Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

Abstract

Introduction: Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.

Methods: In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines.

Results: The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition.

Conclusions: These data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.

Figure 1

Characterisation of basal-like carcinomas (BLCs) and human epidermal growth factor receptor overexpressing (HER2+) carcinomas from human biopsies. (a) Selection and characterisation of human samples by immunohistochemistry (IHC). Thirteen BLCs were first selected as grade III triple-negative ductal carcinomas (negative for oestrogen receptor (ER), progesterone receptor (PR) and HER2 expression) and then characterised for cytokeratin 5/6 (CK5/6), CK14 and epidermal growth factor receptor (EGFR) staining. The control series was composed of 11 hormone receptor negative- and grade III-matched HER2+ carcinomas. Tumours contained between 50% and 90% tumour cells revealed by haematoxylin-eosin-safran staining. (b) Higher HER2 protein expression in HER2+ carcinomas compared with BLCs. The box plot illustrates the expression of total HER2 protein expression measured by reverse phase protein array (RPPA) in human BLCs and HER2+ carcinomas. An outlier is present within the HER2+ population (open circle). The y axis represents logarithmic transformed HER2 relative quantification. The p value (*** p < 0.001) is represented (Mann-Whitney test). Data are representative of three separate RPPA experiments. (c) Tumours selected by immunohistochemistry clustered according to their gene expression signature. A hierarchical clustering was performed on the intrinsic/UNC genes as described [46]. (i) Overview of complete cluster diagram. Each row represents a gene and each column a human tumour. Black is for no change, red for up-regulation and green for down-regulation of gene expression. (ii) Experimental sample-associated dendrogram. Red dendrogram branch represents HER2+ carcinomas and blue designs basal-like carcinomas. (iii) HER2+ signature. A HER2+ expression cluster was observed and contained multiples genes from the 17q11 amplicon including HER2 and growth factor receptor-bound protein 7 (GRB7) (typed in red) as previously described [46]. (iv) Basal-like signature. A basal-like expression cluster was found and contained genes previously identified to be characteristic of basal epithelial cells such as v-kit, FOXC1 and P-cadherin (written in red) [46].

Figure 2

Up-regulated phosphatidylinositol 3-kinase (PI3K) signalling pathway in human basal-like breast cancers. Akt is activated in basal-like carcinomas (BLCs). The expression of (a) total Akt and (b) phosphorylated/active form of Akt (phospho-Akt (S473)) were measured by reverse phase protein array (RPPA) as well as Akt activity determined as the (c) ratio 'phospho/total' in human BLCs and human epidermal growth factor receptor overexpressing (HER2+) carcinomas. mTOR is activated in BLCs. Box plots show the (d) expression of mTOR and (e) its form phosphorylated via the PI3 kinase/Akt signalling pathway (phospho-mTOR (S2448)) determined by Western blotting as well as (f) mTOR activity (phospho/total ratio) in human BLCs and HER2+ carcinomas. Outliers are shown for BLCs (solid circles) and HER2+ carcinomas (open circles). The y axes represent logarithmic transformed relative quantifications. p values (* p < 0.05) are represented (Mann-Whitney test). Data are representative of four and two separate experiments for RPPA and Western-blot, respectively.

Figure 3

Lower phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in human basal-like cancers (BLCs) compared with human epidermal growth factor receptor overexpressing (HER2+) carcinomas. (a) Lower PTEN protein levels in BLCs compared with HER2+ carcinomas. PTEN protein level was quantified by reverse phase protein array (RPPA). Outliers are shown for BLCs (solid circles) and HER2+ carcinomas (open circles). Data are representative of four separate experiments. (b) Lower mRNA PTEN level (probeset 225363_at) in BLCs compared with HER2+ carcinomas. An outlier is present in BLC population (solid circles). (c) Correlation between PTEN protein measured by RPPA and PTEN messenger in the entire tumour population. Linear regression, Spearman correlation c and p value are presented. BLCs (solid circles) and HER2+ carcinomas (open circles) are shown. (d) Stathmin is overexpressed in BLCs compared with HER2+ carcinomas. Box plots indicate the levels of stathmin protein measured by RPPA within the two populations. Data are representative of four separate experiments. P values are shown (a,b,d: Mann-Whitney test). * p < 0.05, ** p < 0.01, *** p < 0.001. Protein and mRNA relative quantifications were logarithmic transformed.

