The Hippo signaling pathway, a conserved regulator of organ size, has emerged as an important regulatory pathway in cancer. The final transducer effectors of this pathway in mammals are the oncoproteins TAZ and YAP1, which are transcriptional coactivators of target genes involved in cell proliferation and survival. TAZ has been previously reported to play a role in tumorigenesis in breast cancer, but detailed analyses of the different breast cancer phenotypes have not been conducted thus far. We analyzed TAZ expression by immunohistochemistry in a retrospective series of 640 invasive breast carcinomas, comprising estrogen/progesterone receptor-positive (ER+/PR+), HER2-positive, and triple-negative (TN) tumors. We found a strong association of TAZ nuclear expression with the TN phenotype (60.5% TAZ-positive, P<0.001), which was strengthened when stratified into the basal-like subtype (70.8% TAZ-positive, P<0.001). Moreover, 90% of metaplastic breast carcinomas with morphological epithelial–mesenchymal transition features were TAZ-positive. We also investigated whether amplification or differential DNA methylation of the TAZ-encoding locus could account for the observed enhanced TAZ protein expression in the TN/basal phenotype. Amplification of the TAZ locus was analyzed by fluorescence in situ hybridization in 30 TN tumors, and we found gene amplification in some cases (6.45%). DNA methylation analysis was performed using the Sequenom MassArray MALDI-TOF platform, and we observed similar low methylation levels both in TN (n=25) and ER+/PR+ (n=26) tumors. These results were further confirmed using a panel of breast cancer cell lines and using the TCGA dataset. Finally, patients with strong TAZ expression showed poorer clinical outcomes with respect to both recurrence and overall survival.
The Hippo pathway acts as a major regulator of tissue growth and organ size, and it has important implications in cancer (Pan 2010, Harvey et al. 2013). The final downstream transducers of the Hippo pathway are the transcriptional coactivators TAZ and YAP, both of which contribute to epithelial–mesenchymal transition (EMT)-mediated cancer progression. A bidirectional relationship between YAP/TAZ and EMT, wherein a loss of cell polarity and cell–cell junctions induces the activation of both factors that in turn sustain the EMT program, has been proposed (Piccolo & Cordenonsi 2013). The activity of TAZ/YAP is mainly inhibited by phosphorylation mediated by the LATS1/2 kinases, which in turn are activated by the MST1/2 kinases (homologues of Drosophila Hippo). Upon phosphorylation, TAZ/YAP undergo ubiquitination-mediated degradation and/or are sequestered in the cytoplasm by anchoring proteins (Pan 2010, Zhao et al. 2011). Elevated TAZ or YAP expression and nuclear localization have been observed in a broad range of different human cancers, and they often correlate with poor patient prognosis (Zender et al. 2006, Chan et al. 2008, Steinhardt et al. 2008, Bhat et al. 2011, de Cristofaro et al. 2011, Xie et al. 2012, Wang et al. 2013). In breast cancer, TAZ seems to be the relevant Hippo effector, because TAZ but not YAP protein expression appeared to be modulated in breast cancer cell lines (Chan et al. 2008). Indeed, TAZ is required for sustaining self-renewal and tumor-initiation capacities in breast cancer stem cells (Cordenonsi et al. 2011), and it is a central mediator of the metastatic ability of those stem cells (Bartucci et al. 2014). Increased protein expression of TAZ has been found in high-grade breast carcinomas, and it has been shown to have an influence on patient prognosis (Bartucci et al. 2014). However, the analysis of TAZ protein expression across different breast cancer phenotypes has not, to our knowledge, been inspected thus far. In order to better understand the role of TAZ in the development and progression of breast cancer, we analyzed TAZ immunohistochemical expression in a large retrospective cohort of 640 tumors, comprising 441 estrogen receptor (ER)-positive (ER+) tumors, 72 HER2-positive (HER2+)/ER-negative (ER−)/progesterone receptor (PR)-negative (PR−) tumors, and 127 triple-negative (TN) tumors. In addition, we evaluated the role of gene amplification and promoter DNA methylation in the regulation of TAZ expression in breast cancer.
A series of 640 formalin-fixed paraffin-embedded (FFPE) human invasive breast carcinomas, including some cases from a previous study (Castilla et al. 2014) and an independent tumor set of 15 metaplastic breast carcinomas (MBC), were obtained from the archives of the Department of Pathology of the Hospital Universitario Virgen del Rocío, Sevilla, Spain. The tumors had been diagnosed between 2007 and 2011, and the only selection criteria were the availability of pathological data and tissue for tissue microarray (TMA) construction. Medical records were retrospectively reviewed for clinicopathologic information (summarized in Table 1). Information regarding stage, grade, tumor size, and nodal status was abstracted. For the mRNA expression and DNA methylation studies, we used frozen sections of a cohort subset of 51 breast tumor specimens, in which the tumor component represented more than 70% of the cells. Both the FFPE and frozen samples were provided by the Biobanco del Sistema Sanitario Público de Andalucía.
