Abnormal APM component expression and/or dysfunction frequently occur in human malignancies [4
], but this mechanism of immune escape has not been thoroughly investigated. In this study, we document that SCCHN cells express low basal levels of pSTAT1 () as a mechanism of APM component downregulation despite intact IFN-γR expression and signaling () and that exogenous IFN-γ-mediated STAT1 activation is required to facilitate STAT1 binding to the TAP1 promoter (), APM protein expression () and TA-specific CTL recognition (). These data support that low basal pSTAT1 levels and not defects in the endogenous IFN-γ-pSTAT1-APM signal transduction pathway are responsible for the low basal APM component expression observed in SCCHN cells.
Complete inhibition of IFN-γ mediated APM component expression was not observed after significant knockdown of total STAT1 protein. Interestingly, treatment of SCCHN cells with a tenfold lower dose of IFN-γ (10 U/ml) upregulated APM components (data not shown) suggesting that a threshold level of pSTAT1 is required to induce APM protein expression. Thus, despite the significant reduction of total STAT1 by siRNA (), residual STAT1 was sufficiently activated by IFN-γ to mediate APM component expression and CTL recognition, albeit at significantly reduced levels (). Interestingly, others have shown reduced IFN-γ levels in the plasma of patients with SCCHN compared to age-matched controls [37
], providing a potential clinical explanation for low basal STAT1 activation and APM-mediated immune escape in SCCHN cells.
Others have shown that STAT3 activation by type I IFNs can inhibit STAT1 target gene expression through formation of STAT1:STAT3 heterodimers [17
]; therefore, we also investigated whether overexpressed pSTAT3 and these heterodimer complexes were responsible for low basal APM component expression in SCCHN cells. Our exclusion for a role of STAT3 in basal APM-mediated immune escape differs from previous reports that provide evidence that STAT3 activation by IL-10 inhibits IFN-γ-mediated STAT1 phosphorylation [20
] and downregulates TAP expression in tumor cells [21
]. In SCCHN cells, the IL-10 receptor is not appreciably expressed, and IL-10 did not activate pSTAT3 in our studies (unpublished data). The biological basis for pSTAT1:pSTAT3 heterodimers in SCCHN is unknown [39
]. We investigated whether pSTAT1:pSTAT3 heterodimers were responsible for low basal APM component expression and SCCHN escape from CTL recognition. STAT1:STAT3 heterodimers were detected in PCI-13 and SCC90 cells at basal, untreated conditions, and these complexes were increased after treatment with IFN-γ and IL-6 (). Interestingly, IFN-γ-mediated pSTAT1 upregulation also induced heterodimer formation with pSTAT3 (), but APM protein was still expressed (). This finding might be due to the abundant activation of STAT1 by IFN-γ, inducing pSTAT1 homodimerization and APM gene activation and excess pSTAT1 dimerizing with pSTAT3, which is overexpressed in SCCHN cells. Also, STAT3 was found bound to the TAP1 promoter (), and this observation is consistent with the fact that STAT1 and STAT3 can bind to the same consensus gamma-activating sequence (GAS), yet regulate different target genes [40
IL-6 induced pSTAT1:pSTAT3 heterodimerization did not alter IFN-γ-mediated APM protein (). To investigate whether the dose of IFN-γ (100 U/ml) used in our studies might be masking a potential negative regulatory effect of IL-6-mediated STAT1:STAT3 heterodimers, we used a 10-fold lower dose of IFN-γ (10 U/ml). Even at this lower dose, IL-6 treatment did not inhibit IFN-γ-mediated APM component expression (data not shown). Importantly, at baseline, STAT3 depletion by siRNA could not reproduce the beneficial effects of stimulating the IFN-γ-STAT1-APM pathway. Treatment of SCCHN cells with IFN-γ after STAT3 depletion did not augment APM component expression or TA-specific CTL recognition (). These data demonstrate that APM component expression and CTL recognition of SCCHN cells primarily require activation of STAT1 with exogenous IFN-γ independent of STAT3.
The mechanism of how SCCHN cells maintain low basal pSTAT1 and APM component expression is still not known. Deficiencies in TAP expression have been documented to occur through a lack of STAT1 and IRF1 phosphorylation [41
], mutations in the JAK1 kinase that prevent its activation and subsequent STAT1 phosphorylation [42
], and impaired RNA polymerase II recruitment to the TAP1 promoter [43
]. Several investigators have identified that members of a small sub-family of non-receptor protein tyrosine phosphatases (PTPs), Src homology-2 domain-containing phosphatases (SHP)-1 and SHP2 can dephosphorylate JAK1 [44
] and STAT1 [45
]. Providing a stimulus for STAT1 activation such as IFN-γ can correct APM downregulation and enhance CTL lysis in vitro, but perhaps a more efficacious therapeutic approach would be a targeted therapy against the negative regulators of STAT1 phosphorylation in SCCHN. A greater understanding of the mechanisms responsible for low basal pSTAT1 and APM expression could augment current T-cell-based immunotherapies by enhancing the immunogenicity of its tumor cell target.
In summary, these studies identified low pSTAT1 in SCCHN cells as a critical mediator of APM component downregulation and CTL escape. We also investigated a potential role for pSTAT1:pSTAT3 heterodimers in APM component downregulation of SCCHN cells. Our data demonstrate that pSTAT1:pSTAT3 heterodimers do not alter the IFN-γ-pSTAT1-APM signaling axis. The function of the pSTAT1:pSTAT3 heterodimer remains unknown, but these findings indicate a need to directly stimulate the STAT1 pathway to enhance APM expression and reverse CTL evasion by tumor cells. Future studies might include investigating the negative regulators of STAT1 activation whose dysregulated activity might be responsible for low basal pSTAT1 and APM component levels in SCCHN.