Reduced inducibility of SOCS3 by interferon gamma associates with higher resistance of human breast cancer lines as compared to normal mammary epithelial cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation

Interferon-alpha treatment may negatively influence disease progression in melanoma patients by hyperactivation of STAT3 protein.

Interferon-alpha (IFN-alpha) is an important drug used in anti-melanoma therapy. However, metastases eventually reappear in almost 60% of melanoma patients, who have received adjuvant cytokine therapy suggesting that IFN-alpha can paradoxically promote disease progression in some cases, at least. In this study, we have investigated the possibility that a growth-promoting STAT3 protein might be activated by interferon-alpha in melanoma cells. We examined 24 primary cultures established from node metastases of melanoma patients who were monitored in a 5-year clinical follow-up. The patients differed in the course of disease and survival end-points. Using Western blot analyses, we show that interferon-alpha stimulated STAT3 phosphorylation at tyrosine (Y705) residue in 17% of cases. These over-reactive cell populations originated from patients who had the shortest disease-free intervals. A significant correlation was obtained between the length of survival end-points and a lack of STAT3 activation by IFN-alpha. No STAT3 induction was observed in normal melanocytes. The STAT1 activation at tyrosine (Y701) occurred at a similar frequency as that of STAT3 (17%) albeit in different patients, no clear correlation with the clinical status could be made. The interferon-alpha/beta receptors (IRFARs) were expressed irrespective to the signal transducers and activators of transcription (STATs) inducibility suggesting that signalling defects occur downstream from IRFAR. We propose that in some cases the application of IFN-alpha could increase the probability of disease progression via overactive STAT3. The tests for STAT3 inducibility prior to cytokine immunotherapy in the clinic are therefore warranted.

Defective IFNα/γ-induced STAT3 protein activation in human malignant melanoma cells.

Signal transducer and activator of transcription 3 (STAT3) protein has been documented as a significant mediator of interferon (IFN) signaling. Physiological STAT3 phosphorylation involves tyrosine (Y705) and serine (S727) activation. Impairment of STAT3 protein levels and/or of STAT3 phosphorylation after IFN treatment has been found in many pathological conditions such as cancer, immunopathy and inflammatory disease. To analyze tumor-associated defective STAT3 response to IFNs, the induction of S727 and Y705 STAT3 activation after IFN exposure was evaluated in 18 human malignant melanoma cell lines and 68 primary cell cultures established from the lymph node metastases of melanoma patients. STAT3 expression and STAT3 phosphorylated forms were assayed by Western blot analysis employing specific STAT3 antibodies. All melanoma cell lines as well as samples derived from metastatic melanoma patients expressed STAT3 with variable signal intensities depending on the appropriate cell type. Significantly altered IFNγ-induced S727 STAT3 activation was found in both experimental models, with on average 94.1% of patients detected to be non-responders in lymph node cell cultures and 83.3% in melanoma cell lines. Moreover, a deficiency in IFNα-induced S727 induction was detected in 88.9% of melanoma cell lines. Defects in Y705 STAT3 phosphorylation were determined in clinical material (61.8% after IFNγ exposure) as well as in melanoma cell lines (absence of response to IFNα/γ in 83.3 and 55.5%, respectively). Our data clearly confirm STAT3 pathophysiological perturbances in human malignant melanoma cells. Depending on the induction of STAT3-activated phosphoforms by IFNs, three categories of melanoma cells were identified: a) phosphorylation on both the S727 and Y705 amino acid residues; b) STAT3 activation on Y705 only; c) phosphorylation at neither S727 nor Y705. The significance of in vitro STAT3 activation for predicting patient response to immunotherapy will be examined in a prospective clinical study by our group.

Development of IFN-gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

Transcription protein STAT1: biology and relation to cancer.

Cell homeostasis is controlled and regulated by multiple signalling proteins that operate almost in all cellular compartments. Their common task is to process regulatory signals from both the extracellular and intracellular spaces by triggering a cascade of intracellular events leading to modulation of downstream gene activity. One of the important signalling pathways is represented by the STAT multigene family comprising seven members. In general, various STATs act as potent transcription factors delivering signals of diverse polypeptide ligands (i.e. cytokines and growth factors) into the nucleus. This review summarizes some up-to-date data on the role of STAT1 in maintaining cellular homeostasis with the emphasis on its role in the control of cell growth, proliferation, apoptosis, and immune reactions. Part of the review deals with expression and posttranslational abnormalities of this molecule identified in a variety of human pathological conditions including cancer. The direct or indirect involvement of STAT1 in the process of malignant transformation is highlighted in view of these molecular perturbances that may contribute to oncogenesis and that may be potentially used as novel targets for anticancer therapy.

Lack of STAT 1 phosphorylation at TYR 701 by IFNgamma correlates with disease outcome in melanoma patients.

STAT 1, a member of signal transducer and transcription activator family has been implicated as key downstream mediator of interferon (IFN) signaling. Its functional activation requires phosphorylation at Tyr 701 and Ser 727 residues. Various STAT abnormalities have been found in cancer cells but their relation to oncogenesis, tumor behavior and disease outcome remains mostly unknown. We have examined the inducibility of STAT 1 phosphorylation by IFN alpha/gamma in primary cultures established from melanoma lymph node metastases at first progression and correlated our results with disease outcome and overall survival. Forty-four patients at clinical stage I-III at initial diagnosis entered the study. STAT 1 inducibility of phosphorylation by IFNs was assessed in melanoma cell lysates by means of standard immunoprecipitation and Western blotting using polyclonal and monoclonal antibodies. Lack of STAT 1 phosphorylation at Ser 727 after either IFN was recorded in 75% of patients, however, no correlations with disease evolution could be proved. In contrast, STAT 1 phosphorylation response at Tyr 701 after IFNalpha occurred in 13 (29.5%) and after IFNgamma in 32 (73%) patients. Inducibility of STAT 1 activation at Tyr 701 but not at Ser 727 driven by IFNgamma but not by IFNalpha significantly and unfavorably [corrected] influenced disease- free interval and overall survival. In conclusion, these results show that the absence of IFNgamma inducibility of STAT 1 phosphorylation at Tyr 701 positively correlates with disease outcome in malignant melanoma patients and may represent new independent prognostic marker.