The Role Of MCPIP1/MALT1 Axis In Control Of Breast Cancer Progression

Breast cancer is a malignant tumor originated in mammary epithelial cells.breast cancer is the second most common cancer worldwide after lung cancer, the fifth most common cause of cancer death, and the leading cause of cancer death in women, each year 1.7 million women receive a new diagnosis of breast cancer 。 Molecular classifi cation of breast carcinoma includes :Luminal A(ER+, PR+/–, HER2–)(MCF-7, T47 D, SUM185),Luminal B(ER+, PR+/–, HER2+)(BT474, ZR-75),Basal(ER–, PR–, HER2–EGFR+)(MDA-MB-468, SUM190),Claudin-low(ER–, PR–, HER2–)(BT549, MDA-MB-231,Hs578 T, SUM1315),HER2(ER–, PR–, HER2+)( SKBR3, MDA-MB-453),MDA-231 and MCF-7 are still the most commonly used breast cancer cell line in the world.MCPIP1 is a kind of zinc finger protein encoded by the zc3h12 a gene,was initially discovered as the most highly induced mRNA by monocyte chemotactic protein-1(MCP-1) in human peripheral blood monocytes.MCPIP1 is rapidly induced in macrophages upon stimulation with proinflammatory molecules, such as TNF, IL-1β, and LPS.MCPIP1 has RNase activity and inhibits the expression of proinflammatory cytokines(IL-1,IL-6, and IL-12) by binding to their 3’UTRs for mRNA degradation. MCPIP1 is also named as Regnase-1 based on the RNase activity. In addition, MCPIP1 can act like a brake for T cell activation. Therefore, MCPIP1 is believed to be a key negative regulator involved in the control of inflammation and maintenance of homeostasis. The recently research indicates that MCPIP1 can suppress breast cancer cell growth and proliferation through induce apoptosis,the mechanism is to regulate the anti-apoptosis and pro-apoptosis mRNA expression level.MALT1(Mucosa Associated Lymphoid Tissue Lymphoma TranslocationGene 1) located on t(11;18)(q21;q21), expressed in 30% malt Lymphoma.Together with CARMA1(coactivator-associated arginine methyltransferase1,also known as CARD11) and BCL10, MALT1 assembles the CBM(CARMA1/Bcl10/MALT1) complex that bridges proximal antigen receptor signaling events to the IkB kinase(IKK) complex, cause the degradation of IKK(Inhibitor of κB),activated NF-κB pathway. T cell receptor(TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. A tumor-promoting role of MALT1 has been found in a subset of diffuse large B cell lymphoma(DLBCL) and MALT lymphoma. positioning MALT1 as an attractive anticancer drug target. Recently, small molecule MALT1 protease inhibitors MI-2 and MEPAZINE also were described and shown to suppress proliferation of ABC-DLBCL in vitro and in xenotransplanted tumors in vivo,but the effect of MALT1 inhibitors on breast cancer cell have not been reported,This study includes three parts:1. Detect the protein and mRNA expression level in breast cancer cell and normal breast cell.According to a266 patient follow-up study analyze the relationship between MALT1 expression level and breast cancer prognosis.2.Treat breast cancer cell with MI-2 and MEPAZINE to determine the effect of MALT1 inhibitors on breast cancer cell proliferation. Treat MCPIP1 knockdown breast cancer cell line to determine whether MCPIP1 involved in the progress of MALT1 inhibitor suppress breast cancer cell growth.3.Inject MDA-231 cell into NSG mice and treat the mice by MEPAZINE to determine the effects of these MALT1 inhibitors on breast cancer cell proliferation and survival in vivo.PART 1 study of MALT1 expression in breast caner cell lineObjective:MCPIP1 is a newly discovered breast cancer cell suppressor protein, pro-apoptotic genes by regulating mRNA levels and anti- apoptosis related genes induced apoptosis in breast cancer cells, thereby inhibiting proliferation of breast cancer cells, the researchers reported in breast cancer cells MCPIP1 was higher than in normal breast epithelial cells, expression levels in tumor tissue of breast cancer patients are also higher than thesurrounding normal tissue, and MCPIP1 and prognosis in breast cancer