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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2020 Oct 1;10(10):3179–3193.

Livin promotes tumor progression through YAP activation in ovarian cancer

Jianhua Gao 1, Wei Han 1, Yanan He 1, Jun Zhou 1, Jintian Miao 1, Guangmei Zhang 1
PMCID: PMC7642671  PMID: 33163264

Abstract

Ovarian cancer is a gynecological malignant tumor with a high morbidity. Livin is a novel member of the inhibitor of apoptosis protein family, which is expressed in various malignant tumors and is suggested to be a poor prognostic factor. However, the prognostic significance of Livin and the molecular mechanisms by which Livin promotes ovarian cancer progression are poorly understood. In this study, the upregulation of Livin was confirmed both in primary specimens from ovarian cancer patients and in ovarian cancer cell lines compared to normal controls in vitro. Overexpression of specific Livin transcripts promoted cell growth and migration in vitro, while knockdown of Livin expression suppressed these cellular processes. These effects of the Livin gene were also demonstrated in a xenograft mouse model. Mechanistic studies further revealed that Livin promotes the proliferation and invasion of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we revealed that inhibition of YAP by short-hairpin RNA prevents the growth and invasion of ovarian cancer cells in vivo and in vitro. Therefore, Livin may be a potential novel therapeutic target for the treatment of ovarian cancer.

Keywords: Ovarian cancer, Livin, Hippo/YAP signaling pathway, apoptosis

Introduction

Ovarian cancer is a common malignant tumor in women, and its incidence is increasing worldwide [1]. Ovarian cancer has unique characteristics of difficult diagnosis and early metastasis [2]. However, the molecular mechanisms underlying the metastasis of ovarian cancer have not been clarified. Therefore, finding the molecular mechanism of ovarian cancer development is of great significance for the treatment of ovarian cancer patients.

Livin is a member of the inhibitor of apoptosis protein (IAP) family encoded by the BIRC7 gene, which consists of a single baculoviral IAP repeat domain and a RING domain at the C-terminus. The protein inhibits apoptosis by inhibiting the proteolytic activation of caspases, such as caspase-3, -7 and -9, and it plays an important role in tumorigenesis and chemoresistance [3]. Overexpression of Livin has been observed in a variety of cancers, including lung, colon, and prostate cancers, but Livin is rarely detected in normal adult tissues. It is thought that Livin protein expression may be an early event in the occurrence of HCC [4]. Aberrant Livin expression has been demonstrated in human malignancy and tumor tissue cells in most tumors, and the expression of Livin correlated with in vitro drug resistance, advanced tumor stage, and poor outcome [5-9]. Knockdown of Livin has been shown to reduce tumor cell proliferative potential and/or induce sensitization toward proapoptotic stimuli in glioma cells [7], lung cancer cells [10] or neuroblastoma cells [11]. Together, these studies suggest that Livin may be essential for the progression of many tumors. However, the significance of Livin in ovarian cancer cells and the underlying molecular mechanisms have not been investigated.

The Hippo pathway was originally discovered as a key factor in the control of Drosophila organ size and was recently found to be important in human cancer [12-14]. Yes-associated protein 1 (YAP1), a 65-kDa proline-rich phosphoprotein and a transcription coactivator, is a major downstream target of the Hippo signaling pathway [15-17]. Regulation of the Hippo signaling pathway is known to be mediated by phosphorylation and subcellular localization of YAP1. Activation of the Hippo signaling pathway induces phosphorylation of YAP1, which prevents translocation to the nucleus. When the Hippo signaling pathway is inactivated, dephosphorylated YAP1 translocates to the nucleus, interacts with transcription factors, and eventually causes the proliferation of cells of various organ systems [18,19]. Recently, YAP1 has been suggested to be a potent oncogene, and it was found to be elevated in several types of cancers compared to normal controls [19-23]. In addition, YAP1 is implicated in the EMT program in diverse cancers, including liver, colon, prostate, ovarian, and breast cancers [24-26]. However, the expression of YAP1 and its correlation with Livin in ovarian cancer have rarely been reported.

In this study, combining in vitro and in vivo analyses, we investigated i) the difference in Livin expression in both primary specimens from ovarian cancer patients and ovarian cancer cell lines compared to normal controls; ii) the effects of overexpression and downregulation of Livin on ovarian cancer cell progression in vitro and in vivo; and iii) the relationship between Livin and the activation and nuclear translocation of transcriptional coactivator YAP, the key component of the Hippo signaling pathway and the functional role of this relationship. Our study indicates that the activation of YAP/TAZ by Livin drives the progression of ovarian cancer cells in vitro and in vivo. Therefore, Livin could be a potential novel therapeutic target for the treatment of ovarian cancer in the clinic.

Materials and methods

Patients and tissue specimens

A total of 44 ovarian cancer samples and 21 normal samples were collected from the Department of Gynecology, the First Affiliated Hospital of Harbin Medical University. The samples were quickly frozen in liquid N2 after collection immediately and then stored at -80°C before use. The research ethics committee of the First Affiliated Hospital Harbin Medical University approved the research in accordance with the Declaration of Helsinki.

