The MoLAB at the IZI

Breast cancer is the most common type of cancer in women. Although genes involved in the development of breast cancer have been identified, the potential contribution of many others is still under investigation. The research of the lab is focused on signaling pathways underlying normal epithelial differentiation and their contribution to cell transformation when deregulated. In the past, we identified the START domain-containing protein StarD10 to be overexpressed primary breast carcinomas and human breast cancer cell lines, correlating with their ErbB2/Her2 status. Soft agar experiments in mouse fibroblasts demonstrated that StarD10 expression cooperated with ErbB1/epidermal growth factor receptor in stimulating anchorage-independent growth, suggesting a role for this protein in breast oncogenesis.

START (steroidogenic acute regulatory protein-related) domains are known to accommodate a single lipid molecule in a hydrophobic binding pocket, which enables them to transfer lipids between membranes. Our current focus is on the physiological functions of START domain-containing lipid transfer proteins in membrane biology and signalling, and their regulation by post-translational modification, protein-protein and protein-lipid interactions. We have a particular interest in the Deleted in Liver Cancer (DLC) protein family, Rho GTPase-activating proteins that have recently emerged as important tumor suppressors, whose downregulation in various types of cancer including those of the breast may be as common as that of p53. DLC proteins contain a START domain of unknown function, raising the possibility that their tumor suppressor activity is regulated by lipid binding.

DLC tumor suppressor proteins in cellular transformation
DLC1, DLC2 and DLC3 are GTPase activating proteins (GAP) that are frequently downregulated in various types of cancer. In vitro, these proteins specifically inactivate the small GTPases of the Rho family and at least in the case of DLC1 this appears to contribute to its tumor suppressor function in vivo. Ectopic expression of the DLC proteins in carcinoma cell lines has been shown to inhibit cell proliferation, migration and invasion. However, whether the loss of DLC family members is the cause of aberrant Rho signaling in transformed cells is not clear. To address this question we downregulated DLC1 expression in breast cancer cells using a RNA interference approach. DLC1 silencing led to the stabilization of stress fibers and focal adhesions and enhanced directed cell migration. Interestingly, downregulation of DLC2 and DLC3 has distinct cellular consequences, indicating that the proteins have non-redundant functions, likely due to different subcellular localizations and protein binding partners. Our aim is to identify such specific protein partners by yeast-two-hybrid screening and proteomic approaches.

Molecular mechanisms regulating DLC protein function
DLC1 localizes to focal adhesions through interaction with tensin proteins, which is thought to be crucial for biological activity. We have shown that activation of the PKC/PKD pathway promotes association of DLC1 with 14-3-3 adaptor proteins. This interaction inhibits DLC1 GAP activity and facilitates signaling by active Rho, most likely by 14-3-3-mediated cytosolic sequestration. Stimulation of DLC1 GAP activity, on the other hand, is achieved by binding of phosphatidylinositol 4,5-bisphosphate (PIP2) via a previously unrecognized polybasic cluster adjacent to the GAP domain. This polybasic cluster appears to be essential for DLC1's tumor suppressor function, identifying PIP2 as an important cofactor in the regulation of DLC1.

Physiological functions of lipid transfer proteins - The importance of membrane biology for organelle function, signaling and disease
Lipid homeostasis is tightly linked to organelle function. In collaboration with the group of Dr. Angelika Hausser at the institute, we identified the lipid transfer protein CERT as a novel substrate for protein kinase D (PKD) at the Golgi complex. PKD is critically involved in the fission of transport carriers from the Golgi en route to the cell surface. PKD phosphorylates CERT on serine 132 adjacent to the PH domain, whereby PI(4)P binding, Golgi targeting, and ceramide transfer activity are negatively regulated. At the Golgi, ceramide is converted to sphingomyelin and diacylglyerol (DAG), with DAG being essential for the recruitment and activation of PKD. Ectopic expression of CERT increased PKD activity and stimulated the secretory activity of the Golgi, whereas siRNA-mediated knock-down of CERT impaired secretion. Thus, by contributing to DAG production, CERT is required for proper Golgi function and the maintenance of constitutive secretory transport to the plasma membrane. We are now extending our studies to potential global changes of lipid homeostasis in dependence of CERT activity and their impact on membrane proximal signaling events and associated cellular responses.