Signalling networks associated with urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐1 in breast cancer tissues: new insights from protein microarray analysis

Abstract

The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

Recent progress in protein profiling of clinical tissues for next-generation molecular diagnostics

“…Protein levels of uPA and PAI-1 were compared with the protein levels of 21 signaling molecules that play a role in breast cancer. The study revealed that protein levels of uPA correlate with expression levels of ERK and Stat3, whereas PAI-1 levels correlated with the levels of uPA receptor and AKT activation, thereby confirming earlier studies performed with cell culture models [61].…”

Targeted therapies in cancer - challenges and chances offered by newly developed techniques for protein analysis in clinical tissues

“…Another advantage of the RPPA is the possibility to quantify the total amount of protein in the sample via the application of purified recombinant proteins one the same slide 15, 16. This method enables to measure protein expression from FFPE tissues more precisely than it is possible by IHC (e.g., for HER2) and implement the analysis of clinical markers, which until now were only measurable from fresh or frozen tissues (e.g.…”

“…Nevertheless, we successfully established a set of about 50 antibodies for use in RPPA analysis by stringently selecting for specificity and sensitivity by western blot 15, 16 thus providing a starting point for the analysis of major cancer-related signaling pathways from FFPE tissues of cancer patients. Based on this expertise we recently developed an algorithm to validate antibodies for their simultaneous application to extract based and classical morphology based methods (Schuster C, Malinowsky K et al ., in preparation).…”

“…In 2001, another study used RPPAs to show the activation of pro-survival proteins and pathways during prostate cancer progression 45. In our group, we provided insight into the role of uPA/PAI-1 in cancer related signaling 16. Other prominent examples of cancer-specific signaling deregulation namely the activation of the phosphatidylinositol 3-kinase (PI3K) pathway in a significant number of ovarian and colon tumors 53, 54.and, alterations in the mitogen-activated protein kinase (MAPK) pathway or the overexpression of HER2,8, 53-57 were summarized in an earlier review by the authors 17.…”

“…Therefore for clinical determination of diagnostic and therapeutic targets new methods are crucially needed to maximize the data that can be gleaned from this kind of tissue. The recently developed method to extract full-length, immunoreactive proteins from FFPE tissues (for review see 15) is a major progress for such approaches and provides the means to quantitate clinically relevant proteins like HER2 and analyze cancer-related pathways by reverse phase protein microarray (RPPA)16.…”