The Ub/proteasome proteolytic pathway is required for efficient cell-cycle control, stress response, DNA repair, and differentiation (Glickman and Ciechanover (2002) Physiol. Rev. 82:373-428; Pickart (1997) FASEB J. 11:1055-1066; Varshaysky (1997) Trends Biochem. Sci. 22:383-387). Mutations in this pathway can cause pleiotropic defects because of its involvement in virtually all aspects of cell function. Consequently, the characterization of the Ub/proteasome pathway for the development of treatment for cancer and other malignancies is an area of active investigation (Voorhees, et al. (2003) Clin. Cancer Res. 9:6316-6325; Yang, et al. (2004) Clin. Cancer Res. 10:2570-7; Yang, et al. (2004) Clin. Cancer Res. 10:2220-2221; Rossi and Loda (2003) Breast Cancer Res. 5:16-22; Ohta and Fukuda (2004) Oncogene 23:2079-2088).
The 26S proteasome is composed of a catalytic (20S) particle and a regulatory (19S) particle. The structure and function of the 20S catalytic particle is conserved in evolution, and its compartmentalized organization ensures that the proteolytic activities are sequestered within the interior of the proteasome (Baumeister, et al. (1998) Cell 92:367-380). The large 19S regulatory particle interacts with the 20S particle to facilitate recognition, unfolding and degradation of ubiquitinated substrates (Glickman, et al. (1998) Mol. Cell. Biol. 18:3149-3162; Groll, et al. (2000) Nat. Struct. Biol. 7:1062-1067). Ubiquitin (Ub) is covalently attached to lysine side-chains in cellular proteins (Pickart (2000) Trends Biochem. Sci. 25:544-548). The ligation of Ub to proteins requires the action of three enzymes termed Ub-activating (E1), Ub-conjugating (E2), and Ub-ligases (E3) (Glickman and Ciechanover (2002) supra). The sequential addition of Ub moieties results in the formation of a multi-Ub chain, which facilitates protein degradation by promoting translocation of substrates to the proteasome (Gregori, et al. (1990) J. Biol. Chem. 265:8354-8357; Thrower, et al. (2000) EMBO J. 19:94-102).
Malignant conditions are frequently associated with altered abundance and stability of regulatory proteins. It is therefore likely that the expression of a unique repertoire of proteins underlies the transition from normal to abnormal growth. Proteasome activity has been found to be elevated in esophageal cancer and cancer cachexia (Wyke, et al. (2004) Br. J. Cancer 91:1742-1750; Zhang, et al. (2004) World J. Gastroenterol. 10:2779-2784).
It has been shown that the co-translational degradation of newly synthesized misfolded proteins requires the Ub/proteasome system (Schubert, et al. (2000) Nature 404:770-774; Reits, et al. (2000) Nature 404:774-778; Turner and Varshaysky (2000) Science 289:2117-2220). Moreover, translation elongation factor 1-alpha (eEF1A) is required for the efficient degradation of nascent polypeptide chains, especially in ATP-depleting conditions, and in the presence of protein synthesis inhibitors (Chuang, et al. (2005) Mol. Cell. Biol. 25:403-413). eEF1A expression is increased in certain cancers, e.g, T-lymphoblastic cancer (Lamberti, et al. (2004) Amino Acids 26:443-448; Dapas, et al. (2003) Eur. J. Biochem. 270:3251-3262), a result that reflects a more general response to aberrant growth (Ejiri (2002) Biosci. Biotechnol. Biochem. 66:1-21).
BRCA1 and BRCA2 susceptibility factors have been linked to the Ub/proteasome pathway. BRCA1 contains a RING domain that binds Ub-conjugating enzymes and provides Ub-(E3) ligase activity (Cardoso, et al. (2004) Clin. Breast Cancer 5:148-157). The RING domain in BRCA1 can also bind a de-ubiquitinating enzyme, BAP1, and both proteins are coexpressed and colocalized (Orlowski, et al. (2003) Breast Cancer Res. 5:1-7). Mutations in the RING domain of BRCA1 abolishes its ability to function as an E3 ligase, and also blocks its interaction with BAP1, resulting in the loss of tumor suppressing properties of BRCA1. Moreover, the proteasome inhibitor, Velcade, has demonstrable efficacy in breast cancer (Ruffner, et al. (2001) Proc. Natl. Acad. Sci. USA 98:5134-5139; Hashizume, et al. (2001) J. Biol. Chem. 276:14537-14540).
Breast cancer represents one of the primary causes of death in women. This disease describes a range of defects, and the most prevalent forms include ductal carcinomas and local (in situ) or migrant (invasive/malignant) lobular-specific malignancies.
Prognosis for the patient is generally determined by the stage of advancement of the cancer. An important parameter that affects the type of treatment (mastectomy/chemotherapy/radiation therapy, etc.) is based on whether the cancer cells have infiltrated the lymphatic system. The aggregation of peripheral lymph nodes is considered an advanced state of disease. A critical and noteworthy fact is that breast cancer is eminently treatable, if it is detected early.
Currently, the most widespread methods for detection rely on breast self-examination and periodic mammography. However, a typical self-examination is imprecise, and it is unclear how faithfully the population adheres to the recommendation of monthly exams. Nonetheless, the self-exam can reveal abnormal growth of approximately one inch, while a mammogram can reveal smaller masses of aberrant growth.
Another non-invasive method that can detect smaller growths involves ultrasound. The feasibility of MRI-based approaches has also being investigated. Unfortunately, none of these methods is specific, since they do not distinguish between benign and cancerous growth. This is an important consideration because ˜80% of biopsies are considered benign following pathological examination. Because early detection is critical for improving the prognosis for the patient, the availability of diagnostic methods that can identify abnormal growth early, and distinguish between cancer and non-malignant growth is crucial.
There is a general lack of biochemical and molecular methods for early diagnosis of breast cancer. Two methods that are currently available are based on polymerase chain reaction (PCR). For instance, the identification of mutations in the gene encoding the BRCA1 gene is currently in practice. However, these mutations, which are present in familial forms for breast cancer, affect fewer than 5% of patients. A second method is to measure the expression levels of the Her2 gene, which is indicative of increased potential for developing breast cancer. This marker is also representative of a minor fraction of patients. Since most incidence of breast cancer occur spontaneously, without a known genetic/familial origin, a proteomic method that can reveal altered expression of specific proteins, rather than genetic alterations (which affect only a subset of patients), is desirable. Ideally, the proteomic approach reveals changes in both familial and spontaneous classes of breast cancer. With a combination of genomic and proteomic diagnostic assays, early detection of breast cancer can become much more reliable and routine.