α (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd

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1 χ π η ώ ε α, α α Ε Γ Η Ε Ε :. ΘΗΓΗ Η " ο π που α π ο ο υ α ο α α ου ο α α α α ου ο χου ο πο" Θ Φ Η Γ α, α ου ο 2017

2 ε υ υ ευ π π π ου α, ου Ε, α α α οπο ου α, απ α α α π α ε ε α π π π ου α, Επ ου ο α α ου Ε, α α α οπο ου α, απ α α α α α που ο α ο, α Γ α α α ο, Επ ου ο α Γυφ που ο α ο απ α ο πυ, Επ ου ο α

3 Χ Ω. Α Ω Α α ο ία αι ισ ο ο ία ο ο ό ο ύσ ς Α α ο ία σ ο ο ία Ο οθ ια ό α ί α ο ο ό ο ύσ ς ι ιο ο ία- α ά ο ς ι ύ ο ι ι ή ι ό α- ιά σ Αι ιο ο ία- αθο ι οί α ισ οί α ά ις ο ο ο έα FGFR Έ φ ασ ο ο έ EGFR ο ο ο ά ι RAS-MAP ο ο ο ά ι PI K-AKT-mTOR Ο ό ος ο p Ο ό ος ο RB οσ ι ή ασ άθ ια Ο ό ος ς θ ί σ ς ο DNA Ο ό ος ς ο άσ ς α ι ό σ ο οθ ια ώ α ι ά WHO αθο ο οα α ο ι ή σ α ιο οί σ ο οθ ια ώ α ι ά ο ια ή α ι ό σ ο οθ ια ώ α ι ά ο ο ό ο ύσ ς ο σ ι οί α ά ο ς-θ α ία ο ια οί βιο ί ς ιο ί ς ια ιά σ αι ι ή σ θ σ ού ιο ί ς ια ό σ αι όβ α οσ ός α ί ς ι ο ί ς ο α οσ ού α ί ς Α α ιο ά σ α οσ ού α ί ς ΐ ς σ ό ς α ί (Actin Binding Proteins) αι α οσ ός α ί ς α οσ ός α ί ς αι α ι ο έ σ

4 . ί (ezrin) α ι ί (paxillin) ο α ί (cortactin) θο ο ο ία ς α οσοϊσ ο ίας ι ο ή ς θό ο ασι ές α ές θό ο Ο Ο Ο Ο Α Ο Α Α Α Ο Ο σ ι ά ί α α ο οι ασία ισ ώ Α οσοϊσ ο ι ή ώσ ό ο ο α οσοϊσ ο ι ής ώσ ς ια α ί σ ς ϊ ι ής έ φ ασ ς ς ί ς α ι ί ς, ο α ί ς, - α ί ς, β- α ί ς αι p σ α ο οθ ια ά α ι ώ α α ς ο ο ό ο ύσ ς ί σ α οσοϊσ ο ι ής ώσ ς α ισ ι ή Α ά σ Α Ο Α Α ι έ α οσοϊσ ο ι ή έ φ ασ ς ί ς σ α ο οθ ια ά α ι ώ α α ς ο ο ό ο ύσ ς σ ί αι ιθ ι ή βιο ο ι ή σ ιφο ά ι έ α οσοϊσ ο ι ή έ φ ασ ς α ι ί ς σ α ο οθ ια ά α ι ώ α α ς ο ο ό ο ύσ ς σ ί αι ι θ ι ή σ ιφο ά Έ φ ασ ι ώ - α ί ς αι β- α ί ς σ ο ς ο οθ ια ούς ό ο ς ς ο ο ό ο ύσ ς Έ φ ασ ς ΐ ς p σ α ο οθ ια ά α ι ώ α α ς ο ο ό ο ύσ ς α οσοϊσ ο ι ή έ φ ασ ς ί ς σ ί αι σ α ισ ι ώς σ α ι ά έ φ ασ ς - α ί ς αι έ φ ασ ς α ι ί ς Ο Α Α

5 . Ω. σ σ ς. Η υ α α απ α ( α, Richard S. Snell, M. D, Ph. D. α α 1992). α πα α α α α α α α α α α αυ α α α α α α αυ π α α α α υ π φ α ( α, Richard S. Snell, M. D, Ph. D. α α 1992, Histology for Pathologists, Stacey E. Mills, 3 rd Edition). α, α α α α α α α α α (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985). α α π πυ υ απ π υ αυ α π υ π α α απ υ α π α υ α α υ α α ( Anatomy of the lower urinary tract, Tanagho E. In: Campbell MF and Walsh PC, eds. Campbell s Urology. 6 th edition. Philadelphia: WB Saunders: 1992). υ υ α α α π α π α π α α. Η α υ α α πυ α α απ α. α α α α π : υφ,, π φ α, π - π φ α αυ α. Η υφ α π απ φυ α α π α απ πυ α π α ( α, Richard S. Snell, M. D, Ph. D. α α 1992). Η π α π φ α α α π υ υ απ α α υ α. α υ υ α πα α α α υ π υ υ α α π α, π α π α υ υ υ π α υ ( α, Richard S. Snell, M. D, Ph. D. α α 1992, The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985). υ. 3

6 α α α π α π α π α α α π α απ π α π υ α π α υ υ υ υ π α. πα απ α α υ α α α π φ α π α απ π α α π α π α α α α. Η π φ α υ α π α π απ π α α. α υ α α υ π α υ π α υ α α α ( α, Richard S. Snell, M. D, Ph. D. α α 1992) ( 1, 2). π - π φ υ α υ α α π α π π π α α π α π υ υ π α α α α υ π π α ( α, Richard S. Snell, M. D, Ph. D. α α 1992). α 1. α υ υ υ α α ( α, Richard S. Snell, M. D, Ph. D. α α 1992). 4

7 α 2. α υ υ α ( α, Richard S. Snell, M. D, Ph. D. α α 1992) αυ α απ π α υ υ α α α απ υ υ υ α υ υ, υ α α υ α υ. α φ α π α α υ απ υ α υ α υ (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985). α αυ α α υ π π φ α υ π α α υ π α υ υ υ α α α υ π π φ α υ υ αφ α α α υ υ υ υ ( α, Richard S. Snell, M. D, Ph. D. α α 1992, Histology for Pathologists, Stacey E. Mills, 3 rd Edition). απ α α α π υ α υ υ α αυ α. υ υ υ απ α α. π αυ υ α απ υ υα υ π υ α υ α α υ υ α α υ α υ. α α υ υ α π α απ α υ α υ ( υ υ Waldeyer) π π υ υ υ α α υ α α α υ υ α υ. Ό α α α, π υ α π α α υ υ α π α α υ αυ. 5

8 απ φ α πα (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985) ( α 3). Η α α π υ α. α 3. α πα α υ υ ( π π απ Viana R et al, 2007) Η α α α πα α απ α υ α π υ α α α α α. φ α υ υ φ π α π υ π π α φ π α υ π α υ α α φ α. Η φ πα υ α π υ α υ φα α α υ αυ α π α πα α π υ π υ α υ φα. Η α απ υ πα α απ πα α υ πα υ π α υ α υ π α. υ πα υ π α απ 11 α α 2 φυ α πα υ. πα α υ πα υ απ 2 α 4 α φ υ υ υ πυ υ α υ υπ α υ π α. υ α α α α. 6

9 αφ α υπ υ α πα υ υ υ α α π α πα αυ α α α φ α. υπ υ α π π α α πυ α υπ α α α υπ υ α α υ π υ α α α υπ ( α, Richard S. Snell, M. D, Ph. D. α α 1992, The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985 ).. σ Η υ απ α απ : υ α, α υ απ π υ α π α υ α α α. υ (πα α α α α α π ) απ α α α φ π υ α α π υ π α υ α υ π υ υ π υ. π υ π α α α α. Ό α υ α πα, π 6-7 υ α α α α π υ α α 2-3 α α α π α α απ π α υ α π υ πα α α (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985). α α υ : α π φα α, α α α α α α α. α π φα α α α α α π α α π υ π α α π α π α α α α α. Έ υ υ υ πυ, π α α π α π α υ φ φ υ α π α α ( The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985) ( 4, 5). Ό α α α α π π α α α α υ α. Έ α παφ α α α α υπ υ α α φ α α α υ ( α α, L Carlos Junqueira, Jose Carneiro, Robert O Kelly, 6 ). α υ υπ υ υ π υ υ α α α α α α υ απ α π α απ α φ α α (Jost et. 7

10 al, 1989, Truschel et al, 1999). υ α α φα υ α απ α φ α α. Η αυ π φ α π φα α υ α α απ α α π α υ α υ α π π υ π α π α π υ π υ απ α α. α π υ υπ υ α α α υ α α α υ α α αφ α π α α υπ υ α α α α υπ υ (Truschel et al, 1999). α 4. α α απα α υ υ υ ( π φα α, α α α α α). α 5. α φυ υ υ (webpathology. Com). 8

11 Έ α α α αυ π φ α π φα υ, υ - π α π α α, απ πα υ α π α α π α α αυ α α α πα πυ α α α π πυ α. υ α υ π υ πυ, α α (asymmetric unit membrane-aum). Η αυ π π π α α απ, υ α π α α υ π φα α υ υ, α α α π υ α υ υ α π υ α α απ π αυ α π α α α. Ό α α α, π α α υ α α α π φα α α α υ υ α π π α υ α, α α α α α υ υ υ (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985) ( 6, 7). υ α π α α π φα α υ α α α υ π α π απ α α α α π π υ α υ π α απ α α υ α υ (Jenkins D et al, Kindey International 2007;71: ). π αυ α π υ π απ 90% υφα α π φ α π φα α υ. Έ υ π αφ π υ π α, UP1a, UP1b, UPII, α UPIII α 27, 28, 15 α 47 kda α α. UP1a α 1b υ α α π UPII α UPIII υ α. π α αφ π υ α α υ π α α α α α υ α απ απ UP1a/UPII α UP1b/UPIII. π αυ απ υ α υ α υ υ α απ α α υ α υ α υ α π α α υ π υ απ α α α απ πα υ α ( Staack et al, 2005). π υ π π υ π α π α π α απ π π υ π υ υ π α. υ α υ α α π α. 9

12 . π π π αυ υπ υ α α υ α π α α υ α π υ π φ α πα απ α υ υ αυ α α α π α α α α υ υ α φ α α αυ α (Truschel et al, 1999) ( α 6). α 6.. -α ( α απ π ).. α α απα α υ α π φα υ π π υ π α α π υ π α π υ υ α. ( π π απ Kachar et al, 1999). 10

13 α 7. α υ απ π α (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985) Η α α υ φ π π α α α α π α α π α α α π υ α. πυ α υ αυ α π πυ α υ α π α α φ α π. Η υ α π α α α α α α α α αυ π α α α α. Ό α α α α π α α υ αυ υ υ υ α π α α α α α υ. α α α α α υ α α α α α α. α π α πυ πυ α π α α π α υ α π α απ α απ α αυ, α πυ α α α α υ α (The pelvis and Perineum. In: Moore KL, ed Clinically Oriented Anatomy, 2 nd Edition, Baltimore: Williams & Wilkins; 1985, Jost SP et al, J. Anat. 1989;167: , Truschel ST et al, J Biol. Chem 1999;274: ).. 11

14 2. σ ς ς α υ υ α υ α α α Η (US Cancer Statistics Working Group (2010) United States Cancer Statistics: Incidence and Mortality Web-Based Report) α α α α πα (Parkin DM, 2008). π π υ α α υ υ π υ α απ α υ π (Theodorescu D, 2003). π α α α α πα π π υ α υ (National Cancer Institute. SEER Data; 2015). α υ υ ( π υ υ α ) α αυ α φυ (Knowles et al, 2015), υ α φ υ αυ α, 9 υ 10 α α α α α 65 (Yee et al, 2011). υ α α α α α υ π α α υ υ. π υ π π α πα π υ υ υ υ υ φ α υ υ υ. π αυ υ α π α, π αυ υ α 3,89 α 4.65 α υ α α α (Freedman et al, 2011). Ά πα α πα α φ υ (α α α, α, αφ α, α α.α), υ υ π. απ, υ α απ υ φ α α π υ φ φα, α α, α πα υ.α. π π πα π α α Lynch (HNPCC) αυ υ υ φ α α υ υ (Pukkala et al, 2009, Turner et al, 2014, Levin et al, 2015).. 12

15 . - σ Η π υ υ α α α υ υ α α α υ α / α α υ π α α απ υ π ( υ υ α, υ υ ) α π α υ απ υ. α α α α υ υ α α υ υ απ υ π α α (Wein et al 2011). Η υ α α απ α π α, α (90-96%) α π υα (Lokeshwar et al, 2005). Η υα α υ αυ α α υ υ α α α υ α α α α α α α in situ, α α α α αυ π αυ α. Ω α α υ π α υ α α υ α, υ υ π, α α π α απ α (Lokeshwar et al, 2005). π π αφ α υ (Optical Coherence Tomography) α α π α (Confocal Laser Endomicroscopy) α π υα α πα υ π πα α α π φ (Morgan et al, 2010). πα απ α α, π α.. - σ α υ α α α α α απ α πα, πα α α α α υ ( α 8). π υ α α π υ π α α π υ α π υ υ α, α α α α situ, α αυ π υ, α α υπ π α α (Knowles et al, 2015, McConkey et al 2010, Ehdaie et al, 2008). α α α α π υ υ α α α α υ απ α υ υ υ υπ α FGFR3 (Fibroblast Growth Factor Receptor 3), π α υπ α υ, π α α π υ α πα RAS/MAPK. α αυ. 13

16 υ α υ υ α α α 7, 10 α 15 υ υ (Di Martino et al, 2012, Bernard-Pierrot et al, 2006). Η π φ α υ α α π υ υ α α α α απ α α π υ π π π υ υ υ α. υ π α υ υ υ MDM2 / α υ CCND1, απ π υ p53 α / υ RB1 α / υ CDKN1A, υ /υπ φ α υ E2F3 (φυ α α α απ RB1), α υ p16, υπ υ π α (Williams et al, 2004, Mitra et al, 2007). ασία α ού βαθ ού α οήθ ιας ο οθ ια ό α ί α HRAS/FGFR3 σιο ο ι ό ο οθή ιο 9q-/9p- 9q-/9p- σ α σία/ α ί α In situ ού βαθ ού α οήθ ιας ο οθ ια ό α ί α P53, Rb, 8p-. 8p+, 17p- α 8. α πα α υ α α. 14

17 .. ς FGFR3 υπ α FGFR3 α α υπ FGFR π α απ α υ απ α π υ υ α. π α απ α υ α, απ α υ φ α α α απ α υ α υ (Knowles et al, 2007). υπ αυ α υ α υ α π υ, α π υ υ, αφ π α α (Xue-Ru Wu, 2005). α υ α α α α υ α υ FGFR3 π π απ 70% α α α α α α π α α 10-20% α υ υ α α υ α α in situ α α α. υ α υπ α π α α α α π α, α αφ πα απ α α α α in situ (Xue-Ru Wu 2005, Wolff et al, 2005). υ α α αυ π α Ig II α Ig III π 50-80% α α α π α α π 15-40% αυ υ α π π 5-10%. α π α Ig II α Ig III α α α α π υ υ α α υ αυ α υπ υ α υ υπ α α φ φ φ υ υ απ υ α π. υ α π α π α α πα π αυ υ RAS ( α 9). Η π υ πα απ πα in vitro α α α απ α α υ π α α: αυ π υ π α υ π SHC ( SRC-homology-2-domain-containing), GRB2 ( growth factor receptor-bound protein 2) α SOS ( son of sevenless) α αυ π υ π α υ π 80K-H, GRB2, pp66 α SOS ( Xue-Ru Wu, 2005).. 15

18 FGFR3 IgI 50-80% α ά IgII IgIII 15-50% α ά RAS RAS 5-10% α ά RAF SOS GRB 2 pp66 80K- H P P SHC GRB 2 SOS RAF MEK PYK2 JAK PI3K MEK ERK STAT AKT ERK α 9. α π α π υ π α υ υ υ α π υ FGFR υπ α ( π π απ Xue-Ru Wu, 2005)... Έ σ EGFR Η α α α υπ υ EGFR (epidermal growth factor receptor) απ α απ α, υ ErbB1, ErbB2, ErbB3 α ErbB4. ErbB1 α α α α α π π υ υ -α φ π πυ α υ π α α α MAP α ( α 10). TGF-a, EGF, HB-EGF (heparin-binding EGFlike growth factor) απ π υ α π αυ α Erb1 α α EGFR. Έ υ υ υ π υ π 4.6% υ α α α π υπ φ α 23%. α πα απ υ υ α π απ α α υ υ, π υ π φ α α π. π π υ α απ. 16

19 α απ α α υ α α α α π υ υ α (Black et al, 2008, Mhawech-Fauceglia et al, 2006). ErbB2 HER2 απ α α αφ π α αυ α-π α α α. π π «α» α α α α π υ υ α απ α υπ πα. α υφ υ πα RAS-MAP α υ υ υ π υ π ( πα α) α υπ φ α υ π υ πα. υ α υ α π υ α υ υ α α α α (>pt2) α αφ υπ φ α υ απ α π πα α π (Black et al 2008, Crawford et al, 2008). ErbB3 (HER3) α ErbB4 (HER4) α α α α α α α α υ α π α α ( Xue-Ru Wu, Nature 2005;9: ). O Η R3 αφ απ α υπ πα α α α α υ α πα π υ. απ α α α α απ α αυ. HER4 HB-EGF π α «π» απ α π (TACE) π π υ α απ υ α α α α υ α α α υ α υ α, απ π υ α α α πυ α. Η υ α υ α, α π α α α α αφ πα α α α α φ α. α α α υ mrna αφ φ υ HER4 π υ υ α υ απ πα υ α (JM-a) απ υ α (JM-b) π TACE. α υ α α α α υ υ υ α απ φ JM-a (Black et al, 2008). Έ υπ υ α α α EGFR π α α α α υ α α α α φ α υ υπ α π α π α. 17

20 υπ. Έ φ α α υπ απ α π απ υ α υπ α (Mhawech-Fauceglia et al, 2006). EFG, TGF-a, α PI3K φ σφο ί σ ΑΚΤ mtor Α Α Ο Ω STAT GRB2 SOS RAS ERK MEK RAF α ι ός ο α ασιασ ός Α ιο έ σ Α ασ ο ή α ό σ ς α άσ σ, ιήθ σ α 10. π α α υ EGFR... RAS-MAP π π α π π α αφ α αυ πα π υ υπ υ π α α αυ φ φ υ υ α α α π π υ α π υ RAS φ, α πα απ υπ υ υπ φ α υ α π υ απ α, α υπ υ α RAS υπ φ α υ π α απ υ α π.. 18

21 φ π RAS, α α α πυ α α MAPK/ERK (mitogen activated protein kinase/ extracellular signal-regulated kinase). υ πα αυ απ MSK 1 ( mitogen-activated and stress activated protein kinase 1) π α α α H3. Η υ α α π α α α α α α π α α α α αφ π α α. υ πα υ υ c-myc π υ α α φ α υ π υ π α ( α 11). Myc π α αφ πα π υ π α υ υ α υ. Έ, π φ α υ D α υ α CDK4 π υ α π π υ φ φ υ RB. υ α, α α φ φ υ απ υ α αφ πα α E2F π α υ α π απ α α. π π Myc α α υ α α υ α α, υ π α α α π p21, αυ α α π π α υ φ φ υ υ RB ( α 11). π α α υ α πυ α υπ υ α π υ υ υ α α α α α α π α α υ π π υ α α. Έ α α α RASSF1A π υ φυ α α RAS π α α π α π π RAS απ π α υ π π υ α α. π π DAPK ( death-associated protein kinase) π υ φυ α ERK π α π απ υ α π α α πυ α, π π υ α α απ π α π υ ( α 11) (Wolff et al, 2005).. 19

