The Molecular Mechanism Of TmTNF-α Promoting Chemoresistance In Breast Cancer Cells And Cytotoxic Effect Of TmTNF-α And Influenza Specific CAR-T On Breast Cancer

PART ONE The molecular mechanism of tmTNF-α promoting chemoresistance in breast cancer cellsBreast cancer is the most frequently diagnosed malignancy among females,cytotoxic chemotherapy is of limited benefit and chemoresistance remains a major obstacle.Tumor necrosis factor-α(TNF-α)has two bioactive forms: transmembrane TNF-α(tmTNF-α)and secretory TNF-α(s TNF-α).tmTNF-α is cleaved by a metalloproteinase TNF-α-converting enzyme(TACE)to generate s TNF-α,leaving an N-terminal fragmenta(NTF)of TNF-α on the cells surface.We previously found that nearly 70% patients with ductal breast cancer expressed tmTNF-α,knockdown of tmTNF-α expression on primary breast cancer cells leads to a switch of tumor cells from resistance to sensitivity to DOX in vitro.However,the mechanism of these kind of drug-resistance still unclear.The aim of the study is to explore the molecular and signal pathway mechanism of resistance to doxorubicin,which provides an idea for targeting tmTNF-α in the treatment of breast cancer.The main results are as follows:1.Expression high level of tmTNF-α in breast cancer mediates DOX resistance: MTT assay was used to confirm high level expression of tmTNF-α leads to resistance to DOX in MDA-MB-231 cell line,knockdown of tmTNF-α expression reverse the sensitivity of this cell line to DOX.Conversely,tmTNF-α-negative MCF-7 cells were more sensitive to DOX-induced cell death,stably transfected the NTF of tmTNF-α in MCF-7 cells resulting in a conversion from sensitivity to resistance to DOX,demaonstrate that expression of tmTNF-α confers the DOX resistance in breast cancer cells.2.ERK contributes to tmTNF-α-mediated DOX resistance: Western blot assay was used to confirm an enhanced ERK phosphorylation in MDA-MB-231 and NTF-MCF-7 cells,knockdown of tmTNF-α expression blocked ERK phosphorylation.ERK inhibitor PD98059 could partially suppress ERK phosphorylation and increase an DOX-induced apoptosis and cytotoxicity.3.ERK contributes to tmTNF-α-mediated DOX resistance: Realtime-PCR assay was used to detected the expression of GST-π,knockdown of tmTNF-α expression suppress the expression of GST-π.FACS was used to detect the fluorescence intensity of DOX,high level of expression of tmTNF-α inhibited intracellular DOX MFI,and promoted resistance to DOX-induced apoptosis;si RNA was used to knockdown expression of in breast cancer cells,which could upregulate expression of intracellular DOX MFI in tmTNF-α-positive cells and increased the sensitivity to DOX.These data indicate that tmTNF-α induced DOX resistance partially through GST-π mediated detoxification.4.GST-π/ ERK positive feedback loop contributes to tmTNF-α-mediated DOX resistance: ERK inhibitors PD98059 and SCH772984 supressed GST-π transcription and protein expression in tmTNF-α-positive cells,indicate the ability of ERK signal pathway promoting expression of GST-π;Coversely,in the presence of DOX,knockdown of GST-π inhibited tmTNF-α/NTF-induced ERK phospraylation.These data indicate that tmTNF-α leads an expression of GST-π through ERK pathway,and GST-π contributes to ERK phospraylation,creating a positive feedback loop to promote DOX resistance.5.NF-κB is necessary for tmTNF-α-mediated DOX resistance: Western blot assay was used to confirm elevated constitutive NF-κB activation in tmTNF-α-positive cells,and upregulated expression of antiapoptic molecules,knockdown of tmTNF-α expression suppress the activity of NF-κB pathway with suppressing apoptisis molecules.NF-κB inhibitor reversed the DOX resistance of breast cancer cells,demonstrate that NF-κB activation