Activation status of Wnt/ß-catenin signaling in normal and neoplastic breast tissues: relationship to HER2/neu expression in human and mouse

Abstract

Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma in situ (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, P<0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P = 0.04) and estrogen receptor-negative status (P = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (P = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The Axin2(NLSlacZ) mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, Axin2(NLSlacZ) mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.

Figure 1. ß-catenin staining patterns of benign human tissues.

The 2^nd Generation Breast Cancer Progression TMA purchased from the NCI Cancer Diagnosis Program was subjected to ß-catenin IHC using BD Transduction Labs anti-ß-catenin antibody Clone 14 as previously described and counterstained with hematoxylin. As a control, serial sections were stained in parallel omitting primary antibody (right-hand panels). (A) Normal colon. (B–E) Normal breast. (F) Ductal carcinoma in situ. The image in (E) illustrates the positive nuclear ß-catenin staining observed in myoepithelial cells in some benign breast cores (inset, enlargement of boxed area; red arrowheads indicate positively-staining nuclei).

Figure 2. ß-catenin signal as a function of breast cancer progression.

Each core was assigned a separate score (0, +/−, 1+, 2+, or 3+) for membrane ß-catenin signal (A) and nucleocytoplasmic ß-catenin signal (B), and the percentage of cores with each score was separately calculated for benign breast tissue, DCIS, and invasive carcinomas. Of note, the staining pattern denoted as +/− in this study appeared visually similar to the staining pattern scored as 1 by Khramtzov and colleagues . A statistically significant reduction in membrane signal was observed in invasive cancers relative to benign breast tissues (P<0.001). A numerical increase in the proportion of DCIS tumors with nucleocytoplasmic ß-catenin compared with normal breast tissue was observed.

Figure 3. ß-catenin stabilization is evident in mammary precancers in HER2/neu transgenic mice.

Mammary gland tissue sections from virgin female MMTV/NDL mice were subjected to ß-catenin IHC as previously described and counterstained with hematoxylin. As a control, serial sections were stained in parallel omitting primary antibody (right-hand panel). Diffuse cytoplasmic ß-catenin was detected in DCIS-like lesions in MMTV/NDL MGs.

Figure 4. Discrete hyperplastic foci with robust ß-catenin/TCF signaling activity are present in MMTV/neu mouse mammary glands.

Abdominal (#4) mammary glands were harvested post-mortem from virgin female mice that were MMTV/neu or bigenic MMTV/neu, Axin2^+/NLSlacZ. MGs were stained with X-gal and wholemounted as previously described . Pea3^+/NLSlacZ (Pea3LacZ) samples were processed in parallel for comparison. (A–D) Wholemounted glands from bigenic MMTV/neu, Axin2^NLSlacZ mice (38–44 weeks old), viewed at 4× magnification. (E,F) Higher power images of lesion seen in Panel A, viewed at 20× magnification. (G) Higher power image of lesion seen in Panel C, viewed at 20× magnification. (H) Wholemounted gland from Pea3^+/NLSlacZ virgin female (39 weeks old) stained in parallel with the specimen in Panels C/G, viewed at 20× magnification. Eight focal lesions displaying intense ß-gal activity, all of similar size, were identified in MGs from 26 bigenic MMTV/neu, Axin2^+/NLSlacZ mice. By contrast, no comparable lesions were observed in MGs from MMTV/neu mice lacking the Axin2LacZ allele (not shown). Additionally, in our previous study of Axin2LacZ mice, no such lesions were observed in tissues from Axin2LacZ animals lacking a tumor-promoting transgene . Strikingly, each discrete region of robust ß-gal activity in bigenic MMTV/neu, Axin2^NLSlacZ MGs coincided with a focus of hyperplastic morphology markedly dissimilar to the normal-looking morphology of the immediately adjacent ductal structures. Also notable were the clearly demarcated boundaries between positive and negatively stained epithelium (examples are marked with red arrowheads in Panels A, C, F, G). The cellular staining profile in bigenic MMTV/neu, Axin2^NLSlacZ MGs contrasted with the myoepithelial pattern characteristic of Pea3LacZ MGs (compare Panels G and H), suggesting that Axin2LacZ is expressed in luminal cells within the HER2/neu-induced hyperplastic lesions.