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The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer
© The Author(s). 2017
Received: 10 March 2017
Accepted: 25 May 2017
Published: 2 June 2017
Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial.
Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells.
Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo.
This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.
Triple-negative breast cancers (TNBCs) are a diverse and heterogeneous group of tumors that lack estrogen and progesterone receptors and HER2 gene amplification. Because they do not express these receptors, hormonal (e.g., tamoxifen) and targeted (e.g., Herceptin) therapies are not effective in treating TNBCs. Moreover, the majority of TNBCs are highly malignant, and only a subgroup responds to conventional chemotherapy with favorable prognosis [1–3]. Therefore, drug resistance and metastatic progression are major clinical issues in the successful treatment of TNBCs. Although high-throughput molecular analyses, including sequencing, pathway analyses, and integrated analyses of alterations at the genomic and transcriptomic levels, have improved our understanding of the molecular alterations involved in TNBC development and progression, the proper use of this knowledge for the rational selection of therapy remains challenging.
Gαh is a novel GTP-binding protein that is an inactive form of calcium-dependent tissue transglutaminase; a multifunctional protein that is ubiquitously expressed in various tissues . Gαh transmits signals from activated G protein-coupled receptors, e.g., α1-adrenoceptor  and follicle-stimulating hormone receptor , to phospholipase C-δ1 (PLC-δ1) via protein-protein interactions (PPIs). Activation of the Gαh/PLC-δ1 pathway has been shown to elevate the concentration of intracellular inositol 1,4,5-triphosphate (IP3) and calcium , both of which are important secondary messengers in signal transduction and promote cell survival, growth, and invasion [8, 9]. In contrast, an abnormal increase in intracellular calcium due to cellular damage or other stressors has been shown to activate the transglutaminase feature of Gαh activity to catalyze the crosslinking of cellular proteins and ultimately induce cell death . Recently, Gαh overexpression has been observed in breast [11, 12], colon , and ovarian cancers ; non-small cell lung cancer ; and renal cell carcinoma  and has been shown to be associated with advanced disease stages and metastatic spread. Importantly, the loss of the GTP-binding, but not transamidating, activity of Gαh was shown to inhibit cancer metastasis in malignant tumor cells , implying the critical importance of Gαh/PLC-δ1-dependent signaling in cancer progression. These findings prompted us to evaluate the feasibility of therapeutic inhibition of the PPI of Gαh with PLC-δ1 in combating cancer metastasis.
Here, we find that Gαh expression is causally associated with the metastatic potential of TNBC cells in vitro and in vivo and is strongly correlated with poor distant metastasis-free survival probability in patients with breast cancer. Notably, loss of the G protein function, but not the transglutaminase activity, abolished the Gαh-induced metastatic progression of TNBC cells. Interestingly, blocking the PPI between Gαh and PLC-δ1 using a synthetic peptide blocker corresponding to the binding-interface of Gαh effectively inhibited the metastatic progression of TNBC cells in vitro and in vivo. These findings provide a potential new strategy to develop anti-cancer agents for overcoming cancer metastasis.
Cell lines and cell culture condition
TNBC cell lines MDA-MB-468, MDA-MB-231, and MDA-MB-436 were cultured in Leibovitz’s (L-15) medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and incubated at 37 °C with free gas exchange with atmospheric air. TNBC cell lines HCC1143, HCC1599, HCC70, and HCC38 were cultured in RPMI-1640 medium (Gibco Life Technologies) with 10% FBS and incubated at 37 °C with 5% CO2. HS578T and 293T cells were cultured in DMEM with 10% FBS and incubated at 37 °C with 5% CO2. All cell lines were obtained from American Type Culture Collection (ATCC). All cells were routinely authenticated on the basis of short tandem repeat (STR) analysis, morphologic, and growth characteristics and mycoplasma detection.
Raw data for pathological information and prognostic values of the Gαh gene were downloaded from the PrognoScan database (http://www.prognoscan.org/). Datasets with p value < 0.05 by Cox regression analysis were included in a meta-analysis for Gαh gene expression. Transcriptional profiling for Gαh was obtained from The Cancer Genome Atlas (TCGA) database and subjected to statistical analysis against differential levels of Gαh mRNA in the subgroups. Kaplan-Meier analysis and the differential analysis of Gαh gene expression were performed on the SurvExpress website.