Clinical and pathological findings according to TAZ expression (n=640)
|Characteristics||Analyzable||TAZ expression (IHC)||P|
|Negative (n=496)||Positive (n=145)|
|Ductal carcinoma||573||444 (77.5%)||129 (22.5%)|
|Lobular carcinoma||33||27 (81.8%)||6 (18.2%)|
|Others||28||19 (67.9%)||9 (32.1%)|
|pT (tumor size)||633||0.445|
|1||298||238 (79.9%)||60 (20.1%)|
|2||281||210 (74.7%)||71 (25.3%)|
|3||36||26 (72.2%)||10 (27.8%)|
|4||18||14 (77.8%)||4 (22.2%)|
|N0||327||249 (76.1%)||78 (23.9%)|
|N1/N2/N3||300||236 (78.7%)||64 (21.3%)|
|I||218||172 (78.9%)||46 (21.1%)|
|II||276||205 (74.3%)||71 (25.7%)|
|III||141||114 (80.9%)||27 (19.1%)|
|IV||4||3 (75.0%)||1 (25.0%)|
|Histological grade (differentiation)||625||<0.001|
|1 and 2 (well/moderate)||297||258 (86.9%)||39 (13.1%)|
|3 (poor)||328||225 (68.6%)||103 (31.4%)|
|<14||470||400 (85.1%)||70 (14.9%)|
|≥14||154||83 (53.9%)||71 (46.1%)|
|Negative||203||111 (54.7%)||92 (45.3%)|
|Positive||437||384 (87.9%)||53 (12.1%)|
|Negative||243||146 (60.1%)||97 (39.9%)|
|Positive||392||344 (87.8%)||48 (12.2%)|
|Negative||524||402 (76.7%)||122 (23.3%)|
|Positive||116||93 (80.2%)||23 (19.8%)|
|ER+/PR+||441||388 (88.0%)||53 (12.0%)|
|HER2||72||58 (80.6%)||14 (19.4%)|
|TN||127||49 (38.6%)||78 (61.4%)|
|Luminal A||299||261 (87.3%)||38 (12.7%)|
|Luminal B||98||92 (93.9%)||6 (6.1%)|
|ER+/PR+ HER2+||44||35 (79.5%)||9 (20.5%)|
|HER2||72||58 (80.6%)||14 (19.4%)|
|TN-NOS||62||30 (48.4%)||32 (51.6%)|
|Basal||65||19 (29.2%)||46 (70.8%)|
ER, estrogen receptor; PR, progesterone receptor; TN, triple-negative; TN-NOS, triple-negative not otherwise specified.
Survival analysis was performed on a group of 483 invasive breast carcinoma patients for whom clinical data were available. In that group, 322 (66.6%) patients had ER+/PR+ carcinomas, 48 (9.9%) had HER2+ carcinomas, and 113 (23.4%) had TN carcinomas. After surgery, the patients were treated with chemotherapy, adjuvant radiotherapy, hormonal therapy, and/or trastuzumab according to their respective disease characteristics. Briefly, 295 of the 322 (91.6%) patients with ER+ carcinomas were treated with hormone therapy (tamoxifen or aromatase inhibitor). A total of 289 patients were treated with chemotherapy: 207 patients (71.6%) with anthracyclines plus taxanes, 75 (25.6%) with anthracyclines only, and seven (2.4%) with taxanes. An additional patient received chemotherapy that did not involve anthracycline or taxane administration. Of the patients with HER2+ tumors, 39 of 48 (81%) were treated with trastuzumab.
The present study was performed in accordance with the standard ethical procedures dictated by Spanish law (Ley de Investigación Orgánica Biomédica, 14 July 2007), and it was approved by the ethics committee of the Hospital Virgen del Rocío de Sevilla and the Fundación Pública Andaluza para la Gestión de la Investigación en Salud de Sevilla (FISEVI), Spain. Written informed consent was obtained from all of the patients, and all of the clinical analyses were conducted in accordance with the principles of the Helsinki Declaration.
