Plasminogen activator (PLAU) is a serine protease that converts plasminogen to plasmin, a general protease, which promotes fibrinolysis and degradation of extracellular matrix. PLAU was reported in 1970s as one of the robustly induced enzymatic activities in Rous sarcoma virus (RSV)-transformed chicken cells. More than three decades later, with the completion of the sequencing of the chicken genome and the subsequent availability of Affymetrix GeneChip genome arrays, several laboratories have surveyed the transcriptional program affected by the RSV transformation. Interestingly, the PLAU gene was shown to be the most highly upregulated transcript. The induction of PLAU was a transformation-dependent process because viruses with deleted Src gene did not induce the transcription of the PLAU gene. Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer. Although the activity and structures of these two enzymes are well characterized, the precise molecular function of these gene products in signaling networks is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy, and PLAU gene and its inhibitor have been included as biomarkers with strong prognostic and therapeutic predictive values. This vignette reviews the history of PLAU and Src discovery, and illuminates the complexity of their relationship, but also points to their emerging impact on public health.
We are about to celebrate the centennial of the groundbreaking publication of Peyton Rous reporting on a virus-induced sarcoma in chickens (Rous, 1911). This is one of the seminal papers in biology, which marks the beginning of research that ultimately identified the first oncogene, Src (Stehelin et al., 1976; Brugge and Erikson, 1977; Hunter and Sefton, 1980).
Transformation of primary or established cells in vitro by Rous sarcoma virus (RSV) and RSV-induced tumor formation in animals continue to represent valuable experimental models of cancer (Martin, 2004). The transforming gene of RSV, the viral src oncogene (v-src), is an activated and overexpressed protein-tyrosine kinase responsible for a number of molecular events and phenotypic changes observed in transformed host cells and the virus-induced tumors (Jove and Hanafusa, 1987).
In the past, a number of laboratories reported identification of individual genes that were either induced or repressed by the v-src oncogene in transformed cells (Hendricks and Weintraub, 1981; Bedard et al., 1987; Sugano et al., 1987; Jahner and Hunter, 1991; Frankfort and Gelman, 1995). Plasminogen activator was reported in 1973 as one of the robustly induced enzymatic activities in RSV-transformed chicken cells, and the urokinase-type plasminogen activator (PLAU) gene was considered to encode the secreted enzyme (Unkeless et al., 1973). This work was reported by the laboratory of Edward Reich at The Rockefeller University in New York City, and later confirmed by numerous publications from various laboratories (Sudol, 1985; Bell et al., 1990; Leslie et al., 1990). More than three decades later, with the completion of the sequencing of mouse and chicken genomes and the subsequent availability of Affymetrix GeneChip genome arrays (Affymetrix, Santa Clara, CA, USA), several laboratories, including our team, have surveyed the transcriptional program affected by the RSV transformation (Masker et al., 2007; Maslikowski et al., 2010). We, and others, have confirmed the transcriptional induction of the PLAU gene and showed that it is the most highly upregulated transcript (Masker et al., 2007; Maslikowski et al., 2010). Now, we have decided to look at these data more closely, focusing on unique features and variations in the PLAU gene induction among different experimental models. Interestingly, we also observed several apparent paradoxes by examining viral Src-affected gene expression profiles. We discuss here several of these paradoxes, provide possible explanations, and suggest new experimental strategies that could solve them.
Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer (Martin, 2004; Hildenbrand et al., 2010). Although the activity and structures of these two enzymes are relatively well characterized (Xu et al., 1997; Carriero et al., 2009), the precise molecular function of these gene products in signaling networks and physiology is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy (Hennequin et al., 2006; Huang et al., 2007; Jallal et al., 2007; Saad and Lipton, 2009; Mayer and Krop, 2010), and PLAU has been proposed as a biomarker with strong prognostic and therapeutic predictive values (Annecke et al., 2008).