22 FGF EGF α ό ασ α FGFR3 RASSF1A Ras Raf EGFR MEK ERK DAPK ή ας ERK MSK1 άβ DNA p14 MDM2 C-Myc Cyclin D p16 p53 p21 CDK4 Α ό σ RB P RB P E2F E2F E2F ο ί ια σ ό οι ι ή ασ άθ ια α ι ός ο α ασιασ ός α 11. α απ υ πα RAS-MAP ( ο ο οι έ α ό Wolff et al, ).. PI K-AKT-mTOR Η π αυ πα α υ α υ υπ υ (FGFR, EGFR, HER2 HER3) π RAS π, α, π π Akt, π α υ α π α α. Η απ π υ PTEN α α α PDK1 (phosphoinositide-dependent protein kinase 1) υ α π α φ φ υ Akt, π π α π πα απ π. υ α Akt φ φ υ απ π GSK3 (glycogen synthase kinase 3 ) π α α π - α α απ. π α πα απ α αυ υ α

23 π απ α α. π π απ π α BAD (BCL-2-associated agonist of cell death) α FOXO (Forkhead box O) π α απ π, υ α π απ α α α απ υ DNA, α α TSC2 (Tuberous Sclerosis 2) π α mtor α α υ RHEB (Ras homologue enriched in brain). mtor π π α υ α α π υ π α αφ πα α 4E-BP1 α eif4e (Grivas et al, 2015). ( α 12). α 12. π PI3K-AKT-mTOR ( π π απ Grivas et al, 2015). 21

24 2.3.5 ς p Η α α α π p53 π α υ υ α υ α α α π υ (G1-S) α υ α DNA, π υ p21 π απ α α α υ α α (CDK1), π α π υ DNA π υ α π α, α αφ α, π απ π, π α α αφ π υ π α αυ, α π α BclX1 ( απ π υ π α α αφ ) (Nishiyama et al, 2008).. 22 α TP53 απα α υ (>50%) α υ α υ α α α α α α α α in situ, α υ απ α πα α α αυ α in situ α α υ α. α απ α α α α, α υ p53 απ α π πα α α υπ π, υ, π α απ απ α (Mitra et al, 2007, Mitra et al 2006). α α α υ α α p21 απ υ p53, α α α α υ p53 π, α φα α υ υ α υ πα α υ p53. π α φ α υ p21 απ α α α α π πα α α υ α α. υ α α α α α α α p53 α p21 φα υ α π υπ π α π αυ α p53 (Mitra et al 2007) ( α 13). Η π Mdm2 π α α αυ υ α α α p53 υ α υ υ α υ. υ α π π α υ p53 π π α υ υ Mdm2 α πα α π Mdm2. Η υ α α π α p53 α αφ π α α απ. α α π π α υ p53 π π Mdm (Mitra et al, 2007). π υ 30% υ α α υπ φ υ Mdm2 α, π α α α α,

25 υπ φ α αυ α π υ απ α α α α υ. υ π, υπ φ α Mdm2 πα α π απ π υ πα υ p53 π υ α α απ α υ υ TP53 ( Xue-Ru Wu, 2005) ( α 12). ο οι ές CBP, TRAF, P300, PCAF, ASPP1 p53 p53 mdm2 ό p53 p53 p21 CDK4 CDK2 E2F Rb1 Cyclin D1 X Cyclin E P Rb1 P P P + E2F α 13. π α α α p53 α RB.. ς RB Η π RB π υ α απ φ G1 φ S υ υ α υ (Mitra et al, 2007). φ α π υ π α υ α π απ α α, αφ π α απ π. υ α α υ α, υ α π υ π π DNA α α αφ πα. φυ α α υ α α α υ α α φ φ υ υ φ ( φ ) α π α α α αφ πα α E2F π α π υ α DNA π π α α αφ, π TS (thymidylate synthase), π υ. 23

26 α απα α α α υ DNA φ S υ υ α υ (Mitra et al, 2007, Wolff et al, 2005). α α α α φ φ υ α απ υ α απ υ πα α E2F π π α υ α α π υ ( α 13). π π, α π α υ υ α αφ πα α AP-2 π φ α E- α υ α α α υ π α φα π υ. π, π α π α α α υ α υ α α α α απ αφ π π υ υ α α α. φ α υ p21 α αφ πα α Sp1 α Sp3. Έ, απ α υ RB α φ α υ p21 (Wolff et al, 2005). υ α α α π α υ π φ α υ υπ φ α φ φ υ υ φ, π υ α π π RB α υ απ α α π α π φυ υ ( Xue-Ru Wu, 2005). α αυ π π υπ υ α π. Η φ φ υ υ υ α απ π α α α. α υ α π υ φ φ υ υ RB α α π α υ α α. α υ α α απ α απ α: α υ. φ φ υ π υ α α α υ α α. π α π α α α φ α υ RB α α αυ α π π α υ D C α π π α α α υ α α ( π p21, p27, p16) α υ α π απ α α. π, απ υ α 13q, π υ π α υ RB, απ α α απ π υ RB. Έ α α απ π υ απ α φ φ υ π α π α φ α απ α υ p16 α / υπ φ α υ D1. υ α 20-80% υ α α υ υ ( Mitra et al, 2007).. 24

27 .. σ σ Η απ α υ α απ α υ α 9 απ υ α ( Mhawech-Fauceglia et al, Cancer 2006;106: ). π α π >50% α α α α α υ α α ( Stephen SG et al, Urol Res 2004;32: ). Η απ α υ α υ α 9 ( LOH υ 9q) υ α α π α α απ α π π (9q-13, 9p22-23, 9q12-13, 9q21-22, 9q34) ( Mhawech-Fauceglia et al, 2006). Έ α α α α α π υ α φυ αυ π α υπ υ α α α π α α α π υ υ α α. υ α υ α α α α α α α π α α α υπ π α α. Ω α υ π α υ α α α in situ. π π α α α πα α υ π α φυ υ. α πα απ υ π α α απ υ α 9 α υ υ α α π α υ α π α α α α απ α α α α πα ( Xue-Ru Wu, 2005). Έ α απ α α α α α π υ α π π αυ α CDKN2A (cyclin-dependent kinase inhibitor 2A) π π α π 9p21 α π π α α α α α, INK4A (p16) α ARF (p14) (Xue-Ru Wu, 2005, Schulz WA, 2006). α υ α α απ α υ υ υ α υ πα υ RB α υ p53 α α. α υ α α α α υ π 30-50% υ α υ α αυ υ υ π α υ α υ απ π υ υ υ ( Xue-Ru Wu, 2005). Έ α υπ φ α TSC1 (tuberous sclerosis 1) π π α π 9q34, π π α α α α α π υ α α φ α α α. α α α α α υ α α α α α φα α α π α υ υ α υ υ υ P27KIP1 π υ απ α α α υ α α. π α υ α 34%. 25

28 υ α α α υ υπ π (Xue-Ru Wu, 2005, Mhawech-Fauceglia et al, 2006). Ά α π α α α PTCH π υ π α π 9q22.3, DBCCR1 π υ π α 9q33.2 α DA41 π υ π α 9q α α ( Mhawech-Fauceglia et al, 2006). Η απ α υ α υ α 8 υ α α 25-30% υ α α α α υ α α α α π υ α υ υ ( Xue-Ru Wu, 2005). α υ α υ υ α 8 υ α υ. α απ π υ π π υ α φ α υ α α α π α α υ α 3, π 3p14.2, π υ φα α π υ α υ α α α α π FHIT (fragile histidine triad). α υ υ αυ υ π α υ α π υ π α, υ α υ α, υ α α υ υ. υ FHIT α α α α υ πα FAS (APO1)- α απ π. π π α α α π υ υ υ απ π α mdm2 α π α υ p53 αυ, α α π υ p53. Η απ π υ FHIT υ α υ υ α α α α ( 25% π π ) α απα α α α υ. π υ υ α π υ α αυ απ π : υ 25% π π α υπ υ υ π α 16% π π. π α φ α π FHIT 60% α υ α α α υ φ π α υ π ( Mhawech-Fauceglia et al, 2006).. 26

29 .. ς ς σ ς DNA Η υ υ DNA α α π α α α π υ π α α α φ α, π α, α α α υ α υ DNA (Egger et al, 2004). π αυ, π α απα α α (Wolff et al, 2010).. 27 υ DNA π α υ υ C-G (α αφ α α C-G) απ υ i % π π υ α π π υ α α, πα υ π αυ π υ π α υ π α, απ α απ π υ α (Venter et al, 2001). Η υ α απ υ α φ (DNMTs) π DNMT1 φ α π α αυ α υ. Η φ α DNMT1 α α α απ α p53 αυ α α αυ α απ. π π DNMT1 α π π α αφ πα α E2F π υ υ π υ RB. Ό α α φ α υ RB αυ α φ α DNMT1 α π α υ α α α (Wolff et al, 2005). υπ υ Η π υ α de novo υ C-P α υ α α α π α απ π π α α π υ υ α α (Salem et al, 2000, Friedrich et al, 2005). υ α 25% α υ α α α, υπ υπ υ π υ π α υ υ sfrp1. Φυ sfrp1 α α α υπ Fz π υ α υπ υ α πα wingless-int (WNT). π αυ π α, αφ π α α stem υ. π α WNT α α π α υ υπ Fz π φ α υ υ υ. Η π αυ απ απα υ α α π υ α α απ - α. π υ α α πα πυ α α υ φ α. π υ α π α υ πα αυ α π υ α υ. Η φ α υ υ sfrp1 α α απ υ

30 πα WNT υπ α α α α α (Mhawech-Fauceglia et al, 2006). π α φ α π sfrp1 α υ α α α, α υ π α υ α α α α. sfrp1 α α α πα α α π α υ α α α, α α α υ υ DNA π α υ απ υ α α α υ α αυ (Mhawech-Fauceglia et al, 2006). π π υπ υ υ π α υ υ CDH1, π π - α, 84% υ α α (Xue-Ru Wu, 2005). π αυ α υ α υ π α π υ α α π, πα α α α α υ α... ς ς σ ς Η (Telomerase Reverse Transcriptase) α α υ α φ α αφ α αυ α α. Η πα απ α α α α α α α α α α α α: α α α α υ απ υ α α π α α π αυ α α (Shay et al, 2011, Günes et al, 2013). Η α α stem α α α α αφ π α α α, α πα π α α υ. Έ υ υ α α π α α υ υ φ υ υ, υ π υ απ α π α απα α, α π α υ α α α α υ υ (Killela et al, 2013.). υ α α α υ υ, Yves Allory et al α υπ υ π α (70%), α α α υ α α α α. α πα απ α TERT υ α α α, α υ α α π α α π α π α α π υ υ α π α (Allory et al, 2014).. 28

31 Η υ υ α α TERT α υ α υ υ, α α α α α α. π π α π α απ α απ υ, π α α α α α α α α π υ υ υ. 2.4 σ ώ WHO 2016 ( Humphrey PA et al, The 2016 WHO Classification of Tumours of the Urinary Systemand Male Genital Organs Part B: Prostate and Bladder Tumours, EAU) υ α α α α φ υ φ π α α α υ α /signet ring/ υ α α Γ α υ α Χα αφ π π α αυ υ α υ α π α α. 29

32 υ α α α in situ υ α α α α α α α υ α α α υ α α α Θ υ α π α α α α υ υ α (LMP) υ α α φ υ α α υ α υπ π α α α α υ α υ υ α υ α υ π α α 2.5 σ σ ώ Η α α α π υ α υ υ υ φα α πα α π α α α α 1. α π α υ υ υ Primary tumor (T) TX T0 Ta Tis T1 T2 Primary tumor cannot be assessed No evidence of primary tumor Noninvasive papillary carcinoma Carcinoma in situ: flat tumor Tumor invades subepithelial connective tissue Tumor invades muscularis propria. 30

33 pt2a pt2b T3 pt3a pt3b T4 T4a T4b Tumor invades superficial muscularis propria (inner half) Tumor invades deep muscularis propria (outer half) Tumor invades perivesical tissue Microscopically Macroscopically (extravesical mass) Tumor invades any of the following: prostatic stroma, seminal vesicles, uterus, vagina, pelvic wall, abdominal wall Tumor invades prostatic stroma, uterus, vagina Tumor invades pelvic wall, abdominal wall Regional lymph nodes (N) Regional lymph nodes include both primary and secondary drainage regions. All other nodes above the aortic bifurcation are considered distant lymph nodes. NX N0 N1 Lymph nodes cannot be assessed No lymph node metastasis Single regional lymph node metastasis in the true pelvis (hypogastric, obturator, external iliac, or presacral lymph node). 31

34 N2 N3 Multiple regional lymph node metastasis in the true pelvis (hypogastric, obturator, external iliac, or presacral lymph node metastasis) Lymph node metastasis to the common iliac lymph nodes Distant metastasis (M) MO M1 No distant metastasis Distant metastasis α α 2. α/π Stage T N M Stage 0a Ta N0 M0 Stage 0is Tis N0 M0 Stage I T1 N0 M0 Stage II T2a N0 T2b N0 Stage III T3a N0 T3b N0 T4a N0 Stage IV T4b N0 M0 M0 M0 M0 M0 M0 Any T N1-3 M0 Any T Any N M1. 32

35 . σ ώ σ ς Έ υ π αφ αφ α υπ υπ α α υ υ α αφ π : Urobasal A, Urobasal B, α α α SCC-like. πα απ υπ υπ α α α απ αφ φ α α π υ αφ υ α, υ α υ, α, υπ υ α α απ α α α απ πα α α α απ α α α. Urobasal A πα υ υ αυ φ α FGFR3, CCDN1, TP63 α α α 5 α α α υ υ π υ α παφ α α α φ α π π υ α υ α υ. Χα α α απ α π υ, υ υ α, α α α α α α π. Urobasal B α α π π, α α πα υ Urobasal A. Έ πα υ υ αυ αυ φ α FGFR3, CCDN1 α P63, π π φα υ α υ TP53 α α φ α α α υπ υπ SCC-like. α α α α α απ α υ TP53, φ α CCNE α ERBB2 α α φ α α. Έ α α π υ υ υ υ, υπ π υ α υπ φ α αυ α α. π π, φ υ α π υ α φ υ α 20. π φ α υ π α α υ α α α υ α φ υ α π α α π υ α υ α υ, αυ π υ α υφα απ φ α. υπ υπ π α α α (SCC-like) α α α απ αυ φ α α π υ α π υ φυ φ α υ α α π α αφ π α α α π (Sjödahl et al, 2013, Eriksson et al, 2015, Sjödahl et al, 2012).. 33

36 . σ ς- π πα υ υ α α α υ α (TNM) α α α α (grade) (Wein et al, 2011). Ά π πα α π υ α α, υ υ, πα υ α in situ α α, υ α α π π υπ π (Netto et al, 2012). Η α α φα απ π α υ π πα (Kaba et al, 2014, Leissner et al, 2003). π υ υ α α α α απ υ π υπ π (50-70%) π α α α π (15%), π υ α π (Holmang et al, 1995). α αυ πα α υ α α υπ π υ υ υ π α υ α. α π υ υ α απα π π απ α υ π π. αυ π π α υ υ υ αυ απ α α α απ α. α α π υ υπ α υ υ, π α π α 69% α υ υ α α 39% α αυ π φα α (Steven et al, 2007). 2.8 ς α TNM α απ α α π υ α υ υ υ, υπ α αφ απ απ α, α α α υ α υ. Γ α αυ, υπ α π α π υ α υ π, α π υ υπ π α απ απ α α α α υ π ( Fei Ye et al, 2014). α α α α π υ πα α απ α, α α α α α α α α α α π π υ π υ υ ( Ming Zhao et al, 2016).. 34

37 .. ς σ σ σ Η π NMP-22 απ υ α υ πυ α π υ α φ α υ, α α α DNA α π α α αφ α φ α. 1996, απ απ α π π α α α α α υ υ υ αυ π (Keesee et al, 1996). α π υ υα α α α υ ( NMP) α α υ α υ υ υ α α π 49% α 87% α α. α, π αυ α 8 απ υ 9 α α υ υ π α φυ υ π (Grossman et al, 2006). Ω, υπ υ υ απ α α π π υ υ π α α υ α (Atsu et al, 2002). Η π αυ α π απ FDA α.. 35 Η α α υ α υ BTA (Bladder Tumor Antigen, BTA-stat, BTA- TRAK ) α α π (Human Complement Factror H) π α α πα α απ υ α α υ υ υ απ π α υ α. υ α φ α φ π (fibronectin), α π υ α υ α, π α π α α υ αυ. α πα απ υ π απ FDA (Ye F et al, 2014). Η α α α α α υ α α απ π α α α α υ υ α (LMP) α α α α α (low grade) Tα α α α υ υ. Έ α πα α, απ α υ α π α α φ α υ CD44, αυ φ α CK20 α υ υ υ α α υ πυ φ α υ p53 α υ π απ α α (ki-67) απ υ υ α π α α (Lopez-Beltran et al, 2013). υ α α α α υ α α, α απ α υ α α. ImmunoCyt ( DiagnCure, Inc., Quebec, Canada) απ α α α α υ

38 α υ α φ π υ υ υ α α φ α α α α α α M344 LDQ10, 19A211 α υ υ π υ α υ α υ (CEA). α ( Comploj et al, 2013) υα α 34.5% α υ α, 68.1% α ImmunoCyt υ α 72.8% α υ υα. Η α 97.9% α υ α, 72.3% α Immunocyt α 71.9% α υ υα. Ω, πα υ α α α υ α, υ α α α υ υπ π α α υ π π α υ υ απ α. UroVysion test (Abbott Molecular, Inc. Des Plaines, IL, USA) απ α α α φ υ υ in situ (FISH) π α α υπ α α α α 3, 7 α 17, α α απ α υ π υ 9p21 π α α α α α α υ α, απ FDA. Dimashkieh et al, 1835 α α, π α 1045 α απ α α α υ υ α α 790 α απ α α α υ α, υ α, α α α α α π π α α α υ υ α α α α 61,9%, 89,7%, 53,9% α 92,4% α α α FISH α 21,9%, 96,9%64,4% α 87,5% α α α υ α α. Η αυ FISH α υ α α α απ α α π π υ α α α υ α α α υ α α α α (Dimashkieh et al, 2013). π π πα απ α α α π α α in situ α occult π υ α α υ π (Ye F et al, 2014). Ω, πα υ α α υ α υ «π α» α υ απ α α υπ α υ DNA υ υ (Wojcik et al, 2000). Έ α υ α α, π α αυ α α α υ υ α π α PCR. Ω, π φα α α α (Ye F et al, 2014).. 36

39 .. ς σ φ α 2 Consultation α α υ υ υπ υ α α π υ υ π α α. α α α α TP53, RB1, FHIT(Fragile HistidineTriad gene) α FGFR3 (Amin MB et al, Eur Urol. 2013;63:16-35).. 37 υ α, α φ α υ p53 α α υ α υ υ, α πυ υ φ α α υ α α α α υ α υ α υ υ (Esrig D et al, N Engl J Med 1994;331: ). α υ TP53 α α α υ RB (Cote RJ et al, Cancer Res 1998;58: ) π π α υ α α α π υ υ α (Cordon-Cardo et al 1992). α α α FHIT α υ υ υ α α α υ α απ α αυ α φ π (Baffa et al 2000, Zhang et al, 2012). Ό αφ FGFR3, φ α, υ α υ απα α υ α α α α υ (Pandith et al, 2013), αυ φ α υ p53 α α υ α α π (Hernández et al, 2006). π π, α υ υ υ α υ π υ D1 α D3 α α p21, p16, p27 α π α α π υ α υ υ υ. υ α, αυ φ α p21, p53, p27 α prb απ α α υ π πα υπ π υ υ π υ α υ α (Shariat et al, 2010) α π απ α Γ α π απ α α απ α υ α υ υ υ, α π π α π α π υ α π φ απ απ α. Η α υπ π υ α π υ α π φ απ απ α α α απ α α υ.

