3. Antiangiogenic activity of PSA (I-V)
3.2 Significance of the antiangiogenic activity of PSA
The antiangiogenic activity of PSA suggests that PSA may prevent or slow down the growth of prostate cancer. However, PSA also exerts activities that may promote tumor growth and metastatic dissemination. Other KLKs (such as KLK2, 5, 6 and 13) have been suggested to affect cancer growth, e.g., by degradation of ECM proteins (Deperthes et al. 1996; Magklara et al. 2003; Kapadia et al.
2004; Michael et al. 2006).
When prostate cancer can be detected on the basis of increased serum PSA levels (> 4 µg/L), the tumor weighs about 0.5 – 1 g and thereafter it usually grows very slowly, with a doubling time of 2 to 3 years, as determined by PSA levels, which correlate with the tumor volume (Schmid et al. 1993;
Stenman et al. 1994). After this it may take 5 to 10 years before the disease surfaces clinically and more than 10 to 15 years before it threatens the life of the patient, unless cured by radical therapy (Stenman et al. 1994; Bill-Axelson et al. 2014).
Assuming that prostate cancer grows at a constant doubling time of 2 years to reach the size of 1 g, it would take over 50 years, as a single tumor cell would need to double about 27 times (Stenman et al. 1999a; Stenman 1997; Del Monte 2009). Obviously, the tumor has to grow more rapidly before reaching this size. However, the growth of prostate cancer usually slows down after this initial rapid growth period. When a tumor reaches a size of ~ 1 mm3 (about 16 -17 doublings), it requires a vasculature to grow
further (Hanahan and Folkman 1996;
Folkman 2006). The switch to slow growth could result from the antiangiogenic activity of PSA (Stenman et al. 2000; Koistinen and Stenman 2012). The lowest PSA concentra-tions at which the antiangiogenic effect was significant in our study in vitro, were of an order of magnitude lower than the concentra-tions measured in the extracellular fluid of the prostate (Denmeade et al. 2001).
Already in men under 40 years of age, serum PSA levels may predict an increased risk of prostate cancer. Higher PSA concen-trations can be detected in men that develop prostate cancer 25 to 35 years later than in men that do not develop prostate cancer (Whittemore et al. 2005; Lilja et al. 2011).
Men of this age may already have micro-scopic tumors, but these tumors are so small that they are not likely to raise serum PSA.
Thus, it is possible that increased circulating PSA levels may, instead of just being a consequence of tumor development, reflect the involvement of PSA in the early development of prostate cancer, e.g., by digestion of IGFBP-3 and release of IGF-I, which is a potent growth factor associated with prostate cancer (Cohen et al. 1994).
PSA has also been shown to increase proliferation and to reduce apoptosis of prostate cancer cells (Niu et al. 2008;
Williams et al. 2011). The expression of PSA in the prostate of transgenic mice has not been found to initiate prostate cancer or to cause any morphological changes in the prostate (Williams et al. 2010). However, the concentrations of PSA in the prostate of these mice were up to 1000-fold lower than those in the human prostate (Denmeade et al.
2001; Williams et al. 2010).
At later stages, when the tumor needs vascularization, PSA may inhibit or slow down tumor growth by its antiangiogenic activity. This dual function would explain why prostate cancer, on the one hand, is so common and, on the other hand, grows so slowly at the stage, when it can be detected on the basis of an increased PSA. Indeed, similar dual functions, involving both cancer promoting and cancer
growth-65 inhibiting activities related to the cancer type
or stage have been suggested also for KLK6 and KLK10 (Zhang et al. 2006; Klucky et al.
2007; Sotiropoulou and Pampalakis 2012).
Several clinical studies have shown that PSA may have a tumor suppressive effect:
low tissue PSA concentrations as well as low PSA expression are associated with aggres-sive cancer and an adverse prognosis (Abrahamsson et al. 1988; Pretlow et al.