TMA construction and immunohistochemistry
Representative areas of the breast tumors were carefully selected on H&E-stained sections, and two 1 mm diameter tissue cores were obtained from each specimen. The cores were precisely arrayed into new paraffin blocks using a TMA workstation (Beecher Instruments, Sun Prairie, WI, USA). immunohistochemistry (IHC) was carried out on TMA sections using the Envision method (Dako, Glostrup, Denmark) with primary antibodies against TAZ, ER, PR, HER2, KI67, CK5/6, CK17, and EGFR. Myoepithelial staining of p63 was used to test the presence of in situ component in a subset of 11 TN tumors. For molecular classification, breast carcinomas were grouped according to immunohistochemical criteria as a surrogate definition of the intrinsic subtypes of breast cancer (Goldhirsch et al. 2011): tumors showing ER or PR expression and no HER2 expression were regarded as luminal A or B depending on their KI67 index (<14 or ≥14 respectively); tumors with ER or PR and HER2 expression were classified as ER+/PR+/HER2+; and TN tumors with CK5/6, CK17, or EGFR expression were ascribed to the basal phenotype, whereas all other TN breast carcinomas were termed TN-not otherwise specified (NOS).
Details of the clones, suppliers, dilutions, and scoring criteria used are provided in Supplementary Table 1, see section on supplementary data given at the end of this article. IHC staining was separately evaluated by two pathologists (J P and M A L). For TAZ, IHC evaluation was carried out for nuclear staining, thus focusing on the transcriptional activity of the factor. Tissue was given a score of 0 (negative) to 3 based on the percentage of positive cells (0: <5%; 1: ≥5% and <30%; 2: ≥30% and <70%; 3: ≥70%). Images of the immunohistochemical markers used in Fig. 1 were captured using an Olympus BX61 microscope and DP72 camera, with CellSens CELL-A Software (Olympus, Hamburg, Germany).
Fluorescence in situ hybridization
We carried out fluorescence in situ hybridization (FISH) analysis on sequential 3 μm sections of TMAs containing 30 TN tumors. To detect TAZ amplification, we used the bacterial artificial chromosome RP11-126i10, chr3: 150 717 532 – 150 892 549 (Children's Hospital Oakland Research Institute, Oakland, CA, USA). A chromosome enumeration probe (CEP) 3 (Vysis, Abbott Laboratories) was included in dual hybridization with the TAZ probe.
Slides were deparaffinized, boiled in a pressure cooker with ethylene diaminetetraacetic acid (1 mM, pH 8.0) for 10 min, and incubated with pepsin at 37 °C for 10 min. The slides were then dehydrated. The probes were denatured at 75 °C for 2 min and simultaneously applied to the sample. After overnight hybridization at 37 °C in a humid chamber, the slides were washed with 0.4×SSC and 0.3% NP40 and counterstained with 0.2 μM 4′, 6′-diamino-2′-phenyl indole. Cell images were captured with a CCD camera (Photometrics SenSys, Tucson, AZ, USA) connected to a PC running Chromofluor image analysis software (Applied Imaging Ltd, Newcastle, UK). Samples showing sufficient FISH efficiency (>90% of nuclei with signals) were evaluated. Signals were scored for at least 50 non-overlapping, intact nuclei. Non-neoplastic cells present in the specimen were used as a control. Specimens were considered to be amplified when the gene–CEP ratio was >2.5 in at least 20% of the tumor cells.
Real-time quantitative RT-PCR
Total RNA was extracted from selected areas of frozen tissue containing >70% tumor cells using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA) after the tissue had been homogenized for 2 min in lysis buffer using an Ultra-turrax (T10 Basic, IKA, Satufen, Germany) according to the manufacturer's protocol. The real-time quantitative RT-PCR (qRT-PCR) for WWTR1 and YAP1 was performed with gene-specific fluorescent Taqman probes (Taqman Gene Expression Assays, Applied Biosystems), using MRPL19 as the endogenous control to normalize for variations in the quantity of the input cDNA. Details of qRT-PCR have been previously described (Castilla et al. 2012, 2014).
DNA methylation analysis by MassARRAY
DNA methylation analysis of TAZ and YAP1 loci by MassARRAY (Sequenom, San Diego, CA, USA) was performed as previously described (Castilla et al. 2012). Three amplicons were designed for each promoter region, covering 1008 pb (TAZ) and 1465 pb (YAP1) respectively. Primer sequences, chromosomal locations, and number of CpG units analyzed are shown in Supplementary Table 2, see section on supplementary data given at the end of this article. The DNA methylation level of the promoter sequences was calculated by averaging the methylation levels of the individual CpG units.
Cell lines and western blot
Six breast cancer cell lines with different phenotypes – MCF7, T47-D, MDA-MB-231, BT-549, BT-474, and SK-BR-3 – were obtained from the American Tissue Culture Collection and cultured in the recommended media. Whole cell extracts and western blotting procedure were performed as described previously (Moreno-Bueno et al. 2009). Signals were visualized using Immun-Star Western C kit (Bio-Rad). For TAZ detection, we used rabbit polyclonal anti-TAZ (Sigma, HPA007415, 1:250 diluted), and as loading control, we used GAPDH (Cell Signaling Technology (Danvers, MA, USA), (D16H11) XP Rabbit mAb, 1:2500 diluted).
