Studying EMT in Breast Cancer
In addition to global proteomic screening strategies, protein profiling also encompasses more focused and informed experimental strategies. Epithelial to mesenchymal transition (EMT) refers to the process by which epithelial cells lose their cell-cell junctions, exhibit spindle cell morphology, and acquire increased cellular motility.
In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer invasiveness, metastasis, and relapse. A group led by Randolph Elble, Ph.D., of Southern Illinois University’s School of Medicine is focusing on the role played in EMT by members of the hCLCA family of calcium-activated chloride channel regulators.
In addition to the role suggested by their name, these proteins also function as secreted metalloproteinases, and their loss indicates an increased risk of metastasis. “Our studies were based on observations of apparent EMT in knockdown cell lines,” he explained.
“To determine whether attenuation of CLCA2 and 4 really caused EMT, we assessed expression levels of proteins associated with epithelial or mesenchymal phenotypes by Western blot.”
The transition from epithelial to mesenchymal phenotype takes about two weeks following knockdown of CLCA2 or 4, and can be confirmed by observing delocalization of E-cadherin from cell-cell junctions. “We could quantify proteins over a wide range of expression using a Li-Cor Odyssey fluorescent scanner,” said Dr. Elble.
The group found that loss of these proteins is associated with transition to a mesenchymal phenotype in breast cancer, raising an inherent paradox: “Most metalloproteases promote the very processes that CLCA2 and 4 inhibit,” Dr. Elble pointed out.
He anticipates that the work may have considerable clinical payoff. “Clinically, we found that the loss of CLCA2 expression in primary breast tumors presages a greater risk of metastasis, and we are currently investigating the therapeutic potential of the secreted form of the protease.”
Manipulating Protein Concentration
In the absence of a sufficiently specific and sensitive clinically approved biomarker for cancer, current methods for diagnosing neoplastic diseases are largely restricted to costly imaging-based procedures, such as computer-assisted tomography or magnetic resonance imaging.
Mauro Ferrari, Ph.D., is president and CEO of the Methodist Hospital Research Institute (MHRI) and professor of internal medicine at Weill Cornell Medical College. Dr. Ferrari and his colleague, Tony Hu, are developing a novel technology that enriches low molecular weight proteins for biomarker discovery.
“Low molecular weight and abundance proteins are excellent candidates for biomarker development, but these proteins can be a billion-fold less concentrated than the most abundant proteins,” said Dr. Ferrari. “In other words, highly abundant proteins often interfere in the detection of lower abundant proteins.”
By precisely controlling the pore geometry and surface chemistry, the MHRI/Weill label-free nanopore-based assay can selectively sort and concentrate proteins of interest from blood samples. Tumor samples are spotted onto a nanoporous silica layer, in which small peptides are trapped, while the larger proteins are removed from the surface after washings. Captured molecules can then be eluted from the pores and analyzed by MALDI TOF MS.
“With no need for additional sample-preparation steps or invasive procedures, this platform provides a rapid, reliable, and low-cost method for novel biomarker discovery,” Hu said.
The nanopore assay has been used to identify subtle changes in low molecular weight protein expression signatures using negative control and diseased serum samples collected from imaging-proven stage I–III cancer mice models, and the application of mass-spectrometry allows for the identification of proteins unique to different stages of cancer development.