The Function And Mechallism Of OPN Transcripts On Breast Cancer Progression

Breast cancer represents the most common cancer in women with1.38million new cases and458000deaths yearly worldwide and becomes a major issue of public health. Due to the lack of nationwide screening program and targets, breast cancers are often found at more advanced stages. Furthermore, the majority of estrogen receptor-positive (ER+) breast cancer patients develop resistance to adjuvant hormonal therapy, and triple negative breast cancers (TNBCs) also lack effective targeted treatment. Therefore, novel therapeutic targets are urgently needed to improve the efficacy of conventional treatments.Osteopontin (OPN), a multifunctional phosphorylated glycoprotein, has important roles in many pathophysiologic process including bone remodeling, cancer and inflammation. Various types of cancers express high levels of OPN and overexpression of OPN into a previously benign cell line is sufficient to produce a metastatic phenotype. In turn, knock-down OPN with siRNA can attenuate the process of cancer in vitro and in vivo. In contrast to tumor per se, immune cells are another main source of OPN. It has been reported that immune cell-derived OPN is crucial for the induction of cell-mediated immune responses and thus contributes to the host anti-tumor defense. Therefore, it has been suggested that OPN may have contrary roles in host defense and tumor process. Furthermore, macrophage-derived OPN can restore the metastatic potential of OPN-knockdown tumor cells which indicates different sources of OPN may interact with each other. We therefore hypothesize that tumor-derived OPN may participate in tumor immune evasion through regulating the differentiation and function of immune cells.Alternative RNA splicing of human OPN engenders three transcripts:OPN-a (full type), OPN-b (with deletion of exon5) and OPN-c (with deletion of exon4). Different from OPN-a and OPN-b, OPN-c is specifically expressed in breast tumors and more effectively supports anchorage independence growth in vitro. Clinical data suggest that OPN-c may be a more valuable diagnostic and prognostic marker than conventional breast cancer markers estrogen receptor, progesterone receptor and HER2. In non-small-cell lung cancer, OPN transcripts differently regulate vascular endothelial growth factor (VEGF) secretion and angiogenesis. Recently, Tilli TM and colleagues demonstrated that the isoform OPNc specifically expressed in ovarian tumor samples and significantly activated OvCar-3cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. All these results prove that different OPN transcripts have distinct roles which may be associated with its distinct roles in host defense and tumor process. But whether OPN transcripts have different roles in breast cancer formation in vivo and whether tumor-derived OPN transcripts have distinct function on immune cells differentiation are totally unknown.In this project, we investigated the function of different OPN transcripts on breast cancer formation in vivo. Furthermore, we detected the roles of tumor derived OPN transcripts on monocytes activation and NK cell mediated cytotoxicity. which may represent another essential mechanism for immune evasion mediated by OPN. Part I All OPN transcripts promoted breast tumor growth in vivoOBJECTIVE:To construct lentiviral Opn transcript vectors and to investigate the potential roles of different OPN transcripts on tumor formation in vivo.METHODS:The constructs for expression of the human OPN splice variants were obtained by reverse transcription-PCR from the malignant breast tumor cell line MDA-MB-435. The amplified products were excised with Age I, and was subcloned into the vector pGC-FU Vector. Sequence fidelity and accurate reading frames were verified by DNA sequencing analysis. Lentiviral stocks were generated by cotransfection of constructed pGC-FU Vector with pHelper1.0and Helper2.0packaging constructs to293T cells using LipofectAmine2000. Viral supernatants were collected48h later. MCF-7cells were transfected with lentiviral expression vectors containing Opn (MOI:100) in the presence of5μg/ml polybrene. By detection GFP positive rate of pGC group we confirmed the transfection efficiency. We verified the successful overexpression of3OPN transcripts in mRNA level By by RT-PCR. We further confirmed the protein level in cell lysate and supernatant by western blotting and ELISA.5×106MCF-7transfected with OPN transcripts or pGC control were injected into armpit of nude mice (n=4/group). Animals were kept in pathogen free conditions and monitored by Kodak2000MM for tumor formation and metastasis by detecting the GFP expression. After eight weeks, mice were sacrificed by cervical dislocation. Primary tumors were dissected out and photograph. One part of the tumors were fixed in10%formalin solution and used for histopathology. Lung and liver were also dissected for metastasis analysis.RESULTS:PCR and sequencing results identified the positive clones of OPN overexpression