| Breast cancer is one of the most common malignancies in females and is the second leading cause of cancer-related mortality in developed countries. Early detection of breast cancer by mammography has been improved recently but the morbidity and mortality continue increasing in most of the countries. Thus, there is a pressing requirement to screen and identify novel biomarkers to diagnose breast cancer at early stage and predict tumor recurrence and metastasis and to develop more novel treatment strategies to effectively control aggressive breast cancer.Cancerous inhibitor of protein phosphatase 2A(PP2A)(CIP2A), also named KIAA1524 or p90 tumor-associated antigen, is a novel oncoprotein that has been known to inhibit PP2 A phosphatase activity via stabilizing the oncogenic c-Myc in human malignancies. Down-regulation of p90/CIP2 A in cancer cells could reduce cell proliferation, and tumor formation in vivo. Although p90/CIP2 A was overexpressed in many different types of cancers and the overexpression is associated with poor prognosis, few studies has revealed the functional roles of p90/CIP2 A in cancer progression.Firstly, in order to evaluate whether autoantibody to a tumor-associated antigen p90/CIP2 A can be used as an immunodiagnostic marker in breast cancer, we examined the prevalence of anti-p90/CIP2 A autoantibody in 168 breast cancer sera and 88 normal human sera by enzyme-linked immunosorbent assay(ELISA). And the localization of p90/CIP2 A in HEp-2 cell was determined by indirect immunofluorescence assay(IIF). Besides, the expression level of p90/CIP2 A in 46 breast cancer tissue specimens and 46 adjacent normal human breast tissues was evaluated by immunohistochemistry(IHC). In addition, we aim to investigate the resulted biological function of p90/CIP2 A in breast cancer. Lentivirus-mediated sh RNA p90/CIP2 A knockdown was performed in two triple-negative breast cancer cell lines(MDA-MB-231 and LM2-4). Cell proliferation assay, colony formation assay and flow cytometry were carried out to evaluate the role of p90/CIP2 A in cell proliferation and apoptosis in vitro. At last, the expression of c-Myc protein and ERK1/2 phosphorylation was examined by Western blotting.Objectives 1 To evaluate whether autoantibody to a tumor-associated antigen p90/CIP2 A can be used as an immunodiagnostic marker in breast cancer. 2 To investigate the biological function of p90/CIP2 A in breast cancer cells.Materials and methods1 Subjects 1.1 Sera and tissues samples: In the current study, total 168 sera from patients with breast cancer and 88 sera from normal individuals were derived from the sera bank in the Cancer Autoimmunity Research Laboratory at University of Texas, El Paso(UTEP). These sera were originally provided by our clinical collaborators. All serum samples were obtained under consented individuals. All breast cancer sera were collected at the initial time of pathological diagnosis, prior to patients being treated with chemotherapy or radiotherapy. Normal human sera were assembled during annual health examinations from adults with no obvious evidence of malignancy. This study was approved by the Institutional Review Board of UTEP and Collaborating Institutions. Tissue array slides were commercially avalible from Biomax company, including 46 human breast cancer tissues and 46 adjacent normal breast tissues. 1.2 Cell lines and cell culture: SK-BR-3, MCF10 A and 293 T cells were purchased from American Type Culture Collection(ATCC). The rest of breast cancer cell lines, MDA-MB-231, LM2-4, T-47 D, and ZR-75-1, were kindly provided by Dr. Giulio Francia at UTEP. MDA-MB-231, LM2-4, and MCF10 A were cultured in DMEM medium containing 10% fetal bovine serum(FBS) and 1% penicillin at 37 with 5% CO2. T-47 D, ZR-75-1, and SKBR3 were cultured under the same condition as other cell lines, except that it is cultured in RPMI-1640 medium.2 Methods 2.1 Autoantibody responses to p90/CIP2 A were evaluated by enzyme-linked immunosorbent assay(ELISA),Western blotting and indirect immunofluorescence assay(IIF) in sera