Abstract
Interest in investigating the role of the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis in the initiation and progression of experimentally induced carcinomas has arisen due to several observations in the human population. First, subjects with Laron syndrome who lack GH signaling have significantly lower rates of cancer than people who have normal GH signaling. Second, epidemiologic studies have found strong associations between elevated circulating IGF-1 and the incidence of several common cancers. Third, women who bear children early in life have a dramatically reduced risk of developing breast cancer, which may be due to differences in hormone levels including GH. These observations have motivated multiple studies that have experimentally altered activity of the GH/IGF-1 axis in the context of experimental carcinoma models in mice and rats. Most of these studies have utilized carcinoma models for four organ systems that are also frequent sites of carcinomas in humans: the mammary gland, prostate gland, liver, and colon. This review focuses on these studies and describes some of the most common genetic models used to alter the activity of the GH/IGF-1 axis in experimentally induced carcinomas. A recurring theme that emerges from these studies is that manipulations that reduce the activity of GH or mediators of GH action also inhibit carcinogenesis in multiple model systems.
Introduction
Investigation of the role of growth hormone (GH) and its downstream mediators including insulin-like growth factor 1 (IGF-1) in the initiation and progression of cancer has been motivated by several factors including low cancer rates in individuals with Laron syndrome (LS) who lack GH-induced signaling and an association between elevated circulating IGF-1 and increased cancer risk (reviewed by accompanying articles in this collection). This review focuses on experiments in mice and rats where changes in the activity of GH and/or mediators of GH action have altered the initiation and/or progression of experimentally induced carcinomas. Early evidence that GH could promote carcinogenesis came from chronic injection of rats with GH, leading to neoplastic changes and some tumors in multiple organ systems (Moon et al. 1950a,b,c). Subsequent studies have investigated the effects of altering activity of the GH/IGF-1 axis in the context of chemically induced and/or genetically induced carcinoma models in several contexts. This review highlights studies of carcinoma models from four organ systems that are also frequent sites of carcinomas in humans: the mammary gland, prostate gland, liver, and colon (Siegel et al. 2021).
The GH/IGF-1 axis is often considered to consist of the upstream regulator(s) of GH secretion, GH itself and its actions on the GH receptor (GHR), the downstream effectors of GHR activation including IGF-1, and the feedback loops that modulate GH secretion. Hypothalamic neurons secrete two regulatory hormones with opposing actions into the hypothalamo–hypophyseal portal system where they travel to the anterior pituitary and regulate GH secretion. GH-releasing hormone (GHRH) from a subset of hypothalamus neurons promotes GH secretion (Guillemin et al. 1982), while somatostatin from a different subset of hypothalamus neurons inhibits GH secretion (Brazeau et al. 1973). In rats, these hormones control a sexually dimorphic pattern of GH secretion. Male rats have a highly pulsatile secretion pattern of GH with high peak levels and low interpulse GH levels, whereas female rats have a lower peak amplitude and higher interpulse GH level, making GH secretion more continuous and less varying (Eden 1979). Sex differences in GH secretion are also likely to occur in mice, but they have been technically more challenging to investigate (Xu et al. 2011). Once GH is released from the anterior pituitary gland, it circulates throughout the body and activates transmembrane GHR, particularly in the liver where GHR is abundant. Binding of GH to the extracellular domains of GHR homodimers results in the transphosphorylation of the homodimers’ cytoplasmic tails and the tyrosine phosphorylation of GHR-associated JAK2 and STAT5 (Waters et al. 2006). GH–GHR binding also activates MAPK/ERK, PI3K (phosphatidylinositol-3 kinase)/AKT (unkown origin), and focal adhesion kinases (Zhu et al. 2002, Sharp & Bartke 2005, Brooks et al. 2008, Goñi et al. 2014). These transduction mechanisms vary by cell type. For example, prostate GHR activation results in the activation of JAK2/STAT5 and MAPK, whereas mammary tissues mediate GHR activity primarily through STAT5 activation (Liu et al. 1997, Weiss-Messer et al. 2004, Chun et al. 2005, Unterberger et al. 2022a). A major outcome of these signal transduction mechanisms is the production and secretion of IGF-1 by the liver and other tissues, which circulates throughout the body and acts on tissues globally (in an endocrine manner) by its binding to and activation of the IGF-1 receptor (IGF-1R) present in most tissues (Fig. 1A). Binding to IGF-1R by IGF-1 triggers the autophosphorylation of the receptor that is followed by the activation of the MAPK/ERK and PI3K/AKT signaling pathways (Wilker et al. 2005, Goto et al. 2009). MAPK/ERK mediates the mitogenic effects of IGF-1 signaling, whereas PI3K/AKT promotes mTOR-controlled cell growth and metabolism (Goto et al. 2009). GH is also negatively regulated by IGF-1 signaling in the pituitary as a major feedback regulatory mechanism (Fig. 1A). The activity of the GH/IGF-1 axis can be genetically manipulated in rodent models to affect some aspect of the GH/IGF-1 axis. Mouse and rat genetic models used by studies discussed in this review are listed and described in Table 1.
