3.2. Identification of Altered Protein Production Between Progression States
Based on comparative analysis of the proteomes of the three PHEC cell lines, the liquid fractions were identified for further characterization using for bottom-up proteomics. Retention time was used to determine the well address for each fraction of interest, and comparisons were made only between the exact two-dimensional positions between proteomes. The selected samples underwent tryptic digestion and mass spectrometry for protein identification. In , the circled differences were identified in this manner, and a condensed summary of proteins identified is given as . Full quantitative assessment of total expression levels of proteins was not performed. The identification and expression of proteins discussed refers to relative qualitative expression between cancer progression states.
Table 1 A summary of proteins identified by mass spectrometry after separation by 2D HPLC. Information pertaining to location in first and second dimension plates of the PF2D run, MASCOT score, accession number and number of identified peptides is also given (more ...)
An overview of reveals that the some proteins have been identified in more than one sample, or at multiple elution points during the 2D chromatography separation process. This can be understood by classifying the identifications and localizations into three general groups: 1) proteins that are localized to the same first dimension fraction but have different retention times in the second dimension, categorized as hydrophobic effects, 2) proteins in adjacent first dimension fractions having similar retention times in the second dimension, categorized as focusing effects, and 3) proteins with the same identification that eluted in different fractions in both first and second dimensions, categorized as modification effects. As these separations use liquid chromatography to resolve complex protein mixtures, the resulting protein fractions have a range of elution points, similar to a normal distribution curve. The fractionation ranges possible due to hydrophobic and focusing effects are compounded by the two separate dimensions of the PF2D separation, creating mixtures of proteins with multiple final endpoints.
Proteins eluted from the PF2D were fractionated by defined intervals, using pH units for the first dimension and time for the second dimension. If a protein began to elute at a pH or time near the end of one fraction qualifier, it could finish in a second fraction qualifier, thereby splitting the protein into multiple fractions. This can be confounded further if the protein elutes into two fractions in the first dimension and two in the second dimension, essentially allowing it to be identified in four separate fractions. Depending on the biochemical and conformational properties of the protein, elution can occur across a very narrow fraction range or a wider one. In this study, one example of impact of these effects was the protein eukaryotic translation elongation factor (eEF1A1). EEF1A1 was found in fractions 13, 17 and 18 (), indicating elution over a 1.5 minute period, from retention times 16.5–18 minutes. Since the second dimension collects a fraction every 30 seconds, eEF1A1 was identified in 3 separate fractions. This is also an example of a protein with focusing effects, as it is found in first dimension fractions 12 and 13 (pI 7.4–7.7 and 7.1–7.4, respectively).
Figure 3 Sites of PF2D where eukaryotic translation elongation factor (eEF1A1) Proteomic difference maps for the comparison of the PHECNT, PHECT and PHECM cell lines show the PF2D profiles for the respective cell lines from the pI range of 4.0 to 8.3. Between (more ...)
Proteins with multiple elution points can also originate from modification effects, the third classification. This general term refers to proteins with structural or conformational changes that impact the chemistry of separation. Included in this category are proteins with various post-translational modifications (PTMs) different isoforms of one protein, analogous proteins, and proteins with point mutations. PTMs and point mutations can lead to subtle or drastic changes in pI, depending on the amount and nature of the alteration15
. Proteins that have non-covalently bound species, such as histones, experience an overall change in pI due to molecular complexation. Protein degradation products are also commonly identified in proteomic analyses. While samples were lysed with buffer containing protease inhibitors to prevent proteolysis during sample preparation, protein degradation occurs regularly within cells, so degradation products were expected. Degradation can lead to changes in tertiary or quaternary structures, so a single degraded protein could have several conformational structures present within a sample. Each conformation interacts with the chromatographic column matrix and mobile phases with slight variations. Depending on the level of degradation present, a single protein could be identified, but with multiple elution points due to changes in chemical interactions with the columns used in the PF2D separation. Similarly, each fraction contains multiple proteins in the same eluent, so all comparative analyses are based upon MS/MS identification of single proteins within complex mixtures.
Further analysis of the results in indicates that, in most cases, more than one protein was identified in each fraction. This can be attributed to the biochemical properties of the protein separation and fraction collection methods. Because the separation involves two-dimensional chromatography, as opposed to a traditional single dimensional separation, the elution parameters cannot be optimized simultaneously for both separation chemistries. However, as the chromatography was designed to separate proteins for the subsequent identification by mass spectroscopy, the separation parameters do not need to resolve individual species per peak or fraction collected. Multiplicity of proteins within peaks is balanced by the power of the detection system, so the separation method serves only to enhance the resolution and sensitivity of the subsequent identification.
There are multiple advantages to this technique over more routine methods, such as single dimension chromatography or gel electrophoresis. The basis of the separation is liquid chromatography, so samples are injected in solution and collected as fractions of eluent. This allows the proteins to remain in solution throughout the full separation process, eliminating dry-down and reconstitution steps that can lead to sample loss. The liquid-based separation procedure allows for preservation of PTMs for subsequent identification. Collecting fractions in solution leads to characterization of modifications directly by mass spectrometry, providing a more accurate picture of the nature of the proteins within the cells of origin. This creates a more reliable picture of the proteins within the cells than can be generate from gel-based separation techniques routinely used in proteomic evaluations.
