Fuction And Mechanism Analysis Of Neutrophil Specific Antigen CD177

CD 177 gene encodes two isoforms NB1 and PRV-1, with four amino acids difference at their c-terminal ends. Here we collectively refer to as CD 177. CD 177 is glycosylphosphatidylinositol (GPI)-linked N-glycosylated cell surface glycoprotein Nearly all literatures related to CD 177 (160 papers in Pubmed) have been under the context of human neutrophils or myeloproliferative diseases, with one exception regarding colon cancer where loss of CD 177 together with expression of cytokeratin 20 (CK20) is an important prognostic factor. Our initial transcriptome analysis implicates a potential impact of TICs on tumor behavior, including up regulation of pathways related to epithelial-mesenchymal transition, cancer metastasis, and basal type of breast cancer in basal-TIC formed tumors.Among all the genes that are differentially expressed comparing basal and luminal TIC-formed tumors, we performed a complex bioinformatics analysis and identified the loss of CD 177 expression in basal TIC-tumors. In addition, loss of CD 177 expression correlates with shorter metastasis-free survival.Using data deposited in ONCOMINE, we identified loss of CD 177 in other cancer types as well, including cervical cancer, prostate cancer, colorectal cancer, and leukemia, indicating that CD 177 might be a potential tumor-and metastasis-suppressor. Here we will use different approaches to examine the role of CD 177 in different stages of breast cancer progression.We already analyzed two published Affymetrix datasets and found a correlation between CD 177 expression and metastasis-free survival.We had initial success to stain CD 177 on invasive breast carcinoma using a mouse monoclonal antibody (clone 4C4 from Sigma-Aldrich).We did observe stronger staining on apical surface of normal ducts, the predicted location where GPI-linked proteins are localized; the staining is relative weaker in the adjacent cancer cells.To examine the role of CD 177 in breast cancer, we first used established breast cancer cell lines to screen the expression of CD 177 and found that more than 80% cancer cell lines (9/11) exhibited loss of CD177 expression compared to relative normal cells HMLE,We silenced CD 177 expression using small hairpin (sh) RNA in high CD177-expressing cells; or overexpressed ectopic CD177 in low CD177-expressing cell lines. We found that overexpression of CD 177 inhibited in vitro colony growth in soft agar and silencing CD 177 promoted it, indicating a suppressive role of CD 177 in tumor growth.Using a syngeneic transplant model in Balb/C genetic background, we silenced CD 177 expression in 4T07, a mouse breast cancer cells known not to colonize in the lung.and found that loss of CD 177 significantly increased pμLmonary metastasis. To explore the role of CD 177 in different stages of tumor development under a more physiological condition, we established a novel genetic knockout model of CD 177. The CD177-/- mice are globally normal but female mice exhibit abnormal mammary gland development when examined at 7 weeks of age. The ductal tree of CD177-/- mammary gland penetrates into the mammary fat pad faster than the ductal trees from wild type (WT) or CD177-/+/- littermates, We also observed an increased basal/luminal ratio in CD17-/- mice relative to their WT littermates, indicating that CD 177 plays a role in suppressing basal cell expansion. Since we found that basal cells are the major TIC for ErbB2-induced tumorigenesis, we are crossing MMTV-ErbB2 transgenic (Tg) with CD1777’to generate ErbB2/WT, ErbB2/CD177+/-, or ErbB2/CD177-/- female mice.Since there is nothing known about the role of CD177 in cancer, we used an unbiased biochemical approach to identify interacting proteins of CD177 in breast cancer cells. We overexpressed Myc-tagged CD 177 in SKBR3, an ErbB2-positive human breast cancer cell line and confirmed its GPI-linked and membrane-targeting property by using phospse C (PLC) treatment of intact cells (PLC can release protein from GPI anchor). Using the anti-Myc antibody and mlgG1 as an isotype control, we immunoprecipitated Myc-CD177 and subjected the immunocomplex to Mass spectrometry. Among all the proteins being p μLled down, we found 4 core enzymes including phosphofructosekinase 1 (PFK1), aldolase A, glyceraldehyde phosphate dehydrogenase (GAPDH), and enolase. We confirmed the interaction between CD 177 and some enzymes using co-immunoprecipitation followed by SDS-PAGE and found that the interaction is localized to the non-lipid raft membrane (not shown). We also found a significant loss of glucose uptake in CD177-overexpressing SKBR3 cells under normoxic condition (21% oxygen); the difference is even more pronounced when cells were exposed to hypoxia (2% oxygen), indicating a reduced glycolysis rate in CD177-overexpressing cells. It has been known for decades that cancer cells use aerobic glycolysis for their growth and progression, a phenotype observed by Otto Warburg in 1924 resulting in his Nobel Prize in 1931. We hypothesize that CD 177 regulates glycolysis by sequestrating the core enzyme complex to plasma membrane, thus limiting the consumption of glucose by cancer cells.