2. CELULA MADRE EN CANCER DE PROSTATA IMPLICACIONES CLINICAS Y TERAPEUTICAS Prof. Dr. L. M. Antón Aparicio Jefe Servicio Oncología Médica Complejo Hospitalario Universitario Coruña

3. SUMMARY (I) Normal prostate gland development requires many coordinated cellular process through prostate stem cells, including epithelial proliferation, mesenchymal- epithelial interaction, ductal branching morphogenesis, and ductal canalisation. Stem cells are defined functionally as cells that have the capacity to self-renew as well as the ability to generate differentiated cells. Stem cells can generate daughter cells identical to their mother (self-renewal) as well as produce progeny with more restricted potential (differentiation). Self-renewal is achieved by symmetrical cell division while maintaining pluripotency; and this can be modulated by extrinsic factors, transcriptional regulator, and effectors. The regenerative capacity of prostate gland has been attributed to stem/progenitor cells within adult prostatic epithelium. It was hypothesized that the adult prostate contains stem, transit/amplifying, and postmitotic cells and that the stem cells were androgen-independent for survival.

4. SUMMARY (II) In the normal prostate gland epithelium exist at many stages a spectrum of cells expressing a continuum of differentiation markers, biological properties, all them beginning from stem/progenitor cells through multiple intermediate cell types along different lineages to terminally differentiated cells. There is now strong evidence that the stem cells of many tissues reside in physically delineated as well as physiologically specialized structures termed niches. Inside the niche, stem cells are often quiescent; outside the niche, stem cells must either possess sufficient intrinsic factors to overcome differentiation or succumb too much of fate. The signal that controls which daughter cell or an adult stem cell remains as stem cell and which begins the process of determination may be mediated through a number of signalling pathways including Wnt, Hedgehog (Hh), Notch, Bone Morphogenic Protein (BMP), Oct-4. The signal that controls which daughter cell of an adult stem cell remains a stem cell and which begind the process of determination may be mediated through a number of signalling pathways including, Wnt, Hh, Notch, Oct-4, BMP, JAK/Stat, or other signalling pathways.

5. PROSTATE GLAND The prostate is located at the base of the bladder in males, surrounding the urethra (Cunha et al. 1987). The gland is composed of tubules that have an epithelial compartment surrounded by stromal cells that include fibroblasts, smooth muscle, and myofibroblasts. 1. The epithelium consists of two cellular compartments made up of three mophologically, functionally, and molecularly distinct cell types. 1.a. Androgen-independent flat basal cells are attached to the basement membrane, where they maintain the homeostasis of the organ and express the high-molecular- weight cytokeratins (CK) 5 and CK14. A subpopulation of basal cells also expresses the p53-family-related gene p63. 1.b. Luminal cells are CK8/18-positive androgen-dependent columnar cells that lay above the basal layer facing the lumen of each tubule where they secrete prostatic proteins. 1.c. Neuroendocrine cells reside largely in the basal compartment, where they secrete neuroendocrine peptides such as synaptophysin and chromogranin A that support epithelial viability (Bonkhoff 1998; Abrahmsson 1999). 1.d. A fourth population of epithelial cells, named transit-amplifying cells, that coexpress basal (CK5) and luminal (CK8) markers (Isaacs and Coffey 1989), as well as prostate stem cell antigen (PSCA) in later stages (Tran et al. 2002).

6. ADULT PROSTATE STEM CELLS The existence of PrSCs was determined by the observation that the rodent prostate can undergo up to 30 cycles of involution and regeneration in response to androgen cycling (English et al. 1987). Two prevalent models have emerged to explain how these PrSCs give rise to the different cell types of the prostate. 1.The linear model prosposes that the PrSCs reside among the CK5- positive basal cells, where they can differentiate into the double- positive intermediate/transit-amplifying population and the finally into the CK8-positive luminal phenotype (Isaacs and Coffey 1989; Hudson et al. 2001) 2.The branched model of differentiation where the luminal and basal cells are in separate lineages, maintained by separate progenitor cells.

7. Location of stem cells: niche.

8. Multiple growth factor and cytokins are involved in stem-niche interactions. These include SCF/Kit, SDF- 1/CXCR4, Jagged/Notch, Androgen/Receptor, etc. BMP4 is expressed in stromal cells, but the type of receptor expressed in prostate stem cells is unknown. The Wnt signal is important for stem cell self- renewal, but the Wnts present in the niche are unknown. The source is true for FGF and Hh.

