Nerve growth factor receptor limits inflammation to promote remodeling and repair of osteoarthritic joints.

Osteoarthritis (OA) is a painful, incurable disease affecting over 500 million people. Recent clinical trials of the nerve growth factor (NGF) inhibitors in OA patients have suggested adverse effects of NGF inhibition on joint structure. Here we report that nerve growth factor receptor (NGFR) is upregulated in skeletal cells during OA and plays an essential role in the remodeling and repair of osteoarthritic joints. Specifically, NGFR is expressed in osteochondral cells but not in skeletal progenitor cells and induced by TNFα to attenuate NF-κB activation, maintaining proper BMP-SMAD1 signaling and suppressing RANKL expression in mice. NGFR deficiency hyper-activates NF-κB in murine osteoarthritic joints, which impairs bone formation and enhances bone resorption as exemplified by a reduction in subchondral bone and osteophytes. In human OA cartilage, NGFR is also negatively associated with NF-κB activation. Together, this study suggests a role of NGFR in limiting inflammation for repair of diseased skeletal tissues.

APC mutations disrupt ß-catenin destruction complex condensates organized by Axin phase separation.

The Wnt/ß-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid-liquid-phase separation (LLPS) of Axin organized the ß-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of ß-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the ß-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3ß and CK1α were unsuccessfully recruited, preventing ß-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of ß-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway.

IGF2BP3 promotes the progression of colorectal cancer and mediates cetuximab resistance by stabilizing EGFR mRNA in an m6A-dependent manner.

Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an RNA-binding protein, is associated with tumorigenesis and progression. However, the exact molecular mechanisms of IGF2BP3 in colorectal cancer (CRC) oncogenesis, progression, and drug resistance remain unclear. This study found that IGF2BP3 was upregulated in CRC tissues. Clinically, the elevated IGF2BP3 level is predictive of a poor prognosis. Functionally, IGF2BP3 enhances CRC tumorigenesis and progression both in vitro and in vivo. Mechanistically, IGF2BP3 promotes epidermal growth factor receptor (EGFR) mRNA stability and translation and further activates the EGFR pathway by serving as a reader in an N6-methyladenosine (m6A)-dependent manner by cooperating with METTL14. Furthermore, IGF2BP3 increases the drug resistance of CRC cells to the EGFR-targeted antibody cetuximab. Taken together, our results demonstrated that IGF2BP3 was a functional and clinical oncogene of CRC. Targeting IGF2BP3 and m6A modification may therefore offer rational therapeutic targets for patients with CRC.

ASCL2 induces an immune excluded microenvironment by activating cancer-associated fibroblasts in microsatellite stable colorectal cancer.

Proficient mismatch repair or microsatellite stable (pMMR/MSS) colorectal cancers (CRCs) are vastly outnumbered by deficient mismatch repair or microsatellite instability-high (dMMR/MSI-H) tumors and lack a response to immune checkpoint inhibitors (ICIs). In this study, we reported two distinct expression patterns of ASCL2 in pMMR/MSS and dMMR/MSI-H CRCs. ASCL2 is overexpressed in pMMR/MSS CRCs and maintains a stemness phenotype, accompanied by a lower density of tumor-infiltrating lymphocytes (TILs) than those in dMMR/MSI CRCs. In addition, coadministration of anti-PD-L1 antibodies facilitated T cell infiltration and provoked strong antitumor immunity and tumor regression in the MC38/shASCL2 mouse CRC model. Furthermore, overexpression of ASCL2 was associated with increased TGFB levels, which stimulate local Cancer-associated fibroblasts (CAFs) activation, inducing an immune-excluded microenvironment. Consistently, mice with deletion of Ascl2 specifically in the intestine (Villin-Cre+, Ascl2 flox/flox, named Ascl2 CKO) revealed fewer activated CAFs and higher proportions of infiltrating CD8+ T cells; We further intercrossed Ascl2 CKO with ApcMin/+ model suggesting that Ascl2-deficient expression in intestinal represented an immune infiltrating environment associated with a good prognosis. Together, our findings indicated ASCL2 induces an immune excluded microenvironment by activating CAFs through transcriptionally activating TGFB, and targeting ASCL2 combined with ICIs could present a therapeutic opportunity for MSS CRCs.

COPA A-to-I RNA editing hijacks endoplasmic reticulum stress to promote metastasis in colorectal cancer.

