CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation.
Background Disorders of the Ras/MAPK pathway have an overlapping skeletal phenotype (eg. scoliosis, osteopenia). The Ras proteins regulate cell proliferation and differentiation and NF1 individuals have osteoclast hyperactivity and increased bone resorption as measured by urine pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Pyd and Dpd are hydroxylysine derived cross-links of collagen found in bone and cartilage and excreted in the urine. Dpd is most abundant in bone. The aim of this study was to evaluate if other syndromes of the Ras/MAPK pathway have increased bone resorption, which may impact the skeletal phenotype. Methods and Results Participants: [Noonan syndrome (n=14), Costello syndrome (n=21), and cardiofaciocutaneous (CFC) syndrome (n=14)]. Pyridinium cross-links from two consecutive first morning urines were extracted after acid hydrolysis and analyzed by High Performance Liquid Chromotography. Three separate analyses of covariance (ANCOVA) were performed to compare Pyd, Dpd, and Dpd/Pyd ratio of each group to controls after controlling for age. Data were compared to 99 healthy controls. Conclusions The Dpd and the Dpd/Pyd ratio were elevated (p<0.0001) in all 3 conditions compared to controls suggesting that collagen degradation was predominantly from bone. The data suggest that the Ras/MAPK signal transduction pathway is important in bone homeostasis.
Background Expression of CD44 receptor is associated with the onset of several tumors. The intracellular domain of CD44 (CD44-ICD) has been implicated as a co-transcription factor for RUNX2 in the regulation of expression of MMP-9 in breast carcinoma cells. Previous studies from our laboratory demonstrated the role of CD44 in migration and invasion of PC3 prostate cells through activation of MMP-9. CD44 signaling regulates the phosphorylation and hence the localization of RUNX2 in the nucleus. The role of CD44-ICD has not been studied in prostate cancer cells. This study aimed to explore the role of CD44-ICD and RUNX2 in the regulation of expression of metastasis-related genes. Methods PC3 and PC3 cells overexpressing RUNX2 protein were analyzed for RUNX2/CD44-ICD interaction by immunoprecipitation, immunoblotting, and Immunofluorescence analyses. Wound healing and tumorsphere formation analyses were also done in these cells. The real-time PCR analysis was used to detect the expression levels of different genes. Results Expression of CD44 and RUNX2 was observed only in PC3 cells (androgen receptor positive) and not in LNCaP or PCa2b cells (androgen receptor negative). Therefore, CD44-ICD fragment (~ 15-16 kDa) was observed in PC3 cells. Moreover, localization of CD44-ICD was more in the nucleus than in the cytoplasm of PC3 cells. Inhibition of cleavage of CD44 with a γ-secretase inhibitor, DAPT reduced the formation of CD44-ICD; however, accumulation of CD44–external truncation fragments (~ 20 and ~ 25 kDa) was detected. RUNX2 and CD44-ICD interact in the nucleus of PC3 cells, and this interaction was more in PC3 cells transfected with RUNX2 cDNA. Overexpression of RUNX2 augments the expression of metastasis-related genes (e.g., MMP-9 and osteopontin) which resulted in increased migration and tumorsphere formation. Conclusions We have shown here a strong functional relationship between CD44-ICD and RUNX2 in PC3 cells. RUNX2 forms a complex with CD44-ICD as a co-transcriptional factor, and this complex formation not only activates the expression of metastasis-related genes but also contributes to migration and tumorsphere formation. Therefore, RUNX2 and CD44-ICD are potential targets for anti-cancer therapy, and attenuation of their interaction may validate the regulatory effects of these proteins on cancer migration and progression.
Background Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria’s outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. Results Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. Conclusion TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.
Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the "molecular signature" of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa.
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