- Email: [email protected]
Oestrogen and the colon: potential mechanisms for cancer prevention
Review
Oestrogen and the colon: potential mechanisms for cancer prevention Rory Kennelly, Dara O Kavanagh, Aisling M Hogan, Desmond C Winter
The role of oestrogen in oncogenesis has been examined extensively, especially in the context of breast cancer, and receptor modulators are an integral part of targeted treatment in this disease. The role of oestrogen signalling in colonic carcinoma is poorly understood. Men are more susceptible than women to colon cancer. Furthermore, hormone-replacement therapy affords an additive protective effect for postmenopausal women, and when these women do develop cancer, they typically have less aggressive disease. The discovery of a second oestrogen receptor (ERβ) and its over expression in healthy human colon coupled with reduced expression in colon cancer suggests that this receptor might be involved. The underlying mechanism, however, remains largely unknown. In this Review, we discuss the various hypotheses presented in the published literature. We examine the cellular and molecular mechanisms through which oestrogen is purported to exert its protective influence, and we review the evidence available to support these claims.
Introduction The oncogenic effects of oestrogens have been investigated extensively in breast cancer where hormonereceptor modulators are now an integral part of targeted treatment. Little is known about oestrogen signalling in colorectal cancer (figure 1) although women are less susceptible to this cancer than men.1 These findings extend to benign colonic adenomas. Large-scale population analysis found that hormone-replacement therapy has an additive protective effect for postmenopausal women at all concentrations and durations of exposure.2 Individual benefits of oestrogen or progesterone are difficult to separate,3 but the protective effects seen with oestrogen treatment alone are compelling.4 Since the identification of two types of oestrogen receptor (ERα and ERβ) in 1996,5 the complex effects of oestrogen on various tissue types has become apparent. Overexpression of ERβ in the human colon coupled with negligible expression of ERα suggest that ERβ is involved in the protective effect of hormone-replacement therapy on colonic carcinogenesis. In this Review, we examine the role of oestrogen in protection from colorectal cancer.
isoforms numbered 2–5.9 In this Review, ERβ is the wildtype receptor, ERβ1, unless stated otherwise. The interior of the ligand-binding pocket is greatly conserved with only a difference of two contact residues.10 Although this explains the similar affinities of ERβ and ERα for endogenous oestrogen originally described by Kuiper and co-workers,11 early ligandbinding assays and ligand-competition experiments showed that phyto-oestrogens have a higher affinity for ERβ than for ERα and five other compounds seem to have selectivity for the ERβ receptor: diarylpropionitrile,12 8β-VE2,13 ERB-041,14 WAY-202196,15 WAY-200070.16 Oestrogens affect the growth, differentiation, and function of target tissues. Oestrogen diffuses freely from serum through the lipid bilayer to the intracellular milieu where oestrogen receptors are maintained in a state of transcriptional silence within the cell through interaction with co-repressor molecules.17 Binding of oestrogens causes a conformational change allowing disassociation from co-repressors and recruitment of coactivator molecules.18 This activated complex binds to an oestrogen-response element on target DNA
Lancet Oncol 2008; 9: 385–91 Department of Surgery, St Vincent’s University Hospital, Dublin, Ireland (R Kennelly Mb, D O Kavanagh MCh, A M Hogan Mb, D C Winter MD) Correspondence to: Dr Rory Kennelly, Institute for Clinical Outcomes in Research and Education, Department of Surgery, St Vincent’s University Hospital, Dublin 4, Ireland [email protected]
Oestrogen-receptor structure and function Oestrogens are steroid hormones, historically associated solely with the human female reproductive cycle. A nuclear receptor, ERα, for 17β oestradiol, the primary oestrogen, was first identified in rat uteri in 1966.6 The gene encoding ERα, first cloned in 1986,7 is located on human chromosome 6q25. In 1996, Kuiper and colleagues5 found a second oestrogen receptor, ERβ, encoded by a gene on chromosome 14q23–24.1. Like other members of the nuclear-receptor superfamily, oestrogen receptors have six functional domains. ERα and ERβ have similar DNA-binding and ligand-binding domains, but otherwise there is little homology (figure 2).8 ERβ has at least five isoforms: the full length receptor called ERβ1 or (wild-type ERβ), and the other http://oncology.thelancet.com Vol 9 April 2008
Figure 1: Typical appearance of colonic adenocarcinoma in a resection specimen
385
Review
HD 30%
DBD 96%
LBD 60%
TAF1 17·5%
TAF2 18%
NH2
COOH
Figure 2: Oestrogen-receptor structure TAF1=transcriptional activation factor 1. DBD=DNA binding domain. HD=hinge domain. LBD=ligand binding domain. TAF2=transcriptional activation factor 2. Numbers are percentage homology between ERα and ERβ domains.
causing increased transcription of target genes.19 An alternative mechanism of gene transcription involves protein–protein interactions between oestrogen receptors and transcription factors such as c-fos and cjun.20 More recently, oestrogen receptors have been identified in the cell cytosol associated with heat-shock protein 90, which is a ubiquitous chaperone molecule that maintains correct folding of proteins.21 Heat-shock protein 90 plays a pivotal part in cell signalling, and its potential role in carcinogenesis, although under intense investigation, is outside the scope of this review. Transcriptional effects of 17β oestradiol have a latency of 2–8 h.22 These effects can be blocked by inhibitors of transcription such as actinomycin D23 or by specific antagonists of the oestrogen receptors. Non-genomic effects occur more rapidly, in the order of seconds to minutes. Rapid effects of oestrogen have been investigated in both animal and human colonic epithelium. Effects seen include changes in intracellular calcium,24 protein Patients (n)
Immunohistochemistry (ERβ,ERα)
Western blot (ERβ,ERα)
RT-PCR (ERβ,ERα)
Campbell-Thompson36
26
++,+
++,··
··
Jassam38
76
+,··
··
·· +,··
18
··
··
Foley33
11
··
++,+
++,+
Xie34
10
+,–
··
··
12
··
··
+++,–
Konstantinopoulos37
90
++,··
··
··
··
++,trace
++,trace
Qiu35
6
+=presence of receptor with increasing symbols indicating semiquantitative increase in numbers detected. –=no receptor detected. Trace=negligible detection. RT-PCR=reverse transcriptase PCR.
Table 1: Summary of ERβ versus ERα expression in studies of healthy human colon tissue
386
kinase cascades,25 and cellular pH.26 The mechanism by which this occurs is controversial. Until the discovery of the second receptor, the standard test for an oestrogen was stimulation of growth of the uterus. In ERα knockout mice and aromatase deficient mice, uterine growth is disrupted.27,28 ERβ knockout mice, however, do not demonstrate this phenomenon.29 In cell lines in which oestrogens cause proliferation in the presence of ERα, ERβ inhibits cell growth.30 Studies in rat models13 show that most of the classic functions associated with oestrogens can be associated with ERα; however, due to the negligible concentrations of ERα in the colon the seemingly antiproliferative effect of oestrogen cannot be attributed to this receptor.
