Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition and lysis by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-g) restores SCCHN cell susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known.
Because IFN-g activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of pSTAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause-effect relationship, since STAT1 knockdown significantly reduced both IFN-g-mediated APM component expression and TA-specific CTL recognition of IFN-g treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and STAT1:STAT3 heterodimers inhibited IFN-g-induced APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-g induced expression of pSTAT1 and APM components, or recognition of SCCHN cells by TA-specific CTL. Second, STAT1:STAT3 heterodimers did not interfere with IFN-g induced STAT1 binding to the TAP1 promoter or APM component protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-g-pSTAT1-mediated signaling pathway, independent of STAT3 in SCCHN cells.
Interestingly, treatment of SCCHN cells with a broad phosphatase inhibitor, sodium orthovanadate, increased pSTAT1 levels, suggesting that a phosphatase might be responsible for maintaining low basal pSTAT1 and APM component levels, as a mechanism for CTL escape by tumor cells. Indeed, immunohistochemical analyses of 14 SCCHN tumors and paired adjacent normal mucosa demonstrated that src homology-2 domain-containing phosphatase (SHP2) was significantly upregulated in the tumor tissue compared to surrounding mucosa. Moreover, SHP2 specific knockdown using siRNA resulted in significant upregulation of pSTAT1, APM components, and HLA class I in SCCHN cells. Furthermore, SHP2 depletion restored the recognition of SCCHN cells by TA-specific CTL, and induced secretion of Regulation upon Activation, Normal T cell Expressed, and presumably Secreted (RANTES) and IFN-g-inducible protein 10 (IP10). These novel findings identify SHP2 as an important molecular regulator contributing to low basal pSTAT1 levels and APM-mediated immune escape in SCCHN cells, and provide a potential inhibitory strategy for enhancing the clinical activity of T cell-based immunotherapy.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-11242010-131242 |
| Date | 29 November 2010 |
| Creators | Leibowitz, Michael Shinichi |
| Contributors | Jennifer Grandis, Walter J Storkus, Anuradha Ray, Robert L Ferris, Robert Binder |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-11242010-131242/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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