Cyclic-AMP signalling, MYC and hypoxia-inducible factor 1α intersect to regulate angiogenesis in B-cell lymphoma.

Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.

Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.

Proteasome inhibition suppress microgravity elevated RANK signaling during osteoclast differentiation.

Microgravity (µXg) induces bone loss in astronauts during space missions. Therefore, it is necessary to delineate the underlying mechanisms which leads to bone loss for developing countermeasures. Osteoclasts (OCLs) are multinucleated cells, which resorb bone. Previously, we have demonstrated that simulated µXg enhances OCL formation. However, control of osteoclast bone resorption activity under µXg remains unclear. The OCL formation has been shown to be regulated by ubiquitin-proteasome pathway. Hence, we hypothesized that proteasome inhibition could regulate osteoclast differentiation under µXg. In this study, we identified that RAW264.7 preosteoclast cells treated with proteasome inhibitor (MG-132) suppress RANK receptor expression essential for OCL differentiation, but no effect on TRAF-6. We identified that MG-132 treatment abolished K48-linked poly-ubiquitination under µXg. Immunostaining confirms inhibition of protein ubiquitination and RANK expression in preosteoclast cells. Furthermore, proteasome inhibition suppresses the expression of SQSTM1/p62 under both the ground based Xg and µXg conditions. Also, confocal microscopy using Lyso-Tracker demonstrated that proteasomal inhibition suppress the co-localization of p62 and lysosomes. MG-132 inhibited RANKL induced proteasome activity. RAW264.7 cells treated with the proteasome inhibitor showed an increased level of p-c-Jun activity in control cultures, however decreased under µXg. In contrast, c-Fos and NFATc1 expression was decreased. In-addition, mouse bone marrow cultures treated with MG-132 suppress OCL formation and bone resorption activity. Thus, our findings suggest that proteasome inhibition represents a novel therapeutic approach for bone loss under µXg in space environment.

Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.

Microgravity modulation of syncytin-A expression enhance osteoclast formation.

Microgravity (µXg) experienced by astronauts during space flights causes accelerated bone loss. However, the molecular basis of µXg induced bone loss in space is unclear. Osteoclast (OCL) is the primary bone-resorbing cell. We previously demonstrated that simulated µXg promotes OCL formation. In this study, we identified that µXg induces syncytin-A expression in RAW264.7 preosteoclast cells without RANKL stimulation. We further tested the effect of osteotropic factors such as CXCL5 and 1,25(OH)2 D3 to regulate the syncytin-A expression in preosteoclast cells subjected to µXg compared to ground based (Xg) cultures. CXCL5 (25 ng/mL) and 1,25(OH)2 D3 (10 ng/mL) increased syncytin-A expression under Xg conditions. However, µXg alone upregulates syncytin-A expression compared to Xg control preosteoclast cells. Confocal microscopy using Lyso-Tracker identified syncytin-A expression co-localized with lysosomes in preosteoclast cells. Acridine orange staining showed RANKL elevated autophagy activity in these cells. Further, siRNA suppression of syncytin-A significantly inhibits autophagy activity in RAW264.7 cells. In addition, knockdown of syncytin-A expression inhibits µXg increased OCL formation in mouse bone marrow cultures. Thus, our findings suggest that targeting syncytin-A expression may be an effective countermeasure to control bone loss under microgravity conditions.

Inhibition of IKKß by celastrol and its analogues - an in silico and in vitro approach.

CONTEXT: Alzheimer's disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions. OBJECTIVE: This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta1-42 (Aß1-42) induced cytotoxicity and IKKß activity, respectively. MATERIALS AND METHODS: Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 µM) before treatment with Aß1-42 (IC30 10 µM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKß inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects. RESULTS: Celastrol (1 µM) inhibited Aß1-42 (10 µM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKß as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol. DISCUSSION AND CONCLUSION: The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKß activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition.

IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes.

