PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Urol Oncol. Author manuscript; available in PMC 2010 May 1.
Published in final edited form as:
PMCID: PMC2799332
NIHMSID: NIHMS117394

Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens

Abstract

Cancer stem cells (CSCs) are a reservoir of tumor cells which exhibit the properties of self-renewal and the ability to re-establish the heterogeneous cell population of a tumor. They appear therapy resistant and may be the underlying cause of recurrent disease. Using prostate as a model, this review presents the CSC hypothesis and discusses the role of the androgen receptor in CSCs, the methods used for isolating CSCs, and the therapeutic challenges CSCs have for cancer therapy.

Introduction

The concept that adult tissues contained dormant embryonic cells which when activated could develop into cancer was initially proposed in 1875 [1,2]. It required another 122 years to demonstrate that a subpopulation of cells expressing the cell surface markers CD34+CD38- was capable of initiating human acute myeloid leukemia in non-obese diabetic mice with severe combined immunodeficiency disease [3]. These experiments suggested that normal primitive cells could undergo leukemic transformation. In mixed lineage leukemia (MLL), most chromosomal translocations occur in the 8.3 kb breakpoint cluster region between exons 8 and 13 [4]. The resulting in-frame fusion proteins have lost the SET (Su(var)3–9, enhancer of zeste, trithorax) domain required to mediate histone H3 lysine 4 (H3K4) methyltransferase activity, thereby efficiently transforming hematopoietic cells into leukemia stem cells [4]. Thus, epigenetic changes [reviewed in [2,5,6]] may provide important links between normal and cancer stem cells. Hemopoietic stem cell studies have provided the paradigms for identifying and isolating CSCs in solid tumors such as prostate [7,8,9], breast [10], colon [11], brain [12], ovarian [13] and pancreatic [10] cancers. Similar to hematopoietic malignancies, key properties exhibited by solid tumor CSCs include self-renewal and the ability to differentiate into the heterogeneous cancer cell lineages comprising a tumor [14].

Androgen receptor and prostate CSCs

The androgen receptor (AR) appears to play a central role in normal prostate function and in the emergence of therapy resistant disease. Regression/regeneration studies in the rat model have shown that even after 30 rounds of androgen deprivation and replacement, androgen treatment could completely restore the normal architecture of the ventral prostate [15]. During this process, basal cells preferentially survived whereas 90% of luminal epithelial cells were lost through apoptosis [16]. The surviving epithelial cells appeared androgen independent but responsive to exogenous androgen treatment [17]. Normal Prostate Stem Cells (PSCs) are thought to be AR- yet able to undergo transit amplification to generate the epithelial cell lineages of the prostate [18,19]. The signaling pathways which promote AR expression in progenitor and secretory luminal epithelium are not known. Similarly, prostate CSCs do not express AR; however they reconstitute AR+ tumor cell populations in vivo [19]. Thus, it is conceivable that CSCs could clonally expand in an androgen-independent environment, resulting in the development of therapy resistance and recurrent disease [20,21,22].

HPET CSC models

Clonal selection of human prostate cancer epithelial cells can be utilized to study CSCs. Thus, primary Human Prostate Epithelial (HPE) cells obtained from patient samples upon radical retropubic prostatectomy were immortalized utilizing the human telomerase reverse transcriptase (hTERT) gene to generate cell lines [19]. The parental cell line termed HPET (where T indicated hTERT) and 4 clonal cell lines did not express AR or AR-regulated secretory proteins such as Prostate Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP) [19]. Further characterization determined that both parental and clonal lines expressed embryonic stem and early progenitor markers, including Oct-4, Nanog, Sox2, and nestin, as well as CD44, CD133 and c-kit [19]. Utilizing the in vivo tissue recombination assay, these lines reconstituted the histopathology of the original tumor they were derived from, differentiating into the three epithelial cell lineages of the prostate. Indeed, the resulting tumors could be scored using the Gleason system, a method exclusively reserved for the evaluation of human clinical Prostate Cancer (PCa) specimens [19]. Interestingly, AR was re-expressed in the appropriate microenvironment, indicating that the stem cell niche was crucial in regulating CSC phenotype and function [19,23].