I would also like to add a touch of history here and bring to light a relevant but unpublished work of Dr Fritz Lipmann on sarcoma implants and plasma clot liquefaction. As a graduate student in the laboratory of Dr Edward Reich in the early 1980s, I had a chance to re-examine original experiments leading to the discovery of the PLAU induction by RSV (Sudol, 1985), and participate in the protein purification (Sudol and Reich, 1984) and complementary DNA cloning (Nagamine et al., 1983, 1984) of the mammalian PLAU protein and gene, respectively. Through my work on the PLAU gene, I became interested in the Src oncogene itself, and joined the laboratory of Dr Hidesaburo Hanafusa as a postdoctoral fellow. During the exciting transition from the Reich Laboratory to the Hanafusa Laboratory, within The Rockefeller University, I had the privileged opportunity to discuss the phenomenon of plasminogen activation with Dr Fritz Lipmann, who, after attending my thesis defense, wrote me a letter discussing his unpublished work on Rous sarcoma implants and a putative plasminogen activator activity (Supplementary Figure 1). The letter prompted our meetings and discussions. In this short review, I would like to mention the excerpts of my discussions with Dr Lipmann and make the content of the letter available to the readers of ‘Oncogene’ (Supplementary Figure 1). The original letter was deposited in, and is available from the Rockefeller Archive Center.
In sum, in this advanced age of systems biology, which is further accelerated by the increasingly powerful ‘omic’ approaches to physiology and pathology, I would like to tell a story of two cancer-associated and functionally interacting genes in order to illuminate the complexity of their relationship, but also their emerging impact on public health. This vignette is also a minute glimpse into the rich past of my alma mater—The Rockefeller University.
Viral Src induces PLAU gene at the level of transcription
As expected, the PLAU gene was shown to be induced by RSV at the level of transcription (Bell et al., 1990), and two recent reports with Affymetrix Gene chip analyses showed that in wt RSV Schmidt–Ruppin-strain-group-A (RSV-SR-A)-transformed chick embryo fibroblasts (CEFs), PLAU transcript was on the very top of the list of upregulated genes, being at least 40-fold elevated over the level found in cells infected with the non-transforming, control viruses of RSV-SR-A strain (Masker et al., 2007; Maslikowski et al., 2010). In fact, our study showed that the PLAU transcript was upregulated 196 (+/− 69)-fold, using Affymetrix Gene Chip arrays. We confirmed this result by a more precise measurement using real-time PCR tests on two independently isolated RNA samples as 42 (+/−11)-fold upregulation (Masker et al., 2007). Independently from our group, the laboratory of Pierre-Andre Bedard also documented that the PLAU transcript was the most robustly upregulated gene in RSV-transformed CEFs, being more than 44-fold increased over the level detected in non-transformed controls (Maslikowski et al., 2010). The induction of the PLAU transcript was considered v-Src gene- and transformation-dependent process because RSV-SR-A with deleted Src (td106) and the temperature-sensitive mutant of RSV (NY315), at non-permissive temperature, did not induce PLAU gene.
Interestingly, the PLAU gene was also induced by RSV infection in other primary cells, namely chicken neuroretinal cells (Maslikowski et al., 2010).
Explaining variability in genes regulated by RSV transformation
A couple of surprises emerged when we examined the transcriptional profile of transformation of chicken cells by RSV-SR-A. Apart from a dozen of robustly regulated genes, such as PLAU or NOV, which were identified and confirmed in the arrays performed by independent laboratories, many other genes identified as v-src-modulated transcripts showed significant differences in terms of their relative levels (Malek et al., 2002; Paz et al., 2004; Masker et al., 2007; Maslikowski et al., 2010). This could be not only due to differences in the experimental conditions used but also due to the differing time points of RNA isolation from cells after they reached morphological transformation. Perhaps a better solution would be to use a non-leaky, temperature-sensitive mutant of RSV, with many experimental time points for RNA isolation during the switch from non-permissive to permissive temperature and full transformation. Such an approach would provide a dynamic profile of transcriptional changes and most likely could explain at least some of the apparent differences in the current reports. For example, certain transcripts would be changed transiently but others would be changed constitutively. More complex profiles of modulation are possible as well.