In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

MYC, mitochondrial metabolism and O-GlcNAcylation converge to modulate the activity and subcellular localization of DNA and RNA demethylases.

Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.

A RET::GRB2 fusion in pheochromocytoma defies the classic paradigm of RET oncogenic fusions.

The RET kinase receptor is a target of mutations in neural crest tumors, including pheochromocytomas, and of oncogenic fusions in epithelial cancers. We report a RET::GRB2 fusion in a pheochromocytoma in which RET, functioning as the upstream partner, retains its kinase domain but loses critical C-terminal motifs and is fused to GRB2, a physiological RET interacting protein. RET::GRB2 is an oncogenic driver that leads to constitutive, ligand-independent RET signaling; has transforming capability dependent on RET catalytic function; and is sensitive to RET inhibitors. These observations highlight a new driver event in pheochromocytomas potentially amenable for RET-driven therapy.

Regulation of PD-L1 expression is a novel facet of cyclic-AMP-mediated immunosuppression.

Cyclic-AMP (cAMP) exerts suppressive effects in the innate and adaptive immune system. The PD-1/PD-L1 immune checkpoint downregulates T-cell activity. Here, we examined if these two immunosuppressive nodes intersect. Using normal and malignant lymphocytes from humans, and the phosphodiesterase 4b (Pde4b) knockout mouse, we found that cAMP induces PD-L1 transcription and protein expression. Mechanistically, we discovered that the cAMP effectors PKA and CREB induce the transcription/secretion of IL-10, IL-8, and IL-6, which initiate an autocrine loop that activates the JAK/STAT pathway and ultimately increase PD-L1 expression in the cell surface. This signaling axis is disarmed at two specific nodes in subsets of diffuse large B-cell lymphoma, which may help explain the variable PD-L1 expression in these tumors. In vivo, we found that despite its immunosuppressive attributes, the PDE4 inhibitor roflumilast did not decrease the clinical activity of checkpoint inhibitors, an important clinical observation given the approved use of these agents in multiple diseases. In summary, we discovered that PD-L1 induction is a part of the repertoire of immunosuppressive actions mediated by cAMP, defined a cytokine-mediated autocrine loop that executes this action and, reassuringly, showed that PDE4 inhibition does not antagonize immune checkpoint blockade in an in vivo syngeneic lymphoma model.

Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients.

Psoriasis is a chronic inflammatory skin disease characterized by hyper proliferation of keratinocytes. Recent data show that the epidermis thickening in psoriasis may be related to imbalance of homeostasis caused by abnormal apoptotic process. Maintenance of keratinocyte apoptotic process is very important in psoriasis. Methotrexate (MTX) has been used for many years to restore the normal skin in psoriasis condition. However, the exact mechanism of MTX in psoriasis condition is poorly understood. The aim of this study was to examine the role of MTX on keratinocyte apoptosis pathway in psoriasis patients. A total of 58 psoriasis vulgaris patients were recruited for this study. Nonlesional skin biopsies served as control. Skin biopsies of psoriatic patients were collected and analyzed for cytosolic, mitochondria and total cytochrome c by ELISA. Expression of caspase-9, NFκBp65, pAkt1 by western blot, real-time PCR and immunohistochemical analysis of c-FLIP protein was analyzed in nonlesional and lesional skin biopsies before (day 0) and after (at the end of 6 and 12 weeks) MTX treatment. After MTX treatment, a significant increase in cytochrome c was observed when compared with before MTX treatment in psoriasis patients (p < 0.001). Protein and gene expression of cleaved caspase-9 were significantly increased after MTX treatment, whereas the expression of Bcl-xL, c-FLIP, NFκBp65, pAkt1 significantly downregulated after MTX treatment. In conclusion, these results showed that intrinsic apoptotic pathway induced by MTX eventually adds the beneficial therapeutic role of MTX in psoriasis by controlling the acanthosis.