1991; Magklara et al. 2000; Stege et al. 2000;
Paju et al. 2007). Also, high PSA expression has been found to be associated both with reduced microvessel density (Papadopoulos et al. 2001) and low angiogenesis activity, as detected by a CD34 antibody in prostate cancer tissue (Ben Jemaa et al. 2010). In this context, it should be stressed that the PSA expression and protein levels in the prostate do not correlate with circulating PSA levels in cancer patients, as the latter are increased due to leakage of PSA into circulation and not due to higher expression, while the expression of PSA in cancer is decreased as compared to normal prostate (Abrahamsson et al. 1988; Paju et al. 2007). Also the enzymatic activity of PSA in prostatic fluid was shown to be inversely associated with the aggressiveness of prostate cancer (Ahrens et al. 2013). This further supports
the relevance of the proteolytic activity of PSA during cancer growth.
Due to its highly prostate-specific expression and antiangiogenic properties, PSA is a potential therapeutic target for prostate cancer. PSA-stimulating peptides, in addition to being useful research tools for studying the proteolytic activity of PSA, may also be useful lead molecules for develop-ment of novel treatdevelop-ments and imaging agents for prostate cancer. Stimulation of the anti-angiogenic activity of PSA may delay the development of clinical disease and may be sufficient for preventing the cancer from surfacing clinically within the lifetime of the patient.
Although the molecular mechanism behind the antiangiogenic activity of PSA could not be identified, the present study provides a good basis for further studies. We showed that the antiangiogenic effect is mediated in the cell culture medium after inactivation of the proteolytic activity of PSA and ruled out the most obvious mediators, angiostatin and endostatin. The mediators of the antiangiogenic activity of PSA would be feasible to study directly or after fractionation of the cell culture medium by proteomic methods, such as 2D-PAGE or mass spectrometry.
66 SUMMARY AND CONCLUSIONS
This thesis work characterized the antiangio-genic and proteolytic activities of PSA. The proteolytic activity of PSA toward different peptide and protein substrates was examined.
The major physiological substrates, semeno-gelins, were shown to be degraded by PSA much more rapidly than any other of protein substrates studied. Nidogen-1, a protein of the basement membrane, was identified as a novel substrate for PSA. Peptides that stimulate PSA enhanced its proteolytic activity differently toward peptide substrates and protein substrates: the stimulating peptide with less efficient stimulation with peptide substrates was more efficient with protein substrates.
PSA exerted antiangiogenic activity in a dose-dependent fashion in the HUVEC tube formation assay. Based on the experiments with active and inactive forms of PSA, the antiangiogenic activity was shown to be dependent on the enzymatic activity of PSA.
Accordingly, the antiangiogenic effect was enhanced by peptides that stimulated the enzymatic activity of PSA and abolished by small molecule compounds and a monoclo-nal antibody that inhibited PSA activity.
To study the molecular mechanism of the antiangiogenic activity of PSA, the PSA-induced changes in HUVEC gene expression during tube formation were determined.
Most of the gene expression changes were minor and it was not possible to elucidate if these changes were primary or secondary to the antiangiogenic activity. Further experi-ments with the tube formation assay corroborated the view that the antiangio-genic activity of PSA may be mediated by a proteolytic cleavage product of PSA. This proteolytic fragment is, for the moment, elusive. However, the antiangiogenic activity of PSA was not mediated by angiostatin-like fragments generated by the cleavage of plasminogen, as assumed earlier.
As the expression of PSA is mainly prostate-specific, with high concentrations of active PSA being present in the prostate cancer microenvironment, PSA could serve as a therapeutic target for prostate cancer.
Peptides that stimulate the activity of PSA could be useful for reducing tumor angiogenesis and for inhibiting prostate cancer growth.
67 ACKNOWLEDGEMENTS
This work was carried out at the Department of Clinical Chemistry at the University of Helsinki during the years 2006-2014. I wish to thank the present and former heads of the department professor Pirkko Vihko and professor Ulf-Håkan Stenman for providing the excellent working facilities. I also thank professor Pirkko Vihko for kindly accepting the post of the custos.
This thesis work would never have been completed without so many people con-tributing to it in one way or the other. I am most grateful to my supervisors Ulf-Håkan Stenman and Hannu Koistinen for their excellent guidance during these years. First of all, I am greatly thankful to Uffe for giving me the opportunity to work in his research group. I appreciate your visionary mind and your thorough enthusiasm for science. You have always shown a warm and supportive attitude toward me. I am also truly thankful to Hannu for advising me wisely and patiently with all the scientific and practical questions that have come up during these years. Your enthusiasm for science, as well as your determined and positive attitude toward any task at hand, has also encouraged me in my work.