9. Stem cell typeCell typeMarkers UGS epithelium (embryonic)Luminal cellCK8, CK18 Basal cellCK5, CK14, p63 Transitional/ IntermediateCK19, GSTpi Prostate epithelium (adult)Luminal cell/predominantCK8, CK18, Neuroendocrine cellChromogranin-A, Synaptophysin, Neuron-specific enolase, Serotonin Basal cell/predominant subpopulation CK5, CK14, CK19, p63, GSTpi Basal cell /small fractionCK5, CK8, CK14, CK18, CK19, P63, GSTpi Basal cell / subsetCK15, CK17, CK19 CK: cytokeratin; GSTPi: Glutathione-S-transferase-pi. Progenitor/stem cells in embryonic urogenital sinus epithelium and adult prostate epithelium with their respective cell lineage markers.

10. Cell lineage relationships in adult prostate: basal cells (CK5/14/p63/CK19/GSTpi + /CK8/CK18 - ); definitive luminal cells (CK8/18 + /CK5/14/p63 - ); embryonic-like cells co-expressing both luminal and basal cell markers (CK8/18/14/5/p63/CK19/GSTpi + ).

11. Cell typeCharacteristicsCell markersRefs. Luminal epithelial cellsAndrogen-dependent, more differentiated, low proliferative capacity, high apoptotic index., PSA, CK8, CK1836, 37, 38 Basal epithelial cellsAndrogen-independent, undifferentiated profile, high proliferative capacity, anti- apoptotic protein., CK5, CK14, CK15, p63, Bcl-2 36, 39, 40, 41 Murine prostateStem cellsSca-1, CK5, p63, CD4af22, 23, 41, 42 Human prostateStem cellsCK5, CK14, CD133, CD44, CD4af 43, 44, 45 Prostate progenitor/ /stem cells Stem cell markersSca-1 +, CD4af +, CD34 - 36 Basal cell markersCK5, CK14, p6336 Luminal cell markersCK8, CK18, AR36 Cells presents in prostate with characteristics and cell markers associated

12. Developmental proposed model that implicates the different prostate gland epithelial cells genesis from stem/progenitor cells.

13. Prostate stem cell hierarchy.

14. Prostate epithelial cell hierarchy.The stem cells divide, give rise to a new stem cell –self renewal- and more committed progenitor cells (early&late) for the functional exocrine and neuroendocrine cell lineages; the exocrine lineage is critically dependent on DHT, and in fact this population represents >90% of all epithelial cells in the adult prostate gland

15. ORIGIN OF PROSTATE CANCER CELLS The vast majority of prostate cancers are acinar-type adenocarcinomas, which are defined by the proliferation of malignant luminal-type cells and a loss of basal cells. The traditional view is that prostate cancer arises from mature luminal cells of the prostate gland. A subgroup of luminal cells, which are typically characterized by the expresion of a transciption factor called NK3 homeobox 1 (Nkx3-1), also survive androgen depletion. A majority of castration-resistant cells have a basal anatomic location and express basal-cell markers. Castration-resistant Nkx3-1 (CARN) cells express the androgen receptor and the luminal-cell marker CK18 and do not express basal-cell markers. 1. CARN cells could give rise to basal, luminal, and neuroendocrine cells after androgen was restored. 2. CARN cells were able to regenerate ducts and self-renew. 3. After the deletion of Pten, cells with a basal phenotype can initiate prostate cancer in the mouse (Figure 1) 4. Deleted Pten (a tumor-suppressor gene) in CARN cells and observed rapid carcinoma formation after androgen-mediated regeneration If prostate cancers indeed originate from different cell types (e.g., the basal or luminal compartment), the resulting tumors might have different genetic profiles, biologic behavior, and therapeutic responses.

16. Proposed model of the cellular events associated with the initiation and progression of prostatic cancer.

17. Ontogeny of prostate caner stem cells. In primary human prostate tumours three classes can be discriminated; those with a minority of cells (candidate stem cells) similar to the early- or late progentors, and the pre- terminally differentiated phenotype.

18. Hh

19. WNT

20. Wnt signaling pathways regulate a variety of processes, including cell growth, development and oncogenesis (Polakis 2000) Wnt regulate the stability of  -catenin, a Key component of Wnt signaling A protein-protein interaction between th AR and  -catenin has been identified (Yang 2002).  -catenin act as an AR coactivator, increasing AR-mediated transcription (Truica 2000). Wnt 3 a plays an important role in androgen-mediated transcription and cell growth (Verras 2004). Wnt 3 a induces AR activity in the absence of androgens or ehances AR activity in the presence of low concentrataions of androgens (Verras 2004) CONCLUSION AR: Wnt crosstalk adds an additional layer of complexity to the Wnt pathway´s role in prostate biology

