Differential activation of Wnt-β-catenin pathway in triple negative breast cancer increases MMP7 in a PTEN dependent manner

Abstract

Mutations of genes in tumor cells of Triple Negative subset of Breast Cancer (TNBC) deregulate pathways of signal transduction. The loss of tumor suppressor gene PTEN is the most common first event associated with basal-like subtype (Martins, De, Almendro, Gonen, and Park, 2012). Here we report for the first time that the functional upregulation of secreted-MMP7, a transcriptional target of Wnt-β-catenin signature pathway in TNBC is associated to the loss of PTEN. We identified differential expression of mRNAs in several key-components genes, and transcriptional target genes of the Wnt-β-catenin pathway (WP), including beta-catenin, FZD7, DVL1, MMP7, c-MYC, BIRC5, CD44, PPARD, c-MET, and NOTCH1 in FFPE tumors samples from TNBC patients of two independent cohorts. A similar differential upregulation of mRNA/protein for beta-catenin, the functional readout of WP, and for MMP7, a transcriptional target gene of beta-catenin was observed in TNBC cell line models. Genetic or pharmacological attenuation of beta-catenin by SiRNA or WP modulators (XAV939 and sulindac sulfide) and pharmacological mimicking of PTEN following LY294002 treatment downregulated MMP7 levels as well as enzymatic function of the secreted MMP7 in MMP7 positive PTEN-null TNBC cells. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA. In cell lines, a high expression of casein-zymograph-positive MMP7 was distinguished by an absence of functional PTEN. A similar inverse relationship between MMP7 and PTEN mRNA levels was observed in the PAM50 data set (a correlation coefficient of -0.54). The PAM50 subtype and outcome data revealed that the high MMP7 group had low pCR (25%) and High Rd (74%) in clinical stage T3 pathologic response in contrast to the high pCR (40%) and low residual disease (RD) (60%) of the low MMP7 group.

Figure 1. Expression of mRNA for a number of different components of Wnt-β-catenin pathway (WP) and transcriptional targets of WP in Triple Negative subset of Breast Cancer (TNBC) patients.

Figure shows the expression of mRNAs for different components of Wnt-signaling in the Montreal cohort (upper panel). Average DASL signal intensity of Wnt transcriptional targets in Triple Negative (TN) tumors (black) are compared to non-TN (HR+ and HER2+) tumors (white). p-values < 0.05 determined using a t-test for unequal variances and Bonferroni’s correction for 46 Wnt related genes on a custom breast cancer DASL panel used to study the expression profile of the Montreal cohort (upper panel). RT-PCR confirmation of Wnt-signaling components in the Montreal cohort is shown (lower panel). Wnt transcriptional targets were validated using RT-PCR (10 TN and 10 HR+ patients in the Montreal cohort). The Wnt transducer DVL1 and the Wnt agonist BTRC were analyzed by RT-PCR and both increased (indicative of an increased Wnt signaling) in TN tumors. Asterisks (*) indicate p-values < 0.01 of differential regulation using two-sided t-tests for unequal variances and Bonferroni’s correction for multiple hypothesis (lower panel).

Figure 2. Expression of beta-catenin mRNA and protein in patients with TN breast tumors and cell lines.