Evidence for expression of oestrogen receptors in the colon Expression of the ERβ differs with tissue type.31 Kuiper and co-workers5 first characterised ERβ in rat prostate and ovary.5 Further investigation of tissue expression, with in-situ hybridisation, found high titres of ERβ mRNA in human colonic mucosal epithelium.32 Thus, ERβ is the dominant receptor and the most likely mediator of transcriptional oestrogenic effects in human colonic mucosa (table 1).33–38 The muscularis mucosa is devoid of ERβ on western blot,33,34 semiquantitative reverse transcriptase PCR (RT-PCR),35,36 and immunohistochemical techniques.37,38
Evidence for ERβ in colon cancer Hormone-dependent cancers have an inverse relation between tumour progression and ERβ expression.39 The same relation is seen in colorectal cancer in cell lines and human clinical specimens.33,34,36 The findings in clinical specimens were of added value as clear margins of colon adenocarcinoma resection specimens were used as normal colon, which allowed for direct comparison of ERβ expression in paired malignant and normal mucosa from the same patients providing internal validation for examination of malignant specimens. The reduction in number of ERβ receptors in colon cancer might relate to worsening stage and grade of the tumour.37,38 More recent work indicates that differential expression of wild-type ERβ and isoforms ERβ2 and ERβ5 occurs in tumours with microsatellite instability.40 Although this finding suggests a role for ERβ signalling in colon carcinogenesis, the work was done on paraffin embedded histology samples with no follow-up of patients. No group has investigated outcome measures such as TNM (tumour, node, metastasis) staging, formal grading, disease-free survival, or overall survival. Prospective data on ERβ expression and outcome in patients with colon cancer are needed before conclusions can be made on ERβ as a prognostic indicator. However, similar associations between low expression of ERβ and http://oncology.thelancet.com Vol 9 April 2008
Review
advanced disease have been shown in gastric41 and gallbladder carcinoma,42 and there is enough evidence to warrant a full investigation into the physiology of oestrogen action in the colon and how it might relate to carcinogenesis. Oestrogen-receptor knockout mice provide valuable information on the role of ERβ in tissues other than colon. A study involving prostate epithelium showed disruption of normal mechanisms of apoptosis and increased cell proliferation.43 In breast tissue, ERβ does not play a part in the development of the mammary gland; however, there is some evidence it might be involved in full differentiation during lactation.31 Pituitary function and uterine morphology and function are normal in ERβ-knockout mice, confirming that ERα is the primary modulator of oestrogenic effects on the female reproductive system.44 Recent work on colonic mucosa in ERβ-knockout mice showed that a loss of ERβ leads to hyperproliferation, loss of differentiation, and disordered apoptosis of the colonic mucosa.45 A concomitant disorganisation of mucin production with disruption of the epithelial tight junction and a reduction in the number of hemidesmosomes was noted. This loss of protection of the uppermost crypt cells from luminal contents might lead to increased shedding and upregulation of cell production rather than any direct cell-cycle effects. Work from the same laboratory has shown disordered homoeostasis of respiratory epithelium in a double-knockout mouse model;46 however, the researchers did not do detailed analysis of intercellular adherence. Further morphological studies in oestrogen-receptor-knockout models should clarify the role of ERβ in colon tissue. However, a systemic specific modulator of ERβ would have minimum effect on development and normal functioning of the uterus, ovary, breast, or pituitary gland.
Protective genomic effects of oestrogen Research into the protective transcriptional effects of oestrogen has focused on two main topics: inhibition of cell proliferation and induction of apoptosis. Reduction of tumour development or of tumour load due to oestrogen is a consistent finding in both cell-culture and animal models. Many mechanisms might explain this effect. ERα-mediated proliferative effects of oestrogen might be modulated via ERβ activation in breast, ovary, and prostate.47 The relevance of these findings to the colon, where ERα is expressed in very low amounts, is questionable. The possibility that ERβ has antiproliferative activity independent of ERα inhibition was examined in a transgenic mouse model.48 Min/+ mice have a germ line mutation in the adenomatous polyposis coli tumoursuppressor gene and are useful in the modelling of intestinal tumorigenesis. Typically, about 5% of these mice develop colon tumours. Oestrogen-receptor defichttp://oncology.thelancet.com Vol 9 April 2008
iency caused 63% of mice to develop at least one colon tumour. Histological analysis showed increased proliferation and decreased differentiation of the colonic mucosa. Furthermore, the mice had raised crypt fission, which suggests oestrogen coordinates stromal-epithelial interactions that maintain normal colonic crypt architecture.49 Overexpression of ERβ in human-colon-tumour-118 cells lowers cell proliferation related to the expression of cyclin E and cyclin-dependent kinase inhibitor 1A to a fifth of normal levels.50 These effects, while reliant on ERβ in a concentration-dependent fashion, were reproducible in the absence of exogenous E2 in the cell medium. This finding might be explained by oestrogenreceptor activation by other growth factors, such as epidermal growth factor and insulin-like growth factor 1. Polyamines (putrescine, spermine) are a group of polycations found in high concentrations in normal and neoplastic proliferating cells. Synthesis of these occurs early in the G1 phase of the cell cycle. Studies of phyto-oestrogen in DLD-1 colon-cancer cell lines have shown that biosynthesis of polyamine by DLD-1 coloncancer cells was decreased by phyto-oestrogen, as was activity of its rate-limiting enzyme ornithine decarboxylase.51,52 High concentrations of ornithine decarboxylase and polyamine are associated with rapidly dividing and malignant cells.53 The association between diet and colon-cancer prevention is well established.54 Dietary fibre and soy products are high in phytooestrogen, which might contribute to their protective effect. Induction of apoptosis by ERβ has been described via differing mechanisms including increased DNA fragmentation in COLO205 colon cancer cells.34 The mechanism behind this effect might be due to upregulation of the proapoptotic BAXα gene or a shift in the ratio of Bcl-2 to Baxα.51 Other researchers noted that ERβ induces apoptosis in LoVo colon cancer cells due to increased p53 signalling (with subsequent upregulation of caspase 8 and 9) and proposed that reduction in β-catenin proteins is the cause of inhibition of cell proliferation.55 17β-oestradiol–ERβ seems to activate caspase 3, thus inducing the caspase-dependent proapoptotic cascade (table 2).56 Regardless of the exact mechanism, protection of apoptotic mechanisms intuitively protects against disordered cell division and ultimately carcinogenesis
Alternative modes of oestrogen action on the colonic mucosa Non-genomic steroid effects include activation of numerous neural signalling cascades,25 phospholipase C,57 and inositol triphosphate,58 intracellular calcium,24 protein kinase cascade,59 and extracellular-signalregulated kinase (ERK).60 The mechanisms driving these intracellular events, and particularly the receptors 387
Review
Model
Challenge
Finding
Proposed mechanism of action
Clinical significance
Qiu34
COLO205 colon cancer Oestradiol exposure cell line (expressing ERβ only)
Dose-dependent increase in apoptosis (p<0·01)
Oestradiol–ERβ mediated cell fragmentation and nuclear condensation
Oestrogen exposure can inhibit cancer progression
Cho48
Oestrogen-receptordeficient colonic mucosa from Min/+ mice
Cross with oestrogenreceptor deficient mice
Decreased differentiation (p<0·001) Increased proliferation (p<0·001) Increased crypt fission (p=0·617)
Disruption of stromalepithelial interactions due to loss of DPC-4 and BMP signalling
Loss of oestrogen signalling after menopause might contribute to colorectal-cancer development
Martinet50
HCT-118 colon cancer cell line overexpressing ERβ
Induced over expression of Cell proliferation reduced to a ERβ fifth (p<0·05)
Reduction in cyclin E expression Induction of CDKN1A expression
Loss of ERβ expression contributes to colorectal cancer progression
Linsalata51
DLD-1 colon cancer cell line (expressing ERβ only)
Genistein exposure, oestradiol-free system
Reduction in cell proliferation (p<0·05) Induction of apoptosis (p<0·05)
Reduction in polyamine and ODC synthesis Bcl2/baxα ratio shift
Dietary phyto-oestrogens may contribute to colorectal cancer prevention
Hsu55
LoVo colon cancer cell Oestradiol exposure line overexpressing ERβ
Reduction in cell proliferation (p<0·05) Induction of apoptosis (p<0·05)
Upregulation of caspases 8 Overexpression of ERβ and 9 might provide alternative Downregulation of colon-cancer treatment β-catenin
DPC-4=deletion target in pancreatic carcinoma 4. BMP=bone morphogenic protein. CDKN1A=cyclin-dependent kinase inhibitor 1A. ODC=ornithine decarboxylase.
