We previously identified Perp as a crucial component of desmosomes in the oral mucosa and epidermis, where it acts to promote tissue integrity [20
], and as a mediator of p53-dependent apoptosis in response to genotoxic stress [17
]. To gain a greater understanding of the function of Perp in tissue homeostasis in other contexts, we investigated a role for Perp in the mammary epithelium, another p63-dependent tissue in which cell adhesion and apoptosis are key for development and homeostasis. Here, we provided the first demonstration that Perp protein is expressed in the mammary epithelium, in a pattern suggesting an adhesive role for Perp in the mammary epithelium, as in the oral epithelium and skin. In addition, our results demonstrate that Perp
deficiency both perturbs mammary epithelial homeostasis and promotes mammary cancer.
In the mammary epithelium, Perp exhibits a punctate pattern of expression at the plasma membrane and colocalizes with other desmosome components. Our results suggest that Perp is expressed in both layers of the mammary epithelium, in luminal and myoepithelial cells. Perp may be an important factor in regulating desmosome assembly and/or stability in this context, as we found that Perp loss in mammary epithelial cells decreased levels of the desmosome proteins Dp, Dsg, and Dsc, compared with those in wild-type cells. These findings suggest that desmosome function in the mammary epithelium is likely compromised in the absence of Perp.
Akin to global Perp deficiency causing impaired desmosome function in the oral mucosa and epidermis, associated with blistering and early lethality [20
], Perp loss in the mammary epithelium also compromises mammary gland homeostasis in vivo
by promoting immune cell recruitment. We demonstrated further that Perp
-null mammary epithelium is able to realize all stages of mammary gland development without the presence of gross structural abnormalities, suggesting that in vivo
mammary gland morphogenesis per se
is not dependent on normal desmosome function. This finding contrasts with a previous study reporting that impairing desmosome-mediated adhesion by simultaneously interfering with the function of both types of desmosomal cadherins, Dsg and Dsc, inhibited mammary epithelial morphogenesis in culture, as seen by the impaired development of alveolar spheres and the inhibition of positional sorting of luminal and myoepithelial cells in aggregates [9
]. This observation could suggest that inactivation of two desmosomal cadherins may be more severe than the loss of Perp in the context of mammary epithelial development. Differences between our findings and those of this report may also be due to the experimental design used in each study: we examined primary mouse mammary epithelium in vivo
, whereas Runswick et al.
] used a mouse mammary cell line in cell-culture experiments.
Because our mammary-transplant assays were performed in vivo
, we were able to examine the mammary epithelium in its native context. Strikingly, in the context of mammary transplantation, we observed abundant immune cell accumulation around the Perp
-deficient mammary epithelium in 8-week-old virgin females, but not that of wild-type counterparts. This observation is reminiscent of our previous findings that ablation of Perp
in the epidermis induced an inflammatory gene signature and that Perp
deficiency in combination with UV irradiation caused the accumulation of immune cells in the skin [17
]. Inflammatory cells are well established to promote tumor progression by producing such factors as cytokines, chemokines, and matrix metalloproteases that can enhance the malignant characteristics of neoplastic cells as well as remodeling the tumor microenvironment to support tumor growth and progression. Indeed, leukocyte accumulation increases during tumor development and progression, and leukocytes contribute to the development of many solid tumors, including breast cancer [40
]. Genetic studies in mouse models have demonstrated the importance of macrophages and T lymphocytes for breast cancer progression [41
], and the presence of macrophages in human breast tumors correlates with poor prognosis [43
]. Therefore, it is possible that the lymphocyte accumulation observed in the Perp
-deficient mammary epithelium could result in microenvironmental changes that potentiate mammary epithelial tumorigenesis.
We showed previously that loss of Perp in mice promotes both the initiation and progression of UVB-induced skin cancer [17
]. To determine whether Perp contributes to tumor suppression in other epithelial cancers, we investigated the effect of Perp
deficiency on mammary carcinogenesis. To date, few studies have examined the specific connection between desmosomes and breast carcinogenesis. A couple of exceptions are studies in which the desmosomal protein Dsc3 was found to be downregulated during human breast cancer development [44
], and in which Dp expression was observed to be inversely correlated with human breast tumor growth and progression [45
]. To more directly assess how desmosomes contribute to carcinogenesis, it is important to evaluate genetic experimental models in which desmosome gene expression can be manipulated in the context of cancer. Here, we found that decreased Perp
dosage in the mammary epithelium reduces tumor-free survival and tumor latency in K14-Cre/+;p53fl/fl
mice. Although Perp has roles both in intercellular adhesion and p53-dependent apoptosis, the effects of Perp
deficiency on tumor latency and tumor-free survival in this mouse cancer model likely result from altered desmosome-mediated cell-cell adhesion, because the tumors are null for p53
. Collectively, our results therefore indicate that Perp can display tumor-suppressor activities in more than one type of epithelial cancer and that Perp
deficiency can promote tumor development in the context of different tumor-promoting stimuli. In the future, it would be interesting to investigate how combined targeting of adherens junctions and desmosomes would affect tumorigenesis in this model system.
Our results demonstrate reduced expression of Perp protein in a variety of human breast cancer cell lines, as compared with normal cells, suggesting the possibility that Perp downregulation may contribute to cancer progression. Interestingly, a previous study by Neve et al.
] identified characteristics that led to the segregation of different normal mammary and breast cancer cell lines into functionally distinct subtypes, described as Luminal, Basal A, and Basal B. Some of the cell lines were evaluated for invasive behavior in a Boyden chamber assay, with the most samples being of the Basal B subtype. When we overlap our data describing Perp levels in these same cell lines with the Boyden chamber results of Neve et al.
, it suggests that Perp levels may inversely correlate with an invasive phenotype. For example, the nontransformed MCF10A cells exhibited high Perp levels but no invasion, whereas the BT549, MDA-MB-231, and SUM149PT breast cancer cells expressed comparatively low Perp levels and displayed significant invasive activity. Together, the two studies suggest that reduced Perp expression may be one characteristic that contributes to the invasive behavior of breast cancer cells and breast cancer progression in vivo
Analyses of human cancers have suggested the importance of Perp as a prognostic marker, as Perp
downregulation is associated with particularly aggressive uveal melanomas [47
] and is predictive for esophageal cancers that will fail to respond efficiently to preoperative combination chemotherapy and radiation treatment [48
]. Moreover, Perp loss correlates with increased rates of local relapse in human head and neck squamous cell carcinoma patients (personal communication, Quynh-Thu Le, M.D.). Although patient outcome has not yet been correlated with Perp expression levels in human breast cancer, human Perp
(also known as THW
) is downregulated in human mammary carcinoma cell lines compared with nonmalignant mammary epithelium [49
]. In addition, the chromosomal region to which human Perp
maps -- 6q24 -- is deleted in human breast cancer, and loss of heterozygosity at this region has been detected both in breast carcinoma cell lines and in human breast tumors [49
]. Future studies will better elaborate the mechanisms by which Perp suppresses epithelial cancers and will evaluate Perp as a prognostic indicator or therapeutic target in breast cancer.