Circulating insulin-like growth factor-I (IGF-I) levels have been shown to be related to risk of prostate cancer in epidemiologic studies. While specific genetic loci responsible for interindividual variation in circulating IGF-I levels in normal men have not been identified, candidate genes include those involved in the growth hormone (GH)–IGF-I axis such as the hypothalamic factors GH releasing hormone (GHRH) and somatostatin and their receptors. To investigate the role of the GH–IGF-I axis on in vivo prostate carcinogenesis and neoplastic progression, we generated mice genetically predisposed to prostate cancer (the TRAMP model) to be homozygous for lit, a mutation that inactivates the GHRH receptor (GHRH-R) and reduces circulating levels of GH and IGF-I. The lit mutation significantly reduced the percentage of the prostate gland showing neoplastic changes at 35 weeks of age (P=0.0005) and was also associated with improved survival (P<0.01). These data provide an example of a germ line mutation that reduces risk in an experimental prostate carcinogenesis model. The results suggest that prostate carcinogenesis and progression may be influenced by germ line variation of genes encoding signalling molecules in the GH–IGF-I axis.
Recent prospective studies have revealed a positive association between serum insulin-like growth factor-I (IGF-I) and prostate cancer risk (Chan et al., 1998; Wolk et al., 1998; Shaneyfelt et al., 2000; Pollak, 2001; Pollak et al., 2004; Renehan et al., 2004; Stattin et al., 2004). Both genetic and lifestyle factors influence IGF-I levels (Harrela et al., 1996). Epidemiologic data showing variation of prostate cancer risk according to IGF-I levels are biologically plausible given that normal prostate epithelial cells (Cohen et al., 1991) as well as prostate cancer cells (Culig et al., 1994; Pollak et al., 1999; Hellawell et al., 2002) are IGF-I responsive. While specific genetic loci responsible for interindividual variation in circulating IGF-I levels in normal men have not been identified, candidate genes include those involved in the growth hormone (GH)–IGF-I axis such as the hypothalamic factors GH releasing hormone (GHRH) and somatostatin and their receptors. A mutation (lit) in the murine GHRH-R gene is associated with a loss of receptor function that leads to low systemic GH and IGF-I levels and to small body size (Jansson et al., 1986; Godfrey et al., 1993; Lin et al., 1993) but does not affect androgen levels (reviewed in Chandrashekar et al., 2004).
In this study, we used the transgenic adenocarcinoma of the mouse prostate model (TRAMP) that was developed using a –426/+28 bp fragment of the rat probasin regulatory sequence to specifically target expression of SV40 early genes to the prostatic epithelium (Greenberg et al., 1995; Gingrich et al., 1999; Kaplan-Lefko et al., 2003). The changes that occur during prostate carcinogenesis in these mice highly resemble human prostate transformation (Greenberg et al., 1995; Gingrich et al., 1999; Kaplan-Lefko et al., 2003) and take place in the setting of normal serum androgen levels (Wang et al., 2004). We generated TRAMP mice (C57BL/6 background) to be homozygous for the lit mutation and compared prostate incidence and progression in TRAMP and TRAMP/lit/lit animals, over a 35-week period.
All TRAMP control mice (n=19) developed prostate cancer and 74% either died prior to the 35-week time point, or had to be killed due to advanced disease, in contrast to only 18% of TRAMP/lit/lit mice (n=17) (Figure 1a). The difference in survival is significant (P<0.01). All animals surviving to the 35-week time point were weighed and examined for gross organ abnormalities at necropsy. The gross pathology showed that the TRAMP mice displayed gross abnormality of the seminal vesicles and tissue invasion while the TRAMP/lit/lit mice appeared normal without any gross abnormality or tissue invasion of the seminal vesicles. We observed that of the five TRAMP mice that survived to that time-point, 100% showed gross evidence of prostate and seminal vesicle neoplasia, in contrast to only 14% of the surviving 14 TRAMP/lit/lit mice.
