CN118079005A - Application of YAP inhibitor combined with EGFR inhibitor and/or TGFβ1 receptor inhibitor in the treatment of breast cancer - Google Patents

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

Application of YAP inhibitors combined with EGFR inhibitors and/or TGFβ1 receptor inhibitors in the treatment of breast cancer

Technical Field

The present invention relates to the field of biomedicine technology, and more specifically, to the use of a YAP inhibitor combined with an EGFR inhibitor and/or a TGFβ1 receptor inhibitor in the treatment of breast cancer.

Background technique

Breast cancer is still one of the most common cancers in women, seriously endangering women's life and health. Studies have shown that breast cancer is a highly heterogeneous tumor. Breast cancer shows great differences in both tissue morphology and immune phenotype. Different types of breast cancer may have completely different biological behaviors and responses to treatment even with the same tumor stage. At present, according to the expression status of hormone receptors and human epidermal growth factor receptor 2 (Her-2), breast cancer is clinically divided into: estrogen receptors (ER) and progesterone receptors (PR) receptor positive, human epidermal growth factor receptor 2 (HER2) positive and ER/PR/HER2 negative triple negative breast cancer (TNBC). At present, in addition to surgical resection, conventional radiotherapy and chemotherapy, and endocrine therapy, targeted therapy is also used to treat breast cancer. Based on accurate molecular typing of breast cancer, the risk of recurrence and metastasis of breast cancer and its response to treatment can be predicted, which is the basis for the development of targeted therapy for breast cancer and targeted anti-breast cancer drugs. For example, the estrogen receptor analogue Tamoxifen has been used to treat ER/PR-positive breast cancer; Trastuzumab has been used to treat HER2-positive breast cancer; and the poly ADP-ribose polymerase (PARP) inhibitor Olaparib has been used to treat breast cancer with BRCA gene mutations. It is worth noting that, so far, breast cancer patients who use these targeted drugs will eventually develop a certain degree of drug resistance, so the development of some new targeted therapeutic drugs is crucial.

The Hippo signaling pathway is a highly conserved inhibitory signaling pathway first discovered in Drosophila in recent years. It regulates organ development by inhibiting cell proliferation and promoting cell apoptosis. The Hippo signaling pathway consists of a kinase chain and a transcriptional coactivator. It can be divided into three interconnected parts: upstream regulatory components, Hippo core kinase components, and downstream transcriptional mechanisms. Upstream signals activate MST1/2 (mammalian Sterile20-like kinases 1/2) and its regulatory subunit WW45. After binding to each other, they promote the activation of MST1/2 and phosphorylate LATS1/2 (large tumor suppressor kinases 1/2). Phosphorylated LATS1/2 then phosphorylates YAP and localizes it in the cytoplasm to bind to 14-3-3 proteins, which are then ubiquitinated and degraded, thereby causing YAP to lose its function of entering the nucleus to perform transcriptional activation. At the same time, the transcription factor (Transcriptional Enhanced Associate Domain, TEAD) in the nucleus loses its binding and coactivation of YAP/TAZ, which directly leads to the downregulation of downstream transcriptional gene expression, thereby reducing cell proliferation and promoting cell apoptosis. If the pathway is blocked or inactivated, its core downstream transcriptional regulatory molecule YAP enters the cell nucleus and binds to transcription factors such as TEAD family members (TEAD), thereby regulating the expression of target genes and participating in abnormal regulation, including promoting cell proliferation and inhibiting cell apoptosis. More and more studies have shown that abnormalities in the Hippo signaling pathway are associated with the occurrence of various tumors. A study used immunohistochemistry to detect the expression of YAP in 69 breast cancer tissues and found that YAP was expressed in 75.4% of breast cancer samples. In vivo experiments also confirmed that overexpression of YAP can promote tumor formation and growth. Therefore, exploring the role of YAP in the occurrence and development of breast cancer is crucial for the development of new targeted treatments for breast cancer.