In addition to the advantages for characterizing whole proteins, the use of two-dimensional chromatography for proteomic separations introduces a different set of proteins into the proteomes being studied. While gel-based separations exclude high mass, aggregated, and hydrophobic proteins, such as those found in cell membranes and within subcellular architecture, solution-based separations allow the analysis of all cell lysate proteins injected into the chromatograph. As such, a different subset of the proteome becomes available for identification and characterization.
The comparative analysis of the three proteomes of the PHEC cell lines showed identifications of proteins from a variety of cellular locations. As shown in , proteins typically associated with the nucleus increased as the cells progressed toward metastasis. The overall complexity of distribution of protein localizations within the cell changed dramatically as cancer progressed. Cells in the non-tumorigenic state localized to 9 sites, while tumorigenic cellular proteins occurred in 12 locations. Interestingly, the metastatic cellular proteins identified were localized to only 5 cellular places. The combination of the inclusion of higher mass and hydrophobic proteins with the ability to evaluate PTMs and binding partners introduces a powerful tool to the understanding of biomolecular interactions within the cell membrane and within the architecture of the cell. This information would not be available with gel-based methodologies.
Figure 4 Subcellular localization breakdown of identified proteins. Proteins in categories with one or more (+) in them are uniquely counted in that category and not the individual categories comprise up that category. A) Subcellular localization of total proteins (more ...)
With these techniques, we generated a proteomic comparison of the three cancer progression states of the PHEC model. The highest frequencies of proteins identified in the fractions of interest between the three cell lines were members of the cytokeratin family (KRT), filamentous protein polymers responsible for the structural integrity of epithelial cells. Others have shown evidence for the correlation between changes in cytokeratins and cancer progresses to a malignant state. In PRCA, cytokeratins have shown differential expression from human prostate tissue depending on the progression state. A tumor-associated species, specifically present in tumors and absent from normal and most benign prostatic hyperplasia samples, was identified to be a proteolytic fragment of KRT8 that showed a mass reduction and a change in pI value16
. Extensive fragmentation of KRT molecules by these proteases has been suggested to support the participation of cytokeratins in malignancy17
In the PHEC model, cancer progression across the three states showed decreased total expression of keratins, with 42 identifications in 21 fractions of the non-tumorigenic cells, 41 identifications in the same fractions of tumorigenic cells, and 17 identifications in those of metastatic cells. Of the identifications made in the chosen fractions, the non-tumorigenic cells displayed 30 different types of keratins, while only 15 and 9 types of keratins were displayed in the tumorigenic and metastatic cells, respectively, as shown in . Their identities are given in . This level of change in keratin production and variety as cells morph from a healthy state into metastatic cancer cells poses an interesting point of speculation regarding the role of keratins in cancer progression. Keratins perform a predominantly structural role, aligning into large aggregate assemblies within cells. Our data showed the progression to malignancy in the PHEC cell lines leads to altered and inconsistent morphology within tumorigenic and metastatic cell cultures. The observed morphological changes could be the result of a limited expression of keratins, or to the introduction of keratin expression types not typically found in the normally replicating cells. These findings support extensive reports in the literature of the phenotypic changes cells undergo as they transform into fully malignant cancer forms.
Figure 5 A comparative analysis of the distribution of histones and keratins within the three PHEC model cell lines. The colors within each bar represent the number of identifications made in the fractions characterized for each cell line. The size of each colored (more ...)
The second most prevalent protein family found in the comparison of the three progression cell lines was the histone family. These proteins showed a trend opposite to that of the keratin family, with histone expression and variety increasing across the progression states toward metastasis, illustrated in . Seven individual histones representing 6 histone types were identified in the non-tumorigenic cells, as opposed to 20 identifications representing 9 types in the tumorigenic state and 23 identifications representing 10 types in the metastatic state. Histones are predominantly responsible for packaging chromosomes. Most importantly, histones play a crucial role in epigenetic variations, and they are drastically altered in cancer formation. One of the key mechanisms of tumor formation is the silencing of genes associated with cancer by epigenetic regulators18
, and epigenetic variations have been suggested to be indicative of disease prognosis, including prostate cancer19
. Histones are able to alter the degradation processes and signaling mechanisms of the healthy cell through their own modifications, fundamental cellular changes that could lead to cancer progression.
Of those proteins identified from the comparison of the three cancer states, individual proteins of interest have shown to be differentially expressed between the progression states and have previous associations with cancer in the literature. Found in fraction 3, maspin (SERPINB5) is a member of the serpin super family of proteins, known for their antiprotease activity. This protein was discovered through its tumor suppressive properties. Maspin is a non-inhibitory serpin showing importance in preventing metastasis in breast20
cancers. In the PHEC system, the maspin protein elutes at only one pI (6.1–6.4), indicating only one form present. Maspin shows a clear reduction in concentration as the cells develop into cancer and metastasize (). The initial reduction in UV peak area seen in the PF2D was confirmed using immunoblot analysis, as shown in . This behavior reflects that observed in the literature and substantiates the potential of maspin as an indicator of the transformation from tumorigenic to metastatic cancer.
