A cell-penetrating peptide targeting ufl1-akt signal axis for preventing and treating tumors, and application and pharmaceutical composition thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a cell penetrating peptide for preventing and treating tumor by targeting UFL1-AKT signal axis, application and pharmaceutical composition thereof. The amino acid sequence of the cell penetrating peptide is shown as SEQ ID No. 1. The use of the cell penetrating peptide to destroy the UFL1-AKT interaction can significantly inhibit the in vitro and in vivo progression of triple-negative breast cancer, hormone receptor-positive and HER2 receptor-positive and other types of breast cancer. The inventor's discovery reveals the key role of UFL1-AKT feedback loop in triple-negative breast cancer, hormone receptor-positive and HER2 receptor-positive and other types of breast cancer, suggesting that targeting the UFL1-AKT1 axis may provide a new strategy for cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a cell membrane-penetrating peptide that targets the UFL1-AKT signaling axis to prevent and treat tumors, its application, and pharmaceutical compositions thereof. Background Technology

[0002] Triple-negative breast cancer (TNC) is the most aggressive subtype of breast cancer, characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). [1] The clinical management of triple-negative breast cancer is challenging due to the lack of targeted therapies. While commonly used standard chemotherapy drugs such as cisplatin and paclitaxel are used, their effectiveness is limited by the frequent occurrence of resistance and the higher potential of triple-negative breast cancer. [2] Therefore, there is an urgent need for new targeted therapies, which could significantly improve clinical outcomes for patients with triple-negative breast cancer.

[0003] UFMylation is a recently identified ubiquitin-like modification mediated by the E3 ligase UFL1, occurring in endoplasmic reticulum (ER) homeostasis. [3,4] ,immunity [5,6] and DNA damage response [7,8] It plays a crucial role in various cellular processes, including those involving the umbilical cord. Dysfunction of the umbilical cord system is associated with several diseases, including anemia and cancer. [9-12] New evidence suggests that UFL1 can function as a tumor suppressor or oncogene, depending on the specific tumor context. For example, p53 can undergo UFL1-mediated UFMylation modification, thereby antagonizing MDM2-mediated ubiquitination, stabilizing p53, and enhancing its tumor suppressor function in colorectal cancer.

[10] Furthermore, the absence of UFL1 / UFBP1 in hepatocytes is associated with the development of liver cancer.

[13] Conversely, UFL1-mediated UFMylation of the nuclear receptor coactivator ASC1 is crucial for 17β-estradiol-induced ERα transcriptional activation, promoting ERα-mediated tumorigenesis in vivo. [9] Furthermore, UFMylation of ERα enhances its stability and transcriptional activation, contributing to ER... + The development of breast cancer

[14] However, little is known about the substrates of UFMylation and their exact biological functions in triple-negative breast cancer, the most malignant subtype of breast cancer.

[0004] AKT, a key serine / threonine kinase, plays a crucial role in regulating various cellular processes, including cell growth, survival, metabolism, and proliferation. [15,16]In response to growth signals and stress stimuli, the pleckstrin homology (PH) domain of AKT binds to phosphatidylinositol (3,4,5)-triphosphate (PIP3), enabling it to adhere to the membrane.

[17] On the cell membrane, AKT is phosphorylated at Thr308 by phosphatidyl-dependent kinase 1 (PDK1) and at Ser473 by mammalian rapamycin complex 2 (mTORC2), leading to its complete activation. [18,19] Activated AKT then phosphorylates downstream substrates, thereby exerting their effects on cellular processes. [20,21] AKT activation is tightly regulated by multiple mechanisms. The pleckstrin homologous leucine-rich repeat protein phosphatase (PHLPP1 / 2) and protein phosphatase 2A (PP2A) complex specifically inhibits AKT through dephosphorylation.

[20] Specifically, PHLPP1 / 2 dephosphorylates AKT at Ser473. [22,23] PP2A dephosphorylates Thr308. [24,25] Furthermore, phosphatidyl and tensin homolog (PTEN) and SH2-domain-containing inositol 5-phosphatase 1 (SHIP-1) dephosphorylate PIP3, thereby reducing AKT activation and negatively regulating this pathway. [26,27] However, mutations or amplifications of upstream components, such as the loss of PI3K catalytic subunit (PIK3CA), mTOR complex components, oncogenic drivers like KRAS, and negative regulators like receptor tyrosine kinases and PTEN, can lead to increased PIP3 production, dysregulation of AKT activation, and enhanced tumor progression. [28-31] In triple-negative breast cancer, persistently abnormal AKT activation is frequently observed and plays a crucial role in cell proliferation and anti-apoptosis, and is associated with poor prognosis. [32,33] However, pathological mutations in key upstream regulators in triple-negative breast cancer, such as PIK3CA... [34,35] mTOR complex components

[36] PTEN

[35] , and KRAS

[37] It is relatively rare compared to other breast cancer subtypes. The mechanisms driving sustained AKT activation in triple-negative breast cancer are still poorly understood.

[0005] References [1] Leon-Ferre, RA & Goetz, MP BMJ (Clinical Research ed.) 381,e071674 (2023). [2] Garrido-Castro, AC, Lin, NU & Polyak, K. Cancer discovery9,176-198 (2019). [3] Liu, J. et al.Nat Commun 8, 14186 (2017). [4] Liang, JR et al.Cell 180, 1160-1177.e1120 (2020). [5] Tao, Y. et al. Cell Death Differ 30, 16-26 (2023). [6] He, J. et al.Molecular cell 84, 1120-1138.e1128 (2024). [7] Wang, Z. et al. Nucleic Acids Res 47, 4124-4135 (2019). [8] Qin, B. et al.Nat Commun 10, 1242 (2019). [9] Yoo, HM et al. Molecular cell 56, 261-274 (2014).

[10] Liu, J. et al. Nat Cell Biol 22, 1056-1063 (2020).

[11] Zhang, M. et al. Cell Death Differ 22, 1922-1934 (2015).

[12] Tatsumi, K. et al.Nat Commun 2, 181 (2011).

[13] Chen, F . et al.Journal of experimental & clinical cancer research: CR 42, 110 (2023).

[14] Yoo, HM, Park, JH, Kim, JY & Chung, CH Molecules and cells 45, 425-434 (2022).

[15] Lauring, J., Park, BH & Wolff, AC J Natl Compr Canc Netw 11,670-678 (2013).

[16] Hua, H. et al.Journal of hematology & oncology 14, 128 (2021).

[17] Bellacosa, A. et al.Oncogene 17, 313-325 (1998).

[18] Higuchi , M. , Onishi , K. , Kikuchi , C. & Gotoh , Y . Nat Cell Biol 10, 1356–1364 (2008).

[19] Sarbassov , DD , Guertin , DA , Ali , SM & Sabatini , DM Science (New York, NY) Rev. 307, 1098–1101 (2005).

[20] Manning , BD & Toker , A . Cell 169, 381–405 (2017).

[21] Basu , S. , Totty , NF , Irwin , MS , Sudol , M. & Downward , J . Molecular cell 11, 11–23 (2003).

[22] Brognard , J. , Sierecki , E. , Gao , T. & Newton , AC Molecular cell 25, 917–931 (2007).

[23] Gao , T. , Furnari , F. & Newton , AC Molecular cell 18, 13–24(2005).

[24] Padmanabhan, S. (1999). et al.Cell 136, 939–951 (2009).

[25] Ruvolo, PP et al.Leukemia 25, 1711–1717 (2011).

[26] Song , MS , Salmena , L. & Pandolfi , PP Nature reviews. Molecular cell biology 13, 283–296 (2012).

[27] Qin , S . et al. Frontiers in immunology Rev. 11, 307 (2020).

[28] Cement , E. , Hassan , D. , Bellacosa , A. & Testa , JR Cancer Res 84, 4126–4139 (2024).

[29] Miricescu, D. et al.Int J Mol Sci 22 (2020).

[30] Jin, Y. et al. Clin Cancer Res 28, 526-539 (2022).

[31] He, Y. et al.Signal transduction and targeted therapy 6,425 (2021).

[32] Khan, MA, Jain, VK, Rizwanullah, M., Ahmad, J. & Jain, K. Drug discovery today 24, 2181-2191 (2019).

[33] Shi, Z. et al. Clin Cancer Res 28, 993-1003 (2022).

[34] Shah, SP et al.Nature 486, 395-399 (2012).

[35] The Cancer Genome Atlas Network. Nature 490, 61-70 (2012).

