The role of the ubiquitination-proteasome pathway in breast cancer: applying drugs that affect the ubiquitin-proteasome pathway to the therapy of breast cancer

Abstract

The ubiquitin-proteasome pathway is responsible for most eukaryotic intracellular protein degradation. This pathway has been validated as a target for antineoplastic therapy using both in vitro and preclinical models of human malignancies, and is influenced as part of the mechanism of action of certain chemotherapeutic agents. Drugs whose primary action involves modulation of ubiquitin-proteasome activity, most notably the proteasome inhibitor PS-341, are currently being evaluated in clinical trials, and have already been found to have significant antitumor efficacy. On the basis of the known mechanisms by which these agents work, and the available clinical data, they would seem to be well suited for the treatment of breast neoplasms. Such drugs, alone and especially in combination with current chemotherapeutics, may well represent important advances in the therapy of patients with breast cancer.

Figure 1

Protein degradation through the ubiquitin (Ub)-proteasome pathway. Most proteins that are destined for degradation through the Ub-proteasome pathway are first subjected to polyubiquitination. This is accomplished in several stages. (a) The E1 Ub-activating enzyme, in an ATP-dependent reaction, forms an activated complex with Ub and transfers it to the E2 Ub-conjugating protein. (b) The E2 Ub-conjugating protein then transfers Ub to an E3 Ub-ligase protein, which has formed a complex with the target protein. In some cases an E3 Ub-ligase may not be necessary. (c) After several cycles of ubiquitination, the polyubiquitinated target protein is recognized by the proteasomal cap proteins (shaded gray and labeled 19 S cap) through its ubiquitin moieties, which are cleaved off by isopeptidases and recycled. (d) In an ATP-dependent fashion the protein is then unwound and fed into the 20S core through an interior channel, where it is exposed to the active proteolytic enzymes (shaded black). (e) Oligopeptide digestion products (OP) are then released and degraded further to amino acids by oligopeptidases. Some proteins may be subject to proteasomal degradation without the need for prior ubiquitination. Please note that this schematic diagram does not represent the various components to scale. Interested readers are referred to several excellent recent reviews with more detailed descriptions of this pathway [43,44].

Figure 2

The combination of PS-341 and Doxil^® induces enhanced apoptosis in vivo. The impact of vehicle, PS-341 alone, Doxil^® alone, or the combination, was studied in a murine xenograft model of human breast cancer established using BT-474 breast carcinoma cells. Apoptosis was evaluated in tumor sections 24 hours after the indicated treatments by detection of single stranded DNA fragmentation using the murine monoclonal antibody Mab 3299 [45] (Chemicon International, Temecula, CA, USA). Single stranded DNA associated with programmed cell death (red) is shown, along with total nuclear DNA (blue), the latter detected using 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Slides were visualized using an ultraviolet Zeiss Axioplan fluorescence microscope (Carl Zeiss Optical, Inc., Chester, VA, USA). Separate photographs were taken with appropriate filters for blue nuclear staining and red single-stranded-DNA staining, overlayed using Adobe Photoshop software, and displayed as a fusion image at 10 × magnification.