In vitro invasion assay
For invasion assays, Boyden chambers (Neuro Probe, Gaithersburg, MD, USA) were used according to the manufacturer’s protocol. Briefly, a polycarbonate membrane (Neuro Probe) was pre-coated with 10 μg of human fibronectin (Sigma-Aldrich, St Louis, MO, USA) on the lower side and Matrigel on the upper side. Cells (1.5 × 104) were seeded in the top chamber in 50 μl of low-serum medium (0.1% FBS) containing drugs or DMSO. After 16 h, stationary cells were removed from the top surface of the membrane, whereas the migrated cells on the bottom surface of the membrane were fixed in 100% methanol and stained with 10% Giemsa solution (Merck Biosciences, Mendota Heights, MN, USA) for 1 h. The invaded cells were counted under a light microscope (×400, ten random fields from each well). All experiments were performed in triplicate.
Western blot analysis
Proteins (100 μg) were boiled for 5 min in SDS sample buffer (62.5 mM Tris [pH 6.7], 1.25% SDS, 12.5% glycerol, and 2.5% β-mercaptoethanol) and separated on 10% SDS-PAGE gels. After transferring to PVDF membranes, the proteins were incubated with antibodies against Gαh (GeneTex, Hsin-Chu, Taiwan), PLC-δ1 (GeneTex), or GAPDH (AbFrontier, San Diego, CA, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence system (GE Healthcare, Pittsburgh, PA, USA).
Membrane proteins (100 μg) were diluted with 500 μl of 5-fold diluted buffer B (prepared as instructed by the manufacturer of the membrane extraction kit) containing 1 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), and 4% protease inhibitor cocktail (Merck Biosciences). The membrane proteins were then absorbed into GTP-agarose (Sigma-Aldrich) overnight at 4 °C. The GTP-agarose-absorbed complexes/proteins were subsequently analyzed by SDS-PAGE/Western blotting using antibodies against PLC-δ1 or Gαh.
In situ tTG activity assay
A 50 μl aliquot of cell homogenate proteins (10 μg) in coating buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, and 5 mM EGTA) was added to each well of a 96-well ELISA plate (Apogent, Portsmouth, NH, USA) and was incubated at 4 °C overnight. To block the coated wells, a 200 μl aliquot of 5% BSA, 0.01% Tween 20, 0.01% SDS in borate-buffered saline (BBS) was added. After an additional 2 h of incubation at 37 °C, the plate was rinsed once with 1% BSA and 0.01% Tween-20 in BBS. To each well, 100 μl of HRP-conjugated streptavidin (Southern Biotech, Birmingham, AL, USA) (1:1000) in 1% BSA and 0.01% Tween-20 in BBS was added, followed by incubation for another 1 h at room temperature (RT). After the wells were rinsed four times with 1% BSA and 0.01% Tween-20 in BBS, the specimen in each well was incubated with 200 μl of TMB substrate (Sigma-Aldrich) for 10–20 min at RT. The reaction was stopped with 50 μl of 3 N HCl, and the absorbance at 450 nm was measured using a microplate spectrophotometer.
TIPWNSLKQGYRHVHLL, a peptide corresponding to the PLC-δ1 amino acid sequence from 720 to 736, was synthesized with or without myristoylation at the N-terminus and purified to approximately 95% (Mission Biotech, Taipei, Taiwan). Ten millimolar synthetic peptide was dissolved in 1 part of 0.1% trifluoroacetic acid in dd-H2O mixed with 1 part 0.1% trifluoroacetic acid in acetonitrile, aliquoted, and kept at −70 °C as a stock solution.
The genes encoding Gαh and PLC-δ1 were amplified from human cDNA (Invitrogen) using standard polymerase chain reaction (PCR) with paired primers (Additional file 1: Table S1) and sub-cloned into the pLAS3w/Ppuro, pLAS3w/Pbsd, or pIRES2-EGFP vector. The Gαh-containing pIRES2-EGFP plasmid was used as a DNA template for site-directed and deletion mutagenesis. PCR for site-directed mutagenesis was performed with paired primers (Additional file 1: Table S1) using a pfu polymerase kit (Stratagene, La Jolla, CA, USA) (for R580A) or primer extension method  (for W241A). The PCR products were treated with Dpn1 endonuclease (New England BioLabs, Hitchin, Hertfordshire, UK) to digest the methylated parental DNA template. For the construction of a C-terminal deletion mutant of Gαh (∆657-687), a Gαh-containing pIRES2-EGFP plasmid was used as the template to amplify the target sequence using paired primers (Additional file 1: Table S1). The two PCR products were subsequently digested with NheI and EcoRI and then ligated into pLAS3w/Pbsd. The identities of individual clones were verified via double-strand plasmid sequencing.