patients was positively correlated with higher expression levels, the better the prognosis. MALT1 having protease activity, can cause degradation MCPIP1 detected MALT1 expression levels in breast cancer cell lines and patient organizations to explore MALT1 on breast cancer cell proliferation and the impact on the prognosis of breast cancer patients.Methods1 culture cell line for research1.1 Normal human breast epithelial cells MCF-10 A, MCF-12 A Human breast cancer cell line MDA-231, MCF-7, T47 D, MDA-4531.2 normal mouse mammary epithelial cells SK4; mouse breast cancer cell line 4T1 TSA1.3 normal human liver cells IHH; human hepatoma cell line H7, H7.51.4 normal human prostatic epithelial cells HPV7;human prostate cancer DU145 1.5 normal human lung epithelial cells MRC-5; human lung cancer cell line H232.Q-PCRExtract RNA from normal cell and cancer cell line by Trizol,do RT-PCR and Q-PCR to detect MALT1 mRNA expression level in different cell line.3 Western blotExtract protein from normal cell and cancer cell line and run Western to determine MALT1 protein level in different cell line.4 Survival curve analysis : according a followup study of 266 patients to analyze MALT1 expression levels with patient prognosis. According to the degree of malignancy into GradeⅠ, GradeⅡ, Grade Ⅲ three groups and compare MALT1 expression level; according to the MALT1 expression level of 266 patients were divided into high expression group and low expression group, and make survival curve analysis. According to immunohistochemical typing into ER +, PR +, HER2 + compare MALT1 expression level in the three groups and make survival curve analysis.1.Q-PCR: MALT1 mRNA expression level in human breast cancer cell lines, mouse breast cancer cell lines, human lung cancer cell lines are lower than normal cell lines.2.Western Result: MALT1 protein expression level in human breast cancer cell lines, mouse breast cancer cell lines, human prostate cancer cell lines, human lung cancer cell lines in are higher than normal cell lines. In human hepatoma cell lines MALT1 expression level is no significant difference in normal cell line.3 Survival curve analysis : MALT1 expression level in Grade Ⅲ group is higher than GradeⅠand GradeⅡ. There is no significant difference between GradeⅡand GradeⅠ, GradeⅡ; patients with high MALT1 expression shows worse prognosis. HER2 + group MALT1 expression level is higher than ER +,PR + groups, prognosis shows worse than ER +, PR + groups, ER +, PR +MALT1 expression and the prognosis was no significant difference between the two groups.conclusions:1 MALT1 protein expression level in multiple cancer cell lines is higher normal cell lines.2 MALT1 expression and prognosis in breast cancer patients was negatively correlated, the higher expression, the worse prognosis.Part 2 The mechanism of MALT1 inhibitor on breast cancer cellObjective: MALT1 in ABC-DLBCL(activated B cell-like diffuse large B cell lymphoma) is highly expressed.In diffuse large B-cell lymphoma(diffuse large B cell lymphoma) and mucosal metastasis lymphoma(MALT lymphoma) MALT1 as a tumor in growth promoting factor has been confirmed, the study found MALT1 inhibitors MI-2(MALT1 inhibitor 2) and MEPAZINE inhibit the proliferation of ABC-DLBCL by inhibiting NF-kB(nuclear factor kappa-light-chain-enhancer of activated B cells) activity,causing cell cycle arrest and induction of apoptosis, but the effect of MALT1 inhibitor in breast cancer cells has not been reported. The first part of the study have confirmed MALT1 expression and prognosis in breast cancer Results:patients with breast cancer cells, analysis MALT1 inhibitor for breast cancer cells, breast cancer aims to find new targets, develop new ideas, Treat MCPIP1 knockdown breast cancer cell line to determine whether MCPIP1 involved in the progress of MALT1 inhibitor suppress breast cancer cell growthMethod1 Cell line: MDA-231, MCF-7 human breast cancer cell lines. MCF-10 A normal human