Although not directly related to PLAU, another surprise came from the analysis of vasoactive intestinal peptide (VIP)-like transcript in RSV-transformed CEFs (Masker et al., 2007). First, the transcript was present in normal CEFs at the same level as that in RSV-transformed CEFs. However, the VIP-like gene as we would like to name it, scored as a significantly induced transcript because the infection with RSV without v-src repressed the VIP-like transcript. The role of viral proteins, especially Env (Maeda et al., 2008) and possibly Gag per se, on the regulation of gene expression deserves attention and careful evaluation in RSV-modulated transcriptomes. The second surprise was that the VIP-like transcript detected in RSV-transformed cells was coding for a protein of very large molecular weight of 52 kDa (Masker et al., 2007). We could not reconcile this large size even with the sequence of the VIP precursor. The biological role of the gene product encoded by the VIP-like transcript is intriguing, and could illuminate a new facet of virus-mediated neoplastic transformation.
Finally, when we used LA90 ts RSV-transformed mouse 3T3 cells and evaluated the level of PLAU expression in these cells as a function of morphological transformation, we did not observe a significant change in the PLAU mRNA levels. Instead, matrix metalloproteinase 1b, also known as interstitial collagenase, was at least 30-fold upregulated, as if ‘replacing’ the upstream activity of PLAU (Kathy Masker, Mechthild Rosing, Angelika Barnekow and M Sudol, unpublished data). Matrix metalloproteinases are acting downstream of PLAU, and are essential for breaking the barrier of extracellular matrix and initiating metastasis (Kessenbrock et al., 2010). We do not know if this difference in PLAU and matrix metalloproteinase 1b induction is due to different modes by which RSV transforms chicken versus mouse cells or because 3T3 cells are immortalized and therefore reveal a different pattern and range of gene expression when challenged by viral Src.
Experiments of Albert Fischer and Fritz Lipmann with Rous sarcoma implants, the first glimpse of PLAU?
At the beginning of the last century, thanks to the initiatives of the Rockefeller Foundation and the Carlsberg Brewery, the Carlsberg Biological Institute in Copenhagen was built primarily for exploration of organ implants and cells in culture (Bing, 2008). A former student of Alexis Carrel from the Rockefeller Institute in New York City, Albert Fischer was the director of the Carlsberg Biological Institute (Figure 1a, left panel). According to Fritz Lipmann, who worked with Fischer first in Berlin-Dahlem at the beginning of the 1930s, and later at the Carlsberg Biological Institute in Denmark from 1932 to 1939 (Lipmann, 1971) (Figure 2), Fischer was the first to discover the effect of Rous sarcoma on the stability of plasma clot; the clot being used routinely at that time to culture chicken fibroblasts (Fischer, 1925) (Supplementary Figure 1). The plasma clot on which Rous sarcoma implant was placed dissolved in a short time, making it impossible to grow the Rous sarcoma through this method (Figure 1b, right panel). In his article (Fischer, 1925), on page 251, where sarcoma implants are compared with normal control implants of chicken tissues, Fischer writes: ‘Die Sarkomzellen bilden sehr lose Verbindugen mit anderen Zellen und peptonisieren das Plasmakoagulum’. In direct translation: ‘The sarcoma cells build very loose junctions with the neighboring cells and peptonize the plasma-coagulum’. The use of the term peptonization of the plasma clot instead of liquefaction was interesting and intuitively correct in suggesting the action of a proteolytic enzyme in this process. Peptone was known at that time as the water soluble product of animal muscles digested by pepsin, and the resulting lysate was used for media to culture bacteria and fungi. It is most likely that while conducting this experiment, Albert Fischer was the first researcher who indirectly observed the RSV-induced PLAU gene via the enzymatic activity of its protein product toward fibrin, which was manifested in the liquefied plasma clot. Fritz Lipmann found this phenomenon interesting and decided to examine the plasma clot liquefaction by employing the Van Slyke method, which measured free amino groups. This was the only way at that time to detect relatively small effects of proteolysis. No change in free amino groups was detected by Lipmann in the dissolved plasma clots, compared with controls, and he argued that it was apparently only a small amount of fibrin that was digested and therefore the plasma was liquefied (Supplementary Figure 1). These experiments were not published, but the data and conclusions of Fritz Lipmann are in fine agreement with what we know about plasma clot, PLAU activity, release of plasmin from plasminogen substrate and the discrete targeting of fibrin by plasmin (D’Alessio and Blasi, 2009; Hildenbrand et al., 2010). More specifically, plasminogen is known to bind to fibrinogen and even more so to fibrin (Doolittle, 1984). Because of the concentrated and localized complex of the pro-enzyme (plasminogen) and its preferred substrate (fibrin), even a relatively small release of plasminogen activator should result in a restricted but efficient lysis of the area of the clot. In his letter (Supplementary Figure 1) and in our discussions, Lipmann also argued that when describing the activity of PLAU in general physiology, we should avoid the term ‘fibrinolysis’ because plasmin, which is released by PLAU from plasminogen, acts like a general proteolytic enzyme. Again, the work from the group of Edward Reich and from other laboratories provided ample evidence in support of this argument (Ossowski and Reich, 1983; Hildenbrand et al., 2010).