Cell culture and reagents

Immortalized epithelial cell lines (HOSE and FTE188) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). The ovarian cancer cell lines (A2780, HO-8910, HO-8910PM, CoC1, Caov-3, OV-1063, SKOV3 and OVCAR-3) were purchased from National Infrastructure of Cell Line Resource (Beijing, China). Immortalized ovarian epithelial cell lines and ovarian cancer cell lines were cultured as described previously.

Lentivirus vector construction and siRNA transfection

The lentivirus vectors for knockdown and overexpression of Livin were produced by GenePharma (Shanghai, China). The shRNAs against Livin (shLivin) and siRNAs against YAP/TAZ (si-YAP/TAZ) and negative control (NC) were synthesized by GenePharma (Shanghai, China). The siRNA sequence against Livin was 5’-CAGGCCATCAGGACAAGGT-3’. The siRNA sequences against YAP/TAZ were as follows: si-YAP#1: 5’-CCCAGTTAAATGTTCACCAAT-3’; siYAP#2: CAGGTGATACTATCAACCAAA-3’. siTAZ#1: 5’-CAGCCAAATCTCGTGATGAAT-3’; siTAZ#2: 5’-CCAGGAACAAACGTTGACTTA-3’. Ovarian cancer cells were transfected with shRNAs and siRNAs with Lipofectamine 3000 Reagent (Thermo, USA) according to the manufacturer’s protocol.

Real-time PCR

All RNA of tissues and cells was isolated by TRIzol reagent (Invitrogen, USA), and 1 μg of total RNA was used to reverse-synthesize cDNA template following the Reverse Transcription kit instructions (Applied Biosystems Co., Ltd. USA) for the SYBR Green PCR (Takara, Japan) assay. The PCR primers and reaction conditions were produced as described previously, and the expression of Livin, CYR61, CTGF, and CCND1 was normalized to GAPDH. Each PCR experiment was performed at least 3 times independently, and the relative expression value was expressed by 2-ΔΔCt.

Western blotting (WB)

The ovarian cancer tissue and normal tissue were minced, homogenized and digested by using RIPA lysis buffer (with protease and phosphatase inhibitor, Thermo Scientific). The cells were scraped from the culture plate on ice and lysed with RIPA lysis buffer (with protease and phosphatase inhibitor, Thermo Scientific). The resulting suspensions were centrifuged at 13,000 g for 20 minutes at 4°C, and the supernatant of protein was collected. Protein samples were prepared for PAGE. Proteins were then transferred to a PVDF membrane (BioRad) for immunoblotting with relevant antibodies. The following antibodies were used in this study: Livin (ab11983), YAP (sc-271134), pSer127-YAP (PA5-17481), Lamin B (sc-374015), LAST1 (CST#3477), TAZ (ab84927), β-actin (ab115777), and GAPDH (CST#5174).

In vivo lung metastasis model

Six-week-old male BALB/c nude mice were used for the in vivo lung metastasis model. Ovarian cancer cells were injected into the tail vein of mice. The bioluminescence signal of lung metastasis from day 7 to day 28 was determined. After 4 weeks, all mice were sacrificed, and then immunohistochemical analysis and hematoxylin/eosin (HE) staining were performed.

Immunohistochemical (IHC) staining

The tumor tissues were embedded in paraffin, cut into 4 μm sections, and either HE stained or treated with corresponding antibodies for IHC evaluation. Slides were incubated with anti-Ki-67 for 2 h at 37°C. The second antibody was incubated for 1 h at room temperature (RT), and then diaminobenzidine (DAB) was used as chromogen. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was used for apoptosis measurement according to the manufacturer’s protocol. The brown dots within the nucleus were considered Ki-67- and TUNEL-positive cells.

In vivo tumor growth model

One hundred microliters of ovarian cancer cells transfected with shLivin or interference control vector were injected subcutaneously into each nude mouse at a density of 1 × 108 cells/ml in saline. We measured the volume of ovarian tumors every 5 days and then sacrificed the mice and isolated and weighed the tumors after 30 days.

Wound healing assay

The ovarian cancer cells were collected and implanted into a 6-well plate at a density of 5 × 105/mL. The cells were induced and cultivated to a confluence of nearly 90%. The ovarian cancer cells were scraped using a 200-μL pipette tip and cultured in FBS-free medium. Typical images were taken under the microscope 0 and 24 h after the scratches were formed.

Transwell assay

To evaluate the migration ability of ovarian cancer, a polycarbonate membrane filter coated with gelatin and 24-well perforated chambers (Corning) coated with Matrigel were used. The cells were suspended in medium without FBS, and the transformed cell density was 5 × 104/mL; 100 μL of the cell suspension was added to the insert, and 600 μL of medium containing 20% FBS (HyClone, FBS) was added to the lower chamber. After 24 h, the cells were stained with crystal violet (0.1%) for 15 minutes and then washed twice with PBS. ImageJ was used to observe the cells and count. The cells were observed with an optical microscope, and then different fields (6 fields per group) were randomly selected for counting.

Colony formation assay

Ovarian cancer cells were plated into 12-well plates (1 × 103/mL). The colonies were stained with 0.1% crystal violet (Sigma-Aldrich) after 2 weeks of culture and then washed with PBS twice. Visible violet colonies were counted and analyzed.

