Nanolipoproteins to Study Breast Cancer Growth Receptors

Institution: University of California, Davis
Investigator(s): Paul Henderson, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14IB-0075 Award: $99,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2008)

Critical breast cancer growth factors and their biology require further study in defined biochemical environments distinct from their complex physiology within cancer cells. For example, key membrane receptors, such as the Her (EGFR) family cannot be studied in aqueous solutions because they will usually clump together and fall out of solution. New developments in nanotechnology are now poised to facilitate these studies. Nanolipoproteins (NLPs) are discoidal nanoparticles formed when an apolipoprotein and a population of phospholipids self-assemble into a lipid bilayer in an aqueous environment. This bilayer mimics closely the cell membrane, allowing membrane proteins to be functional inside the nanoparticles. NLPs present distinct advantages over currently used model membranes in terms of particle size mono-dispersity and consistency and the ability to form water soluble complexes with membrane proteins with out the need for detergents that frequently denature the protein of interest.

We plan to produce NLP-protein complexes with four important cell surface receptors that are related to breast cancer: HER1, HER2, HER3 and HER4. Collectively, these proteins represent an ideal model system because there exist well developed ligand binding, kinase (signaling) activity and other assays that can be employed to demonstrate reconstitution of fully functional proteins. Unresolved questions concerning these receptors we will study include, (1) structure-function relationships, and (2) novel approaches to targeting these receptors for the benefit of breast cancer patients. We will focus on optimizing HER1-4 synthesis conditions to facilitate proper folding and functional activity. This will be carried out using existing isolated genes and proteins for both the HER tyrosine kinases and the NLPs.

Isolation of large amounts of the HER2 complexes in NLPs could be used for structural studies that have previously been unattainable. A clinical assay for resistance susceptibility to Herceptin treatment or better candidate therapeutics is a potential outcome as a result of this IDEA project. Understanding the effect that proteins binding to HER2 can have on receptor activity will lead to better treatments for breast tumors that over express HER2. For example, screening of small molecules against HER2 with different binding partners contained in NLPs could be a very straightforward method to develop new lead compounds for drug discovery.

Final Report (2010)

This is a basic research project aimed at enabling a better understanding of the biology of cell surface receptors that effect the proliferation of breast cells. One of these receptors, called HER2, is the target of a therapeutic called Herceptin. Breast tumors that over-express HER2 compared to normal breast cells can be dramatically shrunk by Herceptin treatment. Many breast cancer patients on Herceptin treatment have had seemingly miraculous improvements only to be devastated by the development of resistance to the drug and subsequent recurrence of the cancer. Understanding of this problem at the molecular level has been hampered due to difficulty in obtaining purified HER2 and related proteins that are functional and in a water soluble formulation.

HER2 is a large protein that binds to cell membranes (not water soluble)—both large size and water insolubility are challenging characteristics for isolating and studying HER2. We are developing a method to produce HER2 and other proteins using “cell-free expression” combined with use of a nanoparticle that confers water solubility to the large protein. Cell-free expression can produce milligram quantities of pure proteins, provided they are water soluble, enough for many different types of biochemical studies.

Our efforts are taking two approaches: (1) To make DNA vectors that express HER2 that can be used directly in the cell free expression system and (2) to make the nanoparticles in the presence of membranes from cells that overexpress HER2, from which the HER2-containing nanoparticles can be purified. We have made progress in the synthesis of the DNA vectors (approach 1), but have yet to demostrate the successful expression of HER2 in the cell free expression system. This problem is being ovecome by remaking the vectors using different promotor sequences in order to maximize protein expression. For approach 2, we made cell membrane extracts that contain HER2, and synthesized the nanoparticles, but are still in the process of purifying and characterizing whether the resulting nanoparticles contain functional HER2.

Our accomplishments include synthesizing the DNA vectors and making nanoparticles from HER-overexpressing cells. We have suceeded in making functional HER2-NLPs in high purity and at large enough scale to enable drug screening and structural analysis. Furthermore, we extended the approach to include the epidermal growth factor receptor (EGFR), a similar receptor that is also important for breast cancer. This receptor is functional only in the presence of epidermal growth factor, just like the natural receptor as observed in cell-based experiments. Currently, these results are in preparation for publication and are part of an NIH R01 grant submission.

Symposium Abstract (2010)

Over 30% of proteins are bound to cell membranes, which makes them difficult to isolate in a soluble and active form. We have implemented recently developed nanolipoprotein particles (NLPs) to allow the preparation of membrane-bound proteins in an active form. In particular, we are focused on tyrosine kinase receptors, which mediate the growth and spread of breast cancer and are already targets of several chemotherapeutics. The ability to study these receptors outside the cell environment will enable structural and mechanistic studies for improved understanding of breast carcinogenesis and development of novel drug screening strategies. Given that membrane-associated proteins account for the majority of drug targets, such as the targeting of HER2 (also known as ErbB2) by Herceptin, it is important to develop novel technologies to gain access to this important class of proteins. This proposal is focused on developing a new biotechnology application leading to formation of NLPs capable of solubilizing HER2 and other related membrane-bound proteins. The NLPs present a distinct advantage over currently used model membranes in terms of particle size monodispersity and solubility. This poster summarizes our progress towards synthesizing homo- and heterodimers of ErbB receptors in NLPs and characterizing them for structure and function.