40 Η π MRE11 απ υ υ π υ MRN π α π «πα α α» π α υ DNA π υ π α α απ α α υ φ α π αυ π φ α π απ α απ α υ (Choudhury et al, 2010). π π α υ π υ υ α υ α α φ α ERCC1 (excision repair crosscomplementing 1) π φ α π απ α απ α α απ α, α υ υ π π α ERCC1 π α υ (Kawashima et al, 2011). Η φ α υ υ BRCA1 α π υ α υ π υ α υ π υ α π φ απ απ α π α π υ. Έ, α α π π α mrna υ υ BRCA1 α π υα α α απ α (Font et al, 2011).. σ ς ς Ό α α υ α υ α α π υ υ α, π απ α απ π π α π. υ υ α φ υ α υ, φυ α υ υ υ π α πα υ α α υ α α α α α α α ( υ α - α υ, υ α α α α υ υ α π α ). α υ α υ υ α α, α α α α α α α ( α). υ 25nm απ α απ α π, υ π υ, π α απα α απ υπ, α- α - υ π υ. α υ α υ α α α,. 38

41 α αφ α, α υ α α α α α α α α υ υ υ α α α φα. α α α α α υ υ, π π υ 10nm. α α π υ π υπ π π α α π υ α α α α α. α α α α α υ πα α υ 8nm απ α απ α υ φα π (G-α ) υ α απ ( α α -F-α ). α υ α υ α (+) α α (-) φ (π α) α αυ υ π α α α α.. υ π υ α α φ (Pollard et al, 2003). π υ α α, υ υ π υ, υ α α, α α α υ α α υ. υ α α απ α α υ α π α π α α α α. α, απ α υ α α π α π α α υ α, π υ α υ π, α α. υ α π α α π π υ υ α (Actin-binding proteins, ABP) υ υ υ α υ υ α α υ α υ υ α α υ αυ π α α α in vitro, π α α α π α υ α αυ υ α π α α α α π υ α α υ α α. απ 150 π υ π α υ π π α (Remedios et al, 2003).. ς σ ς Η α α α α α π υ υ υ α α α αυ υ α π, α α α υα α απ α α, π π. 39

42 υ α υ π α υ υ υ, α υ υ, υ α α π α υ, π φυ υ α υ α (ECM, Extracellular Matrix), α π υ α πα α α, υ (cytokinesis), α. Η α υ υ απ α υ α α α α, υ α υ α π υ α α π υ, π π α α απ. Η υ α π α π αφ υ α α α α α: π α υ π π υ υ υ, π υπ α, υπ υ π υ υ υ υ υ α απ υ απ υπ α (Pollard et al, 2003). Tα α α α π π υ υ α αυ α α α υ α π υ υ α α α αυ α α. υ α α υ α υ α α α π α α απ π π α π α α π α π -«π». Έπ α υ α α π π α υ α υ (filopodia, lamellipodia), υ α πα α α υ υ υ α π α α απ υ υ α υ α ( Small et al, 1978, Pollard et al, 2003). Γ α α π υ α α υ α α υ : α π α π απ π υ α α α π α π α υ. Η α π α α - υ υ α π υ π α υ υ υ α π α α απ υ (retrogade flow) α υ απ α π α υ υ α υ α υ α υ α υ π α π φυ (Wozniak et al, 2004) ( α 14).. 40

43 ι όνα 14. α ι ή ί σ (Ananthakrishnan et al, 2007, Creative Commons Attribution- NonCommercial 4.0 International Public Licensehttp://atlasgeneticsoncology.org/Genes/GC_PXN.html) Η υ α α υ απ α α α α α υ α αυ α α α απ α π α α απ α α. Έ α φυ α α, π α α π α α α α α υ ( α, α υ υ υ α φ α ) α α α π υ α α α α π α α α α α α α. α α α αυ π α π α π α (Ridley et al, 2003). υ α α α α α α υ α πα α απα α α α α υ υ (Pollard et al, 2003) α π π π (blebs) υ α π α α, α α π α (lamellipodia) α π α (invadipodia).. 41

44 π υ π α υ α α π υ υ α υ α υ π : α α, υ α α α. α α π α α α α υ α π (blebbings) α α απα α απ υ α υ α (Sahai, et al, 2003). υ α π α α α α π υ α α υ α απ απ υ α υ α π υ υ υ, α π α απ α, υ α υ π α υ υ υ α απ α υ π υ υ υ (Sanz-Moreno et al 2010). α π α α α απ υ α παφ υ α υ α απα α απ υ α υ α (Friedl et al, 2010). α α π υ υ α α α υ α α α υ υ α α υ υ (contraction ring) π υ υ α α α π α α (Kaibuchi et al, 1999). α αυ απ α υ απ α α υ, πα αυ π α π υ απα α α α υ υ υ α υ α, α α π α α α α α υ α π α α (Theriot et al, 1997), (Fishkind et al, 1995). π Rho/ROCK φ φ υ αφ α υ α υ, MLC, π υ υ α υ υ α αυ π α υ (Kaibuchi et al, 1999). H, α α υ α α α υ α απ υ υ α υ α, α α α α υπ α απ υ α α (Durrbach et al 1996). α π υ φα, π Fc υπ α GTP Rac α Cdc42 (Cell division control protein 42), (Caron et al, 1998). H π Cdc42 (Cell division control protein 42) υ α α π π α α α π α φ π α π υ φ, Rac υ α α υ (Massol et al, 1998). α, α π υ φα υ α π. 42

45 π α α, α φ α α α «υ α» αυ (Caron et al, 1998). αυ π π π Rho/ROCK α π α - υ υ α π υ α πα (contractile force) α απ α π υ π υ φ υ. π π, υ α α πα α υ. π α α απ π α α α α φ α α α υ α π α υ π π α α α υ αυ (Burgoyne et al, 1987, Bretscher et al, 1991). Χα α πα α α π π π φ α α υ α α απ φ α α α α υ α α α υ α φα υ α PC12, απ π α υ (Charalampopoulos et al, 2005). Eπ π π α υπ α υ α απ π LNCaP, π α α απ α υ α υ υ π, PSA (Prostate Specific Antigen). Η αυ υ PSA α α πα α α υ υ α α (Kampa et al, 2002), (Papakonstanti et al, 2003). υ α LNCaP -BSA, φ φ υ FAK (Focal Adhesion Kinase), α PI-3, απ α π α π GTPα Cdc42(Cell division control protein 42) /Rac1 (Papakonstanti et al, 2003). Aπ π φα αφ α π π, α π υ α π α α α αφ π α υ υ υ α υ. α α φυ α α α α π υ υ α, απα α π απ αυ πα, π φυ υ α υ α (ECM) α α υ υ α α (Walker et al, 2005). α α υ α υ υ α α α π υ υ υ υ α υ α (cell spreading) α πα απ υπ υ (Receptor tyrosine kinases, RTK s) G(1) φ υ υ α υ ( hmer et al., 1996). π π,. 43

46 α α υ π υ α, π ROCK, πα α πα φ φ υ αφ α υ α υ (MLC), πα π πα απ αυ πα υ D1 (cyclin D1) α πα α α (Roovers et al, 2003). α, απ υ α α υ π α α α πα π πα υ υ π υ α υ πα π πα απ αυ πα π υ πα Ras-Raf-MEK- ERK (Roovers et al, 2003). H πα απ π υ ERK πα, απ υ α αυ πα α, α α α απ απ π υ α (Chen et al, 1994, Zhu et al, 1995). H α υ α π υ α π α α α α αφ π. υ α, α αφ υ - π MAL, πα α υ α π α α α α π υ α. π π G-α α α π υ, α απ α υ υ υ πα α MAL πυ α, π υ υ π α α αφ π υ π υ υπ υ α π υ πα α απ (SRF, Serum Response Element) (Miralles et al, 2003). α α αυ π α π α α G(1) φ υ υ α υ α υ - π MAL π φ α α απ αυ, π α πα α υ υ υ υ SRF, υ α π Cyr61 (Cysteine-rich angiogenic inducer 61), (Walker et al, 2005). Eπ π α α υ υ α α α απα α α α απ π υ TNF-α π α α α, α α α α υ α αφ πα α NF- B (nuclear factor kappa-light-chainenhancer of activated B cells) πυ α (Papakonstanti et al, 2004). Η α απ π υ TNF-α α α α π 3 υ α υ πα PI-3, π π Cdc42 (Cell division control protein 42) α PLC- 1 π α α α α α α υ NF- (nuclear factor kappa-light-chain-enhancer of activated B cells) πυ α (Papakonstanti et al, 2004).. 44

47 3.2 σ σ ς π υ α α α α υ α α α α απ π π υ υ α α ( -Actin Binding Proteins). α α α φα π π υ υ ATP-ADP (G-α ). α αυ π υ α α α π (F-α ) απ απ π α υ (Bugyi, et al, 2010, Holmes et al, 1990). α υ α, α in vitro α υ υ π υ α π α α υ 5-7 nm α υ α m (F-α ). α π α π αφ φ υ α α α φ α α. (Egelman, 1985, Carlier, 1991). Tα G-α π α α α α α α α υ, απ α α υ α π α α α α.. 45 α π υ α π α φ : (α) π (activation) υ α α υ π υ (nucleation), ( ) π υ (elongation) υ α υ α ( ) π υ α υ α α α (steady state). α G-α α π Mg 2+, Ca 2+, K + α α α φ υ α α π υ υ α α π υ (Carlier et al, 1986, Zimmerle et al, 1986). Η α υ π υ (nucleation) υ α υ α α α π α υ π υ. π α α υ π υ («πυ») α α α φ υπ υ α υ, α α α α απ α (Korn, 1982, Pollard et al, 1986). α α α πυ α υ α α π α π υ α α. υ α, π α α π α π ATP-α π υ α π α ADP-Pi-α α ADP-α ( rn et al, 1987). H απ υ υ Pi απ π ADP-Pi-α α π α α π υ ADP-Pi-α α π απ αυ ADP-α. Η υ α α υ υ υ ATP α υ α α α

48 απ π υ υ αφ α α ADP-α απ π υ α υ α απ ATP-α αφ υ (Carlier MF, Int. Rev. Cytol. 1989;115: ). H π α π υ πα υ υ α α α α π υ -απ π υ υ. " α α απ υ " (barbed end α ) υ α υ πα υ α υ α α ATP-α απ " α α απ υ " (pointed end α ), α α π υ υ α υ πα α α (treadmilling) α α π υ π α α, α α απ α α απ α (Bonder et al, 1983). O α π υ α α α α απ υ υ υ ATP, υ G-α π α π υ α α α α α α υ α ADP υ υ α α α υ ATP (Stournaras et al, 1988). A α, υ υ υ ATP υ α α π υ υπ α υ α υ α α α α α απ υ α υ α (Pollard et al, 2003). Η υπ φ πα α α α υ α π α α α π υ α α υ (Pollard et al, 2009). To α α α απ 30-50% υ α. Η π α α G- α F-α π υ α υ α α π υ α α π απ υ α, α α π α α υφ α α α α α α υ π α υ α π (monomer actinbinding proteins). H υ α α α υ α π α α α υ υ α π α. π α π α υ α α α π υ α π υ υ α π α α α α α π υ απ π υ. π υ α αφ, υ π α α α α πα υ υ υ α υ π φ υ υπ απ " α α απ υ " π " α. 46

49 α απ υ ". Η α α αυ, υ α α ATP, α α π απα α α α α α απα α, α α α υ α π α υ α απ π υ (capping proteins). Eπ π, αφ υ υ υα πα π α π π υ υ α α (actin binding proteins) π π α α υ α υ α α υ α. υ α, π υ α παφ (+) π α π υ υ α υ α α α απ π υ α α (dos Remedioset al, 2003, Le Clainche et al, 2008). π π υ υ α α α υ α α υπ α π α α, π π υ απ π υ, π α ADF α cofilin α α, υ υ απ (Le Clainche et al, 2008) υ α α α (+) α π α π α π υ ( α 15). α πα απ α υπ υ α α α α α απ α α α α υ α π α αυ α α α υ υ α. α αυ υ π υ α α φα απ υ υ υ α α π υ απα α α α α υ υ α α.. 47

50 α 15. α α απα α π υ α ( π π απ Nürnberg et al, 2011) Ό α υπ α α υ α, π α υ de novo α α π υ, α υ α α α (Chesarone et al, 2009, Campellone et al, 2010). π αυ α π απ υ α πα. α απ αυ α α Rho-GTPase π CDC42, Rac α Rho α απ α υ υ υ π υ απ π υ υ υ α α. υ α, π Rho-GTPases υ α formins α π. υ α π υ α (+), υ α π υ α (Goode, et al, 2007). α α υ υ α π α π υ α α, π υ α α π υ. 48

51 α πυ α α. αυ π α α π Arp2/3 α NPFs (Nucleating Formation Proteins-WAVE, WASP, cortactin). π υ α α πα απ, α απ α υ α π υ α π blebs α α π α (lamellipodia υ π α) α π α (invadipodia) α π α π υ υ α α ( α 16). υ α: 1. π (protruding blebs) απ υ α π αυ υ α π π α α υ α α (Fackler et al, 2008). Γ α α υ α α π ( α ERM π υ α π αφ πα α ) α formins π υ υ α π υ υ υ α α. α φ α π (blebs) α α F-α, π α α απ υ α α α α π π π, απ υ πα (Charras et al, 2008). 2. α π α (invadipodia) απ π, π α α υ υ απ υ α υ α. Γ α α υ α υπ υ π N WASP ( α NPFs) α Arp2/3, απα α α α υ α α α π αυ α α cortactin. 3. α υ π α (lamellipodia) απ α α α α υ α α απ π υ α α α α π WAVE α Arp2/3 (Nürnberg et al, 2011).. 49

52 .. 50 α 16. υπ π υ α α α α π α α ( π π απ Nürnberg et al, 2011).. ΐ ς σ ς (Actin Binding Proteins) Η υ α υ υ α α υ α απ α α π s (actin binding proteins) π α π α α : 1. π υ α G α : π π υ υ α -απ υ α α, π υ υ, π υ α G-α α υ (Cc) υ α. αφ α π απ 60 π π α π α π υ υ - 4 α π φ. Η υ - 4 υ α υ -α α απ π υ α α (Carlier et al, 1999). π π φ α υ - 4

53 υ π α υπ π α (Safer et al, 1997, Schutt et al, 1993), π φ π π α α α υ 4- α υ α υ α (Kang et al 1999) 2. π υ α α G-α : π α αυ απ π υ υ α α (-) α α α α, π α α α α π υ α π α α π υ. αυ α υ φ. Η φ υ α α α απ α π α π α α α π υ, πα α π α α α π α απ π υ πα α (Condeelis, 2001, Bamburg et al, 2008). Η φ υ α φ φ υ απ LIM, π υ υ α φ φ υ απ α ROCK. 3. π υ α α π υ α (+) : π α α αφ : α) πα π υ (+) α ) π π υ α π υ (+) (capping proteins) (Campellone et al, 2010). π α α α υ π π υ π υ α α α - π υ α π. π formins (mdia1, mdia2 α mdia3). Ά π π υ π υ π VASP, Mena α Ena/VASP-like (EVL) (Bear et al, 2009). π υ α α υ π υ α α α π α υ π CapZ, adducin α (Zigmond, 2004). 4. π υ π υ υ α α υ π υ (nucleation factors). αυ α α α υ π Arp2/3 α π α WASP/WAVE. πα αυ (Nucleation Promoting Factors-NPFs) π. WASP α Scar/WAVE υ υ α α α υ α α υ π υ υ αυ α α α π π α α α. NPFs α α α π φ α π α, π α α π υ υ. 51

54 α υ (+) ( Carlier et al, 1993, Suetsugu S et al, 1998, Higgs et al, 1999, Egile et al, 1999). 5. π υ απ π υ α α (severing proteins). π αυ π π α φ απ π υ π π α α α α υ α (+) α α π α π α π υ 6. α α π α υ χ α - α α (cross-linking and bundling proteins): α α αυ π υ υ υ α π α α α α αυ π υ υ α π α α α π α α α α. π α α α φ α π α υ α π υ π α α α. α α α φ π α α-α. Η φ π π α α φ π α α α (Bretscher, 1991). Η α-α α π, π υ π α π α α. Η α α π α α αυ. α α π 95 KDa α π π α υ α α α απ α α απ α φ φ α. Έ, α υ α υ α απ α υ α π αυ π α π, α υ υ υ α υ α α, π α π α π α α α. α α υ α φ φ α (Papakonstanti et al, 2000). 7. α υ α (membrane attachment proteins. O π αυ υ υ υ α α α α α υπ α υ π α α π α υ υ υ υπ α π α α π α α υ α. π αυ πα υ α υ α υ υ υ υ α υ α ( α υ ) α υ υ α (π. 52

55 π φυ ). π α π φυ, α-α α α υ υ α α υ α π α α α (Luna et al, 1992, Burridge et al, 1996). π π π υ α απ υ α α π υ π φ α υ α υ, α π α υ π α π π α π υ π φ α υ α υ. α αυ π α π π π π υ π α π α π φυ π α πα, υ α VASP (Vasodilator-stimulated phosphoprotein).. σ ς ς Έ α απ α π α α α α α α α α α απ α π υ υ α απ α φ α α υ α, π α πα α απ α π α απ υ α (Yilmaz, et al, 2009), φα α π υ α α π ( ). Η α α α φ υ υ α α α απ απα α α απ υ π φ υ υ υ α υ α υ π. Η α α α αυ π α α π π α υ υ υ α α. Έ α π απ α απ α α α α α π α υ α α υ υ υ π υ υ π π υ α α., α υ α α α υ π υ α α φα υ υ υ α υ α α υ α α υ α α α α α πυ α. α α α υ υ υ π υ α α υ α α. π π, α υ υ α υ α αφ απ αυ π α υ. Έ, α υ α α α α α υ α α α π α π υ α α υ (Lee et al, 2006).. 53 α πα υ π α α απ π υ, π αυ πα TGF-, υ α υ α