Cell counting Kit-8 (CCK-8) assay

The cell viability of ovarian cancer cells was evaluated by CCK-8 assay according to the manufacturer’s protocol (MCE, China). Ovarian cancer cells were seeded in a 96-well plate at a density of 2 × 103 cells/mL and transfected with shRNA or plasmid 24 h after implantation. After culturing for 2 days, the medium in each well was replaced with 100 ml of culture medium containing 10 ml of CCK-8 solution. The absorbance at 450 nm was measured by a spectrometer (Thermo Fisher, Rockford, IL, USA).

Statistical analysis

Data are presented as the mean ± SEM. Statistical analyses were performed with GraphPad Prism software (version 8.0). Statistical significance was compared via unpaired Student’s t test or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple as appropriate. Differences were considered statistically significant at the level of P < 0.05.

Results

Livin is upregulated in human ovarian carcinoma

We analyzed the expression of Livin in the patient samples collected during ovarian surgery. Compared with normal ovarian tissues, the mRNA level of Livin is higher in ovarian tissue (Figure 1A). Then, we measured the expression of Livin in ovarian carcinoma tissue and normal ovarian tissue from different clinical samples by IHC staining and WB and found that Livin was increased in all ovarian carcinoma samples (Figure 1B and 1C). Next, we detected the expression of Livin in human ovarian surface epithelial (HOSE) cells and fallopian tube epithelial (FTE188) cells and different ovarian carcinoma cell lines (A2780, HO-8910, HO-8910PM, CoC1, Caov-3, OV-1063, SKOV3 and OVCAR-3) at the mRNA and protein levels. The results showed that Livin expression was consistently upregulated in ovarian carcinoma cell lines, especially in A2780 and SKOV3 cells (Figure 1D and 1E). These findings indicate that Livin is increased in ovarian carcinoma.

Figure 1.

Figure 1

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Livin is upregulated in human ovarian carcinoma. A. The mRNA level of Livin in ovarian tissue and normal ovarian tissue. B. IHC staining of Livin in ovarian carcinoma tissue and normal ovarian tissue from different clinical samples. C. WB analysis of Livin in ovarian carcinoma tissue and normal ovarian tissue from different clinical samples. D, E. The mRNA and protein levels of Livin in different ovarian cancer cell lines (HOSE 6-3, FTE188, A2780, HO-8910, HO-8910PM, CoC1, Caov-3, OV-1063, SKOV3 and OVCAR-3). **P < 0.01.

Livin promotes the proliferation and migration of ovarian cancer cells

To explore the function of Livin in ovarian carcinoma, we transfected ovarian carcinoma cell lines with lentivirus carrying Livin or control vector. We measured cell viability at different times (0 h, 24 h, 48 h, 72 h and 96 h) after infection and found that compared with the vector control cells, A2780 and SKOV3 cells infected with Livin showed increased cell viability (Figure 2A). Next, we performed colony formation assays of A2780 and SKOV3 cells infected with lentivirus carrying Livin or vector. Our data indicate that Livin overexpression significantly increased the colony formation of ovarian carcinoma cells (Figure 2B). By measuring the migration and invasion ability of ovarian cancer by Transwell assay and wound healing assay, we found that the upregulation of Livin increased the cell migration and invasion abilities of ovarian cancers (Figure 2C-E).

Figure 2.

Figure 2

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Livin overexpression promotes the proliferation and migration of ovarian cancer cells. A. The cell viability of A2780 and SKOV3 cells was evaluated by CCK-8. B. The colony formation of A2780 and SKOV3 cells transfected with vector and Livin. C, D. The invasive ability of A2780 and SKOV3 cells was measured by Transwell assay. E. The migration potential of A2780 and SKOV3 cells was evaluated by the wound healing test. Scale bars: 200 μm. *P < 0.05, **P < 0.01.

To further explore the function of Livin in ovarian carcinoma progression, we infected A2780 and SKOV3 cells with lentivirus carrying specific short-hairpin RNA (shRNA) targeting Livin (shLivin-1 and shLivin-2), and the knockdown efficiency was verified by WB (Figure 3A). Colony formation assays, Transwell assays and wound healing assays were performed to examine the effects of downregulation of Livin on cell colony formation, migration and invasion of ovarian carcinoma cells. Our results suggest that Livin downregulation suppressed cell viability, colony formation, migration and invasion of ovarian carcinoma cells (Figure 3A-E). These results indicate that Livin plays important roles in the process of cell proliferation, migration, and invasion of ovarian carcinoma cells.

Figure 3.

Figure 3

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Knockdown of Livin impairs the proliferation and migration of ovarian cancer cells. A. The Livin knockdown efficiency measured by WB. B. The effect of Livin knockdown on A2780 and SKOV3 cell proliferation was detected by CCK-8 assay at the indicated times. C. The colony formation of A2780 and SKOV3 cells treated with shNC and shLivin. D. Migration ability and invasion ability were determined after knockdown of Livin in A2780 and SKOV3 cells using a migration assay and a Transwell assay. E. The migration potential of A2780 and SKOV3 cells treated with shNC and shLivin was evaluated by the wound healing test. Scale bars: 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Livin promotes ovarian carcinoma growth and metastasis in vivo

