CN118079005A - Use of YAP inhibitors in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors in the treatment of breast cancer - Google Patents
Use of YAP inhibitors in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors in the treatment of breast cancer Download PDFInfo
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- CN118079005A CN118079005A CN202410197932.3A CN202410197932A CN118079005A CN 118079005 A CN118079005 A CN 118079005A CN 202410197932 A CN202410197932 A CN 202410197932A CN 118079005 A CN118079005 A CN 118079005A
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- yap
- egfr
- breast cancer
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Abstract
The invention discloses the use of YAP inhibitors in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors in the treatment of breast cancer. Studies of the present invention demonstrate that YAP inhibitors can activate the downstream ERK and AKT pathways, as well as the TGF-beta 1-SMAD signaling pathways, by promoting the expression of EGFR and TGF-beta 1 proteins, i.e., YAP inhibitors activate the downstream ERK and AKT pathways, as well as the TGF-beta 1-SMAD signaling pathways, causing them to develop drug resistance and side effects in the treatment of tumors and promoting tumor metastasis. Therefore, the YAP inhibitor, the EGFR inhibitor and/or the TGF beta 1 receptor inhibitor are combined, the drug resistance and tumor metastasis caused by the enhancement of EGFR and TGF beta 1 expression caused by the YAP inhibitor CA3 are overcome, and the synergistic therapeutic effect is achieved.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to an application of YAP inhibitor combined with EGFR inhibitor and/or TGF beta 1 receptor inhibitor in breast cancer treatment.
Background
Breast cancer is still one of the most common cancers in women, severely jeopardizing the life and health of women. Studies have shown that breast cancer is a tumor with a high degree of heterogeneity, exhibiting a great difference in both tissue morphology and immunophenotype, and that different types of breast cancer may differ completely in their biological behavior and response to treatment even with the same tumor stage. Breast cancer is currently classified clinically according to the expression status of hormone receptor and her2 (human epidermal growth factor receptor, her-2): triple negative breast cancers (TRIPLENEGATIVE BREAST CANCER, TNBC) with positive estrogen (estrogen receptors, ER) and progestin (progesterone receptors, PR) receptors, positive for HER2 (HER 2), and negative for ER/PR/HER 2. At present, in addition to surgical excision, conventional radiotherapy and chemotherapy and endocrine treatment, the breast cancer treatment has targeted treatment, and the recurrence and metastasis risks of the breast cancer and the response thereof to the treatment are predicted based on accurate breast cancer molecular typing, so that the method is the basis for the targeted treatment of the breast cancer and the development of targeted medicaments for resisting the breast cancer. For example, the estrogen receptor analog Tamoxifen (Tamoxifen) has been used in ER/PR positive breast cancer treatment; trastuzumab (Trastuzumab) is used for HER 2-positive breast cancer treatment; poly (adenosine diphosphate) ribose polymerase (PARP) inhibitor Olaparib (Olaparib) is used for breast cancer treatment with BRCA gene mutation. It is worth noting that up to now, breast cancer patients eventually develop a certain drug resistance by using these targeted drugs, so that it is important to develop some new targeted therapeutic drugs.
The Hippo signaling pathway is a highly conserved inhibitory signaling pathway first discovered in drosophila in recent years, regulating organ development by inhibiting cell proliferation and promoting apoptosis. The Hippo signal pathway consists of a kinase chain and a transcriptional co-activator. It can be divided into three interrelated parts: an upstream regulatory component, a Hippo core kinase component and a downstream transcription mechanism. The upstream signal activates MST1/2 (MAMMALIAN STERILE-LIKE KINASES 1/2) and its regulatory subunit WW45, which, when combined with each other, promotes MST1/2 activation and phosphorylates LATS1/2 (large tumor suppressor kinases 1/2), the phosphorylated LATS1/2 subsequently re-phosphorylates YAP and localizes it in the cytosol in association with the 14-3-3 protein which is subsequently ubiquitinated and degraded, thereby rendering the YAP useless for transcription activation into the nucleus. At the same time, transcription factors (Transcriptional Enhanced Associate Domain, TEAD) in the nucleus lose YAP/TAZ binding and coactivation, directly leading to down-regulation of downstream transcriptional gene expression, thereby reducing cell proliferation rate and promoting apoptosis. If the pathway is blocked or inactivated, the core downstream transcription regulatory molecule YAP enters the cell nucleus to combine with TEAD family members (TEAdomain family members, TEAD) and other transcription factors, thereby regulating the expression of target genes, participating in abnormal regulation, including processes of promoting cell proliferation, inhibiting cell apoptosis and the like. More and more studies have shown that abnormalities in the Hippo signaling pathway are associated with the development of a variety of tumors. The expression of YAP in 69 breast cancer tissues was examined by immunohistochemical method, and YAP was found to be expressed in 75.4% of breast cancer samples, while in vivo experiments also demonstrated that overexpression of YAP promoted tumor formation and growth. Therefore, exploring the role played by YAP in the development of breast cancer is critical to developing new targeted therapies for breast cancer.