Methods for Identifying and/or Isolating CSC

Only a small number of cultured cells show proliferative potential in various in vitro and in vivo assays [24]. Utilizing stem cell surface markers to isolate these side populations of cells, prostate cell lines as well as primary prostate cells have been subjected to fluorescence activated cell sorting (FACS) (Table 1). Commonly used markers have included CD44 [25], Sca-1 [26], CD133/prominin-1 [27], the ATP-binding cassette transporter/breast cancer resistance protein (ABCG2/BCRP) [28] and integrins [29]. CD44+-expressing prostate cancer cells, including NHP6-hTERT/T, NHP6-DNp53/T, DU-145 and PC-3 cells, displayed increased proliferation, tumorigenicity and metastatic potential compared to CD44- cells [30]. BrdU label-retention, one of the hallmarks of a stem cell, was observed in a small percentage of CD44+ NHP6-hTERT/T cells [30]. Similarly, CD44+/CD24- LNCaP cells formed prostaspheres in vitro [31]. CD44+/CD24- xenografts consistently formed tumors in NOD/SCID with the sc injection of 1000 cells, whereas the depleted cell population did not generate xenografts [31].

Table I
Methods for Identifying and Isolating SC or CSC.

Stem cell antigen-1 (Sca-1) has been extensively used to enrich for murine hematopoietic stem cells [32]. In the prostate, Sca-1+ cells appeared to cluster around the proximal region of the prostatic ducts [33]. These cells demonstrated stem cell characteristics, including multi-lineage differentiation and regeneration of tubular structures containing basal and luminal epithelial cells [33]. Sca-1+ cells exhibited increased proliferative capacity, with a subpopulation of Sca-1+ cells also expressing Bcl-2 and integrin α6, markers observed in stem cells from other organs [26]. Sca-1+ cells appear androgen-independent since castration results in a concomitant enrichment for Sca-1+ cells [33].

CD133 (or prominin-1) is expressed on hematopoietic stem and progenitor cells [34]. In the prostate, CD133+2β1hi expression was detected in approximately 1 % of the basal cell population [27]. CD133+ cells formed prostaspheres in culture and prostatic-like acini in SCID mice [27]. Subpopulations of CD133+ cells also co-expressed cytokeratin 14 or TERT, and generated more numerous and larger branching ducts consisting of luminal and basal cells compared to CD133+ cells [35].

Flow cytometry based on stem cell behavior have also been utilized to isolate side populations. The ATP-binding cassette membrane transporter ABCG2 has been associated with the development of multi-drug resistance [36]. Huss et al. identified a subpopulation of ABCG2+/AR- cells where ABCG2 appeared to isolate the CSC from its microenvironment through the constitutive efflux of androgens, suggesting that this process protected the CSC from androgen deprivation therapy, hypoxia, or adjuvant chemotherapy [28]. In further elucidating the function of ABCG2, Patrawala et al. determined that ABCG2- cells could generate ABCG2+ cells as well as form more and larger clones, [36]. Their study suggested that ABCG2+ cells were fast-cycling progenitors whereas the ABCG2- population contained primitive stem-like cancer cells [36]. Similar to that observed for hemopoietic stem cells [37], prostate cells which did not accumulate Hoechst 33342 dye exhibited stem cell properties such as developing into spheroids and branching structures in 3-dimentional Matrigel culture [38].

Therapeutic approaches to target CSCs

The role CSCs in metastatic and therapy resistant disease is an area of intense research (Table 2). A popular hypothesis is that recurrence is caused by the survival of a small subpopulation of CSCs which have retained or gained the property of self-renewal. If not eliminated by androgen-deprivation and/or chemotherapy, they remain dormant in bone marrow or other target organs until eventually triggered to regenerate the heterogenous cell populations of a tumor [7]. It still needs to be ascertained whether all solid tumors are maintained by CSCs and whether specific markers could differentiate between normal stem and CSCs. Current thinking is that cure rates of prostate and other cancers would be greatly improved by selectively eliminating CSCs while sparing normal stem cells.

Table II
Questions on CSC still to be answered.

Most, if not all, known CSC markers are similarly expressed by a diverse population of stem, progenitor, and differentiated cell types in numerous tissues and organs. Thus, they could be utilized in combination with negative selection against differentiation markers to isolate and characterize CSC populations from tissues or cell culture. Ideally, the identification of a prostate-specific CSC marker would facilitate the development of designer drugs to eliminate CSCs from the tumor cell population.

