Joseph Bekisz

Mini ReviewHuman Interferons Alpha, Beta and Omega

Type I interferons (IFNs), IFN-α, IFN-β, IFN-ω, IFN-δ and IFN-τ are a family of structurally related, species-specific proteins found only in vertebrates. They exhibit a variety of biological functions, including antiviral, antiproliferative, immunomodulatory and developmental activities. Human Type I IFNs interact with the human IFN alpha receptor (IFNAR), which is composed of two identified subunits (IFNAR-1 and IFNAR-2). The interaction of IFN-α/β with its receptor components results in the activation of a number of signaling pathways. The regulation of specific genes and proteins contributes to the numerous biological functions of Type I IFNs.

Antiproliferative Properties of Type I and Type II Interferon

The clinical possibilities of interferon (IFN) became apparent with early studies demonstrating that it was capable of inhibiting tumor cells in culture and in vivo using animal models. IFN gained the distinction of being the first recombinant cytokine to be licensed in the USA for the treatment of a malignancy in 1986, with the approval of IFN-α2a (Hoffman-La Roche) and IFN-α2b (Schering-Plough) for the treatment of Hairy Cell Leukemia. In addition to this application, other approved antitumor applications for IFN-α2a are AIDS-related Kaposi’s Sarcoma and Chronic Myelogenous Leukemia (CML) and other approved antitumor applications for IFN-α2b are Malignant Melanoma, Follicular Lymphoma, and AIDS-related Kapoisi’s Sarcoma. In the ensuing years, a considerable number of studies have been conducted to establish the mechanisms of the induction and action of IFN’s anti-tumor activity. These include identifying the role of Interferon Regulatory Factor 9 (IRF9) as a key factor in eliciting the antiproliferative effects of IFN-α as well as identifying genes induced by IFN that are involved in recognition of tumor cells. Recent studies also show that IFN-activated human monocytes can be used to achieve >95% eradication of select tumor cells. The signaling pathways by which IFN induces apoptosis can vary. IFN treatment induces the tumor suppressor gene p53, which plays a role in apoptosis for some tumors, but it is not essential for the apoptotic response. IFN-α also activates phosphatidylinositol 3-kinase (PI3K), which is associated with cell survival. Downstream of PI3K is the mammalian target of rapamycin (mTOR) which, in conjunction with PI3K, may act in signaling induced by growth factors after IFN treatment. This paper will explore the mechanisms by which IFN acts to elicit its antiproliferative effects and more closely examine the clinical applications for the anti-tumor potential of IFN.

New Function of Type I IFN: Induction of Autophagy

Autophagy is a highly conserved cellular process responsible for recycling of intracellular material. It is induced by different stress signals, including starvation, cytokines, and pathogens. Type I interferons (IFN) are proteins with pleiotropic functions, such as antiviral, antiproliferative, and immunomodulatory activities. Several recent studies showed type I IFN-induced autophagy in multiple cancer cell lines as evidenced by autophagic markers, for example, the conversion of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B, also known as LC3-I) to LC3-II and the formation of autophagosomes by electron microscopy. In addition, studies suggest the involvement of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT) and mechanistic target of rapamycin, serine/threonine kinase (mTOR) pathways in the induction of autophagy. This review highlights a new function of type I IFN as an inducer of autophagy. This new function of type I IFN may play an important role in viral clearance, antigen presentation, inhibition of proliferation, as well as a positive feedback loop for the production of type I IFN.

Type I interferons induce autophagy in certain human cancer cell lines.

Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12–ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.