Table 2: Evidence for genomic protective effects of ERβ in colon tissue
involved, are debated. There seem to be so many signalling pathways and receptor mechanisms that oestrogen action and its modulation are likely to be species, gender, and tissue specific.61 We review the best studied mechanisms. Pappas and colleagues62 were among the first to find membrane-bound oestrogen receptor by use of antibodies in rat pituitary tumour cell lines (GH3/B6). The researchers suggested that the receptors were the same as the classic nuclear receptors because of colocalisation of antibodies against oestrogen receptors and oestrogen-Bovine serum antigen conjugates; however, this work was done before the discovery of ERβ. Therefore, extrapolation of these findings to tissue solely expressing ERβ, such as colon tissue, is difficult. By use of various cell lines,63,64 Razandi and co-workers provided further evidence for a membrane receptor similar to the classic oestrogen receptors; however, most of their work is on ERα and has limited applicability to the colon. Bearing this in mind, the same laboratory showed complete absence of nuclear oestrogen receptors and the putative membrane-bound oestrogen receptors as well as negligible oestrogenic signalling in endothelial cells from DERKO mice.63 Although much remains to be elucidated, such compelling evidence of a membrane-bound receptor cannot be discounted. Recent work has focused on DLD-1 cells, a colon cancer cell line expressing ERβ only.65 Results have suggested that not only does a membrane-bound oestrogen receptor exist, but also that it is dependent on palmitoylation for localisation and therefore its 388
action can be regulated by modulation of the palmitoylation process.66,67 Unlike previous studies investigating membrane-bound oestrogen receptors there is no suggestion that they are the sole mechanism for rapid oestrogenic signalling in the colon.60 The concentration of ERβ shown in DLD cells was small and effects were much less than in similar models involving ERα.63 Different models for rapid oestrogenic signalling independent of the known oestrogen receptors have been proposed. A rapid increase in intracellular calcium in female rat colons caused by oestrogen that was unable to cross cell membranes (due to BSA conjugation) in the presence of a receptor antagonist (ICI 182780) suggests the presence of a membrane-bound receptor other than ERβ.68 The possibility that this is a G-protein coupled receptor was investigated using the known G-protein inhibitors, cholera and pertussis toxins. Cholera toxin but not pertussis toxin abolished all effects caused by 17β oestradiol, suggesting that an unknown G-coupled receptor causes non-genomic effects of 17β oestradiol. By use of specific kinase antagonists, protein kinase C was found to be a mediator of rapid oestrogen effects with the δ isoform emerging as the most likely to be involved.69 Filardo and co-workers70 reported activation of the mitogen-activated-protein-kinase (MAPK) cascade and MAPK3 (ERK-1) and MAPK1 (ERK-2) independent of known oestrogen receptors but again, dependent on a G-protein coupled receptor (GPR-30) in breast-cancer cell lines. GPR-30 is sensitive to pertussis toxin, unlike the putative G-protein coupled receptor responsible for http://oncology.thelancet.com Vol 9 April 2008
Review
non-genomic oestrogen effects in the colon. These results, however, were not always reproducible,71 so whether GPR-30 is the membrane-bound modulator of rapid 17β-oestradiol effects on the cell remains unknown. A rapid calcium flux has been reproduced in a cell line that does not have oestrogen-receptors.63 This calcium flux causes oestrogenic initiation of MAPK– ERK signalling in breast-cancer cells that is independent of oestrogen receptor signalling72 giving credence to the possibility that protein kinase C δ may be an alternative non-genomic effector molecule. These findings raise the possibility that the δ isoform confers resistance to selective oestrogen receptor modulators in breast cancer by providing an alternative mitogenic pathway. If this is the case, and if this is also true in the colon, selective antagonists of protein kinase C δ are potential agents for colon-cancer prevention.
Non-genomic protective effects Acting via membrane-bound ERβ, 17β-oestradiol causes rapid and persistent activation of the p38–MAPK pathway in DLD-1 colon cancer cells, leading to a proapoptotic cascade involving caspase-3.73 After further study of DLD-1 cells, researchers noted that ERβ activation of the p38–MAPK pathway leads to increased expression of ERβ itself by both genomic and nongenomic means66 leading to a self-perpetuating cycle increasing its protective effect. The 17β-oestradiol–ERβ complex probably plays a part in the prevention of methylation of the vitamin-D receptor, a known mediator of colon-cancer growth, thereby allowing for ongoing transcription. A direct increase in expression of the vitamin-D receptor has been described via extracellular MAPK1 or MAPK3 in a rapid non-genomic manner in HT29 colon-cancer cell lines,74 which provides another possible explanation for the protective effect of ERβ on the colon.
Conclusion Epidemiological and scientific data have engendered interest in the role of oestrogen in colon carcinogenesis. The effects of oestrogen seem to be mediated predominantly through ERβ in a combination of genomic and non-genomic mechanisms. Our current knowledge suggests that ERβ, dependent or independent of 17β-oestradiol activation, has a crucial role in coloniccell homeostasis, including modulation of proliferation and organised cell death. An absence of ERβ, via either blockade or genetic manipulation, results in increased cell turnover in the colonic mucosa. The intuitive corollary is that specific ERβ signalling confers protection against colonic mitogenesis. ERβ modulators might provide options for prevention of colorectal cancer, but differences in signalling of ERβ will complicate the development of such treatments. Non-genomic activation of mitogenic cell signalling http://oncology.thelancet.com Vol 9 April 2008
Search strategy and selection criteria References for this Review were identified by searches of MEDLINE, Current Contents, and PubMed with the search terms “oestrogen”, “oestrogen receptor”, and “colon cancer”. References from identified articles were investigated for relevance. Abstracts and reports from meetings were not included. Only papers published in English between 1966 and 2008 were included.
cascades by oestrogen and clarification of receptor independent coupling mediated by protein kinase C δ provide other opportunities for cancer treatment. Conflicts of interest The authors declared no conflicts of interest. References 1 Ries LA, Wingo PA, Miller DS, et al. The annual report to the nation on the status of cancer, 1973-1997, with a special section on colorectal cancer. Cancer 2000; 88: 2398–424. 2 Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. JAMA 2002; 288: 321–33. 3 Lemy A. The relevance of the Women’s Health Initiative results on combined hormone replacement therapy in clinical practice. J Obstet Gynaecol Can 2002; 24: 711–15. 4 Prihartono N, Palmer JR, Louik C, Shapiro S, Rosenberg L. A case-control study of use of postmenopausal female hormone supplements in relation to the risk of large bowel cancer. Cancer Epidemiol Biomarkers Prev 2000; 9: 443–47. 5 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996; 93: 5925–30. 6 Toft D, Gorski J. A receptor molecule for estrogens: isolation from the rat uterus and preliminary characterization. Proc Natl Acad Sci USA 1966; 55: 1574–81. 7 Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science 1986; 231: 1150–54. 8 Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International Union of Pharmacology, LXIV: estrogen receptors. Pharmacol Rev 2006; 58: 773–81. 9 Moore JT, McKee DD, Slentz-Kesler K, et al. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun 1998; 247: 75–78. 10 Pike AC, Brzozowski AM, Hubbard RE, et al. Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999; 18: 4608–18. 11 Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998; 139: 4252–63. 12 Sun J, Baudry J, Katzenellenbogen JA, Katzenellenbogen BS. Molecular basis for the subtype discrimination of the estrogen receptor-beta-selective ligand, diarylpropionitrile. Mol Endocrinol 2003; 17: 247–58. 13 Hillisch A, Peters O, Kosemund D, et al. Dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structure-based design. Mol Endocrinol 2004; 18: 1599–609. 14 Malamas MS, Manas ES, McDevitt RE, et al. Design and synthesis of aryl diphenolic azoles as potent and selective estrogen receptorbeta ligands. J Med Chem 2004; 47: 5021–40. 15 Mewshaw RE, Edsall RJ Jr, Yang C, et al. ERbeta ligands. 3. Exploiting two binding orientations of the 2-phenylnaphthalene scaffold to achieve ERbeta selectivity. J Med Chem 2005; 48: 3953–79. 16 Harris HA. Estrogen receptor-beta: recent lessons from in vivo studies. Mol Endocrinol 2007; 21: 1–13.