EGFR (Epidermal Growth Factor Receptor) are receptors for Epithelial Growth Factor (EGF) cell proliferation and signaling. EGFR receptors activate the MAPK/ERK signaling pathway, as well as the PI3K-AKT signaling pathway. The MAPK/ERK pathway is responsible for controlling gene transcription activities and cell cycle, and participates in proliferation of cells; whereas the PI3K-AKT pathway activates anti-apoptotic signals, promoting cell survival. Thus, EGFR receptor proteins have a very important role in cell proliferation and survival. Meanwhile, gefitinib is a target point of targeted treatment of breast cancer, and is used as a small-molecule reversible EGFR tyrosine kinase inhibitor, and in-vitro experimental research shows that the gefitinib can inhibit the growth of breast cancer of animals, but a plurality of phase II clinical experiments for advanced metastatic breast cancer show that the curative effect is not ideal. Studies have shown that the clinical benefit rate of gefitinib in combination with tamoxifen is higher than that of tamoxifen in combination with placebo in breast cancer patients with metastasis or recurrence after tamoxifen treatment.
Although various clinical trials began using YAP inhibitors for exploring their use in melanoma and lung cancer, YAP inhibitors have not been approved for preclinical trials in breast cancer, perhaps due to drug resistance or poor defenses. Gefitinib, however, is used as an EGFR tyrosine kinase inhibitor (gefitinib) in a plurality of phase II clinical trials for advanced metastatic breast cancer, but the results show that the curative effect is not ideal. Tgfβ1 receptor inhibitors (galunisertib) have made some progress in clinical trials for locally advanced rectal and liver cancers, but have not been used in clinical treatment of breast cancer. There is no report of YAP inhibitors in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors in the treatment of breast cancer.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks and deficiencies of the prior art and to provide the use of YAP inhibitors in combination with EGFR inhibitors and/or tgfβ1 receptor inhibitors in the treatment of breast cancer.
The above object of the present invention is achieved by the following technical solutions:
The core transcription factor YAP in the Hippo signaling pathway has been reported to be highly expressed as an oncogene in a variety of tumors including breast cancer. The exact role of YAP in breast cancer remains complex, so studying the role of YAP in the development of breast carcinogenesis is of great importance for targeted treatment of breast cancer. The invention provides a preclinical experimental basis for enhancing the curative effect of breast cancer by combining other medicines by researching the mechanism of YAP inhibitor medicines in breast cancer treatment. The invention researches find that the YAP inhibitor can promote the expression of EGFR and TGF beta 1 and promote the proliferation and transfer capacity of breast cancer, so that the breast cancer cells have drug resistance to the YAP inhibitor. That is, YAP inhibitors activate downstream ERK and AKT pathways, as well as tgfβ1-SMAD signaling pathways, by promoting expression of EGFR and tgfβ1 proteins, thereby causing YAP inhibitors to develop drug resistance and side effects that promote tumor metastasis during treatment of breast cancer. The remarkable synergistic killing effect of YAP inhibitors on breast cancer with EGFR inhibitors and tgfβ1 receptor inhibitors was subsequently verified in vitro cytology experiments and in vivo animal experiments. For this reason, the present invention proposes the use of YAP inhibitors such as CA3, while using EGFR inhibitors such as gefitinib to inhibit ERK downstream of EGFR and AKT1 phosphorylation, and tgfβ1 receptor inhibitors such as galunisertib to inhibit tgfβ1-SMAD signaling pathway, thereby overcoming unresponsive drug resistance and metastasis of YAP inhibitors and achieving the goal of combined treatment of breast cancer.