Immunomodulatory Effects of Interferons in Malignancies

Investigation of the antitumor and immunomodulatory activities of interferon (IFN) began shortly after the cytokine was discovered in 1957. Early work showed a direct correlation between administration of IFN and inhibition of symptoms associated with virally induced leukemia in mice as well as an increase in their survival time. Subsequent studies with purified IFNs confirmed the direct and indirect stimulation of immune cells, resulting in antitumor activities of IFN. Clinically, IFN-alphas (αs) have been shown to have activity against a variety of tumors. Initially, the U.S. Food and Drug Administration licensed 2 recombinant IFN-αs for the treatment of hairy-cell leukemia and then later for several other cancers. The success rate seen with IFNs and certain tumors has been varied. Unfortunately, some neoplasms show no response to IFN. Monocytes/macrophages play an important role in cancer progression. Monocytes in combination with IFN may be an important therapy for several cancers. This article focuses on the role of IFN and monocytes alone or in combination in affecting malignancies.

Structural variants of IFNα preferentially promote antiviral functions.

IFNα, a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of HIV, exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family members. Although IFNα therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFNα receptor binding sites, we generated IFNα hybrids and mutants and determined that structural changes in the C-helix alter the ability of IFN to limit retroviral activity. Selective IFNα constructs differentially block HIV replication and their directional magnitude of inhibition correlates with APOBEC3 levels. Importantly, certain mutants exhibited reduced toxicity as reflected by induced indoleamine 2,3-dioxygenase (IDO), suggesting discreet and shared intracellular signaling pathways. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects.

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.

BID is a critical factor controlling cell viability regulated by IFN-α

Clinical applications of human interferon (IFN)-&agr; have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-&agr; have not been clearly identified. Our previous study indicated that IFN (&agr;, &bgr;, and &ohgr;) receptor (IFNAR) 1/2- and IFN regulatory factor 9-RNA interference (RNAi) completely restored cell viability after IFN-&agr; treatment in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-&agr;. In this study, IFNAR1/2- and IFN regulatory factor 9-RNAi inhibited the gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not of Fas ligand, after IFN-&agr; treatment. In fact, TRAIL but not Fas ligand inhibited the viability of OVCAR3 cells. IFN-&agr; notably upregulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. After TRAIL signaling, caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-&agr; and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-&agr; and TRAIL from collapsing the mitochondrial membrane potential (&Dgr;&PSgr;m). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability after IFN-&agr; or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-&agr;. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-&agr; which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-&agr; resistant tumors.

Near Eradication of Clinically Relevant Concentrations of Human Tumor Cells by Interferon-Activated Monocytes In Vitro

We have previously reported that low concentrations of interferon (IFN)-activated monocytes exert near-eradicative cytocidal activity against low concentrations of several human tumor cells in vitro. In the present study, we examined 7 human tumor cell lines and 3 diploid lines in the presence or absence of 10 ng/mL IFNα2a and monocytes. The results confirmed strong cytocidal activity against 4 of 7 tumor lines but none against 3 diploid lines. To model larger in vivo tumors, we increased the target cell concentration and determined the concentration of IFNα2a and monocytes, required for cell death. We found that increasing the tumor cell concentration from 10- to 100-fold (10(5) cells/well) required an increase in the concentration of IFNs by over 100-fold and monocytes by 10-fold. High concentrations of monocytes could sometimes kill tumor or diploid cells in the absence of IFN. We may conclude that killing of high concentrations of tumor or diploid cells required high concentrations of monocytes that could sometimes kill in the absence of IFN. Thus, high concentrations of tumor cells required high concentrations of IFN and monocytes to cause near eradication of tumor cells. These findings may have clinical implications.

Gateway cloning is compatible with protein secretion from Pichia pastoris

Abstract Secretion of a recombinant protein from the yeast Pichia pastoris requires the presence of a signal peptide at the amino terminus. Maintaining the full amino acid sequence of the signal peptide is thought to be important for proper signal processing and protein secretion. We show that at least for one protein, a synthetic human interferon, the presence of a Gateway recombination site within the signal peptide is fully compatible with high levels of protein secretion. The amino termini of the secreted interferon proteins cloned with Gateway and cloned with restriction enzymes and ligase are identical, and the proteins were highly active in biological assays. Compatibility with Gateway cloning simplifies construction of plasmids directing secretion of recombinant proteins from P. pastoris.