Wnt modulates MCL1 to control cell survival in triple negative breast cancer
© Yang et al.; licensee BioMed Central Ltd. 2014
Received: 10 December 2013
Accepted: 13 February 2014
Published: 24 February 2014
Triple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells.
We performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test.
WNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival.
All our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics.
KeywordsWNT5B MCL1 WNT/β-catenin pathway Triple negative breast cancer (TNBC)
Triple negative breast cancer (TNBC) is an aggressive form of breast cancer characterized by the lack of estrogen, progesterone receptors (ER, PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2) . With the major contribution of adjuvant targeting therapies, the outcome of breast cancer has been improved dramatically; yet the prognosis of TBNC remains quite poor among the breast cancer subtypes [2–4]. It is largely due to the heterogeneous nature of TNBC and unresponsiveness to the clinic available targeting therapies [5–7]. Many attempts to identify the key oncogenic pathways at the molecular level have been carried out. Aberration of WNT signal is widely recognized as one of the potential pathway that contributes to TNBC tumorigenicity [8–11].
WNT and their downstream responsive genes modulate various processes that are crucial for development and growth, cell fate decision, cell proliferation/differentiation and stem cell self-renewal [12–14]. Activation of WNT signaling cascade is initiated through the binding of WNT with its receptor/co-receptor. WNT/β-catenin is the first indentified WNT pathway that is aberrantly activated in human colorectal cancer [15, 16]. Since then, the complicated signals triggered by WNT, but following distinct pathways have been detected. The complexity of these signals is partially attributed to the multiple members of WNT family and various subtypes of receptor/co-receptor . The cellular response to a given WNT ligand is ultimately context-specific and the dynamic interactions determine the net outcome . Emerging evidence has been demonstrated that WNT signaling is actively involving in many cellular biologic processes via integrating WNT signal to other major cellular pathways, including mitochondrial homeostatic pathway .
Mitochondria engage in various biochemical activities and are the major organelle to generate ATP. In addition to their function as the power plants, they are involving in many other vital cellular processes, such as cell apoptosis, cell cycle control, cell differentiation and cell proliferation [20, 21]. The functional and active mitochondria status is actually essential for cancer cell physiology. Despite frequent mitochondrial gene mutations are detected in human tumor; they don’t turn off the mitochondrial energy metabolism at all. Additionally, they regulate the mitochondrial bioenergetic and biogenetic state . However, how cancer cells modulate mitochondrial status to meet their biological need is under current study. In the current project, we present evidence to demonstrate that MCL1 is a key regulator for TNBC cell survival mediated by controlling mitochondrial biogenesis.
Patients, tissues and serum
All tumor tissues and serum were collected under the Institutional Review Board (IRB)-approved protocols at City of Hope National Medical Center (COH) or Zhejiang University respectively. The patients were given informed consent. One hundred and forty-two breast tumor tissues, including 21 TNBC and 121 Non-TNBC tissues were collected for immunohistochemistry staining. We also collected 30 sera from TNBC and Non-TNBC patients respectively with the assistance from the COH Translational Research Laboratory Core for ELISA assay. Immunohistochemical staining and FISH confirmed that ER/PR/HER2 were negatively expressed, as assessed by pathologists in the Department of Pathology of COH.
For differential expression analysis, differential expression P-values were determined via t-test in R . Significant results are expected to show P-value < 0.05. Differential expression between TNBC and non-TNBC was determined using data from 3 cohorts (Chin et al. , expO (GSE2109), and TCGA . Differential expression between patients that did or did not develop metastatic tumors was determined using 2 cohorts (expO (GSE2109), TCGA  for WNT5B and 1 cohort (Desmedt et al.) for MCL1. For survival analysis, differences in survival between “high” and “low” expression groups were visualized in Kaplan-Meier plots and compared using Cox regression analysis, with P-values calculated via log-rank test, using the ‘survival’ package in R (A Language and Environment for Statistical Computing). The disease-free survival of WNT5B was quantified independently for 2 cohorts (Desmedt et al. and Wang et al. [26, 27]) respectively. And then meta-analysis was conducted by utilizing the same WNT5B probe (so that the signals would be comparable) for an 80 month observation period. The disease-free survival of MCL1 was analyzed by the same method using the cohort of Desmedt et al.
RT-PCR, RT-qPCR and qPCR
Total RNA extraction from MDA-MB-231 was carried out using the RNeasy Mini Kit (Qiagen). For cDNA synthesis, total RNA (1 μg) was transcribed using random hexamers (Invitrogen, Carlsbad, CA), and SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s protocol. For quantification of OXPHOS-related genes, the cDNA amplication program included a denature at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s; 58°C for 30 s. For MtDNA detection, total cellular DNA was isolated with DNAeasy Blood and Tissue Kit (Qiagen). Mitochondrial DNA content was determined by qPCR by using comparing the mitochondrially encoded Cox2 gene to an intron of the nuclear-encoded β-globin (HBB) gene. All qPCR was performed using an iQ5 iCycler (Bio-Rad) according to the manufacturer’s instructions. Data were analyzed using Bio-Rad iQ5 Optical System Software v2.0. All products yielded a single band with the predicted size. All primers are listed in Additional file 1: Table S1 and all products yielded a single band with the predicted size.