Preparation and infection of lentiviral particles
All lentiviral vectors, including pLAS3w/Ppuro and pLAS3w/Pbsd and derivatives of shRNA vector (obtained from the National RNAi Core Facility Platform in Taiwan), were transfected into the packaging cell line 293T along with pMD.G and pCMVΔR8.91 plasmids using a calcium phosphate transfection kit (Invitrogen). After incubation for 48 h, the viral supernatants were collected and transferred to target cells, and the infected cells were cultured in the presence of puromycin and blasticidin (Calbiochem, San Diego, CA, USA) at 5–10 μg/ml in order to select the stably transfected cells.
NOD/SCID mice were obtained from the National Laboratory Animal Center in Taiwan and maintained in compliance with the institutional policy. All animal procedures were approved by the Institutional Animal Care and Use Committee at Taipei Medical University.
For the in vivo lung metastatic colonization assay, cells were implanted into mice through tail vein injection at a concentration of 1 × 106 cells/100 μl of PBS. The mice were sacrificed, and the lungs were dissected for histological analyses at the endpoint. The metastatic lung nodules were quantified after H&E staining using a dissecting microscope.
For orthotopic lung metastasis of breast cancer, GFP/luciferase-expressing MDA-MB-231 cells (5 × 105), which were established by virally transducing cells with an EF1 promoter-driven firefly luciferase vector and an IRES-driven EGFP vector, were suspended in 50 μL of PBS and then were subcutaneously inoculated into the abdominal fat pad of each mouse. In vivo tumor images were captured with an IVIS imaging system (Caliper Life Sciences, Alameda, CA, USA) to measure the signal intensity from the GFP/luciferase vector. The mice were humanely sacrificed at the end of the experiments, and lungs were obtained for histological analyses. The tumors at the primary sites were removed for weight measurement.
Clinical samples and ethics statement
The breast cancer tissues used in this study were from Wan Fang Hospital managed by Taipei Medical University (TMU). Patient information, including gender, age, and histopathological diagnoses, was collected. The surgical specimens had been fixed in formalin and embedded in paraffin before being archived. We used the archived specimens for IHC staining. Follow-up of patients was carried out for up to 60 months. A four-point staining intensity scoring system was devised to determine the relative expression of Gαh and PLC-δ1 in the cancer specimens; the staining intensity score ranged from 0 (no expression) to 3 (maximal expression). All of the IHC staining results were reviewed and scored independently by two pathologists. The study was carried out with the approval of the Institutional Review Boards and with permission from the ethics committees of the institutions involved (TMU-IRB 99049).
Immunohistochemical staining analysis
Paraffin-embedded tumor sections (3 μm thick) were heated and deparaffinized using xylene and then were rehydrated in a graded series of ethanol with a final wash in tap water. Antigen retrieval was performed with a target retrieval solution (DAKO, Woodbridge, VA, USA) in a decloaking chamber (Biocare Medical, Concord, CA, USA). Endogenous peroxidase activity was quenched by hydrogen peroxide. The sections were then incubated with anti-Gαh and anti-PLC-δ1 antibodies (GeneTex) at 4 °C overnight. The Vectastain ABC peroxidase system (Vector Laboratories, Burlingame, CA, USA) was used to detect the reaction products.
SPSS 17.0 software (Informer Technologies, Roseau, Dominica) was used to analyze statistical significance. Paired t test was utilized to compare Gαh gene expression in the cancer tissues and corresponding normal tissues. Pearson’s test was performed to estimate the association between mRNA and protein expression of Gαh and PLC-δ1 by RNA-sequencing and IHC, respectively. Evaluation of survival probabilities were determined by Kaplan-Meier analysis and log-rank test. One-way ANOVA with Tukey’s test was used to estimate the difference in lung colonies and tumor weights in the animal experiments. The Mann-Whitney U test was used to analyze the non-parametric data. p values < 0.05 in all analyses were considered statistically significant.
Gαh upregulation predicts a poor probability of distant metastasis-free survival in breast cancer patients
Next, we examined the transcriptional profiles of Gαh in clinical tissues from breast cancer patients using The Cancer Genome Atlas (TCGA) database . The data showed that the transcriptional activity of Gαh in TNBC and metastatic breast cancer was higher than that of the non-tumor and non-TNBC tissues (Fig. 1c, d). Moreover, the transcriptional activity of Gαh appeared to be enhanced in cancer tissues compared to the adjacent normal tissues derived from breast cancer patients (Fig. 1e).
To validate the clinical relevance of Gαh expression in the metastatic evolution of TNBCs, we evaluated the prognostic significance of Gαh-OE in the lung metastasis of an estrogen receptor (ER)-negative breast cancer cohort (GSE5327) from the SurvExpress database. The data showed that Gαh-OE indicated a poor probability of lung metastasis-free survival (Fig. 1f) and was notable in breast cancer tissues derived from patients with a higher risk of lung metastasis (Fig. 1g).
Gαh overexpression promotes the metastatic potential of TNBC cells
Conversely, we performed Gαh knockdown (KD) in MDA-MB-231 cells (Fig. 2i) to see if the transcriptional inhibition of Gαh could suppress the metastatic potential of TNBCs. Our data showed that silencing Gαh expression by two independent shRNA clones dramatically suppresses the invasive ability of MDA-MB-231 cells (Fig. 2j). Similarly, Gαh-KD appeared to effectively mitigate the metastatic colonization of MDA-MB-231 cells in the lungs of tumor-bearing mice (Fig. 2k–m). These findings demonstrate that Gαh-OE is likely correlated with metastatic progression of TNBCs.
Intracellular GTP-binding activity, but not extracellular transglutaminase activity, of Gαh dictates the metastatic evolution of TNBCs
To validate that the GTP-binding activity of Gαh is critical for mediating invasive activity of TNBCs, we next reconstituted the expression of wild-type and W241A/R580A mutant Gαh in Gαh-silenced MDA-MB-231 cells (Fig. 3e). Our results revealed that restoration of the expression of the wild-type and W241A mutant, but not the R580A mutant, Gαh rescues the GTP-binding activity and invasive ability of the Gαh-silenced MDA-MB-231 cells (Fig. 3e–h). Since restoring the expression of the transamidating activity-deficient W241A mutant was still incapable of recovering the invasive ability of Gαh-KD MDA-MB-231 cells, we concluded that the G protein function of Gαh dictates the metastatic evolution of Gαh-overexpressing TNBC cells.
The upregulation of cytosolic, not extracellular, Gαh predicts poor prognosis and increased metastatic risk in breast cancer
The protein-protein interaction between Gαh and PLCδ1 determines cellular invasion in TNBC cells and serves as a therapeutic target for combating TNBC metastasis
Moreover, IHC results from serial sections demonstrated that the levels of cytosolic Gαh and PLCδ1 are positively correlated (Fig. 5g). Similar to the high level of expression of cytosolic Gαh, increased protein levels of PLCδ1 was identified as poor prognostic factor in patients with breast cancer (Additional file 1: Figure S1A and Table S2) . Moreover, the signature of the combination of high cytosolic Gαh and high PLCδ1 appeared to significantly predict a worse outcome in breast cancer patients (Fig. 5h). In addition, the Cox multivariate analysis of disease-free survival indicated that increased PLCδ1 level and the signature of high cytosolic Gαh/PLCδ1 were independent predictive factors of poor outcomes in breast cancer patients (Additional file 1: Figure S1B and Table S4). Notably, the high level of PLCδ1 and the high cytosolic Gαh/PLCδ1 signature correlated with higher pathologic and N stage in ER-negative breast cancer patients (Fig. 5i). These results highlight the therapeutic value of targeting the PPI of the Gαh/PLCδ1 complex in combating metastasis of TNBC.