56 , π Wnt/ -catenin α Notch, α α φ.α. απα υ α α α π υ α υπ υ (TKR), υπ α TGF- (TGF- R) α (Thiery, 2003). α α α υ π π πυ α π υ π α αφ πα α υ υ πα Snail, Twist α ZEB π π π α α, α π α αφ υ α π π. α α α υ α αφ π α π - α. α πυ α π α φ α π, 3, Wnt/ - α, F- α Notch (Tiwari et al, 2012). α α α α απ - α α - α. α υ α υ α υ α α α υ π - α, π α α α απ α, απ υ α π φυ α υπ α π α π α α α. υ α π α α - α υ α - α, α αυ υ α α- α. Η υ α α υ α F-α, υ α α φυ α π υ υ φυ - α α F- α (Kurisu et al, 2010). πα α α φυ υ α π υ α α α, υ π, α α α α α. Θ α α α α α α υ υ : πα α α α α αφ υ υ φ α α α α, α υ φ α - α α α α in vitro α πα α α π α α α (Vleminckx et al, 1991). Η απ α φ α - α α α α υ α π α π α α α α α π υ α π α α (Christofori et al, 1999). Η απ α - α απ π φυ α απ α υ α π α υ, α α π υ α α π - υ α α α α α α α α α α υ (Thiery, 2002). α υ υ απ α - α αυ α α α. 54

57 α α α υ (Perl et al, 1998). φ α - α α, π α α (υπ υ υ υπ ) αυ α α α αφ υ υ - α α α π π α α υ π α πα α υ (Christofori et al, 1999, Conacci-Sorrell et al, 2002, Dorudi et al, 1993, Van Aken et al, 2001). Η - α α υ α α α α π α απ υ α α φυ α α. α π : (1) υ α υ υ π α α π φυ, υ α υ α υ α α, α π π (2) α α α αφ π πα α πυ α (van Es, et al, 2003). Φ φ υ - α α π 142, α α π α α π BCL9-2 α πυ α π α α υ (Brembeck et al, 2004). α, απ - π α Wnt (Morin, 1999).Η π αυ α α απ - α απ π α απ α πυ υ υ α α υ π υ α αφ πα α TCF/LEF (Kikuchi, 2000). π Wnt π α φ α α υ α π υ φα α πα α α α α υ α υ πα π υ υ υ απ - α π υ APC, α α α α - α α υ α π α π α α (Alexander et al, 2002, Ilyas, 2005, Polakis, 2000, Bienz et al, 2000, Ilyas et al 1997). α - α υ υα TGF- α α π α α α φα υπ π α υ π υ α α α α. π α α αυ φ α - α α, πυ π - α α αυ φ α α αφ πα α π Snail1 (Snail), Snail2 (Slug), Twist, EF1/ZEB1, SIP1/ZEB2, α / E47 π α α υ φ α -. 55

58 α π υ α υ α υ α υ α π α υ (Lee et al2006). Η α α υ υ α α απ α α α υ πα α α α π π υ υ υ α υ υ α α α α α π (Lee et al, 2013). π π π υ υ α υ υ α α π α α N-WASP υ υ α υ α υ α υ α υ υ α α π υ α α α α (Lamouille et al, 2014, Sroka et al, 2016, Truffi et al 2014, Han et al, 2014, Kurisu et al, 2010, Ren et al, 2009, Rajput et al, 2013). α 17. α π υ α α. 56

59 . σ ς ς σ υ υ α α α υ υ υ υ π υ α, υ α α. α π φα π α α α. π π φα αφ α π π o α π απ α α π α α υ α α α α α α α α υ υ α υ, υ α α α α. α α α υ α α υ υ υ υ α π α υ υ π π υ α α α α (Moustakas et al, 1999, Stournaras et al, 1996). Η α υ υ /π υ (G/F) α α α αφ π π α α υ φ φ π α ( υ α, α υ υ, α, α ), α α G/ α α π α α (Stournaras et al, 1996, Stiakaki et al, 1997, Moustakas et al, 1999). π π υπ υ αφ π α α υ α π α α α φα αυ υα α απ α π πα α π υ α π α, π α υ α α, απ α α ( φ α α απ υ α ) α α υ υ α π απ α α ( α απ α α α α υ) (Stournaras et al, 1996, Stiakaki et al, 1997). φα υπ υ φ α α α ( π α υ, αυ πα, π ) πα αφ υ π α G/F-α α α α α π α α υ φυ φα υπ π α α αυ π α α υ (Moustakas et al, 1999, Koukouritaki et al, 1999, Papakonstanti et al, 2000). α π α α α α φα υ α α α π π α φ α π π υ υ α υ α (Vandekerckhove et al, 1990, Chaponnier et al, 1989), π π π φα υπ αυ α φ α α π φ υ υ υ α α φα υπ (Mullauer et al, 1990). α α π π α φ α / π π π υ υ α α υ υ υ α υ υ α α. 57

60 υ φ υ π υ α υ π α π π π α α α αυ αυ α α - α π υ π φ α α π. α π π π υ υ υ υ α α υ αυ π α υπ φ α φ υ π υ α υ (Sahai et al, 2005). α αυ, α π WASP (Wiskott Aldrich syndrom protein), α π Arp2/3, π LIM- / φ α α π α π υ α απ cortactin υ α α α α υ υ α α υ α.. (ezrin) Η α α π ERM π α π α α, α (radixin) α (moesin)., π αυ απ α απ α π FERM α υ, υ π π υ 300 α, π α NERMAD (N-terminal ERM association domain), π α α π υ α. Η υ α αφ α α X απ υ FERM π απ α απ υπ F1, F2 α F3 α A, B α C, π α α π α α α υ α α α α φ υ φυ (Pore et al, 2015). π FERM, υπ α π υ π π υ 200 α, α- α, π α α π π (PKA) (Dransfield et al, 1997) ( α 18 ). α υ υ απ α απ 107 α πα α, α π π F-α, π α α π υ α α (Hamada et al, 2003) ( α 18 ).. 58

61 A NH2- COOH2- F1 F2 ιο ή α-έ ι ας P ιο ή όσ σ ς F-α ί ς F3 B F1 F2 F3 Περιοχή α-έ ι ας P ιο ή όσ σ ς F-α ί ς Ptdins(4,5)P2+ Kinase Phosphatase α 18. α ERM π, -α φ ( π π απ Clucas et al, 2014 ) Ό α α α ERM π υ α π α ( φα ) α α υ υ ( υ ), N- α C-ezrin-radixin-moesin association domains (N-ERMAD α C-ERMAD α α), π α α α υπ υπ α α φα - υ (Pore et al, 2015, Algrain et al, 1993, Pore et al, 2015). Η π N-ERMAD, α π C-ERMAD, απ π α απ πα (Gary et al, 1995). Η α υ α υ α α υ α α α π υ π α π απ α α, α π α α α υ απ α υ α υ α απ α π α α υ υ υ α υ α. π υ π υ υ π ERM π α α, π π φ φ υ υ α α π φ α υ πα υ (Arpin et al, 2011). υ α, π φ φ υ π υ PIP2, υ υ α υ α α. 59

62 α α α φ υ υ α υ. π π, υ α π α π υ υ υ υ α π, υ, α α υ (McClatchey, 2003). π ERM α α, φ α υ α π α α, π υ π π F-α, α α «υ» (Pearson et al, 2000). υ α π, απα α α απ υ π π FERM α α α υ π υ π α F-α, αυ π υ α απ αυ π α υ PIP2 (Fehon et al, 2010) α α φ φ υ α (Bonilha, 2007) ( α 18 ). Η α α PIP2 α π ERM α υπ υ α φ φ υ υ α απ, α α α υ α α (Yonemura et al, 2002). π, π αυ απ π α φ φα α (απ φ φ υ ) α α υ υ υ PIP2. π π, ERM π υ α π FERM FAK (Focal Adhesion Kinase), α υ υ υ π υ υπ α υ α υ α υ α. υ α, π FERM-FAK α π α πυ α., π π p53 α MDM2, α π απ, α α π (Frame et al, 2010) ( α 19).. 60

63 α 19. α ERM π FAK ( π π απ Frame et al,2010) α υ α, α α π υ α π α α α, α υφα α π φ α, υ EMT π υ α α α υ φυ υ π α α α απ α α φα υπ π υ α α π (Sarrio et al, 2006). π α α α, α π α Fes α π, υ α απ α υ α υ α α α υ υ α υ α, π υ α α α απ HGF (Crepaldi et al, 1997, Naba et al, 2008). π π αυ π α π α α υ υ α π υ α α π (Arpin et al, 2011). π π, PKCa π α α φ φ υ ( α ERM) α α α α υπ α CD H. 61

64 φ φ υ υ α αυ CD44 α π α υ α α υ (Legg et al, 2002) ( α 20). π π α υ α υ υ πα υ, π υ α υ L1-CAM (Gavert et al, 2010). α 20. υ π α ERM CD44 φ φ υ α ( π π απ Thorne et al, 2004) π α υ α ( α π α, φ υ α, π α π ) α α φ φ υ αυ φ α ezrin (Elliott et al, 2004, Srivastava et al, 2005, Li et al, 2012) α α υ, π απ α α α απ α α φ α α sirna. Έ π υπ φ α υ α α α α υ π (Valdman et al, 2005, Pang et al, 2004) αυ απ αυ φ α υ υ c-myc (Chuan et al, 2010). υ υ α α α α π υ π π Akt/PI3K, υ α α π π α. 62

65 υ c-myc π υ πα α α υ α (Clucas et al, 2015). υπ φ α α υ πα α, α υ α α α (Hunter, 2004, Meng et al, 2010, Shang et al, 2012), α απ α α φ α π υπ π υ α α α π υ υ α ( Palou et al, 2009).. (paxillin) Η πα απ υ α π π α απ α απ αφ π πα α, paxillin, hic-1 α leupaxin (Schaller, 2001). α α α -π π υ α π α π υ υ, α α υ υ α α, α α π α π απ α α π (Deakin et al, 2008). π α α υ π υ α υ α υ α υ α α. α υ υ υ υ α υ α α π α α (α integrin), π α α α π (>125) υ π π α α. π αυ υ α α α α υ α π υ πα υ φυ υ α α, α υ α υ υ α α α α ( α 21). Η πα υ α «α» π π α π α πα απ α π.. 63

66 α 21. π υ υ α πα α υ ( π π απ Deakin et al, 2008), α υ (C-terminal) α α α π π υ α LIM (Lin11, Isl-1, Mec-3), π α υ υ υ α υ, π α π π (Petrez-Alvarado et al, 1994, Kardamas et al 2004). π LIM2 α LIM3 α απα α α α υ (Brown et al, 1996). π π, π LIM, υ α α π α α υ π π tubulin α PTP-PEST (φ φα ) (Cote et al, 1999;, Herreros et al, 2000). α π αυ πα υ α υ α α υ (Webb et al, 2004). α (N-terminal) υ α α α. π α απ π α LD π α υ α α πα α π α υ α α-π π (Tumbarello et al, 2002). υ α, π LD1 απ α π α ILK (integrin-linked kinase) α α actopaxin ( α π pavrin). α υ πα απ υ π υ υ. 64

67 α π α π α π α α α υ α α α α υ α π, υ π απ α α α φ (Legate et al, 2006). actopaxin α ILK υ υ υ α α υ, π α π Rho GTPase (Clarke et al, 2004, Filipenko et al, 2005, Khyrul et al, 2004). π π LD2 α LD4 π υ FAK α φα α π α υ, αυ α FAK φ φ υ πα α πα υ (Schaller 2001). π LD1 α LD2 π α π vincullin α α FAK, π α α ERK α υ υ α π (Subauste et al, 2004). π π, π α π π α proline π α α αυ π α π SH π υ Scr (Weng et al, 1993) ( α 22). Έ υ π απ φ φ υ α υ πα (Webb et al, 2004). αυ υ α απ (PAK, RACK1, JNK, ERK, MAPK, CDK5.α) π π α α απ π α α α υ α α απ αυ πα. Η φ φ υ πα υ α π π π,, π π φ φ υ υ, πα π π π α α α α α (Brown et al, 2004) ( α 22). π π, α π α φ φα, υ α πα, απ π α υ α α.. 65

68 α 22. α φ φ υ πα (Atlas of Genetics and Cytogenetics in Oncology and Haematology-atlasgeneticoncology.org) Η πα υ α υ α α α π υ α π α α π α υ α υ α, π απ α α, π α υ α α υ. υ υπ υ α απ υ φ α πα α π υ π φ αφ. υ α, α υ α πα π α α α -π α α απ α π (Madan et al 2006), α α φ α α υπ φ α υ HER2 (Sarah et al, 2007). π π, φ α α υ π α υ υ α α (Salgia et al, 1999). Έ π φ φ υ πα υ, υ υ α α υ υ α NBT-II α α υ υ (Petit et al, 2000), π π α α α υ υ υ (Metalli et al, 2010). 3.8 (cortactin) Η α α α π π υ υ α α υ υ α α, υ α υ, υ α αφ, υ α α α π (lamellipodia), υ α α. 66

69 α. αυ π υπ α Scr (Wu et al, 1993)., α α α α (N-terminal) π π π π α Arp2/3, α υ α α υ α π πα α α α α υ, π υ π α F-α (Weed et al, 2000). π α π π α π π φ φ υ υ απ Src, α, π SH3 α υ (C-terminal), π υ α π π α π α α π υ υ α, π N-WASP, dynamin, WIP (Weed et al, 2001) ( α 23). α 23. α α α π π ( π π απ Weed et al, 2001). Η α υ α α π α α π πα υ υ π υ Arp2/3 α υ υ α (Kowalski et al, 2005). Ω, απ α π υ υ π υ υ π N-WASP (Weaver et al, 2001, Uruno et al 2001).. 67

70 υ α π υ υ π υ Arp2/3 α π π, α π α α α α α α υ (Kowalski et al, 2005). π π, α π -WASP υ α υ, α α WIP (WASP-interactin protein), αυ α α α α (Kinley et al, 2003) ( α 24). α 24. π υ υ π υ Arp2/3 απ N-WASP α cortactin α α α α α α ( π π απ Roger, 2004) π, π α π π α (invadipodia) α α α π α (lamellipodia) π υ α α υ υ υ, α α υ α α υ α (Weaver, 2008). υ α, π υ α υ υ υ υ, υ α α π α α π (Bryce et al, 2005). π π, α π α, υ α π α φ α π π α π α π π π N-WASP, WIP, dynamin ASAP1/AMAP1. 68

71 α src kinase (Weaver, 2006) α απ α π π α υ (Bowden et al, 2006). α- αφ α α π π α απ α αφ α π υ π α υ υ (src, fer, abl, fyn, syk, met) α α / (ERK, PAK, MLCK) (Lua et al, 2005, Boylk et al, 2007). α α α α π α α υ α α υ α α υ π απ υ α υ α π υ α α π α α (Ayala et al, 2008). υ α, π α υ π Src kinase, Dynamin α N-WASP α παφ α υ, π υ α απα α α απ υ α υ α. α α, π υ υ π φ φ υ. αυ π α π, π π υ υ π υ Arp2/3 πα α α α α υ 1-4 π υ π α α. Η PAK φ φ υ α π πα α α α α υ α π υ π α α, π υ α α α α α (Webb et al, 2006). Έ υπ φ α α π π υ α υ π π υ π α υ : α φα - α υ, π α α α φ υ, α πα υ α υ, πα υ α α α, α α α, α φ α α (Timpson et al, 2007, Hsu et al, 2008, Ying-Yu et al, 2009, XIAOHAN et al, 2008, Bao-Zhu et al 2003, Dedes et al, 2010, Wang et al, 2009, Xu-Zhi Xu et al, 2010). υ α, υπ φ α υ α υ υ π υ 11q13.3 (Myllykangas et al, 2007), υπ α υπ φ α υ α υπ αυ υ (Greer et al, 2007). αυ π, απ cortactin, υπ υ π α α. π αυ, cyclin D1 α EMS1/cortactin α α υπ φ α π α π α, αφ υπ φ α υ υ π υ (Schuuring, 1995).. 69

72 . ς σ ϊσ ς 4.1 ς Η π φ α π α φ υ. Η α α α α α (α υ ) α απ α α α α π α α α α π α α υ-α α. Έ υ α φα α α υ π π α α α α α. α π α υ α α α υα α α α, αφ α α υ α υα α, α α π α α, απ α π υ α απ α α, π π α πα υ. π π, φα π υ υ α α α υ α π α α α α α α. π α υ α πα υ α α φ α π in situ υπ α, π π α π π υ αυ φ α υ α ( α π π α α α, π α α υ υ υ π α, πυ υ α π α α α ). π π, υ α α υ π φ α πα α α π α αφ π α, α α π α α, α υ α υ π α α α π α υ πα α υ. α α υ απ α υπ α α απ. αυ π α απ απ α απ απ π υ υ α υ, υ φ α α υ α π π πα α.. 70

73 4.2 σ ς ς Η α α α α υ α ( π υ ) υ α υ α α α π υ α in situ υπ. Η α υ υ α α φ υ, π α α, α υ. α α α α υπ π υ, π υ α α α π π υ α υ φα α π α φα α υ απ α υ (background staining). Ω, α π α α υ, α α α υ υ α υ, α α υ α α α υ υ υ α υ. π π υ υ α α α / α υ απ α α α ( υα α). α υ υ π υ υ π α α α α α υπ. Ά α υ α (α α φ φα, υ ) π α υ π π (π. φ υπ ) α π υ α αυ α υ αφ α (doubleimmunostaining). Η υπ α α, πα υ α, πα υ υπ υ υ υ υ α υ. Ω α α π α υ π α α πα υ α ( α). π υ π α α α 3,3- αυ - (DAB) π υ πα α αφ α. α -υπ α α, α. π α α α α υ α π α α α α α υ α α α. α υ υπ α υπ (PAP), α - (ABC) α - π α (B-SA).Η - π α (B-SA) α α π υ α υ φα, α α - υ π α υ α υ α, π υπ π α φα α υα α. 71

74 α υ α -. Η αυ α α α α υ α, υ α α π α, α π α υ 60 kd π υ π α απ α Streptomyces avidinii. Η π α α α π α α π α α υ α α α α α π α π. α α π π α α α υ IgG υ υ π α α υ. υ α π υα υ υ υ α π υ π π, α α α α α α α α π α (π. υπ ) α α α α α α, αυ α α α α. απ α α α α α π α α α αυ α υ υ α α α π υ α α Igs π α υ α α α. υ α υ α π υ π EnVision. Η υ α α α π υ π υ υπ υ υ α α υ α α α α α π α φ υ α π υπ α α φ φα. Η α υ απ α π α α π α υ-υπ α (3,3 α /DAB) π υ α α υ π υ φ α α π υ α α α α. π α αυ α υ α α α α α, α υα α α υ α. α α α π υ υ απ π α υ α α (background) α : 1. π α α υ. Η π υ υ α α υ α υ α υ α π α υ α π, α απ π αυ υ.. Ω, α υ π π α α α α α π υ α α. Έ α υ π π υ α α α φ α, π α α υ π π α α α α υ α υ. π, α π υ α α π α υ. 72