EGFR (Epidermal Growth Factor Receptor) is a receptor for epithelial growth factor (EGF) cell proliferation and signal transduction. EGFR receptors can activate the MAPK/ERK signaling pathway and the PI3K-AKT signaling pathway. The MAPK/ERK pathway is responsible for controlling gene transcription activity and cell cycle, and is involved in cell proliferation; while the PI3K-AKT pathway can activate anti-apoptotic signals and promote cell survival. Therefore, EGFR receptor protein plays a very important role in cell proliferation and survival. At the same time, it is also a target for targeted therapy of breast cancer. Gefitinib, as a small molecule, reversible EGFR tyrosine kinase inhibitor, has been shown in vitro experimental studies to inhibit the growth of animal breast cancer, but the efficacy of multiple phase II clinical trials for advanced metastatic breast cancer is not ideal. Studies have shown that for patients with breast cancer that has metastasized or recurred after tamoxifen treatment, the clinical benefit rate of gefitinib combined with tamoxifen is higher than that of tamoxifen combined with placebo.

Although multiple clinical trials have begun to use YAP inhibitors to explore their use in melanoma and lung cancer, YAP inhibitors have not yet been approved for preclinical experiments in breast cancer, possibly due to drug resistance or poor defense. Gefitinib, as an EGFR tyrosine kinase inhibitor (gefitinib), has been used in multiple phase II clinical trials for advanced metastatic breast cancer, but the results showed that the efficacy was not ideal. TGFβ1 receptor inhibitors (galunisertib) have made some progress in clinical trials for locally advanced rectal cancer and liver cancer, but have not yet been used in the clinical treatment of breast cancer. There are currently no reports on the combination of YAP inhibitors with EGFR inhibitors and/or TGFβ1 receptor inhibitors in the treatment of breast cancer.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide an application of a YAP inhibitor in combination with an EGFR inhibitor and/or a TGFβ1 receptor inhibitor in the treatment of breast cancer.

The above-mentioned object of the present invention is achieved through the following technical solutions:

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. However, the exact role of YAP in breast cancer is still complex, so studying the role of YAP in the occurrence and development of breast cancer is of great significance for the targeted treatment of breast cancer. The present invention provides a preclinical experimental basis for enhancing the therapeutic effect of breast cancer by combining other drugs by studying the mechanism of YAP inhibitor drugs in the treatment of breast cancer. The present invention found that the use of YAP inhibitors to treat breast cancer cells can promote the expression of EGFR and TGFβ1, promote the proliferation and metastasis of breast cancer, and thus make breast cancer cells resistant to YAP inhibitors. That is, YAP inhibitors can promote the expression of EGFR and TGFβ1 proteins, thereby activating downstream ERK and AKT pathways, as well as TGFβ1-SMAD signaling pathways, so that YAP inhibitors produce drug resistance and promote tumor metastasis in the process of treating breast cancer. The significant synergistic killing effect of YAP inhibitors, EGFR inhibitors and TGFβ1 receptor inhibitors on breast cancer was subsequently verified in in vitro cytological experiments and in vivo animal experiments. To this end, the present invention proposes to use YAP inhibitors such as CA3 and EGFR inhibitors such as gefitinib to inhibit the phosphorylation of ERK and AKT1 downstream of EGFR, and use TGFβ1 receptor inhibitors such as galunisertib to inhibit the TGFβ1-SMAD signaling pathway, thereby overcoming the drug resistance and metastasis caused by the lack of response to YAP inhibitors and achieving the purpose of combined treatment of breast cancer.

The combination of YAP inhibitors and EGFR inhibitors, or the combination of YAP inhibitors and EGFR inhibitors, or the combination of YAP inhibitors, EGFR inhibitors and TGFβ1 receptor inhibitors can overcome the drug resistance and tumor metastasis caused by the enhanced expression of EGFR and/or TGFβ1 caused by the YAP inhibitor CA3, and has obvious ability to inhibit proliferation and promote apoptosis in breast cancer, and has a synergistic therapeutic effect.