Figure 4

Loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) DNA copy-number (CN) in human basal-like breast cancers. (a) Basal-like carcinomas (BLCs) and human epidermal growth factor receptor overexpressing (HER2+) carcinomas behaved differently for PTEN CN in a significant manner. Recurrent DNA CN alterations were observed around the PTEN gene (between 72,260,000 and 93,93,000 bp of chromosome 10) in BLCs compared with HER2+ carcinomas. Frequencies of genome CN gain (red) and loss (green) were calculated using the FrAGL (Frequency of Amplicon, Gain and Loss) option of VAMP software (Visualisation and Analysis of array-CGH, transcriptome and other Molecular Profiles) [63]. The vertical blue bar represents PTEN position from 89,613,175 to 89,718,511 bp. Percentages of tumours with loss, normal or gain of PTEN CN are presented within the two populations in the table. p value is shown (** p < 0.01, fisher exact test). (b) Correlation between PTEN protein level and PTEN DNA CN. PTEN protein level was quantified as in Figure 3a. Linear regression, Spearman correlation c and p value (* p < 0.05) are presented. BLCs (solid circles) and HER2+ carcinomas (open circles) are shown. The two vertical black lines (X = 2 ± 0.28) separate loss/normal/gain PTEN CN (forceGL parameter: 0.28).

Figure 5

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-dependent activation of Akt in human basal-like breast cancers. PTEN protein levels are correlated negatively with Akt activity in human basal-like cancer (BLC). Akt activity and PTEN protein levels were measured as in Figures 2c and 3a, respectively. BLCs (solid circles), linear regression, Spearman correlation c and p value (* p < 0.05) are shown.

Figure 6

Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors inhibit basal-like cell line proliferation whereas apoptosis is induced only by PI3K inhibition. (a) Akt activation is associated with low/lack of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in human basal-like cell lines. The expression of Akt, phospho-Akt (S473) and PTEN were analysed by Western blotting in four basal-like (BT20, HCC38, HCC1937 and MDA-MB-468), one human epidermal growth factor receptor overexpressing (HER2+) (SKBr3) and one luminal (MDA-MB-453) human breast cell lines as well as in epidermal growth factor stimulated (EGF) or not (-) A431 cells. (b) PI3K and mTOR inhibition induce cell growth arrest of basal-like cell lines. BT20 (blue triangle), HCC1937 (red square) and MDA-MB-468 (green diamond) cells were exposed continuously for seven days to increasing concentrations of LY294002 (upper panel) or rapamycin (lower panel). Growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) dye conversion and presented as the percentage of control cell growth inhibition obtained from DMSO-treated cells. The x axes represent logarithmic transformed concentration of drugs. (c,d) The inhibition of PI3K, but not mTOR, induces apoptosis in basal-like cell lines. BT20, HCC1937 and MDA-MB-468 were exposed to varying concentrations of LY294002 (0 to 100 μM) or rapamycin (0 to 100 nM) for 24 hours and apoptosis was detected by measuring (c) caspase3/7 activity and the (d) cleavage of PARP (cPARP). (c) Caspase 3/7 activity was normalised by caspase 3/7 activity from vehicle-treated cells. (d) Actin was used as a loading control. The data represented the (b,c) average of three separate experiments performed in triplicates or are representative of (a,d) three separate experiments. Error bars represent standard deviation and p values (** p < 0.01, *** p < 0.001) were calculated by using Student's t test.