75 υ α (Eltoum et al., 2002). υ α φ α α α α π α α α υ α α α: υ, υ, α πα α,, α, υ α υ α (Jones et al., 2002). α α π φ α α α π α φ α (υ α α α α φ α α αφ α υ φ α ) α φ α α α (- Η Η 2 ), α α α αυ υ υ (cross-links) (Dapson et al., 1993). απ α π φ α α α α α α ( α φ ) α, π α α π α α α π (Ags) απ α α α α π α α π. υ α α π π α ( α α α α ) π π α α υ α π α α (Montero et al., 2003) & (Mason et al., 1991). 2. α υ (Antigen Retrieval). αφ α υ π υ α φ υ α α π υ υ α α α π α π υ α α α απ α α α α. Η π π π α α π (α ), α α π φ α α απ α α α. Η α α π α α π α π α α α υ πα α α π π, π α π υ α π π υ υ α απ π φ α α α υ π α απ α π α υ α. αυ π υ φ α α π υ πα αφ π α υ φα υ α α υ υ α ( υ α α ) π υ α «Θ πα α π π υ» (HIER) α α υ π υ α α υ π ( υ πα α π π ). α π υ π α HIER α α υ απ α α α φ α α α α π υ υ α α. 73

76 π. Χ π α α HIER α α α απ αφ υ α α α (π.. α, Tris) α φ α pη (3 10). pη υ α α α α. α α α α α α α α α pη, α α α α υ pη, α α α α α α α α π υ pη υ α α. π π α, α α π α α α, HIER 0.01 υ α α α υ (pη 6.0) α πα α π απ α α. Φ υ, α π α α π π υ υ α αυ π α α α. υ, α α α α 20 π α πα αφ α α π α 20 π. π φ υ α α α υ υ α Η 6.0. α α υ α TRIS-EDTA ph 9.0 α EDTA ph υ υ α α (protein blocking) Η α πα π υ α υ α α υ απ α α α α π υ υ α. π α υ π υ π υ π α υ α. Η background π α α. Η α α α (background) α α π υ α α α υ φ α α υ υ α (Petrelli et al., 1978) & (Wood et al., 1977). υ background α υ φ α π α π α αυ. Η α υ π α α α α π π υ υ π υ α α (protein blocking solution) 4. α - α α υπ. π υ υπ. υ υ α υ α α α α α υ α α υπ α απα α α α υπ π υ α. 74

77 απ φ υ. Η α α υπ π α α π α α α υπ υ π π α π α α. 5. π α π α α 6. Έ α υ α υ α υ α α. 7. Χ α πυ α α υ α πα α φ π.. 75

78 .. Η υ α π α α υ απ α φυ υ α υ π α α α α α α π υ α απ υ α α α α. π υ υ α α α υ υ α α υ υ α α πα υ α α π υ α α υ, π υ α α π ( ), α α α υ. Η, π υ υ υ α υ α, πα α α απ α υ υ α υ υ α α. υ α α α φ α π αυ α α π α πα α α α. α πα απ, υπ α π π υ α π υ α α π α υ α υ α α. υ π, π πα α α α υ φ α υ υ α υ υ α α, πα α α πα α υ - υ α α α α υ υ π α α π αυ πα α υ. Γ α π αυ : 1. Η φ α, πα, α α 104 υ α α α α υ υ α α. 2. Η π α υ υ α φ α πα απ πα α α α α α πα α α πα α υ υ, π. 76

79 π, υ υ α υ υ υ α υ υ α αφ π α υ. 3. Η π α υ π π φ α, πα, α α α π / π α π E-cadherin and -catenin π υ πα υ α π υ α α π. 4. Η π α υ π π φ α, πα, α α φ α υ α α α υ p53 π α πα α π α υ α α α α. 77

80 . 2.1 σ Η πα α α. π α α π φ α υ α α Γ α π α 104 πα α υ α α υ α α υ υ υ α π α π α υ α π, α υ π φ α υ π α. α πα α υ α α α π α υ α φ α 10% α υ α α πα αφ. υ ( πα αφ ) π α απ α υ α α α α υ υ Γ α α υ α «Ά α». α απ υ 104 υ (13.5%) α υ α π α α α α υ υ α (papillary urothelial neoplasms of low malignant potential -PUNLMP), 33/104 (31.7%) α υ α α α α α α α α (low grade urothelial carcinomas ) α 57/104 (54.8%) α υ α α α α υ α α α (high grade urothelial carcinomas). (pt1) 37 π π (35.6%) (3/33 low grade α 34/57 high grade carcinomas) υ α (pt2) 23 π π (22.1%) ( α υ α α υ α α α α). α α απ υ α απ α π. α πα α α α α π α υ α πα υ α α α υ π α α

81 . σ σ ώ α πα α υ α α α π α υ α φ α 10% α υ α α πα αφ. π π α π υ π α α π π υ πα αφ απ π φ α π υ 5 m. αφ π α φ π (Super Frost Plus, Menzel, Freinburg, Germany). α α 1. α πα α α α α υ α α υ υ. υ α (n=104) α α α n(%) π PUNLMP 14/104(13.46%) Χα α α α 33/104(31.73%) α α α 57/104(54.80%) pta 44/104(42.30%) pt1 37/104(35.57%) pt2 23/104(22.11%) * Ό (pta) 44/104(42.30%) α (pt1+ pt2) 60/104(57.69%) υ χ α Ό 81/104(77.88%) α 23/104(22.11%) n=α π α *α αφ α υ υ / α υ υ α. 79

82 . σ ϊσ ώσ Η α π υ φα α α υ π πα, α, - α, - α α p53 πα αφ υ α α α α υ υπ DAB πα αφ π υ (Envision detection kit DAKO, Glostrup, Denmark). 2.4 σ ϊσ ς ώσ ς σ ς ϊ ς σ ς ς ς ς, ς, - ς, - ς p σ ώ ς σ ς Γ α απ πα αφ α α α π π π υ 4 m π α υ α α DAKO target retrieval solution PH 9 α π DAKO pressure cooker (PT, DAKO, Glostrup, Denmark) α 15 min υ 90 0 C. α π α α α π υ π α α υ α αυ π υ υ α α α α υ α α - (background staining) α α α φ α α α υ π α α 2. H υ α α α Envision Detection Kit (DAKO) α DAB. α πυ α α υ, π υ α π α υπ. π α α π α αυ α π α DAKOautostainer (DAKO, Glostrup, Denmark). Γ α υ π α α υ (negative control), π υ π α φ α π π α α α α π υ π α φυ πα α α υ υ ( α control). Ω υ π α απ α α α πα υ.. 80

83 α α 2. π α α πα υ α α α α α π υ π α α α π α α υ. α υ α Χ π α anti-ezrin anti-paxillin Anti-cortactin Mouse monoclonal 1: min at RT Neomarkers, CA, USA Mouse monoclonal 1: min at RT Neomarkers, CA, USA Mouse monoclonal 1:50 45 min at RT Santa Cruz Biotechnology anti-e-cadherin Mouse monoclonal 1: min at RT BD Biosciences CA, USA Anti- -catenin Mouse monoclonal 1: min at RT BD Biosciences CA, USA Anti-p53 Mouse monoclonal 1:20 1. min at DAKO Glostrup, RT Denmark. 81

84 2.5 σ σ ϊσ ς ώσ ς Η α φ π υ. Η α φ α υ p53 α >10% υ υ υ φ πυ α α p53. Η π υ υ υ π υ α π υ π α αφ υ φ α υ p53 υ 10% υ υ υ α υ π α α πα υ α α υ p53 (Esrig et al, 1993, Esrig et al,, Sarkis et al, 1993, Malats et al, 2005, Shariat et al, 2010). Γ α π πυ, υ α π α α α α α π υ πα α α, α α. α α π υ ( α α ) α α α απ 0-3 α. Η α α 0: α, 1: α α, 2: α α α 3:. Η α α (π υ ) α 0 (<1% υ υ υ), 1 (1-25% π υ υ υ), 2 (26-50%), 3 (51-75%) α 4 (76-100%). α α α α (score) α α α. (Immunoreactivity score, IR) : 0: α, 1: α ( α α α = 1,2), 2: ( α α α = 3, 4, 6), 3: ( α α α = 8,9,12). π IR score 0 α α π π IR score 1,2 3 α. π π α - α α - α α IR <3 α πα α π α α υ φ α α α α α α α αυ π 3. π π υ α π α α - α α - α αυ υ α π α α φ α IR>1 α πα α π α α υ φ α α α α υ α π α α α α αυ π

85 . σ σ Η α α υ απ υ α πα υ SPSS v.21 α Windows. Γ α α α αφ α αφ πα α α πα α π π, α αφ π α πα υ α π φ α πα, α, - α, - α α p53, α α α α α α πα α Kruskal- Wallis ( α k α α α α) α Mann Whitney ( α 2 α α α α). Η υ α φ α αφ π (IR scores) Spearman Rank Order Correlation. Γ α π π α α π αυ α αφ π, α ( αυ π α π π πα α ) π Multivariate regression analysis υ Enter. πα π υ υ π φ α α υ multivariate regression α π (predictor variables) α αυ π υ α α α υ α α α -πα α. Γ α α α α p<0.05 α α α σ ϊσ σ ς ς σ ώ ς σ ς σ σ. πα α π α α υ α α α υ α α α υ α π α α ( α 1 ). Θ α α πα α α α α υ υ, α α α α α α α α φ ( α α φ α α). Θ α φ α πα α 103/104 (99%) π π υ α α υ υ. Η α α α / α υ α π α α. α α α 103/104 (99%) π π α υ α π α α 87/104 (83.7%) υ. πα α α α α α αφ υ α π α α. 83

86 α φ α πα α πα α υ π υ α. Ω υπ α α αφ α φ α α υ α α αφ α αφ π α αφ υ π φ ( φ α 4) (π α α 1). α υ α, α α φ α ( α α α ) α α α α υ α α α α υ α α α υ α α α α α α α α (p=0.02) ( φ α 1) απ α α αφ α υ α α α υ α π α α α υ υ α (PUNLMP). α υ α α α α πα υ α α π α α α φ α υ υ υ α υ (p=0.04) ( φ α 2). π π υ α α π υ φ α υ υ α υ υ πα υ α α α α α α φ α (p=0.02) ( φ α 3). α α α υ α π α α α α απ α α α υ υ π υ α υ α. Η π υπα α α υ (multivariate regression analysis) α α φ α απ α π π πα α υ υ α α υ α α α α υ υ ( =0.268, P =0.04) α π α π α α α αφ π, π α υ α α.. 84

87 Γ φ α 1. Η α α φ α α υ α α α α α αφ π Γ φ α 2. Η α α φ α α υ α α α α πα υ α υ υ. 85

88 Γ φ α 3. Η α α φ α υ υ α υ υ υ α υ υ Γ φ α 4. Η α α φ α υ υ α υ pt. 86

89 Α. 87

90 α 1. Η α χ φ α α υ α υ υ χ υ (A) υ α α α υ α π α α α α πα α π α α υ ( ). π π υ π α υ υ α π α α α υ υ α υ α α υ α π α α α α.( Γ). α φ α π α υ α α α π υ υ α υ υ. α α 1. Η α φ α πα α α πα α υ υ α υ υ. Ezrin ( α ) a N p-value c n (%) n (%) n (%) n (%) υ α 104 1/104 10/104 25/104 68/104 ( ) (1.0%) (9.6%) (24.0%) (65.4%) PUNLMP 14 0/14 1/14 3/14 10/14 (0%) (7.1%) (21.4%) (71.4%) Low grade 33 0/33 1/33 6/33 26/ carcinomas (0%) (3.0%) (18.2%) (78.8%) High grade 57 1/57 8/57 16/57 32/ carcinomas (1.8%) (14.0%) (28.1%) (56.1%) 0.06 pta 44 0/44 2/44 9/44 33/44 (0%) (4.5%) (20.5%) (75.0%) pt1 37 0/37 5/37 8/37 24/27 (0%) (13.5%) (21.6%) (64.9%). 88

91 pt2 23 1/23 3/23 8/23 11/23 (4.3%) (13%) (34.8%) (47.8%) * 0.04 Όχ (pta) 44 0/44 2/44 9/44 33/44 (0%) (4.5%) (20.5%) (75.0%) α (pt1 α pt2) 60 1/60 8/60 16/60 35/60 (1.7%) (13.3%) (26.7%) (58.3%) υ χ α 0.02 Όχ 81 0/81 7/81 17/81 57/81 (0%) (8.6%) (21.0%) (70.4%) α 23 1/23 3/23 8/23 11/23 (4.3%) (13.0%) (34.8%) (47.8%) a, b Η α α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α *α αφ α υ υ / α υ υ 3.2 σ ϊσ σ ς ς σ ώ ς σ ς σ σ. πα α π α α υ α α πα α α α υ α π α α ( α 2). Θ α α πα πα α α α α υ υ, α α α α α α α α φ ( α α φ α α). Η α α πα υ α π α α ( υ ) α α π υ π α α π φ α υ. Θ α φ α. 89

92 πα πα α 92/96 (95.8%) π π υ α υ υ. Η α α πα α υ υ α π α α 90/96 (93.7%) π π υ α υ υ. π α α αφ α φ α πα ( α / α υ α π α α ) α υ α α α υ α π α α α υ υ α (p=0.001 α υ α π α α α p=0.005 α ). π π α α αφ φ α πα α υ α α υ α α α υ α α α α α α α α (p=0.026 α ). Όπ φα α απ α αφ α α 5, 6, 7, 8 α 9 α υ π α 2 α 3 α φ α πα α α α υ α α α α α α α α υ α π α α α α υ υ α α α α υ α α α α υ α α α α α α α α. π απ α α αφ α φ α πα ( α / α υ α π α α ) α υ α α αφ α υ (p=0.025 α υ α π α α α p=0.019 α ) ( αφ α α 14 α 15). α υ α α α α π υ α υ α φ α α α φ α πα υ υ υ (p=0.008 α υ α π α α α p=0.005 α ) ( αφ α α 10, 11, 12 α 13) Ω π υπα α α υ φ α πα απ α π π πα α α αφ π, υ α υ.. 90

93 Γ φ α 5. Η α φ α πα ( υ α π α α / α α ) υ υ α υ υ υ. 91

94 Γ φ α 6. Η υ α π α α α φ α πα υ υ α υ υ υ Γ φ α 7. Η α α φ α πα υ υ α υ υ υ. 92

95 Γ φ α 8. Η α φ α πα ( ) α υ α α α α υ υ α αφ π Γ φ α 9. Η υ α π α α α φ α πα α υ α α α α υ υ α αφ π. 93

96 Γ φ α 10. Η α φ α πα ( ) υ υ α υ υ υ πα υ α υ υ υ υ α Γ φ α 11. Η υ α π α α α φ α πα υ υ α υ υ υ πα υ α. 94

97 Γ φ α 12. Η α φ α πα ( ) υ υ α υ υ υ πα υ α υ υ α Γ φ α 13. Η υ α π α α α φ α πα υ υ α υ υ υυ πα υ α υ υ α. 95

98 Γ φ α 14. Η α φ α πα ( ) υ υ α υ υ υ (pt) Γ φ α 15. Η υ α π α α α φ α πα υ υ α υ υ υ (pt). 96

99 Α. 97

100 α 2. Η α χ φ α πα α υ α υ υ χ υ (A, ) υ υ α π α α α α α α πα πα α π α α υ ( ) α α π π υ π α υ υ α π α α α υ υ α ( ). ( Γ). π π υ π α υ α α α pt2 υ α α α α α φ α πα α α α υ υ. α α υ φ α α α α α α. α α 2. Η α φ α πα πα α α πα α υ υ α υ υ. Paxillin ( : υ α π α α α α ) b p-value c n (%) n (%) n (%) n (%) υ α 96 4/96 43/96 27/96 22/96 ( ) (4.2%) (44.8%) (28.1%) (22.9%) PUNLMP 12 0/12 1/12 6/12 5/12 (0%) (8.3%) (50%) (41.7%) Low grade 33 1/33 13/33 8/33 11/ carcinomas (3%) (39.5%) (24.2%) (33.3%) High grade 51 3/51 29/51 13/51 6/ carcinomas (5.9%) (56.9%) (25.5%) (11.7%) pta 42 1/42 13/42 14/42 14/42 (2.4%) (31%) (33.3%) (33.3%). 98

101 pt1 33 1/33 20/33 8/33 4/33 (3%) (60.7%) (24.2%) (12.1%) pt2 21 2/21 10/21 5/21 4/21 (9.5%) (47.6%) (23.8%) (19%) Ό (pta) 42 1/42 13/42 14/42 14/42 (2.4%) (31%) (33.3%) (33.3%) α (pt1 or 54 3/54 30/54 13/54 8/54 pt2) (5.6%) (55.6%) (24.1%) (14.8%) υ χ α /75 33/75 22/75 18/75 (2.7%) (44%) (29.3%) (24%) α 21 2/21 10/21 5/21 4/21 (9.5%) (47.6%) (23.8%) (19.0%) a, b Η α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α α α 3. Η υ α π α α α φ α πα πα α α πα α υ υ α υ υ.. 99

102 Paxillin ( υ α π α α ) b p-value c n (%) n (%) n (%) n (%) υ α 96 6/96 31/96 27/96 32/96 ( ) (6.3%) (32.3%) (28.1%) (33.3%) PUNLMP 12 0/12 1/12 1/12 10/12 (0%) (8.3%) (8.3%) (83.3%) Low grade 33 2/33 10/33 8/33 13/ carcinomas (6.1%) (30.3%) (24.2%) (39.4%) High grade 51 4/51 20/51 18/51 9/ carcinomas (7.8%) (39.2%) (35.3%) (17.6%) pta 42 2/42 10/42 9/42 21/42 (4.8%) (23.8%) (21.4%) (50.0%) pt1 33 2/33 16/33 8/33 7/33 (6.1%) (48.7%) (24.2%) (21.2%) pt2 21 2/21 5/21 10/21 4/21 (9.5%) (23.8%) (47.6%) (19%) * Ό (pta) 42 2/42 10/42 9/42 21/42 (4.8%) (23.8%) (21.4%) (50.0%) α (pt1 or 54 4/54 21/54 18/54 11/54 pt2). 100

103 (7.4%) (38.9%) (33.3%) (20.4%) υ χ α /75 26/75 17/75 28/75 (5.3%) (34.7%) (22.7%) (37.3%) α 21 2/21 5/21 10/21 4/21 (9.5%) (23.8%) (47.6%) (19.0%) a, b Η α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α *α αφ α υ υ / α υ υ α 3.3 Η α φ α α αυ α υ α α α α υ υ α α π υ π φ. πα α π α α υ α α α α α υ α π α α ( α 3). Θ α α α πα α α α α υ υ α α α α α α. Η α α α α α υ α π α α π. Θ α φ α α πα α 104/104 (100%) π π υ α υ υ. υ α π α α α 92/104 (88.5%) α π απ 103/104 (99%) υ α υ υ. π α α αφ α φ α α ( υ, α α υ α π α α ) α υ α α α υ α π α α α υ υ α (p=0.014 α υ, p=0.05 α υ α π α α α p<0.001 α. 101