RNA editing is among the most common RNA level modifications for generating amino acid changes. We identified a COPA A-to-I RNA editing event in CRC metastasis. Our results showed that the COPA A-to-I RNA editing rate was significantly increased in metastatic CRC tissues and was closely associated with aggressive tumors in the T and N stages. The COPA I164V protein damaged the Golgi-ER reverse transport function, induced ER stress, promoted the translocation of the transcription factors ATF6, XBP1 and ATF4 into the nucleus, and activated the expression of MALAT1, MET, ZEB1, and lead to CRC cell invasion and metastasis. Moreover, the COPA A-to-I RNA editing rate was positively correlated with the immune infiltration score. Collectively, the COPA I164V protein hijacked ER stress to promote the metastasis of CRC, and the COPA A-to-I RNA editing rate may be a potential predictor for patient response to immune checkpoint inhibitor (ICIs) treatment.

Nerve Growth Factor Receptor Limits Inflammation to Promote Remodeling and Repair of Osteoarthritic Joints.

Osteoarthritis (OA) is a painful, incurable disease affecting over 500 million people. The need for relieving OA pain is paramount but inadequately addressed, partly due to limited understandings of how pain signaling regulates non-neural tissues. Here we report that nerve growth factor receptor (NGFR) is upregulated in skeletal cells during OA and plays an essential role in the remodeling and repair of osteoarthritic joints. Specifically, NGFR is expressed in osteochondral cells but not in skeletal progenitor cells and induced by TNFα to attenuate NF-κB activation, maintaining proper BMP-SMAD1 signaling and suppressing RANKL expression. NGFR deficiency hyper-activates NF-κB in murine osteoarthritic joints, which impairs bone formation and enhances bone resorption as exemplified by a reduction in subchondral bone and osteophytes. In human OA cartilage, NGFR is also negatively associated with NF-κB activation. Together, this study uncovers a role of NGFR in limiting inflammation for repair of diseased skeletal tissues.

Epigenetic regulation of BAF60A determines efficiency of miniature swine iPSC generation.

Miniature pigs are an ideal animal model for translational research to evaluate stem cell therapies and regenerative applications. While the derivation of induced pluripotent stem cells (iPSCs) from miniature pigs has been demonstrated, there is still a lack of a reliable method to generate and maintain miniature pig iPSCs. In this study, we derived iPSCs from fibroblasts of Wisconsin miniature swine (WMS), Yucatan miniature swine (YMS), and Göttingen minipigs (GM) using our culture medium. By comparing cells of the different pig breeds, we found that YMS fibroblasts were more efficiently reprogrammed into iPSCs, forming colonies with well-defined borders, than WMS and GM fibroblasts. We also demonstrated that YMS iPSC lines with a normal pig karyotype gave rise to cells of the three germ layers in vitro and in vivo. Mesenchymal stromal cells expressing phenotypic characteristics were derived from established iPSC lines as an example of potential applications. In addition, we found that the expression level of the switch/sucrose nonfermentable component BAF60A regulated by STAT3 signaling determined the efficiency of pig iPSC generation. The findings of this study provide insight into the underlying mechanism controlling the reprogramming efficiency of miniature pig cells to develop a viable strategy to enhance the generation of iPSCs for biomedical research.

A-to-I RNA editing of BLCAP promotes cell proliferation by losing the inhibitory of Rb1 in colorectal cancer.

The bladder cancer-associated protein (BLCAP) gene is a tumor-suppressor gene as its encoded protein can inhibit cell proliferation by stimulating apoptosis in many malignant tumors. It is also a novel site of adenosine-to-inosine (A-to-I) RNA editing by ADAR (adenosine deaminase acting on RNA). In this study, we found by exome and transcriptome sequencing that there was an abnormal RNA editing event of the BLCAP gene in colorectal cancer (CRC) tissues compared to adjacent normal tissues. The editing of BLCAP transcripts promoted the degradation of BLCAP by ubiquitination, so BLCAP could not maintain its function as a tumor suppressor gene in CRC. Moreover, our further studies revealed that BLCAP could interact with Rb1 and inhibit its phosphorylation, while the loss of repressive effect due to reduced BLCAP protein levels caused by A-to-I RNA editing facilitates the transition from G1 to S phase of the cell cycle, leading to increased cell proliferation and reduced apoptosis. Thus, A-to-I RNA editing events tend to play an essential role in CRC carcinogenesis.

Reprogrammed Synovial Fluid-Derived Mesenchymal Stem/Stromal Cells Acquire Enhanced Therapeutic Potential for Articular Cartilage Repair.

