Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Different Subtypes of Breast Cancer Cell Lines Without Light Activation

DOI: https://doi.org/10.21203/rs.3.rs-15912/v1

Abstract

Background Breast cancer (BC) can be separated into four molecular subclassifications including Lumina1 A, Lumina1 B, HER-2 overexpression and Basal-like subtype. These classifications are based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2) and cell proliferation antigen (Ki-67). The Hippo signaling pathway plays an indispensable role in BC. The YAP1 gene is a terminal effector of Hippo pathway, and hyperactivation of YAP mediates tumorigenesis. As an inhibitor of YAP, non-photoactivated verteporfin (VP) can inhibit YAP-mediated tumor proliferation and angiogenesis by eliminating its interaction with TEAD. This study set out to determine the effect and molecular mechanisms of VP-mediated inhibition of YAP in different subtypes of BC. Methods Luminal A, Luminal B and Basal-like BC cells were cultivated in vitro in order to study effect of VP on proliferation and apoptosis on these three molecular BC subtypes. Results Our experimental results show that VP inhibits cell proliferation, YAP-TEAD interaction and its downstream target expression. VP also induces tumor cell apoptosis, and promotes the cleavage of Caspase-9 and PARP in various molecular subtypes of BC cells. Conclusion These findings provide a basis for VP as a potential anti-tumor therapeutic for BC by targeting the Hippo pathway effector YAP.

Background

Breast cancer (BC) is a common malignant tumor, with greater than 250,000 newly diagnosed cases every year [1]. The treatment regimen for BC depends primarily on the level of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2) and Ki-67 in tumor patients. In addition to the traditional surgery, different subtypes of BC need endocrine therapy, chemotherapy, radiotherapy, and biologically-targeted treatment [23]. However, early metastasis and drug resistance is associated with a poor prognosis for BC [4. Hence, it is urgent to find reliable molecular targets and targeted drugs for breast cancer patients in order to improve prognosis and treatment efficacy.

The Hippo signaling pathway, whose main function is to inhibit cell proliferation and limit organ overgrowth, is found in Drosophila melanogaster and is highly conserved in mammals [5]. YAP is the final effector of Hippo pathway. When dephosphorylated, YAP localized to the nucleus and binds to TEAD and act as a transcriptional co-activator. YAP-TEAD mediates the expression of different oncogenes including those that regulate tumor cell cycle, epithelial mesenchymal transition, migration, invasion, and chemoresistance [6]. Gene mutations and changes in expression of essential Hippo pathway components encourages the occurrence and development of breast cancer. Overexpression of YAP can significantly stimulate the proliferation of tumor cells [7]. Additionally, the expression of YAP is correlated with specific molecular subtypes of BC [8].

Clinically, Verteporfin (VP) has long been utilized as a porphyrinic photosensitizer in photodynamic treatment for neovascular macular degeneration [9]. According to the previous research, non-photoactivated VP selectively binds to YAP and changes the YAP conformation process, thereby eliminating its interaction with TEAD [10]. In addition, VP blocks YAP function by increasing 14-3-3σ protein in the cytoplasm [11]. Numerous studies have also shown that VP downregulates the transcription of YAP, which then inhibits cell proliferation-related genes including cylinD1, cyclinE1, and TEAD in multiple tumor cells [12].

In this study, we assessed the influence and mechanism of VP on the proliferation and apoptosis of the Luminal A, Luminal B and Basal-like molecular subclassifications of BC in related to YAP. Our findings provide a novel theoretical basis for use of VP in the treatment of BC.

Methods

Cell culture

Human Luminal A breast cancer cell line MCF-7, Luminal B breast cancer cell line BT-474 and Basal-like subtype cell line BT-549 [13] were bought through Zhongqiao xinzhou biology (Shanghai, China). MCF-7, BT-474 and BT-549 were maintained in RPMI-1640 (Hyclone, Logan, Utah, USA) with 10% FBS (Gibco, Carlsbad, CA, USA) and placed in an incubator at 5% CO2 and 37°C. Verteporfin were bought from MCE (St Louis, USA).

Cell viability assay

MCF-7, BT-474 and BT-549 cells were added to a 96-well plate at a 4×104 cells per well density. After incubation with VP, cell viability was assessed utilizing the CCK-8 (DOJINDO) experiment at 24, 48, and 72 hours. In brief, 10 µl of the CCK8 mixture was supplemented into every well and added in a 37°C incubator for 1.5 hours. The absorbance was quantified at a 450 nm wavelength.

