Concerted action of IFN-α and IFN-λ induces local NK cell immunity and halts cancer growth.

Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. No significant improvement has been reported with currently available systemic therapies. IFN-α has been tested in both clinic and animal models and only moderate benefits have been observed. In animal models, similar modest antitumor efficacy has also been reported for IFN-λ, a new type of IFN that acts through its own receptor complex. In the present study, the antitumor efficacy of the combination of IFN-α and IFN-λ was tested in the BNL mouse hepatoma model. This study was accomplished by using either engineered tumor cells (IFN-α/IFN-λ gene therapy) or by directly injecting tumor-bearing mice with IFN-α/IFN-λ. Both approaches demonstrated that IFN-α/IFN-λ combination therapy was more efficacious than IFN monotherapy based on either IFN-α or IFN-λ. In complement to tumor surgery, IFN-α/IFN-λ combination induced complete tumor remission. Highest antitumor efficacy has been obtained following local administration of IFN-α/IFN-λ combination at the tumor site that was associated with strong NK cells tumor infiltration. This supports the use of IFN-α/IFN-λ combination as a new cancer immunotherapy for stimulating antitumor response after cancer surgery.

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion.

In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.

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 (TGFß), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3ß-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 GSK3ß. In addition, GSK3ß could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFß. 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 TGFß 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.

Interferon lambda: a new sword in cancer immunotherapy.

The discovery of the interferon-lambda (IFN-λ) family has considerably contributed to our understanding of the role of interferon not only in viral infections but also in cancer. IFN-λ proteins belong to the new type III IFN group. Type III IFN is structurally similar to type II IFN (IFN-γ) but functionally identical to type I IFN (IFN-α/ß). However, in contrast to type I or type II IFNs, the response to type III IFN is highly cell-type specific. Only epithelial-like cells and to a lesser extent some immune cells respond to IFN-λ. This particular pattern of response is controlled by the differential expression of the IFN-λ receptor, which, in contrast to IFN-α, should result in limited side effects in patients. Recently, we and other groups have shown in several animal models a potent antitumor role of IFN-λ that will open a new challenging era for the current IFN therapy.

Antitumor activity of type I and type III interferons in BNL hepatoma model.

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Characterization of the mouse IFN-lambda ligand-receptor system: IFN-lambdas exhibit antitumor activity against B16 melanoma.

Recently discovered type III IFNs (IFN-lambda) exert their antiviral and immunomodulatory activities through a unique receptor complex composed of IFN-lambdaR1 and interleukin-10 receptor 2. To further study type III IFNs, we cloned and characterized mouse IFN-lambda ligand-receptor system. We showed that, similar to their human orthologues, mIFN-lambda2 and mIFN-lambda3 signal through the IFN-lambda receptor complex, activate IFN stimulated gene factor 3, and are capable of inducing antiviral protection and MHC class I antigen expression in several cell types including B16 melanoma cells. We then used the murine B16 melanoma model to investigate the potential antitumor activities of IFN-lambdas. We developed B16 cells constitutively expressing murine IFN-lambda2 (B16.IFN-lambda2 cells) and evaluated their tumorigenicity in syngeneic C57BL/6 mice. Although constitutive expression of mIFN-lambda2 in melanoma cells did not affect their proliferation in vitro, the growth of B16.IFN-lambda2 cells, when injected s.c. into mice, was either retarded or completely prevented. We found that rejection of the modified tumor cells correlated with their level of IFN-lambda2 expression. We then developed IFN-lambda-resistant B16.IFN-lambda2 cells (B16.IFN-lambda2Res cells) and showed that their tumorigenicity was also highly impaired or completely abolished similar to B16.IFN-lambda2 cells, suggesting that IFN-lambdas engage host mechanisms to inhibit melanoma growth. These in vivo experiments show the antitumor activities of IFN-lambdas and suggest their strong therapeutic potential.