GH works within the GH/IGF-1 signaling axis indirectly by stimulating the production of IGF-1 (A) and directly by binding to GHR on peripheral tissues (B). (A) GH (represented here by blue spheres) is released from the anterior pituitary gland and activates transmembrane GHRs (blue above), which are highly expressed in the liver (A1). The major result of this receptor activation is the production of IGF-1 (A2; green spheres above) whose autocrine and paracrine signaling is mediated through binding to IGF-1R (A3; green above). Negative regulation of GH production is initiated by IGF-1 signaling in the pituitary (A4). IGF-1 can also be generated by non-liver tissue and act locally (A5). (B) GH also acts independently of IGF-1 by activating GHR on target tissues (shown here as prostate, mammary duct, and cancer cells). Figure created at https://www.biorender.com/.
Citation: Endocrine-Related Cancer 30, 5; 10.1530/ERC-22-0403
Rodent models used to alter the activity of the GH/IGF-1 axis.
| Model | Description | References |
|---|---|---|
| GH transgenic mouse | The GH transgenic model expresses the ovine GH1 gene under control of the mouse metallothionein promoter. It can be used to investigate the effects of higher than normal circulating levels of GH. | Snibson et al. (2001) |
| Ghrh-knockout mouse | The Ghrh-knockout mouse has a germline null mutation in the gene encoding GHRH. This results in GH deficiency due to lack of GHRH action in the pituitary. It also likely has additional effects beyond GH deficiency due to the lack of GHRH signaling via the SV1 GHRH receptor variant present in many tissues. | Leone et al. (2020) |
| Laron mouse | The Laron mouse model has an engineered null mutation in the Ghr gene. Mice homozygous for this mutation (Ghr–/– or Laron mice) have no GH signaling beginning at conception. This results in a dwarf mouse. | Zhou et al. (1997); Wang et al. (2005); Zhang et al. (2007) |
| LID mouse | The liver-specific Igf1 deletion (LID) mouse model combines an albumen promoter-driven Cre recombinase with mice homozygous for an Igf1 gene surrounded by loxP sites. This results in mice with hepatocyte-specific deletion of Igf1 that is reported to result in a 70–90% reduction in circulating IGF-1 levels depending on the study. | Yakar et al. (1999); Wu et al. (2003); Anzo et al. (2008); Ealey et al. (2008); Olivo-Marston et al. (2009) |
| Liver-specific Stat5 deletion mouse | The liver-specific Stat5 deletion mouse combines mice with a floxed Stat5ab gene with transgenic Alfp-Cre transgenic mice. Mice homozygous for floxed Stat5ab that also carry the Alfp-Cre transgene lack STAT5 activity in the liver. STAT5 is a downstream mediator of GH signaling in many tissues including the liver. | Kaltenecker et al. (2019) |
| little mouse | The mouse little (lit) mutation is a recessive mutation in the third exon of the gene encoding the GHRH receptor. Receptor isoforms that use this exon are inactive in homozygous lit/lit mice including the receptor isoform expressed in the pituitary that is needed to stimulate GH secretion. The lit mutation does not affect the SV1 receptor isoform that has been implicated in GHRH actions outside the pituitary. | Godfrey et al. (1993); Donahue and Beamer (1993); Majeed et al. (2005); Bugni et al. (2001) |
| Rosa26-CRE-ERT2; Ghr flox mouse | This mouse model allows conditional inactivation of Ghr under control of tamoxifen. The Rosa26-CRE-ERT2 transgene expresses a fusion protein with CRE recombinase fused to a mutant estrogen receptor in all mouse tissues. The fusion protein is sequestered in the cytoplasm and inactive in the absence of tamoxifen. The fusion protein enters the nucleus and has an active CRE recombinase in the presence of tamoxifen. When combined with homozygous Ghr floxed mice that have loxP sites surrounding exon 4 of the Ghr gene, whole body inactivation of Ghr can be achieved with tamoxifen treatment. | Unterberger et al. (2022b) |
