Breast cancer is the second leading cause of cancer death in North American women. There is considerable need for reliable prognostic markers to assist clinicians in making management decisions. Although a variety of factors have been tested, only tumor stage, grade, size, hormone receptor status, and S-phase fraction are used on a routine basis. The cell cycle is governed by a family of cyclin-dependent kinases (cdks), which are regulated by associated cyclins and by phosphorylation. p27Kip1, a cyclin-dependent kinase inhibitor, regulates progression from G1 into S phase by binding and inhibiting cyclin/cdks. p27Kip1 protein levels and/or activity are upregulated by growth inhibitory cytokines including transforming growth factor-beta (TGF-beta) and, thus, provide an important link between extracellular regulators and the cell cycle. Loss of p27Kip1, a negative cell-cycle regulator, may contribute to oncogenesis and tumor progression. However, p27Kip1 mutations in human tumors are extremely rare. We have demonstrated by immunohistochemistry that p27Kip1 protein levels are reduced in primary breast cancers and that this is associated with tumor progression in both in situ and invasive lesions. This was confirmed by western analysis, reflected in increased G1/S-phase cyclin-dependent kinase activities and shown to be regulated posttranscriptionally by in situ hybridization. Furthermore, on multivariate analysis, low p27Kip1 is a predictor of reduced disease-free survival. This simple and reliable immunohistochemical assay may become a routine part of breast cancer evaluation and may influence patient management.

Information on protein-protein interactions is of central importance for many areas of biomedical research. Currently no method exists to systematically and experimentally assess the quality of individual interactions reported in interaction mapping experiments. To provide a standardized confidence-scoring method that can be applied to tens of thousands of protein interactions we have developed an interaction tool-kit consisting of four complementary high-throughput (HT) protein interaction assays. These assays were benchmarked against positive and random reference sets (PRS and RRS) consisting of well documented human interaction pairs and randomly chosen protein pairs, respectively. A logistic regression model was trained using the PRS/RRS data to combine the assay outputs and calculate the probability that any novel interaction pair is a true biophysical interaction once it has been tested in the tool-kit. This general approach will allow a systematic and empirical assignment of confidence scores to all individual protein-protein interactions in interactome networks.

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. Here, we report an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Further, we show that Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. Our work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE.

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K-ras oncogene in these cell lines is associated with marked upregulation of vascular endothelial growth factor͞vascular permeability factor (VEGF͞VPF), we sought to determine whether this potent angiogenesis inducer plays a role in K-ras-dependent tumorigenic competence. Transfection of a VEGF 121 antisense expression vector into DLD-1 and HCT-116 cells resulted in suppression of VEGF͞VPF production by a factor of 3-to 4-fold. The VEGF͞VPF-deficient sublines, unlike the parental population or vector controls, were profoundly suppressed in their ability to form tumors in nude mice for as long as 6 months after cell injection. In contrast, in vitro growth of these sublines was unaffected, thus demonstrating the critical importance of VEGF͞VPF as an angiogenic factor for HCT-116 and DLD-1 cells. Transfection of a full-length VEGF 121 cDNA into two nontumorigenic mutant K-ras knockout sublines resulted in a weak but detectable restoration of tumorigenic ability in vivo in a subset of the transfectants, with no consistent change in growth properties in vitro. The findings indicate that mutant ras-oncogenedependent VEGF͞VPF expression is necessary, but not sufficient, for progressive tumor growth in vivo and highlight the relative contribution of oncogenes, such as mutant K-ras, to the process of tumor angiogenesis.