We next used a xenograft mouse model to explore the effect of Livin inhibition on ovarian cancer cell growth. A2780 cells stably infected with lentivirus encoding shLivin or empty vector were subcutaneously injected into nude mice. After four weeks, we found that shLivin significantly reduced the tumor volume and tumor weight of shLivin mice compared with the control group mice (Figure 4A and 4B). In addition, IHC analysis showed that the proportion of Ki-67-positive cells decreased, while the proportion of TUNEL-positive cells increased in all shLivin groups compared with the control group (Figure 4C). These results indicate that silencing Livin expression inhibits the growth of ovarian cancer cell-derived tumors in the mouse xenograft model. Next, we evaluated whether Livin influences the metastasis of ovarian carcinoma cells in vivo. A2780 cells infected with lentivirus carrying shLivin were injected into the tail vein of BALB/c nude mice. The luciferase signals were measured at day 28 after injection to monitor the xenograft growth of ovarian carcinoma in the lung. We found that the shLivin group showed lower luciferase signals than the control group at day 28 after injection (Figure 4D). HE staining of the lungs of the different groups also showed that shLivin inhibited the occupation of ovarian carcinoma cells in the lungs (Figure 4D). Altogether, we discovered that shLivin effectively inhibits the growth of A2780 cells and their metastasis to the lungs of nude mice compared to the control mice (Figure 4D).

Figure 4.

Figure 4

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Livin knockdown inhibits the growth and metastasis of ovarian cancer cells in vivo. A. Quantitative analysis of the tumor volume at the indicated times. B. Quantitative analysis of the tumor weight. C. HE, Ki-67 and TUNEL immunohistological staining of tumors from shNC and shLivin cells. Scale bars: 100 μm. D. In vivo bioluminescent imaging of nude mice at day 28 after ovarian cancer cell implantation and HE staining for lung occupancy of shLivin- or shNC-expressing ovarian cancer cells. Scale bars: 250 μm. *P < 0.05, **P < 0.01.

Livin activates YAP/TAZ in human ovarian cancer cell lines

To investigate whether Livin promotes the progression of ovarian cancer cells by activating the YAP/TAZ signaling pathway, we measured the protein expression and translocation of YAP from the cytosol to the nucleus in A2780 and SKOV3 cells by immunofluorescence (IF) and WB analysis (Figure 5A-D). IF and WB analysis of biochemically fractionated nuclear and cytoplasmic extracts after Livin upregulation clearly showed the nuclear accumulation of YAP (Figure 5A-D). Accordingly, the expression of the YAP TEAD-regulated genes CTGF, CYR61 and CCND1 was found to be significantly increased when Livin was upregulated (Figure 5E), confirming the functional association between Livin and YAP activity.

Figure 5.

Figure 5

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Livin activates YAP/TAZ in human ovarian cancer cell lines. A, C. Immunofluorescence staining of YAP nuclear localization in A2780 and SKOV3 cells after 24 h transfection with Livin or vector. B, D. WB analysis of YAP in the cytosol and nucleus in A2780 and SKOV3 cells after 24 h transfection with Livin or vector. E. qPCR analysis of the expression of CYR61, CTGF and CCND1 in A2780 and SKOV3 cells after 24 h of treatment. Scale bars: 25 μm. **P < 0.01.

Livin acts through LATS, Rho/ROCK, and actin cytoskeleton to activate YAP/TAZ

The key protein mediating the phosphorylation of YAP is LATS, which is one of the core components of the Hippo pathway [27-29]. LATS regulates target gene expression by phosphorylating, thereby restricting YAP and transcriptional coactivator with PDZ-binding motif (TAZ) in the cytoplasm [28,29]. To investigate whether LATS1 is involved in YAP/TAZ regulation by Livin, we assessed the kinase activity of LATS1 immunopurified from control or Livin-transfected cells. As shown in Figure 6A, Livin overexpression markedly reduced LATS1 kinase activity, which was coincident with YAP/TAZ dephosphorylation. Pretreatment of cells with botulinum toxin C3, a specific inhibitor of Rho GTPases, blocked YAP/TAZ activation by Livin (Figure 6B). We also found that pretreatment with GSK429286 and Y27632, two inhibitors of Rho-associated protein kinase (ROCK), also strongly suppressed Livin-induced YAP/TAZ dephosphorylation (Figure 6C). The Rho/ROCK pathway potently modulates actin cytoskeleton dynamics, particularly stress fiber formation, in response to various GPCR agonists. We therefore examined whether cytoskeletal reorganization contributes to YAP/TAZ activation in response to Livin overexpression. Treatment with latrunculin B (Lat B), an F-actin destabilizing reagent, blocked Livin-induced YAP/TAZ dephosphorylation (Figure 6C). Taken together, the above data support a model in which Livin acts through Rho/ROCK and actin cytoskeleton to regulate the Hippo pathway, leading to YAP/TAZ activation.

Figure 6.

Figure 6

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Livin acts through LATS, Rho/ROCK, and actin cytoskeleton to activate YAP/TAZ. A. Livin overexpression markedly reduced LATS1 kinase activity and dephosphorylated YAP/TAZ. B. Inactivation of Rho GTPase prevents YAP/TAZ dephosphorylation by Livin stimulation. C. Treatment with ROCK inhibitor GSK429286 (0.5 mmol/L) or Y27632 (1 mmol/L) and actin cytoskeleton inhibitor latrunculin B (Lat B; 1 mg/mL) abolished the YAP/TAZ activation induced by Livin.