104 α ). π π α α αφ φ α α α υ α α υ α α α υ α α α α α α α α (p=0.001 α α p<0.001 α υ α π α α α α ). Όπ φα α απ φ α 18 α π α α 4 α φ α α ( υ ) α α α α υ α α α α α υ α π α α α α υ υ α α α α α υ α α α α υ α α α α α α α α. υπ υ α π, απ α αφ α α 16 α 17 α υ π α 5 α 6 φα α α φ α α αυ α υ α π α α π α απ α υ α π α α α α υ υ α α υ α α α α α α α α α υ α α υ α α α υ α α α α. π απ α α αφ α φ α α (, α α υ α π α α ) α υ α α αφ α υ (p<0.001 α, α α υ α π α α ) α α π φα α απ α αφ α α 27, 28 α 29 α π α α 5 α α φ α α αυ α υ α π α α φ α α υ. α υ α α α α φ α α α α φ α α ( α α ) υ υ υ (p<0.001 α α α ) ( αφ α α 21 α 22) α π α υ α α α φ α α α α φ α α ( α α ) υ α υ (p=0.017 α α p<0.001 α α ) ( αφ α α 24 α 25). υ α π α α φ α α α α α α πα υ α (p<0.001) α υ υ α υ υ (p<0.001) ( αφ α α 23 α 26). Η π υπα α (multiple linear regression) α υ α α υ α π α α φ α α απ α π π πα α υ α αφ π, (p=0.019, beta= α. 102

105 υ α π α α α p=0.043, Beta=0.347 α α ) α υ α υ pt (p=0.002, eta= α υ α π α α α p=0.003, eta=0.508 α α ). π, α φ α α απ α π π πα α υ υ / α υ υ α( p=0.002, Beta=0.589) α υ α π α α φ α α απ α π π πα α υ υ α (p=0.002, eta=-0.466). Γ φ α 16. Η α α φ α α υ υ α υ υ υ. 103

106 Γ φ α 17. Η υ α π α α α φ α α υ υ α υ υ υ Γ φ α 18. Η α φ α α ( ) α υ α α α α υ υ α α α. 104

107 Γ φ α 19. Η α α φ α α α υ α α α α υ υ α α α Γ φ α 20. Η υ α π α α α φ α α α υ α α α α υ υ α α α. 105

108 Γ φ α 21. Η α φ α α ( ) α υ α α α α υ υ πα υ α Γ φ α 22. Η α α φ α α α υ α α α α υ υ πα υ α. 106

109 Γ φ α 23. Η υ α π α α α φ α α α υ α α α α υ υ πα υ α Γ φ α 24. Η α φ α α ( ) α υ α α α α υ υ υ υ α. 107

110 Γ φ α 25. Η α α φ α α α υ α α α α υ υ υ υ α Γ φ α 26. Η υ α π α α α φ α α α υ α α α α υ υ υ υ α. 108

111 Γ φ α 27. Η α φ α α ( ) α υ α α α α υ υ pt Γ φ α 28. Η α α φ α α α υ α α α α υ υ pt. 109

112 Γ φ α 29. Η υ α π α α α φ α α α υ α α α α υ υ pt α α 4. Η α φ α α πα α α πα α υ υ α υ υ. α ( ) a N p-value c n (%) n (%) n (%) n (%) υ α 104 0/104 15/104 65/104 24/104 ( ) (0%) (14.4%) (62.5%) (23.1%) PUNLMP 14 0/14 4/14 10/14 0/14 (0%) (28.6%) (71.4%) (0%) Low grade 33 0/33 6/33 25/33 2/ carcinomas (0%) (18.2%) (75.7%) (6.1%). 110

113 High grade 57 0/57 5/57 30/57 22/ carcinomas (0%) (8.8%) (52.6%) (38.6%) <0.001 pta 44 0/44 10/44 32/44 2/44 (0%) (22.7%) (72.7%) (4.5%) pt1 37 0/37 3/37 22/37 12/37 (0%) (8.1%) (59.5%) (32.4%) pt2 23 0/23 2/23 11/23 10/23 (0%) (8.7%) (47.8%) (43.5%) <0.001 Όχ (pta) 44 0/44 10/44 32/44 2/44 (0%) (22.7%) (72.7%) (4.5%) α (pt1 α pt2) 60 0/60 5/60 33/60 22/60 (0%) (8.3%) (55%) (36.7%) υ χ α Όχ 81 0/81 13/81 54/81 14/81 (0%) (16.1%) (66.7%) (17.2%) α 23 0/23 2/23 11/23 10/23 (0%) (8.7%) (47.8%) (43.5%) a, b Η α α α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α. 111

114 α α 5. Η α φ α α α πα α α πα α υ υ α υ υ. α ( α ) a N p-value c n (%) n (%) n (%) n (%) υ α 104 1/104 35/104 36/104 32/104 ( ) (1%) (33.6%) (34.6%) (30.8%) PUNLMP 14 1/14 11/14 2/14 0/14 (7.1%) (78.6%) (14.3%) (0%) Low grade 33 0/33 19/33 13/33 1/33 <0.001 carcinomas (0%) (57.6%) (39.4%) (3%) High grade 57 0/57 5/57 21/57 31/57 <0.001 carcinomas (0%) (8.8%) (36.8%) (54.4%) <0.001 pta 44 1/44 29/44 14/44 0/44 (2.3%) (65.9%) (31.8%) (0%) pt1 37 0/37 4/37 20/37 13/37 (0%) (10.8%) (54.1%) (35.1%) pt2 23 0/23 2/23 2/23 19/23 (0%) (8.7%) (8.7%) (82.6%) <0.001 Όχ (pta) 44 1/44 29/44 14/44 0/44 (2.3%) (65.9%) (31.8%) (0%). 112

115 α (pt1 α pt2) 60 0/60 6/60 22/60 32/60 (0%) (10%) (36.7%) (53.3%) υ χ α <0.001 Όχ 81 1/81 33/81 34/81 13/81 (1.2%) (40.7%) (42%) (16.1%) α 23 0/23 2/23 2/23 19/23 (0%) (8.7%) (8.7%) (82.6%) a, b Η α α α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α α α 6. Η α φ α υ α π α α α πα α α πα α υ υ α υ υ. α ( υ α π α α ) a N p-value c n (%) n (%) n (%) n (%) υ α /104 48/104 39/104 5/104 ( ) (11.5%) (46.2%) (37.5%) (4.8%) PUNLMP 14 0/14 5/14 8/14 1/14 (0%) (35.8%) (57.1%) (7.1%) Low grade 33 0/33 13/33 16/33 4/ carcinomas (0%) (39.4%) (48.5%) (12.1%) High grade 57 12/57 30/57 15/57 0/57 <

116 carcinomas (21%) (52.6%) (26.3%) (0%) <0.001 pta 44 0/44 16/44 24/44 4/44 (0%) (36.4%) (54.98%)(9.1%) pt1 37 2/37 22/37 12/37 1/37 (5.4%) (59.5%) (32.4%) (2.7%) pt /23 10/23 3/23 0/23 (43.5%) (43.5%) (13%) (0%) <0.001 Όχ (pta) 44 0/44 16/44 24/44 4/44 (0%) (36.4%) (54.5%) (9.1%) α (pt1 α pt2) 60 12/60 32/60 15/60 1/60 (20%) (53.3%) (25%) (1.7%) υ χ α <0.001 Όχ 81 2/81 38/81 36/81 5/81 (2.5%) (46.9%) (44.4%) (6.2%) α 23 10/23 10/23 3/23 0/23 (43.5%) (43.5%) (13%) (0%) a, b Η α α α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α. 114

117 Α α 3. Η α χ φ α α υ υ α υ υ χ υ (A, ) υ υ α π α α α α /απ α α α α α α π π υ π α υ α υ α α υ υ α ( ) α υ α α α α α α α ( ). (Γ) υ α α α υ α π α α α α π α υ α α α pt1 υ α α α ( ) π π υ π α υ α α α pt2 υ α α α υ α α α υ α π α α α φ α α.. 115

118 . Έ σ ώ - ς - ς σ ς ς ς ς σ ς πα α π α α υ πα α υ α α α - α α - α υ φ α φυ π π αυ π φυ.. α φ α - α ( α α α <3) πα α 19/55 υ α π α α (34.5%). Η α α υ α α α α α αφ φ α - α πα α α πα α υ π υ α. α φ α - α ( α α α <3) 40/56 (71.4%) π π, υ υ - α υ α π α α (score > 1) 32/56 (57.1%) υ πυ α - α (score > 0) απ 3/56 (5.4%) π π. Η α α υ α α α αφ α φ α - α α αφ π (p=0.02), πα υ α (p=0.058) υ υ α (p<0.001) α υ (p=0.01). Όπ φα α απ υ π α α φ α - α α α υ α α α α υ α α α α α α α α, υ υ υ α α υ υ π υ α υ. υπ α α α α αφ φ α υ α π α α - α πα α α πα α υ π υ α.. 116

119 Α α 4. χ φ α - α χ α υ α α α α.. π π υ π α υ α α α υ α α α - α. απ α α α _ α π α υ α α α υ α α α.. 117

120 α α 7. Η α φ α - α πα α α πα α υ υ α υ υ. E- α χ ( α ) a N υ p-value c φ α Έ φ α (IR=3) (IR<3) n (%) n (%) υ α ( ) (65.5%) 19 (34.5%) PUNLMP 0 Low grade carcinomas (73.3%) (26.7%) High grade carcinomas (62.5%) (37.5%) pta (78.6%) pt (60.9%) pt (61.1%) 3 (21.4%) 9 (39.1%) 7 (38.9%)

121 Όχ (pta) (78.6%) α (pt1 α pt2) (61%) 3 (21.4%) 16 (39%) χ α υ Όχ (67.6%) α (61.1%) 12 (32.4%) 7 (38.9%) a, b Η α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α. 119

122 Α α 5. χ φ α - α α υ α α α α.. π π υ π α υ α α α υ α α α υ α π α α α α - α. υ υ α π α α φ α - α π α υ α α α υ α α α.. 120

123 α α 8. Η α φ α α - α πα α α πα α υ υ α υ υ. - α ( α ) a N p-value c n (%) n (%) n (%) n (%) υ α ( ) (0%) (35.7%) (35.7%) (28.6%) PUNLMP 0 Low grade carcinomas (0%) (6.7%) (40%) (53.3%) 0.02 High grade carcinomas (1.8%) (36.6%) (34.1%) (29.3%) 0.01 pta (0%) (7.1%) (42.9%) (50%) pt (0%) (17.4%) (34.8%) (47.8%) pt (0%) (57.9%) (31.6%) (10.5%) Όχ (pta) (0%) (7.1%) (42.9%) (50%). 121

124 α (pt1 α pt2) (0%) (35.7%) (33.3%) (31%) υ χ α <0.001 Όχ (0%) (48.6%) (37.8%) (13.5%) α (0%) (57.9%) (31.6%) (10.5) a, b Η α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α 3.5 Έ σ ς ΐ ς p σ ώ ς σ ς Θ πυ φ α π p53 90/101 (89.3%) υ. Η φ α π p53 φ α α α αφ π (p=0.015) α π φα α απ φ α 30 α π α α 9 α α φ α π p53 πα α α α α α υ α α α υ α α α α α υ α α α α.. π π α α αφ α φ α π p53 πα υ α α α π φα α απ φ α 31 α π α α 9 φ α π p53 α α α α α α α α (p=0.004) α α α α α α α υ υ α (p=0.017) ( φ α 32). π απ α α αφ α φ α π p53 α υ α α αφ α υ (p=0.007) α α π φα α απ π α α 9 φ α π p53 αυ α υ.. 122

125 Α α 6. χ φ α π p53 υ υ α υ υ χ υ. π π υ π α υ α α ( ) α υ ( ) α α π p

126 Γ φ α 30. Η α φ α π p53 α υ α α α α α αφ π Γ φ α 31. Η α φ α π p53 α υ α π α α. 124

127 Γ φ α 32. Η α φ α π p53 α υ α π α α υ υ α α α 9. Η α φ α π p53 πα α α πα α υ υ α υ υ. p53 a N p-value c n (%) n (%) n (%) n (%) υ α π α α ( ) (10.9%) (22.8%) (36.6%) (29.7%) PUNLMP (16.7%) (33.3%) (41.7%) (8.3%). 125

128 Low grade carcinomas (3.1%) (37.5%) (50%) (9.4%) High grade carcinomas (14%) (12.3%) (28%) (45.6%) pta (7.1%) (38.1%) (45.2%) (9.5%) pt1 26 0/37 5/37 8/37 24/27 (0%) (13.5%) (21.6%) (64.9%) pt2 23 1/23 3/23 8/23 11/23 (4.3%) (13%) (34.8%) (47.8%) Όχ (pta) (7.1%) (38.1%) (45.25%)(9.5%) α (pt1 α pt2) (13.6%) (11.9%) (30.5%) (86.7%) υ χ α Όχ (10.3%) (26.9%) (41.5%) (21.8%) α (13%) (8.7%) (21.7%) (56.6%) a, b Η α π π φ α α υ α υ. c Mann-Whitney test, p<0.05 α α α. 126

129 . σ ϊσ σ ς ς σ σ σ ώς σ σ ς - ς σ ς ς Ό α υ π α υ α α υ α α α υ α α φ α α α φ α - α (r=0.376, p=0.005). π απ α α α υ α α φ α α φ α πα p<0.001). (r=0.402, 3.7 Η α φ α πα α α α φ α π p53 π π πα α α α α υ α φ α πα φ α π p53 (r=-0.202, p=0.053) α α α υ υ α π α α α φ α π p53 (r=-0.237, p=0.017). 127

130 . π α πα υ α υ α α α υ α π α φ π π π π α α α α (McClatchey, 2003, Deakin et al, 2008, Hervy et al, 2006). Η π α υ α α, απ α υ υ α υ υ α α α π υ α α π π υ πα υ α α υ υ (Weaver, 2008). α απ in vitro α in vivo υπ υ αυ π π α φα α π υ α α υ α α α α, π υ α π, υ α π, α υ α (Legg et al, 2002, McClatchey, 2003, Hervy et al, 2006, Weaver, 2008). Η πα α φ α π αυ πα α α πα α υ υ α π α α υ υ. πα α, α π φ α υ π υ α π α α υπ υ α π α α α υ α π α α. υ φ α α α απ α α, υ π α φ α α α υ α π α α π α αφ α α υ α α α υ υ α υ α α α α (Andersson et al, 2014, Palou et al, 2009). α π α υ α π α α α α α π υ α π α α α α α α φ α π α απ φ φ υ α π α φ φα υ α α π α α υ υ α α (Fehon et al, 2010, Bonilha, 2007). α α α α α υ α φ α πα α α πα α υ υ π α α α α υ υ α α α α φ α α α υ υ α α α υ α υ υ. Η π υπα α α. 128

131 α υ α φ α απ α π π πα α υ υ α. υ φ α α υ α α αυ α α π υ α α α α α υ 1 α υ α α α π α υπ υ α α φ α υ π υ υ α α α α π (Palou et al, 2009). π π, α α, π απ α α φ α υ α α α α υ υ, α α π φα υπ υ υ, π α α α α, α α υ π (Andersson et al, 2014). α υ α α αυ υπ υ υ π π φα υπ υ α π α α π α απ α π π υ π φ α π υ α υ α α υ υ. π α υ φ α α απ α α πα α, α α, π α α α π α α. υ α, απ α φ α α π πα α α πα α υ, α π π φα υπ α α, α α υ α υ π υ, α πα α α α (Moilanen J et al, 2003, Bal et al, 2007, Guedj et al, 2016). α πα απ υ α α υ υπ π α υ α -π α. α υπ υ υ α α υ α απ α α αφ υ α υ. υ α, α αφ π υ π υ α υ, υ φ α α υπ φ α αυ α α υ α, π, α α α π. υ α υ φ α α α α π α α (Moilanen et al, 2003), α υ π υ α α υ (Bal et al, 2007) α πα α α α (Guedj et al, 2016)., υπ φ α. 129

132 α α, α α, α α πα υ, πα υ α α α, α α υ α υ α, α α α, π α α α α α α φα α υ, υ α α α π α, α α α υ, α α α πα α π υ α α υ α (Köebel et al 2006, YR et al, 2008, Wang et al, 2009, Li-Juan et al, 2013, Abdou et al, 2016, Kong et al, 2016, Flores-Téllez et al, 2015, Auvinen et al, 2013, Kong et al, 2016, Ma et al, 2013, Gschwantler-Kaulich et al, 2013, Safi et al, 2015, Schlecht et al, 2012, Jin et al 2014, Jin et al, 2012, Piao et al, 2015, Zhang et al, 2015). υ α π απ α α υ υ α in vitro υ α α α υ α α π α α α α α (Chuan et al, 2006, Hiscox et al, 1999). α α υ α αυ α π α α φ α π π απ υ α υ, υ α α υ α υ α π απ α α, π π α αφ υ α αφ π υ, π π α (Gautreau et al, 1999, Parlato et al, 2000). πα α π α φ α π α υ πα α υ α π α α υ υ π. Θ α φ α πα πα α 95.8% π π υ α υ υ α α α πα α υ υ α π α α α α. Η υπ υ α π π πα υ φ α υ α α π α υ υ υ υ α υ α α α υπ. α υ π πα πα α α α π α υ α υ α π α υ α υ υ α α α α αυ π υ α α α α υ. α α α α φ α π πα α υ α α α α, υ α υ α π α α α α υ υ α (PUNLMP) α π α α υ α α α α υ α α α υ α. 130

133 υ α α α α α α α α. π π α φ α πα α α α υ (pt1 - pt2) υ α π α α (pta). α υ α α αυ υπ υ φ α πα π α υ α υ α υ υ π. υ φ α αυ υπ απ π α α α α α υ πα πυ υ υ π p53, π υ πα α α υ υ α α α υ υ α α α π υ α α α υ υ (Esrig et al, 1993, Esrig et al, 1994, Sarkis et al, 1993, Malats et al, 2005, Shariat et al, 2010). υ φ α υπ απ φ α πα α π π υ π φ υ α α π υ πα απ α υ π υ υ α υ α α α α π α π υ α π α α π α υ υ α υ α, π απ α α π α υ α α υ (Deakin et al, 2008). φ α π υπ υ α απ υ φ α paxillin α π υ π φ αφ α π α υ α π υ π α υ α α α υ π α (Madan et al, 2006, Salgia et al, 1999). π α υ φ α α υ α α α φ α πα α αφ α φ υ π υ α υ. υ α, φ α πα α υ α, π υ υ φα (Madan et al, 2006), α α α α πα υ π πα υ πα υ (Ayaki et al, 2001), α π α υ φ α πα α α α α α υ α υπ α απ φ α πα π α υ α υ α α. 131

134 υ πα πα α πα απ π π, α α πα α insulin-like growth factor receptor I (IGF-IR) α υ α α υ α υ υ (Monami et al, 2006, Metalli et al 2010, Petit et al, 2000). π π α α α φ α π α πα α π α α α PTEN α υ α α υ υ υ (Herlevsen et al, 2007). Ω, α α υ α πα α απ π φ α πα πα πα α υ υ α υ υ υ υ α π υ in vivo. Θα π π α υπ υ α α υ α απ α α α υ π υ α υ, π α α πα υ, α α υ α π α α α υ α υπ φ α πα α α αυ φ α αυ α πα α υ α α π (Zhao et al, 2015, Xiao et al, 2014, Chen et al, 2013, Li et al, 2015). π, π φα α α πα υ, α αφ υπ φ α πα απ α π π πα α α, α α π α απ α π πα α υπ π (Yin et al, 2014). π π, π in vitro α α υ πα υ υ α α. Γ α πα α πα α α πα απ π π α υ α υ α α υ υ υ 5637 α insulin-like growth factor receptor I π α α υ α α υ υ υ απ Akt α mitogen-activated protein kinase π πα (Monami et al, 2006, Metalli et al, 2010, Petit et al, 2000). Ω, πα π φ α α υ sirna α α HeLa απ α υ α α π α αυ υ α α υ, α paxillin π α υ α α α α υ α α υ (Yano et al, 2004). α πα απ α υ α απ α α α π α α απ αφ π υ, υ π α α π. 132