Real-time PCR

Total RNA was obtained from VP-incubated cells utilizing the Trizol regent (Invitrogen; Thermo Fisher Scientific, Inc.) as per established protocol. cDNA was made utilizing the PrimeScript reverse transcription reagent kit (TIANGEN BIOTECH Co., Beijing, China) as per established guidelines. cDNA was assessed through qPCR with a SuperReal PreMix Plus (SYBR Green) system (TIANGEN BIOTECH Co.). The amplification conditions include: 95˚C for 15 min, and then 40 cycles of 95˚C for 20 sec, 56˚C for 30 sec and 68˚C for 30 sec. The primer sequences used in qPCR were: YAP forward (F) 5'-TGACCCTCGTTTTGCCATGA-3' and reverse (R), 5'-GTT GCTGCTGGTTGGAGTTG-3'; CTGF F, 5'-TGGAAGAGAACATTAAGAAG GGCA-3' and R, 5'-TGCAGCCAGAAAGCTCAAAC-3'; AXL F, 5'-ACCCC AGAGGTGCTAATGGA-3' and R, 5'-GTGGACTGGCTG TGCTTCC-3'; CYR61 F, 5'- GCAAGGAGCTGGGATTCGAT-3' and R, 5'-ATTCCAAAAACAGGGAGCCG-3'; GAPDH F, 5'-GCACCGTCAAGG CTGAGAAC-3' and R, 5'-TGGTGAAGACGC CAGTGGA-3'. GAPDH functioned as internal control. Gene expression was measured utilizing the 2-ΔΔ Ct method.

Apoptosis assay

In order to determine cell apoptosis, the VP-incubated cells were washed and fixed at 4°C with 4% paraformaldehyde for 30 min. Another wash later, cells were incubated with 0.2% TritonX-100 for 15 min at room temperature, and assessed cytometrically using Principle In Situ Cell Death Detection Kit (Roche, Inc.) according to established guidelines.

Protein extraction and western blot

Total proteins were obtained utilizing RIPA lysis buffer that contained phosphatase and protease inhibitors. Protein concentration was evaluated utilizing a bicinchoniniacid assay (Solarbio, Beijing). The later steps of Western blotting were conducted using standard protocol. Antibody directed towards GAPDH was acquired from Novus Biologicals. Antibodies directed towards YAP, TEAD, Bcl-2, Bax, p-YAP (Ser127), CYR61, CTGF, AXL, Caspase9, Cleaved Caspase9, PARP, Cleaved PARP were bought through Cell Signaling Technology (Danvers, MA).

Statistical analyses

Data was represented as mean ± standard deviation. Comparisons were determined utilizing an unpaired 2-tailed Student t-test in Prism 5. The GraphPad Prism 5 software was utilizedd to assess statistical significance. P<0.05 signifies statistical significance.

Results

YAP levels in different subtypes of BC cells

Western blot results showed that YAP was expressed in MCF-7, BT-474 and BT-549 cells. The expression of YAP in Luminal B BT-474 BC cells and triple negative breast cancer (TNBC) BT-549 cells were substantially increased in Luminal A MCF-7 cells. BT-474 had the highest YAP expression among the three different subtypes of BC cell lines (P<0.001) (Fig 1).

The effect of verteporfin on the proliferation of different molecular subtypes of BC cells

Data from the CCK-8 test demonstrated that VP could suppress MCF-7, BT-474 and BT-549 cell growth in a dose-dependent manner, in comparison to the controls (Fig 2a). Comparison of different molecular subtypes of BC cells under the same drug concentration found that after 24 hours of VP treatment, the proliferation of BT-549 cells decreased most significantly. After treatment with 8 μm and 16 μm VP for 48 h, there was a statistically significant difference in the proliferation rate of different BC subtypes (P <0.05). The most significant reduction after treatment with 8 μm of VP occurred in the BT-549 cells, while the most significant decrease in proliferation after treatment with 16 μm VP occurred in MCF-7 cells. After VP treatment for 48 and 72 h, there was a statistical difference among the different molecular subtypes of BC cells (P <0.05) (Fig 2b).

