We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.

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Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events that occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cellbased assays that monitor changes in cell proliferation, migration and morphogenesis in MCF10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

ABBREVIATIONSDCIS ductal carcinoma in situ ROS reactive oxygen species HEKn human epidermal keratinocytes neonatal NAC N-Acetyl-cysteine NFκB nuclear factor-κB PI3K phosphatidylinositol 3-kinase Research PaperPsoriasin (S100A7) and Calgranulin-B (S100A9) Induction is Dependent on Reactive Oxygen Species and is Downregulated by Bcl-2 and Antioxidants ABSTRACT S-100 proteins are calcium-binding proteins with important growth regulatory functions. Of these proteins, psoriasin and calgranulin-B have been shown to be highly upregulated in ductal carcinoma in situ (DCIS) of the breast and in psoriasis. The purpose of this study was to further elucidate the functional relevance of the overexpression of these two S-100 proteins in psoriasis and DCIS. We report the induction of both proteins by reactive oxygen species, phorbol ester TPA, and the induction of psoriasin in response to the PI3K inhibitor wortmannin. We also demonstrate that Bcl-2 overexpression represses the induction of psoriasin and calgranulin-B under these different conditions. The same effect was obtained with the antioxidant NAC, which indicates that the suppression of psoriasin and calgranulin-B induction is mediated by the antioxidant function of Bcl-2. Furthermore, we demonstrate that overexpression of a dominant negative IKKβ also inhibits the induction of psoriasin suggesting that the NFκB pathway is involved in the induction of this protein. Also, we found NFκB responsive DNA elements in the upstream promoter region of psoriasin. MCF10A cells with a stable retroviral overexpression of psoriasin were significantly more resistant to H 2 O 2 -induced cell death than control cells further supporting the hypothesis that these S-100 proteins may play a role in oxidative stress response.