[36] Pascual, J. & Turner, NC Annals of oncology: official journal of the European Society for Medical Oncology 30, 1051-1060 (2019).

[37] Sánchez-Muñoz, A. et al. BMC cancer 10, 136 (2010). Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a cell-penetrating peptide targeting the UFL1-AKT signaling axis for tumor prevention and treatment, along with its applications and pharmaceutical compositions. Previous research has shown that AKT directly interacts with UFL1 and undergoes UFMylation in triple-negative breast cancer (TNC), a modification crucial for AKT activation and its oncogenic function. Furthermore, AKT phosphorylates UFL1 at Thr426, establishing a positive feedback loop that maintains the high activity of two oncogenic regulators in TNC. Disruption of the UFL1-AKT interaction using the cell-penetrating peptide PDAU significantly inhibits AKT activation and suppresses TNC progression. Clinically, elevated levels of phosphorylated UFL1 (p-T426 UFL1) in TNC specimens are associated with increased pAKT expression. Therefore, the UFL1-AKT feedback loop plays a crucial role in TNC, suggesting that targeting the UFL1-AKT axis may provide a promising therapeutic strategy for cancer treatment.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a cell membrane-penetrating peptide that targets the UFL1-AKT signaling axis to prevent and treat tumors, and the amino acid sequence of the cell membrane-penetrating peptide is shown in SEQ ID No. 1.

[0008] Preferably, the tumor includes triple-negative breast cancer.

[0009] The present invention also provides the application of the cell-penetrating peptide described in the above technical solution in the preparation of drugs for the prevention and treatment of tumors.

[0010] Preferably, the cell-penetrating peptide inhibits tumor growth.

[0011] Preferably, the cell-penetrating peptide enhances the sensitivity of tumor cells to anti-tumor drugs.

[0012] Preferably, the cell-penetrating peptide reduces tumor resistance to anti-tumor drugs.

[0013] The present invention also provides a pharmaceutical composition for preventing and treating tumors by targeting the UFL1-AKT signaling axis, the pharmaceutical composition comprising one or more of the cell-penetrating peptides and AKT inhibitors described in the above technical solutions; The tumors include triple-negative breast cancer, hormone receptor-positive breast cancer, and other types of breast cancer such as HER2 receptor-positive breast cancer.

[0014] Preferred, described AKT inhibitors include MK-2206、MK-2206 dihydrochloride、MK-2206 freebase、Uprosertib、Uprosertib hydrochloride、Capivasertib、Afuresertib、Afuresertibhydrochloride、Borusertib、Ipatasertib dihydrochloride、Miransertib、Miransertib hydrochloride、Vevorisertib、Oridonin、Ipatasertib、Ipatasertib tosylate、Dehydrovomifoliol、Pifusertib hydrochloride、Pifusertib、Borussertib、Oridonin(Standard)、Chrysotoxin、Solenopsin、Perifosin、Alborixin、Isobavachalcone、Akt1 / Akt2-IN-2、Akt1 / Akt2-IN-1、AKT-IN-6、Akt1&PKA-IN-2、AKT-IN-17、AKT-IN-18、A KT-IN-9、AKT-IN-10、AKT-IN-24、AKT-IN-8、AKT-IN-13、AKT-IN-14、AKT-IN-5、AKT-IN-3、AKT-IN-2、AKT-IN-23、A KT-IN-12、AKT-IN-3、Akt1-IN-1、Akt1-IN-4、AKT-IN-7、AKT-IN-21、Akt1-IN-5、Akt1-IN-6、Akt1-IN-7、AKT-IN-14 free base、Akt1&PKA-IN-1、AKT Kinase Inhibitor、AKT-IN-25、AKT-IN-1、AKT-IN-22、AKT Kinase Inhibitor hydrochloride、Akt / ROCK-IN-1、AKT inhibitor VIII、Akt1-IN-2、PF-AKT400、Akt / mTOR-IN-1、AKT-IN-20、Borussertib、AKT inhibitor IV、Akt / SKG Substrate Peptide TFA、Akt / NF-κB / MAPK-IN-1、TAT-TCL1-Akt-in、APN / AKT-IN-1、(E)-Aktinhibitor-IV, STAT3 / AKT-IN-1, Tubulin / AKT1-IN-1, VEGFR-2 / AKT-IN-2, EGFR / Akt-IN-1, VEGFR-2 / AKT-IN-1 , PI3K / AKT-IN-2, PI3K / AKT-IN-1, PI3K / AKT-IN-3, PI3K / Akt / mTOR-IN-2, PI3K / Akt / mTOR-IN-3, PDK1 / Akt / Flt dual pathwayinhibitor, 9-Decyn-1-ol, CCT129254, 2-Chlorophenoxazine, BAY1125976, A-443654, AT7867, AT7867 dihydrochloride, ALM301, Hu7691, Hu7691 free One or more of the following: base, 3CAI, GSK-690693, CCT128930, M2698, PHT-427, TCL1(10-24), BI-69A11, FPA-124, D-87503, API-1, A-674563, AT7867 hydrochloride, SC66, 10-DEBC hydrochloride, TAS0612, HS-345, SH-5, KP372-1, ML-9, and ML-9 Free Base.

[0015] The present invention also provides the application of the pharmaceutical composition described above in the preparation of a product for the prevention and treatment of tumors targeting the UFL1-AKT signaling axis, wherein the tumors include triple-negative breast cancer, hormone receptor-positive breast cancer, and other types of breast cancer such as HER2 receptor-positive breast cancer.

[0016] The beneficial effects of this invention are: Through in-depth research, the inventors discovered that AKT directly interacts with UFL1 and undergoes UFMylation in triple-negative breast cancer. This modification is crucial for AKT activation and its oncogenic function. The inventors found that AKT1 directly interacts with UFL1 and undergoes UFMylation modification at Lys189, Lys276, and Lys297 sites, enhancing AKT phosphorylation and activity, promoting triple-negative breast cancer tumor growth and chemotherapy resistance. Simultaneously, AKT phosphorylates UFL1 at Thr426, forming a positive feedback loop that maintains the pro-cancer activity of both. Disrupting the UFL1-AKT interaction using the specific peptide PDAU (i.e., the amino acid sequence shown in SEQ ID No. 1) significantly inhibited the in vitro and in vivo progression of triple-negative breast cancer. The inventors' findings reveal the crucial role of the UFL1-AKT feedback loop in triple-negative breast cancer, suggesting that targeting the UFL1-AKT1 axis may provide a new strategy for cancer treatment. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Figures to identify UFL1 promoting breast cancer progression; where, a: shows a graph analyzing UFL1 expression in triple-negative breast cancer using the GEPIA database; b: shows a graph of representative immunohistochemical (IHC) staining of UFL1 in normal (n=40) and tumor (n=40) triple-negative breast cancer; c: The left figure shows the Western blotting after UFL1 gene silencing; in MDA-MB-231 and HCC1806 cells, cells were infected with culture medium containing pLKO.1 or sh UFL1 virus, and after selection with puromycin, cell lines with specific UFL1 knockdown were obtained, and the expression of UFL1 was detected by Western blotting. The right figure shows the cell proliferation capacity after UFL1 gene silencing; cell proliferation experiments were performed in MDA-MB-231 and HCC1806 cells, and dynamic counting was used for detection; d: shows the results of cell sensitivity to chemotherapy drugs after UFL1 gene silencing; the CCK8 kit was used to detect the sensitivity of MDA-MB-231 and HCC1806 cells to cisplatin or doxorubicin; e: the top figure shows the Western blotting after UFL1 gene silencing; in MCF-7, T47D, BT-474 and SK-BR-3 cells, cells were infected with culture medium containing pLKO.1 or sh UFL1 virus, and after selection with puromycin, cell lines with specific UFL1 knockdown were obtained, and UFL1 expression was detected by Western blotting. The following figures show the cell proliferation capacity after UFL1 gene silencing; cell proliferation experiments were performed in MCF-7, T47D, BT-474, and SK-BR-3 cells, and dynamic counting was used for detection; f: showing the results of cell sensitivity to chemotherapeutic drugs after UFL1 gene silencing; the CCK8 assay kit was used to detect the sensitivity of MCF-7, T47D, BT-474, and SK-BR-3 cells to tamoxifen, trastuzumab, or paclitaxel. g: showing the effect of UFL1 gene silencing on the growth of mouse subcutaneous xenografts; HCC1806 cells (2 × 10⁻⁶) stably expressing control or UFL1 shRNA were used. 6 Subcutaneous implantation in nude mice; when the tumor volume reaches 100 mm 3 Mice were treated with either saline or cisplatin (5 mg / kg once a week). Tumors were surgically removed and their weight was measured.