The YAP inhibitor and the EGFR inhibitor are combined, or the YAP inhibitor and the EGFR inhibitor, or the YAP inhibitor, the EGFR inhibitor and the TGF beta 1 receptor inhibitor are combined, so that the drug resistance and tumor metastasis caused by the enhancement of EGFR and/or TGF beta 1 expression caused by the YAP inhibitor CA3 can be overcome, the breast cancer has obvious proliferation inhibition capability and apoptosis promotion capability, and the synergistic treatment effect is realized.
Thus, the present invention first provides the use of YAP inhibitors in combination with EGFR inhibitors and/or tgfβ1 receptor inhibitors for the preparation of a medicament for the treatment of breast cancer. Namely, the YAP inhibitor is combined with the EGFR inhibitor to prepare the breast cancer therapeutic drug, or the YAP inhibitor is combined with the TGF beta 1 receptor inhibitor to prepare the breast cancer therapeutic drug, or the YAP inhibitor is combined with the EGFR inhibitor and the TGF beta 1 receptor inhibitor to prepare the breast cancer therapeutic drug. The YAP inhibitor, EGFR inhibitor and TGF-beta 1 receptor inhibitor are used at concentrations according to the recommended concentrations for the drug in the art.
Further, the treatment may be inhibition of proliferation of a tumor, promotion of apoptosis, and/or inhibition of tumor metastasis.
Further, based on the therapeutic mechanisms described above, the YAP inhibitors include, but are not limited to, CA3 (CIL 56), verteporfin (Verteporfin), alcafodin (AICAR).
Preferably, the YAP inhibitor is CA3 (CIL 56).
Further, the EGFR inhibitors include, but are not limited to gefitinib, erlotinib, afatinib (afatinib).
Preferably, the EGFR inhibitor is gefitinib (gefitinib).
Further, the TGF-beta 1 receptor inhibitors include, but are not limited to galunisertib, A-83-01.
Preferably, the tgfβ1 receptor inhibitor is galunisertib.
Preferably, the breast cancer is a high YAP expressing breast cancer.
The invention also provides a breast cancer therapeutic agent comprising a YAP inhibitor and an EGFR inhibitor and/or a tgfβ1 receptor inhibitor; i.e. YAP inhibitor and EGFR inhibitor, or YAP inhibitor and tgfβ1 receptor inhibitor, or YAP inhibitor, EGFR inhibitor and tgfβ1 receptor inhibitor.
Further, the medicament contains YAP inhibitors, EGFR inhibitors and tgfβ1 receptor inhibitors.
Further, the YAP inhibitor is CA3, verteporfin (Verteporfin), alcafodin (AICAR).
Preferably, the YAP inhibitor is CA3.
Further, the EGFR inhibitor is gefitinib (gefitinib), erlotinib (Erlotinib), afatinib (afatinib).
Preferably, the EGFR inhibitor is gefitinib (gefitinib).
Further, the TGF-beta 1 receptor inhibitor is galunisertib, A-01.
Preferably, the tgfβ1 receptor inhibitor is galunisertib.
Further, the medicament takes the YAP inhibitor and the EGFR inhibitor and/or the TGF beta 1 receptor inhibitor as main active ingredients.
Preferably, the medicament further comprises pharmaceutically acceptable excipients.
Preferably, the medicament contains an effective dose of a YAP inhibitor and an EGFR inhibitor and/or a tgfβ1 receptor inhibitor.
Preferably, the YAP inhibitor is used in combination with EGFR inhibitor or TGF-beta 1 receptor inhibitor in a molar concentration ratio of (0.5-1): 5-20.
Further preferred, the YAP inhibitor is used in a molar ratio of 1:10 in combination with an EGFR inhibitor or tgfβ1 receptor inhibitor.
Preferably, the YAP inhibitor is used in combination with EGFR inhibitor and TGF beta 1 receptor inhibitor in a molar concentration ratio of (0.5-1): 5-20: (5-20).
