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Ectopic hCGβ expression by epithelial cancer: Malignant behaviour, metastasis and inhibition of tumor cell apoptosis
Molecular and Cellular Endocrinology 260–262 (2007) 264–270
Review
Ectopic hCG expression by epithelial cancer: Malignant behaviour, metastasis and inhibition of tumor cell apoptosis R.K. Iles ∗ Department of Biomedical Sciences, Institute of Social and Health Research, Middlesex University, Queensway, Enfield Middlesex EN3 4SA, UK Received 15 August 2005; accepted 19 February 2006
Abstract Ectopic expression of the -subunit of human chorionic gonadotropin (hCG) is now a recognized phenomenon in 20–40% of all common epithelial carcinoma arising from mucosal epithelia such as bladder, cervix, lung and naso-pharynx. Recent studies have shown that it acts as an autocrine growth factor by inhibiting apoptosis. Structural homology and in vitro studies suggest that it may achieve this by inhibition of the transforming growth factor  (TGF) receptor complex. Such a molecular mechanism would go some way to explaining ectopic hCG’s association with poor prognosis and tumors that will rapidly progress to metastasis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: hCG; Oncogenesis; TGF receptor; Apoptosis
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectopic hCG expression and poor prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological action of hCG on epithelial tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hCG and the cystine knot growth factor/TGF superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hCG and cross-talk with the TGF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Ectopic human chorionic gonadotropin (hCG) expression by non-gestational tumours was noted as early as 1904 (DeJewitzi reported a case in which he concluded that a gonadotropin was produced by a bladder cancer—reviewed by Iles and Chard, 1991) and was reported by Ascheim and Zondek following the introduction of their seminal bio-assay for hCG in 1927 (Ascheim and Zondek, 1927, 1928; Zondek, 1930, 1935). Up to the late 1970s, the incidence of ectopic hCG expression by common epithelial cancers could be counted in terms of the number of case reports. By the end of the 1980s the incidence had grown exponentially and is now reported in terms of the
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percentage of hCG positive tumors in any given type of epithelial cancers (reviewed by Iles and Butler, 1998). The reason for this apparently dramatic change was the introduction of the subunit hCG radio-immunoassay by Vaitukaitis et al. (1972). This immunoassay detected free -subunit hCG as well as the intact hormone. Extensive immunochemical characterisation has shown that, whilst intact hCG was abundantly produced by the placenta and germ cell tumours, the free -subunit (hCG) – independent of the common glycoprotein hormone ␣-subunit (GPH␣) – is produced by common epithelial tumours (Iles and Chard, 1989; Iles et al., 1990a,b). Intact hCG may be expressed by various common epithelial tumors in an unpredictable fashion, most often by lung cancer and hepatoblastoma (Stenman et al., 2004). In bladder cancer this is associated with carcinomatosis. However, the free -subunit (hCG) is the most abundant form of immunoreactive hCG expressed (Iles and Chard, 1991).
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Table 1 Frequency and significance of hCG expression by common carcinomas summary of large series studies, over the last ten years, where hCG expression by non-germ cell epithelial cancers were reported Tissue of origin
Serum hCG
Urine hCG/cf
IHC hCG
Prognostic
Bladder Dirnhofer et al. (1998) Mora et al. (1996) El-Ahmady et al. (1996) Pectasides et al. (1996) Iles et al. (1996) Halim et al. (1995)
– 76% (33) – 30% (76) – –
– – 73% (237) – 35% (52) 60% (63)
35% (104) – – – – –
– – – Yes Yes –
Lung Dirnhofer et al. (2000) Szturmowicz et al. (1999) Slodowska et al. (1998) Yokotani et al. (1997) Boucher and Yoneda (1995)
– 14% (85) 22% (92) – –
– – – – –
9% (90) 28% (85) – 64% (14) 93% (44)
– Yes – – –
Oral/facial Hedstrom et al. (1999) Bhalang et al. (1999) Scholl et al. (1997)
20% (59) – –
– – –
– 64% (45) 100% (1)
Yes Yes –
Breast Bieche et al. (1998) Schwarz-Roeger et al. (1997) Hoon et al. (1996)
– – –
– 19% (72) –
49% (99) 80% (32)
No – –
Cervical Crawford et al. (1998) Schwarz-Roeger et al. (1997) Grossmann et al. (1995)