389
Review
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35 36
37
38
390
Dobrzycka KM, Townson SM, Jiang S, Oesterreich S. Estrogen receptor corepressors—a role in human breast cancer? Endocr Relat Cancer 2003; 10: 517–36. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001; 276: 36869–72. Huang J, Li X, Yi P, Hilf R, Bambara RA, Muyan M. Targeting estrogen responsive elements (EREs): design of potent transactivators for ERE-containing genes. Mol Cell Endocrinol 2004; 218: 65–78. Kushner PJ, Agard D, Feng WJ, et al. Oestrogen receptor function at classical and alternative response elements. Novartis Found Symp 2000; 230: 20–26. Pratt WB. The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med 1998; 217: 420–34. Harvey BJ, Doolan CM, Condliffe SB, Renard C, Alzamora R, Urbach V. Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon. Steroids 2002; 67: 483–91. Condliffe SB, Doolan CM, Harvey BJ. 17beta-oestradiol acutely regulates Cl-secretion in rat distal colonic epithelium. J Physiol 2001; 530: 47–54. Doolan CM, Harvey BJ. Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon. J Biol Chem 1996; 271: 8763–67. McNamara B, Winter DC, Cuffe J, Taylor C, O’Sullivan GC, Harvey BJ. Rapid activation of basolateral potassium transport in human colon by oestradiol. Br J Pharmacol 2000; 131: 1373–78. Winter DC, Taylor C, O’S C, Harvey BJ. Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon. Br J Surg 2000; 87: 1684–89. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 1993; 90: 11162–66. Korach KS, Emmen JM, Walker VR, et al. Update on animal models developed for analyses of estrogen receptor biological activity. J Steroid Biochem Mol Biol 2003; 86: 387–91. Weihua Z, Saji S, Makinen S, et al. Estrogen receptor (ER) beta, a modulator of ERalpha in the uterus. Proc Natl Acad Sci USA 2000; 97: 5936–41. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor beta inhibits 17beta-estradiolstimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA 2004; 101: 1566–71. Koehler KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA. Reflections on the discovery and significance of estrogen receptor beta. Endocr Rev 2005; 26: 465–78. Enmark E, Pelto-Huikko M, Grandien K, et al. Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 1997; 82: 4258–65. Foley EF, Jazaeri AA, Shupnik MA, Jazaeri O, Rice LW. Selective loss of estrogen receptor beta in malignant human colon. Cancer Res 2000; 60: 245–48. Qiu Y, Waters CE, Lewis AE, Langman MJ, Eggo MC. Oestrogeninduced apoptosis in colonocytes expressing oestrogen receptor beta. J Endocrinol 2002; 174: 369–77. Xie LQ, Yu JP, Luo HS. Expression of estrogen receptor beta in human colorectal cancer. World J Gastroenterol 2004; 10: 214–17. Campbell-Thompson M, Lynch IJ, Bhardwaj B. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Res 2001; 61: 632–40. Konstantinopoulos PA, Kominea A, Vandoros G, et al. OestrogenΩreceptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour’s dedifferentiation. Eur J Cancer 2003; 39: 1251–58. Jassam N, Bell SM, Speirs V, Quirke P. Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes’ staging. Oncol Rep 2005; 14: 17–21.
39
40
41
42
43
44
45
46
47
48
49
50
51
52 53
54 55
56
57
58
59
Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of ERbeta expression as a common step in estrogen-dependent tumor progression. Endocr Relat Cancer 2004; 11: 537–51. Wong NA, Malcomson RD, Jodrell DI, Groome NP, Harrison DJ, Saunders PT. ERbeta isoform expression in colorectal carcinoma: an in vivo and in vitro study of clinicopathological and molecular correlates. J Pathol 2005; 207: 53–60. Wang M, Pan JY, Song GR, Chen HB, An LJ, Qu SX. Altered expression of estrogen receptor alpha and beta in advanced gastric adenocarcinoma: correlation with prothymosin alpha and clinicopathological parameters. Eur J Surg Oncol 2007; 33: 195–201. Sumi K, Matsuyama S, Kitajima Y, Miyazaki K. Loss of estrogen receptor beta expression at cancer front correlates with tumor progression and poor prognosis of gallbladder cancer. Oncol Rep 2004; 12: 979–84. Weihua Z, Makela S, Andersson LC, et al. A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA 2001; 98: 6330–35. Hewitt SC, Korach KS. Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues. Reproduction 2003; 125: 143–49. Wada-Hiraike O, Imamov O, Hiraike H, et al. Role of estrogen receptor beta in colonic epithelium. Proc Natl Acad Sci USA 2006; 103: 2959–64. Patrone C, Cassel TN, Pettersson K, et al. Regulation of postnatal lung development and homeostasis by estrogen receptor beta. Mol Cell Biol 2003; 23: 8542–52. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA. Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 2006; 20: 534–43. Cho NL, Javid SH, Carothers AM, Redston M, Bertagnolli MM. Estrogen receptors alpha and beta are inhibitory modifiers of Apcdependent tumorigenesis in the proximal colon of Min/+ mice. Cancer Res 2007; 67: 2366–72. Sims NA, Dupont S, Krust A, et al. Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males. Bone 2002; 30: 18–25. Martineti V, Picariello L, Tognarini I, et al. ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components. Endocr Relat Cancer 2005; 12: 455–69. Linsalata M, Russo F, Notarnicola M, et al. Effects of genistein on the polyamine metabolism and cell growth in DLD-1 human colon cancer cells. Nutr Cancer 2005; 52: 84–93. Seiler N. Polyamine metabolism. Digestion 1990; 46 (suppl 2): 319–30. Di LA, Linsalata M, Cavallini A, Messa C, Russo F. Sex steroid hormone receptors, epidermal growth factor receptor, and polyamines in human colorectal cancer. Dis Colon Rectum 1992; 35: 305–09. Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002; 3: 364–73. Hsu HH, Cheng SF, Wu CC, et al. Apoptotic effects of overexpressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner. Chin J Physiol 2006; 49: 110–16. Marino M, Galluzzo P, Leone S, Acconcia F, Ascenzi P. Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells. Endocr Relat Cancer 2006; 13: 559–69. Le Mellay V, Grosse B, Lieberherr M. Phospholipase C beta and membrane action of calcitriol and estradiol. J Biol Chem 1997; 272: 11902–07. Lieberherr M, Grosse B, Kachkache M, Balsan S. Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors. J Bone Miner Res 1993; 8: 1365–76. Winter DC, Schneider MF, O’Sullivan GC, Harvey BJ, Geibel JP. Rapid effects of aldosterone on sodium-hydrogen exchange in isolated colonic crypts. J Membr Biol 1999; 170: 17–26.
http://oncology.thelancet.com Vol 9 April 2008
Review
60
61 62
63
64
65
66
67
68
Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol 2004; 18: 2854–65. Nadal A, Diaz M, Valverde MA. The estrogen trinity: membrane, cytosolic, and nuclear effects. News Physiol Sci 2001; 16: 251–55. Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 1995; 9: 404–10. Pedram A, Razandi M, Levin ER. Nature of functional estrogen receptors at the plasma membrane. Mol Endocrinol 2006; 20: 1996–2009. Razandi M, Oh P, Pedram A, Schnitzer J, Levin ER. ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol Endocrinol 2002; 16: 100–15. Fiorelli G, Picariello L, Martineti V, Tonelli F, Brandi ML. Functional estrogen receptor beta in colon cancer cells. Biochem Biophys Res Commun 1999; 261: 521–27. Caiazza F, Galluzzo P, Lorenzetti S, Marino M. 17beta-Estradiol induces ERbeta up-regulation via p38/MAPK activation in colon cancer cells. Biochem Biophys Res Commun 2007; 359: 102–07. Galluzzo P, Caiazza F, Moreno S, Marino M. Role of ERbeta palmitoylation in the inhibition of human colon cancer cell proliferation. Endocr Relat Cancer 2007; 14: 153–67. Doolan CM, Harvey BJ. A Galphas protein-coupled membrane receptor, distinct from the classical oestrogen receptor, transduces rapid effects of oestradiol on [Ca2+]i in female rat distal colon. Mol Cell Endocrinol 2003; 199: 87–103.
http://oncology.thelancet.com Vol 9 April 2008
69
70
71
72
73
74
Doolan CM, Condliffe SB, Harvey BJ. Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon. Br J Pharmacol 2000; 129: 1375–86. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000; 14: 1649–60. Vivacqua A, Bonofiglio D, Albanito L, et al. 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30. Mol Pharmacol 2006; 70: 1414–23. Keshamouni VG, Mattingly RR, Reddy KB. Mechanism of 17-betaestradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem 2002; 277: 22558–65. Marino M, Galluzzo P, Leone S, Acconcia F, Ascenzi P. Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells. Endocr Relat Cancer 2006; 13: 559–69. Gilad LA, Tirosh O, Schwartz B. Phytoestrogens regulate transcription and translation of vitamin D receptor in colon cancer cells. J Endocrinol 2006; 191: 387–98.