breast cells; 4T1, TSA mouse breast cancer cell line, FSK4 normal mouse mammary epithelial cells. MDA-231 Tet-on cells; MDA-231MCPIP1 knock-down cell lines and the corresponding scramble control cell line.2 MTT assay and cell counting: Treat MCF-1OA, MDA-231 MCF-7, with different concentration of MI-2 and MEPAZINE and then do the MTT assay and cell counting at different time points to determine the effect of MALT1 inhibitor on breast cancer cell proliferation.3 PI cell cycle analysis: Treat MDA-231 MCF-7, with different concentration of MI-2 and MEPAZINE and collected cells at different time points with PI staining, cell cycle analysis, to determine the effect of MALT1 inhibitor on cell cycle.4 Q-PCR assay: Treat MDA-231 MCF-7, with MI-2 and MEPAZINE for24 hours,then collect RNA by Trizol and do RT-PCR and Q-PCR to detect the cell cycle-related genes. expression.5 Apoptosis analysis: Treat MDA-231 MCF-7 with different concentration of MI-2 and MEPAZINE for 48 hours and then collected protein. Run Western to detect the apoptosis-related proteins PARP1 expression.The same cells were collected staining by Annexin V-FITC and PI(Propidium Iodide), flow cytometric analysis, observing the percentage of apoptotic cells.6 NF-κB activity analysis: Treat MDA-231 MCF-7 with different concentration of MI-2 and MEPAZINE for 72 hour and then collected nuclear and cytoplasma protein to detect the expression of NF-κb in nuclear and cytoplasm.7 luciferase assay: Luciferase reporter containing the NF-κb, CDK2, CDK63’UTR plasmid were electrical transfected to MDA-231 and MCF-7 cells,24 hours after transfection add MI-2.48 hours later collect protein to detect luciferasr activity to determine the effect of MI 2 on NF-κb activity and CDK2,CDK6 3’UTR activity.8 Western blot: Treat MDA-231 and MCF-7 with different concentration of MI-2 for 72 hours and then extracted protein to detect the effect of MALT1 inhibitor on MCPIP1 expression in breast cancer cells. Using MDA-231Tet-on cells, plus DOX(Doxycycline 100 ng / ml) stimulation for 24 hours and the expression of induced GFP-MCPIP1, then add MEPAZINE or MI-2 for 72 hours and collect the protein for Western blot to determine the effect of MALT1 inhibitor on MCPIP1 Cleavage in breast cancer cell.9 mRNA half-life detectionTreat MDA-231 with 5μmol MI-2 for 24 hours and then add actinomycin D(Act D) and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole(DRB) to block transcription, RNA was collected at different time points and to Q-PCR to detecting cell cycle related gene mRNA expression, draw half-life curves and calculated half-life and compared between the treatment group and control group.10 MALT-1 inhibitor MCPIP1 correlation test.Treat MDA-231 MCPIP1 knock-down and MDA-231 scramble control cell line with different concentration of MI-2 and MEPAZINE, and do the following experiment:10.1 Do MTT assay at different time points to determine whether MCPIP1 involved the progress of MALT-1 inhibitor suppress breast cancer cell growth.10.2 PI assay the cell cycle;10.3 Collected RNA by Trizol to do RT-PCR and Q-PCR to detect the cell cycle-related gene expression.10.4 RIPA lysis protein was collected to detect cell apoptosis related protein expression10.5 Annexin V-FITC / PI apoptosis to detect the percentage of earlyapoptosis cellsResults:1 MTT assay and cell count: MI-2 and MEPAZINE can effectively inhibit MDA-231 and MCF-7 cell proliferation, and the effect was dose-dependent and time-dependent. MDA-231 and MCF-7 cells relative to MCF-10 A cells more sensitive to MALT1 inhibitors.2 Cell Cycle Assay: MI-2 and MEPAZINE could cause significant MDA-231 and MCF-7 cell cycle arrest in G1 phase. Meanwhile G1 cell cycle-related genes CDK2, CDK4, CDK6 expression decreased.3 Apoptosis Analysis: MI-2 in breast cancer cells can not induce significant apoptosis. MEPAZINE can induce apoptosis in breast cancer cells, and the effect was dose-dependent.4 NF-κB activity analysis: The Breast