21. Notch

22. NOTCH Notch is part of an evolutionarily conserved signaling pathway that regulates cell differentiation, proliferation, an growht (Mumm Skopan 2000) Notch signaling has been reported to be a requirement for normal murine prostate re-growth and branching (Wang et al 2004, 2006). Notch-1 signal was seen selectively in the epithelium surrounding the lumen of the budded ductal epithelial units. (Shon et al 2001). Notch-1 expression is associated with basal epithelial cells in prostate (Gluene et al, 2001). The Notch-1 cells appeared to correlate with the population of the bsal cells during prostatic develpment. (Shon et al 2001). Notch-1 signaling may act to repress AR signaling (Belandia et al 2005) Endrogenous Nothc-1 regulates PTEM tumor suppressor gene (Whelan et al 2009). Notch-1 signaling has been linked with regulation of prostate tumor cell motility (Scorey et al 2006). Notch-1 signaling is lost in prostate adenocarcinoma (Whelan et al 2009) CONCLUSION Notch-1 signaling may contribute to the accumulation of genetic alterations that esult in prostate cancer throngh reduced PTEN gene expressions.

23. CROSS TALK / CROSS-REGULATION NOTCH activity inhibits prostate progenitor function ( Shahi et al 2011) The inhibitory role of Notch signaling toward proliferation of prostate epithelial cells is via PTEN induction (Barth et al 1999) Wnt signaling results in proliferation of immature prostate progenitors (Shahi et al 2011) The reported mechanism describe the induction of AR-mediated transcription and enhances prostate cancer cells growth (Verras et al 2004) CONCLUSIONS The ability of Wnt pathway to promote adult stem cell proliferation and self- renewal is associated with is ability to influence the Notch signaling pathway which is upregulated as a result of Wnt pathway induction. (Reya 2003, Duncan 2005) Wnt and Notch pathways have interrelated opposing roles on prostate progenitor cells proliferation and differentiation(Shahi et al 2011).

24. ANDROGEN RECEPTOR The androgen signaling pathway, which is mainly mediated through the AR is importants for the normal and neoplastic development of prostate cells (Gelmann 2002). Multiple mechanisms by which prostate cancer cells progress to androgen-insensitive stages have been reported: AR mutations, anplification (Balk 2002). In particular, it has been shown that PI 3 PK/AKt and PTEN regulate AR. Mediated transcription (Sharma 2002, Wen 2000). Wnt and Notch are able to stimulate AR. Mediated transcription, having interrelated opposing roles on prostate progenitor cell proliferation and differentiation (Shahi 2011).

25. PathwayComponentFunctionSampleExpressionRefs. WntWNT11LigandProstate tumor cell linesAndrogen- -dependent: high with androgens; inhibited without androgens 71 SFRP2Ligand inhibitor Mouse prostate, early/advanced stages of development Activation/ down-regulation in early/advanced stages respectively 63 SFRP4Ligand inhibitor Rat prostate ablation and DHT- dependent re-growth Repressed64 BMPBMP2LigandNormal/tumor prostate tissuesExpressed in both tissues73 BMP2LigandBenign/tumor tissues Decreased in tumor tissue78 BMP3LigandNormal/tumor prostate tissuesExpressed in both samples73 BMP4LigandNormal prostate developmentExpressed75 BMP4LigandNormal/tumor prostate tissuesExpressed in both but predominantly expressed in normal prostate 73 BMP4LigandProstate tumor cell linesInduction under WNT3A, WNT5 administration or DKK1 deletion 72 BMP5LigandNormal/benign prostate tissuesUpregulated in benign tissue74 BMP6LigandProstate tumor cell linesInduction under WNT3A, WNT5 administration or DKK1 deletion 72 BMP6LigandNormal/tumor prostate tissuesExpressed in both tissues73 BMP7LigandNormal prostate developmentExpressed82 BMP9LigandProstate tumorDecreased or absent79 SMAD4Intracellular effector Benign/tumor tissues Decreased in tumor tissue78 SMAD8Intracellular effector Normal/benign/ tumor tissues Expressed in normal/benign; absent in tumor 78

