Background: Cervical squamous cell carcinomas (SCC) represent 95% of cervix cancers and arise from dysplastic squamous intraepithelial lesions (SIL). It is known that 70% of high-grade SILs (HSILs) evolve into SCC. Finding protein markers of HSIL would help understanding the neoplastic mechanisms leading to the early development of cervical SCC. It would also have an impact for clinical diagnosis and practice. Methods: We used the up-to-date laser microdissection (LMD)-based microproteomic method to compare the protein content of HSIL and normal cervical columnar and squamous tissue counterparts, namely endocervix and exocervix. Formalin-fixed and paraffin embedded tissue blocks from conization were selected from five patients with HSIL. From 5 μm tissue sections, 3500 cells of the tissue types described upon were collected by LMD and processed for further microproteomics. Proteolytic digests were nanalyzed by 2D LC-MS/MS. The dataset was normalized using Maxquant and statistical analysis performed with Perseus. We aimed to validate pro- tein biomarkers of HSIL by immunohistochemistry. Results: A total of 3105 different proteins were identified among all samples and 2409 could be quantified in four biological replicates over five in at least one tissue type. The PCA analysis revealed an intermediary profile of HSIL between endo and exocervical tissues. The analysis of variance revealed a subset of 806 proteins significantly different between the tissue types. Hierarchical clustering revealed a cluster of 236 proteins more abundantly present in HSIL compared to the normal counterparts. Gene ontology and Kyoto Encyclopedia of genes and genomes (KEGG) annotations revealed an important implication of HSIL proteins in chromatin remodeling, RNA splicing, RNA transport, mismatch repair, and positive regulation of viral replication. The known marker for HSIL, p16 or cyclin-dependent kinase inhibitor 2 A was found only in HSIL tissues. Three other proteins, histone chaperone ASF, tyrosine-protein kinase Lyn, and DBIRD complex subunit ZNF326 were also found only in HSIL. These proteins represent new potential markers of HSIL. Conclusion: Microproteomics is reliable for tissue-based protein biomarker discovery. It is expected to become an important workflow for future molecular pathology. In the present study, we found new specific biomarkers of HSIL. The description of the biological mechanisms related to the development of HSIL will be supportive for future therapeutic research.
Microproteomic profiling of cervical high-grade squamous intraepithelial lesions
Casadonte R;
2018-01-01
Abstract
Background: Cervical squamous cell carcinomas (SCC) represent 95% of cervix cancers and arise from dysplastic squamous intraepithelial lesions (SIL). It is known that 70% of high-grade SILs (HSILs) evolve into SCC. Finding protein markers of HSIL would help understanding the neoplastic mechanisms leading to the early development of cervical SCC. It would also have an impact for clinical diagnosis and practice. Methods: We used the up-to-date laser microdissection (LMD)-based microproteomic method to compare the protein content of HSIL and normal cervical columnar and squamous tissue counterparts, namely endocervix and exocervix. Formalin-fixed and paraffin embedded tissue blocks from conization were selected from five patients with HSIL. From 5 μm tissue sections, 3500 cells of the tissue types described upon were collected by LMD and processed for further microproteomics. Proteolytic digests were nanalyzed by 2D LC-MS/MS. The dataset was normalized using Maxquant and statistical analysis performed with Perseus. We aimed to validate pro- tein biomarkers of HSIL by immunohistochemistry. Results: A total of 3105 different proteins were identified among all samples and 2409 could be quantified in four biological replicates over five in at least one tissue type. The PCA analysis revealed an intermediary profile of HSIL between endo and exocervical tissues. The analysis of variance revealed a subset of 806 proteins significantly different between the tissue types. Hierarchical clustering revealed a cluster of 236 proteins more abundantly present in HSIL compared to the normal counterparts. Gene ontology and Kyoto Encyclopedia of genes and genomes (KEGG) annotations revealed an important implication of HSIL proteins in chromatin remodeling, RNA splicing, RNA transport, mismatch repair, and positive regulation of viral replication. The known marker for HSIL, p16 or cyclin-dependent kinase inhibitor 2 A was found only in HSIL tissues. Three other proteins, histone chaperone ASF, tyrosine-protein kinase Lyn, and DBIRD complex subunit ZNF326 were also found only in HSIL. These proteins represent new potential markers of HSIL. Conclusion: Microproteomics is reliable for tissue-based protein biomarker discovery. It is expected to become an important workflow for future molecular pathology. In the present study, we found new specific biomarkers of HSIL. The description of the biological mechanisms related to the development of HSIL will be supportive for future therapeutic research.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
