CELULA MADRE EN CANCER DE PROSTATA IMPLICACIONES CLINICAS

Y TERAPEUTICAS

Prof. Dr. L. M. Antón AparicioJefe Servicio Oncología Médica

Complejo Hospitalario Universitario Coruña

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.

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.

PROSTATE GLANDThe 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).

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.

Location of stem cells: niche.

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.

Stem cell type Cell type Markers

UGS epithelium (embryonic) Luminal cell CK8, CK18

Basal cell CK5, CK14, p63

Transitional/ Intermediate CK19, GSTpi

Prostate epithelium (adult) Luminal cell/predominant CK8, CK18,

Neuroendocrine cell Chromogranin-A, Synaptophysin, Neuron-specific enolase, Serotonin

Basal cell/predominant subpopulation

CK5, CK14, CK19, p63, GSTpi

Basal cell /small fraction CK5, CK8, CK14, CK18, CK19, P63, GSTpi

Basal cell / subset CK15, 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.

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+).

Cell type Characteristics Cell markers Refs.

Luminal epithelial cells Androgen-dependent, more differentiated, low proliferative capacity, high apoptotic index.

, PSA, CK8, CK18 36, 37, 38

Basal epithelial cells Androgen-independent, undifferentiated profile, high proliferative capacity, anti-apoptotic protein.

, CK5, CK14, CK15, p63, Bcl-2

36, 39, 40, 41

Murine prostate Stem cells Sca-1, CK5, p63, CD4af 22, 23, 41, 42

Human prostate Stem cells CK5, CK14, CD133, CD44, CD4af

43, 44, 45

Prostate progenitor/ /stem cells

Stem cell markers Sca-1+, CD4af+, CD34- 36

Basal cell markers CK5, CK14, p63 36

Luminal cell markers CK8, CK18, AR 36

Cells presents in prostate with characteristics and cell markers associated

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

Prostate stem cell hierarchy.

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

ORIGIN OF PROSTATE CANCER CELLSThe 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.

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

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.

Hh

WNT

WNT

• 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)

CONCLUSIONAR: Wnt crosstalk adds an additional layer of complexity to the Wnt pathway

´s role in prostate biology

Notch

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.

CROSS TALK / CROSS-REGULATIONNOTCH 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).

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 PI3PK/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).

Pathway Component Function Sample Expression Refs.Wnt WNT11 Ligand Prostate tumor cell lines Androgen-

-dependent: high with androgens; inhibited without androgens

71

SFRP2 Ligand inhibitor

Mouse prostate, early/advanced stages of development

Activation/down-regulation in early/advancedstages respectively

63

SFRP4 Ligand inhibitor

Rat prostate ablation and DHT-dependent re-growth

Repressed 64

BMP BMP2 Ligand Normal/tumor prostate tissues Expressed in both tissues 73BMP2 Ligand Benign/tumor

tissuesDecreased in tumor tissue 78

BMP3 Ligand Normal/tumor prostate tissues Expressed in both samples 73BMP4 Ligand Normal prostate development Expressed 75BMP4 Ligand Normal/tumor prostate tissues Expressed in both but predominantly

expressed in normal prostate73

BMP4 Ligand Prostate tumor cell lines Induction under WNT3A, WNT5 administration orDKK1 deletion

72

BMP5 Ligand Normal/benign prostate tissues Upregulated in benign tissue 74BMP6 Ligand Prostate tumor cell lines Induction under WNT3A, WNT5

administration orDKK1 deletion

72

BMP6 Ligand Normal/tumor prostate tissues Expressed in both tissues 73BMP7 Ligand Normal prostate development Expressed 82BMP9 Ligand Prostate tumor Decreased or absent 79SMAD4 Intracellular

effectorBenign/tumortissues

Decreased in tumor tissue 78

SMAD8 Intracellular effector

Normal/benign/tumor tissues

Expressed in normal/benign; absent in tumor

78

Notch NOTCH1 Receptor Normal prostate development Overexpressed in response to BMP7 82NOTCH1 Receptor Rat prostate ablation and DHT-

dependent re-growthRepressed in rat ventral prostate 83

NOTCH1 Receptor Human normal prostate cell lines: PrEC, BPH-1, PNT2, RWPE-1, PZHPV-1