| Spontaneous dwarf rat (SDR) | The SDR model has a spontaneous mutation in the GH1 gene (dr mutation) that produces an altered spice site in the GH1 pre-mRNA. The mutation results in a frame-shifted mRNA that does not encode functional GH protein. Rats homozygous for this mutation (SDR or dr/dr rats) have no GH signaling beginning at conception which results in dwarfism. | Okuma & Kawashima (1980); Nogami et al. (1989); Takeuchi et al. (1990);Swanson & Unterman (2002); Shen et al. (2007); Wang et al. (2008); Carroll et al. (2009); Lantvit et al. (2021) |
The GH/IGF-1 axis and cancer
The GH/IGF-1 axis plays a role in the normal development of multiple organ systems, including the prostate and mammary gland. While IGF-1 may mediate many of these functions of GH, GHR expression throughout the male reproductive system and female mammary glands (Lobie 1990, Reiter 1992, Kleinberg 1998) suggests that GH also has direct effects in both systems (Fig. 1B), and this is likely true for many other organ systems. For example, our investigation of the effects of the GHR antagonist pegvisomant on prostate cancer cells grown in vivo or in vitro supports the concept that GH alters gene expression in prostate cancer cells in vivo both by directly activating GHR expressed by the prostate cancer cells and indirectly via GH regulation of circulating IGF-1 (Unterberger et al. 2022a). Additionally, our analysis of The Cancer Genome Atlas data identified GHR overexpression in human prostate cancers with ERG-fusion genes or ETV1-fusion genes. Ghr overexpression was then modeled in PTEN-P2 and TRAMP-C2 mouse prostate cancer cells using stable transfectants. The results indicated that Ghr overexpression increased the proliferation of prostate cancer cells in vitro and in vivo and that Ghr overexpression regulated the expression of multiple genes oppositely to Ghr loss-of-function models (Unterberger et al.2022c). The importance of local actions of GH on prostate tumor cells is also supported by genetic experiments using the transgenic adenocarcinoma mouse prostate (Gingrich et al. 1996) and SV40 C3(1) T-antigen (TAg) model (Unterberger et al.2022c) for prostate cancer. One study combined the TRAMP model with the little (lit) mouse (Godfrey et al. 1993). The blood levels of GH in lit/lit mice are reduced ~95% compared to wildtype mice, but other pituitary hormones are unaffected. They also have dramatically reduced circulating IGF-1 levels, body weight, and size (Donahue & Beamer 1993). In this setting, progression of prostate cancer was greatly reduced, including lower tumor burden and increased survival for lit/lit-TRAMP mice compared to controls with normal GH and IGF-1 (Majeed et al. 2005). In contrast, another study combined the TRAMP model with liver-specific IGF-I-deficient (LID) mice, which harbor a liver-specific deletion of Igf1 (Yakar et al. 1999). In this case, the LID–TRAMP mice had a 90% reduction in circulating IGF-1, increased circulating GH, and no significant change in cancer progression relative to controls without liver-specific Igf1 (Anzo et al. 2008). As will be described later in this review, liver-specific Igf1 deletion in LID mice does impair cancer progression in other experimental carcinoma models consistent with an important role for IGF-1 in mediating some pro-tumorigenic actions of GH. In the C3(1)/TAg model, conditional Ghr inactivation was achieved by employing a tamoxifen-inducible Cre and a prostate-specific Cre. In parallel, a transgenic GH antagonist mouse was also used. Pathology, proliferation, and gene expression of 6-month-old mouse prostates were assessed. Loss of function for Ghr globally or in prostatic epithelial cells reduced the proliferation and stratification of the prostatic epithelium in the C3(1)/TAg model (Unterberger et al. 2022c).