Cancer Treated by MEPAZINE nucleus NF-Κb family members P65, C-Rel expression of mRNA expression levels decreased, P65, C-Rel reduced.5 luciferase activity analysis: In the MDA-231 cells treated by MI-2 group,the NF-κB, CDK2 3’UTR, CDK6 3’UTR activity was significantly lower than the control group.6 Western Analysis: MALT1 MCPIP1 inhibitors increase the intracellular expression was observed in MDA-231 Te-on treatment group into the cell lysis MCPIP1 reduced.7 half-life experiment: MI-2 treated group CDK2, CDK4 half-life was significantly lower than the control group.8 MCPIP1 MALT1 inhibitors and related experiments8.1 MTT resultsMI-2 and MEPAZINE of MDA-231 MCPIP1 Knockdown cell inhibition rate was significantly lower than Scramble cells treatment group.8.2 cell cycle resultsCell cyclearrest at G1 phase in MDA-231 MCPIP1 Knockdown treated by MI-2 and MEPAZINE is weaker than in Scramble cells.8.3 Q-PCR resultsMI-2 / MEPAZINE CDK2 for cell cycle related genes in MDA-231MCPIP1 Knockdown cells, CDK4, CDK6 regulation is weaker than Scramble cells.8.4 Western and streaming resultsThe expression of elary apoptotic protein Cleaved-PAPP1 in MCPIP1 Knockdown cell treated by MEPAZINE is much lower than Scramble treatment group. The percentage of early apoptosis cell in MCPIP1 Knockdown cell treated by MEPAZINE is lower than Scramble treatment group.Conclusions1 MALT1 inhibitors can significantly inhibit the proliferation of breast cancer cells, and this effect was concentration-dependent and time-dependent,normal mammary epithelial cell sensitivity MALT1 inhibitors weaker than breast tumor cells.2 MALT1 inhibitors work by reducing the cell cycle-related genes mRNA stability and thus cause cell cycle arrest in G1 phase.3 MALT1 inhibitors block NF-κb from cytoplasm to the nucleus transit and affect the expression of P65, C-Rel’s.4 MALT1 inhibitors increased MCPIP1 expression by inhibit the MALT1 protease activity for MCPIP1-cleavage.5 MCPIP1 involved in the progress of MALT1 inhibitor suppress breast cancer cell growth..6 MALT1 inhibitors cause cell cycle arrest and regulation of cell cycle-related genes have MCPIP1 participation.7 MEPAZINE induce breast cancer cell apoptosis partly through MCPIP1.PART 3 Animal study for MALT1 inhibitorObjective: Research shows that MALT1 inhibitor for ABC-DLBCL inhibition was confirmed in vivo, but the role of MALT1 inhibitor solid tumors has not been reported. By MALT1 inhibitors in vivo experiments on breast cancer cells, to determine its application in the treatment of breastcancer.Methods:1 Subjects: NSG(NOD-SCID IL-2receptor gamma null) mice2 Grouping and administration methods: 6 NSG mice were randomly divided into control and treatment group, each mice was injected with MDA-231 cell in abdomen bilateral breast,40 days after injection initiation of administration, the administration group were intraperitoneally injected MEPAZINE(800μg/each mice), the control group were given intraperitoneal injection of 2% DMSO, administered once every 24 hours, a total of 10 times of administration.3 measuring tumor volume, tumor growth curve, tumors were removed and weighed photographs, extracted tumor tissue protein and RNA and mRNA expression detected related apoptosis-related protein and the cell cycle.4 tumor tissue sections and HE stainingResults:1 Treatment group tumor volume and weight was significantly lower than the control group.2 Treatment groups apoptotic signaling proteins PARP-1 expression and MCPIP1 higher, cell cycle-related genes CDK2, CDK4, CDK6 expression than the control group.3 HE staining: the control group were observed slice visible structural integrity of cell sharp edges, the treatment group was significant damage to tissue structure, cell morphology.conclusions:1 MEPAZINE can inhibit breast cancer cell proliferation in vivo.2 MEPAZINE inhibit breast cancer cell proliferation by inducing apoptosis and cell cycle arrest and induction of expression MCPIP1 in vivo.