Abstract C21: A novel protein microarray analysis for the identification of protein ubiquitination in cancer
Authors: Grotegut Stefan , Rogers Jeff , Sangfelt Olle , Spruck Charles H. 
Published: May 2012

Ubiquitination is a post-translational modification wherein a small (8 kDa) and highly abundant protein called ubiquitin is covalently attached to specific protein substrates in cells. This “glinkage” plays an important role in the regulation of many cellular functions including division, differentiation, protein trafficking, response to DNA damage, and gene transcription, among others. A lysine 48-linked chain of four or more ubiquitins targets a substrate for degradation by the 26S proteasome. A wealth of experimental evidence supports the notion that derangement of ubiquitin-mediated proteolysis contributes to various human malignancies including breast cancer.

During metastasis, breast tumor cells undergo a process that is similar to the epithelial-mesenchymal-transition (EMT). This process is characterized by multiple steps ultimately enabling breast tumor cells to leave their place of origin and to colonize new organs. Little is known about the intrinsic ubiquitination activity during this process and how the ubiquitination pattern differs in metastatic cells compared to their non-malignant counterparts. Furthermore, there are no adequate experimental tools to identify the ubiquitination of protein substrates on a proteome-wide scale. The ubiquitin-proteasome system has been recognized as a valid target for the treatment of some malignancies (e.g. Bortezomib), thus deciphering the change of tumor cells' intrinsic enzymatic activity could warrant for the development of enhanced diagnostic tools and effective treatment of breast cancer patients.

To overcome this shortcoming, we have developed an innovative approach that combines protein microarrays (where &gt;8,300 human recombinant proteins are spotted onto glass microscope slides) with an ubiquitination reaction to determine novel ubiquitinated substrates during EMT. We obtained breast tumor material using the 4T1 mouse model of breast cancer, which consists of five isogenic murine mammary carcinoma cells lines (67NR, 168FARN, 4TO7, 4T1, and 66cl4) that spontaneously arose from a single mammary tumor in BALB/c mice and exhibit differing metastatic abilities resembling the multi-step process of EMT. Lysed tumor tissue was subsequently analyzed for a change of the ubiquitination signature.

More than 230 alterations in specific target protein ubiquitination were detected in the cell lines used, indicating a possible regulatory role of protein uniquitination during the process of breast cancer progression. The identification of the biological consequences of some of these protein modifications will aid in gaining more insight into how the cells' intrinsic enzymatic activity has become distorted and how this could drive the metastatic process of breast cancer. Our novel identification of target protein ubiquitination could ultimately serve as a diagnostic and prognostic tool and refine the regimens for breast cancer therapies.

(This work is supported by California Breast Cancer Research Program grant CBCRP-14FB-0114 to S.G.)

Citation Information: Cancer Res 2009;69(23 Suppl):C21.