Identification of BGN and THBS2 as metastasis-specific biomarkers and poor survival key regulators in human colon cancer by integrated analysis.

BACKGROUND: Colon cancer is the second leading cause of death worldwide. Exploring key regulators in colon cancer metastatic progression could lead to better outcomes for patients. METHODS: Initially, the transcriptional profiles of 681 colonrectal cancer (CRC) cases were used to discover signature genes that were significantly correlated with colon cancer metastasis. These signature genes were then validated using another independent 210 CRC cases' transcriptomics and proteomics profiles, and Kaplan-Meier regression analyses were used to screen the key regulators with patients' survival. Immunohistochemical staining was used to confirm the biomarkers, and transit knockdown was used to explore their implications on colon cancer cells migration and invasion abilities. The impact on the key signalling molecules in epithelial-mesenchymal transition (EMT) process that drive tumour metastasis was tested using Western blot. The response to clinical standard therapeutic drugs was compared to clinical prognosis of key regulators using an ROC plotter. RESULTS: Five genes (BGN, THBS2, SPARC, CDH11 and SPP1) were initially identified as potential biomarkers and therapeutic targets of colon cancer metastasis. The most significant signatures associated with colon cancer metastasis were determined to be BGN and THBS2. Furthermore, highly expression of BGN and THBS2 in tumours was linked to a worse survival rate. BGN and THBS2 knockdown significantly reduced colon cancer cells migration and invasion, as well as down-regulating three EMT-related proteins (Snail, Vimentin and N-cadherin), and increasing the proliferation inhibitory effect of 5-fluorouracil, irinotecan and oxaliplatin treatment. CONCLUSIONS: CRC metastatic progression, EMT phenotypic transition and poor survival time have been linked to BGN and THBS2. They could be utilized as potential diagnostic and therapeutic targets for colon cancer metastatic patients with a better prognosis.

Decorin evokes reversible mitochondrial depolarization in carcinoma and vascular endothelial cells.

Decorin, a small leucine-rich proteoglycan with multiple biological functions, is known to evoke autophagy and mitophagy in both endothelial and cancer cells. Here, we investigated the effects of soluble decorin on mitochondrial homeostasis using live cell imaging and ex vivo angiogenic assays. We discovered that decorin triggers mitochondrial depolarization in triple-negative breast carcinoma, HeLa, and endothelial cells. This bioactivity was mediated by the protein core in a time- and dose-dependent manner and was specific for decorin insofar as biglycan, the closest homolog, failed to trigger depolarization. Mechanistically, we found that the bioactivity of decorin to promote depolarization required the MET receptor and its tyrosine kinase. Moreover, two mitochondrial interacting proteins, mitostatin and mitofusin 2, were essential for downstream decorin effects. Finally, we found that decorin relied on the canonical mitochondrial permeability transition pore to trigger tumor cell mitochondrial depolarization. Collectively, our study implicates decorin as a soluble outside-in regulator of mitochondrial dynamics.

Biglycan, tumor endothelial cell secreting proteoglycan, as possible biomarker for lung cancer.

OBJECTIVES: In lung cancer, surgery remains the most curative treatment and limited resection is beneficial for patients with low cardiopulmonary function and low malignancy tumors. However, there are no biomarkers of low malignancy to select candidates for limited resection without compromising the outcome of treatments. Recently we identified biglycan (BGN) as a tumor endothelial cell (TEC) marker that is associated with tumor progression in various cancers. In this study, we analyzed the association between BGN expression in TECs in lung cancer and cancer progression in patients. MATERIALS AND METHODS: First, we performed immunohistochemistry of BGN with resected lung tumor tissues of 155 patients who had undergone thoracic surgery and analyzed the correlation between BGN-positive vessel density in primary lung tumors and clinicopathological factors. Second, we measured the BGN levels in preoperative serum of other 46 patients with lung cancer by ELISA, and analyzed the correlation between BGN expression in tumor tissues and blood BGN levels. RESULTS: High BGN expression in the TECs was significantly associated with T factor, and was a significant negative predictor. BGN levels in preoperative serum of 46 patients with lung cancer was significantly correlated with BGN expression in the TECs. Preoperative serum BGN level was significantly lower in healthy volunteers and less invasive adenocarcinoma than in invasive adenocarcinoma and other lung carcinomas. These results suggest that low BGN level in preoperative serum in patients with lung cancer might indicate low malignancy. CONCLUSIONS: BGN can be a potential biomarker for lung cancer.