vector. By detecting GFP and Western Blot, we further confirmed the successful expression of OPN in293T cell. Real-time PCR showed that the virus titer we packed up to109TU/ml. Overexpression of OPN transcripts in MCF-7was confirmed by RT-PCR and Western Blot. The secretion level of OPN in the supernatant was detected by ELISA, which further verified the successful expression. All OPN transcripts promoted local tumor formation, but there was no significant difference among transcripts. We did not detect tumor metastasis in liver and lung.CONCLUSIONS:Lentiviral Opn transcript vectors were successfully constructed. OPN promoted breast cancer local growth, but there was no distinct difference among OPN transcripts. Part II Tumor-derived OPN transcripts induced alternative activation of monocytesOBJECTIVE:To investigate the effect and mechanism of tumor-derived OPN transcripts on monocytes activation and to elucidate the potential mechanism for tumor immune escape.METHODS:MCF-7cells transfected with different OPN transcripts and pGC control lentivirus were cultured with RPMI-1640medium at1×105cells/ml for24h, and then collected cell debris free supernatant by centrifugation at10000g for5min. CD14+cells were isolated by using microbeads from PBMCs. The purity of CD14+monocytes was determined by flow cytometry. The isolated monocyteswere coculture with corresponding supernatant from tumor cells for40h, HLA-DR, CD163, CD206levels on cell surface were determined by FACS. The ability of tumor supernatant-treated monocytes to respond to LPS was assayed by detecting TNF-a and IL-10level after stimulation with LPS for6h. MCF-7transfected with different OPN transcripts and pGC lentivirus or stimulated with rhOPN were cultured with RPMI-1640medium for48h, the supernatant was collected by centrifugation, the levels of cytokines were assayed by Bio-plex and ELISA. The surface expression of CD44and αvβ3on MCF-7cells were detected by FACS. The supernatants were pretreated with neutralizing antibody or recombinant cytokines to detect the potential roles of MCP-1and TGF-β1on monocyte activation.RESULTS:Coculture with supernatant overexpressed all3OPN transcripts did not regulate HLA-DR and CD206levels of monocytes. Interestingly, supernatant overexpression OPN-c significantly upregulated CD163level compared with OPN-a and OPN-b. Compared with groups of control or pGC, overexpression of OPN differentially inhibited monocytes TNF-a, but increased IL-10level. However, there was no difference among OPN transcripts on the regulation of TNF-a and IL-10. And overexpression of OPN have no significant effects on the levels of IL-8, IL-12and IL-6. Overexpression of OPN transcripts did not change IL-6, IL-8and G-CSF level, but significantly enhanced TGF-β1and MCP-1levels. OPN transcripts had no difference in TGF-β1and MCP-1regulation. We further confirmed the regulatory effect of OPN on TGF-β1and MCP-1expression by using rhOPN. Furthermore, we found MCF-7expressed high level CD44but undetectable αvβ3, and blocking CD44substantially reduced the upregulation of MCP-1by rhOPN, confirming a partial role of CD44signal pathway in the cytokines expression. By using neutralizing antibody and recombinant cytokines we showed that tumor-derived OPN regulated monocytes TNF-a and IL-10partly via MCP-1and TGF-β1, respectively.CONCLUSIONS:Our results demonstrated that tumor-derived OPN regulated MCP-1and TGF-β1via CD44and induced alternative activation of monocytes partly via TGF-β1and MCP-1, which may represent additional mechanism for tumor immune escape. PartⅢ OPN protected breast tumor from NK cytotoxicity via affecting NKG2D-MICA pathwayOBJECTIVE:To investigate the effect of OPN on NKG2D-MICA pathway and NK cytotoxicity toward beast tumor and enrich the potential mechanism for tumor immune escape.METHODS:rhOPN stimulated MCF-7for24h, RT-PCR detected MICA mRNA level, FACS detected MICA surface level and the soluble MICA in the supernatant was determined by ELISA. MICA-shedding MMPs and ADAM were determined by RT-PCR. The specific inhibitors of protease were used to confirm their potential role in MICA shedding. Coculture rhOPN stimulated MCF-7with NK92cells for24h, FACS detected Annexin V level to determine the cytotoxicity of NK cells. MCF-7was transfected with lentiviral Opn transcript vectors and detected the role of different OPN transcripts on MICA shedding and NK cytotoxicity.RESULTS:rhOPN promoted MICA shedding and inhibited NK cytotoxicity toward MCF-7. RT-PCR showed that rhOPN significantly promoted ADAM-17expression, but had no effects on MMP-2, MMP-9and ADAM-10. Blocking ADAM-17significantly inhibited MICA shedding and partly restored NK92cytotoxicity toward MCF-7. All OPN transcripts promoted MICA shedding and inhibited NK92cytotoxicity, but there was no distinct difference among OPN transcripts.CONCLUSIONS:OPN protected breast cancer cell from NK cytotoxicity through promoting MICA shedding mediated partly by ADAM-17, which may represent another mechanism for OPN in tumor progression.