Ovatodiolide suppresses colon tumorigenesis and prevents polarization of M2 tumor-associated macrophages through YAP oncogenic pathways
- Yan-Jiun Huang1, 2, 3,
- Ching-Kuo Yang4,
- Po-Li Wei2, 3,
- Thanh-Tuan Huynh5,
- Jacqueline Whang-Peng6, 11,
- Tzu-Ching Meng7,
- Michael Hsiao8,
- Yew-Ming Tzeng9, 12,
- Alexander TH Wu†1 and
- Yun Yen†1, 10Email author
© The Author(s). 2017
Received: 21 September 2016
Accepted: 14 February 2017
Published: 28 February 2017
An increased expression of Yes-associated protein (YAP1) has been shown to promote tumorigenesis in many cancer types including colon. However, the role of YAP1 in promoting colon tumorigenesis remains unclear. Here, we demonstrate that YAP1 expression is associated with M2 tumor-associated macrophage polarization and the generation of colon cancer stem-like cells. YAP1 downregulation by gene silencing or a phytochemical, ovatodiolide, not only suppresses colon cancer tumorigenesis but also prevents M2 TAM polarization.
Human monocytic cells, THP-1, and colon cancer cell lines, HCT116 and DLD-1, were co-cultured to mimic the interactions between tumor and its microenvironment. M2 polarization of the THP-1 cells were examined using both flow cytometry and q-PCR technique. The inhibition of YAP1 signaling was achieved by gene-silencing technique or ovatodiolide. The molecular consequences of YAP1 inhibition was demonstrated via colony formation, migration, and colon-sphere formation assays. 5-FU and ovatodiolide were used in drug combination studies. Xenograft and syngeneic mouse models were used to investigate the role of YAP1 in colon tumorigenesis and TAM generation.
An increased YAP1 expression was found to be associated with a poor prognosis in patients with colon cancer using bioinformatics approach. We showed an increased YAP1 expression in the colon spheres, and colon cancer cells co-cultured with M2 TAMs. YAP1-silencing led to the concomitant decreased expression of major oncogenic pathways including Kras, mTOR, β-catenin, and M2-promoting IL-4 and tumor-promoting IL-6 cytokines. TAM co-cultured colon spheres showed a significantly higher tumor-initiating ability in vivo. Ovatodiolide treatment alone and in combination with 5-FU significantly suppressed in vivo tumorigenesis and less TAM infiltration in CT26 syngeneic mouse model.
We have identified the dual function of YAP1 where its suppression not only inhibited tumorigenesis but also prevented the generation of cancer stem-like cells and M2 TAM polarization. Ovatodiolide treatment suppressed YAP1 oncogenic pathways to inhibit colon tumorigenesis and M2 TAM generation both in vitro and in vivo. Ovatodiolide should be considered for its potential for adjuvant therapeutic development.
Colorectal cancer (CRC) represents one of the most prevalent malignancies in the world with an estimated 9% of all cancer incidence . The pathogenesis of CRC is complex due to many factors including environmental risk factors, inherited genetic risks, and nutritional practices . The development of most CRC cases are sporadic from dysplastic adenomatous polyps. These are the results of several oncogenes such as KRas, c-myc, β-catenin, and other less frequent but also powerful tumorigenic pathways [3, 4]. Besides these genetic abnormalities, the formation of an inflammatory microenvironment also plays an essential role for CRC progression. Tumor-associated macrophages (TAMs) as one of the most abundant tumor-infiltrating stromal cell types have been shown to promote metastasis, drug resistance, and associated with a poor prognosis in CRC patients. Recent studies suggest an intimate association between the generation and/or maintenance of cancer stem cells (CSCs) and TAMs . The presence of CSCs potentiates the epithelial-to-mesenchymal transition (EMT), treatment resistance, and tumor repopulation . Thus, targeting TAMs represents an important milieu for anti-cancer drug development.