The Cancer Genome Atlas dataset
mRNA expression z-scores (microarray data), methylation β-values, and putative copy-number alterations (GISTIC) for The Cancer Genome Atlas (TCGA 2012) dataset were obtained from cBioPortal for Cancer Genomics (http://www.cbioportal.org/). Tumors (n=507) were grouped into subtypes according to the PAM50 gene expression signature (Parker et al. 2009) (luminal A, n=234; luminal B, n=134; HER2, n=58; basal, n=81).
The correlation between inmunohistochemical TAZ expression and clinicopathological characteristics was assessed by χ2 tests for the categorical variables (summarized with percentages). Mann–Whitney U tests were used for the analysis of differences with respect to the continuous variable age (summarized with means and s.d.). Breast-cancer-specific survival was defined as the time from surgery to the time of death from breast cancer, with deaths from other causes being censored, whereas in the time to relapse analysis, the endpoint was breast cancer recurrence, either local or distant. Survival curves were estimated using the Kaplan–Meier method, and the differences in survival were evaluated using the log-rank test. Cox's proportional hazards modeling of parameters potentially related to survival was conducted to calculate hazard ratios (HRs) in both univariate and multivariate analyses. All of these statistical analyses were performed using SPSS version 20 (SPSS Inc.) and JMP 10 statistical software (SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered statistically significant.
The statistical significance of relative changes in mRNA expression between different groups of breast tumors was determined by the Wilcoxon rank-sum test, and P values were corrected with the Benjamini–Hochberg algorithm (P<0.05). A moderate t-test was used for analyzing statistical differences in the cell line expression data. These analyses were conducted using the Integromics Real Time StatMiner 4.5 package (Integromics S.L., Granada, Spain). Differences in DNA methylation levels among the distinct breast cancer phenotypes or cell lines were assessed by ANOVA.
TAZ IHC expression is associated with the TN phenotype
We analyzed TAZ expression by IHC in a retrospective series with a total of 640 invasive human breast carcinomas (Table 1). TAZ-expressing tumor cells exhibited intense nuclear staining with a variable fainter signal in the cytoplasm (Fig. 1). TAZ expression was also observed in myofibroblastic cells in TAZ-negative tumor samples, which provided an internal positive control for the IHC determination (Fig. 1B). Table 1 shows the relationship between TAZ IHC expression and clinicopathological factors. TAZ-positive expression was associated with poorly differentiated tumors, high Ki67 index, and ER−/PR− phenotype, whereas no significant association was observed with age at surgery, histological type, tumor size (pT), nodal status, or pTNM stage. Grouping tumor specimens with reference to the receptor phenotype unveiled a clear association of TAZ IHC expression with the TN phenotype (Table 1). Grade distribution among TN and HER2 tumors is biased towards poor differentiation (Supplementary Table 3, see section on supplementary data given at the end of this article), but whereas 61.4% of TN tumors are TAZ-positive, only 19.4% of HER2 tumors showed TAZ expression (Table 1). Moreover, when only grade 3 tumors were compared, the strong association of TAZ IHC expression with the TN phenotype remained, with ER+/PR+ tumors exhibiting a low frequency of TAZ positivity (11.2%, Supplementary Table 4). Therefore, the relationship between TAZ expression and TN phenotype would not be ascribable to an underlying association with the differentiation grade. Interestingly, further stratification of tumor phenotype into a molecular classification revealed a striking association of TAZ expression to the basal phenotype (70.8% TAZ-positive, Table 1). Given the established link between TAZ/YAP and EMT (Piccolo & Cordenonsi 2013), we assessed TAZ expression in an independent tumor set of 15 MBC. Interestingly, 11 out of 12 MBC with histological evidence of EMT toward spindle cell or heterologous sarcomatous differentiation showed TAZ expression; in contrast, two out of three MBC with squamous differentiation were TAZ-negative (Table 2). However, the small sample size precludes detection of a statistically significant difference (P=0.08, Fisher's exact test).
TAZ nuclear expression in metaplastic breast carcinomas (n=15)
|Metaplastic component||n||TAZ expression (IHC)|
|Chondrosarcoma||4||1 (25%)||3 (75%)|
|Osteosarcoma||1||0 (0%)||1 (100%)|
|Spindle cell||7||0 (0%)||7 (100%)|
|Squamous||3||2 (66%)||1 (33%)|
|Total||15||3 (20%)||12 (80%)|
To investigate whether TAZ expression was restricted to the invasive tumor component, we also examined, by IHC, the expression of TAZ in the in situ component of selected TN tumors that had displayed either TAZ-positive (seven specimens) or TAZ-negative (four specimens) expression in the infiltrating component (Fig. 1C and D). We observed a full concordance of TAZ expression between the two components in all of the samples analyzed, with TAZ IHC signal detected in both the in situ and the infiltrating components of TAZ-positive specimens (Fig. 1C and D).
