The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma

Expression of Hyaluronan in human tumor progression

<p>Abstract</p> <p>Background</p> <p>The development and progression of human tumors is accompanied by various cellular, biochemical and genetic alterations. These events include tumor cells interaction with extracellular matrix molecules including hyaluronan (HA). Hyaluronan is a large polysaccharide associated with pericellular matrix of proliferating, migrating cells. Its implication in malignant transformation, tumor progression and with the degree of differentiation in various invasive tumors has well accepted. It has been well known the role HA receptors in tumor growth and metastasis in various cancer tissues. Previously we have observed the unified over expression of Hyaluronic Acid Binding Protein (HABP), H11B2C2 antigen by the tumor cells in various types progressing tumor tissues with different grades. However, the poor understanding of relation between HA and HA-binding protein expression on tumor cells during tumor progression as well as the asymmetric observations of the role of HA expression in tumor progression prompted us to examine the degree of HA expression on tumor cells vs. stroma in various types of human tumors with different grades.</p> <p>Methods</p> <p>In the present study clinically diagnosed tumor tissue samples of different grades were used to screen the histopathological expression of hyaluronan by using b-PG (biotinylated proteoglycan) as a probe and we compared the relative HA expression on tumor cells vs. stroma in well differentiated and poorly differentiated tumors. Specificity of the reaction was confirmed either by pre-digesting the tissue sections with hyaluronidase enzyme or by staining the sections with pre-absorbed complex of the probe and HA-oligomers.</p> <p>Results</p> <p>We show here the down regulation of HA expression in tumor cells is associated with progression of tumor from well differentiated through poorly differentiated stage, despite the constant HA expression in the tumor associated stroma.</p> <p>Conclusion</p> <p>The present finding enlighten the relative roles of HA expression on tumor vs. stroma during the progression of tumors.</p

Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas

Riluzole decreases AKT phosphorylation and activity. A.

<p>Riluzole decreases AKT phosphorylation. Serum-starved melanoma cells were incubated in the absence (−) or presence (+) of 25 µM of riluzole for 4 and 8 hours. Immunoblots were performed using antibodies against AKT phosphorylated at serine 473 (pAKT (S473)) or at threonine 308 (pAKT (T308)). Anti-total AKT and GAPDH antibodies were used for normalization. <b>B.</b> AKT-mediated GSK3 phosphorylation is decreased after riluzole treatment. Serum-starved melanoma cells were incubated in the absence (−) or presence (+) of 25 µM of riluzole for 4, 8 and 16 hours. Immunoblots were performed using antibodies against GSK3β phosphorylated at serine 9, an AKT site; total GSK3β; GAPDH.</p

Genes whose expression was regulated by riluzole in the WM793 cell line using the Human TGFβ/BMP Signaling Pathway RT² Profiler™ PCR Array.

<p>The fold increase or decrease (comparing to control) was above 2 for these four genes. BMP3: bone morphogenetic protein 3; GDF5: growth differentiation factor 5; INHBB: inhibin, beta B; PLAU: plasminogen activator, urokinase.</p

Riluzole upregulates the expression of genes associated with the TGFβ signaling pathway.

<p><b>A.</b> Model of riluzole effect on Smad linker phosphorylation. Riluzole inhibits the phosphorylation of AKT at S473 and T308. As a result of AKT inactivation, GSK3 phosphorylation at the AKT site is decreased, resulting in GSK3 activation. GSK3 phosphorylates Smad2 at the cluster of serines 245/250/255 and Smad3 at serine 204. The consequences of these phosphorylation events on Smad2 and Smad3 activities will likely depend on the promoters of the TGFβ target genes. <b>B and C.</b> Riluzole treatment increases the expression of the INHBB and PLAU genes respectively in the three independent melanoma cell lines A2058, UACC903 and C8161. mRNA from these three melanoma cell lines untreated or treated with riluzole for 24 hours were analyzed by real-time PCR for the expression of the INHBB and PLAU genes. White bars: untreated cells. Dark bars: Riluzole-treated cells. *, P<0.05, compared with untreated cells (t test).</p

Smad linker phosphorylation involves GSK3 in human melanoma lines. A.

<p>Inhibition of Smad2 and Smad3 linker phosphorylation in the presence of LiCl. 24 hours post seeding, WM278, WM793 and 1205LU human melanoma cell lines were serum-starved for about 16 hours, before the addition of 50 mM LiCl for 2 and 5 hours. Cells left with 50 mM NaCl for 2 or 5 hours were used as controls. Whole cell extracts were then prepared. Immunoblots were performed with antibodies against: Phosphorylated β-catenin (pβ-catenin); total β-catenin; Both phosphoSmad3 (Thr179) and phosphoSmad2 (Thr220); phosphoSmad2 (Ser245/250/255); phosphoSmad3 (Ser204); phosphoSmad3 (Ser208); Smad2 and Smad3; GAPDH. <b>B.</b> Inhibition of Smad2 and Smad3 linker phosphorylation in the presence of the specific GSK3 specific inhibitor, CT99021. 24 hours post seeding, WM793 and 1205LU cells were serum-starved for about 16 hours, and incubated in the absence (−) or presence (+) of 2 µM of CT99021 for two hours. Immunoblots were performed as in A. p: Phosphorylated. S: Serine; T: Threonine.</p