Figure 6 Identification and verification of a difference in abundance of the serine protease inhibitor MASPIN. A) Overlaid chromatograms of the fraction containing MASPIN in the three samples. The arrow indicates the peak associated with the identity. B) Sequence (more ...)
Another protein differentially expressed between the three cancer states is HSP70. The HSP70 proteins are a family of proteins, including the two major cytoplasmic isoforms heat shock protein 70 (HSP70) and heat shock 70 kDa protein 8 (HSC70), that assist in a broad set of functions as part of the cellular machinery for protein folding and protection of cells from stress. As one of the chaperone proteins, HSP70 may have a role in cancer because it has been found in increased abundance in cancer cells, though not as a reliable marker to predict the reoccurrence of cancer22
. The poor diagnostic ability of HSP70 in cancer evaluations may be caused by the failure to monitor the precise species. In the PHEC system, both HSP70 and HSC70 were identified. In the PHECNT
cells, HSP70 and HSC70 were identified separately in multiple fractions. In the PHECT
cells, HSP70 and HSC70 were identified together in the same two fractions. However, in the PHECM
cells, only one fraction contained both HSP70 and HSC70. The presence of HSC70 in the metastatic cells corroborates previous findings that HSC70 is over-expressed in the metastatic state23
. Overall, the presence of multiple fractions indicates differing forms of the HSP proteins, suggesting there might be post-translational modifications on these proteins that could change their cellular functions throughout the progression of prostate cancer.
Elongation factor 1 alpha (eEF1A) is also found in several places in the comparative proteomes of the three PHEC cell lines. Physiologically, eEF1A has shown expression in human prostate tumors, while absent in normal human prostate tissue24
, and recently has been shown to interact with phospho-Akt to regulate proliferation, survival and motility of breast cancer cells25
. In addition, overexpression of eEF1A has been demonstrated in cell lines modeling pancreatic cancer, leukemia and osteosarcoma26
. In our PHEC model of prostate cancer progression, eEF1A occurs in all three progression states at multiple pI and hydrophobicity levels, as shown in . Many PTMs have been shown on eEF1A27
, which could explain the various forms eluted in the separations.
The identification of these selected proteins of interest from the comparative analysis of the cancer progression showed one striking feature among all them: the presence of multiple elution points from the two-dimensional chromatography separations employed. As previously described, this could indicate the presence of post-translational modifications, leading to considerations of the purpose of these PTMs. Proteins generally undergo PTMs for regulation of cellular processes. The PHEC model undergoes the full progression of PRCA from non-tumorigenic cells to metastatic cells, so changes in the modifications of proteins across this progression could be suggestive of the regulatory role of the proteins evaluated in the development of cellular metastasis.
The key to understanding the mechanism of cancer metastasis lies in the ability to capture a snapshot of the cell at rapid time intervals as the cells actively convert to the metastatic state, introducing a possible kinetic model of cancer metastasis not previously reported. This allows the disassembly and evaluation of individual aspects of the cell at each time point to consider individually the changes occurring simultaneously physiologically, rather than following only a single molecule or group of species of interest. Gathering such a “cellular snapshot” provides a more complete analysis, allowing a characterization of the molecules and organelles performing together to lead the cell to a metastatic endpoint. While this scenario sounds ideal, it is not trivial experimentally. Such an undertaking requires the ability to capture cells directly at successive time points, from which the lysates undergo total cellular analysis for protein changes. In addition, the cells studied must be capable of transforming fully from healthy prostate cells to metastatic cancer cells.
The purpose of this work is to introduce the model and methodologies by which a “cellular snapshot” study of human prostate cancer metastasis will begin. The PHEC model provides the necessary cellular component – humanized in vitro cell lines with identical genetic backgrounds that provide the full cancer progression to metastasis. The origin of all three PHEC cell lines from the same genetic source provides uniformity to the analysis, eliminating the variable background proteins unavoidable when multiple cell lines are used to cover the full cancer progression. This allows much more sensitive detection of subtle changes in proteins, such as PTMs and isoforms, that could be key facets to understanding metastatic mechanisms.
The combination of the PHEC human prostate cancer model system with an alternate proteomic analysis using two-dimensional HPLC/MS/MS introduces a new aspect to cancer research. This concept could provide a fundamentally unique perspective on the manner in which cellular mechanics are studied. More importantly, these tools together provide a model for human prostate cancer research that has not been studied previously, visualizing differences in individual proteins from the milieu of cellular components changing as cells metastasize.
- Proteomic characterization of a novel in vitro prostate cancer model system
- Progressive cancer states in vivo: non-tumorigenic, tumorigenic, and metastatic
- differential protein expression using PF2D followed by mass spectral identification