Lipid Raft Composition in Deregulated ERBB2 Signaling

Institution: University of California, Los Angeles
Investigator(s): Ralf Landgraf, Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13IB-0077 Award: $99,371
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2007)
Elevated amounts of the ERBB2 (HER2) oncogene growth receptor are clinically important in 25-30% of breast cancers. Yet, despite the availability of drugs (such as Herceptin) that specifically target ERBB2, the therapy may fail because we still do not fully understand the cellular components that control ERBB2 activity. For example, the immediate cell membrane micro-environment of ERBB2 is poorly understood, but would be expected to influence the receptor’s binding and signaling properties. Typically, the cell membrane is assumed to be inert and serves merely to hold ERBB2 in place. However, in reality the membrane surrounding ERBB2 is subdivided into a range of microenvironments, made up of lipids and embedded proteins that have been selectively recruited to specific locations. “Lipid rafts” are a subtype of cell membrane microdomain that generates a favorable packing environment for sterols and saturated fatty acids and some signaling proteins (such as H-Ras), based on the nature of their lipid anchors. Rafts have been shown to modulate both signal transduction and “trafficking” events (i.e., lateral translocations, internalization, and movements within cells). In addition, the composition of lipids of the plasma membrane is subject to changes by external factors (e.g. environmental and dietary fat) and is the most direct, but least understood, point of interaction between ERBB2 signaling and lipid metabolism.

In this project to study the role of lipid rafts on ERBB2 function, we are asking three basic questions:

To address these questions, we will first evaluate the functional linkage between lipid composition, ERBB2 control and Herceptin resistance in our test system using MCF-7 and BT474 breast cancer cell lines. Next, we will determine how changes in lipid profiles alter the microenvironment of ERBB2 and ERBB3, the primary dimerization partner for ERBB2 in signaling. Specifically, we will evaluate the impact of altered lipid compositions on the distribution of signaling partners that co-associate with ERBB2 and ERBB3 in lipid microdomains.

A better understanding of this understudied ERBB2-lipid raft interplay will provide mechanistic insights into the intrinsic control of cell growth, resistance to therapy, and how environmental/dietary factors may influence the course of the disease.

Final Report (2008)
Little is known about the mechanisms by which ERBB2 (HER2) interacts with its immediate cellular micro environment within the cell surface plasma membrane, specifically the consequences of its localization to rafts or glycolipid enriched microdomains (GEMs). Cholesterol, saturated fatty acids and gangliosides are highly enriched in rafts, and changes in the ratio of saturated to unsaturated fatty acids have been reported to correlate with resistance to Herceptin in ERBB2 overexpressing cancers. Gangliosides have recently been implicated in the recruitment of ERBB2 into rafts. This could potentially drive significant shifts in ERBB2 signaling since rafts assemble various signaling partners of ERBB receptors, such as Ras and Src, into functional units.

Results of our study indicate that both ERBB2 and ERBB3, the primary receptor with which ERBB2 forms complexes and which provides most of the pro-survival component in signaling, localize preferentially to raft microdomains, both in Herceptin sensitive and Herceptin resistant cells. This localization is sensitive to ganglioside depletion but surprisingly only for ERBB2, not ERBB3. ERBB2 distribution among plasma membrane fractions is also shifted by Herceptin and the molecular nature of this shift is under investigation. Several lines of evidence also indicate that ERBB2 and ERBB3, and possibly other signaling partners, are localized to distinct subtypes of rafts. This unexpected finding presents both a fascinating and novel mechanistic framework for follow up studies as well as a significant challenge in method development. At present, no methodology is readily available for the characterization of subtypes of rafts and the development of this methodology in the context of Herceptin resistance is a major aspect for the ongoing extension of this CBCRP funded study. ERBB2 overexpressing cells show increased sensitivity to the ganglioside synthesis inhibitor PDMP n(1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a ceramide analog), and Herceptin and PDMP act synergistically in both Herceptin sensitive and resistant cells.

Future studies will analyze whether changes in the ratios of saturated and unsaturated fatty acids drive functionally relevant changes in ganglioside composition of rafts, which in turn may alter ERBB2 recruitment. We see this later study as a critical aspect in our long term effort to integrate ERBB2 regulation, fatty acid and ganglioside composition in a manner that will allow the design of interventions or predictive markers that are based on an experimentally acessible mechanistic framework.

Symposium Abstract (2007)
Elevated cellular levels of the ERBB2 (HER2) growth factor receptor are clinically important in 25-30% of breast cancers. As several lines of targeted therapy are aimed at ERBB2 our ability to understand the molecular basis of responsiveness and emergence of drug resistance has increasingly moved to the forefront. As a membrane embedded receptor, the immediate environment of ERBB2 is made up of lipids and other membrane proteins that are either embedded into or attached to the membrane Mutual interactions of lipids and proteins result in a recruitment and assembly of functionally distinct microenvironments. “Lipid rafts” are critical microdomains within the cell membrane that gathers signaling partners of ERBB2, including its dominant co-receptor ERBB3 and downstream targets. However the crosstalk between lipid metabolism, membrane lipid profile and the assembly of receptor-based signaling platforms is currently poorly understood. In the case of ERBB2, extracellular domains of the receptor that are critical in the control of intrinsic catalytic activity have also been implicated in determining the localization of ERBB receptors to rafts, the interaction with complex glycolipids, such as gangliosides, and are the same domains are known to be the target of Herceptin binding. The raft localization of receptors and complex glycolipids is expected to be influenced by the abundance and distribution of saturated long chain fatty acid containing phospholipids that are, together with cholesterol, critical building blocks of lipid rafts. Raft microdomains are, therefore, not only assembly platforms for ERBB2 signaling, they have the potential to play a key role in the integration of growth factor receptor signaling and lipid metabolism. This is further underscored by the enhanced sensitivity of cancer cells to the all-out inhibition of long chain fatty acid synthesis by FAS or ganglioside production, as well as reported correlations between Herceptin resistance and long chain fatty acid profiles, a correlation for which we currently lack a mechanistic explanation. A better understanding of this understudied interplay between ERBB2 and lipid rafts will provide mechanistic insights into the intrinsic control of cell growth, resistance to therapy, and how environmental/dietary factors may influence the course of the disease and success of treatment.