Age-Dependent Changes of Adipokine and Cytokine Secretion From Rat Adipose Tissue by Endogenous and Exogenous Toll-Like Receptor Agonists.

White adipose tissue but recently also brown adipose tissue have emerged as endocrine organs. Age-associated obesity is accompanied by prolonged and elevated lipopolysaccharide (LPS)-induced sickness symptoms and increased cytokine and adipokine levels in the circulation partially originating from adipose tissue. In the present study, ex vivo fat explants were used to investigate how the exogenous pathogen-associated molecular pattern (PAMP) LPS or the endogenous danger-associated molecular patterns (DAMPs) high mobility group box-1 protein (HMGB1) and biglycan modulate the release of cytokines and adipokines/batokines and, thus, could influence systemic and/or local inflammation. The response of adipose tissue (epididymal, retroperitoneal, subcutaneous, and brown) was compared between young lean and old obese rats (2 vs. 24 months old). LPS induced a strong interleukin (IL)-6 and tumor necrosis factor (TNF) alpha release into the supernatant of all adipose tissue types investigated. HMGB1 (subcutaneous) and biglycan (retroperitoneal) led to an increased release of IL-6 and TNFalpha (HMGB1) and decreased visfatin and adiponectin (biglycan) secretion from epididymal adipose tissue (young rats). Visfatin was also decreased by HMGB1 in retroperitoneal adipose tissue of old rats. We found significantly higher leptin (all fat pads) and adiponectin (subcutaneous) levels in supernatants of adipose tissue from old compared to young rats, whereas visfatin secretion showed the opposite. The expression of the biglycan receptor Toll-like receptor (TLR) 2 as well as the LPS and HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE) were reduced with age (TLR4/RAGE) and by stimulation with their ligands (subcutaneous). Overall, we revealed that adipokines/adipose-tissue released cytokines show some modulation of their release caused by mediators of septic (batokines) and sterile inflammation with potential implication for acute and chronic disease. Moreover, aging may increase or decrease the release of fat-derived mediators. These data show that DAMPS and LPS locally modulate cytokine secretion while only DAMPS but not LPS can locally alter adipokine secretion during inflammation.

Biglycan is a new high-affinity ligand for CD14 in macrophages.

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14-/- mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells.

Changes in the wall of seminiferous tubules in men with impaired spermatogenesis imply sterile inflammation of the testis. We tested the hypothesis that the cells forming the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), orchestrate inflammatory events and that Toll like receptors (TLRs) and danger signals from the extracellular matrix (ECM) of this wall are involved. In cultured HTPCs we detected TLRs, including TLR2. A TLR-2 ligand (PAM) augmented interleukin 6 (IL-6), monocyte chemo-attractant protein-1 (MCP-1) and pentraxin 3 (PTX3) in HTPCs. The ECM-derived proteoglycan biglycan (BGN) is secreted by HTPCs and may be a TLR2-ligand at HTPCs. In support, recombinant human BGN increased PTX3, MCP-1 and IL-6 in HTPCs. Variable endogenous BGN levels in HTPCs derived from different men and differences in BGN levels in the tubular wall in infertile men were observed. In testes of a systemic mouse model for male infertility, testicular sterile inflammation and elevated estradiol (E2) levels, BGN was also elevated. Hence we studied the role of E2 in HTPCs and observed that E2 elevated the levels of BGN. The anti-estrogen ICI 182,780 blocked this action. We conclude that TLR2 and BGN contribute to sterile inflammation and infertility in man.

Biglycan stimulates VEGF expression in endothelial cells by activating the TLR signaling pathway.