TAZ-encoding locus amplification in TN breast cancer
We next investigated whether amplification of WWTR1 (the TAZ-encoding locus) could account for TAZ upregulation in TN/basal tumors. We performed FISH analyses of a subset of 30 TN tumors with strong TAZ IHC expression in which only two tumors (6.5%) exhibited amplification of the WWTR1 locus (Fig. 2). We also interrogated the TCGA dataset for copy number variation in WWTR1 (Supplementary Figure 1, see section on supplementary data given at the end of this article). These tumors are classified according to the PAM50 gene expression signature, and therefore, the different groups do not fully overlap those defined by IHC in the present series. Basal tumors in the TCGA dataset exhibited high-copy gains for the WWTR1 locus with a higher proportion (7.4%) than luminal or HER2 tumors (0.5–0.8%). However, the available data on copy number estimates for WWTR1 in breast cancer cell lines (Reinhold et al. 2012) revealed no evident amplification of the locus either in basal or luminal cell lines (data not shown).
TAZ mRNA expression is not modulated by DNA methylation in breast cancer
We also investigated whether the DNA methylation status of WWTR1, and the closely related transcription coactivator YAP1, is an operating epigenetic regulatory mechanism in breast cancer. First, we quantified mRNA expression levels of TAZ and YAP1 in a series of 51 frozen samples from tumors included in our original cohort. This subset comprised 26 ER+/PR+ tumors and 25 TN tumors. We detected a significant twofold increase of TAZ mRNA in TN tumors (P=0.0134), and a slight but not significant increase was also observed for YAP1 (P=0.0774) (Fig. 3A). Next, we inspected broad enriched CpG regions for DNA methylation of the TAZ and YAP1 loci. Our analysis covered 65 CpG dinucleotides along 1008 bp of the TAZ promoter region and 113 CpG dinucleotides along 1465 bp of the YAP1 promoter region. Minimal differences in the average percentage of CpG methylation levels were detected (TAZ, P=0.0299; YAP1, P=0.0496), with 11 and 13% in the ER+/PR+ and TN groups respectively (Fig. 3B).
We also analyzed TAZ and YAP1 mRNA expression and DNA methylation levels in the breast cancer dataset from the TCGA (2012). In accordance with the observations in our series, we detected upregulation of both TAZ and YAP1 mRNA in basal tumors, with TAZ showing a higher increase (Supplementary Figure 2). Moreover, only minimal differences were observed in the DNA methylation levels of the two loci between different tumor subgroups (Supplementary Figure 2). In fact, no correlation between expression and DNA methylation data was observed in the TCGA dataset or in our tumor series (data not shown).
To further confirm these observations, we analyzed a panel of breast cancer cell lines representing the luminal (MCF7, T47D), HER2 (SK-BR-3, BT-474), and basal phenotypes (MDA-MB-231, BT-549). As previously reported by Chan et al. (2008), we observed higher expression levels of TAZ protein in basal cell lines (Fig. 4A). Moreover, expression levels of TAZ mRNA as well as YAP1 mRNA were higher in basal cell lines (e.g., BT-549 versus T-47D, P<0.0001 and P=0.002625 for TAZ and YAP1 mRNA expression respectively), although YAP1 mRNA modulation across the cell line panel was not as evident as TAZ mRNA variation was (Fig. 4B). Finally, similar DNA methylation levels for both TAZ (below 5%, F=0.432, P=0.826) and YAP1 (below 7%, F=0.508, P=0.770) were observed for the cell lines of different phenotypes (Fig. 4C).
TAZ IHC expression affects survival
Given the association observed between TAZ and the TN/basal phenotype, and because TN/basal phenotypes have a poorer prognosis as compared with luminal phenotypes, we tested the prognostic significance of TAZ expression in our cohort. We retrieved follow-up data for 483 out of 640 patients (median duration of follow-up, 53.7 months). Kaplan–Meier estimates of breast-cancer-specific survival were significantly shorter for the TAZ-positive group as compared with the TAZ-negative group (mean, 69.6 versus 79.6 months, P<0.001, Fig. 5A). Because a similar trend was observed for TAZ IHC scores 0 and 1 versus scores 2 and 3 at the initial follow-up period (Supplementary Figure 3, see section on supplementary data given at the end of this article), we grouped patients into those with strong TAZ expression (scores 2 and 3) and TAZ-negative or weak TAZ expression (scores 0 and 1). This stratification resulted in greater differences in survival estimates between the two patient groups (mean, 66.6 versus 79.2 months, P<0.0001, Fig. 5B). Similarly, TAZ expression also influenced significantly the time to relapse, which was shorter in TAZ-positive patients than it was in TAZ-negative patients (mean, 65.6 versus 74.2 months, P=0.039, Fig. 5C). Again, greater differences were observed when comparing the strong TAZ expression group with the TAZ-negative or weak TAZ expression group (mean, 62.1 versus 74.1 months, P=0.004, Fig. 5D).
