Podocalyxin in the Diagnosis and Treatment of Cancer

Kelly M. McNagny, Michael R. Hughes, Marcia L. Graves, Erin J. DeBruin, Kimberly Snyder, Jane Cipollone, Michelle Turvey, Poh C. Tan, Shaun McColl and Calvin D. Roskelle

The CD34-Related Molecule Podocalyxin Is a Potent Inducer of Microvillus Formation

BACKGROUND: Podocalyxin is a CD34-related transmembrane protein involved in hematopoietic cell homing, kidney morphogenesis, breast cancer progression, and epithelial cell polarization. Although this sialomucin has been shown to block cell adhesion, the mechanisms involved remain enigmatic. It has, however, been postulated that the adaptor proteins NHERF-1 and 2 could regulate apical targeting of Podocalyxin by linking it to the actin cytoskeleton. PRINCIPAL FINDINGS: Here, in contrast, we find that full-length Podocalyxin acts to recruit NHERF-1 to the apical domain. Moreover, we show that ectopic expression of Podocalyxin in epithelial cells leads to microvillus formation along an expanded apical domain that extends laterally to the junctional complexes. Removal of the C-terminal PDZ-binding domain of Podocalyxin abolishes NHERF-1 recruitment but, surprisingly, has no effect on the formation of microvilli. Instead, we find that the extracellular domain and transmembrane region of Podocalyxin are sufficient to direct recruitment of filamentous actin and ezrin to the plasma membrane and induce microvillus formation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that this single molecule can modulate NHERF localization and, independently, act as a key orchestrator of apical cell morphology, thereby lending mechanistic insights into its multiple roles as a polarity regulator, tumor progression marker, and anti-adhesin

Regulation of BRCA1 expression and its relationship to sporadic breast cancer

Germ-line mutations in the BRCA1 tumour suppressor gene contribute to familial breast tumour formation, but there is no evidence for direct mutation of the BRCA1 gene in the sporadic form of the disease. In contrast, decreased expression of the BRCA1 gene has been shown to be common in sporadic tumours, and the magnitude of the decrease correlates with disease progression. BRCA1 expression is also tightly regulated during normal breast development. Determining how these developmental regulators of BRCA1 expression are co-opted during breast tumourigenesis could lead to a better understanding of sporadic breast cancer aetiology and the generation of novel therapeutic strategies aimed at preventing sporadic breast tumour progression

Rat adrenocortical cell differentiation : effects of signal transduction alterations, ras oncogene expression, and parenchymal/stromal interactions

The zones of the adrenal cortex contain distinct subpopulations of cells which share a common mesodermal origin and steroidogenic template. How zonal specializations are overlaid upon this template is unknown. During adrenocortical cytogenesis zonal phenotypes are expressed transiently as a cell moves inward from a subcapsular position toward the medulla. In this study, homogeneous populations of rat adrenocortical parenchymal cells were maintained and manipulated in vitro. This was done in an effort to determine the mechanism responsible for zone-specific differential responsiveness to the trophic hormone angiotensin II (Ang II). In addition, the effects of ras oncogene expression and parenchymal/stromal interactions on overall steroidogenic differentiation were assessed. Initially, a method was devised to separate rat adrenocortical cells by density into populations which retain zone specific properties in primary culture. Two different parenchymal populations were obtained. They were designated FASC (1 .034g/mI, 18.0µ cell diameter) and GLOM (1.069g1m1, 11.7 µ. cell diameter). In freshly isolated cell suspensions the physical characteristics and differential steroidogenic responses to adrenocorticotropin and angiotensin II suggested that FASC cells originated predominantly from the zona fasciculata and GLOM cells from the zona glomerulosa. In primary culture, the two populations exhibited different morphologies. FASC cells retained lipid and formed cohesive epithelial monolayers that remained stationary for three weeks. GLOM cells were initially epithelial but rapidly lost lipid, spread, assumed fibroblastic shapes and expressed vimentin. Both cell types were positive for the steroidogenic cytochrome P-450 side chain cleavage enzyme (P-45Oscc). Therefore, the morphological changes observed in GLOM cultures were due to modulation, not fibroblastic overgrowth. Ang II increased steroidogenesis in GLOM cells but not in FASC cells. To determine the mechanism responsible for this zonal specialization, Ang Il-mediated signal transduction was closely examined. Ang II significantly increased the production of inositol 1,4,5 triphosphate in both cell types. In contrast, the two cell types exhibited very different intracellular release dependent increases in free intracellular calcium ([Ca²⁺]i). Ang II induced [Ca²⁺]i increases of >50nM in 90% of individual GLOM cells, but in only 28% of FASC cells. Also, in populations, Ang II induced dose dependent [Ca²⁺]i increases in GLOM but not FASC cells. Importantly, calcium ionophore treatment increased [Ca2+]i and steroidogenesis in both cell types. These results suggest that FASC cells lack a steroidogenic response to Ang II, at least in part, because of an interruption of the signalling pathway at the level of intracellular calcium release. Therefore, alterations within a specific signal transduction pathway are responsible for one of the cellular phenotypes associated with zonal specialization in the rat adrenal cortex. GLOM and FASC cells were infected with Kirsten murine sarcoma virus, which contains an activated form of the ras oncogene. FASC cells exhibited no discernable morphologic change after infection. In GLOM cultures, discrete foci did not appear. Instead, transient cellular multilayers formed from which rounded cells emerged. After selective passaging these cells (designated KGLOM) acquired a transformed morphology, proliferated rapidly in both 10% and 1% serum, grew to high saturation densities, and they were tumorigenic. They also expressed P-45Oscc and the viral ras protein, p21. In addition, KGLOM cells produced endpoint steroids under conditions in which GLOM cells did not. KGLOM steroidogenesis could be inhibited by lovastatin, a pharmacological inhibitor of p21 ras function. Therefore, expression of the ras oncogene induces transformation and enhances the steroidogenic differentiation of rat glomerulosa cells. This dual function of ras p21 likely reflects the state of the signal transduction pathways present within steroidogenic cells. In longterm culture, FASC and GLOM cells de-differentiated and lost their steroidogenic characteristics. This did not occur in a third density isolated population that consisted of a mixture of cell types (designated MIX). In primary culture, MIX populations formed highly proliferative cellular multilayers that were composed of stromal fibroblasts, endothelial cells and parenchymal cells. From these multilayers rounded cells emerged that contained lipid and the steroidogenic enzymes Δ5,3ßhydroxysteroid dehydrogenase (Δ5,3ß-HSD) and P-45Oscc. This phenomenom was associated with a 10 fold increase in the number of Δ5,3ß-HSD containing cells during the first 21 days in culture. Rounded cells were selectively passaged into secondary culture where they continued to proliferate and remained steroidogenic. Thus, primary MIX cultures provide a microenvironment that produces proliferating, steroidogenic cells independently of trophic hormone treatment. This unique model may prove useful in the quest to further identify specific inducers that are responsible for adrenocortical cytogenesis in general and steroidogenic differentiation in particular.Medicine, Faculty ofGraduat

A hierarchy of ECM-mediated signalling tissue-specific gene expression regulates tissue-specific gene expression

A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution