates OS. We employed qRT-PCR and western blot techniques to quantify circDOCK1, miR-186, and DNMT3A levels. Cell proliferation was assessed using the cell counting kit-8 (CCK-8), 5-Ethynyl-20-deoxyuridine (EdU) assay, and colony formation assay. Cell migration and invasion were evaluated through Transwell and cell scratch assays. Furthermore, we performed dual-luciferase reporter, RIP, and RNA pull-down assays to investigate the association between circUSP48, miR-365, and CYR61. In addition, an in vivo xenograft model was utilized to assess the functional role of circUSP48. High levels of circUSP48 and CYR61 were observed in OS tissues and cells, while miR-365 levels were low. Knockdown of circUSP48 suppressed the multiplication, motility, and invasion of OS cells, thereby reducing carcinoma growth. Moreover, inhibition of miR-365 reversed the OS cell-suppressive effect caused by circUSP48 knockdown through direct interaction with circUSP48. Additionally, circUSP48 upregulated the expression of CYR61 by sponging miR-365. The findings suggest that circUSP48 promotes malignant behavior in OS by regulating the expression of CYR61 through miR-365, making it a potential therapeutic target for OS.

Changes and related factors of blood CCN1 levels in diabetic patients.

Objective: To study the differences in blood cellular communication network factor 1 (CCN1) levels between patients with diabetes mellitus (DM) and healthy individuals and to explore the relationship between CCN1 and diabetic retinopathy (DR). Methods: Plasma CCN1 levels were detected using ELISA in 50 healthy controls, 74 patients with diabetes without diabetic retinopathy (DM group), and 69 patients with diabetic retinopathy (DR group). Correlations between CCN1 levels and age, body mass index, mean arterial pressure, hemoglobin A1c, and other factors were analyzed. The relationship between CCN1 expression and DR was explored using logistic regression after adjusting for confounding factors. Blood mRNA sequencing analysis was performed for all subjects, and the molecular changes that may be related to CCN1 were explored. The retinal vasculature of streptozotocin-induced diabetic rats was examined using fundus fluorescein angiography; in addition, retinal protein expression was examined using western blotting. Results: Plasma CCN1 levels in patients with DR were significantly higher than in the control and DM groups; however, no significant differences were observed between healthy controls and patients with DM. CCN1 levels negatively correlated with body mass index and positively correlated with the duration of diabetes and urea levels. It was observed that high (OR 4.72, 95% CI: 1.10-20.25) and very high (OR 8.54, 95% CI: 2.00-36.51) levels of CCN1 were risk factors for DR. Blood mRNA sequencing analysis revealed that CCN1-related pathways were significantly altered in the DR group. The expression of hypoxia-, oxidative stress-, and dephosphorylation-related proteins were elevated, while that of tight junction proteins were reduced in the retinas of diabetic rats. Conclusion: Blood CCN1 levels are significantly elevated in patients with DR. High and very high levels of plasma CCN1 are risk factors for DR. Blood CCN1 level may be a potential biomarker for diagnosis of DR. The effects of CCN1 on DR may be related to hypoxia, oxidative stress, and dephosphorylation.

Extracellular vesicles in fatty liver promote a metastatic tumor microenvironment.

Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.

CCN1 expression is regulated by mechanical stimuli in tendons.

Tendon overuse injuries are common, but the processes that govern tendon response to mechanical load are not fully understood. A series of experiments of in vitro and in vivo experiments was devised to study to the relationship between mechanical stimuli and the matricellular protein Cellular Communication Network Factor 1 (CCN1) in tenocytes and tendons. First, human and murine tenocytes were subjected to cyclic uniaxial loading in order to evaluate changes in CCN1 gene expression as a response to mechanical stimuli. Then, baseline Ccn1 gene expression in different murine tendons (Achilles, patellar, forearm, and tail) was examined. Finally, changes in Ccn1 expression after in vivo unloading experiments were examined. It was found that CCN1 expression significantly increased in both human and murine tenocytes at 5 and 10% cyclical uniaxial strain, while 2.5% strain did not have any effect on CCN1 expression. At baseline, the Achilles, patellar, and forearm tendons had higher expression levels of Ccn1 as compared to tail tendons. Twenty-four hours of immobilization of the hind-limb resulted in a significant decrease in Ccn1 expression in both the Achilles and patellar tendons. In summary, CCN1 expression is up-regulated in tenocytes subjected to mechanical load and down-regulated by loss of mechanical load in tendons. These results show that CCN1 expression in tendons is at least partially regulated by mechanical stimuli.

[Effect of Cyr61 on Imatinib Resistance in Chronic Myeloid Leukemia and Its Mechanism].

OBJECTIVE: To investigate the effect of Cyr61 on imatinib (IM) resistance in chronic myeloid leukemia (CML) and its mechanism. METHODS: Cyr61 level in cell culture supernatant was determined by enzyme-linked immunosorbent assay. The expression of Cyr61 and Bcl-xL were measured by real-time PCR and Western blot. Cell apoptosis was analyzed using an Annexin V-APC Kit. Expression of signal pathways related proteins was determined by Western blot. RESULTS: The level of Cyr61 obviously increased in K562G cells (IM resistance to CML cell line K562). Down-regulating the expression of Cyr61 decreased the resistance of K562G cells to IM and promoted IM induced apoptosis. In CML mouse model, down-regulating the expression of Cyr61 could increase the sensitivity of K562G cells to IM. The mechanism studies showed that Cyr61 mediated IM resistance in CML cells was related to the regulation of ERK1/2 pathways and apoptosis related molecule Bcl-xL by Cyr61. CONCLUSION: Cyr61 plays an important role in promoting IM resistance of CML cells. Targeting Cyr61 or its related effectors pathways may be one of the ways to overcome IM resistance of CML cells.

Increased CYR61 expression activates CCND1/c-Myc pathway to promote nasal epithelial cells proliferation in chronic rhinosinusitis with nasal polyps.

PURPOSE: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic sinonasal inflammatory disease characterized histologically by hyperplastic nasal epithelium and epithelial cells proliferation. Cysteine-rich angiogenic inducer 61 (CYR61) acts as a positive regulator of cell cycle process. Cyclin D1 (CCND1) and c-Myc play key roles in the processes of cell cycle and cell growth. The purpose of our research was to explore the expression and roles of CYR61, CCND1 and c-Myc in CRSwNP. METHODS: FeaturePlot and vlnPlot functions embedded in the seurat package (version 4.1.1) of R software (version 4.2.0) were applied to explore the cellular distribution of CYR61, CCND1 and c-Myc in the single-cell RNA sequencing (scRNA-seq) dataset of nasal tissue samples. CYR61, CCND1 and c-Myc immunolabeling and mRNA levels in nasal tissue samples were assessed by immunohistochemistry and real-time PCR. Co-localization of CYR61, CCND1 and c-Myc with basal epithelial cell marker P63 was assayed using double-label immunofluorescence staining. Furthermore, we collected and cultured human nasal epithelial cells (HNEC) to assess the regulation and role of CYR61 in vitro study. RESULTS: CYR61, CCND1 and c-Myc were primarily expressed by nasal epithelial cells. Significant upregulation of CYR61, CCND1 and c-Myc positive cells and increased levels of CYR61, CCND1 and c-Myc mRNA were found in nasal polyps in comparison to control samples. Of note, CYR61 mRNA and protein levels were altered by SEB, LPS, IFN-γ, IL-13, IL-17A and TGF-ß1 in HNEC. In addition, CYR61 intervention could increase CCND1 and c-Myc mRNA and protein levels to promote HNEC proliferation, and siRNA against ITGA2 (si-ITGA2) could reverse CYR61 induced upregulation of CCND1 and c-Myc mRNA and protein levels in HNEC and cell proliferation of HNEC. CONCLUSIONS: CYR61, CCND1 and c-Myc were primarily expressed by epithelial cells in nasal mucosa. CYR61, CCND1 and c-Myc expression levels were increased in CRSwNP compared with controls. CYR61 could interact with ITGA2 to enhance HNEC proliferation via upregulating CCND1 and c-Myc levels in the HNEC, leading to hyperplastic nasal epithelium in CRSwNP.

The prognostic implications and tumor-suppressive functions of CYR61 in estrogen receptor-positive breast cancer.

Due to the therapeutic resistance of endocrine therapy and the limited efficacy of immune checkpoint inhibitors in estrogen receptor (ER)-positive breast cancer (BRCA), there is an urgent need to develop novel prognostic markers and understand the regulation of the tumor immune microenvironment (TIME). As a matricellular protein, CYR61 has been shown to either promote or suppress cancer progression depending on cancer types. However, how CYR61 functions in ER-positive BRCA remains elusive. In this study, we comprehensively analyzed the expression of CYR61 in BRCA based on the TCGA and METABRIC databases. Our findings showed that the expression of CYR61 is downregulated in different subtypes of BRCA, which is associated with elevated promoter methylation levels and predicts bad clinical outcomes. By comparing the high or low CYR61 expression groups of ER-positive BRCA patients, we found that CYR61 is intimately linked to the expression of genes involved in tumor-suppressive pathways, such as the TGF-ß and TNF signaling pathways, and genes related to cytokine-receptor interaction that may regulate cancer immunity. Moreover, reduced CYR61 expression is associated with an altered TIME that favors cancer progression. Finally, experimental analyses ascertained that CYR61 is downregulated in clinical BRCA tissues compared to matched normal breast tissues. Furthermore, CYR61 is able to impede the proliferation and colony formation of ER-positive BRCA cells. In summary, our study reveals that CYR61 could serve as a novel prognostic marker for ER-positive BRCA, and function as an inhibitor of cancer progression by both acting on cancer cells and remodeling the TIME.

Acidosis-Induced Regulation of Egr1 and Ccn1 In Vitro and in Experimental Tumours In Vivo.

Extracellular acidosis is a characteristic of solid tumours, resulting from hypoxia-induced glycolytic metabolism as well as from the "Warburg effect" (aerobic glycolysis). The acidic environment has shown to affect functional tumour properties (proliferation, migration, invasion) and thus the aim of the study was to identify signalling mechanisms, mediating these pH-dependent effects. Therefore, the serum response factor (Srf) and the activation of the serum response element (SRE) by acidosis were analysed in AT-1 prostate carcinoma cells. Furthermore, the expression of downstream targets of this cascade, namely the early growth response 1 (Egr1), which seems to be involved in tumour proliferation, and the cellular communication network factor 1 (Ccn1), which both contain SRE in their promotor region were examined in two tumour cell lines. Extracellular acidification led to an upregulation of Srf and a functional activation of the SRE. Egr1 expression was increased by acidosis in AT-1 cells whereas hypoxia had a suppressive effect. In experimental tumours, in vivo Egr1 and Ccn1 were also found to be acidosis-dependent. Also, it turned out that pH regulated expression of Egr1 was followed by comparable changes of p21, which is an important regulator of the cell cycle.This study identifies the Srf-SRE signalling cascade and downstream Egr1 and Ccn1 to be acidosis-regulated in vitro and in vivo, potentially affecting tumour progression. Especially linked expression changes of Egr1 and p21 may mediate acidosis-induced effects on cell proliferation.