Role of Interferon α (IFNα)-inducible Schlafen-5 in Regulation of Anchorage-independent Growth and Invasion of Malignant Melanoma Cells

Abstract

IFN␣ exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon ␣ (IFN␣) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFN␣ resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFN␣. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFN␣ and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.The interferons (IFNs) are cytokines with important pleiotropic biological effects, including generation of antitumor responses and antiviral activities (1-3). The ability of IFNs to induce antitumor responses in selective systems is highly relevant and, over the years, has had a major impact in the management of certain leukemias and solid tumors in humans. Malignant melanoma is one of the most IFN-sensitive solid tumors. There has been extensive clinical evidence on the ability of IFN␣ to generate antitumor effects in vitro in subset groups of patients with advanced metastatic malignant melanoma (4 -7), and IFN␣ is now a Food and Drug Administration-approved agent for the treatment of this malignancy. An important outstanding issue in the IFN research field has been the identification of specific mechanisms that account for differential sensitivity to the effects of IFNs. Despite the advances in the IFN-signaling field over the last 2 decades, the precise mechanisms and specific signals that account for the unique IFN sensitivity that some tumors exhibit remain largely unknown.It is now well established that IFNs regulate transcription of target genes with important functional relevance via engagement of the JAK-STAT pathway (8 -11). In recent years, additional levels of cellular regulation of IFN-inducible genes and their products have been identified, such as involvement of members of the PKC family (12-16), the MAPK cascades (17-22), translational regulation via mammalian target of rapamycin and 4EBP1 (23-28), modulation of histo...

EZH2 Inhibition Blocks Multiple Myeloma Cell Growth through Upregulation of Epithelial Tumor Suppressor Genes

“…Another example is DOCK9 (dedicator of cytokinesis 9), which belongs to the Dock family of evolutionarily conserved exchange factors for the Rho GTPases Rac and Cdc42, regulating actin cytoskeleton, cell adhesion, and migration (48). SLFN5, a protein, was described to have a key role in controlling motility and invasiveness of renal cell carcinoma and melanoma cells (49,50). SLFN5 negatively controls expression of matrix metalloproteinases (MMP), and several other genes involved in the control of malignant cell motility.…”

“…S2C). Cell lines were treated with E7438 at the calculated IC 50 value from proliferation assays, to compare the cycle effects at a similar inhibition level. We observed a general decrease in the percentage of cells in G 2 -M accompanied by an increase in sub-G 1 (apoptotic) cells, except for the U-266 cell line which showed only a minor increase in the G 0 -G 1 fraction.…”

Expression and Regulatory Effects of Murine Schlafen (Slfn) Genes in Malignant Melanoma and Renal Cell Carcinoma

“…Assays using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetra-zolium bromide method were performed after treating cells with IFN␣ for 5 to 7 days, as in our previous studies (9,18).…”

“…In previous studies, we demonstrated that among human SLFNs, SLFN5 is selectively up-regulated in response to IFN treatment in malignant melanoma cells (18). In the present study, we undertook a systematic analysis on the IFN-inducible expression and biological relevance of different mouse Slfn genes in mouse malignant melanoma and renal cell carcinoma (RCC) 2 cells.…”

“…Soft Agar Assays-Anchorage-independent cell growth was analyzed in soft agar assays as described previously (9,18,19). Equal number of cells were plated in soft agar and allowed to form colonies for 14 days, and colony formation was assessed.…”