There are five subspecies of breast cancer, and Triple Negative Breast Cancer(TNBC)is the worst of all due to its drug resistance and high recurrence rate1. It is hard to treat TNBC by using traditional targeted therapy, and treatments like chemotherapy and endocrine therapy have optimistic effectsneither. Thus, discovering new ways to treat TNBC becomes significant for both TNBC patients and medical community. Then, our team focused on a kind of cell death methods that was found recently --- ferroptosis.
Ferroptosis is a natural way for cells to die. There are two independent molecular mechanisms for iron death, namely glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1)2,3.One of these ways is using glutathione. Therefore, we would inhibit glutathione to make ferroptosis happen4,5.To be more specific, ferroptosis needs lipid peroxidation to intrigue cells to die. Generally, cells would consume glutathione to convert cytotoxic lipid peroxides to non-toxic lipid alcohols so as to inhibit ferroptosis. In our project, we aimed at the Gox1 gene. TNBC cells have a high metabolic rate, so they need sufficient nutrition such as glucose. However,under aerobic conditions, Gox1 could effectively digest glucose, which cuts the nutrition income of TNBC cells which will result in ferroptosis.
Figure 1. Project Engineering Schematic Diagram 4
We first multiplied the Gox1 gene.By using ligation, we inserted the Gox1 gene into the pET-28a vector and pCOLA-PesaS-RBS-RFP-rrnbT1-CI857-PR-SRRz-rrnB vector. Then we transformed it into the DH5α E.coli which is also used to store plasmid and transformed it into BL21 E.coli to express protein, respectively.
After obtaining the engineered E.coli with the Gox1 gene, we multiplied it and induced it to express a larger content of protein. A nickel column was also used to purify the protein to remove unnecessary non-target protein. In order to evaluate the protein activity, we used the Reduced Glutathione (GSH) Content Assay kit and Cell Counting Kit-8 with 4T1 cell lines for further evaluation.
We aim to develop a new method based on ferroptosis for the targeted treatment of TNBC and hope this promotes more research on the protein therapy of TNBC.
1.Vagia, Elena et al. “The Landscape of Targeted Therapies in TNBC.” Cancers vol. 12,4 916. 8 Apr. 2020, doi:10.3390/cancers12040916
2. Li, Ke et al. “Multienzyme-like Reactivity Cooperatively Impairs Glutathione Peroxidase 4 and Ferroptosis Suppressor Protein 1 Pathways in Triple-Negative Breast Cancer for Sensitized Ferroptosis Therapy.” ACS nano vol. 16,2 (2022): 2381-2398. doi:10.1021/acsnano.1c08664
3. Yang, Youjing et al. “STAT6 inhibits ferroptosis and alleviates acute lung injury via regulating P53/SLC7A11 pathway.” Cell death & disease vol. 13,6 530. 6 Jun. 2022, doi:10.1038/s41419-022-04971-x
4. Conrad, Marcus et al. “Targeting Ferroptosis: New Hope for As-Yet-Incurable Diseases.” Trends in molecular medicine vol. 27,2 (2021): 113-122. doi:10.1016/j.molmed.2020.08.010
5. Yang, Fan et al. “Ferroptosis heterogeneity in triple-negative breast cancer reveals an innovative immunotherapy combination strategy.” Cell metabolism vol. 35,1 (2023): 84-100.e8. doi:10.1016/j.cmet.2022.09.021