Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells.

Abstract

Effect of c-Ha-ras(V12) on cathepsin trafficking in invasive breast cancer cells. Various mutations of Ha-ras together with lack of p53-related control over cell cycle progression, result in an immortal, tumorigenic phenotype in 50% human epithelial cancers. Unmutated Ha-Ras transiently mediates external growth factor-related signaling, initiating downstream kinase activity that is normally terminated by p53. This protects the cell from immortalization, i.e. uncontrolled proliferation. An MCF10A breast epithelial cell line, derived from a fibrocystic breast mastectomy specimen, spontaneously immortalized in culture, due to a chromosomal deletion (9p21-/-). This gave rise to a non-malignant and non-invasive cell line in which the effects of deletion of upstream control of both p53 and the cell cycle and c-Haras( V12) transfection may be studied. Transfection of this cell line with the c-Haras( V12) oncogene gave rise to the invasive MCF10AneoT premalignant derivate, in which distribution of cathepsin B (CB), cathepsin L (CL) and cathepsin D (CD), membrane-type 1 matrix metalloprotease (MT1-MMP), a membrane-bound collagenase, is altered. The possible role of these proteases in the premalignant invasive phenotype, as well as the role of the V12 mutation and the effect of p53 on vesicle trafficking, was explored. In the MCF10AneoT cell line lack of negative feedback by p53 and other Ha-Ras effectors such as Rac, Rho and CDC42, seems to result in lack of control over the cytoskeleton and thus cell polarity during growth stimulus-related migration. Luminal alkalinization, especially of vesicles distant from the perinuclear region, as well as degradative efficiency seem affected, possibly as functional assembly of the acidifying vacuolar-ATPase proton pump on these vesicles is compromized. In normal cells CB and CD seem discretely located, while a spread of proteases was noted in transfected cells, from a perinuclear position to along the basal plane. Increased association of CB with lysosome-associated membrane protein-2 (LAMP- 2), and of CD with an acidic juxta-nuclear structure (JNS) was also noted, while this structure was observed in two sites in transfected cells, compared to only one in normal cells. In invasive cancers increased levels of both CB and MT1-MMP have been found to correlate with accelerated pathological degradation and invasion of the underlying basement membrane (BM) barrier and extracellular matrix (ECM). MT1- MMP is known to regulate BM turnover, while the manner in which the association of CB with the plasma membrane (PM) supports such turnover, ECM degradation and migration, is not yet clear. The current investigation showed altered distribution of PM-associated CB and MT1-MMP in transformed cells, compared to normal. This phenotype seems explained in terms of the effects of the mutationally activated c-Ha- Ras(V12) on its downstream effectors, Rac and PI3K and their effectors, on cytoskeletal organization and vesicle trafficking, increased calcium and, via Rho, cytoplasmic alkalinization due to proton extrusion by an activated NHE-1 membraneassociated proton pump.