The oncogenic role of extracellular and cytosolic Gαh in promoting cancer progression is still controversial. The increased levels of extracellular Gαh have been shown to promote breast cancer metastasis by recruiting integrin-related signaling cascades [10, 23]. Similar findings were found in ovarian cancer where extracellular Gαh promotes metastasis via activating the NF-κB signaling axis . Conversely, here, we show that the increased levels of extracellular Gαh refers to a favorable prognosis in breast cancer patients. Similarly, an increased level of extracellular Gαh was previously shown to inhibit tumor invasion in TNBCs . Moreover, our data showed that the loss of the transamidating, but not G protein, function of Gαh is capable of rescuing the reduced invasive ability in Gαh-silenced MDA-MB-231 cells. Similarly, it has been demonstrated that the catalytically inactive (C277S) mutant of Gαh activates NF-κB and induces HIF-1α expression as effectively as wild-type Gαh . Since secreted Gαh has been thought to be responsible for catalyzing the crosslinking of ECM proteins via its transamidating activity , our findings suggest that extracellular Gαh likely functions as a tumor suppressor in TNBCs.
Here, we show that the pronounced accumulation of Gαh in the cytoplasm predicts worse prognosis and causally associates with the expression of PLC-δ1 in breast cancer. The loss of both the G protein function of Gαh and its PLC-δ1 interacting domain reduces the Gαh-enhanced metastatic potential of TNBC cells. Previous studies have shown that activation of the Gαh/PLC-δ1 signaling axis by several G protein-coupled receptors (e.g., α1B-adrenergic , oxytocin , and FSH  receptors) induces fluctuations in the levels of intracellular Ca2+ and promotes the metastatic evolution of TNBC cells by modulating their epithelial-to-mesenchymal transition (EMT), which is known as the initial step of cancer metastasis [28, 29]. Notably, the activation of FSH receptor has been shown to induce EMT by triggering the PI3K/Akt-Snail signaling pathway in ovarian cancer cells  and to enhance cellular migration and invasion by modulating actin cytoskeleton activity in breast cancer cells . Accordingly, the activation of oxytocin receptor has been found to elevate the invasive properties of endometrial cancer cells via the PI3K/Akt axis  and to promote the migratory ability of prostate cancer cells by coupling to the Gi-dependent pathway . These findings implicate the possible role of the Gαh/PLC-δ1 signaling axis in these events since the activation of the Gαh/PLC-δ1 pathway has been identified to elicit intracellular Ca2+ elevation, promote IP3 turnover, and modulate myosin and actin dynamics .
Several studies have demonstrated that Gαh-OE correlates with metastatic progression [35–39] as well as drug resistance [40, 41] in different types of cancer, suggesting that the discovery of inhibitors of PPI in the Gαh/PLC-δ1 complex could be a novel and useful strategy to combat malignant tumors. Although therapeutic targeting of the PPI of Gαh/PLC-δ1 is likely to be effective in treating metastatic TNBCs, further experiments are still needed to identify the Gαh-coupled receptor(s) and related signaling cascades in TNBC cells.
Our data delineated the inverse prognostic values of extracellular and cytoplasmic Gαh and validated that the G-protein activity of Gαh is crucial for promoting TNBC metastasis by recruiting the PLC-δ1-associated signaling axis. Blocking the PPI between Gαh and PLC-δ1 displayed an inhibitory effect on the metastatic capacity of TNBC cells in vitro and in vivo, providing a new avenue for drug discovery in cancer.
We like to thank Tzu-Ling Ting for the technical assistance.
This study was supported by Ministry of Science and Technology (MOST 104-2320-B-038-061-MY3) and by Taipei Medical University (TMU103-AE1-B13) to Dr. Yuan-Feng Lin.
Availability of data and materials
SPH, CLC, and YFL contributed to conceptualization and design. SPH, PYL, CJK, CLC, WJL, and YFL helped in the development of methodology. SPH, PYL, CJK, CLC, WJL, YHT, and YFL helped in the acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.). SPH, PYL, CLC, and YFL analyzed and interpretated the data (e.g., statistical analysis, biostatistics, and computational analysis). SPH, CJK, CLC, and YFL wrote, reviewed, and/or revised the manuscript. CJK and CLC performed the administrative, technical, or material support (i.e., reporting or organizing data and constructing databases). CLC and YFL supervised the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The experiments were approved by Institutional Review Board (IRB) (TMU-IRB 99049) of Taipei Medical University.
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