Western blot analysis
Cell protein was extracted from cells using RIPA buffer (Cell Signaling, Danvers, MA) with phosphatase inhibitor (Roche, Indianapolis, IN). Equal amount of protein was loaded and separated by SDS-PAGE. After the protein was transferred onto a membrane, the blot was blocked with 5% non-fat milk in TBS and probed overnight at 4°C using the following antibodies: WNT5B (Sigma-Aldrich, St. Louis, MO), Cyclin E (M20), TOM20 (F-10), Myc (9E10), AIF (E-1), MCL1 (S-19) (Santa Cruz Biotechnology, Santa Cruz, CA), Caspsae-3, Caspase-8,PGC-α, Cyclin D1 (Cell Signaling, Danvers, MA) and β-actin (Bio-Rad, Hercules, CA). Appropriate antibodies were used for secondary antibody reaction. Signal was detected by the ECL Plus Western Blot Detecting System (GE Healthcare, Piscataway, NJ).
Cell culture and growth assays
The triple negative cell lines MDA-MB-231was purchased from ATCC and cultured in the recommended media. Specific lentivirus shRNA (Sigma-Aldrich) was used to disrupt the expression of WNT5B while shRNA targeting non-mammalian sequence (Sigma-Aldrich) served as control. WNT5B expression was determined by immunoblot analysis. MDA-MB-231 cells that expressed WNT5B or control shRNA (shWNT5B or shCtl cells) were cultured in growth medium to observe cell growth. Cells (3 × 104/ml) were seeded into 24-well plates, and cell number was counted every day for five days using a Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA). Independent experiments were performed in triplicate.
Cell morphology, invasion
Cells were infected with shCtl or shWNT5B lentivirus and the morphology was observed and photographed after WNT5B expression was inhibited. Cell mobility was determined by a wound closure assay. Cells were placed onto 6-well plates at 80% confluence and cultured in serum-depleted media for 40 h. A wound was made by scraping the monolayer cells with a plastic pipette tip and fresh serum-free medium was replenished. Images of wound closure were photographed at 0, 16, 24 and 40 h post-scraping.
Cells were trypsinized, resuspend in fresh medium followed by flowcytometry analysis. For cell cycle assay, cells were fixed with 70% ethanol and incubated on ice for 30 min. The cells were then suspended in PBS and treated with RNase A (final concentration 100ug/ml) at 37°C for 30 min. After removing RNase A, the cells were stained with propidium iodide (PI) at 5ug/ml for 10 min and the cell cycle was determined by flowcytometry analysis. For apoptosis assay, FITC Annexin V Apoptosis Detection Kit was used for staining the cells following product’s manual (BD bioscience). All flowcytometry data were analyzed using Summit v4.3 software.
Immunohistochemical (IHC) staining
All the formalin-fixed paraffin-embedded (FFPE) slides were prepared and stained by the Pathology Core Facility at COH using a standard protocol. Antibodies used in this study were: rabbit polyclonal antibodyWNT5B (SAB2900204) (1:20 dilution, Sigma-Aldrich), mouse monoclonal antibody Myc (SC-40) (1:75 dilution, Santa Cruz Biotechnology) and rabbit monoclonal antibody MCL1 (ab32087) (1: 100 dilution, Abcam). All antibodies were titrated with negative and positive controls to obtain optimal staining.
Electon microscope (EM)
The cells infected with shWNT5B or shCtl were collected in 3 days. The electron microscope was done in the core facility at COH following their standard protocol. It has been described in detail elsewhere . The stained sections were subjected to Electron microscopy, which was done on an FEI Tecnai 12 transmission electron microscope equipped with a Gatan Ultrascan 2 K CCD camera.
Oxygen consumption rate (OCR) and ATP measurement
The XF24 flux analyzer (Seahorse Bioscience) was used to measure OCR in 24-well microplates. Six thousand cells transduced with shCtl and 12000 cells infected with shWNT5B lenti-virus were seeded onto 24-well plates and incubated 3 days. The measurement, recording procedure and data analysis were described previously . For cellular ATP measurement, we used ENLITEN ATP Assay System Bioluminescence Detection Kit (Promega Madison, WI). Briefly, the adherent cells in 6-well plate were collected by 2 mM EDTA in PBS on ice, TCA was add at final concentration of 1% and vortex vigorously for 10 sec. It was further diluted to 0.1% TCA by Tris-Acetate (pH = 7.75). The standard as well as the samples were serially diluted by dilution buffer (0.1% TCA/0.08 × PBS/0.9 × Tris-Acetate) and subjected to luminescence measurement.
To measure soluble WNT5B in patients’ serum, we used WNT5B ELISA Kit (USCN Life Science Inc.). The manufacture’s protocol was totally followed for preparing samples and all the reactions. The plate was read by SpectramaxPlus (Molecular Devices).
