Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion.

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

RAGE/SNAIL1 signaling drives epithelial-mesenchymal plasticity in metastatic triple-negative breast cancer.

Epithelial/Mesenchymal (E/M) plasticity plays a fundamental role both in embryogenesis and during tumorigenesis. The receptor for advanced glycation end products (RAGE) is a driver of cell plasticity in fibrotic diseases; however, its role and molecular mechanism in triple-negative breast cancer (TNBC) remains unclear. Here, we demonstrate that RAGE signaling maintains the mesenchymal phenotype of aggressive TNBC cells by enforcing the expression of SNAIL1. Besides, we uncover a crosstalk mechanism between the TGF-ß and RAGE pathways that is required for the acquisition of mesenchymal traits in TNBC cells. Consistently, RAGE inhibition elicits epithelial features that block migration and invasion capacities. Next, since RAGE is a sensor of the tumor microenvironment, we modeled acute acidosis in TNBC cells and showed it promotes enhanced production of RAGE ligands and the activation of RAGE-dependent invasive properties. Furthermore, acute acidosis increases SNAIL1 levels and tumor cell invasion in a RAGE-dependent manner. Finally, we demonstrate that in vivo inhibition of RAGE reduces metastasis incidence and expands survival, consistent with molecular effects that support the relevance of RAGE signaling in E/M plasticity. These results uncover new molecular insights on the regulation of E/M phenotypes in cancer metastasis and provide rationale for pharmacological intervention of this signaling axis.

Circulating SOD2 Is a Candidate Response Biomarker for Neoadjuvant Therapy in Breast Cancer.

There is a great need for non-invasive tools that inform of an early molecular response to cancer therapeutic treatment. Here, we tested the hypothesis that proteolytically resistant proteins could be candidate circulating tumor biomarkers for cancer therapy. Proteins resistant to proteolysis are drastically under-sampled by current proteomic workflows. These proteins could be reliable sensors for the response to therapy since they are likely to stay longer in circulation. We selected manganese superoxide dismutase (SOD2), a mitochondrial redox enzyme, from a screening of proteolytic resistant proteins in breast cancer (BC). First, we confirmed the robustness of SOD2 and determined that its proteolytic resistance is mediated by its quaternary protein structure. We also proved that the release of SOD2 upon chemotherapy treatment correlates with cell death in BC cells. Then, after confirming that SOD2 is very stable in human serum, we sought to measure its circulating levels in a cohort of BC patients undergoing neoadjuvant therapy. The results showed that circulating levels of SOD2 increased when patients responded to the treatment according to the tumor shrinkage during neoadjuvant chemotherapy. Therefore, the measurement of SOD2 levels in plasma could improve the non-invasive monitoring of the therapeutic treatment in breast cancer patients. The identification of circulating biomarkers linked to the tumor cell death induced by treatment could be useful for monitoring the action of the large number of cancer drugs currently used in clinics. We envision that our approach could help uncover candidate tumor biomarkers to measure a tumor's response to cancer therapy in real time by sampling the tumor throughout the course of treatment.

HMGA1, Moonlighting Protein Function, and Cellular Real Estate: Location, Location, Location!

The gene encoding the High Mobility Group A1 (HMGA1) chromatin remodeling protein is upregulated in diverse cancers where high levels portend adverse clinical outcomes. Until recently, HMGA1 was assumed to be a nuclear protein exerting its role in cancer by transcriptionally modulating gene expression and downstream signaling pathways. However, the discovery of an extracellular HMGA1-RAGE autocrine loop in invasive triple-negative breast cancer (TNBC) cell lines implicates HMGA1 as a "moonlighting protein" with different functions depending upon cellular location. Here, we review the role of HMGA1, not only as a chromatin regulator in cancer and stem cells, but also as a potential secreted factor that drives tumor progression. Prior work found that HMGA1 is secreted from TNBC cell lines where it signals through the receptor for advanced glycation end products (RAGE) to foster phenotypes involved in tumor invasion and metastatic progression. Studies in primary TNBC tumors also suggest that HMGA1 secretion associates with distant metastasis in TNBC. Given the therapeutic potential to target extracellular proteins, further work to confirm this role in other contexts is warranted. Indeed, crosstalk between nuclear and secreted HMGA1 could change our understanding of tumor development and reveal novel therapeutic opportunities relevant to diverse human cancers overexpressing HMGA1.

Extracellular HMGA1 Promotes Tumor Invasion and Metastasis in Triple-Negative Breast Cancer.

PURPOSE: The study of the cancer secretome suggests that a fraction of the intracellular proteome could play unanticipated roles in the extracellular space during tumorigenesis. A project aimed at investigating the invasive secretome led us to study the alternative extracellular function of the nuclear protein high mobility group A1 (HMGA1) in breast cancer invasion and metastasis. EXPERIMENTAL DESIGN: Antibodies against HMGA1 were tested in signaling, adhesion, migration, invasion, and metastasis assays using breast cancer cell lines and xenograft models. Fluorescence microscopy was used to determine the subcellular localization of HMGA1 in cell lines, xenograft, and patient-derived xenograft models. A cohort of triple-negative breast cancer (TNBC) patients was used to study the correlation between subcellular localization of HMGA1 and the incidence of metastasis. RESULTS: Our data show that treatment of invasive cells with HMGA1-blocking antibodies in the extracellular space impairs their migration and invasion abilities. We also prove that extracellular HMGA1 (eHMGA1) becomes a ligand for the Advanced glycosylation end product-specific receptor (RAGE), inducing pERK signaling and increasing migration and invasion. Using the cytoplasmic localization of HMGA1 as a surrogate marker of secretion, we showed that eHMGA1 correlates with the incidence of metastasis in a cohort of TNBC patients. Furthermore, we show that HMGA1 is enriched in the cytoplasm of tumor cells at the invasive front of primary tumors and in metastatic lesions in xenograft models. CONCLUSIONS: Our results strongly suggest that eHMGA1 could become a novel drug target in metastatic TNBC and a biomarker predicting the onset of distant metastasis.