Yes-associated protein 1 (YAP1) is a well-established oncogenic factor in hepatocellular carcinoma and recently indicated in colorectal cancer [6–8]. YAP1 was proposed to functioning either partially or in tandem with key oncogenic drivers such as Kras, β-catenin, and Akt/mTOR in colon tumor initiation and progression [6, 8, 9]. However, the role of YAP1 and the tumor microenvironment remains unclear. In this study, we investigated the role of YAP1 in promoting colon tumorigenesis in tumor cells and association with M2 TAM polarization. We hypothesize that oncogenic YAP1 promotes the generation of M2 TAM polarization and in return M2 TAM further enhances tumorigenic phenotypes including enhanced proliferation, colony formation, colon-sphere generation, and metastatic potential. Therefore, agents that target and suppress the expression of YAP1-mediated signaling may act as a dual anti-cancer agent by disrupting the molecular connections between TAMs and tumor cells.
Ovatodiolide (OV) is a bioactive phytochemical purified from Anisomeles indica (L.) Kuntze (Labiatae), a medicinal herb which has been widely used for the treatment of a variety of inflammation-associated diseases and implicated for potential anti-cancer functions by our previous reports [10, 11]. Various oncogenic targets inhibited by ovatodiolide have been implicated; these include TNF-α, NF-κB, β-catenin, MMPs, and others to achieve anti-cancer effects such as the induction of cell cycle arrest, apoptosis, and reduction of metastatic potential [12–15]. Since the connection between inflammation (TAMs) and colon tumorigenesis has been well established, we aimed to examine the potential functions of ovatodiolide (being an anti-inflammatory and anti-cancer agent) in targeting both tumor cells and TAMs.
Increased YAP1 expression is associated with poor prognosis
Tumor-associated macrophages (TAMs) promote the generation of CD133+ and side-population stem-like cells and associated with increased YAP expression
TAM educated CRC cells showed enhanced tumor-initiating ability
Ovatodiolide treatment suppresses tumorigenesis in CRC cells
Ovatodiolide treatment suppresses M2 polarization in vitro
To explore if ovatodiolide treatment can suppress IL-6-induced colon carcinogenesis, exogenous IL-6 was introduced to the culture medium. We showed that the addition of IL-6 significantly increased the expression of multiple oncogenic markers including Kras-MEK, YAP1, NF-kB, vimentin (EMT marker), β-catenin (a marker for both oncogenesis and stemness), and ABCG2 (drug resistance) in both HCT116 and DLD-1 cells. Ovatodiolide treatment significantly suppressed these IL-6-induced pathways (Fig. 6c). For instance, major pro-M2 TAM polarization cytokines, IL-4 and IL-13 expression, were markedly suppressed by the OV treatment even in the presence of IL-6 (Fig. 6c).
IL-6 treatment significantly induced the generation of colon spheres (Fig. 6d), and the addition of OV markedly reduced this effect (Fig. 6d). IL-6-enhanced tumor sphere-forming ability was disrupted significantly as the size and the number of formed spheres was significantly less comparing to IL-6-induced counterparts (Fig. 6d). In support, exogenous IL-6 resulted in an increase in CD133+ HCT116 and DLD-1 cells. The addition of ovatodiolide (in the presence of IL-6) significantly reduced the IL-6-induced effects (Fig. 6e). Furthermore, we tested the possibility of ovatodiolide working in synergy with 5-FU to suppress the viability of colon cancer cells. Different concentration combinations of ovatodiolide and 5-FU were assayed to generate the isobolograms. We found that several combinations of 5-FU and ovatodiolide inhibited both HCT116 and DLD-1 cell viability synergistically (CI index <1, in bold, Fig. 6f).
YAP1-silencing led to decreased CRC tumorigenesis and M2 polarization
Equally important, the number of CD133+ cells were significantly increased in both YAP1-overexpressing DLD-1 and HCT116 cells (Additional file 2: Figure S1A); the tumor sphere-forming ability was also markedly enhanced in YAP1-overexpressing cells (Additional file 2: Figure S1B). In consistent with our proposed role for YAP1, when YAP1 expression is increased so is the level of β-catenin, NF-kB, vimentin, and Kras (Additional file 2: Figure S1C).
The role of YAP1 in M2 polarization was also tested in THP-1 cells by silencing YAP1 (Additional file 1: Figure S2A). We found that the resultant macrophages differentiated from YAP1-silenced THP-1 showed a significantly reduced level of IL-4, TGFB1, and Ym2 (M2 markers) while increased iNOs (M1 marker).