Clinical trials with Src kinase inhibitors, and PLAU as a cancer biomarker with prognostic and predictive value
There are at least three Src kinase inhibitors, namely Dasatinib, Bosutinib and Saracatinib (known also as AZD0530) that are being tested now, in phase I and II of clinical trials, for treatment of invasive breast cancer (Hennequin et al., 2006; Huang et al., 2007; Jallal et al., 2007; Saad and Lipton, 2009). There is also an ongoing phase III clinical trial with Dasatinib for treatment of prostate cancer (Mayer and Krop, 2010). Other agents that inhibit Src, including XL999 and AZM475271, are in preclinical or early phase clinical development (Mayer and Krop, 2010). Although these inhibitors target Src, they also inhibit other protein-tyrosine kinases, notably Dasatinib effectively inhibits ABL kinase and was already approved for the treatment of chronic myelogenous leukemia (Hunter, 2007). Nevertheless, it is hoped that the results of these trials will be as positive as those obtained for inhibitors to other non-receptor and receptor protein-tyrosine kinases, which are now being used as drugs (for example, Imatinib, Gefitinib and Sunitinib) that target leukemias with activated ABL (BCR-ABL) and solid tumors with activated KIT or epidermal growth factor receptor tyrosine kinases (Hunter, 2007).
In contrast to Src, which is being targeted in cancer by inhibitory drugs, PLAU is being used as a diagnostic marker. Elevated levels of PLAU and PAI-1 (plasminogen activator inhibitor-1) in primary tumors of breast cancer patients correlate with tumor aggressiveness and poor clinical outcome (Harbeck et al., 2007). Namely, the patients with high content of PLAU and/or PAI-1 have a worse probability of overall survival than patients with low levels of both of the biomarkers. Initially, the clinical utility of the PLAU and PAI-1 have been proven on the highest level of confidence (so called level of evidence factor 1) by the European consortium of 18 countries (Schmitt et al., 1997, 2004). The Receptor and Biomarker Group of the European Organization for Research and Treatment of Cancer (RBG-EORTC) evaluated findings from the analysis of more that 8000 breast cancer patients (Schmitt et al., 2004). A prospective, randomized multi-center therapy trial in node-negative breast cancer patients showed that the patients with relatively high levels of PLAU and/or PAI-1 are at significantly higher risk of disease recurrence and worse probability of overall survival compared with the patients with low levels of both the biomarkers (Janicke et al., 2001; Schmitt et al., 2004; Harbeck et al., 2007; Annecke et al., 2008). Low levels of PLAU and PAI-1 are indicative of a very good prognosis, and the patients are spared the necessity of harsh treatment in the form of adjuvant chemotherapy. The subgroup of node-negative primary breast cancer patients with relatively high levels of PLAU and/or PAI-1 clearly benefits from the adjuvant chemotherapy. Because of the strong clinical data, the German Working Group of Gynecological Oncology (AGO) has recommended PLAU and PAI-1 as risk-group-classification markers in clinical evaluation of node-negative breast cancer patients. The clinical utility of PLAU and PAI-1 was directly demonstrated by a prospective clinical trial: Chemo-Node-Negative trail (Janicke et al., 2001). A more refined clinical trail is underway, Node Negative Breast Cancer Trial-3 trial that is centered in Germany and France (Harbeck et al., 2007; Annecke et al., 2008). There are also diligent efforts to evaluate feasibility of accurate measuring of the level of PLAU and PAI-1 in core needle biopsies of breast cancer patients (Thomssen et al., 2009). In 2007 guidelines, the American Society of Clinical Oncology recommended PLAU and PAI-1 as biomarkers to be used in practice (Annecke et al., 2008; Hildenbrand et al., 2010).