Mammary cancer
Our interest in the role of GH in carcinogenesis arose from asking why an early full-term pregnancy reduces a woman’s risk of developing breast cancer. McMahon and colleagues studied women from around the world and observed that those who bore their first child when they were younger than 18 had only about one-third the lifetime risk of developing breast cancer compared to those whose first child birth was delayed until the age of 35 years or older (MacMahon et al. 1970). The study was conducted in seven areas of the world, included populations that exhibited a wide range of incidence rates for breast cancer, and involved 4323 cases and 12,699 controls. Others observed that parity provided significant protection from chemically induced mammary cancer in rats (Howell 1960, Moon 1969, Moore et al. 1981). We found that GH was significantly lower in parous rats compared with age-matched virgin controls, while other mammotropic hormones were not significantly altered (Thordarson et al. 1995). This led us to hypothesize that GH may be an important factor modulating cancer susceptibility. We also observed that the spontaneous dwarf rat (SDR; dr/dr), which lacks a functional GH1 gene and does not secrete GH (Takeuchi et al. 1990), was also nearly refractory to 7,12-dimethylbenzanthracene (DMBA)-induced mammary cancer (Swanson & Unterman 2002). The SDR arose in 1977 and has since been bred apart from its founder strain, the Sprague–Dawley (Okuma & Kawashima 1980). To control for factors resulting from genetic drift, Sprague–Dawley rats were purchased from Harlan Sprague Dawley to backcross with the SDR. The progeny of these matings was genotyped using a PCR/RFLP (restriction fragment length polymorphism) method modified from Nogami et al. (1989). Rats were divided into three groups based on their genotype: homozygous wild type (DR/DR, n = 15), heterozygotes (DR/dr, n = 15), and SDR (homozygous mutant dr/dr, n = 14). At 7 weeks of age, each rat received a single treatment with DMBA (20 mg/kg body weight) by gavage. Our results, that dr/dr rats had dramatically lower tumor incidence and multiplicity, were the first direct evidence that GH plays a critical role in mammary carcinogenesis (Swanson & Unterman 2002).
We next asked if the GH-deficient SDR could be made vulnerable to mammary carcinogenesis by administering GH and whether the resulting cancers were dependent on GH for their survival (Shen et al. 2007). Beginning at 3–5 weeks of age, SDRs (Ghdr/dr) were treated with GH twice daily. Other Ghdr/dr rats received vehicle, and wildtype Sprague Dawley rats (Gh+/+) received vehicle. One week later, all rats were exposed to a single injection of N-methyl-N-nitrosourea (MNU). Body weight gain and serum IGF-1 levels were similar in Gh+/+ and GH-treated Ghdr/dr rats. Furthermore, mammary tumor incidence, latency, and multiplicity were similar in Gh+/+ and GH-treated Ghdr/dr rats. Vehicle-treated Ghdr/dr rats developed no tumors. Once advanced (>1 cm3) mammary cancers were established in GH-treated Ghdr/dr rats, GH treatments were halted, and nearly all tumors regressed completely within 2 weeks (Shen et al. 2007). Tumor regression was not associated with alterations in IGF-1, IGF-1R, or GHR. These results indicate that Ghdr/dr rats, which are nearly refractory to mammary carcinogenesis, can be made vulnerable by restoring GH and IGF-1. Furthermore, advanced rat mammary cancers are dependent on GH and/or IGF-1 for their survival, at least on some strain backgrounds (Shen et al. 2007). A follow-up study showed that some MNU-induced mammary tumors on a more outbred SDR line continued to grow after GH supplementation ended. However, these mammary tumors were much more sensitive to doxorubicin than wildtype mammary tumors in that SDR mammary tumors that had been growing in the absence of GH regressed more quickly, had reduced proliferation, and had increased apoptosis even at low doses of doxorubicin when compared to wildtype mammary tumors (Lantvit et al. 2021).
