Increased invasiveness and aggressiveness in breast epithelia with cytoplasmic p63 expression

Abstract

Our previous studies revealed that pregnancy associated breast cancer (PABC) had significantly reduced nuclear p63 expression in myoepithelia, while intense cytoplasmic p63 expression in associated epithelia. Our current study assessed these epithelia using immunohistochemistry with a panel of aggressiveness and invasiveness related markers and comparative genomic hybridization (array-CGH) with over 30,000 DNA probes. These epithelia showed several unique alterations, including (1) immunohistochemical and morphological resemblance to invasive cancer, (2) significant gain in copy numbers of DNA coding genes for morphogenesis, angiogenesis, and metastasis, and (3) significant loss in copy numbers of DNA coding genes for tumor suppressors, cell adhesion, and macromolecular complex assembly or intra-cellular trafficking. Detected array-CGH alterations correlated well with in vivo expression of a number of corresponding proteins tested. These findings suggest that aberrant sub-cellular localization of p63 expression in normal or hyperplastic appearing epithelial cells may significant contribute to increased invasiveness and aggressiveness of these cells.

Keywords: breast epithelia; invasiveness and aggressiveness.; p63 expression; pregnancy associated breast cancer.

Figure 1

Acinar cell clusters or lobules with cytoplasmic p63 expression. Sections from paraffin embedded tissues of PABC were immunostained for p63 (brown or red). Circles identify normal appearing lobules in which all acinar cells show cytoplasmic p63 expression. Thin and thick arrows identify cells with nuclear and cytoplasmic p63 expression, respectively. Asterisks identify invasive tissue components. In tissues without or distant from malignant lesions, both cytoplasmic and nuclear p63 expression cells are seen (a-d), whereas in tissues harbored or adjacent to malignant lesions (e-h), all or nearly all cells within a given lobule uniformly show high levels of cytoplasmic p63 expression. a, c, e, and g: 100X; b, d, f, and h: a higher (300X) magnification of a, c, e, and g, respectively.

Figure 2

Reduced expression of other tumor suppressors in myoepithelium of PABC. A set of 6 consecutive sections of PABC were immunostained for maspin, WT-1, p63, p73, p53, and Ki-67, respectively. Circles identify normal appearing lobules in which all or nearly all acinar cells show cytoplasmic p63 expression. Thick arrows identify the myoepithelial cell layer in different sections. Thin arrows identify isolated tumor cells with p53 and Ki-67 expression. Note that all or nearly all the myoepithelial cells uniformly express maspin, whereas only about 10-30% of maspin positive cells show WT-1, p63, or p73 expression. Also note that these normal appearing lobules harbor some isolated p53 positive tumor cells and have an elevated cell proliferation index. a, c, e, g, I, and k: 80X; b, d, f, h, j, and l: a higher magnification (200X) of a, c, e, g, I, and k, respectively.

Figure 3

Morphological and immunohistochemical resemblance to invasive cancer cells. Sections from 4 PABC cases were immunostained for p63 (brown). Circles identify normal or hyperplastic lobules in which all or nearly all cells show high levels of cytoplasmic p63 expression. Arrows identify adjacent invasive cancer cells. Note that some cells within these normal or hyperplastic appearing lobules and their adjacent invasive counterparts share a very similar immunohistochemical or morphological profile. a, c, e, and g: 100X; b, d, f, and h: a higher (300) magnification of a, c, e, and g, respectively.

Figure 4

Significant gain or loss of DNA copy numbers in lobules with cytoplasmic p63 expression. Microdissected cells from 4 cases of PABC and 4 cases of morphologically similar non-PABC were subjected to array-CGH. Gain or loss of DNA copy numbers were identified arbitrarily by >1.2-fold change in the average of the background-subtracted mean intensity ratios between PABC and control samples. (A) Data normalization; (B) Heat map and clusters; (C) Control samples. The triplicate microarray experiments were performed for each sample.

Figure 5

Elevated expression of angiogenesis and growth-related genes. A set of four consecutive sections adjacent to sections used for array-CGH were immunostained for morphogenesis and angiogenesis-related biomarkers. Arrows identify aberrant expression of D2-40 and CD34 in myoepithelial cell layers, and aberrant expression of tenascin and p53 at the junctions between pre-invasive tissues and invasive lesions. Note that significantly higher expression of these markers were seen in normal appearing lobules adjacent to, compared to those distant from, invasive lesions (asterisks). a, c, e, and g: 80X; b, d, f, and h: a higher (200) magnification of a, c, e, and g, respectively.

Figure 6

Elevated expression of macromolecular complex assembly and intra-cellular trafficking related genes. Two sets of four consecutive sections adjacent to sections used for array-CGH were immunostained for p63, WT-1, and maspin. Circles identify acinal lumen with high level of secretory products. Note that strong p63 and WT-1 immunoreactivities were seen in these luminal secretory products, while maspin is only seen in the myoepithelium. 100X.