MAFB Promotes Cancer Stemness and Tumorigenesis in Osteosarcoma through a Sox9-Mediated Positive Feedback Loop.

Despite the fact that osteosarcoma is one of the most common primary bone malignancies with poor prognosis, the mechanism behind the pathogenesis of osteosarcoma is only partially known. Here we characterized differentially expressed genes by extensive analysis of several publicly available gene expression profile datasets and identified musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) as a key transcriptional regulator in osteosarcoma progression. MAFB was highly expressed in tumor tissues and required for proliferation and tumorigenicity of osteosarcoma cells. MAFB expression was elevated in osteosarcoma stem cells to maintain their self-renewal potential in vitro and in vivo through upregulation of stem cell regulator Sox9 at the transcriptional level. Sox9 in turn activated MAFB expression via direct recognition of its sequence binding enrichment motif on the MAFB locus, thereby forming a positive feedback regulatory loop. Sox9-mediated feedback activation of MAFB was pivotal to tumorsphere-forming and tumor-initiating capacities of osteosarcoma stem cells. Moreover, expression of MAFB and Sox9 was highly correlated in osteosarcoma and associated with disease progression. Combined detection of both MAFB and Sox9 represented a promising prognostic biomarker that stratified a subset of patients with osteosarcoma with shortest overall survival. Taken together, these findings reveal a MAFB-Sox9 reciprocal regulatory axis driving cancer stemness and malignancy in osteosarcoma and identify novel molecular targets that might be therapeutically applicable in clinical settings. SIGNIFICANCE: Transcription factors MAFB and Sox9 form a positive feedback loop to maintain cell stemness and tumor growth in vitro and in vivo, revealing a potential target pathway for therapeutic intervention in osteosarcoma.

Transcription factor MafB is a marker of tumor-associated macrophages in both mouse and humans.

The transcription factor MafB is specifically expressed in macrophages. We have recently demonstrated that MafB is expressed in anti-inflammatory alternatively activated M2 macrophages in vitro. Tumor-associated macrophages (TAMs) are a subset of M2 type macrophages that can promote immunosuppressive activity, induce angiogenesis, and promote tumor cell proliferation. To examine whether MafB express in TAMs, we analyzed green fluorescent protein (GFP) expression in Lewis lung carcinoma tumors of MafB-GFP knock-in heterozygous mice. FACS analysis demonstrated GFP fluorescence in cells positive for macrophage-markers (F4/80, CD11b, CD68, and CD204). Moreover, quantitative RT-PCR analysis with F4/80+GFP+ and F4/80+GFP- sorted cells showed that the GFP-positive macrophages express IL-10, Arg-1, and TNF-α, which were known to be expressed in TAMs. These results indicate that MafB is expressed in TAMs. Furthermore, immunostaining analysis using an anti-MAFB antibody revealed that MAFB is expressed in CD204-and CD68-positive macrophages in human lung cancer samples. In conclusion, MafB can be a suitable marker of TAMs in both mouse and human tumor tissues.

MAFb protein confers intrinsic resistance to proteasome inhibitors in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) patients with t(14;20) have a poor prognosis and their outcome has not improved following the introduction of bortezomib (Bzb). The mechanism underlying the resistance to proteasome inhibitors (PIs) for this subset of patients is unknown. METHODS: IC50 of Bzb and carfilzomib (CFZ) in human myeloma cell lines (HMCLs) were established by MTT assay. Gene Expression profile (GEP) analysis was used to determine gene expression in primary myeloma cells. Immunoblotting analysis was performed for MAFb and caspase family proteins. Immunofluorescence staining was used to detect the location of MAFb protein in MM cells. Lentiviral infections were used to knock-down MAFb expression in two lines. Apoptosis detection by flow cytometry and western blot analysis was performed to determine the molecular mechanism MAFb confers resistance to proteasome inhibitors. RESULTS: We found high levels of MAFb protein in cell lines with t(14;20), in one line with t(6;20), in one with Igλ insertion into MAFb locus, and in primary plasma cells from MM patients with t(14;20). High MAFb protein levels correlated with higher IC50s of PIs in MM cells. Inhibition of GSK3ß activity or treatment with Bzb or CFZ prevented MAFb protein degradation without affecting the corresponding mRNA level indicating a role for GSK3 and proteasome inhibitors in regulation of MAFb stability. Silencing MAFb restored sensitivity to Bzb and CFZ, and enhanced PIs-induced apoptosis and activation of caspase-3, - 8, - 9, PARP and lamin A/C suggesting that high expression of MAFb protein leads to insensitivity to proteasome inhibitors. CONCLUSION: These results highlight the role of post-translational modification of MAFb in maintaining its protein level, and identify a mechanism by which proteasome inhibitors induced stabilization of MAFb confers resistance to proteasome inhibitors, and provide a rationale for the development of targeted therapeutic strategies for this subset of patients.