Building on our work with hormone-dependent rat mammary cancers, we designed studies in mice that develop estrogen-independent mammary cancers (Zhang et al. 2007). We crossed the LS mouse, in which the Ghr gene has been disrupted (Ghr–/–) (Zhou et al. 1997), with the C3(1)/TAg mouse, which develops estrogen receptor-negative mammary cancers (Maroulakou et al. 1994). All mice were heterozygous for the large TAg and either homozygous wild type for GHR (Ghr+/+) or null for GHR (Ghr–/–) and were sacrificed and evaluated at 30 weeks of age. Compared with the TAg/Ghr+/+ mice, the TAg/Ghr–/– mice showed delayed mammary cancer latency with significantly decreased multiplicity (9.8 ± 1.4 vs 3.2 ± 1.2) and volume (776.1 ± 284.4 vs 50.5 ± 8.9 mm3) (Zhang et al. 2007). Furthermore, the frequency of mammary hyperplasias was significantly reduced in the TAg/Ghr–/– mice (15.0 ± 1.7 vs 6.8 ± 1.7) (Zhang et al. 2007). We later evaluated the effects of inactivating Ghr in C3(1)/TAg using the CRE-lox system once mammary tumors were established. In this case, Ghr was inactivated once the largest mammary tumor reached 200 mm3, and the tumors immediately stopped growing and then regressed slightly, whereas vehicle control and CRE-activating tamoxifen control-treated tumors continued to grow (Unterberger et al. 2022b). Another study used LID mice to test the effects of liver-specific Igf1 deletion on mouse mammary tumors in the C3(1)/TAg model and a DMBA-induced mouse mammary tumor model (Wu et al. 2003). In both cases, mammary tumor formation and progression were reduced by liver-specific Igf1 deletion. These experiments in rats and mice in which GH, GHR, or IGF-1 were compromised suggest that the GH/IGF-1 axis plays a key role in mammary carcinogenesis induced chemically or by transgene and in the development of hormone-dependent and -independent mammary cancers.
Prostate cancer
The C3(1)/TAg mouse was originally developed as a model for prostate cancer with TAg expression driven by the regulatory region of the rat prostatic steroid-binding protein (C3(1)) gene (Maroulakou et al. 1994). Therefore, we were able to use the male mice in the crosses described above to study the role of GHR in TAg-driven prostate cancer in the mouse (Wang et al. 2005). The progeny of this cross was genotyped, and TAg/Ghr+/+ and TAg/Ghr–/– mice were sacrificed at 9 months of age. Seven of eight TAg/Ghr+/+ mice harbored prostatic intraepithelial neoplasia lesions of various grades. In contrast, only one of the eight TAg/Ghr–/– mice exhibited atypia (P < 0.01, Fischer’s exact test). Disruption of the GHR gene did not alter prostate androgen receptor expression nor serum testosterone titers. The expression of the TAg oncogene was similar in the prostates of the two mouse strains. Immunohistochemistry revealed a significant decrease in prostate epithelial cell proliferation and an increase in basal apoptotic indices (Wang et al. 2005). These results demonstrate that mouse prostate cancers induced by a powerful oncogene such as TAg require GHR for their progression.
We next asked whether downregulation of GH signaling could block carcinogenesis in the Probasin/TAg rat, a model of aggressive prostate cancer (Wang et al. 2008). The SDR described above, which lacks GH due to a mutation (dr) in its GH gene, was crossed with the Probasin/TAg rat, which develops prostate carcinomas at 100% incidence by 15 weeks of age. The progeny was heterozygous for the TAg oncogene and homozygous for either the wildtype GH gene (TAg/Gh+/+) or the dr mutation (TAg/Ghdr/dr). Prostate tumor incidence and burden were significantly reduced, and tumor latency was delayed in TAg/Ghdr/dr rats relative to TAg/Gh+/+ controls. At 25 weeks of age, loss of GH resulted in a 20 and 80% decrease in the area of microinvasive carcinoma in the dorsal and lateral lobes, respectively. By 52 weeks of age, invasive prostate adenocarcinomas were observed in all Gh+/+ rats, whereas the majority of TAg/Ghdr/dr rats did not develop invasive cancers. Suppression of carcinogenesis could not be attributed to alterations in prostate expression of TAg or androgen receptor or changes in serum testosterone levels. As carcinogenesis progressed in TAg/Gh+/+ rats, prostate GHR mRNA and protein expression increased significantly, but prostate IGF-1R mRNA and protein levels dropped. Furthermore, serum IGF-1 and prostate IGF-1 levels did not change significantly over the course of carcinogenesis. These findings suggest that GH plays an important role in the progression from latent to malignant prostate cancer driven by the powerful Probasin/TAg fusion gene in rats and suggest that GH antagonists may be effective at treating human prostate cancer.
