Mass Spectrometry‐Based Proteomics of Human Breast Milk to Assess Breast Cancer Risk

Objective Breast cancer (BC) as the second common cancer and the second leading cause of cancer death among American women, is one of the major research concerns in the US. One of the potential options for detection of BC is biochemical monitoring of protein markers in different types of body fluids. Breast milk provides access to the breast tissue in the form of exfoliated epithelial cells that are the origins of the most types of BCs; meanwhile, Analysis of breast milk is a noninvasive method for cancer diagnosis. Here we discuss the potential application of mass spectrometry (MS) based proteomics in investigation of human breast milk as a mean for building a biomarker signature for early detection of BC and for monitoring its treatment. Using human breast milk samples from both healthy controls and BC suffering individuals, several alterations in protein expression are identified such as lower expression, overexpression, or post translational modification of some proteins which might be associated with BC risk. Methods The outline for the breast milk proteomics experiment is shown in Figure 1 of the attached file. We performed different analytical techniques of protein digestion of the human breast milk samples, such as in‐gel digestions (1D and 2D‐PAGE; Figures 2 and 3) and in‐solution digestion ( Figure 4) coupled with nanoliquid chromatography tandem mass spectrometry (nanoLC‐MS/MS). To further investigate of differences in protein pattern, the milk samples with the same genotypes were compared. Specifically, we analyzed the breast milk from the breast with BC and from the healthy breast from the same donor. In order to do prediction and risk assessment of BC, we also analyzed some breast milk samples that were donated before being diagnosed with BC by biopsy test and breast milk from healthy controls. Preliminary Results There were several up‐regulated and down‐regulated proteins in BC samples. For instance, some of these proteins are shown in Table 1 of the attached file. The literature review for the possible relationship between these proteins and cancer is also shown in Table 1. In a different set of experiments, we analyzed the individual different protein bands (instead of the whole proteome of one milk sample). These individual differences were visible on the SDS‐PAGE, Some of the proteins that were identified from BC samples are given in Table 2. Currently we are doing different types of analytical experiments such as in solution digestion and 2D gel electrophoresis to complement the results. We also plan to investigate the protein post translational modifications (PTMs) such as phosphorylation and glycosylation that might provide us additional insights on the onset and evolution of BC. Analysis of small molecules and metabolites in the milk samples will also be completed soon. Also, the possible availability of serum and milk samples from the same donors can lead to the comparison of local and systemic diagnosis. Conclusion There were several protein alterations identified in BC samples, some of which were discussed here as examples. The relationship between these proteins and BC needs further investigation and strong validation by different analytical techniques. These alterations could result in a biomarker signature associated to BC, thus giving us information about possible risk or the existence of BC and BC severity. In addition, the biomarker discovery can also be applied in the assessment of responses to the BC treatment and monitoring the progression of disease. 1 Examples of dysregulated proteins in BC samples versus controls, identified in our experiments and their relationship with the current literatureUp‐regulated protein Cancer related overexpression based on Literature review FunctionPlasminogen “Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins.” Ranson et al. Involved in tissue reorganization: mammary gland involution and wound healing Tumors can steal the system to do metastasisZinc finger helicase Expression happens in tumors including breast (mammary gland) “Genes‐to‐Systems Breast Cancer Database.” tumorCould be a global transcriptional regulator. Modifies gene expression by affecting chromatin.Hepatocyte nuclear factor 3, beta “ The hepatocyte nuclear factor 3 α gene , HNF3α (FOXA1), on chromosome band 14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas. ” Lin et al. A transcription factor known to play a role in airway differentiation Endoderm related developmental factorsZinc finger protein 169 Genes‐to‐”Expression happens in tumors including ovarian tumor ” Systems Breast Cancer Database. May be involved in transcriptional regulationE3 ubiquitin protein ligase “ E3 ubiquitin ligases as cancer targets and biomarkers.” Regulates diverse areas such as cell trafficking, DNA repair, and signaling.IgG heavy chain variable region “ Immunoglobulin G heavy‐chain allotypes as possible genetic markers for human cancer.” Nakao et al. One of the components of humoral immunityMyeloperoxidase isoform “Association of myeloperoxidase with ovarian cancer. ” Castillo T. et al. An oxidant generating enzyme, catalyzes reactions that produce HOCI that damages DNAType IV collagenase “ Collagenases in human breast carcinoma cell line. “Kao, Richard T., and Robert Stern. extracellular matrix remodeling and angiogenesis chromatin.Ceruloplasmin (ferroxidase) isoform “ Ceruloplasmin gene expression in human cancer cells . “Kunapuli, Satya P., et al. Involved in angiogenesis and neovascularizationMyosin‐9 “Elevated expression of myosin X in tumors contributes to breast cancer aggressiveness and metastasis “Cao, R., et al. Play a role in cytokinesis: division of cytoplasm to form two daughter cellsDown‐ regulated protein Cancer related low expression based on Literature review Functionbeta‐casein precursor “Lack of β‐Casein production by human breast tumours revealed by monoclonal antibodies. ”Bártková, J., et al. provides the source of aminoacids for growthalpha s1 casein “Antioxidant properties of casein‐phosphopeptides.” Kitts, D. *antioxidant activity has been reported, not down‐regulation yet. Important role in the capacity of milk to transport calcium phosphate Antioxidant activity.lg alpha‐2 chain C region Low levels occur in some types of leukemia based on International Waldenstrom's Macroglobulinemia Foundation. defend against local infection prevent access of foreign antigens to the general Immunologic system.Ig alpha1 Bur Low levels occur in some types of leukemia based on International Waldenstrom's Macroglobulinemia Foundation. defend against local infection prevent access of foreign antigens to the general immunologic system.2 Examples of highly expressed identified proteins in BC samples in individual bands and their relationship with the current literatureProtein Type of cancer related alteration based on literatureCholesterol esterase BC, based on the Genes‐to‐systems breast cancer database CEL (carboxyl ester lipase) BC, based on the Genes‐to‐systems breast cancer database Xanthine dehydrogenase/oxidase BC, Wright, R.M. et al., 1999. Tenascin Different types of cancers SNC66 Alteration of SNC‐γ in BC, Jia, T., et al., 1999 Carbonic anhydrase isozyme VI Different tumors, Nógrádi, A., et al. 1998 Annexin A1 BC, Wang. L., et al., 2009. Lactadherin BC, TAYLOR, M.R., et al., Azurocidin BC, based on the human protein ATLAS HP protein Breast, colorectal and lung cancer, Dowling, P., et al., 2012 HMFG BC, Larocca, D., et al., 1991. Annexin A3 BC. Zeng, C., et al., 2013 Cathepsin G Tumor metastasis, Tan, G.‐J., et al., 2013 Neuropilin‐1 BC, Bachelder, R.E., et al., 2001. Plasminogen BC, Ranson, M., et al., 1998 Zn‐alpha2‐glycoprotein BC, Dubois, V., et al., 2010 Human Neutrophil peptide 1 and 2 BC, Li, D., et al., 2014 Epididymis luminal protein 214 Ovarian Carcinomas, Drapkin, R., et al., 2005 Mucin 4 BC, Workman, H.C., et al., 2009