Further preferred, the YAP inhibitor is used in a molar ratio of 1:10:10 in combination with an EGFR inhibitor and a tgfβ1 receptor inhibitor.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides an application of YAP inhibitor combined with EGFR inhibitor and/or TGF beta 1 receptor inhibitor in breast cancer therapeutic drugs. Studies of the present invention demonstrate that YAP inhibitors can activate the downstream ERK and AKT pathways, as well as the TGF-beta 1-SMAD signaling pathways, by promoting the expression of EGFR and TGF-beta 1 proteins, i.e., YAP inhibitors activate the downstream ERK and AKT pathways, as well as the TGF-beta 1-SMAD signaling pathways, causing them to develop drug resistance and side effects in the treatment of tumors and promoting tumor metastasis. The YAP inhibitor is combined with EGFR inhibitor and/or TGF beta 1 receptor inhibitor, so that drug resistance and tumor metastasis caused by the enhanced expression of EGFR and TGF beta 1 caused by the YAP inhibitor can be overcome, the YAP inhibitor has obvious proliferation inhibition capability and apoptosis promotion capability on breast cancer, and has synergistic treatment effect. The present invention provides a combination targeted therapy tumor regimen, i.e., YAP inhibitors are used in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors to achieve synergistic treatment of breast cancer.
Drawings
FIG. 1 is the effect of YAP inhibitor CA3 on EGFR and TGF-beta 1 protein expression; wherein A is the chemical structural formula of YAP inhibitor CA3, B is the expression level of EGFR, TGFbeta 1 and downstream signal molecule mRNA after different breast cancer cell strains are treated by the YAP inhibitor CA3 with concentration gradient, C is the protein expression condition of EGFR, TGFbeta 1 and downstream signal molecule after different breast cancer cell strains are treated by the YAP inhibitor CA3 with concentration gradient, D is the expression level of EGFR, TGFbeta 1 and downstream signal molecule mRNA in a stable and transformed breast cancer cell line knocked down by YAP, and E is the protein expression level of EGFR, TGFbeta 1 and downstream signal molecule mRNA in a stable and transformed breast cancer cell line knocked down by YAP.
FIG. 2 is a graph showing the effect of XMU-MP-1 on EGFR and TGF-beta 1 expression; wherein A is the expression level of EGFR, TGF beta 1 and downstream signal molecule mRNA after YAP knockdown steady transfer breast cancer cell line is treated by XMU-MP-1 with concentration gradient, and B is the expression level of EGFR, TGF beta 1 and downstream signal molecule mRNA after different breast cancer cells are treated by XMU-MP-1 with concentration gradient through fluorescence quantitative PCR.
FIG. 3 is the effect of YAP inhibitor (CA 3) and EGFR inhibitor (gefitinib) on proliferation and apoptosis of breast cancer cells; wherein A is the phosphorylation level of EGFR downstream AKT1 and ERK after CA3 and gefitinib treat breast cancer cells independently or jointly, B is the cell activity level after CA3 and gefitinib treat breast cancer cells independently or jointly, C is the cell clone formation condition after CA3 and gefitinib treat breast cancer cells independently or jointly, D is the apoptosis condition after CA3 and gefitinib treat breast cancer cells independently or jointly.
FIG. 4 is a graph showing that YAP knockdown promotes sensitivity of breast cancer cells to EGFR inhibitors (gefitinib); wherein A is the expression level of EGFR downstream AKT1 and ERK after gefitinib is treated and YAP knockdown steady transfer cell line is knocked down, B is the cell activity level after gefitinib is treated with different breast cancer cells, and C is the cell clone formation condition after gefitinib is treated with different breast cancer cells.
FIG. 5 is a graph showing that YAP inhibitor CA3 and TGF-beta 1 receptor inhibitor galunisertib act synergistically to inhibit migration of breast cancer cells; wherein, A is the expression level of TGF beta 1 downstream signal molecule after CA3 and galunisertib are treated separately or jointly for different breast cancer cells, B is the expression level of mRNA of TGF beta 1 downstream signal molecule after CA3 or CA3 and galunisertib are treated jointly for YAP knockdown stable transfer breast cancer cell line, C is the expression of TGF beta 1 downstream signal molecule after CA3 and galunisertib are treated separately or jointly for breast cancer cells, D is the migration condition of crystal violet observation cell after CA3 and galunisertib are treated separately or jointly for breast cancer cells.