A brief clarification is appropriate here. It seems somewhat contradictory that when an enzyme and/or its inhibitor are coordinately or independently upregulated, this event leads to a specific single outcome. One has to emphasize that PAI-1 has multiple functions and, apart from the inhibitory activity directed toward the PLAU enzyme, has a crucial role in regulating cell adhesion processes, therefore contributing to the metastatic potential of the tumor (Stefansson et al., 2003). Interestingly, in RSV-transformed CEFs, both PLAU and PAI-1 are upregulated in unison (Masker et al., 2007; Maslikowski et al., 2010). However, the PAI-1 is upregulated only several fold and always less than the transcript of PLAU.
It is also important to place PLAU in a broader perspective of signaling and physiology. PLAU is one of the components of a serine protease system that is involved in the degradation of extracellular matrix, and therefore contributes to cancer cell adhesion, migration and metastasis (Blasi and Sidenius, 2010). The PLAU system, apart from the PLAU itself and PAI-1, also includes PLAU receptor and type-2 plasminogen activator inhibitor (Mekkawy et al., 2009). Members of the PLAU system, especially PLAU and PLAUR, are overexpressed in several malignant tumors. Therefore, the PLAU system has been widely explored as a target for anticancer drug therapy (Ulisse et al., 2009; Hildenbrand et al., 2010). Various strategies have been pursued, including ribozymes, RNA interference, soluble PLAU receptor, antagonist peptides that block PLAU–PLAU receptor complex and PLAU enzyme inhibitors to reduce or abrogate the expression or activity of PLAU and PLAUR in cancer cells (Mekkawy et al., 2009; Ulisse et al., 2009). All these strategies have been tested in cell culture and animal models, and some showed promising results, but they need confirmation in humans. The good news is that a few PLAU inhibitors have entered early clinical trials (Ulisse et al., 2009). On the basis of the tight transcriptional regulation of PLAU gene by activated Src, it is perhaps not too far-fetched to speculate that a select group of cancer patients could benefit in the future from the availability of drugs that would inhibit Src and PLAU, as the effect of such drugs could be synergistic.
The basic research on PLAU has been tightly connected with the research on RSV and its transforming gene Src. To better illustrate that connection, I depicted two parallel timelines of major research hallmarks and clinical developments in the Src and PLAU fields (Figure 3). The timelines are presented as closely parallel lines, but in reality they should be fused into one avenue of cancer research that started with the discovery of RSV.
Interesting serendipities: WW domain and RSV budding, YAP oncogene
While studying Src and Yes oncogenes, my group has delineated a small modular protein domain known as the WW domain, which mediates specific protein–protein interactions (Bork and Sudol, 1994; Sudol et al., 1995). We also identified the specific peptide ligand that is cognate for WW domains and contains proline–proline–any-amino-acid–tyrosine (PPxY) consensus sequence (Chen and Sudol, 1995). Since the delineation of the WW domain–ligand complex, the research in my group has been on the signaling role of the WW domain in the human proteome, with a special focus on human diseases (Sudol and Harvey, 2010; Tapia et al., 2010). Two interesting and serendipitous developments connected the WW domain back to RSV biology and cancer. The WW domains of several proteins, including that of human YAP protein were shown to regulate the RSV budding process by interacting with viral Gag proteins through their PPxY motifs (Garnier et al., 1996). A lucid report from the laboratory of John Wills has described a successful interference of the RSV budding process by cis overexpression of the YAP WW domain that targeted PPxY motif on viral Gag (Patnaik and Wills, 2002). Most likely, the function of YAP WW domain is not specific here, and as a highly expressed module it competes with WW domains of NEDD4 family of E3 ubiquitin ligases that are known to form physiological complexes with avian sarcoma virus Gag protein to facilitate viral particle release (Pincetic et al., 2008).