I wish to thank the reviewers of the thesis, professor Tero Soukka and adjunct professor Kaisa Lehti, for the careful exami-nation, valuable comments and criticism on the thesis work. I also thank Robert Paul for an excellent language revision of the thesis.
I wish to thank all the co-authors and collaborators involved in this thesis work.
Pirjo Laakkonen for introducing me to this project and for the enthusiastic guidance into the world of cancer biology during the early years of the project. Leena Valmu for con-tributing to this study with her outstanding skills in mass spectrometry. Ale Närvänen for his work on the PSA-binding peptides.
Can Hekim for creating a good atmosphere in the laboratory and in the office, and for being always helpful when there were problems with computers or any other
devises. Suvi Ravela for skillful mass spec-trometry analysis and for your friendship in the office. I also thank all the other co-authors: Sami Kilpinen, Ping Wu, Magnus Jonsson, Johan Malm, Juhani Lahdenperä and Gerd Wohlfahrt for their excellent work in the research projects.
I also wish to thank all the other collaborators involved in the PSA project, especially Erik Wallén and Kristian Meinander at the University of Helsinki and Henna Ylikangas, Maija Lahtela-Kakkonen, me the way forward in the project.
I warmly thank all the present and former colleagues and friends in the labora-tory: Laura Hautala, Anna Lempiäinen, Elina Keikkala, Kristiina Hotakainen, Kati Räsänen, Nick Domanskyy, Anne Kaukinen, Riitta Koistinen, Outi Itkonen, Annukka Paju, Susanna Lintula, Jakob Stenman, Jari Leinonen, Anna-Kaisa Herrala, Ileana Quintero, César Araujo and all the other colleagues in the departments of Clinical Chemistry and of Obstetrics and Gynecology with whom I have had the pleasure to work with. I also wish to thank the members of the Laakkonen laboratory, especially Piia-Riitta Karhemo, Juulia Enbäck and Marika Waltari. I wish to thank all the colleagues who have kept me fun company during the scientific meetings and symposia both in Finland and abroad.
I am truly grateful for all the practical help and guidance I have received during these years in the laboratory from Krisse Nokelainen, Marianne Niemelä, Maarit Leinimaa, Helena Taskinen, Annikki Löfhjelm, Taina Grönholm and Anne Ahmanheimo. Without you this work really would never have been completed.
Especially I wish to thank Krisse Nokelainen
68 and Marianne Niemelä for your invaluable
help and practical tips in so many experiments. I also warmly thank Ansa Karlberg for taking good care of all the practical things in the office.
I direct my warmest thanks to all my friends, who have brought joy and meaning to my life. I thank my friends within science Laura, Hanski and Outi, for sharing both the enthusiasm and the moments of frustration that are closely related to the scientific work.
Special thanks to my lifelong friend Hanna for her warm friendship.
I am most grateful for my family for always believing in me, even at the moments I did not believe in myself. I thank my mother Tuula, and Kari, and my father Pertti, and Arja, for supporting me every way in my life. I express my deepest gratitude for my beloved brother Juhani, who died tragically only four weeks before writing these words.
I thank you for your warm companionship during our journey together through the universe on this planet earth; for sharing all the good times and hard times we went through together. You will always have a very special place in my heart. My dearest thoughts are also with my sister-in-law Johanna and the little girls Kerttu and Siiri. I
thank my dear sisters Päivi and Suvi; I want you to know how much you mean to me. I also thank my dear grandmother Anna and all the other relatives and in-laws for being there.
My loving thoughts are devoted to the three most important men in my life. I am grateful for my beloved husband Jani for walking there by my side, for your endless love and compassion, and for your perceptive mind. I thank you for your patience with this thesis project and for your support in both mental and down-to-earth matters. I thank our charming little boys Sampo and Eero; you remind me every day what is most important in life. I admire your imagination and your fascinated attitude toward the wonders of this world.
I am grateful for the financial support from Finska Läkaresällskapet, Biomedicum Helsinki Foundation, Maud Kuistila Memo-rial Foundation, Ida Montin Foundation, Orion-Farmos Research Foundation, the Finnish Cancer Organisations, Helsinki University Central Hospital and University of Helsinki.
Espoo, October 2014
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