Livin promotes cell migration and cell proliferation through YAP/TAZ

Because YAP/TAZ activities are significantly elevated upon Livin overexpression in ovarian cancer cells, we investigated the role of YAP/TAZ in Livin-regulated cellular functions by knocking down YAP/TAZ expression. The knockdown efficiency was confirmed by WB (Figure 7A). Livin promoted the proliferation of A2780 cells as determined by the colony formation assay, and this effect was largely abolished by YAP/TAZ knockdown (Figure 7B), suggesting an essential role of YAP/TAZ in mediating Livin-induced ovarian cancer cell proliferation. Our results showed that Livin overexpression enhanced cell proliferation, whereas knockdown of YAP/TAZ significantly blocked the promigratory and proinvasive effects of Livin (Figure 7C). These observations indicated that YAP/TAZ play a critical role in vin-induced cell growth.

Figure 7.

Figure 7

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Livin promotes cell migration and cell proliferation through YAP/TAZ. A. The knockdown efficiency of YAP and TAZ was confirmed by WB. B. Livin overexpression promoted the proliferation of A2780 cells as determined by the colony formation assay. C. Knockdown of YAP/TAZ significantly blocked the promigration and proinvasion effect of Livin. Scale bars: 200 μm. *P < 0.05, **P < 0.01.

Livin promotes tumorigenesis and metastasis of ovarian cancer through YAP/TAZ

To determine whether Livin promotes the tumorigenesis and metastasis of ovarian cancer through YAP/TAZ in vivo, tumor growth in response to treatment was investigated in a xenograft mouse model. A2780 cells stably transfected with Livin or empty vector were injected into nude mice treated with or without shYAP/TAZ. After four weeks, we found that Livin overexpression significantly increased tumor volume and tumor weight compared with those in the vector group, while these effects were abolished when YAP/TAZ was downregulated (Figure 8A). In addition, YAP/TAZ knockdown reversed the increased numbers of Ki-67-positive cells induced by Livin overexpression. In addition, TUNEL staining showed that YAP/TAZ knockdown partially abolished the apoptosis inhibition induced by Livin (Figure 8B). Next, we evaluated whether YAP/TAZ knockdown can inhibit the effects of Livin on ovarian carcinoma metastasis in vivo. SKOV3 cells infected with Livin overexpression lentivirus were injected into the tail vein of BALB/c nude mice with or without YAP/TAZ knockdown, and luciferase signals were measured at day 28 to observe the growth of ovarian cancer xenografts in the lung (Figure 8C). We found that Livin significantly increased tumor metastasis in vivo. Significantly, the tumor metastasis effects of Livin were partially reversed when YAP/TAZ was downregulated. Taken together, these data revealed that Livin promotes the tumorigenesis and metastasis of ovarian cancer in vivo in a YAP/TAZ-dependent manner.

Figure 8.

Figure 8

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Livin promotes tumorigenesis and metastasis of ovarian cancer through YAP/TAZ. A. Quantitative analysis of the tumor volume at the indicated time and quantitative analysis of the tumor weight. B. HE, Ki-67 and TUNEL immunohistological staining of tumors from different groups. Scale bars: 100 μm. C. In vivo bioluminescent imaging of nude mice at day 28 after ovarian cancer cell implantation and HE staining for lung occupancy of different groups. Scale bars: 250 μm. **P < 0.01, ***P < 0.001.

Discussion

Ovarian cancer has the highest mortality rate of malignant tumors in women [30]. Until now, the molecular mechanisms underlying the metastasis of ovarian cancer have not been clarified. In this study, by combining in vitro and in vivo analyses, we demonstrated Livin as a tumor promoter in ovarian cancer. Livin is upregulated both in primary specimens from ovarian cancer patients and in ovarian cancer cell lines compared to controls, suggesting that Livin may function as a promoter of ovarian cancer and may have clinical value for ovarian cancer therapy. Furthermore, Livin overexpression promotes ovarian cancer cell growth and migration in vitro, while downregulation shows an inhibitive effect during these cellular processes. These effects of the Livin gene were also demonstrated in a xenograft mouse model. Further mechanistic studies revealed that Livin promotes the proliferation and invasion of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we revealed that knockdown of YAP prevented the growth and invasion of ovarian cancer cells. Our study revealed that Livin may be a potential therapeutic target for the treatment of ovarian cancer.

Livin is a member of the IAP family and has been shown to be expressed in multiple malignant tumors, such as superficial bladder cancer tumors [31,32], neuroblastoma [11,33], adrenocortical tumors [34], colorectal tumors [35,36], melanoma and acute lymphoblastic leukemia [37]. Our study proved that Livin is significantly overexpressed in ovarian cancer patients and related cell lines compared to controls, and similar results were demonstrated by other authors. It has been reported that Livin plays an important role in the regulation of apoptosis with subsequent modulation of the cell cycle and cell proliferation. The antiapoptotic activity of Livin is mainly mediated by the direct inhibition of apoptosis executers, such as caspase-3, -7 and -9 [34]. Using shRNA to downregulate the expression of Livin, we also demonstrated that Livin can inhibit apoptosis while promoting the proliferation of ovarian cancer cells in vivo.