Verteporfin regulates the expression of YAP, TEAD and YAP-TEAD downstream targets

As shown in Western blot results, VP blocks YAP, p-YAP and TEAD protein expression in MCF-7, BT-474 and BT-549 cells (Fig 3a). As the concentration of VP is increased, the expression of AXL and CYR61 proteins in different subtypes of breast cancer cells is downregulated. In addition, VP inhibited the expression of CTGF in BT-474 and BT-549 cells (Fig 3b). VP also inhibited YAP-TEAD transcription in MCF-7, BT-474 and BT-549 cells in vitro. RT-PCR results show that, in comparison to the controls, YAP, AXL and CYR61 mRNA expression in MCF-7, BT-474 and BT-549 cells were significantly reduced after treatment with 4, 8 and 12 μmol/mL VP. Treatment with VP also downregulated the mRNA expression of CTGF in BT-474 and BT-549 cells (Fig 3c).

Verteporfin induces cell apoptosis by disrupting YAP-TEAD contact

TUNEL experiments showed that VP induces apoptosis in MCF-7, BT-474 and BT-549 cells (Fig 4a). The Western blot data indicates that treatment VP leads to apoptosis of different subtypes of BC cells by decreasing expression of the YAP downstream target gene Survivin (Fig 4b), Bcl-2 and the ratio of Bcl-2/Bax compared to the controls. Additionally, VP led to an increase in levels of Bax (Fig 4c), cleaved Caspase-9 and cleaved PARP proteins (Fig 4d). These results demonstrate that VP, an inhibitor of Hippo YAP signaling pathway, is highly conducive for inducing apoptosis among different molecular subtypes of MCF-7, BT-474 and BT-549 BC cells.

Discussion

BC is the leading malignant tumor among females worldwide. In order to identify targeted treatment of BC, it is important to analyze the genetic differences between normal and tumor cells. BC can be separated into four subclassifications: Luminal A, Luminal B, HER-2 overexpression and Basal like type as per expression of ER, PR, HER-2 and Ki-67. The expression of PR and HER-2 are the further indicators for classification of Luminal A and Luminal B [1415]. Although the clinical treatment of BC has made great progress, there is still limited treatment options for patients who experience chemotherapy resistance and metastasis [16]. Moreover, TNBC, which is described by deficiency of ER, PR, and HER-2, is associated with the worst prognosis due to increased rates of recurrence and a lack of effective targeted therapies among all major subtypes of BC [17]. Recently, large scale genetic sequencing research has identified many mutations related to the occurrence and development of BC, and gradually outlined the interaction network of breast cancer oncogenes [1819].

Hippo is a complex tumor regulation pathway, and its deregulation is associated with changing normal state into a pathological cancer state. As the main effector of Hippo, YAP is phosphorylated by the Hippo Core complex and degraded in the cytoplasm [20]. When the Hippo upstream pathway is suppressed, hyperactivation of TEAD as a transcription factor requires the involvement of YAP as a coactivator to mediate downstream target gene transcriptional activity. Thus, YAP translocates into the nucleus and promotes proliferation, metastatic development, and stem cell maintenance of cancer cells [21]. Knockdown of AXL, one of YAP-TEAD target genes, reduces the growth and invasiveness of tumor xenographs [22]. Similarly, dysregulation of CTGF and CYR61 are strongly correlated with development of BC, prostate cancer and malignant melanoma [2325]. Survivin has a key role in controlling cell apoptosis, cell cycle and drug resistance [2627]. All of these functions depend on the interaction of YAP and TEAD transcription factors. Therefore, YAP exerts its oncogenic function by combining with the transcription factor TEAD to encourage expression of genes that have a function in the progression and metastasis of cancer.

Several studies demonstrated that VP decreases transcriptional activity of YAP by being competitively binding to YAP and abrogating the connection of YAP with TEAD, which blocks YAP-TEAD-stimulated tumorigenesis [12]. Our data demonstrated that VP inhibited the growth of different subtypes of BC cells, and that VP treatment efficiently prevented YAP-TEAD transcriptional activity. VP treatment led to YAP, AXL, CYR61 and/or CTGF downregulation in different subtypes of BC cell lines, suggesting that these effectors could be their own target genes as described in MDA-MB-231 cells [2830]. Recently, studies have shown that non-photoactivated VP could induce apoptosis of melanoma and pancreatic cancer cells by activating apoptosis-related proteins by preventing YAP-TEAD interaction [3133]. Our present results have demonstrated that VP induced the apoptosis of MCF-7, BT-474, and BT-549 cells by upregulating Bax, cleaved Caspase-9, and cleaved PARP, and downregulating Bcl-2. Although initially regarded as an FDA-approved photo-sensitizer drug in photodynamic treatment, VP has been described as having anti-tumor behaviors by preventing YAP-TEAD connection [34]. In agreement with prior studies that showed that VP suppressed proliferation of lung tumor [35], prostate tumor [36] and mesothelioma tumor [37], our results show that VP also inhibited the proliferation of Luminal A MCF-7, Luminal B BT-474 and TNBC BT-549 cells, and stimulated apoptosis in vitro. VP treatment led to downregulation of YAP-TEAD as well as the expression of the YAP-TEAD target genes CYR61, AXL, Survinvin and/or CTGF, regulation of Bcl-2 proteins, and stimulation of PARP and Caspase-9. These encouraging results suggest that use of VP could be a promising strategy for treatment of BC.