OV treatment suppressed CRC tumorigenesis and M2 infiltration in vivo
Tumor-associated macrophages or TAMs (the M2-polarized TMAs particularly) have been indicated to promote tumorigenesis and drug resistance [27, 28]. The increased abundance of tumoring infiltrating M2 TAMs has been correlated with poor prognosis in various types of human cancers including colon [24, 29, 30]. The modification and/or disruption between the molecular communications between colon cancer cells and its microenvironment (TAMs) thus represents an important milieu for intervention development. Here, we first demonstrated that the presence of M2 TAMs promotes the generation of cancer stem-like phenotypes in both HCT116 and DLD-1 colon cancer cells; M2 TAM co-cultured cancer cells demonstrates increased CD133 expression, colon sphere-forming ability in association with a significantly resistant 5-FU resistance.
M2 TAMs have been shown to promote tumorigenesis via the secretion of a plethora of cytokines . One of the major M2 TAM-secreted oncogenic cytokines, IL-6, has been implicated in contributing metastatic potential and therapeutic interventions in different cancer types including colon [26, 32]. Our study adds another dimension of IL-6’s role in colon tumorigenesis where IL-6 from M2 TAMs or exogenous source enhanced the generation of colon stem-like cells (or colon spheres); the addition of IL-6 was associated with an increased expression of several oncogenic pathways including YAP1, Kras, β-catenin, NF-κB, and mTOR and the increased expression of IL-4 and IL-13, both of which have been established for the promotion of M2 phenotype .
Experimental and clinical evidence indicate that targeting and eliminating TAMs represent a potential therapeutic strategy of cancer therapy [34, 35]. However, previous studies suggested that targeting M2 TAMs systemically carries the risk of compromising the host’s immunity [27, 36, 37]. Here, we identified YAP1 as a potential molecule whose expression is associated with M2 polarization and colon tumorigenesis. First, an increased YAP1 expression was detected in colon spheres and M2 TAMs. Second, the downregulated YAP1 expression in colon cancer cells by either gene-silencing technique or phytochemical treatment of ovatodiolide led to the decreased tumorigenesis (colony formation, migration, and tumor sphere formation) accompanied by the decreased M2-polarizing cytokines such as IL-4 (the major one affected by YAP1 silencing) and IL-13 (to a lesser extent) as well as mTOR/Akt signaling. Our observation was supported by a study indicating that IL-4/IL-13/Akt [38–40] signaling axes are important for M2 phenotype generation.
In parallel, ovatodiolide treatment suppressed YAP1 along with other major oncogenic pathways and prevented the THP-1 monocytes from M2 polarization in association with a decreased oncogenic cytokine IL-6 expression. Notably, IL-6 has been shown to contribute to proliferation and promote EMT and self-renewal in breast cancer cells  establishing an essential communication between cancer cells and M2 TAMs. We showed that exogenous IL-6 led to the increased generation of colon tumor spheres with concomitant elevated YAP1 expression, establishing a novel link between YAP1 and IL-6 network. Moreover, bioinformatics analysis (data not shown) indicated that downregulation of YAP1 is associated with a decreased level of IL-10RB (Interleukin-10 receptor subunit beta) implicating their collaborative roles in M2 polarization process. Together, our data suggests that YAP1’s function in promoting colon tumorigenesis not only derives from its intrinsic oncogenic properties but also functions to promote M2 TAM polarization. Interestingly, previous report indicated that oncogenic Kras, YAP1, and β-catenin serve similar functions in cell cycle control in tumor initiation . A recent seminal study showed that acquired resistance against Kras inhibition in a Kras-driven mouse lung cancer model was associated with an increased YAP1 signaling; Kras and YAP1 signaling converges and activates the cellular EMT machinery . This is in agreement of our finding that YAP1 suppression either by ovatodiolide or gene silencing significantly reduced the expression of Kras and oncogenic properties (including sphere-forming ability).