FIG. 6 shows the combined effect of YAP inhibitor CA3 and EGFR inhibition (gefitinib) in nude mice tumor-bearing experiments; wherein A is the volume of subcutaneous tumor measured during administration, B is the weight after the final subcutaneous tumor is removed, C is the photographing result of subcutaneous tumor, and D is the expression condition of each protein in the subcutaneous tumor tissue of nude mice.
FIG. 7 is a graph showing the synergistic effect of YAP inhibitor CA3 in combination with EGFR inhibitor (gefitinib) and TGF beta 1 receptor inhibitor galunisertib on inhibiting proliferation and metastasis of breast cancer cells; wherein A is the expression level and apoptosis signal detection of EGFR and TGF beta 1 downstream signal molecules after CA3 and galunisertib and Gefitinib are treated separately or jointly, B is the observation of cell migration condition by crystal violet after CA3 or CA3 and galunisertib and Gefitinib are treated separately or jointly, C is the quantitative condition and statistical analysis of B, D is the quantitative condition and statistical analysis of EdU staining condition after CA3 or CA3 and galunisertib and Gefitinib are treated separately or jointly to represent the inhibition of tumor cell proliferation, E is the quantitative condition and statistical analysis of D, F is the quantitative condition and statistical analysis of EdU staining condition after CA3 or CA3 and galunisertib and Gefitinib are treated separately or jointly to represent the inhibition of tumor cell proliferation after CA3 and galunisertib are treated separately or jointly to breast cancer cell apoptosis condition, and F is the quantitative condition and statistical analysis of G.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1YAP inhibitor CA3 promotes expression of EGFR and TGF beta 1 proteins
YAP, a core transcription factor in the Hippo signaling pathway, has been reported to be highly expressed as an oncogene in a variety of tumors including breast cancer. The exact role of YAP in breast cancer remains complex, so studying the role of YAP in the development of breast carcinogenesis is of great importance for targeted treatment of breast cancer. The method comprises the following steps:
(1) The effect of CA3 on EGFR and TGF-beta 1 expression was studied. Treating cells in various breast cancer cells (MDA-MB-231, MDA-MB-468, SKBR3 and T47D) with concentration gradients (0 mu M, 0.5 mu M, 1 mu M and 1.5 mu M) of CA3 for 24 hours respectively, and collecting cell extract proteins to detect the expression of EGFR, TGF beta 1 and downstream signal molecules thereof by western blot; meanwhile, the cells treated by CA3 are also collected to extract total RNA, and the expression level of EGFR, TGF beta 1 and downstream signal molecule mRNA thereof is detected by fluorescent quantitative PCR after reverse transcription into cDNA.
(2) The effect of down-regulating YAP on EGFR and tgfβ1 expression was studied. pLKO-shYAP1 was packaged into lentiviral infected breast cancer cell lines SKBR3 and MDA-MB-468 and screened with puromycin to form stable cell clones. And then collecting a wild cell line and a YAP knock-down stable transgenic cell line to extract protein and total RNA, and detecting the expression levels of EGFR, TGF beta 1 and downstream signal molecules thereof through western blot and fluorescent quantitative PCR experiments respectively.
As shown in fig. 1, the present invention studied the treatment of various breast cancer cells with YAP inhibitor CA3, and found that CA3 can promote expression of EGFR and tgfβ1, and promote phosphorylation of ERK and AKT1 downstream of EGFR, and promote phosphorylation of SMAD2 downstream of tgfβ1 and expression of EMT-related genes such as snail, vimentin (fig. 1B to C). Consistent with this, EGFR and tgfβ1 expression was also significantly enhanced in YAP knockdown stable breast cancer cell lines (fig. 1D-E).
The above results show how does YAP inhibit promote expression of EGFR and tgfβ1, which in turn is? Further treatment of breast cancer cells with inhibitors XMU-MP-1 of the upstream kinase MST1/2 in the Hippo signaling pathway, as inhibitors of MST1/2, XMU-MP-1 may promote its nuclear transcription function by inhibiting phosphorylation of LAST1/2 and thereby inhibiting phosphorylation of YAP. The method comprises the following steps:
Study XMU-MP-1 effects on EGFR and TGF-beta 1 expression: treating a plurality of breast cancer cells (MDA-MB-231, MDA-MB-468, SKBR3, T47D) respectively by using XMU-MP-1 with concentration gradients (0 mu M, 1 mu M, 2 mu M and 4 mu M), collecting cell extract proteins after 24 hours, and detecting the expression of EGFR, TGF beta 1 and downstream signal molecules thereof by using western blot; meanwhile, XMU-MP-1 treated cells are also collected to extract total RNA, and after reverse transcription into cDNA, the expression level of EGFR, TGF beta 1 and downstream signal molecule mRNA is detected by fluorescent quantitative PCR.