– – 35% (40)
33% (46) 29% (49) –
26% (46) – –
Yes – –
Ovarian Ind et al. (1997) Grossmann et al. (1995)
36% (73) 41% (27)
– –
– –
Yes –
Endometrial Schwarz-Roeger et al. (1997) Grossmann et al. (1995)
– 30% (39)
32% (25) –
– –
– –
Vulval/vaginal De Bruijn et al. (1997) Schwarz-Roeger et al. (1997) Carter et al. (1995)
10% (118) – 38% (50)
– 17% (12) –
– – –
Yes – Yes
Colorectal Louhimo et al. (2002) Lundin et al. (2000) Kido et al. (1996) Webb et al. (1995)
16% (204) 17% (232) – –
– – – –
– 22% (232) 37% (123) 54% (377)
Yes Yes Yes Yes
Prostate Span et al. (2002) Daja et al. (2000) Sheaff et al. (1996)
mRNA study, tumor mRNA study, tumor –
And controls And controls –
Positive Positive 15% (80)
No No Yes
Pancreas Louhimo et al. (2004) Louhimo et al. (2001) Syrigos et al. (1998)
50% (160) – 42% (36)
– – –
– 56% (107) –
Yes Yes Yes
Renal Berzal-Cantalejo et al. (2004) Jiang et al. (2003) Hotakainen et al. (2002)
– mRNA study, tumor 23% (177)
– And controls –
0% (55) Positive –
No No Yes
Case reports, which are numerous, have been omitted, as they do not add information as to the frequency of ectopic hCG expression by common epithelial cancers. Thirty-seven of 40 reports investigated the frequency of elevated levels of hCG (or hCGcf) in serum or urine and/or detection in tissue sections by immunohistochemistry (IHC). Three of the studies investigated mRNA levels. Positive detection ranges from 0% in renal cell cancer to 93% in small cell (SC) lung cancer. Twenty-three of the studies specifically investigated cancer prognosis and 18 (78%) report a strong association between hCG detection and poor patient prognosis–metastatic spread. Interestingly the mRNA studies were compromised by high-level hCG mRNA detection in normal tissues. Values in parenthesis denote n values.
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Ectopic production of free hCG by bladder carcinoma is well described (reviewed by Iles and Butler, 1998). Expression of hCG has also been shown in cervical and endometrial carcinoma as well as many other non-germ cell tumours of the ovary, vulva, breast, prostate, lung, colon, oral/facial tissue and stomach (see Table 1). As hCG expression by bladder cancers has been extensively reported it serves as a good model for ectopic expression by other common epithelial cancers. 2. Ectopic hCG expression and poor prognosis The common consensus of multiple studies is that 30–40% of bladder cancer patients have elevated immunoreactive hCG in serum and urine or immunohistochemically positive tumours (see Table 1) (reviewed by Iles and Butler, 1998). Although the incidence is too low for screening purposes there is a strong correlation between free hCG expression and tumour grade, stage and patient survival (Iles and Chard, 1991; Iles, 1995; Moutzouris et al., 1993). An association between hCG expression and anaplastic and advanced disease has been noted by virtually all authors (see Table 1). Many authors have commented on the aggressive nature of the hCG positive tumours. Martin and co-workers compared the response rates to radiotherapy between hCG positive (n = 29) and hCG negative (n = 71) bladder tumors and found a statistically significant lower response rate for the hCG positive tumour group (24% versus 59%, p < 0.0005) (Martin et al., 1989). Similarly it was noted that patients with tumours that do not express hCG survived longer (Moutzouris et al., 1993). Other studies reported that serial measurement of hCG levels in bladder cancer patients predicted recurrence and relapse prior to clinical changes (Marcillac et al., 1993) and that serial hCG monitoring could predict the superficial or invasive nature of the disease (Dobrowolski et al., 1994). Significantly, a prospective study of pre-operative urinary immunoreactive hCG found that those patient with invasive transitional cell carcinoma of the bladder (T2–T4) who had elevated hCG levels were much more likely to develop metastasis than those with tumors matched for stage and grade but who were hCG negative (p < 0.01). Survival analyses also showed a strong association between hCG expression and early death (p < 0.001) (Iles et al., 1996) (see Fig. 1).
Fig. 1. Survival plot of patients with T2–T4 bladder tumours positive for hCG x-axis indicates percentage survival of patients with normal (- - -) and elevated (—) hCG as a function of months post treatment (y-axis) (Iles et al., 1996).
