Breast cancer is one of the most aggressive malignant tumors in women. According to the expression differences of estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2 (HER-2) and cell proliferation antigen Ki-67, breast cancer can be divided into four molecular subtypes: Luminal A, Luminal B, HER-2 overexpression and Basal-like. Yes-associated protein (YAP), a downstream effector of the Hippo pathway, is overexpressed in human cancers and is associated with proliferation, apoptosis, migration, invasion and resistance to chemotherapy drugs in breast cancer cells. Verteporfin (VP) is used as a photosensitizer in the treatment of neovascular macular degeneration. VP is also identified as an inhibitor of YAP/TEA domain transcription factor (TEAD) interaction in the absence of light activation. However, detailed structural information about VP and YAP interactions is relatively scarce and VP research targeting YAP in different molecular subtypes of breast cancer cells is also rare. The aims of the present study were to structurally describe the VP binding site in the YAP crystal structure and to verify the non-photoreactive VP effect targeting YAP on the migration of different molecular subtypes of breast cancer cells. The crystal structure of VP and YAP was calculated by AutoDock 4.2 and the result was illustrated using PyMOL. The non-photoactivated VP effect on the migration of Luminal A MCF-7, Luminal B BT-474 and triple-negative breast cancer BT-549 breast cancer cells was evaluated by wound healing and Transwell migration experiments. Results from molecular docking experiments demonstrated that VP could interact through hydrogen bonds and hydrophobic interactions with important YAP residues involved in TEADs binding (Gln82, Val84, Met86 and Arg89). Migration experiments revealed that the non-photoinduced VP could inhibit the migration of different molecular subtypes of breast cancer cells. The results of the present study indicated that VP may be a novel repositioned drug for breast cancer treatment in the future.
The Hippo signaling pathway serves an important role in the occurrence and progression of breast cancer (
Verteporfin (VP) was originally designed as a photosensitizer for macular degeneration (
Non-photoactivated VP can disrupt the interaction and transcriptional activity of YAP/TEAD and thus inhibit YAP-mediated tumor proliferation, induce tumor cell apoptosis and restore sensitivity to chemotherapy drugs (
VP (MedChemExpress) was dissolved in DMSO as a stock solution at a concentration of 10 mM, stored at −20°C and protected from light.
Luminal A MCF-7, Luminal B BT-474 and TNBC BT-549 cells (
All molecular docking studies were performed using AutoDock 4.2.6 software package (
Three parallel lines were drawn in advance on the bottom of a 24-well plate. MCF-7, BT-474 and BT-549 cells were placed in the plates at 1.2×105 cells per well. After the cells reached 85–90% confluence, a 10 µl pipette tip was used to draw a light, straight line. The floating cells in the wells were carefully washed off with PBS buffer and images captured under the light microscope at the 0 h time point. The blank control group cells were cultured in fresh medium without FBS and the treatment group was treated with 8 µM VP. After 24 h of culture, the cells were washed with PBS buffer three times and images captured under the microscope. Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) was used to quantify the migration distance of cells before and after scratches and to calculate the wound healing distance.
For these experiments, the cells were cultured at a density of 3×105 in FBS-free medium. In the control group, 10% FBS medium was dripped along the side wall of the lower Transwell chamber. In the treatment group, 10% FBS medium containing 8 µM VP was added to the lower Transwell chamber. The 3×104 cell suspensions were carefully added to the upper Transwell chamber and then cultured at 37°C in the incubator for 24 h. The medium was then removed, and the cells were washed twice with PBS and fixed for 20 min with 4% paraformaldehyde at room temperature. The sample were stained with DAPI for 15 min in the dark and images captured under a fluorescence microscope (magnification, ×100). A total of five visual fields were randomly selected and the results were quantified by Image Pro Plus 6.0 software (Media Cybernetics, Inc.).
Data are presented as the mean ± standard deviation. Comparisons between VP-treated and -untreated control groups were made by unpaired t-test. P<0.05 was considered to indicate a statistically significant difference.
The crystal structure of YAP (PDB code 3KYS) was used as docking receptor for VP in docking molecular experiments and was prepared by adding hydrogen atoms, removing waters and adding Gasteiger charge. The best-scored pose of VP was selected as the possible bind conformation for the docking analysis (
The expression of YAP target proteins in MCF-7, BT-474 and BT-549 cells was evaluated. Western blotting demonstrated that 8 µM VP treatment could notably downregulate the protein expression levels of YAP, TEAD, and CYR61. The expression of CTGF were differential in three subtypes of breast cancer and no CTGF expression was detected in MCF-7 cells (
Transwell migration assays revealed that 8 µM VP could inhibit the number of migratory cells of Luminal A MCF-7, Luminal B BT-474 and TNBC BT-549 cells (P<0.01;
The core components of the Hippo signaling pathway are involved in the regulation of proliferation, migration, invasion and chemoresistance of breast cancer cells (
YAP and TEAD proteins are the best molecular target candidates to regulate the Hippo pathway, as the formation and activation of the YAP/TEAD complex is the last step of the Hippo-YAP pathway. VP is a compound that directly destroys the formation of YAP/TEAD complexes and inhibits the most essential part of the Hippo pathway (
YAP is highly expressed in many solid tumors and its carcinogenic function is mediated by its nuclear localization and interaction with TEAD transcription factors (
Abnormal expression of CTGF and Cyr61 proteins is associated with the progression of breast cancer, prostate cancer and malignant melanoma (
In conclusion, VP is an FDA-approved photosensitizer for the treatment of age-related macular degeneration and can inhibit the occurrence of tumor by destroying the formation of YAP/TEAD complex in its non-photoactivated form (
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The present study was supported by The National Natural Science Foundation of China (grant no. 81473687), the Shandong Provincial Natural Science Foundation, China (grant nos. ZR2009CM039 and ZR2013HM038), the High Level Project Cultivation Program of Shandong First Medical University, China (grant no. 2018GCC14) and the Academic promotion of Shandong First Medical University, China (grant no. 2019QL017).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
CW acquired and analyzed the data, and drafted and revised the manuscript. XL conceptualized the study, acquired and analyzed the data, and drafted and revised the manuscript. Both authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
Molecular docking analysis (A) Docking results of verteporfin interaction with protein surface and (B) surrounding residues of YAP (PDB code 3KYS). VP is shown in green stick model and key residues of YAP were shown in cyan stick models. The nitrogen, oxygen and sulfur atoms were shown in blue, red and yellow, respectively. The hydrogen bonds are represented by red dashed lines. YAP, yes-associated protein.
Effects of VP on the protein expression levels of downstream YAP targets. CTGF, connective tissue growth factor; CYR61, cysteine-rich angiogenic inducer; TEAD, TEA domain transcription factor; YAP, yes-associated protein; VP, verteporfin.
VP affects the migratory ability of three breast cancer cell subtypes: MCF-7, BT-474 and BT-549. **P<0.01, ***P<0.001 vs. control group. VP, verteporfin.
VP effect on the wound healing ability of three breast can cell subtypes: MCF-7, BT-474 and BT-549. Magnification, ×100. ***P<0.001 vs. control group. VP, verteporfin.