[0019] Figure 2Figures illustrating how UFL1 promotes the progression of triple-negative breast cancer and other subtypes of breast cancer by activating AKT; where: a: a graph of UFL1-related proteins identified by mass spectrometry; MDA-MB-231 cells stably expressing Flag-S-UFL1 were generated, and UFL1 interacting proteins were identified by affinity mass spectrometry analysis; b: a Western blotting graph showing the interaction between endogenous UFL1 and AKT; HCC1806 cell lysates were immunoprecipitated with control IgG, anti-UFL1, anti-AKT (pan-) or anti-AKT1 antibodies. Western blotting was used to detect the expression of related proteins; c: GST pulldown and Western blotting plots showing the interaction between purified GST, GST-AKT1, GST-AKT2 or GST-AKT3 and purified His-UFL1 protein; d: A plot showing the co-localization of UFL1 and AKT1 in triple-negative breast cancer; Immunofluorescence (IF) staining was used to detect the co-localization of endogenous UFL1 and AKT1 in MDA-MB-231 cells. e: Western blotting plot showing the expression levels of AKT and its downstream proteins; MDA-MB-231, HCC1806, MCF-7, T47D, BT-474, and SK-BR-3 cells were infected with culture medium containing control or UFL1 shRNA virus, and cell lines with specific UFL1 knockdown were obtained after selection with puromycin. Western blotting was used to detect the expression of related proteins; f: The left image shows the Western blotting plot showing the expression levels of AKT and its downstream proteins; MDA-MB-231 and HCC1806 cells were transfected with empty vector or Flag-UFL1; Western blotting was used to detect the expression of related proteins. The right figure shows the cell proliferation capacity after UFL1 overexpression; cell proliferation experiments were performed in MDA-MB-231 and HCC1806 cells, and dynamic counting was used for detection; g: shows the results of cell sensitivity to chemotherapy drugs after UFL1 overexpression; the sensitivity of MDA-MB-231 and HCC1806 cells to cisplatin or doxorubicin was detected by the CCK8 kit; h: shows the Western blotting plot of AKT and its downstream protein expression levels; MCF-7, T47D, BT-474 and SK-BR-3 cells were transfected with empty vector or Flag-UFL1; Western blotting was used to detect the expression of related proteins.

[0020] Figure 3A plot showing that UFL1-mediated UFMylation can activate AKT in triple-negative breast cancer; where, a: Western blotting plot showing AKT1 UFMylation levels; the corresponding plasmid was stably expressed in 293T cells; immunoprecipitated with S-protein agarose.b: Western blotting of AKT1 UFMylation levels; b: Western blotting plot showing in vitro AKT1 UFMylation levels; purified UFMylation components (His-UBA5, His-UFC1, His-UFL1, His-UFBP1, and His-UFM1ΔC2) were incubated with GST or GST-AKT1 in UFMylation buffer at 30°C for 90 min; Western blotting of AKT1 UFMylation levels in vitro; c: Western blotting plot showing intracellular AKT1 UFMylation levels; immunoprecipitation using anti-AKT1 antibody; Western blotting of AKT1 UFMylation levels in vivo; de: Western blotting plot showing AKT1-UFL1 interaction fragments; d shows full-length UFL1 (FL), 1-247 aa, and 248-C aa fragments; e shows stable expression of the corresponding plasmids in cells; immunoprecipitation with S-protein agarose; Western blotting of AKT1 UFMylation levels in vivo; Blotting to detect the expression of related proteins; fg: Western blotting plot showing the interaction fragments of UFL1 and AKT1; f shows AKT1 (FL), 1-148 aa, 148-410 aa, and 410-C aa fragments; g shows the stable expression of the corresponding plasmids in cells; immunoprecipitation with S-protein agarose; Western blotting to detect the expression of related proteins; h: Western blotting plot showing the fragments of AKT1 that can undergo UFMylation; stable expression of the corresponding plasmids in 293T cells; immunoprecipitation with S-protein agarose; Western blotting to detect the UFMylation level of the full-length AKT1 and different fragments; h: Western blotting plot showing the fragments of AKT1 that can undergo UFMylation; stable expression of the corresponding plasmids in cells; immunoprecipitation with S-protein agarose; Western blotting to detect the UFMylation level of the full-length AKT1 and different fragments; i: showing AKT1 Western blotting of WT and 3KR undergoing UFMylation; the corresponding plasmids are stably expressed in cells.Immunoprecipitation with S-protein agarose; Western blotting to detect UFMylation levels of AKT1 and 3KR; jk: Western blotting plot showing the interaction between AKT1 WT and 3KR and downstream proteins of AKT1; Stable expression of the corresponding plasmids in cells; Immunoprecipitation with S-protein agarose; Western blotting to detect the interaction between AKT1 and its downstream proteins; l: Western blotting plot showing the expression levels of overexpressed AKT or 3KR and its downstream proteins; In MDA-MB-231 and HCC1806 cells, cells were infected with medium containing control or AKT1 / 2 / 3 shRNA virus, and after selection with puromycin, cell lines specifically knocked down AKT1 / 2 / 3 were obtained, and transfected with empty vector or Flag-AKT WT or 3KR. Western blotting was used to detect the expression of related proteins; m: a graph showing the cell proliferation capacity after overexpression of AKT or 3KR; cell proliferation experiments were performed in MDA-MB-231 and HCC1806 cells, and dynamic counting was used for detection; n: a graph showing the results of cell sensitivity to chemotherapeutic drugs after overexpression of AKT or 3KR; the sensitivity of MDA-MB-231 and HCC1806 cells to cisplatin or doxorubicin was detected using the CCK8 kit; op: a graph showing the effect of overexpression of AKT or 3KR on the growth of mouse subcutaneous xenografts; HCC1806 cells (2 × 10⁻⁶) stably expressing control or Flag-AKT WT or 3KR were used. 6 Subcutaneous implantation in nude mice; when the tumor volume reaches 100 mm 3 Mice were treated with either saline or cisplatin (5 mg / kg once a week); tumors were surgically removed and their weight was measured. Figure 4Figures illustrating how AKT phosphorylates UFL1 at T426 to enhance UFMylation and promote the progression of triple-negative breast cancer; where: a: Western blotting plot showing AKT1 UFMylation levels after capacitinib treatment; the corresponding plasmid was stably expressed in 293T cells; immunoprecipitation with S-protein agarose; AKT1 UFMylation levels detected by Western blotting; b: Western blotting plot showing AKT phosphorylation of UFL1; the corresponding plasmid was stably expressed in 293T cells; immunoprecipitation with S-protein agarose; UFL1 phosphorylation levels detected by Western blotting; c: Western blotting plot showing AKT phosphorylation of UFL1; the corresponding plasmid was stably expressed in 293T cells and treated with capacitinib; immunoprecipitation with S-protein agarose; Western blotting; Blotting to detect UFL1 phosphorylation levels; d: Plot showing the amino acid sequence around the T426 residue in UFL1 across different species; e: Western blotting plot showing UFL1 phosphorylation levels; The corresponding plasmid was stably expressed in 293T cells. Immunoprecipitation was performed using anti-Flag antibody.Western blotting was used to detect the phosphorylation level of UFL1; f: Western blotting plot showing the phosphorylation level of UFL1; HCC1806 cells were treated with DMSO or a specified concentration of capapasetinib for 24 hours, and Western blotting was used to detect the phosphorylation level of UFL1; g: Western blotting plot showing the phosphorylation level of UFL1; HCC1806 cells stably expressing control or AKT1 / 2 / 3 shRNA were constructed, and Western blotting was used to detect the phosphorylation level of UFL1; h: Western blotting plot showing the AKT1 UFMylation level; The corresponding plasmid was stably expressed in 293T cells; Immunoprecipitation was performed using S-protein agarose; Western blotting was used to detect the AKT1 UFMylation level; i: Western blotting plot showing the expression level of overexpression of UFL1 or T426A and its downstream proteins; HCC1806 cells were infected with culture medium containing control or Flag-UFL1 or T426A virus, and Western blotting was used to detect the phosphorylation level of UFL1 or T426A. Blotting to detect the expression of related proteins; j: A graph showing the cell proliferation capacity after overexpression of UFL1 or T426A; Cell proliferation experiments were performed in HCC1806 cells, and dynamic counting was used for detection; k: A graph showing the results of cell sensitivity to chemotherapy drugs after overexpression of UFL1 or T426A; CCK8 kit was used to detect the sensitivity of MDA-MB-231 and HCC1806 cells to cisplatin or doxorubicin. Figure 5Figure 1 shows the inhibition of UFL1-AKT interaction on the progression of triple-negative breast cancer. In figure 1, a: Western blotting of AKT1-UFL1 interaction fragments; HA-S-UFL1 (FL), 1-247 aa, 10-247 aa, 20-247 aa, 30-247 aa, and 40-247 aa fragments are stably expressed in cells; immunoprecipitation with S-protein agarose was performed. Western blotting was used to detect the expression of related proteins. Immunoprecipitation with S-protein agarose; Western blotting to detect the expression of related proteins; b: Western blotting plot showing the interaction fragment between AKT1 and UFL1 in vitro; purified recombinant GST, GST-UFL1 truncated mutant and purified His-AKT1 were incubated in vitro. Western blotting to detect the expression of related proteins; c: Plot showing two TAT-Peptides from UFL1; PDAU (20-30 aa) and NP (1-10 aa); d: Plot showing the interaction between UFL1 and AKT after treatment with two TAT-Peptides; stable expression of empty vector or Flag-UFL1 in cells, and treatment of cells with ddH2O, PDAU or NP (10 μM), followed by immunoprecipitation with S-protein agarose.Western blotting was used to detect the expression of related proteins; e: This plot shows the AKT1 UFMylation level after treatment with two TAT-Peptides; The corresponding plasmids were stably expressed in cells and treated with ddH2O, PDAU, or NP (10 μM), followed by S-protein agarose immunoprecipitation; Western blotting was used to detect the expression of related proteins; f: This plot shows the Western blotting of AKT1 and its downstream protein expression levels after treatment with two TAT-Peptides; MDA-MB-231 and HCC1806 cells were treated with ddH2O, PDAU, or NP (10 μM), and Western blotting was used to detect the expression of related proteins; Blotting to detect the expression of related proteins; g: plot showing cell proliferation capacity after treatment with two TAT-Peptides; cell proliferation experiments were performed in MDA-MB-231 and HCC1806 cells, and dynamic counting was used for detection; h: plot showing the results of cell sensitivity to chemotherapy drugs after treatment with two TAT-Peptides; CCK8 kit was used to detect the sensitivity of MDA-MB-231 and HCC1806 cells to cisplatin or doxorubicin; ij: plot showing the effect of treatment with two TAT-Peptides on the growth of subcutaneous xenografts in mice; triple-negative breast cancer patient-derived xenografts (PDXs) were subcutaneously implanted into nude mice; when the tumor volume reached 100 mm. 3Mice were intraperitoneally injected with saline, PDAU, or NP (15 mg / kg) every 2 days; then treated with saline or cisplatin (5 mg / kg once a week) (n=6 per group); tumors were surgically removed and tumor weight was measured; k: shows the AKT1 UFMylation level in subcutaneous xenografts of mice after treatment with two TAT-Peptides; subcutaneous xenografts of mice treated with two TAT-Peptides were lysed and subjected to immunoprecipitation, and the expression of related proteins was detected by Western blotting; k: shows the AKT1 UFMylation level in subcutaneous xenografts of mice after treatment with two TAT-Peptides; subcutaneous xenografts of mice treated with two TAT-Peptides were lysed and subjected to immunoprecipitation, and the expression of related proteins was detected by Western blotting; l: shows pAKT T308, pAKT S473, and pUFL1 in subcutaneous xenografts of mice after treatment with two TAT-Peptides. Plot of T426 levels; lysis of mouse subcutaneous xenografts treated with two TAT-Peptides, and Western blotting to detect the expression of related proteins; m: plot showing the levels of H&E, Ki67, and Cleaved PARP1 in mouse subcutaneous xenografts treated with two TAT-Peptides; IHC detection of the expression of related proteins. Detailed Implementation

