A Novel Mechanism Of 4EBP1 Feedback Activation Of MTORC1 To Promote Tumor Proliferation In Breast Cancer

According to the global cancer statistics in 2022,breast cancer remains the first malignant tumor in women in terms of incidence and second in terms of mortality.More than 70% of breast cancer patients are ER+ patients,for which hormone therapy is mostly used clinically,however,resistance to therapeutic drugs is still the main cause of death.Therefore,the search for effective therapeutic targets for breast cancer remains urgent.In tumors,the mTOR signaling pathway plays an important role in regulating cell growth,proliferation,cycle,apoptosis and autophagy.The mTOR signaling pathway has been found to be aberrantly activated in a variety of tumors,including breast cancer,and promotes malignant progression of tumors.Currently,mTOR signaling pathway has been investigated by many laboratories worldwide,but the specific regulatory mechanisms related to tumorigenesis and development are still not fully understood.Therefore,an indepth study of the mTOR signaling pathway will not only help to reveal the molecular mechanism of tumor development,but also provide an important theoretical basis and molecular targets for clinical diagnosis and treatment of tumors.Eukaryotic initiation factor binding protein 1(4EBP1)is one of the most critical substrates downstream of mTOR and plays an important role in tumor development by competing with the scaffold protein eIF4 G to translate the initiator eIF4 E and inhibit capdependent protein translation(Cap-dependent mRNA translation).Because 4E-BP1 plays a rate-limiting role in cap-dependent translation,it is commonly thought to play an oncogenic role in tumors.However,by comparing cancer tissues with paraneoplastic tissues from patients with multiple tumors,4EBP1 expression levels were found to be significantly increased in tumor tissues,and patients with high 4EBP1 expression had a poorer clinical prognosis and lower long-term survival rates.In a variety of tumor cells,amplification of the 8q12 region of chromosome encoding 4E-BP1 was found,and its mRNA level was positively correlated with tumor malignant progression.It is suggested that 4E-BP1 may promote tumor development and play an oncogene role.The results of our previous immunohistochemical experiments also showed that the expression level of4EBP1 in breast cancer tissues compared with paraneoplastic tissues.The level was significantly elevated,suggesting that 4EBP1 may play an oncogene role in breast cancer tissues,however,the molecular mechanism by which 4EBP1 promotes breast cancer progression needs to be further investigated.Objectives1.To investigate whether the 4EBP1 molecule plays an oncogene role in breast cancer?2.What is the molecular mechanism by which 4EBP1 promotes breast cancer progression ?Methods1.Comparison of 4EBP1 expression in breast cancer tissues and paraneoplastic tissues by IHC assay;2.Construction of 4EBP1 knock-out breast cancer stable transitional cell lines by i.CRISPR Cas9 gene editing and 4EBP1 overexpression breast cancer cell line b)by lentiviral infection;3.Comparison of the effects of 4EBP1 knockdown and overexpression on clone formation by plate cloning experiments;4.Comparison of the effect of 4EBP1 knockdown and overexpression on the valueadded of breast cancer cells by CCK8 cell proliferation assay;5.Comparison of the effect on tumor growth after 4EBP1 knockdown by tumorigenic assay in nude mice;6.Detection of relevant signaling pathways by immunoblotting experiments.7,Detection of the effect of 4EBP1 on cap-dependent translation by dual luciferase reporter gene assay.7.8,Detecting the effect of 4EBP1 on cell cycle and apoptosis after knockout by flow cytometric analysis.8.9,Observation of the effect on autophagy after 4EBP1 knockout by AO staining,electron microscopy and immunoblotting experiments.9.10,Analysis of phosphatase activity changes in cells after 4EBP1 knockdown by proteomic techniques.Results1.The results of immunohistochemistry experiments confirmed that the expression level of 4EBP1 was 69.9% in cancer tissues and 35.5% in paracancerous tissues,and the expression level was significantly higher in cancer tissues.2.The results of plate cloning assay showed that high expression of 4EBP1 could promote clone formation,and the number of clones decreased significantly after knocking down 4EBP1.3.The results of CCK8 experiment showed that high expression of 4EBP1 could promote MCF-7 and ZR-75-1 cells.4.proliferation of breast cancer cells,knockdown of 4EBP1 resulted in diminished proliferation of breast cancer cells.5.Tumorigenesis experiments in nude mice showed that tumor growth was significantly reduced after 4EBP1 knockdown,and tumor size was significantly reduced.6.The results of immunoblotting assay showed that high expression of 4EBP1 would activate mTORC1 and its downstream substrate S6 by feedback;and 4EBP1 still played a hat-dependent translation inhibition role in breast cancer.7.The results by flow cytometry experiments showed that 4EBP1 had no effect on the cycle and apoptosis of breast cancer cells,and it may promote the proliferation of breast cancer cells by inhibiting autophagy of breast cancer cells.8.the study by further experimental results showed that 4EBP1 may promote autophagy of breast cancer cells through the ATP/AMPK/mTORC1/ULK1/LC3 B signaling pathway.ConclusionsThe present experimental study showed that 4EBP1 may play an oncogene role in breast cancer,and high expression of 4EBP1 could promote the proliferation of breast cancer cells,and knockdown of 4EBP1 would inhibit the proliferative effect of the cells;further investigation of the mechanism of 4EBP1 promoting the value-added of breast cancer cells revealed that it had no significant effect on the cycle and apoptosis of breast cancer cells.However,the knockdown of 4EBP1 was found to promote autophagy in breast cancer cells,suggesting that 4EBP1 may promote the proliferation of breast cancer cells by inhibiting autophagy.To further investigate the molecular mechanism of4EBP1 inhibition in breast cancer autophagy,we used proteomic analysis to further demonstrate that 4EBP1 may affect autophagy by influencing the ATP/AMPK/mTORC1/ULK1/LC3 B signaling pathway.