The anti-cancer and self-renewal properties of ovatodiolide have been previously established by our group [10, 15, 22]. The present study furthered the exploration of ovatodiolide’s anti-cancer function by showing its effects on preventing the generation of M2 TAMs. In this study, we identified the elevated YAP1 expression in M2 TAMs and ovatodiolide treatment prominently suppressed the expression of YAP1, M2 markers (ARG1 and CD23), and inflammatory-signaling networks such as NF-κB and Akt. Our findings suggest that YAP1 within the tumor cells may function in promoting colon tumorigenesis via its association with aforementioned oncogenic pathways and generating cancer stem-like cells (as shown in our study) while generating M2 TAMs via increased M2-polarizing IL-4 and IL-13 cytokines into the stroma (Fig. 8d for proposed model). In addition, ovatodiolide-mediated IL-6 suppression in the M2 TAMs represented another important anti-cancer attribute of ovatodiolide; even in the presence of exogenous IL-6 which increased the generation of cancer stem-like cell population in both HCT116 and DLD-1 cell lines, ovatodiolide was able to attenuate IL-6’s effects.
Certainly, ovatodiolide-mediated anti-cancer effects in this study appeared to be multi-targeted and differential between HCT116 and DLD-1 cells. This reflects the heterogeneity of cancer and the importance of precision medicine. HCT116 and DLD-1 cells used in this study share a very similar gene mutation profile. But one of the major difference is that DLD-1 is a TP53 mutant while HCT116 wild-type. This may explain that HCT116 is slightly more sensitive toward ovatodiolide’s treatment; HCT116 also contains a lower intrinsic YAP1 expression level as compared to DLD-1 and demonstrates a lower ability to form tumor spheres as compared to DLD-1. A recently study indicated that primary breast cancers harboring TP53 mutations and expressing high level of YAP1 demonstrate a higher expression level of cyclin A, cyclin B, and CDK1 genes as compared to the TP53 wild-type counterparts . It warrants further investigations to determine if this phenomenon also exists in colon cancer and for establishing a colon cancer signature for ovatodiolide sensitivity.
Here, we demonstrated for the first time that YAP1 played a key role in colon tumorigenesis in a dual dimensional fashion where it acts in cancer cells to promote the malignant phenotypes including cancer stem-cell like properties and in polarizing TAMs toward M2 phenotype. The suppression of YAP1 either by siRNA or ovatodiolide suppressed YAP1-associated oncogenic characteristics. Thus, YAP1 could represent an important druggable target; ovatodiolide could be further evaluated and considered to be used as an adjuvant agent for treating colon cancer.
Cell culture and chemicals
The human colon cancer cell lines HT-29, SW480, DLD-1, HCT116, and monocytic cell line, THP-1, were obtained from the American Type Culture Collection (ATCC), and cells were cultured according to ATCC’s recommended conditions. Colon sphere formation assay was performed according to previously established method . DLD-1 and HCT116 cells were cultured in Serum-Free Medium (SFM) composed of DMEM/Ham’s F12 (1:1), human epidermal growth factor (hEGF, 20 ng/ml), basic fibroblast growth factor (bFGF; 10 ng/ml (PeproTech, NJ, USA), 2 μg/ml of 0.2% heparin (Sigma), and 1% penicillin/streptomycin (P/S, 100 U/ml, Hyclone). Cells were seeded (1000 cells/ml) in 12-well low adhesion plates and incubated at 37 °C and 5% CO2 for 5–7 days. Cell aggregates or spheroids (compact, spherical, non-adherent masses >50 μM in diameter) were counted. 5-FU was purchased from SelleckChem, Taiwan (Cat. No.S1209), and ovatodiolide was isolated and purified as described previously .
Cell viability determination
Cellular viability in this study was determined using the sulforhodamine B (SRB) assay . Briefly, colon cancer cells and/or spheres were seeded in 96-well plates (3.5 × 105 cells/well) and treated with drugs of interest (ovatodiolide or 5-FU) alone or in combination at indicated concentrations and times. Post treatment, the relative cell number was determined using a SRB reagent according to the manufacturer’s protocol (Sigma, USA). For tumor spheres or non-attached cells, the cell viability was quantified using Alamar blue staining (Life Technologies, USA).