[0021] This invention provides a cell membrane-penetrating peptide that targets the UFL1-AKT signaling axis to prevent and treat tumors. The amino acid sequence of the cell membrane-penetrating peptide is shown in SEQ ID No. 1, and is as follows: GRKKRRQRRRGGFAEATQRLSER.

[0022] In this invention, the tumor preferably includes other types of breast cancer such as triple-negative breast cancer, hormone receptor-positive breast cancer, and HER2 receptor-positive breast cancer.

[0023] This invention also provides the application of the cell-penetrating peptide described in the above-mentioned technical solution in the preparation of drugs for the prevention and treatment of tumors. This invention does not impose any special limitations on the dosage form or type of the drug; any medically acceptable dosage form of the cell-penetrating peptide can be used. In this invention, the cell-penetrating peptide inhibits tumor growth. In this invention, the cell-penetrating peptide enhances the sensitivity of tumor cells to anti-tumor drugs. In this invention, the cell-penetrating peptide reduces the drug resistance of tumors to anti-tumor drugs.

[0024] This invention provides a pharmaceutical composition for the prevention and treatment of tumors by targeting the UFL1-AKT signaling axis. The pharmaceutical composition includes one or more of the cell-penetrating peptide, AKT inhibitor, and UFL1 inhibitor described in the above-mentioned technical solution. The tumors include triple-negative breast cancer, hormone receptor-positive breast cancer, and other types of breast cancer such as HER2 receptor-positive breast cancer.

[0025] In the present invention, said AKT inhibitor preferably includes MK-2206, MK-2206 dihydrochloride, MK-2206 free base, Uprosertib, Uprosertib hydrochloride, Capivasertib, Afuresertib, Afuresertib hydrochloride, Borusertib, Ipatasertib dihydrochloride, Miransertib, Miransertib hydrochloride, Vevorsertib, Oridonin, Ipatasertib, Ipatasertib. tosylate、Dehydrovomifoliol、Pifusertib hydrochloride、Pifusertib、Borussertib、Oridonin (Standard)、Chrysotoxine、Solenopsin、Perifosine、Alborixin、Isobavachalcone、Akt1 / Akt2-IN-2、Akt1 / Akt2-IN-1、AKT-IN-6、Akt1&PKA-IN-2、AKT-IN-17、AKT-IN-18、AKT-IN-9、AKT-IN-10、AKT-I N-24、ACT-IN-8、ACT-IN-13、ACT-IN-14、ACT-IN-5、ACT1-IN-3、ACT-IN-2、ACT-IN-23、ACT-IN-12、AK T-IN-3、Akt1-IN-1、Akt1-IN-4、ACT-IN-7、ACT-IN-21、Akt1-IN-5、Akt1-IN-6、Akt1-IN-7、ACT-IN-14 free base、Akt1&PKA-IN-1、AKT Kinase Inhibitor、AKT-IN-25、AKT-IN-1、AKT-IN-22、AKT Kinase Inhibitorhydrochloride、Akt / ROCK-IN-1、AKT Inhibitor VIII、Akt1-IN-2、PF-AKT400、Akt / mTOR-IN-1、AKT-IN-20、Borussertib、AKT Inhibitor IV、Akt / SKG Substrate PeptideTFA, Akt / NF-κB / MAPK-IN-1, TAT-TCL1-Akt-in, APN / AKT-IN-1, (E)-Akt inhibitor-IV, STAT3 / AKT-IN-1, Tubulin / AKT1-IN-1, VEGFR-2 / AKT-IN-2, EGFR / Akt-IN-1, VEGFR-2 / AKT-IN-1 , PI3K / AKT-IN-2, PI3K / AKT-IN-1, PI3K / AKT-IN-3, PI3K / Akt / mTOR-IN-2, PI3K / Akt / mTOR-IN-3, PDK1 / Akt / Flt dual pathway Inhibitor, 9-Decyn-1-ol, CCT129254, 2-Chlorophenoxazine, BAY1125976, A-443654, AT7867, AT7867 dihydrochloride, ALM301, Hu7691, Hu7691 free base, 3CAI, GSK-690693, CCT128930, M2698, PHT-427, TCL1(10-24), BI-69A11, FPA-124, D-87503, API-1, A-674563 hydrochloride, AT7867 hydrochloride, SC66, 10-DEBC hydrochloride, TAS0612, HS-345, SH-5, KP372-1, ML-9 and ML-9 Free Base, one or more of these.

[0026] The present invention also provides the application of the pharmaceutical composition described above in the preparation of a product for the prevention and treatment of tumors targeting the UFL1-AKT signaling axis, wherein the tumors include triple-negative breast cancer, hormone receptor-positive breast cancer, and other types of breast cancer such as HER2 receptor-positive breast cancer.

