Global Analysis of Protein Ubiquitination in Breast Cancer

Institution: Sidney Kimmel Cancer Center
Investigator(s): Stefan Grotegut, Ph.D. - Stefan Grotegut, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14FB-0114 Award: $45,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

This is a collaboration with: 14FB-0114A -

Initial Award Abstract (2008)

Ubiquitin is a small protein found in all eukaryotic cells. Its main function is to “mark” other proteins for destruction, a process known as proteolysis. Normally the ubiquitinated protein is recognized by a protein complex called the proteasome, a barrel-shaped structure where the proteolysis occurs. In terms of cell physiology, ubiquitination plays an important role in the regulation of many functions including division, differentiation, protein trafficking, the response to DNA damage, and gene transcription, among others.

A wealth of experimental evidence supports the notion that changes in ubiquitin-mediated proteolysis contribute to various human malignancies, including breast cancer. However, there are currently no adequate experimental tools to identify the substrates of enzymes involved in protein ubiquitination on a proteome-wide (i.e. global) scale.

This project undertakes a completely new systematic approach to identify proteins that are subject to ubiquitin-mediated proteolysis and to unravel the relevant protein ubiquitination involved in breast tumor progression and metastasis. To achieve this, we have developed an innovative assay that combines protein microarrays (where >8,300 human recombinant proteins are spotted onto glass microscope slides) with an ubiquitination reaction to determine novel ubiquitinated substrates. If successful, this method will enable us to define key molecular changes in a breast tumor cell’s intrinsic enzymatic activity that drives the metastatic process. We plan to place particular emphasis on the ubiquitination processes leading to the epithelial-mesenchymal transition (EMT), a precursor step of metastasis, and the change of cell-cell and cell-matrix adhesion. In our planned studies we will first obtain mouse breast tumor specimens from mammary cancer cell lines with distinctive metastasizing potential and define changes in ubiquitin ligase activity associated with breast tumor progression. Next, we will use biochemical and cell-based assays to validate the connection between ubiquitin ligase activity and its substrates. We hope to establish a mechanistic model for changes in the ubiquitination pattern occurring during breast cancer metastasis.

The successful employment of our innovative protein microarray ubiquitination assay in these endeavors could lead to an understanding of how protein-ubiquitination drives breast cancer progression. This would undoubtedly contribute to a better understanding of basic cellular function and the molecular events that ensue as breast epithelial cells become metastatic. The ubiquitin proteasome system has been recognized as a valid target for the treatment of some malignancies (e.g. Bortezomib), so it is our hope that this research will lead to the discovery of a novel set of diagnostic and prognostic markers based on the tumor cell’s intrinsic enzymatic activity, which could greatly impact the diagnosis and effective treatment of breast cancer.




Final Report (2011)

Despite the tremendous efforts in elucidating the causes of breast cancer, there clearly are gaps in our understanding of the molecular features special to metastatic breast carcinomas. In particular, the breast tumor cells' intrinsic enzymatic activities that support their motile and invasive behavior are complex and poorly defined. Among these are post-translational protein modifications that severely alter the function, localization and lifetime of proteins.

Ubiquitination, a process in which the small and abundant protein, ubiquitin, is attached to target proteins, regulates a variety of cellular processes including apoptosis, cell division, DNA transcription and repair, differentiation, development, and modulation of cell surface receptors, among others. It has previously been demonstrated that a change of ubiquitination activity is associated with breast tumorigenesis and might also be involved in breast tumor progression. However, insight into how dysregulation of ubiquitin in conjugation with other proteins contributes to breast cancer has proven problematic primarily due to the lack of appropriate tools capable of detecting these activities in complex biological specimens on a proteome-wide level.

In this research project we applied a novel high-throughput screen in which protein micro arrays (glass slides with >9,000 human recombinant proteins spotted in duplicates) are overlaid with a cocktail containing lysates from primary breast tumor samples and ubiquitination reaction buffer, to identify changes of ubi quit in conjugating activity associated with the transformation of breast tumor cells to metastatic entities. Several differences in target protein ubiquitination could be identified and biochemically validated, indicating a key regulatory role of protein ubiquitination during the process of breast tumor progression. Furthermore, the ubiquitin conjugating activity captured by the protein micro arrays could be linked to an ubiquitination signature unique to the metastatic status of breast tumors.

The results of this study are warranting for further investigations of the biological consequences of some of the newly identified posttranslational modifications. This is an important step since it and will ultimately help to not only unravel the molecular mechanism of breast cancer metastasis but to also identify advanced therapeutic regimens, which collectively will help to lower the mortality associated with the disease. Furthermore, knowledge gained from this study could also be translated into novel tools for diagnostic and prognostic biomarkers leading to earlier detection of breast cancer, which will enhance the effective treatment of breast cancer patients.




Symposium Abstract (2010)

Stefan Grotegut, Sonia del Rincon, Jeff Rogers, Charles H. Spruck

This project is aimed to identify changes in the breast tumor cell’s intrinsic enzymatic activity that facilitate these cells to leave their place of origin and colonize new organs (i.e. metastasize).

During metastasis, breast tumor cells undergo a multi-step process ultimately enabling them to leave their primary site and spread throughout the body to find new niches. In this process, many proteins are being altered (‘post-translational modified’) and subsequently change their biological function, sub-cellular localization, or stability. One of the well-known post-translational modifications is ubiquitination, in which a highly abundant protein called ubiquitin is covalently attached to specific protein substrates. This ‘linkage’ 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 wealth of experimental evidence supports the notion that derangement of ubiquitin-mediated proteolysis contributes to various human malignancies including breast cancer.

To date, little is known about how this ubiquitination activity can contribute to breast cancer cells’ invasive growth 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. Importantly, the ubiquitin-proteasome system has been recognized as a valid target for the treatment of some malignancies, thus deciphering the change of breast tumor cells’ intrinsic enzymatic activity could warrant for the development of enhanced diagnostic tools and effective treatment of breast cancer patients.

To assess the breast tumor cell’s ubiquitination activity, we have developed a novel proteomic approach by combining protein microarrays (where >8,300 human recombinant proteins are spotted onto glass microscope slides) with an ubiquitination reaction of cellular extracts. This innovative technique is capable of identifying substrates of ubiquitination in breast tumors as they progress to a malignant phenotype. Using the 4T1 mouse model of breast cancer, which consists of five isogenic murine mammary carcinoma cells lines at different metastatic levels, more than 100 alterations in specific target protein ubiquitination were detected, indicating a possible regulatory role of protein ubiquitination during the process of breast cancer progression.

The identification of the biological consequences of some of these protein modifications is currently being investigated and will aid in gaining insight into how the cells’ intrinsic enzymatic activity has become distorted and how this could drive the metastatic process of breast cancer. It is our hope that this study will greatly impact the early diagnosis and the refinement of breast cancer treatment and prevent breast cancer mortalities, which occur primarily due to metastasis.