Accordingly, using Cox regression univariate analyses (Table 3) it was determined that strong TAZ expression was significantly correlated with breast cancer–specific survival and the time to relapse, with the unadjusted HRs being 3.739 (P<0.001) and 2.244 (P=0.004) respectively. A significant correlation with survival and time to relapse was also observed for the classical prognostic factors analyzed (Table 3). These significant variables were all included simultaneously in order to assess the independent prognostic significance based on multivariate analysis (Table 3). The adjusted HR of strong TAZ expression for breast cancer–specific survival was 2.191 (P=0.044) after controlling the Cox regression model for the effects of stage, grade, ER, PR, tumor size, nodal status, and Ki index (Table 3). This indicates that TAZ expression is an independent and significant predictor of poorer survival. We could not definitely conclude the same for time to relapse, even though the level of significance nearly reached 0.05 (Table 3).
Prognostic value of TAZ IHC expression in relation to traditional prognostic variables in breast cancer (n=483). Adjusted HR refers to the multivariate model after controlling for the significant factors in the univariate analysis
|Factors||Breast-cancer-specific survival||Time to relapse|
|Unadjusted HR (95% CI)||P||Adjusted HR (95% CI)||P||Unadjusted HR (95% CI)||P||Adjusted HR (95% CI)||P|
|TAZ (strong versus negative/weak)||3.739 (1.898–7.366)||<0.001||2.191 (1.021–4.707)||0.044||2.244 (1.291–3.900)||0.004||1.755 (0.931–3.306)||0.082|
|Age||0.994 (0.971–1.018)||0.633||0.985 (0.967–1.002)||0.09|
|ER (negative versus positive)||3.986 (2.116–7.506)||<0.001||3.087 (0.483–19.744)||0.234||2.335 (1.489–3.660)||<0.001||1.189 (0.436–3.243)||0.735|
|PR (negative versus positive)||3.444 (1.816–6.530)||<0.001||0.364 (0.053–2.514)||0.305||2.238 (1.428–3.508)||<0.001||0.939 (0.336–2.262)||0.905|
|HER2 (positive versus negative)||1.207 (0.552–2.640)||0.637||1.424 (0.821–2.470)||0.209|
|Tumor size (pT1 versus pT2+pT3+pT4)||8.699 (3.098–24.430)||<0.001||4.132 (1.240–13.775)||0.021||3.963 (2.249–6.981)||<0.001||2.240 (1.159–4.327)||0.016|
|Node status (pN0 versus pN1,2,3)||5.062 (2.338–10.960)||<0.001||2.179 (0.874–5.430)||0.095||4.163 (2.452–7.067)||<0.001||1.870 (0.988–3.539)||0.055|
|Clinical stage (I+II versus III+IV)||6.605 (3.525–12.378)||<0.001||3.435 (1.643–7.184)||0.001||5.022 (3.210–7.859)||<0.001||2.740 (1.574–4.771)||<0.001|
|Grade (poorly versus well/moderately differentiated)||12.615 (3.890–40.912)||<0.001||6.266 (1.414–27.771)||0.016||4.630 (2.590–8.277)||<0.001||2.803 (1.408–5.586)||0.003|
|Ki67 (<14 versus ≥14)||6.025 (3.150–11.523)||<0.001||2.447 (1.153–5.194)||0.020||2.724 (1.717–4.321)||<0.001||1.320 (0.759–2.296)||0.326|
ER, estrogen receptor; PR, progesterone receptor.
The results of the present study clearly establish that TAZ oncoprotein expression is associated with the TN phenotype of breast cancer, and this correlation is not ascribable to the differentiation grade. Hence, the association of TAZ IHC expression with high-grade breast cancer that has been reported in previous studies (Cordenonsi et al. 2011, Bartucci et al. 2014) reflects instead the actual correlation with the receptor status. Skibinski et al. (2014) identified TAZ in a screen of transcription factors that regulate the differentiation state of mammary epithelial cells. Overexpression of TAZ was sufficient to induce the transition of luminal cells to a basal cell fate, and conversely, TAZ depletion in basal cells led to luminal differentiation (Skibinski et al. 2014).