Expression of beta-catenin mRNA in patients with TN breast tumors (A), the expression of beta-catenin in two of our data sets (Montreal-91 [GSE17650], Georgia-137 [GSE18539]) and another published data set (MSK-96 [GSE2603]) (B), the expression of beta-catenin mRNA in different breast cancer cell line (C), and the expression of beta-catenin protein in different breast cancer cell line (D) are shown. (A) Expression of CTNNB1 probes that code the beta-catenin protein in the Montreal cohort. Mean expressions of three CTNNB1 probes (Montreal cohort) for TN (black), HER2+ (grey), and luminal (white) patients indicate greater levels of expression in the TN subtype as compared to luminal and HER2+ subtypes. Error bars represent one standard error of the mean and p-values are determined using t-test for unequal sample sizes and means. (B) Expression of beta-catenin (CTNNB1 encodes beta-catenin) with respect to immunohistochemistry (IHC) subtype in two of our data sets (Montreal-91 [GSE17650], Georgia-137 [GSE18539]) and another published data set (MSK-96 [GSE2603]). beta-catenin is up-regulated in TN as compared to the HER2+ group in the Montreal cohort (p = 0.020) and as compared to luminal (HR+) group in the Georgia-137 cohort (p = 0.000316) (C i). Expression of CTNNB1 in four BT cell lines. Expression of CTNNB1 in four cell lines, including three TN breast cancer cell lines HCC70 (black), MDA-MB-468 (dark grey), HCC38 (light grey), and one luminal-like cell line MCF7 (white). Values represent the mean of three replicates, and error bars represent one standard error of the mean. P-values are determined using a t-test for unequal sample sizes and means (C ii). RT-PCR expression of mRNA for beta-catenin in different BT cell lines (TNBC and Non-TNBC). Values represent the mean of three replicates, and error bars represent one standard error of the mean. (D) Differential expression of beta-catenin protein in different TN breast cancer cells (lanes 9-22) as compared to HER2+ (lanes 1-4), and luminal-like (lanes 5-8) breast cancer (BC) cell lines (beta-actin as the loading control). Upper panel shows the quantification of beta-catenin protein using imageJ program (absorbance ratio of beta-catenin to beta-actin).

Figure 3. Expression of MMP7, one of the transcriptional targets of the WP as compared to other members of the MMP family in different subtypes of breast tumor samples from the Montreal and Georgia cohorts.

(A) Figure shows MMP7 expression in the Montreal cohort. Expression of MMP7 is higher in the TN subtype. Expression is sorted from the highest to the lowest level for TN (black), HER2+ (grey), and luminal (white) breast tumor subtypes as classified by IHC. Inset shows average expression of MMP7 by subtype in the cohort. Average expression of MMP7 is significantly higher (P< 0.05) in the TN (black) subtype as compared to both the HER+ (grey) and luminal (white) subtypes as indicated by the asterisk. Numbers within bracket are the patients in each subtype. (B) Figure shows selective upregulation of mRNA for MMP7 in TN tumors of the Montreal cohort as evident from the DASL assay average intensity data for different metalloproteinases. Expression of MMP1, MMP2, MMP3, MMP9, MMP11 and MMP7 grouped by hormone receptor and HER2 status; HR+/HER2-, HER2+, and TN. HR+ (hormone receptor+) is ER+ and/or PR+; HER2+ is HER2-positive and HR±; TN is triple negative. Error bars represent STDEV. Level of transcript expression is represented by DASL average intensity. *P< 0.05, and n represents number of samples in each subset. (C) Figure shows the expression of MMP7 with respect to IHC subtypes in two of our data sets (Montreal-91 [GSE17650], Georgia-137 [GSE18539]) and another published data set (MSK-96 [GSE2603]). The expression of mRNA for MMP7 is significantly upregulated in TN as compared to the luminal subtype (HR+) in all the sets and HER2+ subtype in the Georgia-137 cohort (p = 6.00×10^-22). The expression of the mRNA for the gene is significant in univariate analysis (p < 0.01). (D) The upregulation of MMP7 mRNA is observed in a subset of TNBC specimens as shown by the bar graph of the expression of MMP7 in each tumor in the cohort. Bars represent MMP7 mRNA expression in 87 FFPE breast tumor specimens (36 HR+/HER2- tumors, 27 HER2+/ HR± tumors and 24-TN tumors from the Montréal cohort) grouped by hormone receptor and HER2 status. MMP7 expression was upregulated in a subset ~40% (9/24) of TN tumor samples (shown in red) but not in any of the other (0/63) tumor samples (shown in blue).

Figure 4. Expression of mRNA, protein and enzymatic activity of MMP7 in TNBC cell lines as compared to HER2+ and luminal BC cell lines.

