These authors contributed equally to this work.

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IntroductionInterferon-␥ (IFN-␥), produced by T and natural killer (NK) cells, is considered the principal effector cytokine of cell-mediated immunity and exerts its effects on target cells through a highaffinity receptor complex linked to a specific Janus kinase (Jak)/ signal transducer and activator of transcription (STAT) signaling cascade. 1,2 The IFN-␥ receptor (IFN-␥R) complex consists of 2 chains: an IFN-␥R1 binding chain and an IFN-␥R2 signaling chain. 1 The intracellular portions of the 2 chains provide the Jak1 and Jak2 docking sites. Upon phosphorylation, Jak1 and Jak2 activate STAT1: Following phosphorylation and dimerization, STAT1 is translocated into the nucleus where it activates transcription of numerous sets of IFN-␥-inducible genes. 2 The IFN-␥/STAT1 pathway plays an essential role in controlling the expansion of normal and neoplastic cell types of different origin. Activation of this pathway switches on many proapoptotic and antiproliferative genes such as interferon regulatory factor 1 (IRF1), p21 waf/cip , Fas and FasL, and activates caspases. [3][4][5][6][7][8][9][10] However, the signals transduced by IFN-␥ do not always induce apoptosis or block proliferation, and lymphoid cells become resistant to the antiproliferative effects of IFN-␥. Resting, malignant, or normal T cells that develop toward the T helper 1 (Th1) pathway become resistant to the antiproliferative effects of the IFN-␥/STAT1 pathway 9,11,12 or rather, IFN-␥ favors their proliferation and differentiation. [13][14][15] The refractoriness of T cells to the IFN-␥/STAT1 pathway has been attributed mainly to down-regulation of the IFN-␥R chains, especially IFN-␥R2, which protects these cells from the antiproliferative/proapoptotic effects of [16][17][18][19] Both IFN-␥-dependent and -independent mechanisms have been reported to downregulate IFN-␥R2 expression in T lymphocytes. During murine Th cell differentiation, IFN-␥ itself induces IFN-␥ resistance by down-regulating IFN-␥R2, 16 whereas in human T lymphocytes, IFN-␥R2 internalization occurs mostly in clathrin-coated pits independently from IFN-␥ 17 and is selectively induced by insulinlike growth factor 1 (IGF1). 20 Since the IFN-␥/STAT1 pathway is usually down-regulated in T lymphocytes, information on the mechanisms that maintain low IFN-␥R2 expression in these cells might prove useful for devising therapeutic approaches centered on selectively reinstating the IFN-␥/STAT1 apoptotic signaling pathway in autoreactive or neoplastic T cells.Besides T-cell receptor (TCR) engagement, 9,12 up-regulation of surface IFN-␥R2 in T cells may also be induced by serum deprivation, 5 exposure to nitric oxide (NO), 21 Among the plethora of factors present in serum, iron has profound effects on numerous critical cell functions, such as electron and oxygen transport, mitochondrial energy metabolism, and detoxification, thus requiring tight homeostatic regulation. 24 Iron binds to cytoplasmic iron regulatory protein 1 (IRP1) and IRP2 which in turn regulate expression of proteins such as fe...

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STAT1 and STAT3 are the main mediators of the signaling of interferons (IFNs) and of gp130 cytokines, respectively. Neoplastic T lymphocytes frequently become resistant to the IFN-c/ STAT1 apoptotic pathway, often because of the downregulation of the IFN-cR2 receptor chain. Many studies suggest that crossregulation between different STATs, in particular between STAT1 and STAT3, may profoundly affect cytokine/growth factor signaling. Here, the function of STAT3 in the negative regulation of STAT1 apoptotic pathway was investigated by RNA interference-mediated STAT3 silencing in human malignant T lymphocytes. In STAT3-depleted cells, interleukin (IL)-6 acquired the capacity to induce apoptosis, correlating with prolonged STAT1 activation and the induction of major histocompatibility complex (MHC) class I expression. In contrast, in the absence of STAT3, IFN-c could slightly enhance apoptosis but its ability to induce MHC class I expression was unchanged. Accordingly, IL-6, but not IFN-c, could significantly impair the in vivo growth of STAT3-depleted human neoplastic T lymphocytes transplanted into severe combined immunodeficient mice. Therefore, treatment with IL-6 and simultaneous STAT3 silencing may represent a potential therapeutic approach to control the expansion of IFN-c-unresponsive neoplastic T cells.