As expected, TRAMP/lit/lit mice were smaller in size than TRAMP mice. The mean body weight for TRAMP/lit/lit group was found to be 14.26±0.28 versus 30.77±1.24 g of the control group (P=0.001). The weight of the genito-urinary organs relative to body weight was reduced in TRAMP/lit/lit animals as compared to controls (mean ratio 0.04±0.01 versus 0.10±0.01 respectively, P=0.0019).
Microscopic examination of the prostate glands revealed a significant impact of the lit mutation on prostate neoplasia (Figure 1b and c). Among TRAMP controls, 22.9±2.2% of the prostate gland at 35 weeks of age was found to be histologically normal, 19.9±4.5% exhibited prostatic intraepithelial neoplasia (PIN), and 57.3±5.8% showed invasive cancer, as compared to 41.6±5.5, 31.0±3.4 and 27.4±4.8% respectively in the homozygous lit mice (Figure 2a). The difference between the percentage of the prostate gland with invasive cancer between the TRAMP control and the TRAMP/lit/lit groups is significant (P=0.0005). While the TRAMP mice homozygous for the lit mutation had far less invasive cancer at the 35-week time point than the TRAMP controls, we noted a slight increase in PIN in the former as compared to the latter group. This may suggest that the IGF-I deficiency reduced the rate of neoplastic progression from PIN to frank neoplasia.
The histopathology of the seminal vesicles also varied significantly as a function of genotype (Figures 1d, e and 2b). In the TRAMP mice, the seminal vesicles were significantly enlarged while TRAMP/lit/lit seminal vesicles were normal in size. Among TRAMP control mice, 64.6±8.8% of the seminal vesicles were scored as normal, as compared to 93.6±1.9% in the TRAMP/lit/lit group (P=0.001). The presence of invasive adenocarcinoma was rare in the seminal vesicles, and was confined to the TRAMP controls, where it involved on average 2.5±2.1% of the tissue. Phylloides-like histopathology, indicative of obstruction, was found to be 32.9±8.8% in TRAMP controls, but only 6.4±1.9% in TRAMP/lit/lit animals (P=0.0024).
As anticipated, TRAMP/lit/lit mice had lower serum IGF-I and insulin-like growth factor binding protein-3 (IGFBP-3) levels than TRAMP control mice. The mean serum IGF-I levels of TRAMP/lit/lit measured 19.41±8.64 versus 305.83±81.40 ng/ml in controls (P=0.025). Western ligand blot analysis of serum IGFBP-3 showed a very weak doublet band representing the anticipated low serum levels of IGFBP-3 in the IGF-I-deficient TRAMP mice (data not shown).
As a secondary end point, we used Ki-67 labeling to compare proliferative rate between TRAMP controls and TRAMP/lit/lit mice in both normal and neoplastic tissue at the 35-week time point. Although we had insufficient tissue for formal quantitative analysis of this end point, Ki-67 staining showed reduced tumor cell proliferation in the TRAMP/lit/lit animals compared to the TRAMP controls (Figure 3c and d). The difference in proliferation in the untransformed (at-risk) tissue between controls and TRAMP/lit/lit animals was in the same direction, but less marked (Figure 3a and b).
Our data identify lit as a germ-line mutation that confers protection against prostate carcinogenesis. Although it has been suggested that GHRH-R is present and functional in prostate cells (Chopin and Herington, 2001; Letsch et al., 2003), we were unable to detect GHRH-R transcripts in either the normal or the neoplastic prostate tissue of TRAMP or TRAMP/lit/lit mice (Figure 4). We therefore speculate that in contrast to classic oncogenes and tumor suppressor genes, which act at the cellular level, GHRH-R influences carcinogenesis indirectly, acting at the level of whole organism by influencing the levels of GH and IGF-I.