391
Oestrogen and the colon: potential mechanisms for cancer prevention Rory Kennelly, Dara O Kavanagh, Aisling M Hogan, Desmond C Winter
The role of oestrogen in oncogenesis has been examined extensively, especially in the context of breast cancer, and receptor modulators are an integral part of targeted treatment in this disease. The role of oestrogen signalling in colonic carcinoma is poorly understood. Men are more susceptible than women to colon cancer. Furthermore, hormone-replacement therapy affords an additive protective effect for postmenopausal women, and when these women do develop cancer, they typically have less aggressive disease. The discovery of a second oestrogen receptor (ERβ) and its over expression in healthy human colon coupled with reduced expression in colon cancer suggests that this receptor might be involved. The underlying mechanism, however, remains largely unknown. In this Review, we discuss the various hypotheses presented in the published literature. We examine the cellular and molecular mechanisms through which oestrogen is purported to exert its protective influence, and we review the evidence available to support these claims.
Introduction The oncogenic effects of oestrogens have been investigated extensively in breast cancer where hormonereceptor modulators are now an integral part of targeted treatment. Little is known about oestrogen signalling in colorectal cancer (figure 1) although women are less susceptible to this cancer than men.1 These findings extend to benign colonic adenomas. Large-scale population analysis found that hormone-replacement therapy has an additive protective effect for postmenopausal women at all concentrations and durations of exposure.2 Individual benefits of oestrogen or progesterone are difficult to separate,3 but the protective effects seen with oestrogen treatment alone are compelling.4 Since the identification of two types of oestrogen receptor (ERα and ERβ) in 1996,5 the complex effects of oestrogen on various tissue types has become apparent. Overexpression of ERβ in the human colon coupled with negligible expression of ERα suggest that ERβ is involved in the protective effect of hormone-replacement therapy on colonic carcinogenesis. In this Review, we examine the role of oestrogen in protection from colorectal cancer.
isoforms numbered 2–5.9 In this Review, ERβ is the wildtype receptor, ERβ1, unless stated otherwise. The interior of the ligand-binding pocket is greatly conserved with only a difference of two contact residues.10 Although this explains the similar affinities of ERβ and ERα for endogenous oestrogen originally described by Kuiper and co-workers,11 early ligandbinding assays and ligand-competition experiments showed that phyto-oestrogens have a higher affinity for ERβ than for ERα and five other compounds seem to have selectivity for the ERβ receptor: diarylpropionitrile,12 8β-VE2,13 ERB-041,14 WAY-202196,15 WAY-200070.16 Oestrogens affect the growth, differentiation, and function of target tissues. Oestrogen diffuses freely from serum through the lipid bilayer to the intracellular milieu where oestrogen receptors are maintained in a state of transcriptional silence within the cell through interaction with co-repressor molecules.17 Binding of oestrogens causes a conformational change allowing disassociation from co-repressors and recruitment of coactivator molecules.18 This activated complex binds to an oestrogen-response element on target DNA
Lancet Oncol 2008; 9: 385–91 Department of Surgery, St Vincent’s University Hospital, Dublin, Ireland (R Kennelly Mb, D O Kavanagh MCh, A M Hogan Mb, D C Winter MD) Correspondence to: Dr Rory Kennelly, Institute for Clinical Outcomes in Research and Education, Department of Surgery, St Vincent’s University Hospital, Dublin 4, Ireland [email protected]
Oestrogen-receptor structure and function Oestrogens are steroid hormones, historically associated solely with the human female reproductive cycle. A nuclear receptor, ERα, for 17β oestradiol, the primary oestrogen, was first identified in rat uteri in 1966.6 The gene encoding ERα, first cloned in 1986,7 is located on human chromosome 6q25. In 1996, Kuiper and colleagues5 found a second oestrogen receptor, ERβ, encoded by a gene on chromosome 14q23–24.1. Like other members of the nuclear-receptor superfamily, oestrogen receptors have six functional domains. ERα and ERβ have similar DNA-binding and ligand-binding domains, but otherwise there is little homology (figure 2).8 ERβ has at least five isoforms: the full length receptor called ERβ1 or (wild-type ERβ), and the other http://oncology.thelancet.com Vol 9 April 2008
Figure 1: Typical appearance of colonic adenocarcinoma in a resection specimen
385
Review
HD 30%
DBD 96%
LBD 60%
TAF1 17·5%
TAF2 18%
NH2
COOH
Figure 2: Oestrogen-receptor structure TAF1=transcriptional activation factor 1. DBD=DNA binding domain. HD=hinge domain. LBD=ligand binding domain. TAF2=transcriptional activation factor 2. Numbers are percentage homology between ERα and ERβ domains.
causing increased transcription of target genes.19 An alternative mechanism of gene transcription involves protein–protein interactions between oestrogen receptors and transcription factors such as c-fos and cjun.20 More recently, oestrogen receptors have been identified in the cell cytosol associated with heat-shock protein 90, which is a ubiquitous chaperone molecule that maintains correct folding of proteins.21 Heat-shock protein 90 plays a pivotal part in cell signalling, and its potential role in carcinogenesis, although under intense investigation, is outside the scope of this review. Transcriptional effects of 17β oestradiol have a latency of 2–8 h.22 These effects can be blocked by inhibitors of transcription such as actinomycin D23 or by specific antagonists of the oestrogen receptors. Non-genomic effects occur more rapidly, in the order of seconds to minutes. Rapid effects of oestrogen have been investigated in both animal and human colonic epithelium. Effects seen include changes in intracellular calcium,24 protein Patients (n)
Immunohistochemistry (ERβ,ERα)
Western blot (ERβ,ERα)
RT-PCR (ERβ,ERα)
Campbell-Thompson36
26
++,+
++,··
··
Jassam38
76
+,··
··
·· +,··
18
··
··
Foley33
11
··
++,+
++,+
Xie34
10
+,–
··
··
12
··
··
+++,–
Konstantinopoulos37
90
++,··
··
··
··
++,trace
++,trace
Qiu35
6
+=presence of receptor with increasing symbols indicating semiquantitative increase in numbers detected. –=no receptor detected. Trace=negligible detection. RT-PCR=reverse transcriptase PCR.
Table 1: Summary of ERβ versus ERα expression in studies of healthy human colon tissue
386
kinase cascades,25 and cellular pH.26 The mechanism by which this occurs is controversial. Until the discovery of the second receptor, the standard test for an oestrogen was stimulation of growth of the uterus. In ERα knockout mice and aromatase deficient mice, uterine growth is disrupted.27,28 ERβ knockout mice, however, do not demonstrate this phenomenon.29 In cell lines in which oestrogens cause proliferation in the presence of ERα, ERβ inhibits cell growth.30 Studies in rat models13 show that most of the classic functions associated with oestrogens can be associated with ERα; however, due to the negligible concentrations of ERα in the colon the seemingly antiproliferative effect of oestrogen cannot be attributed to this receptor.