As shown in FIG. 2, XMU-MP-1 was found to inhibit EGFR and TGF-beta 1 expression by promoting YAP activation according to western blot and real-time qPCR experiments (FIGS. 2A-B), which is contrary to the effect of CA 3.
EXAMPLE 2 Effect study of the combination of CA3 and EGFR inhibitor or TGF-beta 1 receptor inhibitor
Example 1 studies indicate that YAP inhibitors (CA 3) can significantly promote expression of EGFR and tgfβ1 proteins, thereby activating downstream ERK and AKT pathways, as well as tgfβ1-SMAD signaling pathways. This finding may be an important reason for the development of YAP inhibitors for the treatment of breast cancer resistance. To this end, the invention contemplates the use of EGFR inhibitors to inhibit activation of ERK and AKT signaling pathways by CA3, and TGF-beta 1 receptor inhibitors to inhibit activation of downstream signaling pathways resulting from increased TGF-beta 1 expression by CA 3. The specific method comprises the following steps:
synergism study of yap inhibitor CA3 with EGFR inhibitor gefitinib on proliferation and apoptosis of breast cancer cells:
(1) Breast cancer cells were treated with CA3 and gefitinib, either alone or together, wherein the working concentration of CA3 was 0.5 μm and the working concentration of gefitinib was 5 μm. Cell extracts were collected 24 hours after treatment of the cells to detect the phosphorylation levels of AKT1 and ERK downstream of EGFR.
(2) Cell proliferation-toxicity experiments examined the synergistic inhibition of the breast cancer cells by the YAP inhibitor CA3 and the EGFR inhibitor gefitinib. The viability of breast cancer cells was detected with CCK8 kit using CA3 alone or in combination with gefitinib treatment (0.5. Mu.M for CA3 and 5. Mu.M for gefitinib) for 72 h.
(3) The clone formation assay detects the synergistic inhibition of breast cancer cells by the YAP inhibitor CA3 and gefitinib. 1000 breast cancer cells were inoculated into each well of a 6-well plate, CA3 and gefitinib-treated cells (working concentration of CA3 is 0.5. Mu.M, working concentration of gefitinib is 5. Mu.M) were used singly or in combination, the medium containing the drug was discarded after 24 hours, the cells were cultured to macroscopic cell clones with complete medium, the cells were fixed and stained with crystal violet, and the cell clone formation was observed.
(4) The synergistic killing effect of YAP inhibitor CA3 and gefitinib on breast cancer cells was examined. Different breast cancer cells were treated with CA3 and gefitinib alone or in combination (working concentration of CA3 was 0.5. Mu.M, working concentration of gefitinib was 5. Mu.M), cells were collected after 48h and tested for apoptosis with Annexin V-FITC/PI apoptosis kit (Vazyme, A211-01).
Study of tgfβ1 receptor inhibitor galunisertib to inhibit migration of breast cancer cells caused by YAP inhibitor CA 3:
Breast cancer cells were treated with CA3 and galunisertib either individually or together, with a working concentration of CA3 of 0.5 μm and galunisertib of 5 μm. Cells were collected and counted 24 hours after treatment, the same number (3 ten thousand cells) of cells were seeded into a transwell chamber with a serum-free medium, a normal medium was contained under the chamber, the cells under the chamber were fixed after culturing for 24 to 48 hours and stained with crystal violet, and the migration of the cells was observed.
3. Analysis of results:
In a cellular experiment, it was first determined that the EGFR inhibitor gefitinib was able to inhibit CA 3-induced activation of ERK and AKT signaling pathways (fig. 3A). And then, through a cell proliferation experiment, a clone formation experiment and an apoptosis experiment, the CA3 and gefitinib are found to obviously inhibit proliferation capacity of breast cancer cells, promote apoptosis capacity of the breast cancer cells, and have good synergistic effect (figures 3B-D). This finding was also confirmed in YAP knockdown stable cell lines (fig. 4A-C).
