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Cell compartment specific Ras signaling and breast cancer
Ras GTPases frequently act as master switches for numerous functions, such as growth and differentiation control, apoptosis, and cytoskeleton organization, that must be reprogrammed for tumorigenesis. Thus mutations rendering Ras constitutively active are among the most common genetic alterations in tumors. There is evidence that some Ras pathways can enhance breast tumorigenesis while other Ras pathways may block it. This in part explains why constitutively active ras mutations are rare in breast tumors, and underscores the importance of deciphering the complexity of the Ras pathways to better define their roles in breast tumorigenesis. The mammalian Ras pathways are complicated. There are four structurally similar Ras proteins, which although not functionally redundant in vivo, can each interact with nearly all known effectors in vitro. These Ras proteins are almost identical in their N-termini, but highly divergent in the C-termini, which undergo various lipidations to selectively influence association with different cell membranes. These observations support a model whereby different Ras proteins, or even the same Ras protein, can control different functions by localizing to a different membrane compartment. To unambiguously investigate this, we turned to the much simpler Ras system in fission yeast, which contains only one Ras protein that controls two separate evolutionarily conserved pathways. We have firmly demonstrated that yeast Ras selectively regulates a MAP kinase pathway from the plasma membrane (PM) to mediate mating, and a Cdc42 pathway from the endomembrane to mediate cell polarity and mitosis. In ongoing studies with mammalian cells, we found that while endomembrane-targeted Ras can efficiently transform cells, PM-targeted Ras cannot. Thus the mammalian Ras pathways seem compartmentalized like those of fission yeast and some Ras pathways may transform cells better than others. To fully understand how each compartmentalized Ras pathway influences tumorigenesis, it is imperative to identify Ras effectors that interact with Ras in a compartment-specific manner. We have thus modified the biomolecular fluorescence complementation (BIFC) method to develop a system in which the N-terminus of YFP is fused to Ras and C-terminus of YFP is fused to Ras effectors so that the binding between Ras and its effectors in human cells reconstitutes the YFP signal that can be readily identified by FACS. In this project we hypothesize that deciphering cell compartment-specific Ras pathways can better elucidate the complex roles of Ras in breast tumorigenesis. In Aim 1, we will isolate compartment specific Ras effectors by BIFC and FACS, and in Aim 2 we will analyze their tumorigenicities by the MCF10A cell 3D culture system and xenografts in nude mice. Our results may lead to better, more targeted interventions by focusing on spatially segregated Ras-effector interactions.
This project centers on Ras proteins that act as On-Off switches to control whether a cell should continue to grow or to remain dormant or to differentiate into a different cell. Ras also plays an important "quality control" role to switch on a self-destruction program to remove abnormal cells that arise when they fail to follow the instructions to properly grow and differentiate. Since cancer cells need to tamper with these surveillance mechanisms that are monitored by Ras, it is not surprising that mutations in ras genes are among the most frequently found genetic alterations in human tumors. Overexpression of Ras and its downstream targets called effectors can be readily found in almost half of all human breast cancers. Furthermore, during breast tumorigenesis, one of the most dangerous steps is when tumor cells lose their dependency on estrogen and estrogen receptor (ER) for growth while adapting to use growth factor pathways that often require Ras. These "ER-negative" breast tumors are very aggressive and cannot be treated using compounds that target the ER, such as tamoxifen. These findings underscore the importance of Ras signaling pathways for breast tumorigenesis. A major challenge in Ras study is the complexity of its signaling pathways. There are four structurally similar Ras proteins in humans, each of which can activate a long list of effectors. However, how a given Ras protein activates a specific effector in the cell to induce breast cancer formation is unknown. We have evidence that Ras can control unique functions by localizing to different regions in the same cell, and Ras proteins restricted to a specific region can transform cells with strikingly different efficiencies. Consistent with the idea that Ras needs to be properly localized to induce tumor formation, farnesyl transferase inhibitors, which disrupt normal Ras localization, have emerged as a promising treatment for breast cancer. In this project, we plan to identify Ras effectors that interact with Ras in a unique region of cell and then test their ability to influence tumorigenesis. The identification of these specific Ras effectors can not only better dissect the complex functions of Ras in tumorigensis but also allow for a more targeted design of new drugs for effective therapeutic interventions.