OBJECTIVES: Functions of mesenchymal stem/stromal cells (MSCs) are affected by patient-dependent factors such as age and health condition. To tackle this problem, we used the cellular reprogramming technique to epigenetically alter human MSCs derived from the synovial fluid of joints with osteoarthritis (OA) to explore the potential of reprogrammed MSCs for repairing articular cartilage. MATERIALS AND METHODS: MSCs isolated from the synovial fluid of three patients' OA knees (Pa-MSCs) were reprogrammed through overexpression of pluripotency factors and then induced for differentiation to establish reprogrammed MSC (Re-MSC) lines. We compared the in vitro growth characteristics, chondrogenesis for articular cartilage chondrocytes, and immunomodulatory capacity. We also evaluated the capability of Re-MSCs to repair articular cartilage damage in an animal model with spontaneous OA. RESULTS: Our results showed that Re-MSCs increased the in vitro proliferative capacity and improved chondrogenic differentiation toward articular cartilage-like chondrocyte phenotypes with increased THBS4 and SIX1 and decreased ALPL and COL10A1, compared to Pa-MSCs. In addition, Re-MSC-derived chondrocytes expressing elevated COL2A and COL2B were more mature than parental cell-derived ones. The enhancement in chondrogenesis of Re-MSC involves the upregulation of sonic hedgehog signaling. Moreover, Re-MSCs improved the repair of articular cartilage in an animal model of spontaneous OA. CONCLUSIONS: Epigenetic reprogramming promotes MSCs harvested from OA patients to increase phenotypic characteristics and gain robust functions. In addition, Re-MSCs acquire an enhanced potential for articular cartilage repair. Our study here demonstrates that the reprogramming strategy provides a potential solution to the challenge of variation in MSC quality.

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration.

Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.

FOXO1 expression in chondrocytes modulates cartilage production and removal in fracture healing.

Fracture healing is a multistage process characterized by inflammation, cartilage formation, bone deposition, and remodeling. Chondrocytes are important in producing cartilage that forms the initial anlagen for the hard callus needed to stabilize the fracture site. We examined the role of FOXO1 by selective ablation of FOXO1 in chondrocytes mediated by Col2α1 driven Cre recombinase. Experimental mice with lineage-specific FOXO1 deletion (Col2α1Cre+FOXO1L/L) and negative control littermates (Col2α1Cre-FOXO1L/L) were used for in vivo, closed fracture studies. Unexpectedly, we found that in the early phases of fracture healing, FOXO1 deletion significantly increased the amount of cartilage formed, whereas, in later periods, FOXO1 deletion led to a greater loss of cartilage. FOXO1 was functionally important as its deletion in chondrocytes led to diminished bone formation on day 22. Mechanistically, the early effects of FOXO1 deletion were linked to increased proliferation of chondrocytes through enhanced expression of cell cycle genes that promote proliferation and reduced expression of those that inhibit it and increased expression of cartilage matrix genes. At later time points experimental mice with FOXO1 deletion had greater loss of cartilage, enhanced formation of osteoclasts, increased IL-6 and reduced numbers of M2 macrophages. These results identify FOXO1 as a transcription factor that regulates chondrocyte behavior by limiting the early expansion of cartilage and preventing rapid cartilage loss at later phases.

GATA6 regulates aging of human mesenchymal stem/stromal cells.

Cellular reprogramming forcing the expression of pluripotency markers can reverse aging of cells, but how molecular mechanisms through which reprogrammed cells alter aging-related cellular activities still remains largely unclear. In this study, we reprogrammed human synovial fluid-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs) using six reprogramming factors and reverted the iPSCs back to MSCs, as an approach to cell rejuvenation. Using the parental and reprogrammed MSCs as control nonrejuvenated and rejuvenated cells, respectively, for comparative analysis, we found that aging-related activities were greatly reduced in reprogrammed MSCs compared with those in their parental lines, indicating reversal of cell aging. Global transcriptome analysis revealed differences in activities of regulatory networks associated with inflammation and proliferation. Mechanistically, we demonstrated that, compared with control cells, the expression of GATA binding protein 6 (GATA6) in reprogrammed cells was attenuated, resulting in an increase in the activity of sonic hedgehog signaling and the expression level of downstream forkhead box P1 (FOXP1), in turn ameliorating cellular hallmarks of aging. Lower levels of GATA6 expression were also found in cells harvested from younger mice or lower passage cultures. Our findings suggest that GATA6 is a critical regulator increased in aged MSCs that controls the downstream sonic hedgehog signaling and FOXP1 pathway to modulate cellular senescence and aging-related activities.