Therefore, the present invention first provides the use of a YAP inhibitor in combination with an EGFR inhibitor and/or a TGFβ1 receptor inhibitor in the preparation of a breast cancer therapeutic drug. That is, the use of a YAP inhibitor in combination with an EGFR inhibitor in the preparation of a breast cancer therapeutic drug, or the use of a YAP inhibitor in combination with a TGFβ1 receptor inhibitor in the preparation of a breast cancer therapeutic drug, or the use of a YAP inhibitor in combination with an EGFR inhibitor and a TGFβ1 receptor inhibitor in the preparation of a breast cancer therapeutic drug. The concentrations of the YAP inhibitor, EGFR inhibitor and TGFβ1 receptor inhibitor are in accordance with the recommended concentrations of the drug in the art.

Furthermore, the treatment may be to inhibit tumor proliferation, promote apoptosis and/or inhibit tumor metastasis.

Based on the above treatment mechanism, further, the YAP inhibitors include but are not limited to CA3 (CIL56), Verteporfin, and Acadesin (AICAR).

Preferably, the YAP inhibitor is CA3 (CIL56).

Furthermore, the EGFR inhibitors include but are not limited to gefitinib, erlotinib, and afatinib.

Preferably, the EGFR inhibitor is gefitinib.

Furthermore, the TGFβ1 receptor inhibitor includes but is not limited to galunisertib and A83-01.

Preferably, the TGFβ1 receptor inhibitor is galunisertib.

Preferably, the breast cancer is breast cancer that highly expresses YAP.

The present invention also provides a drug for treating breast cancer, which contains a YAP inhibitor and an EGFR inhibitor and/or a TGFβ1 receptor inhibitor; that is, it contains a YAP inhibitor and an EGFR inhibitor, or contains a YAP inhibitor and a TGFβ1 receptor inhibitor, or contains a YAP inhibitor, an EGFR inhibitor and a TGFβ1 receptor inhibitor.

Furthermore, the drug contains a YAP inhibitor, an EGFR inhibitor and a TGFβ1 receptor inhibitor.

Furthermore, the YAP inhibitor is CA3, Verteporfin, or Acadesin (AICAR).

Preferably, the YAP inhibitor is CA3.

Furthermore, the EGFR inhibitor is gefitinib, erlotinib, or afatinib.

Preferably, the EGFR inhibitor is gefitinib.

Furthermore, the TGFβ1 receptor inhibitor is galunisertib or A83-01.

Preferably, the TGFβ1 receptor inhibitor is galunisertib.

Furthermore, the drug has a YAP inhibitor and an EGFR inhibitor and/or a TGFβ1 receptor inhibitor as main active ingredients.

Preferably, the drug further comprises a pharmaceutically acceptable excipient.

Preferably, the drug contains an effective dose of a YAP inhibitor and an EGFR inhibitor and/or a TGFβ1 receptor inhibitor.

Preferably, the molar concentration ratio of the YAP inhibitor and the EGFR inhibitor or TGFβ1 receptor inhibitor when used in combination is (0.5-1):(5-20).

Further preferably, the molar concentration ratio of the YAP inhibitor and the EGFR inhibitor or the TGFβ1 receptor inhibitor when used in combination is 1:10.

Preferably, the molar concentration ratio of the YAP inhibitor, EGFR inhibitor and TGFβ1 receptor inhibitor when used in combination is (0.5-1):(5-20):(5-20).