135 αφ α πα. Γ α πα α, α π α paxillin kinase linker απ π υ Rac1 απ π α α π πα υ α α (Yano et al, 2004, West et al, 2001), α FAK α src φα α α α πα απ αυ πα α α α απ πα α α α υ α υ υ (Monami et al, 2006, Metalli et al, 2010). π π, πα φα α π φ φ υ α α π υ Bcl-2 π ERK, π α α π α α αυ α α α α υ α α πα υ (Huang et al, 2015). πα α, α α φ α / π α πα π υ π φ υ α π α υ υ. α α υ α α α αφ α α α α (Palou et al, 2009, Andersson et al, 2014) α α απ in vitro π υ πα α α α υ α υ υ (Monami et al, 2006, Metalli et al, 2010). Ω, π υ α α π αυ α α α α α υ α υ υ υ, απα α π π π α π α υ α α υ 3, 4 α 1, α π α in vitro π α α α. π π, π υ α υ πα α α απ π α υ α α α υ υ υ α π υ, α α α α π π in vitro α π α α α υ π α α (gain and loss of function). πα α α π υ υ α υ υ α α, π α, υ υ υ υ π..η π α α υ π υ α α α α π α α π α α π υ π υ υ α α α π α α π α π. 133

136 (invadipodia) (Oser et al, 2009, Mader et al, 2011, Oser et al, 2010).Η α π π υ α π α α απ υ α υ α, π α α α α α α α α (Clark et al, 2008, Kirkbride et al, 2011). Θ φ α α 100 % υ α π α υ υ πα α π α α α φ α α π α α α α α απ α. α π αυ φ α α υ υ α υ υ υ υ α πα α υ, π α αφ π, π υ, υ υ / α υ υ α. α υ α α υπ υ αυ φ α α α υ α π α α υ υ π α υ υ υ α απ π π υ π φ. υ α φ α α υ π υ υ α α α. υ α α α π α α π α π α Arp2/3 α N-WASP π υ α α α π α α υ υ α α α πα α υ α α α π π α υ α π α α α π α (Kowalski et al, 2005, Kowalski et al, 2005). π π α πα π α α α υ α π α α π π υ υ α α απ υ α υ α α π α, α π α π α α α α α υ (Weaver, 2006, 2008). υ φ α α απ α α α α υ α υ α π α υ α α υ υ, α π α υ α υ υ, απ π υ υ α απ α α π, α α α φ α sh RNA α απ α φ α MMP-2 α απ υ α υ α (Tokui et al, 2014). α α υ α α α αφ α α α α α π α α. 134

137 υ απ υ α α α α υ υ φ α π α α α α α α π α απ π υ υ α φ α α π, α α α α (Imanish et al, 2014). π α (EMS1) π α π 11q13 π υ φα υ α α α α υ υ α α υ α υ υ π υ αυ αυ α α α α υ 1. α υ υ α α υ υ υ α υ υα α α υ D1 (CCND) α FGF3, α FGF4 α υ 68% π π υ υ υ α απ 1.9% π α (Zaharieva et al, 2003). α π π α υπ φ α α α υ α α α α α φ α υ υ υ π α π π α υ φα α υ α α α α (Schuuring, 1995, Hui et al, 1998, Luo et al, 2006) α α υ π υ α υ πα α α υπ φ α α π α φ α αφ α απ αυ υ υ υ (Yuan et al, 2003, Greer et al 2007). π α υ φ α α υ α α π υ υπ υ υπ φ α α π α π υ π φ υ α α α, π π α π α α υ υπ φ α α πα πα α υ. υ α, πα υ α α α α α απ α π π πα α π α α φ π (Gang et al, 2013, Zhao et al, 2013). Η υ α υ υ α α υπ φ α α υ α α α α υ α υ φα α υ (Luo et al, 2006) α απ α π α φα α α υ α α υ α α υπ (Ormandy et al, 2003, Rodrigo et al, 2000). π π, α, α α υ πα α α υ φ α Vater, α υ π α υ α α α π α, αυ φ α α α π, α π, υ α φ π. 135

138 α α (Folio et al, , Tsai et al 2013, Hou et al, 2012, Noh et al, 2013). υ φ α α π υ α α α υ υ α α α α α α α α φα (Jing et al, 2016), υπ φ α α π π υ α α π α αυ (Kim et al, 2012). π, πα α υ α α α υ πα υ, αυ φ α α α υ υ, α φα α υ α φ π, α α π α α α απ υ υ (Ni et al, 2015). π π αυ φ α α α α α α α α π αφ α α (prostatic foamy gland α α), α φ π (Li et al, 2016, Hou et al, 2015)., υ φ α αυ, πα α αυ φ α α α φ υ (Chen et al, 2010) α α α α πα υ (Ni et al, 2015), υ πα α φυ. υπ υ α π α, α α α α π α α υ α π α α. π π α α φ α α αυ α α υ α π α α φ α α αφ π, υ α α υ υ α. υ α πα π α υ α α π α, α αυ π α α α α υ α α α α α π α απ υ α π α α α π π α α υ π υ π υ α υ α, π α π α. πα απ υπ υπ υ π π υ υ α π α π α α α π α α α απα α α υ α α υ α αυ α υ (Clark et al, 2007, Weaver, 2006;, Linder, 2007, Artym et al, 2006, Bowden et al, 1999, Webb et al, 2006, Webb et al, 2007, Oser et al, 2009). φα α α υ υ π α α. 136

139 υ απ υ υ α α α α υ υ α υ α υ α α απα α α υ α π α υ π α α α υ υ υ υ υ υ α α α α απ α υ α (Tokui et al, 2014, Imanish et al, 2014). π α υ φ α α α α α υ α υ α υ υ, π υπα α α υ α α α π π πα α υ α αφ π, υ α υ pt α. π π, απ α α α α υ υ α π α α α πυ υ υ π p53, π υ α α υ α α α υ π υ υ α α α α π υ α α α υ υ. Ω απα α π α α α π α α π φ υπ π, υ α π α π υ α π α α π υ α α α υ υ. Η π υ α α π ( ) απ α α α α π υ π α α α α α α α - α α π α α α υ α υ υ απ α απ α α απ α α α υ, απ α υ α φα υπ. Η α π π α υ υ υ α απα α φ α, υ α α α π α α υ. α πα απ α υ α υ υ φ α - α, α π π φυ α α α α α υ υ υ α υ π υ π αυ α α α υ. α α φ α π α α αυ α φ α υ α. υ π π - υ α α π α φ α - α υ α α π π α υ α υ (π υ ), αυ φ α

140 α (N-Cadherin) α (Vimentin), πυ π - α ( -catenin) α α αυ φ α α αφ πα α π Snail1 (Snail), Snail2 (Slug), Twist, EF1/ZEB1, SIP1/ZEB2, α / E47 π υ α α πα α - α (E-Cadherin) (Lee et al, 2006). πα α α, - α α - α α α φ α υ α υ α α α υ πα α π α α. π π α α φ α α - α α α φ α α α α α υ α π α. υ φ α υπ π α π α πα α υ α π α, π υ απ α φ α - α φ α α υ α α υ α α α α α π υ (McConkey et al, 2009). υ π α α α α π α, α, πα φ α - α α - α α πα πα α υ π α α α α, υ α α α α υ υ (Baumgart et al, 2007). α π υ π πα α υ α α υ α π υ (Singh et al, 2016, Hensley et al, 2016) α α α υ α απ υ α α α α υ υ α π υ (Beat et al, 2016) α υ υ πα υ α α α α α α υ υ (Hensley et al, 2016). Ω, α πα α π π πυ φ α - α, α π υ υπ α π υ πα - α α υ α α α α, α π. υ υ α π α α π α φ α - α, π α απα α α απ αφ υ υ - α α α υ α α α α υ υ π.. 138

141 α α α α α απ α υ α υ α α α υ υ α α απ α α α υ. Η υ α υ υ α α π α α α υ α α υ α α υ. π υ υ υ α υ υ α α α α. Γ α πα α α υ α α α π υ α α π α α υ α π υ α υ υ α α α υ π α αυ α α α α α α υ (Lee et al, 2013). π π, α α π π α υ π υ α υ υ α α, α α α α α πα α φ α α, α πα υ α - π α α π ( ) υπ α υ α α α α απα α α (Shankar et al, 2010). π, υ α υ υ α LIMK/cofilin π α π π α α υ α φ α α π π LIMK απα α α υ α α πα απ TGF- (Lamouille et al, 2014). π π υ υ υ α α α φ υ υ α α α υ α α π - π α π α, π π υ α π α π α α N-WASP πα υ α υ α α α π υ α υ (Sroka et al, 2016, Truffi et al 2014, Han et al, 2014, Kurisu et al, 2010) α π π α φα α πα α υ υ α π α υ α υ υ α α α α υ α α υ (Ren et al, 2009). π α υ φ α υπ υ υ α υ υ α α π α, υ υ π Arp 2/3, υ υ N- WASP α α, α α απ - α υ α π α α α απ (Rajput et al, 2013). υ π π υ υ α υ υ α α π α α N-WASP υ υ α. 139

142 υ α υ α υ α υ υ α, α π υ α α α α. α πα απ α π α υ π π υ α π υ α α, πα α α φ α - α α - α., π α υ α α α υ E-cadherin α -catenin α υ α υ α α α απ ezrin α α α υ (Hiscox et al, 1999). α πα υ α α φ α antisense υ, απ α υ α υ α αυ α α α α. α π α υ υ α υ α π α E- α α - α. π π πα π - α απ υ α υ υ α π α α υ απ α α (Hiscox et al, 1999). υ φ α α πα απ, πα α απ α υ α α α α E- α α υ α α α α υ α π υ. Ω, υπ α α φ α α πα - α. π π α υπ υ πα α υ α α απ α υ α υ. π υ α υα α - (zebrafish), φ πα π α α α υ α υ π υ υπ α, π α π υ α α α φα α πα α α υ α α απ απ α α α υ α υ α φ (Crawford et al, 2003). π, πα π α υ α υ π α υ υ α α α υ π (Surgue et al, 1997, Chenn et al, 1998). Η α π α πα α π α α, αφ E-cadherin α π α α υ α π πα (Brown et al, 1998),, υ (Tong et al, 1997). π π, απ π πα α. 140

143 α α HeLa, απ α α α α απ N-cadherin υ (Yano et al, 2004). πα απ πα α, απ πα α α, π α απ α υ α υ, π α π α α φα υπ. Ω, πα α α α υ α φ α E- α α πα α υ α α α α, α αυ υπ. π α π α υ α φ α α α φ α - α α - α. υ α α - α α πα α π φυ υ α α. π α υ α π π Arp2/3 α WAVE υ α υ - π φυ (Stroka et al, 2016, Han et al, 2014) α αφ α π π α φ φ υ απ π D1 (PKD1) RhoA, α π α α - α α υ α απ π φυ, υπ α α α υ α υ α, α α α υ α υ α υ (Sroka et al, 2016). αφ α π α α α π υ υ α υ π α - α α - α α φ α α π α α α α α α α α π α υ α α α α α υ α, απ α α α υ υ α α α α αυ υ α π (Rossum et al, 2006). υ φ α α υ α υ υ α α α α υ α - α υ α υ π υ α αυ υ π α, α π α πα απ TGF-beta1 (Zhang et al, 2009). π π, α α π α α α π υ φα υ π α α, α α α π α υ α α π υ υπ α φα υ π α α π α φ α α (Takkunen et al, 2010). α α πα απ α π υ π υ α. 141

144 α α απ - α α - α υ α υ α α α α α υ α φ α α α - α α - α. α απα α α απ αφ υ υ α υ α υ α α υ α α α α υ υ π. α TP53 απα α υ (>50%) α υ α α α α υ α α α α α α α in situ, α απ α α α α, α υ p53 απ α π πα α α υπ π, υ, π α απ απ α (Anirban et al, 2007, Mitra et al, 2006). Η πυ φ α υ π υ υ p53 α α, π α α α π υ α α υ υ α π α πα υ α α υ υ TP53 (Esrig et al, 1993, Esrig et al 1994, Sarkis et al, 1993, Malats et al, 2005, Shariat et al, 2010). Γ α αυ, πα α πυ φ α υ p53 α α υπ φ α υ φ α π, πα α α. υ φ α π υ, φ α υ p53 α α α α α α α υ υ α. Ω, α α α α υ α φ α α υ p53, υπ α α α α υ α φ α υ p53 α πα. π απ α υ α α α p53. α π φ α πα α υ υ p53 π α πα απ φ α α υ HeLa (Zhao et al, 2016). υ αυ υ υπ π αυ π υ α π / α υ υ υ α υ υ α α π α α -π α υ α α υ υ.. 142

145 , πα α α υ α α φ α α πα α υ α α α α υ α π υ. α α υ, α α πα υ, φ α απ α α α υ υ - υ α υ α α υ, π α α υ α α αυ π π υ α π υ α α. Ω, α, α π α πα α α α (Hiscox et al, 1999). Ω α α υ υ π α α α π αυ π υ α απ π υ π φ α υ α α α α υ υ π. υ π α α, φ α ( α ) α πα α αυ φ α ( α ) α α π α α υ α α υ υ υ α π υ. α π α υ απ υ α υ α α υ, α υ α υ, π απ υπ α απ α φ α α π φ α E- α. π π, υ υ φ α αυ πα αυ, π α π α α υ α α α π π υ υ υ F-α, α α α α α υ υ., α πα απ α, α π α α π α υ α α α α, π α υ α υ π υ υ υπ π, α α υ.. 143

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180 . 178

181 Ε ΙΛΗΨΗ υ α α α υ υ απ α υ α α α πα υ π α υ υ υ α. πα υ π α α π α: απ φυ υ π α α α α in situ α α α α υ υ α α α, υπ π α α α α α α α α υ α π α α, α α α α α. π π α α α α α π α α HRAS α FGFR, α υ α α α π α υ α p53 α Rb. Η π α α υ α αφ π α υ α υ, α υ α α α α υπ π υ α α πα υ υ α π π α π (5%). Ο υ α α απ α υ α απ απ α π υ φα α α α απ π υ π. Ο υ α α α υ α πα α υ α α, π α -π φυ υ α υ α, α υ α α π υ, υ α υ. Ο π υ α α π π υ α αυ, α απ α υ α π υ α, π α α π α α π α, α π α π υ υ α α. Επ π, α α α φ υ υ α α α απ απα α α απ υ π φ υ υ υ α υ π υ α α π α υ π. π πα α α α υ φ α π π υ α π υ α α α υ α α α α υ υ πα α πα α υ υ π φ αυ π α. υ π, α φ α π, πα α α 104 υ α π α α υ υ α α α α π α υ π αυ πα α α πα α υ υ π υ υ α α α αυ

182 π α υ, α υ πα π - υ α α π E- α, - α α π p53. Η α φ α π υ π φ α π π π υπα α α υ α α φ α απ α π π πα α υ υ α α υ α α α α υ υ. Η α φ α πα υ π φ, α φ α α α α α υ α α α α υ υ α π υ π φ. Επ π, π υπα α α υ α α υ α π α α φ α α απ α π π πα α υ α αφ π. υ α υ pt α. Επ, α α υ α α α α α αφ φ α Ε- α πα α α πα α υ π υ α, α α α αφ α φ α - α α αφ π, πα υ α, υ υ α α υ. Επ, πα α α α αφ φ α υ p53 πα υ α α α υ., α φ α α α α α φ α Ε- α α φ α πα, α φ α πα α α α α φ α π ΐ p53. υ π α α, φ α α πα α αυ φ α α α π α α υ α α υ υ υ α π υ. Μ α π α υ απ υ α υ α α υ, α υ α υ, π απ υπ α απ α φ α α π φ α E- α. Επ π, υ υ φ α αυ πα αυ, π α π α α υ α α α π π υ υ υ F-α, α α α α α υ υ., α πα απ α, α π α α π α

183 υ α α α α, π α υ α υ π υ υ υπ π, α α υ. ABSTRACT Urothelial bladder carcinoma consists the 9 th most common malignancy worldwide. There are two distinct molecular pathways implicated in the pathogenesis of urothelial carcinomas. Thus, the normal urothelium can turn into myoinvasive urothelial carcinoma through carcinoma in situ, or into low grade urothelial carcinoma through hyperplasia, with distinct molecular features for each pathway. Mutations of HRAS and FGFR are implicated in the pathogenesis of low grade urothelial carcinomas, whereas mutations of p53 and Rb are encountered in the myoinvasive urothelial carcinomas. The prognosis is associated with grade and stage, and the 5-years survival is low (5%) for those carcinomas which invade the muscularis propria. Actin cytoskeleton is a dynamic structure composed of F-actin filaments and actin binding proteins (ABPs). Polymerization of actin filaments is a tightly regulated process serving several important functions including cell movement, communication with/adhesion to the extracellular matrix, maintenance of cell shape and polarity and ecto/endo-cytosis. Actin polymerization can lead to the creation of protruding membrane structures such as blebs, invadipodia and lamellipodia which account for cell invasiveness. Moreover, remodeling of actin cytoskeleton is essential for cell migration and invasion, as well as for epithelial-mesenchymal transformation (EMT). The aim of the study was to investigate the protein expression of actin regulators in human urothelial bladder tumors in relation to pathologic parameters of tumor progression, mainly invasion. We thus studied the protein expression of ezrin, paxillin and cortactin by immunohistochemistry in 104 human urothelial tumors and evaluated their role in urothelial carcinomas progression. We also explored a possible correlation of these proteins with each other, with EMT regulators E-cadherin and catenin, as well as with the prognostic index p53. Decreased ezrin expression in human urothelial carcinomas of the bladder was correlated with aggressive tumors features and was shown by multivariant analysis to represent an independent predictor of muscularis propria invasion. Paxillin

184 expression was significantly decreased in urothelial carcinomas compared with tumors of low malignant potential and low paxillin levels also correlated with advancing tumor stage and invasion. Cortactin expression was increased in urothelial carcinomas of the bladder and was also correlated with aggressive tumor behavior. In addition, multivariant analysis indicated tha cortactin expression is an independent predictor of grade, stage (pt) and invasion. Expression of EMT marker E-cadherin did not differ significantly among the examined pathological parameters while membranous expression of -catentin was significantly downregulated with advancing grade, stage and invasion. Moreover, high p53 expression associated with advanced stage and the presence of invasion. Finally, a statistically significant correlation was found between membranous ezrin and E-cadherin as well as between ezrin and paxillin expression in urothelial tumors, while paxillin and cortactin expression were found to have a statistically significant correlation with the expression of p53. In conclusion, decreased ezrin and paxillin expression and increased cortactin expression in human urothelial bladder tumors correlate with aggressive tumor features suggesting their implication in bladder cancer progression. Moreover ezrin and cortactin immunohistochemical expression were shown to be independent predictors of invasive disease. Decreased cell adhesion probably accounts for ezrin implication in the process of bladder cancer invasion as shown from the strong correlation between ezrin and E-cadherin expression. In addition, the statistically significant correlation between ezrin and paxillin in this study, could propose a possible signaling interaction among the F-actin regulatory proteins during the process of bladder cancer invasion. Finally, these actin regulatory proteins could probably be used as biomarkers in the future, defining the patients that are in high risk of recurrence, progression and metastasis.