Interferon ß improves the efficacy of low dose cisplatin by inhibiting NF-κB/p-Akt signaling on HeLa cells.

The purpose of this study was to evaluate the anticancer efficacy of interferon ß in combination with low dose of cisplatin on human cervical cancer progression, as well as its principal action mechanism. The combination treatment synergistically potentiated the effect of interferon ß on cell growth inhibition and DNA damage on HeLa cells by repressing NF-κB/p-Akt signaling. Synergistic targeting of these pathways has a therapeutic potential. Further, the combination treatment ameliorated the expression of pro-apoptotic Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Additionally, the expression of active PARP was significantly increased and MMP-9 level was decreased in combination group as compared to the expression seen for the treatment with interferon ß or cisplatin alone. Results demonstrate that the synergistic inhibitory effects of interferon ß and low dose of cisplatin on human cervical cancer cells and also suggest that the inhibition of NF-κB/p-Akt signaling pathway plays a critical role in the anticancer effects of combination treatment along with the induction of PARP. Therefore, the combination of interferon ß and cisplatin may be a useful treatment for human cervical cancer, with a greater effectiveness than other treatments.

Inhibitory effects of interferon-ß on hepatocellular carcinoma HepG2 via Akt/STAT phosphorylation.

Conventional chemotherapy fails to cure metastatic hepatoma mainly due to its high hepatotoxicity. Currently, doxorubicin is the most widely used drug against liver cancer either as single agent or in combination with other chemotherapeutics such as cisplatin. It is limited due to their severe toxicity on normal hepatocytes. Therefore, alternative therapeutic agents without or with low hepatotoxicity are highly desirable. Interferons are a family of cytokines that potently demonstrate antiviral, immunomodulatory, and antiproliferative activities. It also exerts direct cytotoxic effects on tumor cells. The purpose of this study was to examine the in vitro cytotoxicity of interferon-ß on HepG2 cells. We revealed the presence of binding receptor of interferon-ß in HepG2 cells. The dose-dependent inhibition on cell proliferation was observed. We demonstrated that IFN-ß exhibited significant cytotoxicity in HepG2 cells mainly through phosphorylation of signal transducers and activators of transcription 2. The activation of Akt was suppressed. The stimulation of pro-apoptotic protein expression of Bax, inhibition of anti-apoptotic protein expression of Bcl-2, activation of cleaved caspases 9 and 3 was found at increasing concentrations. In conclusion, our results suggest that interferon-ß has potential to inhibit cell proliferation dose dependently. Increased concentrations of interferon-ß influenced apoptosis via mitochondrial pathway through inhibition of p-Akt.

Synergistic anti-carcinogenic effect of interferon-ß with cisplatin on human breast adenocarcinoma MDA MB231 cells.

Cisplatin is one of the most commonly used chemotherapeutic agents for breast cancer treatment. However, its efficacy is greatly limited by its toxic side effects. The present study investigated the synergistic effect of interferon ß with cisplatin on MDA MB231 cells. The antiproliferative effect was measured by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) was calculated using the method of Chou and Talalay. Cytotoxicity was determined by trypan blue and clonogenic assay. Genotoxic and cytostatic effects were studied using micronucleus assay and nuclear division index (NDI). Protein expression was analyzed using immunoblotting. Interferon ß (100-2500 IU/mL) and Cisplatin (0.01-100 µM) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 1500 IU/mL and 20 µM for interferon ß and cisplatin, respectively. Western blot analysis revealed expression of interferon ß binding receptor in MDA MB231 cells. More interestingly, synergistic, cytotoxic and genotoxic effects were observed after treatment with a combination of interferon ß with reduced dosage of cisplatin. Decreased expression of Bcl-2 and increased expression of Bax stimulated the cytochrome c release, which triggers caspase-9 and -3 activation significantly increased in the combinational group. In conclusion the combination of interferon ß with reduced dose of cisplatin results synergistically improved growth-inhibition and apoptosis-inducing effect on MDA MB231 cells.