Liver cancer
Early evidence that the GH/IGF-1 axis may play a role in liver carcinogenesis came from investigation of an N-2-fluorenyldiacetamide-induced liver cancer model in A × C rats (Reuber 1968). In this model, repeated dosing with GH increased the incidence of hyperplastic and neoplastic lesions in both castrated males and castrated females. Several subsequent studies have investigated the potential role of the GH/IGF-1 axis in liver tumorigenesis using both mouse and rat chemically induced models. In several of these studies, there is a sexually dimorphic response with more robust tumorigenesis in males than females (Blanck et al. 1984, Porsch Hallstrom et al. 1991, Schulien & Hasselblatt 2021) that parallels the higher incidence of liver cancer in men relative to women (Siegel et al. 2021). The sexually dimorphic, varying pulsatory patterns of GH secretion in male vs. female rodents need to be considered as manipulations are made to the GH/IGF-1 axis in these models.
In the mouse, perinatal treatment with N-nitrosodiethylamine (DEN) results in a high incidence of liver cancer as mice age to 1 year. An additional feature of this model is a higher incidence of liver tumors in males than females. One study using this model (Bugni et al. 2001) tested the role of the GH/IGF-1 axis in liver tumorigenesis using mice homozygous for the lit mutation, which results in an inactive GHRH receptor (Godfrey et al. 1993). When Bugni and colleagues crossed the lit mutation onto three mouse strains that varied in sensitivity to DEN, tumor formation was strongly suppressed in both male and female lit/lit mice compared to strain-matched wildtype controls with strong reductions in tumor incidence, multiplicity, and volume. These data demonstrate a requirement for the GH/IGF-1 in DEN-induced liver tumorigenesis. One known mediator of GH action in the liver is signal transducer and activator of transcription 5 (STAT5) (Rowland et al. 2005). Hepatocyte-specific deletion of STAT5 also dramatically reduces liver tumorigenesis in the DEN-induced model (Kaltenecker et al. 2019).
While an intact GH/IGF-1 axis is required for liver tumorigenesis in the DEN model, it is less clear whether the axis has a role in driving liver tumorigenesis rather than simply playing a permissive role. Supraphysiologic expression of ovine GH in transgenic mice accelerates tumorigenesis in the DEN model (Snibson et al. 2001). A caveat to this study is that GH levels in the transgenic mice are many-fold higher than any naturally occurring context so the relevance of this finding for naturally occurring liver cancers is uncertain. There is also a sexually dimorphic pattern of GH secretion that leads to differential activation of GH target genes in the liver that correlate with different rates of liver tumorigenesis in males and females. However, studies using estrogen receptor alpha (ERα) and prolactin receptor mutant mice (Bigsby & Caperell-Grant 2011) showed that ERα mutant females had the male pattern of GH target gene expression in the liver but not the male pattern of liver tumorigenesis in response to DEN. They concluded that the sexually dimorphic GH secretion pattern is not responsible for sex differences in liver tumorigenesis in the mouse DEN model. A caveat of this conclusion is that the authors did not directly measure GH levels in the mice, and only six GH-regulated genes in the liver were evaluated as surrogates for the GH secretion pattern. However, GH is known to regulate numerous genes through a multitude of mechanisms.