[0027] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 Experimental methods 1.1 Cell Culture Breast cancer cell lines (MDA-MB-231, MCF-7, BT-474) and HEK293T were cultured in DMEM medium containing 10% FBS. Breast cancer cells HCC1806 and T47D were cultured in RPMI-1640 medium containing 10% FBS. SK-BR-3 cells were cultured in McCoy's 5A medium containing 10% FBS. All cells were cultured in a 37°C incubator containing 5% CO2. Cells were passaged and cultured according to their growth rate and condition, and cells with good growth and a clean background were used for subsequent experiments.

[0029] 1.2 Cell cryopreservation Centrifuge cells in good condition and at appropriate density at 800 rpm for 5 min, and discard the supernatant. Prepare cryopreservation solution by mixing culture medium containing 10% FBS and DMSO at a volume ratio of 9:1. Add about 1 mL of cryopreservation solution to the tube, then mix the cells evenly and transfer the solution to the cryopreservation tube. Place the tube in a cryopreservation box containing isopropanol and store it at -80 °C for about 24 hours. After that, transfer the cryopreservation tube to liquid nitrogen for long-term storage.

[0030] 1.3 Cell resuscitation Remove the frozen cells from liquid nitrogen and thaw them in a 37 ℃ water bath for 5 min. Centrifuge at 800 rpm for 5 min, discard the supernatant, add an appropriate amount of culture medium to resuspend the cells and transfer them to a culture dish. Add an appropriate amount of culture medium to the culture dish and mix well using the cross-hatching method.

[0031] 1.4 Preparation of Virus Add 1 mL of Opti-MEM to each 1.5 mL EP tube, and add 30 μL of PEI (1 mg / mL) at a PEI:plasmid ratio of 3:1. Mix by inversion and let stand for 5 min. Then add 5 µg of the target plasmid, 3.75 μg of the packaging plasmid PSPAX3, and 1.25 μg of the packaging plasmid PMD2G (the specific ratio is (g): packaging plasmid: target plasmid 1:1; PSPAX3:PMD2G in the packaging plasmid 3:1), for a total of 10 µg of plasmid. Mix by inversion and let stand for 20 min. Replace the 293T cells with serum-free DMEM medium, and then add the above MIX dropwise to the 293T cells that are in good growth condition and have a density of 80%. After 12-16 h, replace the 293T cell medium with DMEM medium containing 10% FBS and continue culturing. Forty-eight hours after transfection, the virus-containing supernatant was carefully aspirated using a sterile 10 mL syringe, filtered through a sterile 0.45 µm microporous membrane, collected, and aliquoted into sterile 4 mL centrifuge tubes. Store at -20°C or -80°C for later use, thawing at 4°C before use.

[0032] 1.5 Plasmid transient transformation HEK293T cells in good growth condition were digested with trypsin, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 2 mL of fresh culture medium, counted, and the cell density was adjusted before plating. After 24 h of plating, the medium was changed. 12 h after the medium change, the medium was replaced with serum-free medium. Simultaneously, sterile 1.5 mL EP tubes were labeled, and 300 μL of Opti-MEM was added to each tube. 9 μL of PEI (1 mg / mL) was added to each tube at a PEI:plasmid ratio of 3:1. The mixture was inverted and incubated for 5 min. Then, 3 µg of the target plasmid was added to each tube, inverted and incubated for 15 min. The plasmid was then added dropwise to cell culture dishes, which were shaken well and placed in a cell culture incubator. 12 h after transfection, the medium was discarded, and the cells were cultured in medium containing 10% FBS. Cells were collected 48 h after transfection for subsequent experiments.

[0033] 1.6 Lentiviral infection of cells Cells were seeded in six-well plates and allowed to adhere for 24 hours until the cells reached approximately 50% confluence. The original culture medium was then aspirated, and 1 mL of normal culture medium and 1 mL of virus solution were added. Next, 30 µL of 1M HEPES (pH 7.4) and 3 µL of Polybrene (8 mg / mL) were added (1 M HEPES: medium = 1:100, Polybrene: medium = 1:1000). The mixture was thoroughly mixed and incubated. The medium was changed 16 hours after infection. 24 hours after infection, the culture medium was aspirated, and 1 mL of culture medium, 1 mL of virus solution, and 2 µL of polybrene were added for a second infection. 24 hours later, puromycin (1 mg / mL) was added to select cells (puromycin: medium (µL) = 1:1000).

[0034] 1.7 Protein sample preparation and immunoblotting Aspirate the intracellular culture medium, wash once with PBS, discard the PBS, then add 1 mL of PBS solution. Scrape the cells off with a spatula and collect them in a 1.5 mL EP tube. Centrifuge at 3500 rpm for 5 min at room temperature, discarding the supernatant. Depending on the size of the cell clumps, resuspend the cells in the appropriate volume of NETN lysis buffer and lyse them on ice for 30 min. Sonicate the cells on ice at a typical intensity of 20%, sonicating for 5 s, pausing for 3 s, until the cell suspension is clear. Centrifuge at 12000 rpm for 20 min at 4 °C, collecting the supernatant. Add 6× Loading buffer and incubate at 96 °C for 5 min. After gently shaking in a handheld centrifuge, perform Western blotting. Transfer the protein to a PVDF membrane at a constant voltage of 100 V for 90 min, then at a constant current of 250 mA for 90 min. Block with 5% skim milk for 60 min. Wash three times with TBST and incubate with primary antibody overnight at 4 °C. The membrane was then washed three times with TBST, incubated with secondary antibody for 60 min, and then washed three more times with TBST. The membrane was scanned using an AI 600 ultrasensitive multi-functional imager, developed with ultrasensitive ECL chemiluminescence solution, and the band information was labeled.

[0035] 1.8 GST-pulldown Experiment 1) Transformation: Use the plasmids required for BL21 competent transformation (generally Pet28α vector with His tag; 4T-1 vector with GST tag respectively), spread on solid LB culture plates, and incubate at 37 ℃ for 16 h; 2) Use a sterile pipette tip to select the single clones grown on the LB solid culture dish, add them to a sterile 50mL centrifuge tube, add another 25mL of sterile liquid LB medium, and add the antibiotic corresponding to the target plasmid. 3) Expand the culture on a shaker at 37 ℃ until the OD value at 450 nm reaches 0.4-0.6, then stop shaking. At this point, freeze the bacteria (bacterial solution: 50% glycerol = 1:1), label the frozen bacteria as BL21 transformed, and store them at -80 ℃ for later use. Also, extract protein from 1 mL of the bacterial solution and prepare a protein sample as a control before IPTG induction. 4) The remaining protein expression was induced by adding 0.1% IPTG (IPTG: liquid LB medium = 1:1000) and inducing in a shaker at 18 ℃ for 16 h; 5) After induction, aspirate 1 mL of bacterial culture, extract protein and prepare protein sample. Perform Western blotting verification together with the control group before IPTG induction. Immerse the gel in Coomassie Brilliant Blue staining solution and stain on a shaker at room temperature for 1 h. After staining, wash the gel with Coomassie Brilliant Blue elution solution and incubate on a shaker at room temperature overnight to verify whether the target protein is expressed. Then perform immunoprecipitation. 6) Centrifuge the remaining bacterial suspension at 4000 rpm for 10 min, and discard the supernatant after centrifugation. Add 4 mL of lysis buffer and lyse on ice for 30 min. Then, sonicate on ice at 40% intensity for 10 min, sonicating for 5 s, pausing for 3 s. After the bacterial suspension becomes clear, aliquot the bacterial suspension into two 2 mL EP tubes. 7) Centrifuge at 12000 rpm for 20 min at 4 ℃ and collect the supernatant. Add 20-30 μL of GST beads to the bacterial lysate labeled with GST and incubate on a shaker at 4 ℃ for 4 h; 8) Wash the beads three times with NETN stock solution in a centrifuge at 4 ℃. The centrifuge speed is 6800 rpm for 30 s. Then rotate the EP tube 180° and centrifuge again under the same conditions. Discard the NETN stock solution and add 500 μL of fresh NETN stock solution. This counts as one bead wash. Wash the beads three more times with PBS following the above procedure. 9) Add 300 μL of PBS to the washed beads and resuspend them. Take 80 μL of the resuspended liquid. 10) Centrifuge the above liquid at 4 ℃ and 6800 rpm for 30 s, discard the supernatant, add 50 μL of 2×LoadingBuffer, and boil in a metal bath at 95 ℃ for 5 min. 11) After boiling, perform Western blotting to verify whether the target protein is expressed. After electrophoresis, carefully cut the SDS-PAGE and soak it in Coomassie Brilliant Blue staining solution. Stain on a shaker at room temperature for 1 h. After staining, recover the Coomassie Brilliant Blue solution, wash the gel with Coomassie Brilliant Blue elution buffer, and shake on a shaker at room temperature overnight. Verify whether the target protein has been purified according to the molecular weight of the bands, and adjust the concentration to be consistent with the expression intensity using PBS. 12) Add the remaining beads to the His-tagged bacterial lysate in the same volume, incubate at 4 °C on a shaker for 4 h, then wash the beads using the steps in 8). After the last wash, discard the supernatant and add 50 μL of 2×Loading Buffer. Boil in a 95 °C metal bath for 5 min. 13) Western blotting to verify the existence of in vitro interactions.