Furthermore, TAZ IHC staining was observed both in the in situ and in the infiltrating components of TAZ-positive tumor specimens. Most in situ breast carcinomas (without an invasive component) exhibit an ER+/PR+ or an HER2+ phenotype, and therefore low TAZ positivity is observed among them. This may have led to the notion that TAZ expression is only associated with invasive ductal carcinomas (Chan et al. 2008). The stratification of TN tumors into basal and non-basal (TN-NOS) groups brought out preferential TAZ positivity in basal tumors (70.8%, P<0.001). In our series, 50% (65/129) of the TN tumors were identified as basal tumors using additional IHC markers. There is a substantial overlap between TN and basal-like definitions, and thus TN has often been assimilated as a surrogate for basal-like breast cancer (Foulkes et al. 2010, Badve et al. 2011). However, not all TN tumors would be identified as basal-like by their gene expression, and not all basal-like tumors are TN. It has been shown that 71–78% of TN cancers exhibit a basal gene expression signature (Bertucci et al. 2008, Prat et al. 2013). Hence, our TN group of tumors might include unidentified basal specimens that may exhibit TAZ expression, and thus the ratio of TAZ-positive specimens in our basal group could be even higher than was observed (Table 1). This striking association of TAZ protein expression with the basal phenotype is a relevant finding, because it may contribute to the immunohistochemical/molecular characterization of these tumors. Patients with TN/basal breast cancer have a poor outcome because of the intrinsic aggressiveness of the cancer and the lack of effective targeted therapies (Duffy et al. 2012, Bayraktar & Gluck 2013). Therefore, the identification of specific biomarkers/molecular targets is imperative. In this regard, we have recently reported that Vgll1, a transcriptional cofactor with structural similarities to TAZ and YAP1, is mainly expressed in a subset of sporadic and BRCA1-associated TN/basal breast carcinomas (Castilla et al. 2014). In a recent report by Bartucci et al. (2014), TAZ IHC expression in a cohort of 99 tumors was not significantly correlated with receptor status, although the authors did observe an association between high tumor grade and Ki67 index, as we have observed in the present study. The limited size of the series or the different antibodies used could account for this discrepancy.
The mechanisms responsible for TAZ overexpression in human tumors are poorly understood, although both gene amplification and promoter methylation have been reported in some subsets of tumors (Bhat et al. 2011, Cordenonsi et al. 2011). The present study is the first to use FISH to analyze the role of gene amplification, and our results indicated, as previously reported by Cordenonsi et al. (2011), that amplification of the TAZ-encoding locus occurred only in a small proportion of breast carcinomas. Also in agreement with the present results, an analysis of the TCGA dataset indicated that high-level amplification of the WWTR1 locus occurs in only 2% of breast carcinomas, although within the basal subgroup, the frequency increases to 7.4% (Supplementary Figure 1), a frequency similar to that observed in the present study but in contrast with the 60–70% of TAZ-positive TN/basal tumors observed by IHC. In fact, upregulation of TAZ mRNA levels were observed in 34 out of the 81 TN tumors (44%) in the TGCA dataset, which greatly exceeds the proportion of TAZ-amplified cases (Supplementary Figure 1).
To date, few somatic or germline mutations have been discovered in Hippo pathway genes (Harvey et al. 2013), which indicates that the frequent perturbation of the activity of the pathway in human cancer results from other mechanisms. In breast cancer, only three TAZ mutations in five unique samples are registered in the Catalogue of Somatic Mutations in Cancer, which represents only 0.51% of the total unique samples (Ding et al. 2010, Shah et al. 2012). Epigenetic regulation of TAZ by DNA methylation also did not explain the differential expression of TAZ among different phenotypes of breast cancer (Fig. 3, Supplementary Figure 2, see section on supplementary data given at the end of this article). This contrasts with results described in a recent report on glioblastoma, which indicated that TAZ expression correlated with the DNA methylation status of the promoter sequences (Bhat et al. 2011). This difference in TAZ deregulation might indicate that the mechanisms that modulate Hippo signaling are tissue- or cell-type-specific. It has been proposed that enrichment of YAP/TAZ target gene expression in high-grade breast cancer may be mainly attributed to TAZ protein stabilization (Cordenonsi et al. 2011). Delocalization from the cell membrane of Scribble, an upstream component of the Hippo pathway, relieves LATS-dependent TAZ inactivation and consequently induces TAZ stabilization and nuclear translocation. Recently, integrin-linked kinase (ILK) has been shown to be involved in the suppression of the Hippo pathway. Knockdown of ILK in breast, prostate, and colon cancer cells results in the activation of the core kinases MST and LAST and the cytoplasm sequestration of TAZ and YAP1 (Serrano et al. 2013). In light of these results, it seems that enhanced TAZ protein expression in TN/basal breast cancer may result from post-transcriptional/translational regulatory mechanisms, although transcriptional regulation also clearly contributes to this process (Fig. 3A, Supplementary Figure 2, see section on supplementary data given at the end of this article).