Gillott et al. (1996) (Fig. 2) showed that bladder cancer cell numbers increased following incubation with hCG in a dosedependent manner. No effect could be seen following treatment with intact hCG, GPH␣ or hCGcf, but the growth stimulation was abolished by co-incubation with anti-hCG anti-serum. Furthermore, addition of these antibodies to the culture media of bladder cancer cell lines inhibited the growth of the cell lines that produced endogenous free hCG. These same antibodies did not affect the growth of the bladder cell line that produced no free hCG (Gillott et al., 1996). It was also noted that the cell lines least affected by the -subunit were those that secreted the highest concentrations of the same molecule, suggesting autocrine stimulation. The hCG/hLH gene cluster has been shown not to be amplified or rearranged in bladder tumour cells, indicating that ectopic hCG expression is more likely to be the result of altered gene regulation (Iles et al., 1989). Further studies failed to show hCG stimulation of cell replication, but the clear increase in cancer cell population was brought about by inhibition of apoptosis (Butler et al., 2000).
3. Biological action of hCG on epithelial tumors Why hCG expression by bladder cancers should be associated with the tendency for the tumour to resist radiotherapy and develop metastases had been largely ignored as an epiphenomenona of no molecular consequence. Expression of fetal proteins by cancers is well recognised and an established understanding exists that cancer is a form of cellular regression; a legacy of the Luterian/Trophoblast theory of cancer (Krebs and Krebs, 1950; Acevedo, 2002). Thus, hCG and by association hCG, expression has largely been regarded as a marker of pluripotent stem/germ cells, but of no functional significance in oncogenesis and tumour spread.
Fig. 2. hCG growth effect on four bladder cancer cell lines. Bladder cancer cell numbers were measured by the MTT reduction assay after exposure to hCG and its related fragments and normalised to untreated control cells. Cell line T24, which does not express hCG, shows the greatest growth response. The growth effect is reduced in the lines where endogenous hCG is produced, in particular SCaBER and RT112 (Gillott et al., 1996).
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Fig. 3. Opposing effects of hCG and TGF on bladder cancer cell line growth. (A) The percentage change in cell number from the control (marked as 100% and dashed line) as a result of incubation with hCG (top) and TGF1 (bottom) in five bladder cancer cell lines. (B) The percentage change in nucleosome concentration from control as a result of incubation with hCG (bottom) and TGF1 (Butler et al., 2000).
Furthermore, given that the LH/hCG receptor is not expressed by those tissues, and that only intact hCG can stimulate the LH/hCG receptor, any activity observed must be mediated by an as yet unidentified pathway (Davies, 2001). 4. hCG and the cystine knot growth factor/TGF superfamily Lapthorn et al. (1994) determined the three-dimensional structure of hCG. The most striking feature was the arrangement of three disulphide bridges in the centre of each subunit. The positions of the three cystines are almost identical in both subunits, where two disulphides bridge the anti-parallel strands of the peptide chain forming a central loop, through which the third disulphide passes. This structure has been identified before in a group of growth factors which are designated by its name; the cystine knot. The cystine knot growth factor (CKGF) family includes transforming growth factor  (TGF), platelet derived growth factor B (PDGFB) and nerve growth factor (NGF), and despite sharing structure similarities they carry out quite distinct functions. The TGFs are a ubiquitous family of proteins that includes the inhibins and the activins, TGF 1 and 2 have been extensively described, and are multifunctional growth factors with both stimulatory and inhibitory activities. These opposing actions are largely dependent on the embryonic origin of the target tissue. PDGF B is an autocrine and paracrine mitogenic stimulator of mesenchymal and glial cells, whilst NGF is a potent apoptotic inhibitor of both central and peripheral nervous system neuronal cells (reviewed by Sun and Davies, 1995). TGF, NGF and PDGFB are biologically active as dimers, bringing about intracellular signalling by binding multiple receptor components and bringing their intracellular domains into close association. 5. hCG and cross-talk with the TGF receptor The absence of a receptor for free hCG, but structural homology with the cystine knot growth factors, suggests that
free hCG cross-interaction with other cystine knot growth factor receptors may occur. Given that the growth modulation action of hCG on epithelial cancer occurs via inhibition of apoptosis, whilst the induction of epithelial cell apoptosis is a well-established action of TGF and that we have previously highlighted the topological homology of hCG with TGF (Gillott et al., 1996; Butler et al., 2000); this anti-apoptotic effect of hCG could potentially occur by blockade of the TGF receptor.
Fig. 4. Inhibition of the apoptotic signalling of TGF by hCG in vitro. The percentage change in nuclecsome concentration from the control following coincubation of the bladder carcinoma cell line 5637 with TGF1 (to initiate apoptosis) and increasing concentrations of hCG (to negate the TGF1 effect whilst the apoptotic factor was still present) 00 contained no hCG or TGF1 and represents the control to which the other values were corrected and further indicated by the dashed line (Butler et al., 2000).
