Protein profiling has existed in one form or another for several decades and has its origins in enzyme purification strategies of the mid 20th century.
The advent of computational technologies in biomedical science has catalyzed the development of myriad high-throughput experimental platforms and the birth of the “omics” age.
The proteome represents the identity, expression levels, interacting partners, and post-translational modifications of proteins expressed within any given cell. Global protein profiling—or proteomics—aims to census the quantitative and qualitative factors regulating the biological relationships of proteins acting in concert as functional cellular networks.
In addition to the broader, discovery-driven scope of proteomics, however, more focused protein-profiling approaches are still widely used to validate hypotheses and add finer brush strokes to the mechanistic and functional characterization of protein biology. This year’s “American Association of Cancer Research” conference included sessions focusing on the application of protein-profiling techniques and strategies for basic and translational aspects of cancer biology.
“Chemotherapeutic sensitivity encompasses both sensitivity of the tumor resulting in response to therapy and sensitivity of the individual to the toxic side effects of the chemotherapy,” said Eileen Dolan, Ph.D., professor of medicine at the University of Chicago.
“Identifying individuals with genetic variants that make them more sensitive to a particular adverse event associated with chemotherapy, or less likely to respond, will help clinicians provide alternative therapies or a lower dose of chemotherapy to prevent toxicity.”
Dr. Dolan’s group is interested in characterizing the role of transcription factors and signaling molecules in mediating chemotherapeutic sensitivity, with a view of ultimately harnessing these proteins as novel therapeutic leverage points. Dr. Dolan’s colleague, Richard Jones, Ph.D., an assistant professor in the University of Chicago’s Ben May department for cancer research, described a novel technique, referred to as microwestern, that the group is using to identify candidate proteins.
“Microwestern technology can be thought of as DNA expression arrays for proteins with a twist,” said Dr. Jones. “The twist is that biological samples examined with the platform undergo a chromatographic separation following their deposition onto microwestern gels, whereby small proteins migrate through a gel matrix at a quicker speed than do large proteins.”
While DNA microarrays work well through the exquisite selectivity afforded by the predictable complementary base pairing between labeled mRNAs and immobilized DNA oligonucleotides, there is no comparable complementarity for proteins, and unique affinity reagents must be designed specifically for each protein that a researcher wishes to pursue.
Dr. Jones cited two disadvantages of existing Western-based protein profiling that microwesterns address. “Antibodies obtained through traditional processes typically cross-react to some degree with proteins other than their intended targets. In addition to this, standard Western blots require the use of large amounts of cellular material, large amounts of antibody reagents, and a large amount of human intervention.
“By electrophoretically resolving microarrayed cell lysate proteins through a gel matrix, protein isoforms representing intended targets can be visually discerned from unintended targets. Microwesterns also reduce starting material and antibodies required by up to two orders of magnitude.” Dr. Jones added that microwesterns can outperform mass spectrometry-based methods in metrics of sensitivity and reproducibility of detection of low-abundance proteins.
“The ability of the microwestern array to measure the levels of large numbers of unique proteins across populations of individuals makes it a wonderful platform for the identification of variants that affect protein function,” noted Drs. Jones and Dolan.
The group applied the technology in a study that evaluated the relationship between protein abundances, genetic variation, microRNA and mRNA expression, and chemotherapeutic-induced cytotoxicity and apoptosis in lymphoblastoid cell lines. They identified over 70 proteins whose levels were associated with cytarabine-induced cytotoxicity and apoptosis, knockdown of one of which, ATF2, was associated with a restoration of apoptosis in response to the drug in the cell lines.
VEGF and Drug Resistance
VEGFA (or simply VEGF) is a master regulator of angiogenesis, a critical process in tumor development, and is well established as a mitogen, survival factor, and permeability factor for endothelial cells. A group led by Gillian Tozer, Ph.D., of the University of Sheffield is using mouse models of cancer to shed light on the role that VEGF isoforms play in mediating resistance to therapeutic regimens.
“The most common splice variants in mice are VEGF120, 164, and 188, and these different isoforms have unique roles in tumor vascular maturation processes. For example, VEGF188 has been found to be associated with a relatively mature tumor vasculature compared with the other main isoforms in the mouse,” Dr. Tozer explained.
Her group recently found that tumor expression of VEGF120 in mice makes tumor vasculature particularly susceptible to damage by the tumor vascular-disrupting agent, combretastatin A4 phosphate (CA4P) but, compared to VEGF188-expressing tumors, resistant to radiotherapy.
To identify potential players mediating these discrepancies, the group carried out differential proteomic profiling of wild-type and VEGF isoform-specific tumor cell lines and solid tumor lysates using commercial angiogenesis and phosphoreceptor tyrosine kinase arrays (R&D Systems) combined with iTRAQ technology (Life Technologies).
They found that fibroblast growth factor (FGF-2), neuropilin 1 (NRP1), and platelet-derived growth factor receptor (PDGFR) were upregulated in VEGF188-specific tumor cells, while Neuropilin 2 (NRP2) was downregulated in the same tumor extracts relative to VEGF164 and VEGF120. While stressing that VEGF-based therapeutic strategies are in the early days of development, Dr. Tozer does see promise ahead.
“In Roche and Genentech’s AVAGAST (gastric cancer) trial, the ELISA was more sensitive to low molecular weight VEGF than 165 and 189, and when used to screen blood samples, was capable of predicting which patients would benefit from bevacizumab treatment, i.e., the higher the levels, the more benefit,” she pointed out.