[0036] 1.9 Immunoprecipitation assay 1) Seed HEK293T, triple-negative breast cancer HCC1806, or MDA-MB-231 cells in 60 mm culture dishes. When the cell density is about 80-90%, collect the cells into 1.5 mL EP tubes. 2) Centrifuge the collected cells at 3000 rpm at room temperature for 5 min, discard the supernatant, and wash once with PBS; 3) Centrifuge the cells at 4000 rpm at room temperature for 5 min, discard the PBS, then add 500 μL of NETN lysis buffer and mix by pipetting. Incubate on ice for 30 min to lyse. 4) Disrupt cells by sonication, then centrifuge at 13000 rpm at 4 ℃ for 10 min; 5) Add 400 μL of cell lysis buffer to a new 1.5 mL EP tube. Add antibody (2 μg) (IgG for the control group) and 15 μL of protein A / G beads to the cell lysis buffer and incubate overnight at 4 °C with a shaker to obtain the IP sample. Take 100 μL of cell lysis buffer to prepare a protein sample for Input (whole cell lysis buffer). 6) Wash the beads 4 times with NETN stock solution in a centrifuge at 4 ℃. The centrifuge speed is 6800 rpm for 30 s. Then rotate the EP tube 180 degrees and centrifuge again under the same conditions. Discard the NETN stock solution and add 500 μL of fresh NETN stock solution; this counts as one bead wash. Perform the entire process on ice. 7) During the last wash of the beads, centrifuge at 4 ℃, 6800 rpm for 30 s, discard the supernatant, add 50 μL of 2×Loading Buffer, and heat in a 95 ℃ metal bath for 5 min to denature. 8) Western blotting verification.

[0037] 1.10 UFMylation Experiment 1) In vivo UFMylation assay: HEK293T cells were transfected with the AKT1 construct and UFMylation system components (Myc-UBA5, Myc-UFC1, Myc-UFL1, Myc-UFBP1, HA-UFM1ΔC2). Endogenous AKT1 UFMylation was detected. MDA-MB-231 and HCC1806 cells were treated with MG132 (10 μM) for 10 h before harvest. After 48 h, cells were centrifuged at 4000 rpm for 5 min and washed with PBS. The PBS was discarded, and the cells were boiled in 50 μL of Buffer 1 for 10 min. Then, 950 μL of Buffer A was added to dilute the cells 20-fold, and immunoprecipitation was performed using indicated antibody beads. Western blotting was used to detect the UFMylation level of the precipitated proteins. 2) In vitro UFMylation experiment: GST-AKT1 was expressed in BL21 competent cells, induced by IPTG, and purified using glutathione agarose. His-UBA5, His-UFC1, His-UFL1, and His-UFM1 ΔC2 were expressed in BL21 cells, induced by IPTG, and purified using Ni-NTA agarose. The purified His-UBA5 (0.1 μM), His-UFC1 (0.1 μM), His-UFL1 (0.1 μM), His-UFBP1 (0.1 μM), His-UFM1 ΔC2 (0.1 μM), and GST-AKT1 (0.1 μM) were mixed in a reaction buffer containing 5 mM ATP and 10 mM MgCl2 (containing 0.05% bovine serum albumin, 50 mM HEPES, pH 7.5) and incubated at 30 °C for 90 min. Add 2×Loading Buffer and boil for 10 minutes.

[0038] 1.11 Dynamic Counting Experiment 1) After trypsin digestion, stable triple-negative breast cancer cell lines were resuspended in DMEM medium containing 10% FBS and collected in sterile 4 mL centrifuge tubes. The cells were centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in 2 mL of DMEM medium. 2) Count the cells and adjust the cell density; seed 4 × 10⁶ cells per well in a 6-well plate. 4 Add 2 mL of DMEM medium to each cell and mix well. Repeat the experiment in 3 replicates per group. 3) Starting from the day of plating, denote day 0. Count cells at the same time interval on days 1, 2, 3, 4, 5, 6, 7, and 8 after plating. Digest and resuspend cells, counting 3-4 times per well. Statistical analysis and plot a curve.

[0039] 1.12 Cell proliferation-toxicity assay 1) Perform cell proliferation-toxicity assays according to the CCK-8 assay instructions. 2 × 10⁶ cells were placed in a container... 3 The target stable cell lines were plated in 96-well plates, and 100 μL of the corresponding cell culture medium containing 10% FBS was added to each well. Each group had 3-4 auxiliary wells, and the cells were incubated at 37 ℃ for 24 h. 2) Add 50 μL of different concentrations of the drug (diluted 3-fold with DMEM containing 10% serum) to each well of a 96-well plate and incubate at 37°C for 72 h. 3) Add 15 μL of CCK-8 solution to each well and incubate at 37 ℃ for 1-4 h; 4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at a wavelength of 450 nm.

[0040] 1.13 Mouse subcutaneous tumor experiment 1) Mouse subcutaneous tumor assay (CDX) HCC1806 cells (2×10⁻⁶) 6 Each tumor cell was resuspended in 100 μL of PBS and then subcutaneously injected into the flank of 4- to 6-week-old female BALB / c nude mice. Tumor volume was determined every two days by measuring the major and minor diameters of the tumor using calipers, calculated as follows: V = a (minor diameter) 2 ×b (long diameter) × 0.4. When the tumor volume reaches 100mm. 3 Mice were randomly divided into two groups and injected intraperitoneally once a week with either saline or cisplatin (5 mg / kg) (n = 6). After six weeks, the mice were euthanized and tumor weight was measured.

[0041] 2) Xenograft models (PDX) derived from triple-negative breast cancer patients. PDX from triple-negative breast cancer patients was cut into 3×3×3mm pieces using sterile surgical instruments. 3 Small tumor fragments were implanted into the backs of mice using an inoculation needle. Tumor volume was measured three times weekly using calipers. When the tumor volume reached 100 mm², [the tumor was considered complete]. 3 Mice were intraperitoneally injected every two days with saline, PDAU, or NP (15 mg / kg). Then, mice (n=6) were treated with saline or cisplatin (weekly, 5 mg / kg). Mice were euthanized at designated times, and tumor weight was measured. Serum from each mouse was collected and sent to Wuhan Saiwei Biotechnology Co., Ltd., China, for liver function (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBIL), total bilirubin (TBIL)) and kidney function (creatinine (CRE), uric acid (UA)) analysis.

[0042] 1.14 Plasmid Transformation Remove DH5α or BL21 competent E. coli cells from a -80 ℃ freezer (DN5α for plasmid amplification, BL21 for protein expression), and quickly place them on ice. Once the competent cells are completely thawed, transfer 30 µL to a 1.5 mL sterile EP tube, then add 2 µL of the target plasmid and mix well. Incubate on ice for 30 min. Place the mixture in a water bath and heat-shock at 42 ℃ for 90 s. Allow to recover on ice for 3-5 min. Add 200 µL of sterile, antibiotic-free LB medium and incubate at 37 ℃ with shaking for 1 h. Finally, transfer the bacterial culture from the 1.5 mL EP tube to a 50 mL sterile centrifuge tube, add approximately 25 mL of sterile LB medium, and add ampicillin or kanamycin corresponding to the target plasmid at a 1:1000 ratio. Incubate with shaking for 16 h.