26. NotchNOTCH1ReceptorNormal prostate developmentOverexpressed in response to BMP782 NOTCH1ReceptorRat prostate ablation and DHT- dependent re-growth Repressed in rat ventral prostate83 NOTCH1ReceptorHuman normal prostate cell lines: PrEC, BPH-1, PNT2, RWPE-1, PZHPV-1 Expressed83 NOTCH1ReceptorMouse prostate ablation and androgen- dependent re- growth Induction after castration. Return to normal levels during re-growth 85 NOTCH2ReceptorHuman normal prostate cell line: PNT2Expressed83 NOTCH2ReceptorRat prostate developmentExpressed in specific areas of stromal mesenchyme 88 NOTCH3ReceptorHuman normal prostate cell line: PNT2Not expressed83 NOTCH4ReceptorHuman normal prostate cell line: PNT2Not expressed83 JAG1LigandHuman normal prostate cell lines: PrEC, PNT2 Expressed83 JAG1LigandRat prostate ablation and DHT- dependent re-growth Repressed in rat ventral prostate64 JAG2LigandHuman normal prostate cell lines: PrEC, PNT2 Expressed83 DLL1LigandHuman normal prostate cell lines: PrEC, PNT2 Expressed83 DLL1LigandRat prostate developmentExpressed in specific areas of stromal mesenchyme 88 DLL4LigandHuman normal prostate cell line: PrECNot expressed83 SEL1LInhibitorRat prostate development in response to DHT Induced in rat ventral prostate64 DTX (Deltex) Intermediary in signaling Human normal prostate cell line: PNT2Not expressed83 HES1Intermediary target Human normal prostate cell line: PNT2Expressed83 HES1Intermediary target Normal prostate developmentInhibited in presence of BMP782 HEY1Intermediary target Human normal prostate cell lines: PrEC, RWPE-1, PZHPV-1 Expressed83

27. HedgehogSHHLigandMouse UGSExpressed in epithelial cells80 SHHLigandDeveloping mouse prostateExpressed in epithelial cells in tips of buds 91 SHHLigandHuman fetal prostatesExpressed in epithelial ducts97 IHHLigandMouse prostate developmentUp-regulated in SHH nulls95 DHHLigandHuman fetal prostatesExpressed in epithelial ducts97 PTCH1ReceptorMouse developing prostateExpressed in mesenchyme around tip buds 91 PTCH1ReceptorRat developing prostateExpressed in mesenchyme stromal cells adjacent to epithelium of tip buds 92 PTCH1ReceptorHuman fetal prostatesExpressed in epithelial ducts97 PTCH1ReceptorAdult prostateNot expressed in epithelial basal cells98 PTCH2ReceptorRat developing prostateExpressed in mesenchyme stromal cells adjacent to epithelium of tip buds 92 PTCH2ReceptorHuman fetal prostatesExpressed in epithelium and stroma97 SMOTrans- -membrane effector Human fetal prostatesExpressed in epithelial ducts97 GLI1Intracellular effector Mouse developing prostateMesenchyme around tip buds91 GLI1Intracellular effector Human fetal prostatesExpressed in epithelial ducts97 GLI1Intracellular effector Adult prostateNot expressed in epithelial basal cells98 GLI2Intracellular effector Mouse developing prostateExpressed in mesenchyme around tip buds 91 GLI2Intracellular effector Adult prostateNot expressed in epithelial basal cells98 GLI3Intracellular effector Mouse developing prostateExpressed throughout mesenchyme91 GLI3Intracellular effector Adult prostateNot expressed in epithelial basal cells98

29. Scheme showing the possible mitogenic and antiapoptotic cascades induced through the EGF–EGFR, hedgehog, Wnt and other growth factor signaling pathway elements co-localized in caveolea.

30. Scheme showing the possible oncogenic cascades involved in the stimulation of sustained growth, survival,migration and drug resistance of cancer progenitor cells.

31. STRATEGIES FOR ELIMINATION OF CSCs IN THE PROSTATE (I) Therapeutic strategies to repair or eliminate mutated stem cells are in at their stages, and the dangers associated with targeting stemness remain to be assessed. In determining the phenotypic differences between normal and malignant stem cells in prostate, there was a signature that could not only differentiate cancer from benign, but also stem from TA cells. The cancer element is more tractable, and overlaps the stem and TA compartiments: destruction of stem and TA cells would provide a more lasting therapy than just elimination of the more differentiated progeny cells as at present.

32. Thus, to target the stem cell compartment for elimination of the CSCs will require new strategies and array systems that are quite distinct form those used to derive antiproliferatives that are the currently favored targets of the pharmaceutical industry. First, arrays systemics for cancer drug development require the availability of large numbers of cells, and are currently based on a selected number of cell lines for most tumor types. Second, because rapid proliferation is not necessary in the stem-cell compartment, the assays required for elimination of the CSCs cannot be measured by a slowing of growth rat or metabolism. Third, there is also the likelihood that the CSCs have inherent resistance mechanisms. STRATEGIES FOR ELIMINATION OF CSCs IN THE PROSTATE (II)