Macrophage generation and differentiation
Macrophage generation and differentiation from THP-1 cells were performed according to previously established method . In brief, for M2-polarized macrophages, THP-1 cells were first treated with 320 nM PMA for 6 h, followed by cultured by the addition of IL-4 and IL-13 (20 ng/ml) for another 18 h. For M1-polarized THP-1 macrophages, LPS (100 ng/ml) and IFN-γ (20 ng/ml) were used instead. THP-1 cells were also co-cultured with CRC cell lines. CRC cells were seeded in the upper insert of a six-well Transwell apparatus (0.4 μM pore size, Corning, Lowell, MA) while THP-1 with 320 nM PMA in the lower chamber. After 48 h, both macrophages and CRC cells were then harvested for further biochemical analyses. In the colon cancer co-culture experiments, only PMA was added to the THP-1 cells (M0 macrophage progenitors) without the addition of IL-4 and IL-13. After 48 h of co-culture, both colon cancer cells and differentiated macrophages were harvested for further analyses.
Flow cytometry analysis
Flow cytometry was used to profile CRC stem-like cells using the BD Accuri™ C6 personal flow cytometer. CD133/1 (AC133) antibodies conjugated to APC (Miltenyi Biotec, Auburn, CA, USA) were used to determine CD133+ CRC cells. Side-population analysis was performed according to previously established method . In brief, side-population HCT116 and DLD-1 cells were identified using FACSAria™ III sorter (BD Biosciences, Taiwan). Verapamil (100 μM final concentration) was added 15 min before Hoechst incubation and was used as a control. SP cells which express ATP-binding cassette ABCG2 and Hoechst 33342 efflux activity was identified and determined to be SP cells.
Real-time PCR reaction
(additional primer sequences may be found in Additional file 1: Figure S2).
YAP1 silencing and overexpression
YAP1 expression was downregulated using siGNEOME SMARTpool YAP1 siRNAs (Catalog No. M-012200-00, Dharmacon). The gene-silencing experiments were carried out according to vendor’s instructions. After each gene-silencing experiment, western blots were used to verify that YAP1 gene product was successfully knocked down. YAP1 overexpression was performed by transfecting the cells with YAP1 open-reading frame vector (human, Cat. No. LV805163, Applied Biological Materials Inc., BC, Canada). The transfection protocol was performed by following vendor’s instructions.
Western blot analysis
Total colon cancer cell lysates obtained from different experiments were separated using the SDS-PAGE using Mini-Protean III system (Bio-Rad, Taiwan) and transferred onto PVDF membranes using Trans-Blot Turbo Transfer System (Bio-Rad, Taiwan). The majority of the primary antibodies used in this study were listed. YAP1 (#4912), mTOR (#2983), and β-catenin (#9562) from Cell Signaling Techology (Taipei, Taiwan) and Kras (12063-1-AP), IL-6 (21865-1-AP), and IL-4 (16545-1-AP) from Protein Tech (Taipei, Taiwan). Secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Protein of interests were detected and visualized using ECL detection kit. Images were obtained and analyzed using UVP BioDoc-It system (Upland, CA, USA).
Colony formation and migration assays
The colony-forming assay was performed using an established protocol  with modifications. Briefly, 500 HCT116 and DLD-1 cells were seeded in six-well plates with (2.8 μM ovatodiolide, equivalent of IC10 values) and without ovatodiolide. The plates were then stained using 0.005% crystal violet, and the colonies were counted. The cells were allowed to grow for another week. The cells were then harvested, fixed, and counted. The migratory ability of the cells was examined using Transwell migration assay (ThermoFisher, Taipei, Taiwan). In short, cells were trypsinized and re-suspended in a serum-free DMEM medium with or without treatments (siYAP or ovatodiolide of different dosing regimens). The cells were subsequently seeded into the upper chamber polycarbonate filters (8 μm pore size). A serum-containing DMEM medium (500 μL) was added to the lower chambers. The experiment was performed for either 24 or 48 h depending on the cell line. The cells were fixed with 3.7% formaldehyde and stained with crystal violet. The non-migratory cells on the upper side of the membrane were removed. The migratory ability was determined as the number of cells calculated on the lower side of the membrane.