Biglycan (BGN) is an important component of the extracellular matrix (ECM) that is implicated in a variety of human cancers. In our previous study, we reported that BGN was overexpressed in gastric cancer (GC) tissues and promoted cancer metastasis. Moreover, the tubular formation capacity in HUVECs was promoted by stimulation with culture media from BGN-overexpressing GC cells, but the exact underlying mechanism is still unknown. The purpose of this study was to determine the role and molecular mechanism of BGN in VEGF expression in endothelial cells. We found that BGN stimulation of endothelial cells increased the interaction between NF-kB and the HIF-1α promoter, leading to enhanced promoter activity and increased HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. All of this was dependent on the interaction of BGN with its receptors, TLR2 and TLR4. Moreover, we found that BGN enhanced endothelial cell migration and proliferation, as well as tube formation, in a TLR signaling pathway-dependent manner. In addition, endothelial cell-derived VEGF in turn was found to act on GC cells and promotes their migration. The combined findings of our current and previous studies suggest that BGN secreted from GC cells into the tumor stroma promotes GC development, as well as its progression, potentially through the chronic activation of tumor angiogenesis.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes.

AIMS: Exogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. METHODS AND RESULTS: A pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. CONCLUSIONS: The core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

Biglycan up-regulated vascular endothelial growth factor (VEGF) expression and promoted angiogenesis in colon cancer.

Biglycan is an important component of the extracellular matrix, which belongs to the small leucine-rich proteoglycan family. Recent studies have shown that biglycan expression is elevated in many tumor tissues and implies poor prognosis, such as colon cancer. However, the molecular mechanism of biglycan in colon cancer has not been investigated. The present study aimed to investigate the effects of biglycan on vascular endothelial growth factor (VEGF) expression in colon cancer cells and on tumor angiogenesis in vivo. Biglycan overexpression vectors were constructed, and the stable biglycan overexpression in human colon cancer cell lines (HCT116 cells) was established by G418 screening. The stable cell clones were subsequently used to initiate tumor xenografts in nude mice. Our results showed that biglycan overexpression notably up-regulated the levels of VEGF in colon cancer cells, which was further confirmed by immunohistochemistry analysis in the xenograft colon tumors. Moreover, high levels of biglycan promoted angiogenesis and colon tumor growth, as evidenced by the increased cell viability, colon tumor size, and weight, as well as the CD34 expression. Additionally, we found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated by biglycan in colon cancer cells. The ERK inhibitor PD98059 dramatically reversed the increased expression of VEGF induced by biglycan. Taken together, our results indicated that biglycan up-regulated VEGF expression in colon cancer cells and promoted tumor angiogenesis. Biglycan-mediated VEGF regulation may correlate with the activation of the ERK signaling pathway. Therefore, biglycan may be a promising target for anti-angiogenic therapy for cancer.

Recombinant biglycan promotes bone morphogenetic protein-induced osteogenesis.

The aim of this study was to determine the effects of glutathione-S-transferase-fused recombinant biglycan (GST-BGN) on craniofacial bone regeneration. We recently demonstrated a positive effect of tissue-derived BGN on bone morphogenetic protein 2 (BMP-2) function, which is exerted likely via the BGN core protein. Here, we investigated the effects of GST-BGN lacking any posttranslational modifications on BMP-2 function in vitro and in vivo. In the C2C12 cell culture system, BMP-2-induced Smad 1/5/8 phosphorylation and alkaline phosphatase activity were both enhanced by the addition of GST-BGN. For the in vivo effect, we employed a Sprague-Dawley rat mandible defect model utilizing 1 µg (optimal) or 0.1 µg (suboptimal) of BMP-2 combined with 0, 2, 4, or 8 µg of GST-BGN. At 2 weeks post-surgery, newly formed bone was evaluated by microcomputed tomography and histologic analyses. The results revealed that the greatest amounts of bone within the defect were formed in the groups of suboptimal BMP-2 combined with 4 or 8 µg of GST-BGN. Also, bone was well organized versus that formed by the optimal dose of BMP. These results indicate that recombinant BGN is an efficient substrate to promote low-dose BMP-induced osteogenesis.