The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer

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.

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.

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.

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 [18] (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.

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.

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).

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.

To evaluate the clinical relevance of Gαh overexpression (OE), we performed a global prognostic meta-analysis for the Gαh gene (TGM2) on microarray data from clinical cohorts with different cancer types using PrognoScan [19]. The data demonstrated that the upregulation of Gαh correlated with poor prognosis as judged by an increased hazard ratio in patients with bladder, brain, breast, lung, or ovarian cancers but not in patients with blood or colorectal cancer (Fig. 1a). Although the lower expression of Gαh transcripts referred to a poor outcome in a few studies, the clinical cohorts with highly invasive breast and lung cancers that expressed an increased level of Gαh transcripts accounted for the reduction in the probabilities of distant metastasis or recurrence-free survival (Fig. 1b). Based on these findings, this study was focused on investigating the oncogenic effect of Gαh overexpression (OE) on promoting the metastatic progression of breast cancer.

Next, we examined the transcriptional profiles of Gαh in clinical tissues from breast cancer patients using The Cancer Genome Atlas (TCGA) database [20]. 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).

To investigate the association between the expression of Gαh and the metastatic potential of TNBC, we performed immunoblotting to survey the endogenous level of Gαh in a panel of TNBC cell lines, including BT-20, BT549, HCC38, HCC1806, HCC1937, and MDA-MB-231. HCC1806 cells expressed the lowest Gαh level, whereas MDA-MB-231 cells displayed a relatively high level of Gαh expression (Fig. 2a). Accordingly, invasion assay revealed that HCC1806 cells exhibit a reduced invasive ability, and the MDA-MB-231 cells display an elevated invasive property among the tested TNBC cell lines (Fig. 2b). Statistical analysis showed that the endogenous protein levels of Gαh significantly (p = 0.042) correlates with cellular invasion ability in the tested TNBC cells (Fig. 2c). To understand if Gαh-OE is capable of promoting the invasiveness of TNBC cells, we ectopically expressed Gαh in HCC1806 cells (Fig. 2d). The data showed that Gαh-OE robustly increased the invasive ability of HCC1806 cells (Fig. 2e). Moreover, Gαh-OE appeared to significantly enhance the metastatic colony formation of HCC1806 cells in the lungs after tail vein injection into mice (Fig. 2f–h).

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.

To delineate which Gαh activity is required for promoting the metastatic progression of TNBCs, we next performed site-directed mutagenesis to generate Gαh constructs with mutations at residues 241 (W to A) and 580 (R to A), which have been shown to result in defects in its transamidating and GTP-binding activities, respectively [17]. Our data showed that compared to wild-type Gαh, ectopic expression of the W241A mutant abolishes the transamidating, but not GTP-binding, activity of Gαh; conversely, the overexpression of R580A mutant inhibits the GTP-binding, but not transamidating, activity of Gαh in HCC1806 cells (Fig. 3a, b). Whereas the overexpression of the wild-type and W241A mutant Gαh appeared to enhance the invasive ability, ectopic expression of the R580A mutant failed to promote the invasive ability of HCC1806 cells (Fig. 3c, d).

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.

We next performed immunohistochemical (IHC) staining analysis to examine the protein level and distribution of Gαh in clinical specimens of breast cancers. Intriguingly, we found a pronounced accumulation of Gαh in either the extracellular matrix (ECM) or the cytosol in a subset of breast cancer tissues (Fig. 4a). Moreover, Gαh expression in the ECM and cytosol was significantly inversely correlated in the analyzed breast cancer tissues (Fig. 4a). Whereas increased levels of Gαh in the ECM predicted a favorable outcome, the accumulation of Gαh in the cytosol predicted a poor prognosis in breast cancer patients (Fig. 4b). In contrast to the signature of high extracellular and low cytosolic Gαh expression, low extracellular and high cytosolic Gαh levels appeared to be strongly correlated with a worse probability of disease-free survival in breast cancer patients (Fig. 4b). Cox regression analyses of disease-free survival demonstrated that increased levels of cytosolic Gαh, the signature of low extracellular/high cytosolic Gαh and higher T and N stages significantly correlate with an unfavorable hazard ratio, whereas the elevated levels of extracellular Gαh serves as a good prognostic factor in a univariate model (Additional file 1: Table S2). Notably, in the multivariate analysis adjusted by the prognostic power of higher T and N stages, high cytosolic Gαh and the low extracellular/high cytosolic Gαh signature remained as an independent risk factors for poor outcome among the studied breast cancer patients (Fig. 4c and Additional file 1: Table S3). Furthermore, in contrast to the high extracellular Gαh levels, our data revealed that the high cytosolic Gαh levels and the low extracellular/high cytosolic Gαh signature closely associates with lymph node metastasis in ER-negative breast cancer (Fig. 4d).

The coupling of activated Gαh to PLCδ1 signaling cascades has been identified previously [21]. To understand the importance of the protein-protein interaction (PPI) between Gαh and PLCδ1 in Gαh-promoted cellular invasion in TNBC cells, we next generated a Gαh variant (Δ657_687) with deletion of the PLCδ1-binding interface (between amino acids 657 and 677) (Fig. 5a). Notably, the ectopic expression of the Gαh/Δ657_687 mutant did not have any effect, whereas the wild-type Gαh increased the invasive ability of HCC1806 cells (Fig. 5b, c). We also synthesized a peptide fragment corresponding to the PLCδ1 amino acid sequence from 720 to 736, which has been identified previously as the binding-interface of Gαh [7], with or without myristoylation (Myr) at the N-terminus (Fig. 5d) in order to facilitate its penetration across the cell membrane. Cell invasion assay showed that the Myr-PLCδ1 peptide compared to the unmodified peptide suppressed the invasiveness of MDA-MB-231 cells in a dose-dependent manner (Fig. 5e, f). These findings suggest that the PPI between Gαh and PLCδ1 likely plays a central role in regulating the invasion of TNBC cells.

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) [22]. 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.

To validate the therapeutic utility of disrupting the PPI between Gαh and PLCδ1 to suppress TNBC metastasis, we next generated an orthotropic mouse model with luciferase-expressing MDA-MB-231 cells (Fig. 6a). Our data showed that administration of Myr-PLCδ1 compared to that of the control peptide robustly inhibited distant lung metastasis (Fig. 6a, b) accompanied with a reduced tumor growth (Fig. 6b) of MDA-MB-231 cells at the primary site in tumor-bearing mice 3 weeks post-treatment. Moreover, histological analysis revealed that the formation of tumor colonies in the lungs was clearly suppressed in the orthotropic mouse model of breast cancer after the treatment with Myr-PLCδ1 peptide compared to that with the control peptide for 3 weeks (Fig. 6c, d). Significantly, in comparison with the control groups, the administration of Myr-PLCδ1 peptide appeared to reduce tumor weight at the end of treatment (Fig. 6e). Therefore, the PPI of the Gαh/PLCδ1 complex is not only critical for relaying TNBC metastasis but also serves as a useful target for developing anti-cancer agents against metastatic TNBCs.