In vivo experiments
Female NOD/SCID and BALB/c mice were purchased from BioLASCO Taiwan Co., Ltd. The experiments were conducted strictly in compliance to the Affidavit of Approval of Animal Use Protocol Taipei Medical University (protocol LAC-2014-0170). First, the tumor-initiating ability test was performed using the tumor spheres generated from the naïve DLD-1 and M2 TAM co-cultured DLD-1 cells. Spheroids cells (1 × 105 cells/injection) were subcutaneously injected into the right flank of NOD/SCID mice. DLD-1 expressing GFP and firefly luciferase dual reporter system (L2G, a generous gift from Dr. Sanjiv Sam Gambhir, Stanford University) was used for the bioluminescence imaging experiments. Tumorigenesis was monitored using bioluminescence (IVIS 200 system, Caliper) on a weekly basis and quantified using Living Imaging software. The tumor growth was indicated by the fold change in bioluminescence intensity plotted over time. Second, for drug treatment test and examination of TAM infiltration, subcutaneous tumor model was established using murine colon cancer cell line, CT26 (1 × 106 cells/20 μL/injection) in BALB/c mice (4–6-weeks old). The treatments commenced when the tumor size reached approximately 100 mm3 measured by a standard caliper. Dosing regimens are as the following: 5-FU alone (30 mg/kg, two times/week), ovatodiolide alone (5 mg/kg, five times/week), and 5-FU + ovatodiolide combination (30 mg/kg, two times/week; 5 mg/kg five times/week, respectively). Both agents were given intraperitoneally. The change in tumor burden was expressed in fold change in cubic millimeter as compared to its starting volume. Mice were humanely sacrificed upon completion of experiment, and tumor biopsies were collected for further analyses.
For preliminary screening, commercially available tissue microarray slides (Category # BA5 and CDA3) were purchased from SuperBioChips (Seoul, Korea). The staining protocol was followed as per the vendor’s published instructions (UltravVision Quanto Detection System HRP DAB manual, Thermo Sicentific, CA, USA). In short, the immunostaining was performed on 5-μm-thick tissue sections cut from the TMA (both commercially purchased and in vivo tumor samples). The sections were dewaxed and deparaffinized in xylene and rehydrated in graded alcohol solutions. Antigen-retrieval process was performed by heat-induction for 30 min in Tris-EDTA buffer. Slides were subsequently stained with primary antibodies of YAP1 (#4912, 1:400), mTOR (#2983, 1:500), β-catenin (#9562, 1:400, Cell Signaling technology, Taiwan), CD206 (anti-mannose receptor antibody, ab64693, 1: 400, Abcam, Taiwan), and their respective secondary antibodies. The sections were then counterstained with hematoxylin, followed by dehydration and mounting. The images were captured and recorded using Tissue FAXS viewer software (TissueGnotics, GmBH, Vienna, Austria).
All experiments were performed and repeated at least three times. For in vitro experiments, bar charts and graphs represent mean values, and error bar indicates standard deviation (s.d). A paired “one-tailed” or ‘two-tailed” Student’s t test was performed using GraphPad Prism software. The result was considered significant when *P < 0.05, **P < 0.001, and ***P < 0.001, respectively.
We are grateful to the excellent technical supports from Hui-Hsien Chiang, Li-Wen Wang, Min-Hao Ho, Cian-Ru Yang, and Po-Yang Huang.
This study was funded by the following agencies. (1) TMUTOP103003-8 from Taipei Medical University, The Aim for the Top University Project, the Ministry of Education, Taiwan, to Alexander TH Wu and Yan-Jun Huang. (2) MOST104-2314-B-038-077-MY3 and MOHW105-TDU-B-212-134001 (Health and welfare surcharge of tobacco products, Ministry of Health and Welfare, Taiwan) to Dr. Whang-Peng Jacqueline and Excellence for Cancer Research (TMU-CECR).
Availability of data and materials
Please contact the author for data requests.
YJH performed experiments and drafted the manuscript. ATHW, MH, TCM, and YY designed the experiments and wrote the manuscript. CKY, PLW, TTH, and JWP contributed to the study design and data analyses. YMT isolated/purified the ovatodiolide and contributed to the study design. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The experiments were conducted strictly in compliance to the Affidavit of Approval of Animal Use Protocol Taipei Medical University (protocol LAC-2014-0170).
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