Meanwhile, the invention also determines that the TGF-beta 1 receptor inhibitor (galunisertib) can inhibit the activation of a TGF-beta 1 downstream signal channel caused by CA3 (figures 5A-C), and the trans-well experiment proves that galunisertib obviously inhibits the EMT formation and migration of breast cancer cells caused by CA3 (figure 5D).
Therefore, the research result shows that the YAP inhibitor CA3 and the EGFR inhibitor gefitinib have synergistic effect on proliferation and apoptosis of breast cancer cells, and the TGF beta 1 receptor inhibitor galunisertib can also remarkably inhibit migration of the breast cancer cells caused by the YAP inhibitor CA 3.
EXAMPLE 3 animal Experimental study of the combination of CA3 and EGFR inhibitor
The specific research method is as follows:
(1) Effects of CA3 in combination with gefitinib (gefitinib) on tumor-bearing mouse models. The Balb/c immunodeficient mice were inoculated subcutaneously with 5X 10 6 breast cancer cells MDA-MB-468, and after the tumor volume reached 50-100 cubic millimeters (mm 3), gefitinib and oral CA3 were injected intraperitoneally, either alone or in combination, wherein gefitinib was intraperitoneally injected daily at a dose of 75mg/kg, and CA3 was perfused every two days at a dose of 1mg/kg, the tumor-bearing mice were weighed every 2-3 days and the subcutaneous tumor size was measured. After the subcutaneous tumor of the control tumor-bearing mice grows to a certain size, the mice are euthanized, the subcutaneous tumor is removed and the tumor is weighed. Tumor volume was calculated using the formula (length x width 2 x 0.52).
(2) The expression of various proteins in tumor tissues in the step (1) is detected by western blot. And (3) carrying out splitting and protein extraction on the tumor tissue obtained in the step (1), and then carrying out western blot analysis on the expression condition of each protein.
Analysis of results:
according to the invention, mouse tumor-bearing experiments prove that YAP inhibitor CA3 and EGFR inhibitor (gefitinib) are combined, so that the tumor-forming capacity of breast cancer cells in nude mice can be obviously inhibited (figures 6A-C), and meanwhile, inhibition effects on downstream ERK and AKT channels are accompanied (figure 6D). Thus, the results indicate that YAP inhibitor CA3 and EGFR inhibitor gefitinib synergistically inhibit the growth of breast tumors in vivo.
EXAMPLE 4 cell experiment study of the combination of CA3 and EGFR inhibitor gefitinib and TGF-beta 1 receptor inhibitor galunisertib
Example 1 studies indicate that YAP inhibitors (CA 3) can significantly promote expression of EGFR and tgfβ1 proteins, thereby activating downstream ERK and AKT pathways, as well as tgfβ1-SMAD signaling pathways. Examples 2-4 show that YAP inhibitors can be used with TGF beta 1 receptor inhibitor galunisertib to inhibit tumor metastasis and with EGFR inhibitor gefitinib to inhibit breast tumor growth in vitro and in vivo. To this end, the present invention contemplates the use of EGFR inhibitors in combination with TGF-beta 1 receptor inhibitors galunisertib to inhibit activation of ERK and AKT signaling pathways by CA3, and TGF-beta 1 receptor inhibitors to inhibit activation of downstream signaling pathways resulting from increased TGF-beta 1 expression by CA 3. The specific method comprises the following steps:
Synergistic inhibition studies of proliferation and metastasis of breast cancer cells by yap inhibitor CA3 in combination with EGFR inhibitor gefitinib and tgfβ1 receptor inhibitor galunisertib:
(1) Breast cancer cells were treated with CA3 and gefitinib and galunisertib either individually or together, wherein the working concentration of CA3 was 0.5 μm, the working concentration of gefitinib was 5 μm, and the working concentration of galunisertib was 5 μm. Cells were treated for 24 hours and then cell extracts were collected to detect the phosphorylation levels of AKT1 and ERK downstream of EGFR and tgfβ1 downstream signals and metastasis (E-cad, snail, vimentin) and apoptosis signals (CLEAVED PARP).