Most recently, the YAP gene, whose cloning helped to identify the WW domain as a differentially spliced repeat of 38-amino-acid-long region (Bork and Sudol, 1994; Sudol, 1994), was shown to act as a bona fide oncogene and the main effector of the tumor suppressor pathway known as Hippo (Harvey and Tapon, 2007; Sudol and Harvey, 2010). Moreover, the WW domains of YAP were shown as critical elements in modulating the oncogenic potential of YAP (Grusche et al., 2010). Serendipities indeed.
Over time, the study of RSV has resulted in an avalanche of paradigm shifting discoveries (Martin, 2004; Vogt, 2010), which included the discovery of the first oncogene, Src (Stehelin et al., 1976; Brugge and Erikson, 1977), the identification of its protein-tyrosine kinase activity (Hunter and Sefton, 1980) and the purification of reverse transcriptase that broke the DNA–RNA–Protein dogma of molecular biology (Baltimore, 1970; Temin and Mizutani, 1970) The study of Src protein resulted in the delineation of first modular protein domains, such us SH2 (Sadowski et al., 1986), SH3 (Mayer et al., 1988; Ren et al., 1993) and WW domains (Bork and Sudol, 1994; Chen and Sudol, 1995), which led to the subsequent identification of new modules plus their ligands, and revolutionized our understanding of signal transduction at the level of proteomes. Current success in cancer therapy via drugs, which inhibit protein-tyrosine kinases could be traced directly to the Rous sarcoma virus (Hunter, 2007). So could be the PLAU gene that serves as a biomarker in the management of breast cancer patients (Annecke et al., 2008).
In 1979, as a graduate student at The Rockefeller University, I had a chance to participate in the symposium celebrating the hundredth anniversary of the birth of Peyton Rous. At the centennial held in the Caspary Auditorium, Lewis Thomas, Howard Temin and David Baltimore discussed eloquently the impact of the Rous virus discovery on the understanding of the molecular mechanisms of cancer (Supplementary Figure 2). They also summarized the current state of the RNA tumor virus research. According to Rene Dubos, who co-organized the symposium, Peyton Rous always strived for simplicity and was disturbed by complicated experiments that involved, in his words, ‘wheels within wheels’ (Dubos, 1979). Indeed, the experiments described in his two seminal papers on transmissible avian sarcoma (Rous, 1910, 1911) were relatively simple, if not elegant. And for some of us reading those papers is as exhilarating as reading the wrought iron prose of Samuel Beckett (Sudol, 2006).
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Special thanks are to Mario Gimona for translating the 1925 publication of Fischer from German to English and for valuable comments on the text, Bethany Antos and Lee Hiltzik from the Rockefeller Archive Center for the photographs and information about 1979 Rous Centennial. Kathy Sheridan and Peter K Vogt are thanked for valuable comments on the early version of the manuscript, and Angelika Barnekow for a fruitful collaboration and permission to mention our unpublished data. I am grateful to my PhD mentors Ed Reich and Wolf-Dieter Schleuning, my senior colleagues Mary Rifkin, Tony Cerami, Sidney Strickland, Jean-Dominique Vassalli, Yoshi Nagamine, Michael W Young, and my peers Michael E Greenberg, Michael Yamin and Ned Waller for their guidance throughout my Rockefeller tenure. My postdoctoral mentor, the late Hidesaburo Hanafusa, is remembered with warmth. This work was supported by PA Breast Cancer Coalition grants and by Geisinger Clinic.
This mini-review is not meant to comprehensively discuss all relevant publications. The references are intentionally selective and the author apologizes for omission of many pertinent publications, and for the myopic focus of this article.
The author declares no conflict of interest.
Supplementary information accompanies the paper on the Oncogene website
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Sudol, M. From Rous sarcoma virus to plasminogen activator, src oncogene and cancer management. Oncogene 30, 3003–3010 (2011). https://doi.org/10.1038/onc.2011.38
- Rous sarcoma virus
- The Rockefeller University
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