from patients with breast cancer and normal human individuals. The expression of p90/CIP2 A in breast cancer tissues were determined by immunohistochemistry(IHC) with tissue array slides.2.2 Lentivirus-mediated sh RNA p90/CIP2 A knockdown was performed in MDA-MB-231 and LM2-4 cell lines. Cell proliferation assay, colony formation assay and flow cytometry were carried out to evaluate the role of p90/CIP2 A in cell proliferation and apoptosis in vitro.3 Statistical analysis Statistical analysis was performed using IBM SPSS21.0. Data were analyzed with χ2 test from ELISA and IHC. The immunodignostic value of autoantibody to p90/CIP2 A was evaluated by ROC cuve. Two-independent-samples t test was used in MTT, Colony Formation Assay and Apoptosis Detection. M±SD was demonstrated in MTT, Colony Formation Assay and Apoptosis Detection. P< 0.05 was considered to indicate statistical significance.Results 1 Frequency anti-p90/CIP2 A autoantibody in sera from patients with breast cancer The prevalence of autoantibody against p90/CIP2 A was 19.1% in breast cancer, which was higher than that in normal human sera(2.3%)(P<0.001). The ELISA results were also confirmed by Western blotting analysis. The representative breast cancer serum with positive reaction to p90/CIP2 A in ELISA also has strong reactivity in Western blotting compared to normal serum. 2 Immunofluorescence staining pattern of p90/CIP2 A in HEp-2 cells HEp-2 cell slides were used in IIF to detect breast cancer sera with anti-p90/CIP2 A positive in ELISA. A representative anti-p90/CIP2 A positive breast cancer serum had the predominantly cytoplasmic staining pattern. The fluorescent staining was significantly reduced when the same breast cancer serum was pre-absorbed with recombinant p90/CIP2 A protein. 3 Expression of p90/CIP2 A in breast cancer tissues and adjacent normal breast tissues by IHC All of the breast cancer tissues were positively stained(100%), 13 of the 46 adjacent normal breast tissues were positively stained(28%). The positive rate of p90/CIP2 A expression in breast cancer tissues was higher than that in adjacent normal tissues(P<0.001).4 Establishment of stable breast cancer cell lines expressing p90/CIP2 A sh RNA The MDA-MB-231 and LM2-4 cell lines were used as our research model to generate cell lines stably expressing p90/CIP2 A sh RNA. p90/CIP2 A sh RNA decreased p90/CIP2 A expression in both MDA-MB-231 and LM2-4 cell lines after transduction. 5 p90/CIP2 A knockdown inhibits cell proliferation and clonogenic ability in breast cancer cell lines A significant decrease in proliferation was observed in MDA-MB-231 and LM2-4 cell lines treated with p90/CIP2 A sh RNA compared with the vector control(P<0.05). Consistent with the MTT results, colony formation assay showed that the down-regulation of p90/CIP2 A in MDA-MB-231 and LM2-4 cell lines decreased the number of foci significantly as compared to vector control cell lines by plating cells on the petri-dish for 15 days(P<0.05). 6 p90/CIP2 A ablation enhances paclitaxel-induced apoptosis in breast cancer cells A significant population of early and late apoptosis was observed in cells with p90/CIP2 A depletion compared with sh RNA controls, demonstrating that p90/CIP2 A knockdown results in paclitaxel-induced apoptosis in breast cancer cells(P<0.05). 7 p90/CIP2 A depletion down-regulates c-Myc expression and inhibits ERK1/2 phosphorylation in breast cancer cells Western blotting analysis revealed that the knockdown of p90/CIP2 A decreased the protein expression levels of c-Myc in both cell lines. Besides, the level of ERK1/2 phosphorylation was also decreased in both cell lines, while the expression of ERK1/2 was not changed.Conclusions 1 Autoantibody against p90/CIP2 A may be a potential serum biomarker for breast cancer screening and immonodiagnosis. 2 p90/CIP2 A as a crucial oncoprotein has been involved in breast cancer cell proliferation and apoptosis, which could serve as a novel therapeutic target in breast cancer treatment. |
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