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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Prostate. 2022 Dec 22;83(5):416–429. doi: 10.1002/pros.24474

Conditional gene regulation models demonstrate a pro-proliferative role for growth hormone receptor in prostate cancer

Christopher J Unterberger 1, Sean J Mcilwain 2, Philippos K Tsourkas 2, Vilena I Maklakova 1, Jordyn L Prince 1, Abigail Onesti 1, Rong Hu 3, John J Kopchick 4, Steven M Swanson 1, Paul C Marker 1
PMCID: PMC9974633  NIHMSID: NIHMS1858851  PMID: 36562110

Abstract

Background:

Humans with inactivating mutations in growth hormone receptor (GHR) have lower rates of cancer, including prostate cancer. Similarly, mice with inactivating Ghr mutations are protected from prostatic intraepithelial neoplasia in the C3(1)/TAg prostate cancer model. However, gaps in clinical relevance in those models persist. The current study addresses these gaps and the ongoing role of Ghr in prostate cancer using loss-of-function and gain-of-function models.

Methods:

Conditional Ghr inactivation was achieved in the C3(1)/TAg model by employing a tamoxifen-inducible Cre and a prostate-specific Cre. In parallel, a transgenic GH antagonist was also used. Pathology, proliferation, and gene expression of 6-month old mouse prostates were assessed. Analysis of The Cancer Genome Atlas data was conducted to identify GHR overexpression in a subset of human prostate cancers. Ghr overexpression was modeled in PTEN-P2 and TRAMP-C2 mouse prostate cancer cells using stable transfectants. The growth, proliferation, and gene expression effects of Ghr overexpression was assessed in vitro and in vivo.

Results:

Loss-of-function for Ghr globally or in prostatic epithelial cells reduced proliferation and stratification of the prostatic epithelium in the C3(1)/TAg model. Genes and gene sets involved in the immune system and tumorigenesis, for example, were dysregulated upon global Ghr disruption. Analysis of The Cancer Genome Atlas revealed higher GHR expression in human prostate cancers with ERG-fusion genes or ETV1-fusion genes. Modeling the GHR overexpression observed in these human prostate cancers by overexpressing Ghr in mouse prostate cancer cells with mutant Pten or T-antigen driver genes increased proliferation of prostate cancer cells in vitro and in vivo. Ghr overexpression regulated the expression of multiple genes oppositely to Ghr loss-of-function models.

Conclusions:

Loss-of-function and gain-of-function Ghr models, including prostatic epithelial cell specific alterations in Ghr, altered proliferation and gene expression. These data suggest that changes in GHR activity in human prostatic epithelial cells play a role in proliferation and gene regulation in prostate cancer, suggesting the potential for disrupting GH signaling, for example by the FDA approved GH antagonist pegvisomant, may be beneficial in treating prostate cancer.

Keywords: GHR, GH, IGF-1, RNA-seq, T-antigen, Pten

INTRODUCTION

Prostate cancer is the most common non-cutaneous cancer affecting men in the United States (1). Common chemotherapeutic agents used to treat a variety of cancers, including prostate cancer, exert a majority of their effects by inducing apoptosis. Insulin-like growth factor 1 (IGF-1) possesses anti-apoptotic properties that can contribute to resistance to chemotherapy (2). However, promising results seen in preclinical lab testing have not shown the same benefit in humans, as blocking IGF-1 signaling alongside other treatments has been shown to have no effect on cancer progression in clinical trials, possibly due to feedback loop mechanisms involving GH that would be hyperactivated upon IGF-1 antagonism (3–6).

Human Laron syndrome is caused by mutations in the growth hormone receptor (GHR) gene that disrupt the GH/IGF-1 axis. Humans and mouse models with GHR mutations possess protections against diseases including cancer—including breast and prostate cancer—and diabetes (7–9). Research focusing on this protection has studied the role of GH and IGF-1 in cancer progression. Higher serum IGF-1 levels correlate with poor prognoses, and although much of the available evidence suggests GH effects in prostate cancer are largely mediated through IGF-1, some effects may be through direct GHR activation in the prostate (10,11). GHR expression has been shown to be differentially regulated in the human prostate cancer cell lines PC3, DU145, and LNCaP depending on their androgen sensitivity (12). The SV40 C3(1) T-antigen (C3(1)/TAg) mouse model of prostate cancer fails to develop PIN lesions when the GH/IGF-1 axis is heritably disrupted, mimicking the cancer protection seen in human Laron patients (9). However, this study fails in modelling the more clinically relevant situation of existing PIN and identifying the therapeutic potential of GH disruption after lesions have formed.