Streptococcus gordonii promotes rapid differentiation of monocytes into dendritic cells through interaction with the sialic acid-binding adhesin.

Infective endocarditis is frequently attributed to oral streptococci. Although the pathogenetic mechanisms are not well understood, interaction between streptococci and phagocytes is thought to be important for infective endocarditis. In this study, HL-60 cell-derived monocytes were characterized following interaction with Streptococcus gordonii DL1. Exposure of monocytes to S. gordonii DL1 induced up-regulation of the dendritic cell (DC) markers CD83, CD1a, CD86, and interleukin-12, while monocyte markers PU.1 and MafB, which are typically present at low levels in mature DCs, were down-regulated. Interaction of HL-60-derived monocytes with S. gordonii DL1 was instructive for DC differentiation in the absence of released cytokines. Furthermore, neither the filtered culture medium of S. gordonii nor the hsa mutant, deficient in sialic acid-binding activity, was able to induce the differentiation of HL-60 cells. Taken together, these data suggest that monocytes stimulated with S. gordonii DL1 rapidly undergo monocyte-to-DC differentiation through interaction with the bacterial surface receptor Hsa and that this response may be the initial step in infective endocarditis by oral streptococci.

Immunohistochemical staining for transcription factor MafB in alveolar macrophages is correlated with spirometric measures of airflow limitation in smokers.

BACKGROUND AND OBJECTIVE: Alveolar macrophages (AM) play an important role in the pathogenesis of COPD, and their numbers are significantly increased in patients with COPD. We previously demonstrated that expression of the transcription factor, MafB, was upregulated in AM of mice exposed to cigarette smoke. The aim of this study was to investigate whether the expression of MafB is associated with the degree of airflow limitation (AFL) in smokers. METHODS: Lung tissue specimens were obtained from male patients undergoing resection of small peripheral lung tumours. The patients were classified into three groups according to smoking index and FEV1 /FVC: (i) non-smokers or non-heavy ex-smokers without AFL (FEV1 /FVC ≥ 0.7, smoking index ≤ 400) (n=8); (ii) heavy current smokers without AFL (FEV1 /FVC ≥ 0.7, smoking index ≥ 800) (n=8); and (iii) heavy current smokers with AFL (FEV1 /FVC < 0.6, smoking index ≥ 800) (n=8). The intensity of immunostaining for MafB in AM was quantified by image analysis. RESULTS: Immunostaining for MafB was significantly enhanced in AM of smokers with AFL compared with AM of subjects without AFL. Smoking index, FEV1/FVC and FEF(25-75%) (% predicted) were significantly correlated with the intensity of MafB immunostaining. Multiple linear regression analysis revealed that FEV1 % was also an independent negative predictor of the intensity of MafB immunostaining. CONCLUSIONS: The degree of immunostaining for MafB in AM was correlated with the degree of AFL in smokers. MafB may be involved in the pathophysiology of COPD.

Potential roles of large mafs in cell lineages and developing pancreas.

OBJECTIVES: Maf is a family of transcription factor proteins characterized by a typical bZip structure, and mafA, a member of the large-maf family, is a strong transactivator of insulin in cell lines. The present study investigated the expression profiles of the large-maf family proteins in porcine pancreatic tissue and in primary culture cells. METHODS: Immunohistochemical staining was performed to localize each maf protein. Messenger RNA expression was quantitated by real-time polymerase chain reaction, and protein expression was assessed by Western blotting. RESULTS: Islet formation was not as clear in newborn pancreatic tissue as in adult pancreatic tissue. MafA- and c-maf-positive cells were more diffusely localized in pancreatic tissue with fewer mafB-positive cell clusters scattered throughout. By contrast, islet formation was clearer, and positive staining for mafA and c-maf tended to be more prominent in the islets of adult pancreatic tissue. Messenger RNA and protein expressions were consistent with the immunohistochemical findings. MafA, mafB, and c-maf coexpressed with insulin-positive cells, and c-maf coexpressed with glucagon-positive cells in adult porcine pancreas based on the results of a double-staining study. CONCLUSIONS: Large mafs were identified in normal porcine and human pancreas, and the expression levels and localizations of the large mafs in newborn and adult pancreatic tissues differed. Mafs may play important roles in establishing endocrine function during pancreatic cell differentiation.