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TGF is coded for by a locus adjacent to that of the hCG–hLH gene cluster on chromosome 19 (Fujii, 1986), and high levels of TGF and its receptors are expressed by urothelial tumors, despite their growth inhibitory effects (Coombs et al., 1993; Eder et al., 1996, 1997). This has long been a puzzle to cancer researchers and disruption of the TGF apoptosis signalling pathway has long been considered an important feature of oncogenisis, i.e. escape from a fundamental mechanism of regulating normal tissue epithelial cell population (Barcellos-Hoff and Ewan, 2000). We have previously shown that exogenous addition of hCG to bladder cancer cell cultures reverses the apoptotic inducing effects of TGF (Butler et al., 2000; see Figs. 3 and 4). Thus, ectopic expression of free hCG may be acting as an autocrine/paracrine growth-stimulating factor by virtue of its ability to block TGF’s action. Its structural homology to TGF (and its monomeric rather than dimeric nature) suggests that free hCG may bind to a component of the TGF receptor complex (for example, receptor chain II), block its ligand binding site and thereby prevent any further interactions with other receptor components that are necessary for initiating cytoplasmic signalling that leads to apoptosis (see Fig. 5). This hypothesis, if proven, would go some way to explaining
Fig. 5. A theoretical model depicting hCG blockade of the TGF receptor. Cartoon representing: (A) the consequences of TGF-dimer binding to the TGF receptor component chains (only RI and RII are indicated), intracellular domain interaction and subsequent 2◦ signalling that results in epithelial cell apoptosis. The presence of free hCG (monomer) is proposed; (B) to blockade the binding site of one of the TGF receptor chains, preventing TGF induced receptor association, intracellular signalling and apoptosis. Thus, the cell survives; population increases and these cells can potentially undergo further replication.
the aggressive nature of tumours that produce hCG and why they have such poor prognoses.
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Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.K., Issacs, N.W., 1994. Crystal structure of human chorionic gonadotrophin. Nature 369, 455–461. Louhimo, J., Nordling, S., Alfthan, H., Von Boguslawski, K., Stenman, U.H., Haglund, C., 2001. Specific staining of human chorionic gonadotropin beta in benign and malignant gastrointestinal tissues with monoclonal antibodies. Histopathology 38, 418–424. Louhimo, J., Carpelan-Holmstrom, M., Alfthan, H., Stenman, U.H., Jarvinen, H.J., Haglund, C., 2002. Serum HCG beta, CA 72-4 and CEA are independent prognostic factors in colorectal cancer. Int. J. Cancer 101, 545– 548. Louhimo, J., Alfthan, H., Stenman, U.H., Haglund, C., 2004. Serum HCG beta and CA 72-4 are stronger prognostic factors than CEA, CA 19-9 and CA 242 in pancreatic cancer. Oncology 66, 126–131. Lundin, M., Nording, S., Carpelan-Holmstrom, M., Louhimo, J., Alfthan, H., Stenman, U.H., Haglund, C., 2000. A comparison of serum and tissue hCG beta as prognostic markers in colorectal cancer. Anticancer Res. 20, 4949–4951. Marcillac, I., Cottu, P., Theodore, C., Terrier-Lacombe, M.J., Bellet, D., Droz, J.P., 1993. Free hCG- subunit as tumor marker in urothelial cancer. Lancet 341, 1354–1355. Martin, J.E., Jenkins, B.J., Zuk, R.J., Oliver, R.T., Baithun, S.I., 1989. Human chorionic gonadotrophin expression and histological findings as predictors of response to radiotherapy in carcinoma of the bladder. Virch. Arch. Path. Anat. 414, 273– 277. Mora, J., Gascon, N., Tabernero, J.M., Rodriguez-Espinosa, J., Gonzalez-Sastre, F., 1996. Different hCG assays to measure ectopic hCG secretion in bladder carcinoma patients. Br. J. Cancer 74, 1081–1084. Moutzouris, G., Yannapoulos, D., Barbatis, C., Zaharof, A., Theodorou, C., 1993. Is  human chorionic gonadotrophin production by transitional cell carcinoma of the bladder a marker of aggressive disease and resistance to radiotherapy? Br. J. Urol. 72, 907–909. Pectasides, D., Bafaloucos, D., Antoniou, F., Gogou, L., Economides, N., Varthalitis, J., Dimitriades, M., Kosmidis, P., Athanassiou, A., 1996. TPA, TATI, CEA, AFP, -hCG, PSA, SCC and CA 19-9 for monitoring transitional cell carcinoma of the bladder. Am. J. Clin. Oncol. 19, 271–277. Scholl, P.D., Jurco, S., Austin, J.R., 1997. Ectopic production of beta-HCG by a maxillary squamous cell carcinoma. Head Neck 19, 701–705. Schwarz-Roeger, U., Petzoldt, B., Waldschmidt, R., Walker, R.P., Bauknecht, T., Kiechle, M., 1997. UGP- a tumor marker of gynecologic and breast malignancies? Specificity and sensitivity in pretherapeutic patients and the influence of hormonal substitution on the expression of UGP. Anticancer Res. 17, 3041–3045. Sheaff, M.T., Martin, J.E., Badenoch, D.F., Baithun, S.I., 1996. hCG as a prognostic marker in adenocarcinoma of the prostate. J. Clin. Pathol. 