A tamoxifen-inducible Cre was created via the chimeric fusion of Cre to estrogen receptor T2 (Cre-ERT2). Cre-ERT2 is normally sequestered in the cytoplasm, however, tamoxifen binding induces a conformational change in the ER (13–15). This binding releases Cre to translocate to the nucleus and freely recombine loxP sites throughout the genome (16,17). When linked to the constitutively active ROSA26 promoter (ROSA βgeo gene trap), Cre-ERT2 becomes a globally inducible, tamoxifen-dependent recombination tool (18,19). The use of tamoxifen inducible Cre to specifically delete functional GHR from mice has been standardized and described elsewhere (20–22). Cre expressed under control of the rat probasin promoter directs expression of Cre to the prostate epithelium in the PbCre4 model (23). The bGH-A122D (GHA) mouse expresses a transgene that creates a competitive antagonist to GH. These mice have lower circulating serum IGF-1 which results in stunted growth early in life (24,25). These three genetic models of GH signaling disruption provided us with the tools to further investigate the role of GH signaling in the C3(1)/TAg transgenic prostate cancer model previously described as developing low-grade PIN at two months, high-grade PIN at five months, and invasive carcinoma at seven months (26).

In this study, we investigated the effects of conditionally deleting Ghr in the C3(1)/TAg mouse model of prostate cancer globally and specifically in the prostate epithelium in the C3(1)/TAg model. In both cases, the extent of epithelial stratification and proliferation were reduced suggesting that direct actions of GHR in the prostatic epithelium contribute to these cancer-related phenotypes. Examination of The Cancer Genome Atlas (TCGA) project data (27) also showed increased GHR expression in prostate cancers with ERG-fusion genes or ETV1-fusion genes that represent about 50% of prostate cancer cases in man. Modeling this overexpression of Ghr increased the growth rate of prostate cancer cells in vitro and in vivo via increased proliferation and changes in cancer-related gene expression.

MATERIALS AND METHODS

Animal Work Methodology

Mice were housed in polysulfide cages containing corn cob bedding and maintained on a 12-hour light and dark cycle at 20.5 ± 5°C and 30–70% relative humidity. Mice were fed a 5015 Diet (PMI Nutrition International, Brentwood MO) from conception through weaning (post-natal day 21) and an 8604 Teklad Rodent Diet thereafter (Harlan Laboratories, Madison WI). Feed and water were available ad libitum. All procedures were approved by the University of Wisconsin Animal Care and Use Committee and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Mice were provided from the Swanson lab from the University of Illinois Chicago and used to establish a breeding colony in our laboratory. C3(1)/TAg mice were maintained on a FVB background while Ghrflox/flox (provided by the Kopchick Lab), Rosa-Cre-ER, Probasin-Cre, and GHA (Kopchick Lab) mice were maintained on a C57BL/6J background.

Tamoxifen was compounded to a 75 mg/mL stock concentration in a 5:1 corn oil:ethanol mixture and 250 mg/kg or vehicle control was delivered intraperitoneally for five consecutive days as previously described (20) beginning at 5 months of age. When applicable, mice were sacrificed one month after first dose of tamoxifen or vehicle treatment. Untreated mice were sacrificed at 6 months of age.

Trunk blood was collected at the time of tissue harvest for analysis of serum IGF-1 as described previously (28). Steroid hormone analysis via LC-MS/MS and was adapted from methods previously described (29).

Xenograft surgeries were performed as described previously (28). Briefly, 10-week old Balb/C nu/nu male mice were used as hosts for ~3.5 × 105 PTEN-P2 and TRAMP-C2 cells that were transfected with a mouse Ghr overexpressing plasmid or an empty vector (EV) control. Cells in collagen were grafted bilaterally under the kidney capsules and harvested after 5 weeks.