Further preferably, the molar concentration ratio of the YAP inhibitor, the EGFR inhibitor and the TGFβ1 receptor inhibitor when used in combination is 1:10:10.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides the use of YAP inhibitors in combination with EGFR inhibitors and/or TGFβ1 receptor inhibitors in breast cancer treatment drugs. The present invention shows that YAP inhibitors can activate downstream ERK and AKT pathways, as well as TGFβ1-SMAD signaling pathways, by promoting the expression of EGFR and TGFβ1 proteins, that is, YAP inhibitors activate downstream ERK and AKT pathways, as well as TGFβ1-SMAD signaling pathways, so that they produce drug resistance and promote tumor metastasis side effects during the treatment of tumors. By using YAP inhibitors in combination with EGFR inhibitors and/or TGFβ1 receptor inhibitors, the drug resistance and tumor metastasis caused by the enhanced expression of EGFR and TGFβ1 caused by YAP inhibitors can be overcome, and breast cancer has obvious ability to inhibit proliferation and promote apoptosis, and has a synergistic therapeutic effect. The present invention provides a combined targeted tumor treatment scheme, that is, YAP inhibitors are used in combination with EGFR inhibitors and/or TGFβ1 receptor inhibitors to achieve synergistic treatment of breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of YAP inhibitor CA3 on EGFR and TGFβ1 protein expression; wherein A is the chemical structure of YAP inhibitor CA3, B is the mRNA expression level of EGFR, TGFβ1 and its downstream signaling molecules after different breast cancer cell lines were treated with YAP inhibitor CA3 with concentration gradient, C is the protein expression of EGFR, TGFβ1 and its downstream signaling molecules after different breast cancer cell lines were treated with YAP inhibitor CA3 with concentration gradient, D is the mRNA expression level of EGFR, TGFβ1 and its downstream signaling molecules in YAP knockdown stable breast cancer cell lines, and E is the protein expression level of EGFR, TGFβ1 and its downstream signaling molecules in YAP knockdown stable breast cancer cell lines.

Figure 2 shows the effect of XMU-MP-1 on the expression of EGFR and TGFβ1; wherein A is the expression level of EGFR, TGFβ1 and its downstream signaling molecule mRNA after the YAP knockdown stable breast cancer cell line was treated with XMU-MP-1 at a concentration gradient, and B is the expression level of EGFR, TGFβ1 and its downstream signaling molecule mRNA detected by fluorescence quantitative PCR after different breast cancer cells were treated with XMU-MP-1 at a concentration gradient.

Figure 3 shows the effects of YAP inhibitor (CA3) and EGFR inhibition (gefitinib) on the proliferation and apoptosis of breast cancer cells; wherein, A is the phosphorylation level of AKT1 and ERK downstream of EGFR after breast cancer cells were treated with CA3 and gefitinib alone or together, B is the cell viability level after breast cancer cells were treated with CA3 and gefitinib alone or together, C is the cell clone formation after breast cancer cells were treated with CA3 and gefitinib alone or together, and D is the cell apoptosis after breast cancer cells were treated with CA3 and gefitinib alone or together.

Figure 4 shows that knocking down YAP promotes the sensitivity of breast cancer cells to the EGFR inhibitor (gefitinib); wherein, A is the expression level of AKT1 and ERK downstream of EGFR after gefitinib treatment of the stable transgenic cell line with YAP knockdown, B is the cell viability level after gefitinib treatment of different breast cancer cells, and C is the cell clone formation after gefitinib treatment of different breast cancer cells.

Figure 5 shows that the YAP inhibitor CA3 and the TGFβ1 receptor inhibitor galunisertib have a synergistic effect in inhibiting the migration of breast cancer cells; wherein, A is the expression level of TGFβ1 downstream signaling molecules after different breast cancer cells are treated with CA3 or galunisertib alone or together, B is the quantitative detection of the expression level of TGFβ1 downstream signaling molecule mRNA after YAP knockdown stable breast cancer cell lines are treated with CA3 or CA3 and galunisertib together, C is the immunofluorescence detection of TGFβ1 downstream signaling molecules after breast cancer cells are treated with CA3 or galunisertib alone or together, and D is the crystal violet observation of cell migration after breast cancer cells are treated with CA3 or galunisertib alone or together.