185 Urologic Oncology: Seminars and Original Investigations 31 (2013) Original article Decreased ezrin and paxillin expression in human urothelial bladder tumors correlate with tumor progression Afrodite Athanasopoulou, M.D. a,b, Panagiotis Aroukatos, M.D. b, Dimitrios Nakas, M.D. b, Maria Repanti, M.D., Ph.D. b, Helen Papadaki, M.D., Ph.D. a, Vasiliki Bravou, M.D., Ph.D. a, * a Department of Anatomy-Histology-Embryology, School of Medicine, University of Patras, Patras, Greece b Department of Pathology, General Hospital Agios Andreas, Patras, Greece Received 2 May 2011; received in revised form 29 June 2011; accepted 7 July 2011 Abstract Objectives: F-actin binding proteins ezrin and paxillin are involved in cell adhesion and cell migration/invasion. The aim of the study was to investigate their role in urothelial bladder carcinogenesis. Materials and methods: Expression of ezrin and paxillin was studied by immunohistochemistry in 104 and 96 cases of urothelial bladder tumors, respectively. Correlations with clinicopathologic data and expression of p53, E-cadherin, and -catenin were examined. Results: Positive ezrin and paxillin protein expression was found in 99% and 93.7% of cases, respectively. Membranous expression of ezrin was significantly lower in high grade tumors and correlated with invasion. Multivariate analysis showed that ezrin is an independent predictor of muscularis propria invasion. Paxillin expression was significantly decreased in urothelial carcinomas compared with tumors of low malignant potential and low paxillin levels also correlated with advancing tumor stage and invasion. A statistically significant correlation was found between membranous ezrin and E-cadherin as well as between ezrin and paxillin expression in urothelial tumors. Conclusions: Down-regulation of ezrin and paxillin in urothelial bladder tumors is associated with aggressive tumor features and invasiveness Elsevier Inc. All rights reserved. Keywords: E-cadherin; Ezrin; Invasion; Paxillin; Tumor progression; Urothelial tumors 1. Introduction Cell adhesion and cell migration are important normal cellular processes exploited by tumors cell as they invade surrounding tissues and metastasize. Several F-actin binding proteins involved in these cellular activities have been implicated in tumor invasion and metastasis [1]. Ezrin, a member of ezrin/radixin/moesin (ERM) protein family, highly expressed in epithelial cells, interacts with both membrane proteins and the actin cytoskeleton [2]. Ezrin resides in the cytosol in a dormant closed conformation [3]. When activated upon phosphorylation in response to various stimuli or following association with phospoinositides, ezrin links the plasma membrane to the cortical cytoskeleton [4]. Ezrin has been shown to control actin cytoskeleton * Corresponding author. Tel.: ; fax: address: vibra@upatras.gr (V. Bravou). remodeling and also to participate in signaling pathways regulating survival, adhesion, and migration/invasion [2,5]. Several in vitro studies link ezrin to cell motility and spreading of various cell types [6,7]. Also, altered ezrin expression, both up-regulation and down-regulation, has been reported in many human tumors and correlated with cancer progression and poor outcome [8 10]. In addition to its role in regulating cytoskeleton dynamics, cell-matrix adhesion, and motility, there is evidence that ezrin is also involved in cell cell adhesion by interacting with cell adhesion molecules E-cadherin and -catenin [7]. Paxillin is a multi-domain scaffold protein that localizes to the sites of cell adhesion to the extracellular matrix [11]. This focal adhesion F-actin binding protein was originally identified as a substrate for the nonreceptor tyrosine kinase oncogene pp 60v-src in Rous sarcoma virus-transformed fibroblasts [12]. Through interactions with numerous regulatory and structural proteins, paxillin controls cytoskeletal reorganization and signaling critically involved in cell ad /$ see front matter 2013 Elsevier Inc. All rights reserved. doi: /j.urolonc

186 A. Athanasopoulou et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) hesion, cell migration, and survival [13]. Several studies have found an association between deregulated paxillin expression and invasive tumor behavior [14,15]. Bladder urothelial cancer is a common neoplasia characterized by recurrences and poor prognosis when tumors invade the muscularis propria [16]. Understanding the mechanisms of urothelial carcinoma invasion may help identify new prognostic biomarkers and therapeutic targets. Several in vitro studies implicate paxillin in motility and invasion of bladder cancer cell lines [17 19]. However, apart from a single study of ezrin expression in T1 high grade urothelial carcinomas [20], to the best of our knowledge, little is known regarding ezrin and paxillin protein expression in relation to pathologic parameters in human bladder urothelial tumors. In this study, expression of ezrin and paxillin was examined by immunohistochemistry in 104 and 96 cases of urothelial tumors, respectively, in relation to clinicopathologic data. Since nuclear overexpression of p53 by immunohistochemistry has been commonly regarded to relate with adverse prognosis of urothelial bladder tumors [21 25], we examined the association of ezrin and paxillin expression with p53 immunostaining. In addition, based on evidence that both ezrin and paxillin may be involved in cadherin-mediated cell cell adhesion, we also evaluated potential correlations with cell adhesion molecules E- cadherin and -catenin [7,26]. 2. Materials and methods 2.1. Tissue samples Formalin-fixed, paraffin-embedded tissue samples from 104 primary human urothelial carcinomas cases were retrieved from the archives of the Department of Pathology, General Hospital Agios Andreas Patras, Greece. Fourteen out of 104 tumors (13.5%) were papillary urothelial neoplasms of low malignant potential (PUNLMP), 33/104 (31.7%) were low grade urothelial carcinomas, and 57/104 (54.8%) were high grade urothelial carcinomas. Tumor invaded the lamina propria (pt1) in 37 of the cases (35.6%) (3/33 low grade and 34/57 high grade carcinomas) and the muscularis propria (pt2) in 23 of the cases (22.1%) (all cases were high grade carcinomas). All tissue samples were from previously untreated cases. The study was performed in accordance with the institutional ethical guidelines Immunohistochemistry Immunostaining was performed using an automatic staining system (DAKOautostainer; DAKO, Glostrup, Denmark). For deparaffinization and antigen retrieval, representative 4 m tissue sections were treated with DAKO target retrieval solution PH 9 in a DAKO pressure cooker (PT; DAKO, Glostrup, Denmark) for 15 min at 90 C. Primary antibodies used were mouse monoclonal anti-ezrin (1:300; NeoMarkers, Fremont, CA), mouse monoclonal anti-paxillin (1:400; NeoMarkers), mouse monoclonal anti-p53 (1:20; DAKO), mouse monoclonal anti E-cadherin (1:1000; BD Biosciences, San Jose, CA) and mouse monoclonal -catenin (1:1000; BD Biosciences) for 30 min at RT. Detection was performed using the Envision detection kit (DAKO) according to the manufacturer s instructions. Diaminobenzidine (DAB) was used as the chromogen for visualization. Slides were counterstained with hematoxylin, dehydrated and mounted. Colon cancer cases were used as positive control. For negative controls, blocking solution was added instead of the primary antibody Immunohistochemical evaluation All slides were assessed blinded to the case. Expression of p53 was considered positive when 10% of tumor cells showed nuclear p53 immunoreactivity. This cutoff was selected based on previous reports showing that p53 expression in 10% of tumor cells has prognostic significance and correlates with p53 mutational status [21 25]. For all other proteins, cytoplasmic, membranous, and nuclear staining, when observed, was evaluated separately. Immunoreactivity was scored on a scale of 0 3 depending on the intensity of staining and the percentage of positive cells. Staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of positive cells was scored as 0 ( 1%), 1 (1% 25%), 2 (26% 50%), and 3 (51% 75%), and 4 (76% 100%). The 2 scores were multiplied and the immunoreactivity score (values 0 12) was classified as follows: score 0 as negative; score 1 (multiplication values 1, 2) as weakly positive; score 2 (multiplication values 3, 4, 6) as moderately positive, and score 3 (multiplication values 8, 9, 12) as strongly positive. Cases with score 0 were considered negative and cases with scores 1, 2, or 3 were considered positive. In cases of membranous -catenin and membranous E-cadherin, decreased membranous expression was defined as score 3 since adjacent non-neoplastic urothelium had a score 3 for membranous localization of both proteins. In case of cytoplasmic -catenin, increased cytoplasmic expression was defined as score 1 since in adjacent non neoplastic urothelium, expression of cytoplasmic -catenin had a score 1 [27,28] Statistical analysis To test the significance of differences among groups of clinicopathologic parameters, such as tumor type, grade and invasion, ordinal data were analyzed using the nonparametric Kruskal-Wallis or Mann-Whitney tests. Correlations between expression of proteins (immunoreactivity scores) were investigated using the Spearman rank-order correlation coefficient. Correction for ties was performed for all ranking tests. Multivariate regression analysis using the Enter method was performed in order to identify independent predictors of grade, stage, and invasion. Variables

187 838 A. Athanasopoulou et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) that showed significant correlation with the dependent variable under question by non-parametric tests were included as predictor variables in the multivariate analysis. The significance level was defined as P Statistical analysis was performed with the SPSS for Windows, release 12.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Decreased ezrin expression in human urothelial bladder tumors correlates with aggressive tumor features Positive ezrin expression was observed in tumor cells, in blood vessel endothelium and inflammatory cells (mainly lymphocytes). In non-neoplastic urothelium, ezrin showed strong membranous and weak cytoplasmic localization (Fig. 1A). Positive ezrin expression was found in 103/104 (99%) urothelial bladder tumors. Immunoreactivity was membranous and/or cytoplasmic. Membranous immunoreactivity was observed in 103/104 (99%) cases while cytoplasmic localization was found in 87/104 (83.7%) tumors. While there was no significant correlation between cytoplasmic ezrin and any of the clinicopathologic parameters under evaluation, membranous ezrin immunoreactivity differed significantly among cases. Membranous ezrin expression was significantly lower in high grade compared with low grade urothelial carcinomas (P 0.02), while there was no significant difference between PUNLMP and urothelial carcinomas. Reduced membranous ezrin expression was also found in invasive tumors (pt1 and pt2) compared to noninvasive tumors (pta) (P 0.04) (Fig. 1B, C) and cases with invasion of muscularis propria showed significantly lower levels of membranous ezrin protein expression (P 0.02). Notably, there was a significant down-regulation of ezrin expression (both cytoplasmic and membranous) in invasive tumor areas (Fig. 1C). Results are shown in Table 1. Multivariate analysis showed that membranous ezrin is a significant independent predictor of muscularis propria invasion ( 0.268, P 0.04) but not of grade or stage. Fig. 1. Loss of ezrin and paxillin protein expression in urothelial tumor invasion. (A) Strong membranous and weak cytoplasmic ezrin expression in non-tumoral urothelium. (B) A representative case of an urothelial tumor of low malignant potential with strong membranous and cytoplasmic expression of ezrin. (C) Weak ezrin expression in an invasive pt2 tumor. (D) Strong membranous and cytoplasmic paxillin expression in non-tumoral urothelium. (E) Strong paxillin expression in an urothelial tumor of low malignant potential. (F) A representative case of an invasive pt2 tumor with weak paxillin expression in tumor cells ( 400). (Color version of figure is available online.)

188 A. Athanasopoulou et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) Table 1 Expression of ezrin and paxillin in relation to clinicopathologic characteristics in urothelial bladder tumors N Ezrin (membranous) a P N Paxillin (cytoplasmic and membranous) b P value c value c 0 n (%) 1 n (%) 2 n (%) 3 n (%) 0 n (%) 1 n (%) 2 n (%) 3 n (%) Urothelial tumors Total 104 1/104 (1.0%) 10/104 (9.6%) 25/104 (24.0%) 68/104 (65.4%) 96 6/96 (6.3%) 31/96 (32.3%) 27/96 (28.1%) 32/96 (33.3%) PUNLMP 14 0/14 (0%) 1/14 (7.1%) 3/14 (21.4%) 10/14 (71.4%) /12 (0%) 1/12 (8.3%) 1/12 (8.3%) 10/12 (83.3%) 0.02 Low grade carcinomas 33 0/33 (0%) 1/33 (3.0%) 6/33 (18.2%) 26/33 (78.8%) /33 (6.1%) 10/33 (30.3%) 8/33 (24.2%) 13/33 (39.4%) 0.1 High grade carcinomas 57 1/57 (1.8%) 8/57 (14.0%) 16/57 (28.1%) 32/57 (56.1%) 51 4/51 (7.8%) 20/51 (39.2%) 18/51 (35.3%) 9/51 (17.6%) Stage pta 44 0/44 (0%) 2/44 (4.5%) 9/44 (20.5%) 33/44 (75.0%) 42 2/42 (4.8%) 10/42 (23.8%) 9/42 (21.4%) 21/42 (50.0%) pt1 37 0/37 (0%) 5/37 (13.5%) 8/37 (21.6%) 24/27 (64.9%) 33 2/33 (6.1%) 16/33 (48.7%) 8/33 (24.2%) 7/33 (21.2%) pt2 23 1/23 (4.3%) 3/23 (13%) 8/23 (34.8%) 11/23 (47.8%) 21 2/21 (9.5%) 5/21 (23.8%) 10/21 (47.6%) 4/21 (19%) Invasion No (pta) 44 0/44 (0%) 2/44 (4.5%) 9/44 (20.5%) 33/44 (75.0%) 42 2/42 (4.8%) 10/42 (23.8%) 9/42 (21.4%) 21/42 (50.0%) Yes (pt1 or pt2) 60 1/60 (1.7%) 8/60 (13.3%) 16/60 (26.7%) 35/60 (58.3%) 54 4/54 (7.4%) 21/54 (38.9%) 18/54 (33.3%) 11/54 (20.4%) Muscularis propria invasion No 81 0/81 (0%) 7/81 (8.6%) 17/81 (21.0%) 57/81 (70.4%) 75 4/75 (5.3%) 26/75 (34.7%) 17/75 (22.7%) 28/75 (37.3%) Yes 23 1/23 (4.3%) 3/23 (13.0%) 8/23 (34.8%) 11/23 (47.8%) 21 2/21 (9.5%) 5/21 (23.8%) 10/21 (47.6%) 4/21 (19.0%) a,b Scoring of ezrin and paxillin immunostaining was performed as described in Materials and Methods. c Mann-Whitney test, P 0.05 is considered statistical significant Down-regulation of paxillin in urothelial bladder tumors correlates with invasion Paxillin expression was observed in tumor cells, endothelial cells, and lymphocytes. Immunoreactivity of paxillin in epithelial cells was cytoplasmic and membranous, frequently showing attenuation in cells of the basal layer. In non-neoplastic urothelium, there was a strong membranous and a moderate cytoplasmic localization of paxillin (Fig. 1D). Positive membranous and cytoplasmic paxillin expression was found in 90/96 (93.7%) urothelial tumors. Paxillin expression (cytoplasmic and membranous) was significantly lower in low grade and high grade urothelial carcinomas compared with PUNLMP (P 0.02 and P 0.001, respectively), while there was no significant difference between low and high grade tumors. Expression of paxillin was also significantly lower with advancing tumor stage (P 0.02). Tumors that invaded the lamina propria and muscular wall showed a statistical significant lower paxillin expression (cytoplasmic and membranous) compared with noninvasive tumors (P 0.008) (Table 1) (Fig. 1E, F). However paxillin was not shown, by multivariate analysis, to be an independent predictor of grade, stage, or invasion Ezrin expression in human urothelial bladder tumors significantly correlates with E-cadherin and paxillin expression Expression of p53 was positive in 90/101 (89.3%) tumors (Fig. 2A, B) and positively correlated with grade (P 0.004) and invasion of the muscularis propria (P 0.017). However, statistical analysis failed to show any significant association between ezrin and p53 immunostaining, while there was a marginally significant negative correlation between p53 and paxilin expression (P 0.05, r 0.252). E-cadherin and -catenin expression was evaluated in 55 and 56 representative cases of urothelial carcinomas respectively. Decreased membranous E-cadherin (score 3) was found in 19/55 urothelial carcinomas (34.5%) (Fig. 2C, D). Decreased membranous -catenin (score 3) was observed in 40/56 (71.4%) cases. Cytoplasmic accumulation of -catenin (score 1) was demonstrated in 32/55 (57.1%) tumors (Fig. 2E, F), while nuclear -catenin (score 0) was found in only 3/56 (5.4%) cases. There was no statistically significant correlation between E-cadherin or -catenin expression levels with any of the clinicopathologic parameters neither with expression of paxillin. However membranous ezrin expression significantly correlated with membranous E-cadherin (r 0.376, P 0.005). Importantly, there was a significant positive correlation between ezrin and paxillin expression (r 0.402, P 0.001) in urothelial tumors.

189 840 A. Athanasopoulou et al. / Urologic Oncology: Seminars and Original Investigations 31 (2013) Fig. 2. Expression of p53, E-cadherin, and -catenin in urothelial bladder tumors. (A), (B) Representative cases of urothelial tumors with negative (A) and positive (B) p53 expression. (C), (D) Representative cases of urothelial tumors with normal strong membranous (C) and decreased E-cadherin expression (D). (E), (F) Strong membranous (E) and cytoplasmic (F) expression of -catenin ( 400). (Color version of figure is available online.) 4. Discussion Actin cytoskeleton-associated proteins ezrin and paxillin function as structural scaffolds and signaling molecules [5,11,13]. Both proteins have been implicated in many aspects of cancer cell biology such as cell survival, adhesion, migration, and invasion [5,13]. In this study, we provide evidence that altered expression of ezrin and paxillin are involved in human urothelial tumor progression. In our study, ezrin was expressed in urothelial tumors with a membranous and cytoplasmic localization as previously described [20]. Interestingly, membranous ezrin was significantly lower in high grade, invasive tumors and it was also shown to be a significant independent predictor of muscularis propria invasion. Consistent with our findings, a previous study in urothelial carcinomas reported correlation of low membranous ezrin with progression to muscleinvasive disease and decreased overall survival [20]. In the latter study, however, only T1 high grade tumors were included. In further agreement with our results, downregulation of ezrin has been previously reported in other tumors such as ovarian carcinomas and diffuse gastric carcinomas, where weak ezrin expression also correlated with clinicopathologic parameters and adverse prognosis [9,10]. These results suggest that down-regulation of ezrin in human urothelial carcinomas may be involved in tumor progression. It should be noted, however, that several contradictory results have been published with regards to the role of ezrin in cancer progression. Both down-regulation [9,10] and overexpression of ezrin [29 31] in relation to increased metastatic potential and adverse prognostic parameters have been reported in human tumors. Controversial data also come from in vitro studies in cell lines [6,7]. This apparent discrepancy may be due to the fact that ezrin shows multiple functions in cell adhesion, cell migration, survival, and proliferation that may differ among various cell types or tissues as previously suggested [32,33]. We also showed low paxillin protein expression in urothelial carcinomas compared with PUNLMP and a significantly lower expression in invasive tumors. There was also a marginally significant negative correlation of paxillin with nuclear accumulation of p53 that is commonly regarded as an adverse prognostic indicator in bladder cancer [21 25]. Consistently, decreased paxillin expression has