INTERNATIONAL JOURNAL OF ONCOLOGY 57: 409-432, 2020 Biomarkers of tumor invasiveness in proteomics (Review) DANIEL L. POULIQUEN1, ALICE BOISSARD2, OLIVIER COQUERET1 and CATHERINE GUETTE2 1CRCINA, Inserm, Université d'Angers, F-44000 Nantes; 2Paul Papin ICO Cancer Center, CRCINA, Inserm, Université d'Angers, F-44000 Nantes, France Received February 19, 2020; Accepted May 7, 2020 DOI: 10.3892/ijo.2020.5075 Abstract. Over the past two decades, quantitative proteomics 4. Plasma proteins has emerged as an important tool for deciphering the complex 5. Membrane proteins molecular events involved in cancers. The number of refer- 6. Cytoplasm/cytosol ences involving studies on the cancer metastatic process 7. Cytoskeleton has doubled since 2010, while the last 5 years have seen the 8. Endoplasmic reticulum development of novel technologies combining deep proteome 9. Mitochondrion and nucleus coverage capabilities with quantitative consistency and accu- 10. Miscellaneous racy. To highlight key findings within this huge amount of 11. Extension to and links with other proteins of interest not information, the present review identified a list of tumor inva- included in the list sive biomarkers based on both the literature and data collected 12. Conclusions on a biocollection of experimental cell lines, tumor models of increasing invasiveness and tumor samples from patients with colorectal or breast cancer. Crossing these different data 1. Introduction sources led to 76 proteins of interest out of 1,245 mentioned in the literature. Information on these proteins can potentially be The development of molecular characterization of tumors translated into clinical prospects, since they represent potential two decades ago led to a revolution in cancer research and targets for the development and evaluation of innovative thera- therapeutic approaches. In the case of breast cancer, ‘molec- pies, alone or in combination. Herein, a systematical review of ular portraits’ provided by the quantitative analysis of gene the biology of each of these proteins, including their specific expression patterns (1) have helped define different intrinsic subcellular/extracellular or multiple localizations is presented. subtypes while multiparameter molecular tests such as the Finally, as an important advantage of quantitative proteomics PAM-50 provide useful prognostic information (2). However, is the ability to provide data on all these molecules simultane- despite the usefulness of mRNA‑based classifications, inte- ously in cell pellets, body fluids or paraffin‑embedded sections grated proteogenomic analyses of breast or colon tumors of tumors/invaded tissues, the significance of some of their have revealed that protein abundance cannot be reliably interconnections is discussed. predicted from DNA- or RNA-level measurements as they are only modestly correlated. In particular, proteomics identify colorectal cancer subtypes similar to those detectable by tran- Contents scriptome profiles, but also reveal features not detectable in transcript profiles, probably as a result of post‑transcriptional 1. Introduction regulation (3). Further evidence of this unique potential of 2. Data collection methods proteomics-based subtyping was recently provided in the study 3. Extracellular matrix (ECM) by Johansson et al who identified protein products mapping to non-coding genomic regions, potentially leading to a new class of tumor‑specific immunotherapeutic targets (4). High-throughput proteomics is still an underdeveloped field compared to transcriptomics (4) and its contribution to Correspondence to: Dr Daniel L. Pouliquen, Paul Papin ICO Cancer Center, CRCINA, Inserm UMR 1232, 15 Rue André Boquel, oncology has probably not yet been fully realized. However, 49055 Angers, France in two decades, quantitative proteomics has rapidly evolved E-mail: [email protected] both technologically (5) and strategically, allowing researchers to explore the complexity of protein interaction networks in a Key words: proteomics biomarkers, tumor invasiveness, extracellular wide variety of situations, but also to formulate new hypoth- matrix remodeling, plasma proteins, membrane proteins, cytoplasmic eses to be further functionally tested (6). To identify tumor enzymes, cytoskeletal proteins, endoplasmic reticulum, mitochondrial biomarkers to assist in individualizing treatments for certain proteins, nuclear proteins types of cancer, many complementary technologies have also been developed (7). As an example, for the majority of the common causes of cancer-related mortality worldwide, such as 410 POULIQUEN et al: BIOMARKERS OF TUMOR INVASIVENESS IN PROTEOMICS lung cancer, these technologies have led to the identification of mesotheliomas (MMs) differing by their invasiveness (21), predictive biomarkers of drug resistance, candidate biomarkers and then with clinical data from cohorts of patients with for diagnosis and prognostic biomarkers (8). To deal with the colon adenocarcinoma (22) or breast cancer (23). As a result limited success of ‘targeted therapies’, quantitative proteomics, of technological improvements that occurred between these together with other major technological and conceptual devel- two studies, the number of specific biomarkers identified in opments, has reinforced the search for characteristic features breast cancer was higher than that in colon cancer. Although of the adhesive-migratory phenotype of malignant cells (9). our team is primarily focused on breast cancer, the authors Some other examples of important contributions include the wished to extend the present review to other types of malignant study of RNA-protein complexes (10), stem cell plasticity (11), tumors originating in two different tissues in order to validate chromatin remodeling (12) and more recently, the regulation of the most robust and generalizable biomarkers. mitochondrial function and dynamics (13), shed microvesicles This led to a final list of 76 upregulated or downregulated biology (14) or mechanisms of radioresistance (15). proteins common to the three sources of data, which represent The advent of data-independent acquisition methods potential tumor invasive biomarkers. The present review is (DIA) (16), such as sequential window acquisition of all based on i) articles selected from the procedure illustrated in theoretical mass spectra (SWATH-MS) that combines deep Fig. 1; and ii) the screening of literature for each individual proteome coverage capabilities with quantitative consistency protein listed in Table I with the following keywords ʻname and accuracy (17), has further extended this potential. In of the proteinʼ and ʻcancerʼ, without or with ʻinvasivenessʼ. particular, SWATH-MS does not rely on precursor intensity Finally, some articles combining two or more of these protein for fragmentation selection, but rather analyzes all precursors names with these keywords were also analyzed. in specified mass ranges (18). This overcomes the limitations of previous data-dependent acquisition (DDA) methods in 3. Extracellular matrix (ECM) mass spectrometry related to the large dynamic concentration range observed in body fluids, and the fact that less abundant A number of the proteins in the list of potential biomarkers proteins were usually masked by highly abundant proteins. of invasiveness are localized in the ECM, either specifically With regard to relative quantification, strong correlations or in parallel to other intracellular localizations, which are have been found between protein abundances and functional summarized in Fig. 2. activities of a set of drug metabolizing enzymes in human liver microsomes (19). Additionally, although mass spectrometry S100 proteins. Communications between stromal cells and is not commonly used in clinical laboratories, the validation cancer cells represent a key parameter of invasiveness. The of cancer biomarkers found by this technology compared S100 family of proteins in particular is involved in the forma- with western blot analysis or ELISA has already been estab- tion and maintenance of a pro‑inflammatory environment. lished (20). Among the 21 members of this family present in humans, all It was recently demonstrated that proteomes and transcrip- regulated by Ca2+ binding, 11 are described in the literature in tomes were better associated in highly proliferative tumors relation to the subject of the present review (S100A4, S100A6, than in lowly proliferative tumors (4). Herein, data on cancer S100A7, S100A8, S100A9, S100A10, S100A11, S100A13, invasiveness are reviewed with the aim of highlighting key S100A14, S100A16 and S100P). S100A4 and S100A6 are the findings within the huge amount of information available in most extensively documented; however, S100A8, which forms the literature. The present review focuses in particular on heterodimers with S100A9 [the overexpression of which is very a list of 76 proteins of interest, selected after crossing with useful for the identification of circulating tumor cells (24)], is SWATH-MS data collected by our team on experimental also involved in the regulation of inflammatory responses and models and human tumor samples. pre-metastatic niche formation, together with S100A4 (25,26). The heterodimeric protein, named calprotectin, is released 2. Data collection methods by activated granulocytes, and functions in a cytokine-like manner by triggering signaling pathways involved in inflam- The procedure used to identify the list of proteins of interest matory processes. A recent review revealed the complex