ShWNT5B- or shCtl- virus transduced MDA-MB-231 cells were distributed into 12-well plates the day before transfection. Cells at 80% confluence were co-transfected with TCF-driven Topflash reporter plasmid (Millipore) (1 μg) and control Renilla luciferease (20 ng) using 2.5 μl of Lipofectamine 2000 (Invitrogen). Cells were lysed in 1X passive lysis buffer in 48 h and the supernatant was collected for Dual-luciferase activity measurement (Promega, Madison, WI). For each sample, firefly luciferase activity was normalized with an internal control, Renilla luciferase activity.
WNT5B was upregulated in triple negative breast cancer
ShWNT5B led to impairment of cancerous features in TNBC cells
ShWNT5B induced cell cycle arrest and caspase-independent cell death
Knockdown of WNT5B attenuated mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) in MDA-MB-231 cells
Possible mechanism for shWNT5B-induced suppresion of mitochondrial physiology
Clinical significance of WNT5B in metastasis and disease-free survival of TNBC
We have previously reported that aberrant activation of WNT signaling contributed to proliferation and metastasis in TNBC cells and in animal model . To carefully address the wider role of WNT signaling playing in the TNBC, we extended the study population from the data in our institute to the public arrays. Firstly, WNT5B was identified as the possible ligand for activation of WNT signaling in TNBC. In the functional study, we found that WNT5B played a crucial role for TNBC cells. It empowered cell growth through regulation of the cellular energy plant, mitochondria. Most importantly, this delicate regulation by WNT5B didn’t limited in a particular cell model; it was fundamentally associated with patients’ metastasis and disease-free survival in a larger population with breast cancer. These strong evidences highlighted the promising effect that WNT5B exerts on TNBC.
The WNT effect is highly dynamic and tissue context-specific in human cancers. For instance, the elevated WNT3A promotes the growth of myeloma cells in vitro  and prostate tumor in mouse model , while it dramatically decreases the growth of melanoma cells transplanted in the mice . Most likely, each WNT exhibits unique sensitivities and the response upon a particular tissue-derived cancer, which might be true for WNT5B in TNBC. Recently, it has been noted that WNT signal promoted mitochondrial biogenesis in mouse skeletal myoblasts ; it was also observed that mitochondrial function and oxidative phosphorylation were impaired in hepatocytes of β-catenin knockout mice ; and the adipocyte mitochondrial metabolism was suppressed through WNT inhibition . Collectively, we speculate that convergence on the mitochondria might be a mechanism of WNT controlling diverse process in some cancer cells. Despite the multitude of reports, the mechanism of how WNT modulate mitochondrial physiology in TNBC remains unexplored. In the current study, MCL1 was verified as the responsive protein which opposed cell death through controlling mitochondrial homeostasis. Among the Bcl-2 pro-survival protein family members, MCL1 was the one that raised particular attention because of its high expression in extensive cancer subtypes and its functions that extended beyond apoptosis regulation, but contributed to diverse biological process, such as malignancy and autophagy [38, 39]. Increased MCL1 levels in cancer cells can result from elevated transcription or translation and decelerated degradation [40, 41]. A genome-wide study of somatic copy number amplification (SCNA) uncovered that MCL1 was enriched in over 3000 tumor samples collected from 26 histological types. The increased copy number of MCL1 was found in more than 10% of cancers, but the amplification was higher in lung and breast cancers . Recent research progression of TNBC indicated that Myc and MCL1 are both upregulated in TNBC and they play important role in cell survival . In the current study, we demonstrated that WNT5B-stimulated WNT/β-catenin signaling held MCL1 at high level via its target protein, Myc. It was also reported that GSK3 controlled MCL1 degradation by phosphorylation of MCL1 for ubiquitylation-dependent degradation. Impaired phosphorylation of GSKs induced by activation of WNT/β-catenin might corporate with Myc to stabilize MCL1 in TNBC. We will address it in the future. Taken together, our study provided wider insight into the deeper role of WNT5B-triggered WNT/β-catenin signaling; it might regulate breast tumor progression and outcome by modulating mitochondrial physiology through MCL1.
Taken together, the data suggest that WNT5B plays an important role in aberrant activation of WNT canonical pathway in TNBC. Inhibition of WNT5B induces cell cycle arrest and caspase-independent apoptosis, which is caused by attenuated mitochondrial biogenesis. WNT5B modulates mitochondrial biogenesis through MCL1, which is regulated by WNT/β-catenin responsive gene, Myc. These findings provide promising evidences to target WNT5B-indeced WNT/β-catenin signaling in TNBC.
Triple negative breast cancer
Epidermal growth factor receptor 2
Mouse recombinant WNT5B
Cytochrome c 1
ATP synthase γ subunit
Cellular oxygen consumption rate
Translocase of outer mitochondrial membrane 20 homolog (yeast).
We thank Mariko Lee in the Light Microscopy and Digital Imaging Core for assistance with photography, Sofia Loera in the Pathology Core for IHC staining, Zhuo Li in the Molecular and Cellular Department for EM images and Lucy Brown in the Analytical Cytometry Core for running flow samples. The helper vectors for retrovirus generation are from Richard Mulligan (Harvard University) and MSCV-IRES-PURO-MCS vector is from Martine Roussel (St. Jude Children’s Research Hospital).
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