49, 329–332. Slodowska, J., Szturmowicz, M., Rudzinski, P., Giedronowicz, D., Sakowitcz, A., Androsiuk, W., Zakrzewska-Rowinska, E., 1998. Expression of CEA and trophoblastic cell markers by lung carcinoma in association with histological characteristics and serum marker levels. Eur. J. Cancer Prev. 7, 51–60. Span, P.N., Thomas, C.M., Heuvel, J.J., Bosch, R.R., Schalken, J.A., Vd Locht, L., Mensink, E.J., Sweep, C.G., 2002. Analysis of expression of chorionic gonadotrophin transcripts in prostate cancer by quantitative Taqman and a modified molecular beacon RT-PCR. J. Endocrinol. 172, 489–495. Stenman, U.H., Alfthan, H., Hotakainen, K., 2004. Human chorionic gonadotropin in cancer. Clin. Biochem. 37, 549–561. Sun, P.D., Davies, D.R., 1995. The cystine-knot growth-factor superfamily. Ann. Rev. Biophys. Biomol. Struct. 24, 269–291. Syrigos, K.N., Fyssas, I., Konstandoulakis, M.M., Harrington, K.J., Papadopoulos, S., Milingos, N., Peveretos, P., Golematis, B.C., 1998. Beta human chorionic gonadotropin concentrations in serum of patients with pancreatic adenocarcinoma. Gut 42, 88–91. Szturmowicz, M., Wiatr, E., Sakowicz, A., Slodkowska, K., Filipecki, S., Rowinska-Zakrzewska, E.R., 1999. The role of human chorionic gonadotropin beta subunit elevation in small-cell lung cancer patients. Tumor Biol. 20, 99–104.
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Review
Ectopic hCG expression by epithelial cancer: Malignant behaviour, metastasis and inhibition of tumor cell apoptosis R.K. Iles ∗ Department of Biomedical Sciences, Institute of Social and Health Research, Middlesex University, Queensway, Enfield Middlesex EN3 4SA, UK Received 15 August 2005; accepted 19 February 2006
Abstract Ectopic expression of the -subunit of human chorionic gonadotropin (hCG) is now a recognized phenomenon in 20–40% of all common epithelial carcinoma arising from mucosal epithelia such as bladder, cervix, lung and naso-pharynx. Recent studies have shown that it acts as an autocrine growth factor by inhibiting apoptosis. Structural homology and in vitro studies suggest that it may achieve this by inhibition of the transforming growth factor  (TGF) receptor complex. Such a molecular mechanism would go some way to explaining ectopic hCG’s association with poor prognosis and tumors that will rapidly progress to metastasis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: hCG; Oncogenesis; TGF receptor; Apoptosis
Contents 1. 2. 3. 4. 5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ectopic hCG expression and poor prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological action of hCG on epithelial tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hCG and the cystine knot growth factor/TGF superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hCG and cross-talk with the TGF receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Ectopic human chorionic gonadotropin (hCG) expression by non-gestational tumours was noted as early as 1904 (DeJewitzi reported a case in which he concluded that a gonadotropin was produced by a bladder cancer—reviewed by Iles and Chard, 1991) and was reported by Ascheim and Zondek following the introduction of their seminal bio-assay for hCG in 1927 (Ascheim and Zondek, 1927, 1928; Zondek, 1930, 1935). Up to the late 1970s, the incidence of ectopic hCG expression by common epithelial cancers could be counted in terms of the number of case reports. By the end of the 1980s the incidence had grown exponentially and is now reported in terms of the
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percentage of hCG positive tumors in any given type of epithelial cancers (reviewed by Iles and Butler, 1998). The reason for this apparently dramatic change was the introduction of the subunit hCG radio-immunoassay by Vaitukaitis et al. (1972). This immunoassay detected free -subunit hCG as well as the intact hormone. Extensive immunochemical characterisation has shown that, whilst intact hCG was abundantly produced by the placenta and germ cell tumours, the free -subunit (hCG) – independent of the common glycoprotein hormone ␣-subunit (GPH␣) – is produced by common epithelial tumours (Iles and Chard, 1989; Iles et al., 1990a,b). Intact hCG may be expressed by various common epithelial tumors in an unpredictable fashion, most often by lung cancer and hepatoblastoma (Stenman et al., 2004). In bladder cancer this is associated with carcinomatosis. However, the free -subunit (hCG) is the most abundant form of immunoreactive hCG expressed (Iles and Chard, 1991).
