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...

The precise STAT-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I interferons (IFNs) remain unknown. We provide evidence that Type I IFNs regulate expression of Schlafens (SLFNs), a group of genes involved in the control of cell cycle progression and growth inhibitory responses. Using cells with targeted disruption of different STAT proteins and/or the p38 MAP kinase, we demonstrate that the IFN-dependent expression of distinct Schlafen genes is differentially regulated by STAT complexes and the p38 MAP kinase pathway. We also provide evidence for a key functional role of a member of the SLFN family, SLFN2, in the induction of the growth-suppressive effects of IFNs. This is shown in studies demonstrating that knockdown of SLFN2 enhances hematopoietic progenitor colony formation and reverses the growth-suppressive effects of IFN␣ on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form more colonies in soft agar, implicating this protein in the regulation of anchorage-independent growth. Altogether, our data implicate SLFN2 as a negative regulator of the metastatic and growth potential of malignant cells and strongly suggest a role for the SLFN family of proteins in the generation of the antiproliferative effects of Type I IFNs.

Purpose: Statins are pharmacologic inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase with potent regulatory effects on cholesterol biosynthesis in vitro and in vivo. There is accumulating evidence that, beyond their cholesterol-lowering properties, statins inhibit cell proliferation and promote apoptosis of malignant cells in vitro, but the mechanisms by which they generate such responses remain to be defined. Experimental Design: Combinations of experimental approaches were used, including immunoblotting and cell proliferation and apoptosis assays. Results: We provide evidence that fluvastatin is a potent inducer of apoptosis and suppresses proliferation of renal cell carcinoma (RCC) cells in vitro. Such effects are mediated by direct targeting of the Akt/mammalian target of rapamycin (mTOR) pathway, as evidenced by the suppression of phosphorylation/activation of Akt, resulting in inhibition of its downstream effectors, mTOR and p70 S6 kinase. In addition, fluvastatin blocks the mTOR-dependent phosphorylation/ deactivation of the translational repressor eukaryotic initiation factor 4E (eIF4E)-binding protein, leading to the formation of eIF4E-binding protein-eIF4E complexes that suppress initiation of cap-dependent mRNA translation. Importantly, inhibition of p70 S6 kinase activity by fluvastatin results in the up-regulation of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein with inhibitory effects on the translation initiation factor eIF4A, suggesting a mechanism for the generation of antitumor responses. Conclusions: Altogether, our findings establish that fluvastatin exhibits potent anti-RCC activities via inhibitory effects on the Akt/mTOR pathway and raise the possibility that combinations of statins and Akt inhibitors may be of future therapeutic value in the treatment of RCC.