Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1–Cullin-1–F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin–proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11 –deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11 , along with CIITA , is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.
<div>Abstract<p>TRIM29 (tripartite motif-containing protein 29) is a TRIM family protein that has been implicated in breast, colorectal, and pancreatic cancers. However, its role in stratified squamous epithelial cells and tumors has not been elucidated. Here, we investigate the expression of TRIM29 in cutaneous head and neck squamous cell carcinomas (SCC) and its functions in the tumorigenesis of such cancers. TRIM29 expression was lower in malignant SCC lesions than in adjacent normal epithelial tissue or benign tumors. Lower expression of TRIM29 was associated with higher SCC invasiveness. Primary tumors of cutaneous SCC showed aberrant hypermethylation of <i>TRIM29</i>. Depletion of TRIM29 increased cancer cell migration and invasion; conversely, overexpression of TRIM29 suppressed these. Comprehensive proteomics and immunoprecipitation analyses identified keratins and keratin-interacting protein FAM83H as TRIM29 interactors. Knockdown of TRIM29 led to ectopic keratin localization of keratinocytes. In primary tumors, lower TRIM29 expression correlated with the altered expression of keratins. Our findings reveal an unexpected role for TRIM29 in regulating the distribution of keratins, as well as in the migration and invasion of SCC. They also suggest that the TRIM29–keratin axis could serve as a diagnostic and prognostic marker in stratified epithelial tumors and may provide a target for SCC therapeutics.</p>Significance:<p>These findings identify TRIM29 as a novel diagnostic and prognostic marker in stratified epithelial tissues.</p></div>
<p>Supplemental Table S1. Patients attributes.</p>
<p>Supplemental figures Supplemental Fig. 1. TRIM29 is highly expressed in stratified epithelial tissues. Supplemental Fig. 2. The expression levels of TRIM29 protein are as high in benign skin tumors as they are in normal epidermis. Supplemental Fig. 3. The expression level of TRIM29 is low in keratoacanthoma-like SCC, but not in keratoacanthoma. Supplemental Fig. 4. Reduced TRIM29 RNA expression is associated with DNA methylation in cutaneous SCC. Supplemental Fig. 5. TRIM29 expression is silenced in cutaneous SCC cells. Supplemental Fig. 6. The cell proliferation of TRIM29-knockdown cancer cells does not differ significantly from that of the control cells. Supplemental Fig. 7. siRNA-mediated TRIM29-knockdown does not affect cell proliferation. Supplemental Fig. 8. TRIM29 regulates migration of SCC cells and immortalized epidermal keratinocytes. Supplemental Fig. 9. TRIM29 regulates migration of SCC cells and immortalized epidermal keratinocytes. Supplemental Fig. 10. TRIM29 knockdown increases cell invasion in SCC cells and immortalized epidermal keratinocytes. Supplemental Fig. 11. TRIM29 knockdown increases cell invasion in oral SCC cells. Supplemental Fig. 12. TRIM29 knockdown enhances the cancer cell metastasis. Supplemental Fig. 13. The top 20 list of proteins bound with FLAG-TRIM29 in mass spectrometry analysis. Supplemental Fig. 14. TRIM29 co-localizes with FAM83H. Supplemental Fig. 15. TRIM29 co-localizes with FAM83H. Supplemental Fig. 16. The zinc finger, B-box, coiled-coil, and C-terminal domains of TRIM29 are necessary for the formation of the TRIM29-keratin-FAM83H complex. Supplemental Fig. 17. Schema of the keratin distribution patterns. Supplemental Fig. 18. Knockdown of TRIM29 alters the distribution of keratins.</p>
<p>Supplemental Table S1. Patients attributes.</p>
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