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Table 1 Frequency and significance of hCG expression by common carcinomas summary of large series studies, over the last ten years, where hCG expression by non-germ cell epithelial cancers were reported Tissue of origin
Serum hCG
Urine hCG/cf
IHC hCG
Prognostic
Bladder Dirnhofer et al. (1998) Mora et al. (1996) El-Ahmady et al. (1996) Pectasides et al. (1996) Iles et al. (1996) Halim et al. (1995)
– 76% (33) – 30% (76) – –
– – 73% (237) – 35% (52) 60% (63)
35% (104) – – – – –
– – – Yes Yes –
Lung Dirnhofer et al. (2000) Szturmowicz et al. (1999) Slodowska et al. (1998) Yokotani et al. (1997) Boucher and Yoneda (1995)
– 14% (85) 22% (92) – –
– – – – –
9% (90) 28% (85) – 64% (14) 93% (44)
– Yes – – –
Oral/facial Hedstrom et al. (1999) Bhalang et al. (1999) Scholl et al. (1997)
20% (59) – –
– – –
– 64% (45) 100% (1)
Yes Yes –
Breast Bieche et al. (1998) Schwarz-Roeger et al. (1997) Hoon et al. (1996)
– – –
– 19% (72) –
49% (99) 80% (32)
No – –
Cervical Crawford et al. (1998) Schwarz-Roeger et al. (1997) Grossmann et al. (1995)
– – 35% (40)
33% (46) 29% (49) –
26% (46) – –
Yes – –
Ovarian Ind et al. (1997) Grossmann et al. (1995)
36% (73) 41% (27)
– –
– –
Yes –
Endometrial Schwarz-Roeger et al. (1997) Grossmann et al. (1995)
– 30% (39)
32% (25) –
– –
– –
Vulval/vaginal De Bruijn et al. (1997) Schwarz-Roeger et al. (1997) Carter et al. (1995)
10% (118) – 38% (50)
– 17% (12) –
– – –
Yes – Yes
Colorectal Louhimo et al. (2002) Lundin et al. (2000) Kido et al. (1996) Webb et al. (1995)
16% (204) 17% (232) – –
– – – –
– 22% (232) 37% (123) 54% (377)
Yes Yes Yes Yes
Prostate Span et al. (2002) Daja et al. (2000) Sheaff et al. (1996)
mRNA study, tumor mRNA study, tumor –
And controls And controls –
Positive Positive 15% (80)
No No Yes
Pancreas Louhimo et al. (2004) Louhimo et al. (2001) Syrigos et al. (1998)
50% (160) – 42% (36)
– – –
– 56% (107) –
Yes Yes Yes
Renal Berzal-Cantalejo et al. (2004) Jiang et al. (2003) Hotakainen et al. (2002)
– mRNA study, tumor 23% (177)
– And controls –
0% (55) Positive –
No No Yes
Case reports, which are numerous, have been omitted, as they do not add information as to the frequency of ectopic hCG expression by common epithelial cancers. Thirty-seven of 40 reports investigated the frequency of elevated levels of hCG (or hCGcf) in serum or urine and/or detection in tissue sections by immunohistochemistry (IHC). Three of the studies investigated mRNA levels. Positive detection ranges from 0% in renal cell cancer to 93% in small cell (SC) lung cancer. Twenty-three of the studies specifically investigated cancer prognosis and 18 (78%) report a strong association between hCG detection and poor patient prognosis–metastatic spread. Interestingly the mRNA studies were compromised by high-level hCG mRNA detection in normal tissues. Values in parenthesis denote n values.