VCAM1 Promotes Tumor Cell Invasion and Metastasis by Inducing EMT and Transendothelial Migration in Colorectal Cancer.

Vascular cell adhesion molecular 1 (VCAM1), an important member of the immunoglobulin superfamily, is related to the development of malignant tumors, such as breast cancer, melanoma, and renal clear cell carcinoma. However, the molecular role and mechanism of VCAM1 in the regulation of the progression of colorectal cancer (CRC) has rarely been studied. The results of IHC and RT-PCR analyses proved that VCAM1 was upregulated in human CRC tissues compared with matched adjacent normal intestinal epithelial tissues. Moreover, analysis of data from the TCGA and Gene Expression Omnibus (GEO) databases revealed that a higher level of VCAM1 was strongly correlated with poor differentiation, metastasis, and short survival in CRC patients. Furthermore, VCAM1 significantly influenced the invasion and metastasis of CRC cells in vitro and in vivo and activated the EMT program, by which cancer cells adhere to the endothelium and cross the vessel wall by forming pseudopodia and invadopodia. The current findings demonstrate that VCAM1 promotes tumor progression in CRC.

Upregulation of OSBPL3 by HIF1A promotes colorectal cancer progression through activation of RAS signaling pathway.

Oxysterol-binding protein like protein 3 (OSBPL3) has been shown involving in the development of several human cancers. However, the relationship between OSBPL3 and colorectal cancer (CRC), particularly the role of OSBPL3 in the proliferation, invasion and metastasis of CRC remains unclear. In this study, we investigated the role of OSBPL3 in CRC and found that its expression was significantly higher in CRC tissues than that in normal tissues. In addition, high expression of OSBPL3 was closely related to poor differentiation, advanced TNM stage and poor prognosis of CRC. Further experiments showed that over-expression of OSBPL3 promoted the proliferation, invasion and metastasis of CRC in vitro and in vivo models. Moreover, we revealed that OSBPL3 promoted CRC progression through activation of RAS signaling pathway. Furthermore, we demonstrated that hypoxia induced factor 1 (HIF-1A) can regulate the expression of OSBPL3 via binding to the hypoxia response element (HRE) in the promoter of OSBPL3. In summary, Upregulation of OSBPL3 by HIF1A promotes colorectal cancer progression through activation of RAS signaling pathway. This novel mechanism provides a comprehensive understanding of both OSBPL3 and the RAS signaling pathway in the progression of CRC and indicates that the HIF1A-OSBPL3-RAS axis is a potential target for early therapeutic intervention in CRC progression.

Downregulation of Siah1 promotes colorectal cancer cell proliferation and migration by regulating AKT and YAP ubiquitylation and proteasome degradation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Siah E3 ubiquitin protein ligase 1 (Siah1) has been identified as a tumor suppressor gene and plays an important role in the development of malignant tumors. However, the potential role and molecular mechanism of Siah1 in the development and progression of CRC is still unclear. METHODS: To explore the role and molecular mechanism of Siah1 in the development and progression of CRC, we examined the expression of Siah1 in CRC tissue samples and analyzed its association with progression and prognosis in CRC. In addition, overexpression and knockdown of Siah1 was used to investigate its activity in CRC cells. We also use bioinformatics to analyze and verify the significant roles of Siah1 in critical signaling pathways of CRC. RESULTS: We found that the expression of Siah1 was significantly downregulated in CRC tissues, and low expression of Siah1 was associated with aggressive TNM staging and poor survival of CRC patients. Moreover, we revealed that overexpression of Siah1 in CRC cells markedly inhibited CRC cell proliferation and invasion in vitro and in vivo, while knockdown of Siah1 enhanced CRC cell proliferation and invasion. Furthermore, we found that Siah1 prohibited cell proliferation and invasion in CRC partially through promoting AKT (the serine-threonine protein kinase) and YAP (yes associated protein) ubiquitylation and proteasome degradation to regulate the activity of MAPK(mitogen-activated protein kinase 1), PI3K-AKT (phosphatidylinositol 3-kinase-the serine-threonine protein kinase) and Hippo signaling pathways. CONCLUSIONS: These findings suggested that Siah1 is a novel potential prognostic biomarker and plays a tumor suppressor role in the development and progression of CRC.