Expressed 83

NOTCH1 Receptor Mouse prostate ablation and androgen- dependent re- growth

Induction after castration. Return to normal levels during re-growth

85

NOTCH2 Receptor Human normal prostate cell line: PNT2 Expressed 83NOTCH2 Receptor Rat prostate development Expressed in specific areas of stromal

mesenchyme88

NOTCH3 Receptor Human normal prostate cell line: PNT2 Not expressed 83NOTCH4 Receptor Human normal prostate cell line: PNT2 Not expressed 83JAG1 Ligand Human normal prostate cell lines:

PrEC, PNT2Expressed 83

JAG1 Ligand Rat prostate ablation and DHT-dependent re-growth

Repressed in rat ventral prostate 64

JAG2 Ligand Human normal prostate cell lines: PrEC, PNT2

Expressed 83

DLL1 Ligand Human normal prostate cell lines: PrEC, PNT2

Expressed 83

DLL1 Ligand Rat prostate development Expressed in specific areas of stromal mesenchyme

88

DLL4 Ligand Human normal prostate cell line: PrEC Not expressed 83SEL1L Inhibitor Rat prostate development in response

to DHTInduced in rat ventral prostate 64

DTX (Deltex)

Intermediary in signaling

Human normal prostate cell line: PNT2 Not expressed 83

HES1 Intermediary target

Human normal prostate cell line: PNT2 Expressed 83

HES1 Intermediary target

Normal prostate development Inhibited in presence of BMP7 82

HEY1 Intermediary target

Human normal prostate cell lines: PrEC, RWPE-1, PZHPV-1

Expressed 83

Hedgehog SHH Ligand Mouse UGS Expressed in epithelial cells 80SHH Ligand Developing mouse prostate Expressed in epithelial cells in tips of

buds91

SHH Ligand Human fetal prostates Expressed in epithelial ducts 97IHH Ligand Mouse prostate development Up-regulated in SHH nulls 95DHH Ligand Human fetal prostates Expressed in epithelial ducts 97PTCH1 Receptor Mouse developing prostate Expressed in mesenchyme around tip

buds91

PTCH1 Receptor Rat developing prostate Expressed in mesenchyme stromal cells adjacent to epithelium of tip buds

92

PTCH1 Receptor Human fetal prostates Expressed in epithelial ducts 97PTCH1 Receptor Adult prostate Not expressed in epithelial basal cells 98PTCH2 Receptor Rat developing prostate Expressed in mesenchyme stromal cells

adjacent to epithelium of tip buds92

PTCH2 Receptor Human fetal prostates Expressed in epithelium and stroma 97SMO Trans-

-membrane effector

Human fetal prostates Expressed in epithelial ducts 97

GLI1 Intracellular effector

Mouse developing prostate Mesenchyme around tip buds 91

GLI1 Intracellular effector

Human fetal prostates Expressed in epithelial ducts 97

GLI1 Intracellular effector

Adult prostate Not expressed in epithelial basal cells 98

GLI2 Intracellular effector

Mouse developing prostate Expressed in mesenchyme around tip buds

91

GLI2 Intracellular effector

Adult prostate Not expressed in epithelial basal cells 98

GLI3 Intracellular effector

Mouse developing prostate Expressed throughout mesenchyme 91

GLI3 Intracellular effector

Adult prostate Not expressed in epithelial basal cells 98

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.

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

STRATEGIES FOR ELIMINATIONOF 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.

• 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 ELIMINATIONOF CSCs IN THE PROSTATE (II)