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Ectopic production of free hCG by bladder carcinoma is well described (reviewed by Iles and Butler, 1998). Expression of hCG has also been shown in cervical and endometrial carcinoma as well as many other non-germ cell tumours of the ovary, vulva, breast, prostate, lung, colon, oral/facial tissue and stomach (see Table 1). As hCG expression by bladder cancers has been extensively reported it serves as a good model for ectopic expression by other common epithelial cancers. 2. Ectopic hCG expression and poor prognosis The common consensus of multiple studies is that 30–40% of bladder cancer patients have elevated immunoreactive hCG in serum and urine or immunohistochemically positive tumours (see Table 1) (reviewed by Iles and Butler, 1998). Although the incidence is too low for screening purposes there is a strong correlation between free hCG expression and tumour grade, stage and patient survival (Iles and Chard, 1991; Iles, 1995; Moutzouris et al., 1993). An association between hCG expression and anaplastic and advanced disease has been noted by virtually all authors (see Table 1). Many authors have commented on the aggressive nature of the hCG positive tumours. Martin and co-workers compared the response rates to radiotherapy between hCG positive (n = 29) and hCG negative (n = 71) bladder tumors and found a statistically significant lower response rate for the hCG positive tumour group (24% versus 59%, p < 0.0005) (Martin et al., 1989). Similarly it was noted that patients with tumours that do not express hCG survived longer (Moutzouris et al., 1993). Other studies reported that serial measurement of hCG levels in bladder cancer patients predicted recurrence and relapse prior to clinical changes (Marcillac et al., 1993) and that serial hCG monitoring could predict the superficial or invasive nature of the disease (Dobrowolski et al., 1994). Significantly, a prospective study of pre-operative urinary immunoreactive hCG found that those patient with invasive transitional cell carcinoma of the bladder (T2–T4) who had elevated hCG levels were much more likely to develop metastasis than those with tumors matched for stage and grade but who were hCG negative (p < 0.01). Survival analyses also showed a strong association between hCG expression and early death (p < 0.001) (Iles et al., 1996) (see Fig. 1).
Fig. 1. Survival plot of patients with T2–T4 bladder tumours positive for hCG x-axis indicates percentage survival of patients with normal (- - -) and elevated (—) hCG as a function of months post treatment (y-axis) (Iles et al., 1996).
Gillott et al. (1996) (Fig. 2) showed that bladder cancer cell numbers increased following incubation with hCG in a dosedependent manner. No effect could be seen following treatment with intact hCG, GPH␣ or hCGcf, but the growth stimulation was abolished by co-incubation with anti-hCG anti-serum. Furthermore, addition of these antibodies to the culture media of bladder cancer cell lines inhibited the growth of the cell lines that produced endogenous free hCG. These same antibodies did not affect the growth of the bladder cell line that produced no free hCG (Gillott et al., 1996). It was also noted that the cell lines least affected by the -subunit were those that secreted the highest concentrations of the same molecule, suggesting autocrine stimulation. The hCG/hLH gene cluster has been shown not to be amplified or rearranged in bladder tumour cells, indicating that ectopic hCG expression is more likely to be the result of altered gene regulation (Iles et al., 1989). Further studies failed to show hCG stimulation of cell replication, but the clear increase in cancer cell population was brought about by inhibition of apoptosis (Butler et al., 2000).
3. Biological action of hCG on epithelial tumors Why hCG expression by bladder cancers should be associated with the tendency for the tumour to resist radiotherapy and develop metastases had been largely ignored as an epiphenomenona of no molecular consequence. Expression of fetal proteins by cancers is well recognised and an established understanding exists that cancer is a form of cellular regression; a legacy of the Luterian/Trophoblast theory of cancer (Krebs and Krebs, 1950; Acevedo, 2002). Thus, hCG and by association hCG, expression has largely been regarded as a marker of pluripotent stem/germ cells, but of no functional significance in oncogenesis and tumour spread.
Fig. 2. hCG growth effect on four bladder cancer cell lines. Bladder cancer cell numbers were measured by the MTT reduction assay after exposure to hCG and its related fragments and normalised to untreated control cells. Cell line T24, which does not express hCG, shows the greatest growth response. The growth effect is reduced in the lines where endogenous hCG is produced, in particular SCaBER and RT112 (Gillott et al., 1996).
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Fig. 3. Opposing effects of hCG and TGF on bladder cancer cell line growth. (A) The percentage change in cell number from the control (marked as 100% and dashed line) as a result of incubation with hCG (top) and TGF1 (bottom) in five bladder cancer cell lines. (B) The percentage change in nucleosome concentration from control as a result of incubation with hCG (bottom) and TGF1 (Butler et al., 2000).