The Hippo-YAP/TAZ pathway plays an important role in regulating ovarian development and carcinogenesis [38-40]. Importantly, the Hippo pathway acts as an important inhibitor of tissue growth and reduces organ size by controlling cell proliferation, apoptosis, and migration in response to a variety of extracellular and intracellular signals [41,42]. Therefore, we speculate that the promotion of tumor progression by Livin in ovarian cancer is related to abnormal activation of the Hippo-YAP/TAZ pathway. In this study, we first identified the role of YAP/TAZ in Livin-mediated tumor promotion: Livin promotes ovarian cancer proliferation and invasion by activating YAP, while knockdown of YAP/TAZ prevents the progrowth and proinvasive effects of Livin in ovarian cancer cells.

The core components of the Hippo pathway consist of a series of kinases, including MST1/2, MAP4Ks, and LATS1/2; MST1/2 or MAP4Ks phosphorylate and activate LATS. The activation of LATS regulates target gene expression by inhibiting YAP and TAZ. Inhibition of LATS activity causes dephosphorylation of YAP/TAZ, which allows YAP/TAZ to translocate to the nucleus and promote the expression of target genes [15,16,28]. In this study, we found that LATS is involved in YAP/TAZ regulation by Livin, which can markedly reduce LATS1 kinase activity with dephosphorylation of YAP/TAZ. We further found that a specific inhibitor of Rho GTPases blocked YAP/TAZ activation by Livin, and pretreatment with GSK429286 and Y27632, inhibitors of Rho-associated protein kinase (ROCK), strongly suppressed Livin-induced YAP/TAZ dephosphorylation. The Rho/ROCK pathway potently modulates actin cytoskeleton dynamics, particularly stress fiber formation [43]. We therefore examined whether cytoskeletal reorganization contributes to YAP/TAZ activation in response to Livin overexpression. Treatment with Lat B, an F-actin destabilizing reagent, blocked Livin-induced YAP/TAZ dephosphorylation. Our data support a model in which Livin acts through Rho/ROCK and the actin cytoskeleton to regulate the Hippo pathway, leading to YAP/TAZ activation during ovarian cancer cell progression.

In summary, our findings indicate that the activation of YAP/TAZ by Livin drives the invasion and proliferation of ovarian cancer cells in vitro and in vivo. We predict that Livin is a potential novel therapeutic target for the treatment of ovarian cancer that may be applied in the future.

Acknowledgements

This work was supported by the Research and innovation fund of the First Affiliated Hospital of Harbin Medical University [grant number: 2020M09].

Disclosure of conflict of interest

None.