Figure 6 shows the combined effect of YAP inhibitor CA3 and EGFR inhibitor (gefitinib) in nude mouse tumor-bearing experiments; wherein A is the subcutaneous tumor volume measured during the administration period, B is the weight of the subcutaneous tumor after removal, C is the photographic result of the subcutaneous tumor, and D is the expression of each protein in the subcutaneous tumor tissue of nude mice.

Figure 7 shows the synergistic effect of the YAP inhibitor CA3 in combination with EGFR inhibitor (gefitinib) and TGFβ1 receptor inhibitor galunisertib on inhibiting breast cancer cell proliferation and metastasis; A is the expression level of EGFR and TGFβ1 downstream signaling molecules and apoptosis signal detection after CA3, galunisertib and Gefitinib are treated with breast cancer cells alone or together, B is the detection of crystal violet to observe cell migration after CA3 or CA3, galunisertib and Gefitinib are treated with breast cancer cell lines alone or together, and C is the quantitative expression of B The results are as follows: the results of the quantitative analysis and statistical analysis of the data; D is the EdU staining results after CA3 or CA3 and galunisertib or Gefitinib were treated alone or together with breast cancer cell lines, to indicate the inhibitory effect on tumor cell proliferation; E is the quantitative results and statistical analysis of D; F is the EdU staining results after CA3 or CA3 and galunisertib or Gefitinib were treated alone or together with breast cancer cell lines, to indicate the inhibitory effect on tumor cell proliferation; and F is the quantitative results and statistical analysis of G.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 YAP inhibitor CA3 promotes the expression of EGFR and TGFβ1 proteins

As a core transcription factor in the Hippo signaling pathway, YAP has been reported to be highly expressed as an oncogene in a variety of tumors, including breast cancer. However, the exact role of YAP in breast cancer remains complex, so studying the role of YAP in the occurrence and development of breast cancer is of great significance for the targeted treatment of breast cancer. Specifically:

(1) To study the effect of CA3 on the expression of EGFR and TGFβ1. In various breast cancer cells (MDA-MB-231, MDA-MB-468, SKBR3, T47D), CA3 was used to treat the cells with a concentration gradient (0μM, 0.5μM, 1μM, 1.5μM) for 24 hours, and then the cell extracts were collected to detect the expression of EGFR, TGFβ1 and their downstream signaling molecules by western blot; at the same time, total RNA was extracted from CA3-treated cells, reverse transcribed into cDNA, and then fluorescence quantitative PCR was used to detect the expression levels of EGFR, TGFβ1 and their downstream signaling molecules mRNA.

(2) The effect of downregulating YAP on the expression of EGFR and TGFβ1 was studied. pLKO-shYAP1 was packaged into lentivirus to infect breast cancer cell lines SKBR3 and MDA-MB-468, and stable cell clones were formed by screening with puromycin. Then, wild-type cell lines and YAP knock-down stable cell lines were collected to extract proteins and total RNA, and the expression levels of EGFR, TGFβ1 and their downstream signaling molecules were detected by western blot and fluorescence quantitative PCR experiments, respectively.

The results are shown in Figure 1. The present study treated a variety of breast cancer cells with the YAP inhibitor CA3 and found that CA3 can promote the expression of EGFR and TGFβ1, promote the phosphorylation of ERK and AKT1 downstream of EGFR, and promote the phosphorylation of SMAD2 downstream of TGFβ1 and the expression of EMT-related genes such as snail and vimentin (Figure 1B-C). Consistent with this, the expression of EGFR and TGFβ1 was also significantly enhanced in the YAP knockdown stable breast cancer cell line (Figure 1D-E).