Furthermore, given that the LH/hCG receptor is not expressed by those tissues, and that only intact hCG can stimulate the LH/hCG receptor, any activity observed must be mediated by an as yet unidentified pathway (Davies, 2001). 4. hCG and the cystine knot growth factor/TGF superfamily Lapthorn et al. (1994) determined the three-dimensional structure of hCG. The most striking feature was the arrangement of three disulphide bridges in the centre of each subunit. The positions of the three cystines are almost identical in both subunits, where two disulphides bridge the anti-parallel strands of the peptide chain forming a central loop, through which the third disulphide passes. This structure has been identified before in a group of growth factors which are designated by its name; the cystine knot. The cystine knot growth factor (CKGF) family includes transforming growth factor  (TGF), platelet derived growth factor B (PDGFB) and nerve growth factor (NGF), and despite sharing structure similarities they carry out quite distinct functions. The TGFs are a ubiquitous family of proteins that includes the inhibins and the activins, TGF 1 and 2 have been extensively described, and are multifunctional growth factors with both stimulatory and inhibitory activities. These opposing actions are largely dependent on the embryonic origin of the target tissue. PDGF B is an autocrine and paracrine mitogenic stimulator of mesenchymal and glial cells, whilst NGF is a potent apoptotic inhibitor of both central and peripheral nervous system neuronal cells (reviewed by Sun and Davies, 1995). TGF, NGF and PDGFB are biologically active as dimers, bringing about intracellular signalling by binding multiple receptor components and bringing their intracellular domains into close association. 5. hCG and cross-talk with the TGF receptor The absence of a receptor for free hCG, but structural homology with the cystine knot growth factors, suggests that
free hCG cross-interaction with other cystine knot growth factor receptors may occur. Given that the growth modulation action of hCG on epithelial cancer occurs via inhibition of apoptosis, whilst the induction of epithelial cell apoptosis is a well-established action of TGF and that we have previously highlighted the topological homology of hCG with TGF (Gillott et al., 1996; Butler et al., 2000); this anti-apoptotic effect of hCG could potentially occur by blockade of the TGF receptor.
Fig. 4. Inhibition of the apoptotic signalling of TGF by hCG in vitro. The percentage change in nuclecsome concentration from the control following coincubation of the bladder carcinoma cell line 5637 with TGF1 (to initiate apoptosis) and increasing concentrations of hCG (to negate the TGF1 effect whilst the apoptotic factor was still present) 00 contained no hCG or TGF1 and represents the control to which the other values were corrected and further indicated by the dashed line (Butler et al., 2000).
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TGF is coded for by a locus adjacent to that of the hCG–hLH gene cluster on chromosome 19 (Fujii, 1986), and high levels of TGF and its receptors are expressed by urothelial tumors, despite their growth inhibitory effects (Coombs et al., 1993; Eder et al., 1996, 1997). This has long been a puzzle to cancer researchers and disruption of the TGF apoptosis signalling pathway has long been considered an important feature of oncogenisis, i.e. escape from a fundamental mechanism of regulating normal tissue epithelial cell population (Barcellos-Hoff and Ewan, 2000). We have previously shown that exogenous addition of hCG to bladder cancer cell cultures reverses the apoptotic inducing effects of TGF (Butler et al., 2000; see Figs. 3 and 4). Thus, ectopic expression of free hCG may be acting as an autocrine/paracrine growth-stimulating factor by virtue of its ability to block TGF’s action. Its structural homology to TGF (and its monomeric rather than dimeric nature) suggests that free hCG may bind to a component of the TGF receptor complex (for example, receptor chain II), block its ligand binding site and thereby prevent any further interactions with other receptor components that are necessary for initiating cytoplasmic signalling that leads to apoptosis (see Fig. 5). This hypothesis, if proven, would go some way to explaining
Fig. 5. A theoretical model depicting hCG blockade of the TGF receptor. Cartoon representing: (A) the consequences of TGF-dimer binding to the TGF receptor component chains (only RI and RII are indicated), intracellular domain interaction and subsequent 2◦ signalling that results in epithelial cell apoptosis. The presence of free hCG (monomer) is proposed; (B) to blockade the binding site of one of the TGF receptor chains, preventing TGF induced receptor association, intracellular signalling and apoptosis. Thus, the cell survives; population increases and these cells can potentially undergo further replication.
the aggressive nature of tumours that produce hCG and why they have such poor prognoses.
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