While IGF-I signalling has been found to favor survival at the cellular level (O'Connor et al., 2000; Pollak et al., 2004), activation of the insulin/IGF-I signalling pathway leads to accelerated aging in a variety of model organisms (Guarente and Kenyon, 2000; Kenyon 2001; Dillin et al., 2002; Holzenberger et al., 2002; Tissenbaum and Guarente, 2002; Arantes-Oliveira et al., 2003; Longo and Finch, 2003; Tatar et al., 2003; Pollak et al., 2004). The lit mutation provides an interesting contrast to the recently described p53+/m mutation (Tyner et al., 2002). Both are associated with substantial reduction in tumor incidence, but the former leads to enhanced longevity (Flurkey et al., 2001) while animals with the latter mutation display an accelerated aging phenotype (Tyner et al., 2002).
Higher IGF-I levels may facilitate carcinogenesis and/or early neoplastic progression by increasing the turnover rate of at-risk epithelial cells of the prostate, and/or by decreasing the probability of apoptosis of partially transformed cells. It is of interest that a rare human mutation analogous to the murine lit mutation has been described and also causes growth failure (Wajnrajch et al., 1996; Maheshwari et al., 1998). It is likely, however, that polymorphic variation of genes related to the GH–IGF-I axis has a greater impact on cancer risk and cancer aggressivity in human populations than inactivating mutations. The GH–IGF-I axis may offer novel molecular targets for prevention and treatment of prostate cancer.
Note added in Proof
Drs L Murphy and J Dodd (University of Manitoba) have recently observed that prostate cancer growth in a similar model is reduced when prostate cancer-prone mice are crossed with IGFBP-3 transgenic mice (personal communication).
insulin-like growth factor I
growth hormone releasing hormone receptor
insulin-like growth factor binding protein-3
transgenic adenocarcinoma of the mouse prostate
prostatic intraepithelial neoplasia
Aleppo G, Moskal II SF, De Grandis PA, Kineman RD and Frohman LA . (1997). Endocrinology, 138, 1058–1065.
Arantes-Oliveira N, Berman JR and Kenyon C . (2003). Science, 302, 611.
Chan JM, Stampfer MJ, Giovannucci E, Gann PH, Ma J, Wilkinson P, Hennekens CH and Pollak M . (1998). Science, 279, 563–566.
Chandrashekar V, Zaczek D and Bartke A . (2004). Biol. Reprod., 71, 17–27.
Chopin LK and Herington AC . (2001). Prostate, 49, 116–121.
Cohen P, Peehl DM, Lamson G and Rosenfeld RG . (1991). J. Clin. Endocrinol. Metab., 73, 401–407.
Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J, Hittmair A, Bartsch G and Klocker H . (1994). Cancer Res., 54, 5474–5478.
Dillin A, Crawford DK and Kenyon C . (2002). Science, 298, 830–834.
Flurkey K, Papaconstantinou J, Miller RA and Harrison DE . (2001). Proc. Natl. Acad. Sci. USA, 98, 6736–6741.
Gingrich JR, Barrios RJ, Foster BA and Greenberg NM . (1999). Prostate Cancer Prostatic Dis., 2, 70–75.
Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA and Mayo KE . (1993). Nat. Genet., 4, 227–232.
Greenberg NM, DeMayo FJ, Finegold MJ, Medina D, Tilley WD, Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ and Rosen JM . (1995). Proc. Natl. Acad. Sci. USA, 92, 3439–3443.
Guarente L and Kenyon C . (2000). Nature, 408, 255–262.
Harrela M, Koinstinen H, Kaprio J, Lehtovirta M, Tuomilehto J, Eriksson J, Toivanen L, Koskenvuo M, Leinonen P, Koistinene R and Seppala M . (1996). J. Clin. Invest., 98, 2612–2615.
Hellawell GO, Turner GD, Davies DR, Poulsom R, Brewster SF and Macaulay VM . (2002). Cancer Res., 62, 2942–2950.
Holzenberger M, Dupont J, Ducos B, Leneuve P, Geleon A, Even PC, Cervera P and Le Bouc Y . (2002). Nature, 421, 125–126.