Experimental verification of various tumors indicates that targeting Hippo pathway is an effective way for treating cancers [38]. Proteins of the CCN (CTGF/CYR61/NOV) family exhibit different levels of expression and transcription in different tumor tissues. Changes in the transcriptional activity of CCN are also extremely important during tumorigenesis. CCN intervenes in embryonic development, angiogenesis, tumor heterogeneity and progression through various signaling pathways, including the Hippo pathway [3940]. Genetic studies have indicated that stable levels of CYR61 in MCF-7 cells is able to significantly counteract paclitaxel-induced apoptosis and increase chemotherapy resistance to doxorubicin [41]. In our study, we found that the treatment with VP of Luminal A MCF-7 cells downregulated the expression of CYR61, thereby suppressing the proliferation of MCF-7 cells and providing a new treatment strategy for Luminal A endocrine patients. Our study found differential expression of CTGF in three subtypes of BC. Interestingly, high expression of CTGF was observed in BT-549 cells, and no CTGF expression was detected in MCF-7 cells, which was concordant with previous data [42]. The activation of CTGF and CYR61 may exert divergent roles in the processes of different subtypes of BC.

Clinical studies have shown that YAP is overexpressed and related to poor patient prognosis in various cancers [43]. However, some researchers doubt the role of YAP as an oncogene in the developmental pathogenesis of BC. It has also been suggested that YAP is associated with a specific internal environment and the tumor itself as an oncogene or tumor suppressor gene [44]. Our study found that expression of YAP was significantly different in Luminal A-type BC cell MCF-7, Luminal B-type BC cell BT-474 and TNBC cell BT-549. Most studies have shown that inhibition of YAP expression can induce apoptosis. However, studies have also shown that YAP can migrate to the nucleus, bind to p73 tumor suppressor and induce apoptosis by activating the pro-apoptotic gene (PUMA) [45]. Another study showed that the tumor suppressive effect of YAP is dysregulated by YAP-induced apoptosis and anti-tumor immune surveillance response in BC cells. Furthermore, YAP stimulates the expression of anti-apoptotic genes and apoptosis-inducing genes may depend on the downstream transcription target genes expressed by intracellular environmental stimulation. Therefore, it may be possible for YAP to selectively induce p73-mediated apoptosis only under specific conditions such as DNA damage [46]. Our data show that VP also induced apoptosis of Luminal A MCF-7, Luminal B B-474 and TNBC BT-549 cells in vitro. VP treatment led to a decrease of Survivin and Bcl-2/Bax ratio, and cleavage of PARP and Caspase-9.

Although based on theoretical concepts, there are still several problems with the use of VP as an inhibitor of YAP/TEAD interaction. VP is a photosensitizer drug that is already in clinical use. It must be strictly controlled during exposure to cells in order to avoid exposure to light. Furthermore, the exact mechanism by which VP is able to affect Hippo signaling pathway still needs to be evaluated in greater depth. More importantly, our study paves the way for further drug development for BC.

Conclusions

In conclusion, we demonstrated that VP treatment inhibits the proliferation of MCF-7, BT-474 and BT-549 cells and YAP-TEAD transcriptional activity, and efficiently induces BC cell apoptosis. VP targeting of YAP-TEAD activity can provide theoretical support for progress of VP as a novel bio-therapeutic targeted treatment and the precision treatment of breast cancer.

Abbreviations

estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER -2) , breast cancer (BC), triple negative breast cancer (TNBC).

Declarations

Compliance with Ethical Standards

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors report no conflicts of interest in this work.

Funding

This work was supported by the following projects: the National Natural Science Foundation of China (No81473687), Shandong Provincial Natural Science Foundation, China (No ZR2009CM039 and No ZR2013HM038). High level project cultivation program of Shandong First Medical University, China (No 2018GCC14).

Authors' contributions

X.Q.L.,C.R.W., designed experiments, wrote the main manuscript text, X.Q.L.,C.R.W.,  performed experiments and prepared the figures. All authors have read and approved the manuscript.

Acknowledgements

Not applicable

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