(A) Bar diagram (upper panel) shows mRNA expressions for MMP7 in HCC70 (black), MDA-MB468 (grey), and MCF7 (white) BT-like cell lines. Error bars represent one standard error of the mean and p-values are determined using a t-test for unequal sample sizes and means. Bar diagram in the lower panel (left) shows the validation of the expression of mRNA for MMP7 by RT-PCR in the BT cell lines. Expression of MMP7 measured by RT-PCR is concordant with microarray data for the HCC70, MDA-MB-468, and MCF7 cell lines. Expression is determined by the average of three replicates and bars represent one standard error of the mean (*p < 0.05). Bar diagram in the lower panel (right) shows RT-PCR expression of mRNA for MMP7 in different BT cell lines (MCF7, SKBr3, HCC38, SUM149). Values represent the mean of three replicates, and error bars represent one standard error of the mean (*p < 0.05). (B) MMP7 mRNA expression was upregulated in only a subset of TN breast cancer cell lines (shown in red) and not in any other types of BC-like cell lines. MMP7 affymatrix expression data from Neve et al. [36], grouped by hormone receptor and HER2 status. (C) Immunoblots showing differential expression of MMP7 protein (lanes 5-7, 12, 13, 14–16, 18-19) in TN breast cancer (T) cell lines as compared to HER2+ (H) and luminal (L) BC-like cell lines. Levels of MMPs 2, 9 and 14 are determined as references. Inset shows a differential expression of expression of MMP7 protein in a subset of TN breast cancer cell lines as determined by densitometry semi-quantitative analyses (imageJ program; absorbance ratio of MMP7 to beta-actin) of respective immunoblots. (D) Caesin-zymogram of the secreted-MMP7 and corresponding immunoblots of the cellular-MMP7 protein levels show both an increased expression and enzymatic activity of MMP7 (lanes 3, 5, 6, 9, 10) in TN breast cancer cell line (T) as compared to HER2+ (H) and luminal (L)-like cell lines.

Figure 5. Relative levels of expression of MMP7 and PTEN mRNAs from tumors of Montreal cohort.

Relative levels of expression of MMP7 and PTEN mRNAs (DASL intensities) using standard (A, B) as well as custom (C, D) panels from tumors of the Montreal cohort Data show (A & C) a differential expression of high MMP7 in the TN (NNN) tumors as compared to HR+(PPP, PPN, PNN) and HER2-enriched (NNP, PNP) groups. Analyses of levels of expression of MMP7 to the PTEN mRNAs (DASL intensities) in TN samples show that (1) MMP7 mRNA is expressed in only a subset of TN tumors (A & C), and (2) the presence of a higher level of MMP7 within the individual tumors of TN subset are associated with relatively lower levels of PTEN mRNA (B & D). These reciprocal levels of expression of MMP7 to the PTEN mRNAs (DASL intensities) were observed from the MMP7 to PTEN ratios, and heat maps of expressions in both standard (B) as well as custom (D) panels of the Montreal cohort.

Figure 6. Relationship between MMP7 expression and PTEN expression in TN tumors and cell lines, and MMP7 intensity correlation with PAM50 designation.

