Tissue top down microproteomics gives insight on the tumor m

Tissue top-down microproteomics gives insight on the tumor microenvironment with the identification of proteins involved in cancer processes, diagnosis and/or progression (Table 1). For example, the C-terminal fragment (aa425–483) of Cytokeratin-8 (KRT8) has been detected in our experiments in the necrotic/fibrotic tumor and tumor regions (Fig. 1d). KRT8 was previously referenced as a potential biomarker for ovarian cancer (Wang et al., 2012). We demonstrate here that in cancer regions, it is not the complete protein that is present but a C-terminal fragment of 58 amino mCAP residues. We previously obtained similar results for the C-terminal fragment of the immunoproteasome 11S, PA28 or Reg alpha, a marker for Grade III-IV serous ovarian cancer (Lemaire et al., 2007), as well as for Grade I and tumor relapse (Longuespée et al., 2012). Similarly, a fragment (aa55–72) of Cytokeratin-7 (KRT7) was detected in the tumor region. KRT7 is already a marker for ovarian adenocarcinoma (Chu et al., 2000; Waldemarson et al., 2012), but here we demonstrate that, in fact, the fragment composed of 17 amino acid residues is potentially the actual marker in ovarian tumor. KRT8 and 7 were also reported to be highly expressed in ovarian cancer cell lines (Chu et al., 2000). Protein S100-A11 was detected in the tumor and necrotic/fibrotic tumor regions and was also observed as being particularly highly expressed in ovarian cancer (Liu et al., 2015). The pro-inflammatory cytokine Macrophage migration inhibitory factor (MIF) was detected in the necrotic/fibrotic tumor and tumor regions (Fig. 3d). MIF is already a potential biomarker for ovarian cancer and is associated with tumor growth, metastasis and poor prognosis (Simpson et al., 2012). This protein is also a serum biomarker that distinguishes benign from malignant ovarian tumors in combination with other biomarkers (Agarwal et al., 2007; Krockenberger et al., 2008), and is associated with loss of p53 suppressor activity (Hudson et al., 1999), inhibiting apoptosis and DNA damage repair. Several other proteins already linked to cancer were also identified, including nitrilase-1(Nit1), melanoma antigen family D 2 (MAGED2), Zyxin (ZYX), and ATX1 antioxidant protein 1 homolog (ATOX1). Nit1 is a negative regulator in primary T cells and is classified as a tumor suppressor in association with the fragile histidine-triad protein Fhit (Semba et al., 2006) over-produced in non-small cell lung cancer (NSCLC) and may be a therapeutic target in ovarian cancer (Croce et al., 1999). MAGED2 is also over-expressed in NSCLC (Sienel et al., 2004). Zyxin, a Smad3-mediated TGF-β1 signaling target, regulates cancer cell motility and epithelial-mesenchymal transition during lung cancer development and progression (Beaino et al., 2014; Mise et al., 2012). Interestingly, some proteins identified in the present work have not yet been identified by the Cancer Network Galaxy (TCNG) e.g. Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 4 (OST4), Signal recognition particle receptor subunit alpha (SRPRA), and U6 snRNA-associated Sm-like protein LSm8 (LSM8). In addition to reference proteins, we identified altprots by top-down microproteomics. 6 altprots were detected in the benign region, 5 in the necrotic/fibrotic tumor region, and 4 in the tumor region. None of these 15 altprots were previously identified. Genes coding for these altprots are annotated as genes coding for receptors (TLR5, LTB4R, AGAP1R), enzymes (CMBL, SERPINE1, MTHF, CSNK1A1L, TMP1), or cytoplasmic or nuclear proteins (Apol6, GRAMD4, GNL1, PKHD1L1). AltLARS2-AS1 and AltRP11-576E20.1 are expressed from genes annotated as non-coding genes, and thus should be re-annotated. We focused our interest on AltProts detected in the cancer region, specifically AltGNL1. Indeed, the reference GNL1 protein was previously detected in ovarian cancer according to the Protein Atlas. None of the other 13 reference proteins were previously identified in proteomic or genomic large-scale studies on ovarian cancer. We validated the co-expression of the reference GNL1 protein with its AltProt AltGNL1 (Fig. 5b–c). Our results clearly demonstrate that both proteins are co-expressed from a single mRNA expressed from a cDNA construct. Immunofluorescence experiments showed that AltGNL1 displays nuclear localization whereas GNL1 is present in the cytosol (Fig. 5c). Our results confirm the presence of a hidden proteome which can constitute a reservoir of potential biomarkers and therapeutic targets.