One of the unpleasant survival tactics shown by cancer cells is their ability to develop resistance to previously efficacious chemotherapy. Scientists know this happens in part from kinome reprogramming, where the malignant cell increases kinase activity in enzymes unaffected by treatment, radically altering behavior and activity. When resistance occurs, oncologists switch to a broad-spectrum chemotherapy regime in an attempt to avoid further deleterious development. Unfortunately, this is often associated with debilitating side effects.
Instead of blasting cancer cells with broad-spectrum drugs, the use of a targeted approach personalized to the individual malignancy seems sensible; however, this requires a detailed characterization of the tumor to determine cell susceptibility. Some of the most promising targets for consideration are the plasma membrane proteins. These proteins—usually receptors, including tyrosine kinases—regulate cell adhesion, proliferation and migration, all key factors in cancer pathogenesis. Their positioning on the outside of the cell also means they are highly accessible to drugs.
Researchers have already found that by targeting relevant plasma membrane proteins, they can decrease metastatic spread and decrease tumor growth. Combining specific, targeted treatments works well in prostate cancer1 and melanoma,2 with effective tumor control.
Ziegler et al. (2014) characterized plasma membrane proteins in several breast cancer-derived cell lines.3 They used SK-BR-3 cells that overexpressed the ErbB2 receptor, and triple-negative MDA-MB-231 cells that did not contain estrogen, progesterone or ErbB2 receptors. They compared them with the benign MCF-10A cell line. The research team chose to work with cell lines rather than tissue in order to avoid tumor heterogeneity and also to maximize sample availability.
The team grew the cell lines in standard culture conditions before harvesting them and using differential centrifugation to purify plasma membranes preparations. Once separated, the plasma membrane preps underwent trypsin digestion followed by MudPIT (Multidimensional Protein Identification Technology) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. The research team prepared the MudPIT column in-house and completed proteomic analysis using an LTQ Orbitrap XL hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific).
Using the SK-BR-3 cells, Ziegler and co-workers used Western immunoblotting to verify that the plasma membrane preparation procedure excluded proteins from other cellular locations. Once satisfied, they proceeded with proteomic analysis. In total, the researchers identified 13,650 plasma membrane proteins, ranging from 7,075 in DT22 cells to only 5,660 in the benign control MCF-10A cell line. Characterizing the proteins found among the cell lines, the scientists observed that the malignant cell lines shared more plasma membrane proteins with each other than they did with the control cells. They confirmed this finding with an analysis (using PatternLab software) of the derived proteomes, which showed a high degree of similarity among the malignant cell types.
Further examination using a semi-quantitative proteomics approach based on spectral counts yielded plasma membrane protein groups showing differential expression with malignant phenotype. The researchers confirmed these data using Western immunoblotting, immunofluorescent staining to show cellular abundance and localization, and RT-PCR.
Ziegler et al. grouped the proteins into specific clusters, focusing on those already known to play a role in oncogenesis. These protein groups included tyrosine kinases, major histocompatibility complex class-I proteins, cell adhesion molecules, G-protein coupled receptors, and cytoskeletal proteins.
In summary, the authors propose that their data show evidence that breast cancer cells evolve to promote growth and metastatic spread. Moreover, the results seen in this study are similar to those observed in other types of cancer, suggesting a commonality in cell strategy during malignant transformation. In developing novel therapeutics, the authors strongly advise that, for personalization of an individual patient’s onward treatment, typing of the tumor is the proper route.
1. Smith, D.C., et al. (2013) “Cabozantinib in patients with advanced prostate cancer: Results of a phase II randomized discontinuation trial,” Journal of Clinical Oncology, 31 (pp. 412–19).
2. Cooper, Z.A., et al. (2013) “Combining checkpoint inhibitors and BRAF-targeted agents against metastatic melanoma,” Oncoimmunology, 2 (p. e24320).
3. Ziegler, Y.S., et al. (2014, July) “Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment,” PLOS ONE, 9(7) (p. e102341), doi: 10.1371/journal.pone.0102341.
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