In the present study, we showed that TAZ protein expression is significantly associated with a decreased survival rate and time to relapse. Results of multivariate analysis indicated that strong TAZ IHC expression had prognostic value for breast-cancer–specific survival, independent from classical factors such as tumor size, nodal status, histological grade, and pathological stage. Accordingly, Bartucci et al. (2014) similarly reported that TAZ expression had an effect on disease-free survival. Thus, TAZ may represent a potential selective therapeutic target for TN/basal breast cancer, which has a poor outcome because of the intrinsic aggressiveness of the disease and the lack of effective targeted therapies (Toft & Cryns 2011, Duffy et al. 2012, Bayraktar & Gluck 2013). The association between TAZ overexpression and poor prognosis in breast cancer could be related to the intrinsic properties acquired by neoplastic cells when the Hippo pathway is inactivated. Indeed, a direct link between stemness, EMT, cell polarity, and Hippo signaling has been demonstrated in breast cancer. Cordenonsi et al. (2011) demonstrated that EMT induces TAZ protein stabilization, which is required to sustain self-renewal and tumor-initiation capacities in breast cancer stem cells. Furthermore, the different signaling pathways that are involved in EMT, such as the transforming growth factor-β pathway and the Wnt pathway, are highly interconnected with the Hippo pathway, and there is a substantial degree of crosstalk between them (Varelas & Wrana 2012, Hiemer et al. 2014). In addition, it has recently been shown that TAZ endows metastatic abilities to breast cancer stem cells (Bartucci et al. 2014). We previously reported that basal tumors undergo EMT-like processes (Sarrio et al. 2008) that may be related to the high aggressiveness and the characteristic metastatic spreading of these tumors. Thus, we speculate that upregulation of TAZ protein in basal tumors could be regarded as part of the EMT program that occurs in this tumor subtype. Indeed, we have shown that MBC with sarcomatous differentiation exhibited TAZ protein expression (Table 2). In this context, Lehmann et al. (2011) reported that TAZ is upregulated in the mesenchymal-like subtype and downregulated in the luminal AR subtype (as compared with the rest of the subtypes; Supplementary Table 3 in Lehmann et al. 2011). In the present paper, the mesenchymal-like subtype shared similar features with MBC. Therefore, our results for MBC (Table 2) were in accordance with this observation.
In summary, in this large series of breast carcinomas, we found a striking association of TAZ expression, determined by IHC, with the TN and basal phenotypes of breast cancer that was not ascribable to the underlying grade distribution in these tumor subtypes. Moreover, TAZ overexpression was even more frequent in those tumors with morphological EMT features, such as metaplastic carcinomas with spindle cell or hetrologous sarcomatous differentiation. TAZ-enhanced expression in TN/basal tumors was not correlated with DNA methylation, but amplification could contribute to TAZ upregulation in a small number of cases. Using Cox regression analyses, which included the classical prognostic parameters, it was determined that TAZ IHC expression could be a relevant prognostic biomarker in breast cancer.
This is linked to the online version of the paper at http://dx.doi.org/10.1530/ERC-14-0456.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work was supported by grants to J Palacios from the Instituto de Salud Carlos III (ISCIII, grant number PI080971), the Ministerio de Ciencia e Innovación (MICINN), the European Development Regional Fund (grant number RD06/0020/0013), the Junta de Andalucía (Consejería de Salud, grant numbers PI-0384/2007 and PI0581/2009); and the ISCIII-Ministerio de Economía y Competitividad (MINECO, grant numbers RD12/0036/0064 and PI13/02477). J Díaz-Martín and M A Castilla are PhD researchers funded by the ISCIII (Sara Borrell and RD06/0020/0013 contracts respectively). L Romero-Pérez is a PhD student recipient of a Predoctoral de Formación en Investigación en Salud (PFIS) fellowship (grant number F109/ 00193). M L Pecero works as a laboratory technician supported by the ISCIII (PI080971). M R Atienza-Amores is an MD funded by the ISCIII (Río Ortega program).
Author contribution statement
J Díaz-Martín was responsible for the experimental design, the performance of the experiments, and the statistical analysis. M Biscuola and A Santón performed the statistical analyses. L Romero-Pérez, M L Pecero, and M A Castilla carried out some of the experiments. J Palacios and M A López-García reviewed the pathological and immunohistochemical analyses. M R Atienza-Amores reviewed the clinical data. J Díaz-Martín and J Palacios designed the study and drafted the manuscript. All of the authors contributed to the editing of the manuscript and gave their approval of the final version.
The authors thank the donors and the Andalusian Public Health System Biobank (ISCIII-Red de Biobancos RD09/0076/00085) for the human specimens used in this study.
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