References

  • 1.Jayson GC, Kohn EC, Kitchener HC, Ledermann JA. Ovarian cancer. Lancet. 2014;384:1376–1388. doi: 10.1016/S0140-6736(13)62146-7. [DOI] [PubMed] [Google Scholar]
  • 2.Bast RC Jr, Hennessy B, Mills GB. The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer. 2009;9:415–428. doi: 10.1038/nrc2644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kasof GM, Gomes BC. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem. 2001;276:3238–3246. doi: 10.1074/jbc.M003670200. [DOI] [PubMed] [Google Scholar]
  • 4.Lazar I, Perlman R, Lotem M, Peretz T, Ben-Yehuda D, Kadouri L. The clinical effect of the inhibitor of apopotosis protein livin in melanoma. Oncology. 2012;82:197–204. doi: 10.1159/000334234. [DOI] [PubMed] [Google Scholar]
  • 5.Wang X, Xu J, Ju S, Ni H, Zhu J, Wang H. Livin gene plays a role in drug resistance of colon cancer cells. Clin Biochem. 2010;43:655–660. doi: 10.1016/j.clinbiochem.2010.02.004. [DOI] [PubMed] [Google Scholar]
  • 6.Nachmias B, Ashhab Y, Bucholtz V, Drize O, Kadouri L, Lotem M, Peretz T, Mandelboim O, Ben-Yehuda D. Caspase-mediated cleavage converts Livin from an antiapoptotic to a proapoptotic factor: implications for drug-resistant melanoma. Cancer Res. 2003;63:6340–6349. [PubMed] [Google Scholar]
  • 7.Liu Y, Guo Q, Zhang H, Li GH, Feng S, Yu XZ, Kong LS, Zhao L, Jin F. Effect of siRNA-Livin on drug resistance to chemotherapy in glioma U251 cells and CD133(+) stem cells. Exp Ther Med. 2015;10:1317–1323. doi: 10.3892/etm.2015.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Crnkovic-Mertens I, Muley T, Meister M, Hartenstein B, Semzow J, Butz K, Hoppe-Seyler F. The anti-apoptotic livin gene is an important determinant for the apoptotic resistance of non-small cell lung cancer cells. Lung Cancer. 2006;54:135–142. doi: 10.1016/j.lungcan.2006.07.018. [DOI] [PubMed] [Google Scholar]
  • 9.Sun JG, Liao RX, Chen ZT, Wang ZX, Zhang Q, Hu YD, Wang DL. Gene transfection of Livin isoforms into A549 cell line and its effect on cell growth and sensitivity to chemotherapy and radiotherapy. Zhonghua Jie He He Hu Xi Za Zhi. 2005;28:836–840. [PubMed] [Google Scholar]
  • 10.Zhao X, Yuan Y, Zhang Z, Feng X, Zhang J, Yuan X, Li J. Effects of shRNA-silenced livin and survivin on lung cancer cell proliferation and apoptosis. J BUON. 2014;19:757–762. [PubMed] [Google Scholar]
  • 11.Kim DK, Alvarado CS, Abramowsky CR, Gu L, Zhou M, Soe MM, Sullivan K, George B, Schemankewitz E, Findley HW. Expression of inhibitor-of-apoptosis protein (IAP) livin by neuroblastoma cells: correlation with prognostic factors and outcome. Pediatr Dev Pathol. 2005;8:621–629. doi: 10.1007/s10024-005-4108-3. [DOI] [PubMed] [Google Scholar]
  • 12.Harvey KF, Zhang X, Thomas DM. The Hippo pathway and human cancer. Nat Rev Cancer. 2013;13:246–257. doi: 10.1038/nrc3458. [DOI] [PubMed] [Google Scholar]
  • 13.Pan D. The hippo signaling pathway in development and cancer. Dev Cell. 2010;19:491–505. doi: 10.1016/j.devcel.2010.09.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zheng Y, Pan D. The hippo signaling pathway in development and disease. Dev Cell. 2019;50:264–282. doi: 10.1016/j.devcel.2019.06.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev. 2014;94:1287–1312. doi: 10.1152/physrev.00005.2014. [DOI] [PubMed] [Google Scholar]
  • 16.Crawford JJ, Bronner SM, Zbieg JR. Hippo pathway inhibition by blocking the YAP/TAZ-TEAD interface: a patent review. Expert Opin Ther Pat. 2018;28:867–873. doi: 10.1080/13543776.2018.1549226. [DOI] [PubMed] [Google Scholar]
  • 17.Varelas X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development. 2014;141:1614–1626. doi: 10.1242/dev.102376. [DOI] [PubMed] [Google Scholar]
  • 18.Yu FX, Guan KL. The Hippo pathway: regulators and regulations. Genes Dev. 2013;27:355–371. doi: 10.1101/gad.210773.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J, Xie J, Ikenoue T, Yu J, Li L, Zheng P, Ye K, Chinnaiyan A, Halder G, Lai ZC, Guan KL. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 2007;21:2747–2761. doi: 10.1101/gad.1602907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Sudol M. YAP1 oncogene and its eight isoforms. Oncogene. 2013;32:3922. doi: 10.1038/onc.2012.520. [DOI] [PubMed] [Google Scholar]
  • 21.Lee KW, Lee SS, Kim SB, Sohn BH, Lee HS, Jang HJ, Park YY, Kopetz S, Kim SS, Oh SC, Lee JS. Significant association of oncogene YAP1 with poor prognosis and cetuximab resistance in colorectal cancer patients. Clin Cancer Res. 2015;21:357–364. doi: 10.1158/1078-0432.CCR-14-1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lorenzetto E, Brenca M, Boeri M, Verri C, Piccinin E, Gasparini P, Facchinetti F, Rossi S, Salvatore G, Massimino M, Sozzi G, Maestro R, Modena P. YAP1 acts as oncogenic target of 11q22 amplification in multiple cancer subtypes. Oncotarget. 2014;5:2608–2621. doi: 10.18632/oncotarget.1844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Tu B, Yao J, Ferri-Borgogno S, Zhao J, Chen S, Wang Q, Yan L, Zhou X, Zhu C, Bang S, Chang Q, Bristow CA, Kang Y, Zheng H, Wang H, Fleming JB, Kim M, Heffernan TP, Draetta GF, Pan D, Maitra A, Yao W, Gupta S, Ying H. YAP1 oncogene is a context-specific driver for pancreatic ductal adenocarcinoma. JCI Insight. 2019;4:e130811. doi: 10.1172/jci.insight.130811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Shao DD, Xue W, Krall EB, Bhutkar A, Piccioni F, Wang X, Schinzel AC, Sood S, Rosenbluh J, Kim JW, Zwang Y, Roberts TM, Root DE, Jacks T, Hahn WC. KRAS and YAP1 converge to regulate EMT and tumor survival. Cell. 2014;158:171–184. doi: 10.1016/j.cell.2014.