Relationship between MMP7 expression and PTEN expression in TN tumors and cell lines (A and B), and MMP7 intensity correlation with PAM50 designation (C and D) are presented. A. Cell lines were downloaded from GEO (GD3285) with replicates averaged for each cell line. HCC38 and SUM149PT did not have replicates. Error bars represent the coefficient of variation between each of the replicates. MDA-MB231 and MCF7 cell lines had two distinct groups of replicates that are included on this graph to demonstrate the variability standard cell lines can have (most likely due to passage number differences). Relative levels of expression of MMP7 and PTEN mRNAs (published DASL intensities) from different TN cell lines showing a differential expression of high MMP7 in the TN (HCC38, HCC70, MDA-MB231, BT20, DKAT and SUM149T) cell lines as compared to non-TN cell lines (MCF7, T47D) as shown in the upper panel. Normalized mRNA levels for breast cancer cell lines show that the presence of a higher level of MMP7 mRNA in the individual TN cell lines is associated with relatively lower levels of PTEN mRNA. These reciprocal levels of expression of MMP7 to the PTEN mRNAs (DASL intensities) were observed in heat map of gene expression as shown in the lower panel. Cell line intensity data generated from Affymetrix Gene Expression arrays (Upper panel). RMA normalized intensity values shows relative inter-gene levels of each mRNA in a specific cell line with the (Lower panel) heat map using Z’ values calculated for each specific gene to visualize intra-gene mRNA levels. B (i). Expression of MMP7 protein and the secretion of functional MMP7 were determined in the PTEN-null and PTEN expressing TNBC cells. High levels of expression of MMP7 protein and the secretion of functional MMP7 (casein zymogram from the conditioned media) in TNBC cells is inversely related to the presence of functional PTEN (protein expression as well as pAKT). B (ii). Expression of MMP7 protein and the secretion of functional MMP7 were determined in the PTEN expressing non-TNBC cells (HR+ and HER2 over-expressed). C. Patient downloaded from GEO (GSE25055). Top panel showing PAM50 designations used to separate the patients into each subtype. Each subtype was then sorted from left to right by increasing MMP7 intensity. Bottom panel is sorted by increasing MMP7 from left to right. Bold vertical line separates pCR on the left with residual disease (RD) on the right. D. Heat Map of expression of mRNAs for MMP7, PTEN, CTNNB1, c-MYC genes (PAM50 data) in Basal, HER2E and HR+ subsets of BC. Table (Lower panel) shows the outcome data for triple negative breast cancer patients from PAM50 data set with high MMP7, and low MMP7. Patient data from Fig. 6C were further examined for basal-like subtype using Z’ scores. Top panel shows increasing MMP7 Z’ score from left to right for all patient samples. Middle panel displays only patients with Z’ scores over 2.0 for MMP7, which shows the association with high MMP7 levels and low PTEN levels. The outcome data for the patients expressing low-MMP7 and high-MMP7 in their tumors are shown in the table (Lower panel).

Figure 7. An attenuation of WP caused downregulation of MMP7 in PTEN null TNBC cells.

PTEN null MMP7 expressing TNBC cell line, SUM149 was transiently transfected with beta-catenin SiRNA and MMP7 protein and its enzymatic activity were determined by Western blot and casein zymography respectively (A). Upper bar diagram showed the relative desitometric expressions of beta-catenin and MMP7. Pharmacological attenuation of beta-catenin in PTEN null MMP7 expressing TNBC cell lines, MDA-MB468 (B i) and SUM149 (B ii) following the treatment with XAV939 (B i) and sulindac sulfide (B ii) caused a decrease in MMP7 protein expression as well as an abrogation of its enzymatic activity. Upper bar diagrams showed the relative desitometric expressions of beta-catenin, active beta-catenin and MMP7. Relative luciferase activity (TOP Flash over FOP Flash) measured in SUM149 cells following sulindac sulfide was plotted (three different experiments). Error bars represent standard error of the means (SDs), and statistical significance was determined by paired t-test. *P < 0.05. The lower panels showed images of Western blot measuring β-actin (Inset of B ii). Sulindac sulfide treatment substantially reduced nuclear immune-fluorescence of beta-catenin (active form) in TNBC cells (B iii). To find out the involvement of MMP7 in metastasis-associated phenotypes, fibronectin-directed migration (C, upper panel) and matrigel-invasion (C, lower panel) were determined in MMP7 expressing MDA-MB468 cells and MMP7 null BT20 cells. Error bars represent standard error of the means (SDs), and statistical significance was determined by paired t-test. *P < 0.05. Since the pan PI3K inhibitor, LY294002 has been shown to closely mimic the effects of PTEN reconstitution in PTEN-deficient cancer cells, to understand the role of PTEN on the expression/enzymatic activity of MMP7 in TNBC cells, we have treated PTEN null MMP7 expressing MDA-MB468 cells with LY294002 (D). Data showed a decrease in MMP7 following LY294002 treatment in MDA-MB468 cells.