[0043] 1.15 Plasmid Extraction Centrifuge the transformed bacterial culture at 4000 rpm for 10 min and discard the supernatant. Add 250 µL of Buffer BL to the adsorption column AC and centrifuge at 12000 g for 1 min to activate the adsorption column. Add 200 µL of Buffer S1 containing RNase A enzyme to a 50 mL centrifuge tube containing E. coli, and repeatedly pipet to resuspend and mix the bacterial clumps. Transfer the mixture to a 1.5 mL EP tube. Add 200 µL of Buffer S2 to the mixture and gently invert and mix 4-6 times to ensure complete bacterial lysis. Add 200 µL of Buffer S3 and gently invert and mix 4-6 times until a white flocculent precipitate is visible. Centrifuge at 12000 g for 10 min. Transfer the supernatant from centrifugation to the activated adsorption column AC and centrifuge at 12000 g for 1 min. Discard the waste liquid and return the adsorption column to the empty collection tube. Add 700 µL of Buffer W2 (with the appropriate volume of anhydrous ethanol) to the adsorption column and centrifuge at 12000 g for 1 min. Discard the waste liquid and return the adsorption column to the empty collection tube. Wash the adsorption column once more with 700 µL of Buffer W2, following the same steps. Discard the waste liquid and centrifuge the empty column at 12000 g for 2 min. Remove the adsorption column and place it in a sterile 1.5 mL EP tube. Incubate at room temperature for 2 min, add 50 µL of Eluent, and incubate at room temperature for approximately 2 min. Centrifuge at 12000 g for 2 min. After centrifugation, discard the adsorption column and collect the eluent. Run a gel electrophoresis to verify that the plasmid band positions are as expected and measure the concentration for later use.

[0044] 1.16 Immunohistochemical staining First, bake the slides in an oven at 60°C for 30 minutes. Then, soak the slides in xylene I for 10 minutes, xylene II for 10 minutes, and xylene III for 10 minutes. Next, elute with a gradient of ethanol: soak in 100% ethanol I for 3 minutes, 100% ethanol II for 3 minutes, 95% ethanol for 3 minutes, 85% ethanol for 3 minutes, and 75% ethanol for 3 minutes. Wash twice with double-distilled water for 2 minutes each time. Wash once with PBS for 5 minutes each time. Then, perform antigen retrieval: place the slides in 1× sodium citrate tissue antigen retrieval solution (100× sodium citrate tissue antigen retrieval solution diluted with double-distilled water), and microwave on high for about 5 minutes until small bubbles appear in the retrieval solution. Then heat on high for 5 seconds, pause for 30 seconds as one cycle, and repeat 18-25 times. Cool to room temperature. Wash twice with double-distilled water for 2 minutes each time, and wash three times with PBS for 3 minutes each time. Endogenous peroxides were blocked in 3% hydrogen peroxide for 10 min. The slides were washed twice with double-distilled water for 3 min each time, and three times with PBS for 5 min each time. Blocked with 0.5% BSA for 1 h. Primary antibody was applied, and the slides were incubated overnight at 4 °C. The next day, the slides were removed and allowed to reach room temperature for about 10 min. The slides were washed twice with double-distilled water for 2 min each time, and three times with PBS for 3 min each time. Secondary antibody was applied for 1 h. The slides were washed twice with double-distilled water for 5 min each time, and three times with PBS for 5 min each time. DAB staining was performed in the dark, and the reaction was stopped by soaking in water as needed based on the staining progress. The slides were washed once with double-distilled water for 5 min each time. Hematoxylin counterstaining was performed for 1 min. The slides were rinsed with running water for 30 min to achieve blue staining. The tissue was then dehydrated: soaked in 75% ethanol for 3 min, 95% ethanol for 3 min, and 100% ethanol for 3 min. The slides were then mounted in xylene.

[0045] Statistical analysis All data in this paper were analyzed using GraphPad Prism 9.3 software. Each experiment was repeated three times to ensure reproducibility. Experimental data are presented as mean ± standard deviation (Mean ± SD). Differences between two groups were compared using a t-test, and differences between multiple groups were compared using one-way ANOVA analysis of variance and Tukey's test: compare all pairs of columns. A p-value < 0.05 was considered statistically significant.

[0046] Experimental Example 1 UFL1 promotes the progression of triple-negative breast cancer by activating AKT. To investigate the role of UFL1 in triple-negative breast cancer, the inventors analyzed data from the Cancer Genome Atlas (TCGA) and found that UFL1 expression was significantly upregulated in triple-negative breast cancer samples compared to normal breast tissue. Figure 1 (a). Immunohistochemistry further confirmed the increased UFL1 protein level in triple-negative breast cancer samples. Figure 1 (b) To explore the functional role of UFL1 in triple-negative breast cancer, the inventors depleted UFL1 in the triple-negative breast cancer cell lines MDA-MB-231 and HCC1806, and found that UFL1 depletion significantly inhibited cell proliferation and enhanced sensitivity to chemotherapeutic drugs. Figure 1 c and Figure 1 (d). It is also noteworthy that these results are consistent across other breast cancer subtypes, including estrogen receptor-positive (MCF-7 and T47D) and HER2-positive (BT-474 and SK-BR-3) cell lines. Figure 1 China and Figure 1 (f). In vivo xenograft experiments also showed that UFL1 depletion significantly inhibited the progression of triple-negative breast cancer (f). Figure 1 (g). These findings highlight UFL1 as a promising therapeutic target for overcoming chemotherapy resistance in triple-negative breast cancer.

[0047] Despite mounting evidence suggesting the role of UFL1-mediated UFMylation in cancer and other diseases, specific inhibitors targeting UFMylation have yet to emerge. Therefore, identifying UFL1-specific substrates for triple-negative breast cancer and targeting the UFL1-mediated oncogenic pathway in triple-negative breast cancer holds promise as a novel therapeutic strategy for this aggressive malignancy. To identify potential triple-negative breast cancer-specific substrates for UFL1, the inventors performed tandem affinity purification and mass spectrometry analysis using MDA-MB-231 cells stably expressing Flag-S-UFL1. The inventors discovered that both AKT1 and AKT2 were identified as potential UFL1 interacting proteins. Figure 2 (a) Co-immunoprecipitation assays confirmed the interaction between UFL1 and pan-AKT, and further validation in HCC1806 cells showed an endogenous interaction between UFL1 and AKT1 ( Figure 2 (b) In vitro binding assays showed that GST-AKT1, rather than GST alone, interacted directly with recombinant His-UFL1, while the interaction between UFL1 and AKT2 / AKT3 was relatively weak. Figure 2 (c). Immunofluorescence staining also showed that UFL1 and AKT1 co-localized in MDA-MB-231 cells. Figure 2 (d).

[0048] Given AKT's central role in cellular processes such as cell proliferation and apoptosis, and its dysregulation in various cancers, the inventors next investigated whether UFL1 regulates AKT stability or activation in triple-negative breast cancer. Figure 2 As shown in Figure e, UFL1 depletion in MDA-MB-231 and HCC1806 cells led to a significant reduction in AKT phosphorylation, as well as reduced phosphorylation of downstream targets GSK-3β and PRAS40, without affecting their total protein levels. These results are consistent across various breast cancer subtypes, including estrogen receptor-positive (MCF-7 and T47D) and HER2-positive (BT-474 and SK-BR-3) breast cancer cell lines. Figure 2 (e) indicates that UFL1-driven AKT activation is a common regulatory mechanism in breast cancer. Conversely, overexpression of Flag-UFL1 enhances phosphorylation of AKT, GSK-3β, and PRAS40 ( Figure 2 f and Figure 2 (h).

[0049] To assess the functional significance of UFL1-mediated AKT activation in the progression of triple-negative breast cancer, the inventors treated UFL1-overexpressing cells with carpacitinib (a pan-AKT kinase inhibitor). Figure 2 f and Figure 2 As shown in Figure g, inhibiting AKT activity significantly suppressed cell proliferation and enhanced chemosensitivity to cisplatin and doxorubicin. These results collectively suggest that UFL1 promotes the progression of triple-negative breast cancer by activating AKT.

[0050] Experimental Example 2 UFL1 was used for UFMylation at Lys189, Lys276, and Lys297 sites of AKT1. Since UFL1 functions as an E3 ligase responsible for UFMylation, the inventors investigated whether UFL1 modifies AKT1 through this mechanism. In cells co-transfected with components of the UFMylation system, including UBA5, UFC1, UFL1, UFM1 ΔC2 (the active form of UFM1), and UFBP1, UFMylation signaling in AKT1 was detected by immunoprecipitation, showing a 20 kDa shift compared to unmodified AKT1. Figure 3 (a). Similar results were also observed in in vitro UFMylation analysis ( Figure 3 (b) UFMylation of endogenous AKT1 was also observed in MDA-MB-231 and HCC1806 cells, and this modification was significantly reduced after UFL1 depletion. Figure 3(c) To determine the UFMylation site on AKT1, the inventors discovered that the N-terminal region of UFL1 (amino acids 1-247) interacts with AKT1. Figure 3 (middle d and 3 middle e). Further analysis showed that the kinase domain of AKT1 (amino acids 148-410) is responsible for this function. Figure 3 (f and g in 3). Furthermore, UFMylation exists on both the full-length AKT1 and the kinase domain (amino acids 148-410). Figure 3 The inventors discovered that mutants of K189R, K276R, and K297R (3KR) almost completely eliminated AKT1 UFMylation. Figure 3 (i). These results indicate that AKT1 can be UFMylated both in vivo and in vitro, with Lys189, Lys276, and Lys297 being key sites for UFMylation.