Evidence for expression of oestrogen receptors in the colon Expression of the ERβ differs with tissue type.31 Kuiper and co-workers5 first characterised ERβ in rat prostate and ovary.5 Further investigation of tissue expression, with in-situ hybridisation, found high titres of ERβ mRNA in human colonic mucosal epithelium.32 Thus, ERβ is the dominant receptor and the most likely mediator of transcriptional oestrogenic effects in human colonic mucosa (table 1).33–38 The muscularis mucosa is devoid of ERβ on western blot,33,34 semiquantitative reverse transcriptase PCR (RT-PCR),35,36 and immunohistochemical techniques.37,38
Evidence for ERβ in colon cancer Hormone-dependent cancers have an inverse relation between tumour progression and ERβ expression.39 The same relation is seen in colorectal cancer in cell lines and human clinical specimens.33,34,36 The findings in clinical specimens were of added value as clear margins of colon adenocarcinoma resection specimens were used as normal colon, which allowed for direct comparison of ERβ expression in paired malignant and normal mucosa from the same patients providing internal validation for examination of malignant specimens. The reduction in number of ERβ receptors in colon cancer might relate to worsening stage and grade of the tumour.37,38 More recent work indicates that differential expression of wild-type ERβ and isoforms ERβ2 and ERβ5 occurs in tumours with microsatellite instability.40 Although this finding suggests a role for ERβ signalling in colon carcinogenesis, the work was done on paraffin embedded histology samples with no follow-up of patients. No group has investigated outcome measures such as TNM (tumour, node, metastasis) staging, formal grading, disease-free survival, or overall survival. Prospective data on ERβ expression and outcome in patients with colon cancer are needed before conclusions can be made on ERβ as a prognostic indicator. However, similar associations between low expression of ERβ and http://oncology.thelancet.com Vol 9 April 2008
Review
advanced disease have been shown in gastric41 and gallbladder carcinoma,42 and there is enough evidence to warrant a full investigation into the physiology of oestrogen action in the colon and how it might relate to carcinogenesis. Oestrogen-receptor knockout mice provide valuable information on the role of ERβ in tissues other than colon. A study involving prostate epithelium showed disruption of normal mechanisms of apoptosis and increased cell proliferation.43 In breast tissue, ERβ does not play a part in the development of the mammary gland; however, there is some evidence it might be involved in full differentiation during lactation.31 Pituitary function and uterine morphology and function are normal in ERβ-knockout mice, confirming that ERα is the primary modulator of oestrogenic effects on the female reproductive system.44 Recent work on colonic mucosa in ERβ-knockout mice showed that a loss of ERβ leads to hyperproliferation, loss of differentiation, and disordered apoptosis of the colonic mucosa.45 A concomitant disorganisation of mucin production with disruption of the epithelial tight junction and a reduction in the number of hemidesmosomes was noted. This loss of protection of the uppermost crypt cells from luminal contents might lead to increased shedding and upregulation of cell production rather than any direct cell-cycle effects. Work from the same laboratory has shown disordered homoeostasis of respiratory epithelium in a double-knockout mouse model;46 however, the researchers did not do detailed analysis of intercellular adherence. Further morphological studies in oestrogen-receptor-knockout models should clarify the role of ERβ in colon tissue. However, a systemic specific modulator of ERβ would have minimum effect on development and normal functioning of the uterus, ovary, breast, or pituitary gland.
Protective genomic effects of oestrogen Research into the protective transcriptional effects of oestrogen has focused on two main topics: inhibition of cell proliferation and induction of apoptosis. Reduction of tumour development or of tumour load due to oestrogen is a consistent finding in both cell-culture and animal models. Many mechanisms might explain this effect. ERα-mediated proliferative effects of oestrogen might be modulated via ERβ activation in breast, ovary, and prostate.47 The relevance of these findings to the colon, where ERα is expressed in very low amounts, is questionable. The possibility that ERβ has antiproliferative activity independent of ERα inhibition was examined in a transgenic mouse model.48 Min/+ mice have a germ line mutation in the adenomatous polyposis coli tumoursuppressor gene and are useful in the modelling of intestinal tumorigenesis. Typically, about 5% of these mice develop colon tumours. Oestrogen-receptor defichttp://oncology.thelancet.com Vol 9 April 2008
iency caused 63% of mice to develop at least one colon tumour. Histological analysis showed increased proliferation and decreased differentiation of the colonic mucosa. Furthermore, the mice had raised crypt fission, which suggests oestrogen coordinates stromal-epithelial interactions that maintain normal colonic crypt architecture.49 Overexpression of ERβ in human-colon-tumour-118 cells lowers cell proliferation related to the expression of cyclin E and cyclin-dependent kinase inhibitor 1A to a fifth of normal levels.50 These effects, while reliant on ERβ in a concentration-dependent fashion, were reproducible in the absence of exogenous E2 in the cell medium. This finding might be explained by oestrogenreceptor activation by other growth factors, such as epidermal growth factor and insulin-like growth factor 1. Polyamines (putrescine, spermine) are a group of polycations found in high concentrations in normal and neoplastic proliferating cells. Synthesis of these occurs early in the G1 phase of the cell cycle. Studies of phyto-oestrogen in DLD-1 colon-cancer cell lines have shown that biosynthesis of polyamine by DLD-1 coloncancer cells was decreased by phyto-oestrogen, as was activity of its rate-limiting enzyme ornithine decarboxylase.51,52 High concentrations of ornithine decarboxylase and polyamine are associated with rapidly dividing and malignant cells.53 The association between diet and colon-cancer prevention is well established.54 Dietary fibre and soy products are high in phytooestrogen, which might contribute to their protective effect. Induction of apoptosis by ERβ has been described via differing mechanisms including increased DNA fragmentation in COLO205 colon cancer cells.34 The mechanism behind this effect might be due to upregulation of the proapoptotic BAXα gene or a shift in the ratio of Bcl-2 to Baxα.51 Other researchers noted that ERβ induces apoptosis in LoVo colon cancer cells due to increased p53 signalling (with subsequent upregulation of caspase 8 and 9) and proposed that reduction in β-catenin proteins is the cause of inhibition of cell proliferation.55 17β-oestradiol–ERβ seems to activate caspase 3, thus inducing the caspase-dependent proapoptotic cascade (table 2).56 Regardless of the exact mechanism, protection of apoptotic mechanisms intuitively protects against disordered cell division and ultimately carcinogenesis
Alternative modes of oestrogen action on the colonic mucosa Non-genomic steroid effects include activation of numerous neural signalling cascades,25 phospholipase C,57 and inositol triphosphate,58 intracellular calcium,24 protein kinase cascade,59 and extracellular-signalregulated kinase (ERK).60 The mechanisms driving these intracellular events, and particularly the receptors 387
Review
Model
Challenge
Finding
Proposed mechanism of action
Clinical significance
Qiu34
COLO205 colon cancer Oestradiol exposure cell line (expressing ERβ only)
Dose-dependent increase in apoptosis (p<0·01)
Oestradiol–ERβ mediated cell fragmentation and nuclear condensation
Oestrogen exposure can inhibit cancer progression
Cho48
Oestrogen-receptordeficient colonic mucosa from Min/+ mice
Cross with oestrogenreceptor deficient mice
Decreased differentiation (p<0·001) Increased proliferation (p<0·001) Increased crypt fission (p=0·617)
Disruption of stromalepithelial interactions due to loss of DPC-4 and BMP signalling
Loss of oestrogen signalling after menopause might contribute to colorectal-cancer development
Martinet50
HCT-118 colon cancer cell line overexpressing ERβ
Induced over expression of Cell proliferation reduced to a ERβ fifth (p<0·05)
Reduction in cyclin E expression Induction of CDKN1A expression
Loss of ERβ expression contributes to colorectal cancer progression
Linsalata51
DLD-1 colon cancer cell line (expressing ERβ only)
Genistein exposure, oestradiol-free system
Reduction in cell proliferation (p<0·05) Induction of apoptosis (p<0·05)
Reduction in polyamine and ODC synthesis Bcl2/baxα ratio shift
Dietary phyto-oestrogens may contribute to colorectal cancer prevention
Hsu55
LoVo colon cancer cell Oestradiol exposure line overexpressing ERβ
Reduction in cell proliferation (p<0·05) Induction of apoptosis (p<0·05)
Upregulation of caspases 8 Overexpression of ERβ and 9 might provide alternative Downregulation of colon-cancer treatment β-catenin
DPC-4=deletion target in pancreatic carcinoma 4. BMP=bone morphogenic protein. CDKN1A=cyclin-dependent kinase inhibitor 1A. ODC=ornithine decarboxylase.