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kulkarni M, Tan TZ, Syed Sulaiman NB, Lamar JM, Bansal P, Cui J, Qiao Y, Ito Y. RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer. Oncotarget. 2018;9:14175–14192. doi: 10.18632/oncotarget.24419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Liu Y, He K, Hu Y, Guo X, Wang D, Shi W, Li J, Song J. YAP modulates TGF-beta1-induced simultaneous apoptosis and EMT through upregulation of the EGF receptor. Sci Rep. 2017;7:45523. doi: 10.1038/srep45523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, Brummelkamp TR. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr Biol. 2007;17:2054–2060. doi: 10.1016/j.cub.2007.10.039. [DOI] [PubMed] [Google Scholar]
  • 28.He C, Lv X, Huang C, Hua G, Ma B, Chen X, Angeletti PC, Dong J, Zhou J, Wang Z, Rueda BR, Davis JS, Wang C. YAP1-LATS2 feedback loop dictates senescent or malignant cell fate to maintain tissue homeostasis. EMBO Rep. 2019;20:e44948. doi: 10.15252/embr.201744948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Oka T, Mazack V, Sudol M. Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP) J Biol Chem. 2008;283:27534–27546. doi: 10.1074/jbc.M804380200. [DOI] [PubMed] [Google Scholar]
  • 30.Grunewald T, Ledermann JA. Targeted therapies for ovarian cancer. Best Pract Res Clin Obstet Gynaecol. 2017;41:139–152. doi: 10.1016/j.bpobgyn.2016.12.001. [DOI] [PubMed] [Google Scholar]
  • 31.Gazzaniga P, Gradilone A, Giuliani L, Gandini O, Silvestri I, Nofroni I, Saccani G, Frati L, Agliano AM. Expression and prognostic significance of LIVIN, SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer. Ann Oncol. 2003;14:85–90. doi: 10.1093/annonc/mdg002. [DOI] [PubMed] [Google Scholar]
  • 32.Yin W, Chen N, Zhang Y, Zeng H, Chen X, He Y, Wang X, Zhou Q. Survivin nuclear labeling index: a superior biomarker in superficial urothelial carcinoma of human urinary bladder. Mod Pathol. 2006;19:1487–1497. doi: 10.1038/modpathol.3800675. [DOI] [PubMed] [Google Scholar]
  • 33.Dasgupta A, Alvarado CS, Xu Z, Findley HW. Expression and functional role of inhibitor-of-apoptosis protein livin (BIRC7) in neuroblastoma. Biochem Biophys Res Commun. 2010;400:53–59. doi: 10.1016/j.bbrc.2010.08.001. [DOI] [PubMed] [Google Scholar]
  • 34.Altieri B, Sbiera S, Della Casa S, Weigand I, Wild V, Steinhauer S, Fadda G, Kocot A, Bekteshi M, Mambretti EM, Rosenwald A, Pontecorvi A, Fassnacht M, Ronchi CL. Livin/BIRC7 expression as malignancy marker in adrenocortical tumors. Oncotarget. 2017;8:9323–9338. doi: 10.18632/oncotarget.14067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Pan Y, Tong JHM, Lung RWM, Kang W, Kwan JSH, Chak WP, Tin KY, Chung LY, Wu F, Ng SSM, Mak TWC, Yu J, Lo KW, Chan AWH, To KF. RASAL2 promotes tumor progression through LATS2/YAP1 axis of hippo signaling pathway in colorectal cancer. Mol Cancer. 2018;17:102. doi: 10.1186/s12943-018-0853-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Kim HB, Kim M, Park YS, Park I, Kim T, Yang SY, Cho CJ, Hwang D, Jung JH, Markowitz SD, Hwang SW, Yang SK, Lim DS, Myung SJ. Prostaglandin E2 activates YAP and a positive-signaling loop to promote colon regeneration after colitis but also carcinogenesis in mice. Gastroenterology. 2017;152:616–630. doi: 10.1053/j.gastro.2016.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Choi J, Hwang YK, Sung KW, Lee SH, Yoo KH, Jung HL, Koo HH, Kim HJ, Kang HJ, Shin HY, Ahn HS. Expression of Livin, an antiapoptotic protein, is an independent favorable prognostic factor in childhood acute lymphoblastic leukemia. Blood. 2007;109:471–477. doi: 10.1182/blood-2006-07-032557. [DOI] [PubMed] [Google Scholar]
  • 38.Piccolo S, Cordenonsi M, Dupont S. Molecular pathways: YAP and TAZ take center stage in organ growth and tumorigenesis. Clin Cancer Res. 2013;19:4925–4930. doi: 10.1158/1078-0432.CCR-12-3172. [DOI] [PubMed] [Google Scholar]
  • 39.Jeong GO, Shin SH, Seo EJ, Kwon YW, Heo SC, Kim KH, Yoon MS, Suh DS, Kim JH. TAZ mediates lysophosphatidic acid-induced migration and proliferation of epithelial ovarian cancer cells. Cell Physiol Biochem. 2013;32:253–263. doi: 10.1159/000354434. [DOI] [PubMed] [Google Scholar]
  • 40.Kim W, Khan SK, Gvozdenovic-Jeremic J, Kim Y, Dahlman J, Kim H, Park O, Ishitani T, Jho EH, Gao B, Yang Y. Hippo signaling interactions with Wnt/beta-catenin and Notch signaling repress liver tumorigenesis. J Clin Invest. 2017;127:137–152. doi: 10.1172/JCI88486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Miyamura N, Hata S, Itoh T, Tanaka M, Nishio M, Itoh M, Ogawa Y, Terai S, Sakaida I, Suzuki A, Miyajima A, Nishina H. YAP determines the cell fate of injured mouse hepatocytes in vivo. Nat Commun. 2017;8:16017. doi: 10.1038/ncomms16017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Lange AW, Sridharan A, Xu Y, Stripp BR, Perl AK, Whitsett JA. Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung. J Mol Cell Biol. 2015;7:35–47. doi: 10.1093/jmcb/mju046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Schofield AV, Bernard O. Rho-associated coiled-coil kinase (ROCK) signaling and disease. Crit Rev Biochem Mol Biol. 2013;48:301–316. doi: 10.3109/10409238.2013.786671. [DOI] [PubMed] [Google Scholar]

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