[0051] Experimental Example 3 In triple-negative breast cancer, UFL1-mediated UFMylation can activate AKT. Given that UFL1 catalyzes UFMylation of AKT1 and is crucial for AKT activation in triple-negative breast cancer, the inventors next investigated whether UFL1-mediated AKT1 UFMylation plays a role in AKT activation. The inventors hypothesized that UFMylation might regulate AKT phosphorylation by influencing its interaction with specific kinases or phosphatases. Figure 3 As shown in Figure j, the AKT1 3KR mutant impairs the interaction of AKT1 with mTOR and Rictor, a key component of mTORC2, which is essential for AKT1 phosphorylation in Ser473. However, this mutation does not affect the interaction of AKT1 with PHLPP1, a selective dephosphorylase of AKT in Ser473. Figure 3 (j)23. Furthermore, the AKT1 3KR mutation enhances the interaction between AKT1 and PP2A B5α, a subunit of the PP2A complex, which promotes AKT1 phosphorylation at Thr308. In contrast, the lack of AKT1 UFMylation did not affect its interaction with PDK1 or PP2A B56β. Figure 3 (middle k).

[0052] To examine the biological consequences of UFL1-mediated UFMylation on AKT1 in triple-negative breast cancer, the inventors reintroduced AKT1 WT or AKT1 3KR mutants into AKT1 / 2 / 3-deficient HCC1806 and MDA-MB-231 cells. Compared to AKT1 WT, the AKT1 3KR mutation significantly reduced AKT activation ( Figure 3 In AKT1 / 2 / 3 deficient HCC1806 and MDA-MB-231 cells, reintroduction of AKT1 WT, instead of the 3KR mutant, significantly promoted cell proliferation and reduced cell sensitivity to cisplatin and doxorubicin in vitro and in vivo. Figure 3 m- Figure 3 These results indicate that UFL1-mediated UFMylation is crucial for AKT activation and enhances its oncogenic function in triple-negative breast cancer.

[0053] Test Example 4 AKT phosphorylates UFL1 at T426 to enhance its UFMylation and promote the progression of triple-negative breast cancer. The inventors observed a dose-dependent decrease in AKT UFMylation after cell treatment with capapasetinib. Figure 4 (a) This suggests that AKT inhibition may impair its UFMylation. The inventors hypothesize that activated AKT may phosphorylate UFL1, which in turn may regulate AKT UFMylation. To test this hypothesis, the inventors evaluated the phosphorylation of Flag-S-UFL1 overexpression in HCC1806 cells using a phosphorylated AKT substrate antibody. Figure 4 (b) Inhibition of AKT activity reduced UFL1 phosphorylation ( Figure 4 (c) The inventors discovered a conserved phosphorylation site at Thr426 in the UFL1 amino acid sequence. This site is highly conserved across species and matches the AKT consensus phosphorylation motif (RXRXXS* / T*). Figure 4 (d). The inventors generated a phosphorylated Thr426-specific antibody to study UFL1 phosphorylation at this site in cells. Figure 4 As shown in Figure e, the phosphorylation-deficient T426A mutant almost completely eliminates UFL1 phosphorylation. Use of capapasetinib or depleted AKT1 / 2 / 3 significantly reduced UFL1 phosphorylation at T426. Figure 4 f and Figure 4 (g). Furthermore, compared to UFL1 WT, the UFL1 T426A mutant impairs AKT1 UFMylation ( Figure 4 (h).

[0054] The inventors then examined the biological function of UFL1 phosphorylation in triple-negative breast cancer. Overexpression of Flag-UFL1 WT, rather than the T426A mutant, increased phosphorylation of AKT, GSK-3β, and PRAS40 in HCC1806 cells. Figure 4 (i). UFL1 WT also significantly enhanced cell proliferation and reduced sensitivity to cisplatin and doxorubicin, while the T426A mutant did not ( Figure 4 j and Figure 4 These results indicate that phosphorylation of UFL1 at Thr426 plays a crucial role in maintaining AKT activation and enhancing the oncogenic function of UFL1 and AKT in triple-negative breast cancer.

[0055] Experimental Example 5 Inhibiting UFL1-AKT interaction suppresses the progression of triple-negative breast cancer Given the positive feedback loop between UFL1 and AKT in triple-negative breast cancer, the inventors hypothesized that inhibiting their interaction might suppress AKT activation and inhibit tumor progression. To determine the minimal region where UFL1 binds to AKT1, the inventors tested truncated UFL1 protein fragments and determined that amino acids 20-30 (SEQ ID No. 4: FAEATQRLSER) are crucial for binding to AKT1 in vitro and in vivo. Figure 5 (a in middle and b in middle 5).

[0056] Previous reports have demonstrated the potential of cell-penetrating peptides as tools for targeting specific molecular pathways. To disrupt the UFL1-AKT interaction, the inventors synthesized a peptide corresponding to amino acids 20-30 of UFL1 (SEQ ID No. 1: GRKKRRQRRRGGFAEATQRLSER) and a negative control peptide corresponding to amino acids 1-10 (SEQ ID No. 3: GRKKRRQRRRGGMADAWEEIRR). Both peptides were bound to a TAT peptide (SEQ ID No. 2: GRKKRRQRRGG) to form cell-penetrating peptides named PDAU (a peptide that disrupts the AKT-UFL1 interaction) and NP (a negative control peptide), respectively. Figure 5 (c). The inventors evaluated the ability of these peptides to disrupt UFL1-AKT interactions in HCC1806 cells. Figure 5 As shown in Figure d, PDAU, but not NP, effectively interferes with UFL1-AKT binding. Furthermore, PDAU treatment reduces AKT1 UFMylation, while NP does not have this effect. Figure 5 (e). PDAU treatment also inhibited AKT activation, significantly suppressed cell proliferation, and sensitized breast cancer cells to chemotherapy drugs. Figure 5 f- Figure 5(h).

[0057] The inventors further validated the therapeutic potential of PDAU in cancer cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Figure 5 Zhong i and Figure 5 (j). In the PDX model, PDAU treatment significantly disrupted UFL1-AKT interaction in tumor samples and inhibited AKT phosphorylation ( Figure 5 k and Figure 5 PDAU treatment also led to a significant decrease in Ki67 expression and upregulation of cleaved PARP1 staining in cisplatin-treated PDX tumors. Figure 5 These results indicate that PDAU effectively inhibits the progression of triple-negative breast cancer by disrupting the UFL1-AKT interaction and suppressing AKT activation, highlighting its therapeutic potential for triple-negative breast cancer treatment.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cell-penetrating peptide targeting the UFL1-AKT signaling axis for the prevention and treatment of triple-negative breast cancer, characterized in that, The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID No.

1.

2. The use of the cell-penetrating peptide according to claim 1 in the preparation of drugs for the prevention and treatment of triple-negative breast cancer.

3. The application according to claim 2, characterized in that, The cell-penetrating peptide inhibits the growth of triple-negative breast cancer.

4. The application according to claim 2, characterized in that, The cell-penetrating peptide enhances the sensitivity of triple-negative breast cancer cells to drugs against triple-negative breast cancer, namely doxorubicin and cisplatin.

5. The application according to claim 2, characterized in that, The cell-penetrating peptide reduces the resistance of triple-negative breast cancer to anti-triple-negative breast cancer drugs, namely doxorubicin and cisplatin.

6. A pharmaceutical composition targeting the UFL1-AKT signaling axis for the prevention and treatment of triple-negative breast cancer, characterized in that, The pharmaceutical composition comprises the cell-penetrating peptide of claim 1 and an AKT inhibitor; The AKT inhibitor is capapasetinib.

7. The use of the pharmaceutical composition of claim 6 in the preparation of a product targeting the UFL1-AKT signaling axis for the prevention and treatment of triple-negative breast cancer.

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