Acknowledgments

Funding for this work was provided by the National Institute of Diabetes & Digestive & Kidney Diseases (R01 DK60957).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Polyak K, Hahn WC. Roots and stems: stem cells in cancer. Nat Med. 2006;12:296–300. [PubMed]
2. Sell S. Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol. 2004;51:1–28. [PubMed]
3. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3:730–737. [PubMed]
4. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7:823–833. [PubMed]
5. Bell DR, Van Zant G. Stem cells, aging, and cancer: inevitabilities and outcomes. Oncogene. 2004;23:7290–7296. [PubMed]
6. Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet. 2006;7:21–33. [PubMed]
7. Kasper S. Exploring the origins of the normal prostate and prostate cancer stem cell. Stem Cell Rev. 2008;4:193–201. [PubMed]
8. Maitland NJ, Collins AT. Prostate cancer stem cells: a new target for therapy. J Clin Oncol. 2008;26:2862–2870. [PubMed]
9. Taylor RA, Risbridger GP. The path toward identifying prostatic stem cells. Differentiation. 2008;76:671–681. [PubMed]
10. LaMarca HL, Rosen JM. Minireview: hormones and mammary cell fate--what will I become when I grow up? Endocrinology. 2008;149:4317–4321. [PubMed]
11. Humphries A, Wright NA. Colonic crypt organization and tumorigenesis. Nat Rev Cancer. 2008;8:415–424. [PubMed]
12. Das S, Srikanth M, Kessler JA. Cancer stem cells and glioma. Nat Clin Pract Neurol. 2008;4:427–435. [PubMed]
13. Nieto Y, Jones RB, Shpall EJ. Stem-cell transplantation for the treatment of advanced solid tumors. Springer Semin Immunopathol. 2004;26:31–56. [PubMed]
14. Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006;66:9339–9344. [PubMed]
15. Isaacs JT. Control of cell proliferation and death in the normal and neoplastic prostate: A stem cell model. In: Rogers CH, Coffey DS, Cunha GR, Grayhack JT, Hinman F Jr, Horton R, editors. Benign Prostatic Hyperplasia. Bethesda: National Institutes of Health; 1985. pp. 85–94.
16. Kyprianou N, Isaacs JT. Identification of a cellular receptor for transforming growth factor- beta in rat ventral prostate and its negative regulation by androgens. Endocrinology. 1988;123:2124–2131. [PubMed]
17. De Marzo AM, Nelson WG, Meeker AK, et al. Stem cell features of benign and malignant prostate epithelial cells. J Urol. 1998;160:2381–2392. [PubMed]
18. Rizzo S, Attard G, Hudson DL. Prostate epithelial stem cells. Cell Prolif. 2005;38:363–374. [PubMed]
19. Gu G, Yuan J, Wills ML, et al. Prostate cancer cells with stem cell characteristics reconstitute the original human tumor in vivo. Cancer Res. 2007;67:4807–4815. [PubMed]
20. Isaacs JT. The biology of hormone refractory prostate cancer. Why does it develop? Urol Clin North Am. 1999;26:263–273. [PubMed]
21. Collins AT, Maitland NJ. Prostate cancer stem cells. Eur J Cancer. 2006;42:1213–1218. [PubMed]
22. Robinson EJ, Neal DE, Collins AT. Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium. Prostate. 1998;37:149–160. [PubMed]
23. Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 2005;65:10946–10951. [PubMed]
24. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105–111. [PubMed]
25. Tang DG, Patrawala L, Calhoun T, et al. Prostate cancer stem/progenitor cells: identification, characterization, and implications. Mol Carcinog. 2007;46:1–14. [PubMed]
26. Burger PE, Xiong X, Coetzee S, et al. Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue. Proc Natl Acad Sci U S A. 2005;102:7180–7185. [PubMed]
27. Richardson GD, Robson CN, Lang SH, et al. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci. 2004;117:3539–3545. [PubMed]
28. Huss WJ, Gray DR, Greenberg NM, et al. Breast cancer resistance protein-mediated efflux of androgen in putative benign and malignant prostate stem cells. Cancer Res. 2005;65:6640–6650. [PubMed]
29. Lawson DA, Xin L, Lukacs RU, et al. Isolation and functional characterization of murine prostate stem cells. Proc Natl Acad Sci U S A. 2007;104:181–186. [PubMed]
30. Patrawala L, Calhoun T, Schneider-Broussard R, et al. Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene. 2006;25:1696–1708. [PubMed]
31. Hurt EM, Kawasaki BT, Klarmann GJ, et al. CD44(+)CD24(-) prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br J Cancer. 2008;98:756–765. [PMC free article] [PubMed]
32. Holmes C, Stanford WL. Concise review: stem cell antigen-1: expression, function, and enigma. Stem Cells. 2007;25:1339–1347. [PubMed]
33. Xin L, Lawson DA, Witte ON. The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc Natl Acad Sci U S A. 2005;102:6942–6947. [PubMed]
34. Fargeas CA, Joester A, Missol-Kolka E, et al. Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis. J Cell Sci. 2004;117:4301–4311. [PubMed]
35. Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer. 2002;2:584–593. [PubMed]
36. Patrawala L, Calhoun T, Schneider-Broussard R, et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and A. Cancer Res. 2005;65:6207–6219. [PubMed]
37. Goodell MA, Rosenzweig M, Kim H, et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med. 1997;3:1337–1345. [PubMed]
38. Brown MD, Gilmore PE, Hart CA, et al. Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations. Prostate. 2007;67:1384–1396. [PubMed]