The above results show that since YAP inhibition can promote the expression of EGFR and TGFβ1, what will happen in reverse? Breast cancer cells were further treated with XMU-MP-1, an inhibitor of the upstream kinase MST1/2 in the Hippo signaling pathway. As an inhibitor of MST1/2, XMU-MP-1 can inhibit the phosphorylation of YAP by inhibiting the phosphorylation of LAST1/2, thereby promoting its nuclear entry and transcriptional function. Specifically:

The effect of XMU-MP-1 on the expression of EGFR and TGFβ1 was studied: a variety of breast cancer cells (MDA-MB-231, MDA-MB-468, SKBR3, T47D) were treated with XMU-MP-1 at a concentration gradient (0μM, 1μM, 2μM, 4μM). After 24 hours, the cell extracts were collected and the expression of EGFR, TGFβ1 and their downstream signaling molecules was detected by western blot. At the same time, the total RNA was extracted from the cells treated with XMU-MP-1, and the expression levels of EGFR, TGFβ1 and their downstream signaling molecules mRNA were detected by fluorescence quantitative PCR after reverse transcription into cDNA.

The results are shown in Figure 2 . According to western blot and real-time qPCR experiments, it was found that XMU-MP-1 could inhibit the expression of EGFR and TGFβ1 by promoting YAP activation ( Figure 2A-B ), which was the opposite of the effect of CA3.

Example 2 Study on the effect of CA3 combined with EGFR inhibitor or TGFβ1 receptor inhibitor

Example 1 Studies have shown that YAP inhibitors (CA3) can significantly promote the 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 use of YAP inhibitors in the treatment of breast cancer to produce drug resistance. To this end, the present invention intends to use EGFR inhibitors to inhibit the activation of ERK and AKT signaling pathways caused by CA3, and TGFβ1 receptor inhibitors to inhibit the activation of downstream signaling pathways caused by enhanced TGFβ1 expression caused by CA3. The specific method is as follows:

1. Study on the synergistic effect of YAP inhibitor CA3 and EGFR inhibitor gefitinib on the proliferation and apoptosis of breast cancer cells:

(1) CA3 and gefitinib were used alone or together to treat breast cancer cells, wherein the working concentration of CA3 was 0.5 μM and the working concentration of gefitinib was 5 μM. After 24 hours of treatment, the cell extracts were collected to detect the phosphorylation levels of AKT1 and ERK downstream of EGFR.

(2) Cell proliferation-toxicity experiment detected the synergistic inhibitory effect of YAP inhibitor CA3 and EGFR inhibitor gefitinib on breast cancer cells. Different breast cancer cells were treated with CA3 and gefitinib alone or in combination (the working concentration of CA3 was 0.5 μM and the working concentration of gefitinib was 5 μM) for 72 h, and the viability of breast cancer cells was detected using CCK8 kit.

(3) Clone formation experiment to detect the synergistic inhibitory effect of YAP inhibitor CA3 and gefitinib on breast cancer cells. 1000 breast cancer cells were inoculated in each well of a 6-well plate, and the cells were treated with CA3 and gefitinib alone or in combination (the working concentration of CA3 was 0.5 μM, and the working concentration of gefitinib was 5 μM). After 24 hours, the medium containing the drugs was discarded, and the cells were cultured with complete medium until cell clones were visible to the naked eye. The cells were fixed and stained with crystal violet to observe the cell clone formation.

(4) Detect the synergistic killing effect of YAP inhibitor CA3 and gefitinib on breast cancer cells. Different breast cancer cells were treated with CA3 and gefitinib alone or in combination (the working concentration of CA3 was 0.5 μM and the working concentration of gefitinib was 5 μM). After 48 hours, the cells were collected and the apoptosis of cells was detected using Annexin V-FITC/PI apoptosis kit (Vazyme, A211-01).

2. Study on the inhibition of breast cancer cell migration induced by YAP inhibitor CA3 by TGFβ1 receptor inhibitor galunisertib:

CA3 and galunisertib were used alone or together to treat breast cancer cells, with the working concentration of CA3 being 0.5 μM and the working concentration of galunisertib being 5 μM. After 24 hours of treatment, the cells were collected and counted, and the same number of cells (30,000 cells) were seeded into transwell chambers with serum-free culture medium, with normal culture medium underneath the chambers. After 24 to 48 hours of culture, the cells underneath the chambers were fixed and stained with crystal violet to observe cell migration.