Table 2: Evidence for genomic protective effects of ERβ in colon tissue
involved, are debated. There seem to be so many signalling pathways and receptor mechanisms that oestrogen action and its modulation are likely to be species, gender, and tissue specific.61 We review the best studied mechanisms. Pappas and colleagues62 were among the first to find membrane-bound oestrogen receptor by use of antibodies in rat pituitary tumour cell lines (GH3/B6). The researchers suggested that the receptors were the same as the classic nuclear receptors because of colocalisation of antibodies against oestrogen receptors and oestrogen-Bovine serum antigen conjugates; however, this work was done before the discovery of ERβ. Therefore, extrapolation of these findings to tissue solely expressing ERβ, such as colon tissue, is difficult. By use of various cell lines,63,64 Razandi and co-workers provided further evidence for a membrane receptor similar to the classic oestrogen receptors; however, most of their work is on ERα and has limited applicability to the colon. Bearing this in mind, the same laboratory showed complete absence of nuclear oestrogen receptors and the putative membrane-bound oestrogen receptors as well as negligible oestrogenic signalling in endothelial cells from DERKO mice.63 Although much remains to be elucidated, such compelling evidence of a membrane-bound receptor cannot be discounted. Recent work has focused on DLD-1 cells, a colon cancer cell line expressing ERβ only.65 Results have suggested that not only does a membrane-bound oestrogen receptor exist, but also that it is dependent on palmitoylation for localisation and therefore its 388
action can be regulated by modulation of the palmitoylation process.66,67 Unlike previous studies investigating membrane-bound oestrogen receptors there is no suggestion that they are the sole mechanism for rapid oestrogenic signalling in the colon.60 The concentration of ERβ shown in DLD cells was small and effects were much less than in similar models involving ERα.63 Different models for rapid oestrogenic signalling independent of the known oestrogen receptors have been proposed. A rapid increase in intracellular calcium in female rat colons caused by oestrogen that was unable to cross cell membranes (due to BSA conjugation) in the presence of a receptor antagonist (ICI 182780) suggests the presence of a membrane-bound receptor other than ERβ.68 The possibility that this is a G-protein coupled receptor was investigated using the known G-protein inhibitors, cholera and pertussis toxins. Cholera toxin but not pertussis toxin abolished all effects caused by 17β oestradiol, suggesting that an unknown G-coupled receptor causes non-genomic effects of 17β oestradiol. By use of specific kinase antagonists, protein kinase C was found to be a mediator of rapid oestrogen effects with the δ isoform emerging as the most likely to be involved.69 Filardo and co-workers70 reported activation of the mitogen-activated-protein-kinase (MAPK) cascade and MAPK3 (ERK-1) and MAPK1 (ERK-2) independent of known oestrogen receptors but again, dependent on a G-protein coupled receptor (GPR-30) in breast-cancer cell lines. GPR-30 is sensitive to pertussis toxin, unlike the putative G-protein coupled receptor responsible for http://oncology.thelancet.com Vol 9 April 2008
Review
non-genomic oestrogen effects in the colon. These results, however, were not always reproducible,71 so whether GPR-30 is the membrane-bound modulator of rapid 17β-oestradiol effects on the cell remains unknown. A rapid calcium flux has been reproduced in a cell line that does not have oestrogen-receptors.63 This calcium flux causes oestrogenic initiation of MAPK– ERK signalling in breast-cancer cells that is independent of oestrogen receptor signalling72 giving credence to the possibility that protein kinase C δ may be an alternative non-genomic effector molecule. These findings raise the possibility that the δ isoform confers resistance to selective oestrogen receptor modulators in breast cancer by providing an alternative mitogenic pathway. If this is the case, and if this is also true in the colon, selective antagonists of protein kinase C δ are potential agents for colon-cancer prevention.
Non-genomic protective effects Acting via membrane-bound ERβ, 17β-oestradiol causes rapid and persistent activation of the p38–MAPK pathway in DLD-1 colon cancer cells, leading to a proapoptotic cascade involving caspase-3.73 After further study of DLD-1 cells, researchers noted that ERβ activation of the p38–MAPK pathway leads to increased expression of ERβ itself by both genomic and nongenomic means66 leading to a self-perpetuating cycle increasing its protective effect. The 17β-oestradiol–ERβ complex probably plays a part in the prevention of methylation of the vitamin-D receptor, a known mediator of colon-cancer growth, thereby allowing for ongoing transcription. A direct increase in expression of the vitamin-D receptor has been described via extracellular MAPK1 or MAPK3 in a rapid non-genomic manner in HT29 colon-cancer cell lines,74 which provides another possible explanation for the protective effect of ERβ on the colon.
Conclusion Epidemiological and scientific data have engendered interest in the role of oestrogen in colon carcinogenesis. The effects of oestrogen seem to be mediated predominantly through ERβ in a combination of genomic and non-genomic mechanisms. Our current knowledge suggests that ERβ, dependent or independent of 17β-oestradiol activation, has a crucial role in coloniccell homeostasis, including modulation of proliferation and organised cell death. An absence of ERβ, via either blockade or genetic manipulation, results in increased cell turnover in the colonic mucosa. The intuitive corollary is that specific ERβ signalling confers protection against colonic mitogenesis. ERβ modulators might provide options for prevention of colorectal cancer, but differences in signalling of ERβ will complicate the development of such treatments. Non-genomic activation of mitogenic cell signalling http://oncology.thelancet.com Vol 9 April 2008
Search strategy and selection criteria References for this Review were identified by searches of MEDLINE, Current Contents, and PubMed with the search terms “oestrogen”, “oestrogen receptor”, and “colon cancer”. References from identified articles were investigated for relevance. Abstracts and reports from meetings were not included. Only papers published in English between 1966 and 2008 were included.
cascades by oestrogen and clarification of receptor independent coupling mediated by protein kinase C δ provide other opportunities for cancer treatment. Conflicts of interest The authors declared no conflicts of interest. References 1 Ries LA, Wingo PA, Miller DS, et al. The annual report to the nation on the status of cancer, 1973-1997, with a special section on colorectal cancer. Cancer 2000; 88: 2398–424. 2 Rossouw JE, Anderson GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women’s Health Initiative randomized controlled trial. JAMA 2002; 288: 321–33. 3 Lemy A. The relevance of the Women’s Health Initiative results on combined hormone replacement therapy in clinical practice. J Obstet Gynaecol Can 2002; 24: 711–15. 4 Prihartono N, Palmer JR, Louik C, Shapiro S, Rosenberg L. A case-control study of use of postmenopausal female hormone supplements in relation to the risk of large bowel cancer. Cancer Epidemiol Biomarkers Prev 2000; 9: 443–47. 5 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996; 93: 5925–30. 6 Toft D, Gorski J. A receptor molecule for estrogens: isolation from the rat uterus and preliminary characterization. Proc Natl Acad Sci USA 1966; 55: 1574–81. 7 Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science 1986; 231: 1150–54. 8 Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International Union of Pharmacology, LXIV: estrogen receptors. Pharmacol Rev 2006; 58: 773–81. 9 Moore JT, McKee DD, Slentz-Kesler K, et al. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun 1998; 247: 75–78. 10 Pike AC, Brzozowski AM, Hubbard RE, et al. Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999; 18: 4608–18. 11 Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 1998; 139: 4252–63. 12 Sun J, Baudry J, Katzenellenbogen JA, Katzenellenbogen BS. Molecular basis for the subtype discrimination of the estrogen receptor-beta-selective ligand, diarylpropionitrile. Mol Endocrinol 2003; 17: 247–58. 13 Hillisch A, Peters O, Kosemund D, et al. Dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structure-based design. Mol Endocrinol 2004; 18: 1599–609. 14 Malamas MS, Manas ES, McDevitt RE, et al. Design and synthesis of aryl diphenolic azoles as potent and selective estrogen receptorbeta ligands. J Med Chem 2004; 47: 5021–40. 15 Mewshaw RE, Edsall RJ Jr, Yang C, et al. ERbeta ligands. 3. Exploiting two binding orientations of the 2-phenylnaphthalene scaffold to achieve ERbeta selectivity. J Med Chem 2005; 48: 3953–79. 16 Harris HA. Estrogen receptor-beta: recent lessons from in vivo studies. Mol Endocrinol 2007; 21: 1–13.