(2) Breast cancer cells were treated with CA3 and gefitinib and galunisertib either individually or together, wherein the working concentration of CA3 was 0.5 μm, the working concentration of gefitinib was 5 μm, and the working concentration of galunisertib was 5 μm. Cells were collected and counted 24 hours after treatment, the same number (3 ten thousand cells) of cells were seeded into a transwell chamber with a serum-free medium, a normal medium was contained under the chamber, the cells under the chamber were fixed after culturing for 24 to 48 hours and stained with crystal violet, and the migration of the cells was observed.
(3) Cell proliferation experiments examined the synergistic inhibition of YAP inhibitor CA3 with gefitinib and galunisertib on breast cancer cells. Breast cancer cells were treated with CA3 and gefitinib alone or in combination and galunisertib (working concentration of CA3 was 0.5 μm, working concentration of gefitinib was 5 μm, working concentration of galunisertib was 5 μm) for 24h, and viability of the breast cancer cells was detected with BdU kit.
(4) The synergistic killing effect of YAP inhibitor CA3 and gefitinib galunisertib on breast cancer cells was examined. CA3 and gefitinib treatment and galunisertib (CA 3 working concentration 0.5. Mu.M, gefitinib working concentration 5. Mu.M, galunisertib working concentration) were used alone or in combination for 48h after breast cancer cells were collected and tested for apoptosis using an Annexin V-FITC/PI apoptosis kit (Vazyme, A211-01).
2. Analysis of results:
in the cell experiments, it was first determined that the combination of CA3 with gefitinib treatment and galunisertib (three-drug combination) not only inhibited the cell proliferation pathway, promoted apoptosis, but also inhibited the cell EMT formation and metastasis pathway (fig. 7A). We have then shown by trans-well experiments that the combination of the three drugs significantly inhibited migration of breast cancer cells caused by CA3 (fig. 7B-C). The cell proliferation experiment and the apoptosis experiment show that the combination of the three medicines remarkably inhibits the proliferation capacity of breast cancer cells, promotes the apoptosis capacity of the breast cancer cells, and has good synergistic effect (figures 7D-F).
Thus, the results of this study demonstrate that YAP inhibitor CA3 in combination with EGFR inhibitor gefitinib and/or tgfβ1 receptor inhibitor galunisertib has a synergistic effect on the inhibition of proliferation and metastasis of breast cancer cells. In addition, it is reasonably expected by those skilled in the art that the use of other YAP inhibitors (e.g., verteporfin, acadesine) in combination with other EGFR inhibitors (e.g., erlotinib, afatinib) and/or other TGF-beta 1 receptor inhibitors (e.g., A83-01) would also have a synergistic effect on the treatment of breast cancer according to the therapeutic mechanisms of the present invention.
Claims (10)
- Use of yap inhibitors in combination with EGFR inhibitors and/or tgfβ1 receptor inhibitors for the preparation of a medicament for the treatment of breast cancer.
- 2. The use according to claim 1, wherein the YAP inhibitor is CA3, verteporfin or acadesine.
- 3. The use of claim 1, wherein the EGFR inhibitor is gefitinib, erlotinib, or afatinib.
- 4. The use according to claim 1, wherein the tgfβ1 receptor inhibitor is galunisertib or a83-01.
- 5. A breast cancer therapeutic agent comprising a YAP inhibitor and an EGFR inhibitor and/or a tgfβ1 receptor inhibitor.
- 6. The medicine according to claim 5, wherein the molar concentration ratio of YAP inhibitor to EGFR inhibitor or TGF-beta 1 receptor inhibitor is (0.5-1): 5-20; the molar concentration ratio of YAP inhibitor to EGFR inhibitor and TGF beta 1 receptor inhibitor is (0.5-1): 5-20.
- 7. The medicament of claim 5, wherein the YAP inhibitor is CA3, verteporfin or acadesine.
- 8. The medicament of claim 5, wherein the EGFR inhibitor is gefitinib, erlotinib, or afatinib.
- 9. The medicament of claim 5, wherein the tgfβ1 receptor inhibitor is galunisertib or a83-01.
- 10. The medicament of claim 5, further comprising pharmaceutically acceptable excipients.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410197932.3A CN118079005A (en) | 2024-02-22 | 2024-02-22 | Use of YAP inhibitors in combination with EGFR inhibitors and/or TGF-beta 1 receptor inhibitors in the treatment of breast cancer |
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