Study On Simultaneous Targeting Of The Entire ErbB Receptor Tyrosine Kinases For Degradation By The Protein Knockout System In Breast Cancer

Objective:To Study the engineered ubiquitin ligase strategy for simulating the consequences of pharmacological eradication of the entire family of activated ErbB RTKs in ErbB-overexpressing tumor cells, and further assess the impact on cell proliferation, sensitivity to chemotherapy, and cell malignant transformation, providing a versatile and powerful investigative tool for developing the next generation of targeted therapies against ErbB-positive tumors.Methods:1.(1) To confirm the function of pcDNA3.0-F-TrCP-Shc and pcDNA3.0-F-TrCP-Shc (F198V), FLAG tagged chimeric constructs were transiently transfected into293T cells and co-immunoprecipitation, Western blotting experiments were performed.(2) The plasmid of pcDNA3.0-F-TrCP-Shc and pcDNA3.0-F-TrCP-Shc (F198V) were subcloned into the BamHI/NotI sites of the retroviral pBMN-GFP vector to generate recombinant retroviruses for infection of SKBR3, BT474cells.(3) FACS analysis was carried out to isolate infected cells based on the GFP expression, selected by the puromycin for7days, and western blot analysis were carried out to test the protein expression of FLAG.2.(1) The protein and mRNA expression of SKBR3and BT474cells infected with pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc (m) or pBMN-GFP retroviruses were tested by western blotting and real-time PCR. Flow cytometric analysis of cell membrane ErbB2was performed on live cells.(2) The Inhibition of Engineered F-TrCP-shc induced ErbB2degradation by proteasome inhibitor MG132and lysosome inhibitor chloroquine were tested by western blotting.(3) The attenuation of MAPK and PI3K signaling upon degradation of ErbB RTKs by F-TrCP-Shc was tested by western blotting.3. The impact of degradation of ErbB RTKs by F-TrCP-Shc on cell proliferation, apoptosis, sensitivity to chemotherapy, and cell malignant transformation were analysied in cell and nude mice model.4. The non-malignant mammary epithelial cells MCF-10A infected by recombinant retroviruses was isolated by FACS analysis based on the GFP expression, selected by the puromycin for7days, and western blot analysis were carried out to test the protein expression of p-ErbB2, and the impact on cell proliferation, cell apoptosis, cell cycle, and sensitivity to chemotherapy were assessed.Results:1.(1) The engineered F-TrCP-Shc ubiquitin ligase interacted with ErbB2and induces its degradation in a dose-dependent manner.(2) Enzyme digestion and sequencing confirmed the successful construction of recombinant retroviruses pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc (m).(3) The retroviral vector-infected BT474and SKBR3cells were constructed by FACS analysis and puromycin selection, and their FLAG proteins expressions were well.2.(1) The engineered F-TrCP-Shc E3ligase selectively targeted the activated ErbB family members for degradation and depletes ErbB2RTK on the cell surface.(2) Treatment of F-TrCP-Shc-expressing cells with the proteasome inhibitor MG132blocked ErbB2degradation, whereas the lysosomal inhibitor chloroquine had no discernable effect.(3) Targeted degradation of ErbB RTKs by F-TrCP-Shc led to the attenuation of MAPK and PI3K signaling.3.(1) Targeted degradation of ErbB RTKs by F-TrCP-Shc triggered apoptosis, led to the cell cycle arrest, inhibition of proliferation, sensitized breast cancer cells to cytotoxic killing by cisplatin and decreased their transforming ability in SKBR3and BT474breast cancer cells.(2) The engineered F-TrCP-Shc E3ligase suppressed BT474xenograft tumor growth in nude mice.4. The engineered F-TrCP-Shc E3ligase selectively targeted the activated ErbB family members for degradation but had no effect on the cell proliferation, cell apoptosis, cell cycle, and sensitivity to chemotherapy in non-cancerous MCF-10A mammary epithelial cells.Conclusions:1.(1) The engineered F-TrCP-Shc ubiquitin ligase interacted with ErbB2and induced its degradation.(2) The recombinant retroviruses pBMN-F-TrCP-Shc, pBMN-F-TrCP-Shc (m) were generated.(3) The retroviral vector-infected BT474and SKBR3cells were constructed.2.(1) The engineered F-TrCP-Shc E3ligase effectively binds the activated ErbB family members and recruits them to the SCF machinery for ubiquitination and proteasome-dependent degradation.(2) The targeted depletion of ErbB RTKs effectively attenuates the downstream MAPK and PI3K/AKT signaling cascade.3.(1) In the breast cancer cell model, degradation of ErbB RTKs by F-TrCP-Shc led to inhibition of proliferation, sensitized breast cancer cells to cytotoxic killing by cisplatin and decreases their transforming ability.(2) The engineered F-TrCP-Shc E3ligase suppressed BT474xenograft tumor growth in nude mice.4. Targeted depletion of ErbB RTKs had no discernable effect on non-cancerous MCF-10A mammary epithelial cells indicating that the engineered F-TrCP-Shc ligase specifically inhibits the proliferation of ErbB-overexpressing breast cancer cells. It is a feasible intervention strategy for breast cancer.5. In this study, we study on simultaneous capturing all activated EGFR/ErbB family proteins and recruit them to the SCF machinery for degradation in breast cancer. Future studies should directly compare the therapeutic effects of eradicating a single or multiple ErbB family members in pre-clinical models, and evaluate the potential of pan-ErbB suppression as a feasible strategy for the development of the next generation of targeted therapies against ErbB-positive tumors.