IntroductionInterferon-␥ (IFN-␥), a cytokine produced by T and natural killer (NK) cells, plays an essential role in cell-mediated immunity, and it contributes to the control of the expansion of many normal and neoplastic cell types. 1 It exerts its biological activities by interacting with its specific cell-surface receptor (IFN-␥R), which consists of 2 IFN-␥R1 binding chains and 2 IFN-␥R2 signal-transducing chains. 2 The binding of IFN-␥ to its receptor complex activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal transduction pathway [3][4][5] and induces the transcription of numerous sets of IFN-␥-inducible proapoptotic genes. [6][7][8][9][10][11] However, IFN-␥ does not always cause apoptosis or block proliferation. T lymphocytes that undergo T helper 1 (Th1) polarization or malignant transformation become resistant to the antiproliferative/apoptotic effects of the IFN-␥/STAT1 pathway. [12][13][14] This refractoriness is mainly due to IFN-␥R2 downregulation, 12-17 due to ligand-independent internalization within clathrin-coated pits. [18][19][20] By inducing IFN-␥R2 internalization, both insulin-like growth factor-1 (IGF-1) and iron are critical factors in limiting the IFN-␥/STAT1 pathway in T lymphocytes. [20][21][22] Since IGF-1 induces cell-membrane accumulation of the transferrin receptor (TfR), 23 which plays an obligatory role in the iron-induced IFN-␥R2 internalization, 22 blockade of IGF-1 signaling in T cells could perhaps hinder their intracellular IFN-␥R2 trafficking and reinstate their sensitivity to IFN-␥/STAT1 apoptotic signaling.IGF-1 is a growth factor mainly produced by the liver that plays an important role in the promotion of mitogenesis, transformation, and protection from apoptosis of many cell types. [24][25][26] It acts by binding to IGF-1R, a type II tyrosine kinase receptor composed of 2 extracellular ␣ subunits that bind ligand, and 2 trans-membrane catalytic ␤ subunits. 24,27,28 Upon ligand binding, the receptor is autophosphorylated on several tyrosine residues located in the intracellular domain of the ␤ subunits, and this provides binding sites for Shc, insulin receptor substrate-1 (IRS-1) and IRS-,2 and other signaling molecules. IRS-1/IRS-2 and Shc are thus phosphorylated and activate the phosphoinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways. 29 Preclinical studies have demonstrated that genetic or pharmacological inhibition of IGF-1 signaling reverses the neoplastic phenotype of many tumor cells. 30 The effects of specific blockade of IGF-1 signaling on malignant T cells, however, have not been investigated.The effects of this blockade, alone or in combination with IFN-␥ administration, on the growth and apoptosis of human malignant T cells have now been evaluated. A retrovirus-based approach was exploited to stably express a dominant-negative (DN) form of the IGF-1R in malignant ST4 T cells. Expression of IGF-1R DN did not affect their ability to proliferate in the presence of serum in vitro, ...

STAT3, a pleiotropic transcription factor acting downstream of cytokines and growth factors, is known to enhance proliferation, migration, invasion and aerobic glycolysis in tumors upon aberrant activation. In the murine epidermis, STAT3 is necessary for experimentally induced carcinogenesis. Skin tumorigenesis is conversely enhanced by overexpression in keratinocytes of the constitutively active STAT3C mutant, which also induces robust, psoriasis-like epidermal hyperplasia. We show here that STAT3C expression at physiological levels in knock-in mice leads to mild epidermal hyperplasia and attenuated expression of terminal differentiation markers. Altered differentiation is confirmed in isolated primary epidermal keratinocytes in vitro, correlating with enhanced proliferative and clonogenic potential, attenuated senescence and, strikingly, high-frequency spontaneous immortalization. These results suggest that moderate levels of continuous STAT3 activation, which closely resemble those triggered by chronic inflammation or persistent growth factor stimulation, may establish a preneoplastic state in part by promoting the escape of epidermal progenitor cells from differentiation and senescence checkpoints.