is required for drug resistance through tmTNF-α-mediated reverse signaling.6.tmTNF-α promoting DOX resistance in breast cancer in vivo: we injected MDA-MB-231 cells stably transfected with Control sh RNA or tmTNF-α sh RNA into mamry fat pad of the female athymic nude mice.After tumor formation(about 100 mm3),the animals were received DOX for 3 weeks.Data shows knockdown of tmTNF-α expression upregulated sensitivity of DOX,suppressed tumor size and prolonged survial time.Consistent with results in vitro,Knockdown of tmTNF-α inhibited ERK and NF-κB pathway,reduced expression of GST-π and antiapoptotic molecules.Here,we found that tmTNF-α-ERK / GST-π axis and tmTNF-α-NF-κB pathway play significant roles in tmTNF-α-positive cells resistance to DOX.Combination of TNF-α antibody and chemotherapy may be a successful strategy for treatment of chemo-resistant advanced breast cancer.PART TWO Cytotoxic effect of tmTNF-α and influenza specific CAR-T on breast cancerCAR-T cells have demonstrated tremendous success in iradicating hematologic malignancies.Different from those of hematological tumors,such success has yet to be extrapolated to solid tumors.In addition,the restriction of persistence of CAR-T cells in vivo is one of the reasons for the recurrence of the tumor.tmTNF-α antibody,made in house,could mediate ADCC and CDC effectively killing tmTNF-α positive breast cancer cells,suggesting that tmTNF-α can used as TAA in the treatment of CAR-T therapy.The aim of the study is to construct the second generation tmTNF-α-scFv-4-1BB chemiric antigen receptor,transduce the tmTNF-α-CAR into mouse memory T lymphocytes using retrovirus,and investigate the cytotoxicity of CAR-T cells target tmTNF-α or H1N1-HA positive breast cancer cells.The desire to treatment is eventually eliminating tumor cells through one-time adoptive transferring CAR-T cells,combinig with influenza vaccine used to stimulate the proliferation of CAR-T cells in vivo.The main results are as follows:1.Genetic ngineering technology was used to possess and recombine tmTNF-α-sc Fv、CD8α transmembrane domain、4-1BB constimulation domain and CD3ζ signal domain,successfully constructing the tmTNF-α-sc Fv CAR.A retrovirus packaging cell line was stably transduced tmTNF-α-sc Fv CAR,and obtain high titer virus particles.2.Successfully constructing stably transduced tmTNF-α or H1N1-HA 4T-1 cell lines,constructing stably transduced tmTNF-α 4T1-LUC cell lines,FACS was used to evaluate tmTNF-α or H1N1-HA expression as target cells.3.The second generation of tmTNF-α-CAR specific for tmTNF-α successfully transduced into mouse primary T lymphocytes using retrovirus and highly expressed on T cells,which specifically recognize tmTNF-α positive 4T-1 cells.4.Vaccination of influenza virus vaccine for 45 days,higher expression of effect T cells and memory T cells were determined by flow cytometry.The maximum quality influenza virus specific memory T cells obtained from spleen were stimulated with influenza vaccine and CD3/CD28 antibody,which were transduced with tmTNF-α-sc Fv CAR.5.influenza virus/tmTNF-α specific CAR-T cells could specifically recognize tmTNF-α positive breast cancer cells and H1N1-HA positive cells,which have showed specifically cytotoxic against target cells using LDH release cytotoxicity assay and upregulating expression of effect molecules CD107a、Perforin、Granzyme B、Fas and Fas L in vitro.Influenza virus/tmTNF-α specific CAR-T cells were successfully constructed and specifically recognizeg tmTNF-α or H1N1-HA positive breast cancer cells.Lay the foundation for in vivo cytolytic effect of influenza virus/tmTNF-α dual pecific CAR-T cells and eliminating tumor cells through vaccination induced CAR-T cells expansion in vivo.