The possible role of sexually dimorphic GH secretion as a contributor to sex differences in chemically induced liver tumorigenesis has been extensively evaluated in rat models. In a model that sequentially exposes rats to DEN, 2-acetylaminofluorene (2-AAF), and partial hepatectomy (PH), liver tumorigenesis is more robust in males. Furthermore, experiments that included ectopic pituitary grafts implicated the hypothalamo–pituitary axis in regulating sex-specific susceptibility to liver tumorigenesis (Blanck et al. 1984). In this model, enzyme-altered foci in the liver serve as an early endpoint that is more extensive and rapidly growing in males than females. The foci are later followed by hepatoma formation that have a shorter latency in males than females. Subsequent studies (Blanck et al. 1987) involved treatment of rats with continuous infusion of either bovine GH (bGH) or ovine prolactin. Continuous infusion with bGH in males mimicked the pattern of GH secretion in females (Eden 1979), and males that received continuous bGH infusion became ‘feminized’ in their response to the DEN/2-AAF/PH protocol with a lower total area of enzyme-altered foci 6 weeks after tumor initiation compared with controls. Prolactin infusion had no effect on the extent of enzyme-altered foci. These data clearly implicated sex differences in GH secretion as critical for sex differences in liver tumorigenesis in response to the DEN/2-AAF/PH protocol. However, these findings may not be generalizable for other causes of liver cancer as the GH secretion pattern also regulated the sex-specific metabolism of the carcinogen 2-AAF (Blanck et al. 1987). Another study (Porsch Hallstrom et al. 1991) addressed this limitation by using a different rat liver tumorigenesis model where tumorigenesis was initiated with DEN followed by dietary deoxycholic acid (DCA) and PH. Similar to the DEN/2-AAF/PH model, the DEN/DCA/PH protocol produces enzyme-altered foci in the liver that are considered early markers of carcinogenesis and are more extensive and rapidly growing in males than females. Male enzyme-altered foci also have higher expression of the oncogene c-myc compared to females. Continuous infusion with human GH (hGH) in males mimicked the pattern of GH secretion in females (Eden 1979), and males that received continuous hGH infusion became ‘feminized’ in their response to the DEN/DCA/PH protocol with a lower total area of enzyme-altered foci 8 weeks after tumor initiation. Males treated with hGH also had lower c-myc expression in the enzyme-altered foci comparable to females. Altogether, the rat studies are consistent with the concept that the sex differences in GH secretion have a causal role in the more robust response of male rats to chemically induced liver tumorigenesis protocols.
Colon cancer
Four studies have investigated the potential role of the GH/IGF-1 axis in colon tumorigenesis using mouse or rat chemically induced models. These include investigations using LID mice (Ealey et al. 2008, Olivo-Marston et al. 2009), rats with a spontaneous mutation that inactivates the GH1 gene encoding GH (Carroll et al. 2009), and mice with an engineered inactivating mutation in the Ghrh gene that encodes GHRH (Leone et al. 2020). In the first three studies, colon tumorigenesis was reduced by mutations reducing the activity of the GH/IGF-1 axis, while the fourth study found increased colon tumorigenesis when Ghrh was inactivated. The following section of this review will describe these studies in detail and discuss the possible reasons for the different study outcomes.
Two studies (Ealey et al. 2008, Olivo-Marston et al. 2009) have used LID mice (Yakar et al. 1999) exposed to azoxymethane (AOM) to induce colon carcinogenesis. LID mice in these studies had a 70% reduction in circulating IGF-1 and increased circulating GH due to reduced feedback inhibition of GH secretion by IGF-1. Unlike mice with GH deficiency, LID mice have normal growth and development (Yakar et al. 1999). The study by Ealey and colleagues evaluated both aberrant crypt foci (ACF) and colon tumors in response to AOM carcinogenesis protocols. They observed significantly fewer ACF in female LID mice relative to controls, but no significant differences in ACF number were observed for LID vs control males. Tumor size was also significantly reduced for both female and male LID mice without differences in tumor incidence or multiplicity (Ealey et al. 2008). The study by Olivo-Marston and colleagues observed reduced colon tumor multiplicity in LID mice relative to controls particularly in the proximal colon in response to AOM treatment. Colon tumors in LID mice also had fewer proliferating cells as measured by PCNA (proliferating cell nuclear antigen) labeling index and increased apoptosis as measured by the TUNEL assay (Olivo-Marston et al. 2009). The SDR, a rat model for GH deficiency, has a mutation inactivating the GH1 gene encoding GH (Takeuchi et al. 1990) and has been used to investigate the role of the GH/IGF-1 axis in AOM-induced colon carcinogenesis (Carroll et al. 2009). The study by Carroll and colleagues found that the incidence of ACFs was dramatically lower in SDRs compared to controls at multiple time points in response to an AOM carcinogenesis protocol. Similarly, tumor incidence, number, and weight were reduced in SDRs relative to controls, and these changes were accompanied by reduced proliferation in SDR distal colons relative to controls (Carroll et al. 2009).