Jansson JO, Downs TR, Beamer WG and Frohman LA . (1986). Science, 232, 511–512.
Kaplan-Lefko PJ, Chen T-M, Ittmann MM, Barrios RJ, Ayala GE, Huss WJ, Maddison LA, Foster BA and Greenberg NM . (2003). Prostate, 55, 219–237.
Kenyon C . (2001). Cell, 105, 165–168.
Letsch M, Schally AV, Busto R, Bajo AM and Varga JL . (2003). Proc. Natl. Acad. Sci. USA, 100, 1250–1255.
Lin SC, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE and Rosenfeld MG . (1993). Nature, 364, 208–213.
Longo VD and Finch CE . (2003). Science, 299, 1342–1346.
Maheshwari HG, Silverman BL, Dupuis J and Baumann G . (1998). J. Clin. Endocrinol. Metab., 83, 4065–4074.
O'Connor R, Fennelly C and Krausse D . (2000). Biochem. Soc. Trans., 28, 47–51.
Peng XD, Park S, Gadelha MR, Coschigano KT, Kopchick JJ, Frohman LA and Kineman RD . (2001). Endocrinology, 142, 1117–1123.
Pollak M, Beamer W and Zhang JC . (1999). Cancer Metastasis Rev., 17, 383–390.
Pollak M, Schernhammer ES and Hankinson SE . (2004). Nat. Rev. Cancer, 4, 505–518.
Pollak M . (2001). Epidemiol. Rev., 23, 59–66.
Renehan AG, Zwahlen M, Minder C, O'Dwyer ST, Shalet SM and Egger M . (2004). Lancet, 363, 1346–1353.
Shaneyfelt T, Husein R, Bubley GJ and Mantzoros CS . (2000). J. Clin. Oncol., 18, 847–853.
Stattin P, Rinaldi S, Biessy C, Stenman UH, Hallmans G and Kaaks R . (2004). J. Clin. Oncol., 22, 3104–3112.
Tatar M, Bartke A and Antebi A . (2003). Science, 299, 1346–1351.
Tissenbaum HA and Guarente L . (2002). Dev. Cell, 1, 9–19.
Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G, Cooper B, Brayton C, Hee Park S, Thompson T, Karsenty G, Bradley A and Donehower LA . (2002). Nature, 415, 45–53.
Wajnrajch MP, Gertner JM, Harbison MD, Chua SC and Leibel RL . (1996). Nat. Genet., 12, 88–90.
Wang J, Eltoum IE and Lamartiniere CA . (2004). Mol. Cell Endocrinol., 219, 171–180.
Wolk A, Mantzoros CS, Andersson SO, Bergstrom R, Signorello LB, Lagiou P, Adami HO and Trichopoulos D . (1998). J. Natl. Cancer Inst., 90, 911–915.
We thank Dr Michael Ittmann for assistance with pathological examination of the mouse tissues, Julie Bédard for the work on GHRH-R mRNA detection, Kathy-Ann Forner, Scott Hartigan and Danielle Couture for technical assistance with the breeding program, and Martine Bourdeau for assistance in Ki-67 immunostains. This work was funded by CPCRI-IDEA to Dr M Pollak. P Gaudreau is recipient of a scholarship chercheur-boursier national from FRSQ.
About this article
Cite this article
Majeed, N., Blouin, M., Kaplan-Lefko, P. et al. A germ line mutation that delays prostate cancer progression and prolongs survival in a murine prostate cancer model. Oncogene 24, 4736–4740 (2005). https://doi.org/10.1038/sj.onc.1208572
- prostate carcinogenesis
Molecular Biology (2016)
EphA4-deleted microenvironment regulates cancer development and leukemoid reaction of the isografted 4T1 murine breast cancer via reduction of an IGF1 signal
Cancer Medicine (2016)
BioMed Research International (2015)
The Role of the Insulin/IGF System in Cancer: Lessons Learned from Clinical Trials and the Energy Balance-Cancer Link
Frontiers in Endocrinology (2015)