389
Review
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35 36
37
38
390
Dobrzycka KM, Townson SM, Jiang S, Oesterreich S. Estrogen receptor corepressors—a role in human breast cancer? Endocr Relat Cancer 2003; 10: 517–36. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001; 276: 36869–72. Huang J, Li X, Yi P, Hilf R, Bambara RA, Muyan M. Targeting estrogen responsive elements (EREs): design of potent transactivators for ERE-containing genes. Mol Cell Endocrinol 2004; 218: 65–78. Kushner PJ, Agard D, Feng WJ, et al. Oestrogen receptor function at classical and alternative response elements. Novartis Found Symp 2000; 230: 20–26. Pratt WB. The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med 1998; 217: 420–34. Harvey BJ, Doolan CM, Condliffe SB, Renard C, Alzamora R, Urbach V. Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon. Steroids 2002; 67: 483–91. Condliffe SB, Doolan CM, Harvey BJ. 17beta-oestradiol acutely regulates Cl-secretion in rat distal colonic epithelium. J Physiol 2001; 530: 47–54. Doolan CM, Harvey BJ. Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon. J Biol Chem 1996; 271: 8763–67. McNamara B, Winter DC, Cuffe J, Taylor C, O’Sullivan GC, Harvey BJ. Rapid activation of basolateral potassium transport in human colon by oestradiol. Br J Pharmacol 2000; 131: 1373–78. Winter DC, Taylor C, O’S C, Harvey BJ. Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon. Br J Surg 2000; 87: 1684–89. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 1993; 90: 11162–66. Korach KS, Emmen JM, Walker VR, et al. Update on animal models developed for analyses of estrogen receptor biological activity. J Steroid Biochem Mol Biol 2003; 86: 387–91. Weihua Z, Saji S, Makinen S, et al. Estrogen receptor (ER) beta, a modulator of ERalpha in the uterus. Proc Natl Acad Sci USA 2000; 97: 5936–41. Strom A, Hartman J, Foster JS, Kietz S, Wimalasena J, Gustafsson JA. Estrogen receptor beta inhibits 17beta-estradiolstimulated proliferation of the breast cancer cell line T47D. Proc Natl Acad Sci USA 2004; 101: 1566–71. Koehler KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA. Reflections on the discovery and significance of estrogen receptor beta. Endocr Rev 2005; 26: 465–78. Enmark E, Pelto-Huikko M, Grandien K, et al. Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 1997; 82: 4258–65. Foley EF, Jazaeri AA, Shupnik MA, Jazaeri O, Rice LW. Selective loss of estrogen receptor beta in malignant human colon. Cancer Res 2000; 60: 245–48. Qiu Y, Waters CE, Lewis AE, Langman MJ, Eggo MC. Oestrogeninduced apoptosis in colonocytes expressing oestrogen receptor beta. J Endocrinol 2002; 174: 369–77. Xie LQ, Yu JP, Luo HS. Expression of estrogen receptor beta in human colorectal cancer. World J Gastroenterol 2004; 10: 214–17. Campbell-Thompson M, Lynch IJ, Bhardwaj B. Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Res 2001; 61: 632–40. Konstantinopoulos PA, Kominea A, Vandoros G, et al. OestrogenΩreceptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour’s dedifferentiation. Eur J Cancer 2003; 39: 1251–58. Jassam N, Bell SM, Speirs V, Quirke P. Loss of expression of oestrogen receptor beta in colon cancer and its association with Dukes’ staging. Oncol Rep 2005; 14: 17–21.
39
40
41
42
43
44
45
46
47
48
49
50
51
52 53
54 55
56
57
58
59
Bardin A, Boulle N, Lazennec G, Vignon F, Pujol P. Loss of ERbeta expression as a common step in estrogen-dependent tumor progression. Endocr Relat Cancer 2004; 11: 537–51. Wong NA, Malcomson RD, Jodrell DI, Groome NP, Harrison DJ, Saunders PT. ERbeta isoform expression in colorectal carcinoma: an in vivo and in vitro study of clinicopathological and molecular correlates. J Pathol 2005; 207: 53–60. Wang M, Pan JY, Song GR, Chen HB, An LJ, Qu SX. Altered expression of estrogen receptor alpha and beta in advanced gastric adenocarcinoma: correlation with prothymosin alpha and clinicopathological parameters. Eur J Surg Oncol 2007; 33: 195–201. Sumi K, Matsuyama S, Kitajima Y, Miyazaki K. Loss of estrogen receptor beta expression at cancer front correlates with tumor progression and poor prognosis of gallbladder cancer. Oncol Rep 2004; 12: 979–84. Weihua Z, Makela S, Andersson LC, et al. A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA 2001; 98: 6330–35. Hewitt SC, Korach KS. Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues. Reproduction 2003; 125: 143–49. Wada-Hiraike O, Imamov O, Hiraike H, et al. Role of estrogen receptor beta in colonic epithelium. Proc Natl Acad Sci USA 2006; 103: 2959–64. Patrone C, Cassel TN, Pettersson K, et al. Regulation of postnatal lung development and homeostasis by estrogen receptor beta. Mol Cell Biol 2003; 23: 8542–52. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA. Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 2006; 20: 534–43. Cho NL, Javid SH, Carothers AM, Redston M, Bertagnolli MM. Estrogen receptors alpha and beta are inhibitory modifiers of Apcdependent tumorigenesis in the proximal colon of Min/+ mice. Cancer Res 2007; 67: 2366–72. Sims NA, Dupont S, Krust A, et al. Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males. Bone 2002; 30: 18–25. Martineti V, Picariello L, Tognarini I, et al. ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components. Endocr Relat Cancer 2005; 12: 455–69. Linsalata M, Russo F, Notarnicola M, et al. Effects of genistein on the polyamine metabolism and cell growth in DLD-1 human colon cancer cells. Nutr Cancer 2005; 52: 84–93. Seiler N. Polyamine metabolism. Digestion 1990; 46 (suppl 2): 319–30. Di LA, Linsalata M, Cavallini A, Messa C, Russo F. Sex steroid hormone receptors, epidermal growth factor receptor, and polyamines in human colorectal cancer. Dis Colon Rectum 1992; 35: 305–09. Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002; 3: 364–73. Hsu HH, Cheng SF, Wu CC, et al. Apoptotic effects of overexpressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner. Chin J Physiol 2006; 49: 110–16. Marino M, Galluzzo P, Leone S, Acconcia F, Ascenzi P. Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells. Endocr Relat Cancer 2006; 13: 559–69. Le Mellay V, Grosse B, Lieberherr M. Phospholipase C beta and membrane action of calcitriol and estradiol. J Biol Chem 1997; 272: 11902–07. Lieberherr M, Grosse B, Kachkache M, Balsan S. Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors. J Bone Miner Res 1993; 8: 1365–76. Winter DC, Schneider MF, O’Sullivan GC, Harvey BJ, Geibel JP. Rapid effects of aldosterone on sodium-hydrogen exchange in isolated colonic crypts. J Membr Biol 1999; 170: 17–26.
http://oncology.thelancet.com Vol 9 April 2008
Review
60
61 62
63
64
65
66
67
68
Razandi M, Pedram A, Merchenthaler I, Greene GL, Levin ER. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol 2004; 18: 2854–65. Nadal A, Diaz M, Valverde MA. The estrogen trinity: membrane, cytosolic, and nuclear effects. News Physiol Sci 2001; 16: 251–55. Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 1995; 9: 404–10. Pedram A, Razandi M, Levin ER. Nature of functional estrogen receptors at the plasma membrane. Mol Endocrinol 2006; 20: 1996–2009. Razandi M, Oh P, Pedram A, Schnitzer J, Levin ER. ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol Endocrinol 2002; 16: 100–15. Fiorelli G, Picariello L, Martineti V, Tonelli F, Brandi ML. Functional estrogen receptor beta in colon cancer cells. Biochem Biophys Res Commun 1999; 261: 521–27. Caiazza F, Galluzzo P, Lorenzetti S, Marino M. 17beta-Estradiol induces ERbeta up-regulation via p38/MAPK activation in colon cancer cells. Biochem Biophys Res Commun 2007; 359: 102–07. Galluzzo P, Caiazza F, Moreno S, Marino M. Role of ERbeta palmitoylation in the inhibition of human colon cancer cell proliferation. Endocr Relat Cancer 2007; 14: 153–67. Doolan CM, Harvey BJ. A Galphas protein-coupled membrane receptor, distinct from the classical oestrogen receptor, transduces rapid effects of oestradiol on [Ca2+]i in female rat distal colon. Mol Cell Endocrinol 2003; 199: 87–103.
http://oncology.thelancet.com Vol 9 April 2008
69
70
71
72
73
74
Doolan CM, Condliffe SB, Harvey BJ. Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon. Br J Pharmacol 2000; 129: 1375–86. Filardo EJ, Quinn JA, Bland KI, Frackelton AR Jr. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 2000; 14: 1649–60. Vivacqua A, Bonofiglio D, Albanito L, et al. 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30. Mol Pharmacol 2006; 70: 1414–23. Keshamouni VG, Mattingly RR, Reddy KB. Mechanism of 17-betaestradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem 2002; 277: 22558–65. Marino M, Galluzzo P, Leone S, Acconcia F, Ascenzi P. Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells. Endocr Relat Cancer 2006; 13: 559–69. Gilad LA, Tirosh O, Schwartz B. Phytoestrogens regulate transcription and translation of vitamin D receptor in colon cancer cells. J Endocrinol 2006; 191: 387–98.
391