NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer.

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), l-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p = .02, OS: p = .04). CYB5R3 gene knock-down using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFß and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.

Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells.

Recent studies have shown that Abs that target the cell-surface enzyme CD73 (ecto-5'-nucleotidase) reduce growth of primary tumors and metastasis in syngenic mice by inhibiting the catalytic activity of CD73, and thus increasing the activity of cytotoxic T lymphocytes. In this article, we report another anticancer mechanism of anti-CD73 Abs and show that an anti-CD73 mAb (AD2) inhibits metastasis formation by a mechanism independent of CD73 catalytic activity and inhibition of primary tumor growth. This mechanism involves clustering and internalization of CD73, but does not require cross-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab' fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73 internalization and metastasis inhibition. Furthermore, anti-CD73 AD2 mAb inhibited development of metastasis in a spontaneous animal model of human metastatic breast cancer. Our study shows that some anti-CD73 mAbs cause cell-surface clustering of CD73 followed by internalization, thus inhibiting the ability of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.

Plasma membrane proteomics and its application in clinical cancer biomarker discovery.

Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions is altered on cancer cells, and these proteins may also be subject to post-translational modification, such as altered phosphorylation and glycosylation. Additional protein alterations on cancer cells confer metastatic capacities, and some of these cell surface proteins have already been successfully targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal of this research is to improve individualized treatment and increase cancer patient survival, further development is crucial to direct analysis of clinically relevant patient samples. These efforts include optimized specimen handling and preparation as well as improved proteomics platforms. Identification of potentially useful proteomics-based biomarkers must be validated in larger, well defined retrospective and prospective clinical studies, and these combined efforts should result in identification of biomarkers that will greatly improve early detection, prognosis, and prediction of treatment response.

A novel approach to evaluate ELISA antibody coverage of host cell proteins-combining ELISA-based immunocapture and mass spectrometry.

Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.

miR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model, inhibits cancer cell extravasation and colonization in vivo and causes significant signaling alterations.

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered expression between isogenic metastasizing and non-metastasizing cancer cells, with miR-155 being the most differentially expressed. Highly metastatic mesenchymal-like CL16 cancer cells showed very low miR-155 expression, and miR-155 overexpression in these cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. Our experiments addressing the underlying mechanism of the altered tumor burden revealed that miR-155-overexpressing CL16 cells were less invasive than CL16 control cells in vitro, while miR-155 overexpression had no effect on cancer cell proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis-associated proteins ALDH1A1, PIR and PDCD4 in miR-155-overexpressing tumors was validated by immunohistochemistry. Our results demonstrate that miR-155 inhibits the ability of cancer cells to extravasate and/or colonize at distant organs and brings additional insight into the complexity of miR-155 regulation in metastatic seeding.

Identification of markers associated with highly aggressive metastatic phenotypes using quantitative comparative proteomics.

BACKGROUND: The spread of cancer cells from a primary tumor to form metastases at distant sites is a complex process that remains poorly defined. Certain tumor cells are more aggressive and thus lead to rapid development of multiple distant metastases. Here, we identify proteins associated with these aggressive phenotypes. MATERIALS AND METHODS: To identify proteins associated with cancer cell aggressiveness, we used comparative, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteome analysis of a unique metastasis model comprised of three isogenic human breast cancer cell lines that are equally tumorigenic in mice, but display different metastatic potentials ranging from non-metastatic, intermediate-metastatic and highly-metastatic. The altered expression of selected proteins was subsequently confirmed by immunocyto- and immunohistochemistry. RESULTS: The difference in metastatic capabilities was initially confirmed using live animal imaging. Comparative, quantitative proteomics identified 414 proteins, out of which 44 exhibited altered expression between the metastatic and non-metastatic cell lines. The proteins correlating with the aggressiveness of metastasis included leucine-rich repeat containing 59 (LRRC59), while CD59 and chondroitin sulfate proteoglycan 4 (CSPG4) exhibited an inverse correlation with metastatic capability. The altered expression levels of these proteins were biochemically confirmed, as well as demonstrated in xenografts generated from these cell lines. This analysis further demonstrated that the three proteins were associated with the aggressiveness of metastasis rather than metastasis colonization per se. CONCLUSION: Our study provides novel insights into key proteins associated with the metastatic potential of breast cancer cells and identified LRRC59, CD59 and CSPG4 as candidates that merit further study.