3. Result analysis:

In the cell experiment, it was first determined that the EGFR inhibitor gefitinib could inhibit the activation of the ERK and AKT signaling pathways caused by CA3 (Figure 3A). Then, through cell proliferation experiments, clone formation experiments, and apoptosis experiments, it was found that CA3 and gefitinib significantly inhibited the proliferation ability of breast cancer cells and promoted the apoptosis ability of breast cancer cells, with a good synergistic effect (Figure 3B-D). This finding was also confirmed in the stable transfection cell line with YAP knockdown (Figure 4A-C).

At the same time, the present invention also determined that the TGFβ1 receptor inhibitor (galunisertib) can inhibit the activation of the TGFβ1 downstream signaling pathway caused by CA3 (Figure 5A~C), and confirmed through trans-well experiments that galunisertib significantly inhibited the EMT formation and migration of breast cancer cells caused by CA3 (Figure 5D).

Therefore, the results of this study showed that the YAP inhibitor CA3 and the EGFR inhibitor gefitinib had a synergistic effect on the proliferation and apoptosis of breast cancer cells, and the TGFβ1 receptor inhibitor galunisertib could also significantly inhibit the migration of breast cancer cells caused by the YAP inhibitor CA3.

Example 3 Animal Experimental Study on the Combination of CA3 and EGFR Inhibitors

The specific research methods are as follows:

(1) Effect of CA3 combined with gefitinib on tumor-bearing mouse model. 5×10 ⁶ breast cancer cells MDA-MB-468 were subcutaneously inoculated into Balb/c immunodeficient mice. When the tumor volume reached 50-100 cubic millimeters (mm ³ ), gefitinib and CA3 were intraperitoneally injected orally alone or in combination. Gefitinib was intraperitoneally injected at a dose of 75 mg/kg per day, and CA3 was gavaged at a dose of 1 mg/kg every two days. The weight of tumor-bearing mice and the size of subcutaneous tumors were measured every 2-3 days. When the subcutaneous tumors of control tumor-bearing mice grew to a certain size, the mice were euthanized, the subcutaneous tumors were removed and the weight of the tumors was weighed. The tumor volume was calculated using the formula (length × width ² × 0.52).

(2) Western blot was used to detect the expression of various proteins in the tumor tissues in (1). The tumor tissues obtained in (1) were lysed to extract proteins, and then the expression of various proteins was analyzed by western blot.

Result analysis:

The present invention uses mouse tumor-bearing experiments to confirm that the combination of YAP inhibitor CA3 and EGFR inhibitor (gefitinib) can significantly inhibit the tumorigenicity of breast cancer cells in nude mice (Figure 6A-C), accompanied by an inhibitory effect on the downstream ERK and AKT pathways (Figure 6D). Therefore, the results show that the YAP inhibitor CA3 and the EGFR inhibitor gefitinib synergistically inhibit the growth of breast tumors in vivo.

Example 4 Cellular Experimental Study on the Combination of CA3 with EGFR Inhibitor Gefitinib and TGFβ1 Receptor Inhibitor Galunisertib

The study in Example 1 shows that YAP inhibitors (CA3) can significantly promote the expression of EGFR and TGFβ1 proteins, thereby activating downstream ERK and AKT pathways, as well as TGFβ1-SMAD signaling pathways. The studies in Examples 2 to 4 show that YAP inhibitors can be used in combination with the TGFβ1 receptor inhibitor galunisertib to inhibit tumor metastasis, and synergistically with the EGFR inhibitor gefitinib to inhibit the in vitro and in vivo growth of breast tumors. To this end, the present invention intends to use EGFR inhibitors and TGFβ1 receptor inhibitors galunisertib to jointly inhibit the activation of ERK and AKT signaling pathways caused by CA3, and TGFβ1 receptor inhibitors to inhibit the activation of downstream signaling pathways caused by enhanced TGFβ1 expression caused by CA3. The specific method is as follows:

1. Study on the synergistic inhibitory effect of YAP inhibitor CA3 combined with EGFR inhibitor gefitinib and TGFβ1 receptor inhibitor galunisertib on the proliferation and metastasis of breast cancer cells:

(1) CA3, gefitinib and galunisertib were used alone or together to treat breast cancer cells, where 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. After 24 hours of treatment, the cell extracts were collected to detect the phosphorylation levels of AKT1 and ERK downstream of EGFR, and the downstream signals and transfer of TGFβ1 (E-cad, Snail, Vimentin) and apoptosis signals (cleaved PARP).

(2) CA3, gefitinib, and galunisertib were used alone or together to treat breast cancer cells, where 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. After 24 hours of treatment, the cells were collected and counted, and the same number of cells (30,000 cells) were seeded into transwell chambers with serum-free culture medium, and the bottom of the chamber contained normal culture medium. After culturing for 24 to 48 hours, the cells under the chamber were fixed and stained with crystal violet to observe cell migration.

(3) Cell proliferation assay to detect the synergistic inhibitory effect of YAP inhibitor CA3 with gefitinib and galunisertib on breast cancer cells. Breast cancer cells were treated with CA3 alone or in combination with gefitinib and galunisertib (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) for 24 h, and the viability of breast cancer cells was detected using a BdU kit.

(4) Detect the synergistic killing effect of YAP inhibitor CA3 with gefitinib and galunisertib on breast cancer cells. Breast cancer cells were treated with CA3 alone or in combination with gefitinib and galunisertib (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) for 48 hours, and the cells were collected and the apoptosis of cells was detected using Annexin V-FITC/PI apoptosis kit (Vazyme, A211-01).

2. Result analysis:

In the cell experiment, we first determined that the combined use of CA3, gefitinib treatment and galunisertib (three-drug combination) can not only inhibit cell proliferation pathways and promote apoptosis, but also inhibit cell EMT formation and metastasis pathways (Figure 7A). We then confirmed through trans-well experiments that the three-drug combination can significantly inhibit the migration of breast cancer cells caused by CA3 (Figure 7B-C). Cell proliferation experiments and apoptosis experiments found that the three-drug combination significantly inhibited the proliferation ability of breast cancer cells and promoted the apoptosis ability of breast cancer cells, with a good synergistic effect (Figure 7D-F).

Therefore, the results of this study show that the YAP inhibitor CA3 combined with the EGFR inhibitor gefitinib and/or the TGFβ1 receptor inhibitor galunisertib has a synergistic effect on the inhibition of breast cancer cell proliferation and metastasis. In addition, those skilled in the art can reasonably expect that the use of other YAP inhibitors (such as verteporfin, acadexin) combined with other EGFR inhibitors (such as erlotinib, afatinib) and/or other TGFβ1 receptor inhibitors (such as A83-01) in the treatment of breast cancer also has a synergistic effect based on the treatment mechanism of the present invention.

Claims (10)

1. 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. 2. The use according to claim 1, wherein the YAP inhibitor is CA3, verteporfin or acadesine.
3. 3. The use of claim 1, wherein the EGFR inhibitor is gefitinib, erlotinib, or afatinib.
4. 4. The use according to claim 1, wherein the tgfβ1 receptor inhibitor is galunisertib or a83-01.
5. 5. A breast cancer therapeutic agent comprising a YAP inhibitor and an EGFR inhibitor and/or a tgfβ1 receptor inhibitor.
6. 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. 7. The medicament of claim 5, wherein the YAP inhibitor is CA3, verteporfin or acadesine.
8. 8. The medicament of claim 5, wherein the EGFR inhibitor is gefitinib, erlotinib, or afatinib.
9. 9. The medicament of claim 5, wherein the tgfβ1 receptor inhibitor is galunisertib or a83-01.
10. 10. The medicament of claim 5, further comprising pharmaceutically acceptable excipients.