Another study by Leone and colleagues investigated colon carcinogenesis in male mice genetically deficient for GHRH (Leone et al. 2020). This study used a carcinogenesis protocol with AOM followed by the pro-inflammatory agent dextran sodium sulfate (DSS). In contrast to the other manipulations of the GH/IGF-1 axis, deficiency for GHRH accelerated colon tumorigenesis with larger and more invasive colon tumors in homozygous Ghrh-knockout males relative to wildtype male controls. This seemingly contradictory study might be due to several differences from other studies. First, it is the only study to use the pro-inflammatory DSS treatment, which may affect the biology of the resultant tumors. In addition, GHRH is thought to have actions outside its regulation of GH secretion and the GH/IGF-1 axis. The expression of GHRH and a splice variant of its receptor have been reported for multiple normal tissues outside the hypothalamus/pituitary including the colon (Christodoulou et al. 2006) as well as in cancers and cancer models from several organ systems including breast cancer (Kovari et al. 2017) and breast cancer cell lines (Garcia-Fernandez et al. 2003) and xenografts of colorectal, gastric, prostate, and pancreatic cancers (Busto et al. 2002, Plonowski et al. 2002). For the potential actions of GHRH outside the pituitary, the SV1 spice variant of Ghrhr is proposed to encode the receptor responsible for direct actions of GHRH. In the SV1 variant, the initial part of the extracellular protein, corresponding to the portion of the receptor encoded by the first three exons of the gene, has been replaced by a sequence encoded by the third intron (Kiaris et al. 2002). The implication of this is that Ghrh-knockout mice are not specific for effects of reduced GH secretion and GH/IGF-1 axis activity as the knockout also lacks peripheral GHRH signaling via the SV1 variant receptor. This is notably different than the mouse lit mutation affecting the GHRH receptor previously discussed in the context of prostate and liver cancer studies. The lit mutation is in exon 3 of the pituitary-expressed Ghrhr receptor transcript variant (Godfrey et al. 1993), and this exon is not part of the SV1 transcript variant making lit/lit mice more specific for the effects of reduced GH section and GH/IGF-1 axis activity.
Conclusions
The GH/IGF-1 axis has been disrupted in multiple experimentally induced carcinoma models in vivo. This review describes experiments using both chemically induced and genetically induced carcinoma models from four organ systems that are also frequent sites of carcinomas in humans: the mammary gland, prostate gland, liver, and colon (Siegel et al. 2021). A recurrent theme of these studies is that multiple methods of reducing the activity of the GH/IGF-1 axis including genetic disruption of GH production as in SDRs, genetic disruption of GHR as in LS mice, genetic disruption of the GHRH receptor as in lit/lit mice, and genetic disruption of liver IGF-1 secretion as in LID mice all impair the incidence and/or progression of chemically or genetically induced carcinomas. It should be noted that when GH is reduced, insulin resistance and hyperinsulinemia are reduced and can explain some of the reduction in tumor burden observed in the studies described in this review. In most cases, it remains unclear whether the GH/IGF-1 axis has a role in driving tumorigenesis or is simply playing a permissive role. However, rat liver carcinogenesis studies support the concept that sex differences in GH secretion have a causal role in the sex-specific responses to liver carcinogenesis protocols.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work was funded by grants from the National Cancer Institute of the National Institutes of Health (R03 AG20820; R21CA238105-01; R01CA099904; R21CA238105), Department of Defense, United States Army Medical Research, and Materiel Command (09-1-0399).
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