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. 2013 Nov 10;13:537.
doi: 10.1186/1471-2407-13-537.

Wnt Signaling in Triple Negative Breast Cancer Is Associated With Metastasis

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Free PMC article

Wnt Signaling in Triple Negative Breast Cancer Is Associated With Metastasis

Nandini Dey et al. BMC Cancer. .
Free PMC article

Abstract

Background: Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies.

Methods: We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease.

Results: The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin (functional read out of Wnt/β-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases.

Conclusion: These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.

Figures

Figure 1
Figure 1
Unsupervised hierarchical clustering of Wnt3A induced genes from the LWS-81 signature (shown in the figure as Wnt+) [24] common to the Breast Cancer DASL panel segregated triple negative tumors (shown in black) from other subtypes in both A) Quebec (QC-BCP) and B) Georgia (GA-BCP) cohorts. Each row represents a transcript, and each column represents a tumor sample, with triple negative denoted in black, HER2+ in grey, and HR + in white. Yellow indicates genes upregulated in response to Wnt3A and blue indicates genes down regulated. High expression is represented by red and low expression by green.
Figure 2
Figure 2
Lung cancer Wnt induced (LWS-81) genes [24] used to assess Wnt transcriptional regulation in the Quebec cohort profiled on the A) human cancer panel (QC-HCP) and B) breast cancer panel (QC-BCP) as well as C) Georgia (GABCP), D) MSKCC-99 [29], E) UNCCH-186 [30], and F) Stockholm-159 (STH-159) cohorts [31]. Significant p-values after Bonferroni’s correction are shown relative to the TNBC subtypes.
Figure 3
Figure 3
Wnt/β-catenin pathway plays a critical role in the regulation of metastasis-associated phenotypes in TNBC cell line based models. Functional blockade of Wnt/β-catenin pathway following the administration of Wnt-antagonist, WntC59 (10nM) (A), and transient transfection of β-catenin SiRNA (B) caused a decrease in fibronectin-mediated migration (left panel), and invasion (right panel) in MDA-MB231 TNBC cells. Migration assays (scratch assay), and invasion assays were performed on WntC59 treated or β-catenin SiRNA transfected cells. Crystal violet stain was used for the semi-quantification (Olympus DP72; X10) (A). Functional assays (transwell migration assay, and invasion assay) were carried out at 48 hours following transfection. Lysates from cells transiently transfected with scrambled SiRNA, and β-catenin SiRNA at 24, 48, and 72 hours were quantified for β-catenin expression by Western Blot (B Inset). Attenuator of Wnt-beta catenin signaling (sulindac sulfide) substantially abrogated the F-actin cytoskeletal organization in HCC38 TNBC cell line (Cii) as compared to the control (Ci) as well as in MDA-MB231 TNBC cell line (Ciii). Active beta-catenin levels were semi-quantified in arbitrary units (Image J) following Western blot analyses from the clarified MDA-MB231 cell lysates (beta-catenin SiRNA transfected cells and sulindac sulfide treated cells. Upper bar diagrams showed the relative desitometric expressions of beta-catenin, and active beta-catenin. Beta-actin was used as the loading controls (D left panel). Relative luciferase activity (TOP Flash over FOP Flash) measured in MDA-MB231 cells following beta-catenin SiRNA transfection and sulindac sulfide treatment 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 (D right panel).
Figure 4
Figure 4
Kaplan-Meier survival curves of Wnt/β-catenin positive (Wnt+: Wnt classifier signature) and Wnt/β- catenin negative (Wnt-) patients with respect to A) overall survival, B) recurrence-free survival, C) metastasis-free survival, D) lung metastasis-free survival, E) brain metastasis-free survival, and F) lung metastasis-free survival in TNBC patients. P-values were calculated by the log-rank test.
Figure 5
Figure 5
Wnt signaling schematic identifying well known mechanisms of interaction overlaid with gene expression regulation in TNBC tumors in the Quebec, Georgia, and MSKCC-99 cohorts and basal-like carcinomas in the UNCCH-186 and Stockholm-159 cohorts. Upregulated components include frizzled receptors, which when bound by Wnt ligands sequester disheveled (DVL1), breaking up a β-catenin phosphorylating complex composed of Axin, APC, and GSK-3β. This complex, when intact phosphorylates β- catenin on Serines 33, 37, and Threonine 41. Phosphorylated β-catenin can either become poly-ubiquitinated by the phosphate dependent ubiquitin ligase, BTRC, and subsequently degraded by the proteasome, or phosphorylated β-catenin can bind to P, N, and E type cadherins that enhance cytosolic β-catenin turnover. When the DVL, Axin, GSK-3β, and APC β-catenin phosporylating complex is not intact β-catenin does not become phosphorylated leading to transcriptional active β-catenin which can translocate to the nucleus and transcribes Wnt transcriptional target in combination with other co-activators including TCF7L2 (also known as TCF4), BCL9, and CREB binding protein (CBP).
Figure 6
Figure 6
Kaplan-Meier survival analyses were performed on the data. Kaplan-Meier survival analyses for i) overall, ii) recurrence-free, iii) metastasis-free,Metastasis-free, iv) lung metastasis-free, v) brain metastasis-free, and vi) bone metastasis-free survival for patients classified as Wnt + (Wnt classifier signature) and Wnt- for A) all patients and patients stratified by B) pathological determined subtype, C) intrinsic subtype, D) histological grade, and E) lymph node status. Only significant (p < 0.05) p-values are shown and are color-coded to correspond to the respective stratum.

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