RT-qPCR

Complementary DNA (cDNA) was reverse transcribed from RNA extracted from mouse liver and dorsolateral prostate. Gene expression was assessed as an inverse function of cycle threshold (Ct) read by StepOnePlus™ Real-Time PCR System, (4376600; ThermoFisher, Waltham, MA). To determine gene expression relative to the housekeeping gene, TATA Box Binding Protein (TBP), the mean Ct, TBP was subtracted from the mean Ct, gene. Relative gene copy was two raised to the negative of that difference, like such: 2−(Ct,gene−Ct,TBP).

Histology and Immunohistochemistry

Tissues were fixed in 4% paraformaldehyde, dehydrated in alcohol, cleared in Citrisolv, and infiltrated with paraffin. Hematoxylin & eosin (H&E) stains of 5 μm sections of tissue mounted on Superfrost™ Plus Gold Slides (Thermo Fisher Scientific; Waltham, MA) were assessed by a histopathologist.

H&E-stained tissues were assessed blindly according to the following criteria by Dr. Hu, a board-certified pathologist specializing in genitourinary pathology. Epithelial hyperplasia was defined as an increase in epithelial cells within the normal-appearing gland architecture that stand out from the background glands but without cytologic atypia. The increase in epithelial cells was recognized as tufting, nuclear crowding, micropapillary, or even cribriform architecture. PIN was recognized as proliferation of epithelial cells with cytologic atypia such as hyperchromasia, nuclear enlargement, nuclear membrane irregularity or prominent nucleoli within the preexisting glands. PIN was graded as PIN1-PIN4 similarly to previously described (30). Invasive carcinoma was recognized as malignant epithelial proliferation with destructive invasion to stroma.

Cell proliferation was measured via Ki67 immunohistochemistry (IHC) staining as described previously (28).

In Vitro Plasmid Transfection, Selection, and Cell Viability

To create stable transfectants with overexpression of Ghr in mouse prostate cancer cells, PTEN-P2 and TRAMP-C2 cells were plated and grown to 70% confluency at which point 1.6 μg of plasmid vector DNA containing mouse Ghr (MG50043-UT, Sino biological, Wayne, PA) or pcDNA3.1+ empty vector control was incubated with 4 μg of Lipofectamine 2000 in Transfectgro base media as described by the manufacturer. After 24 hours, cells were split 1:10, incubated for 24 hours, and selected using neomycin for seven days. Transfection and selection was confirmed using qPCR to identify an upregulation in relative Ghr expression in cells treated with Ghr-expressing plasmids.

Cell viability in culture was assessed using the Promega CellTiter-Blue Cell Viability Assay (G808A; Promega, Madison, WI). Cells were plated at 250 cells/well in 96-well plates 24 hours before the addition of 20 μL of CellTiter-Blue reagent was added to each well and fluorescence signal was read on a Fluostar Omega fluorescence plate reader (BMG Labtech) at 544/590 nm.

RNA Sequencing

Total RNA submitted to the University of Wisconsin-Madison Biotechnology Center was assayed for purity and integrity and gene expression was assessed as described previously (28). Raw BAM files produced by sequencing can be found at NCBI Gene Expression Omnibus with accession numbers GSE197640 and GSE197683 for WT and C3(1)/TAg prostate RNAseq data, respectively. Bioinformatic analysis of transcriptomic data adhere to recommended ENCODE guidelines and best practices for RNA-Seq and analyzed as described previously (28).

Statistical Analysis

For all histological stains, an ANOVA with Tukey’s multiple comparisons test was conducted to identify differences among means. Results are reported as mean ± standard error of the mean (SEM). A difference of p < 0.05 was considered significant. All statistics were performed using GraphPad software (GraphPad Software, San Diego, CA).

RESULTS

C3(1)/TAg transgenic mice were bred to mice with a conditionally null allele of Ghr in which the 4th exon is flanked by loxP sites (Ghrflox mice), mice that ubiquitously express an estrogen-receptor-T2-Cre-recombinase fusion protein from the Rosa26 locus (Rosa-Cre-ERT2 mice), PB-Cre4 transgenic mice that expresses Cre recombinase specifically in prostate epithelial cells (PbCre mice), and/or mice expressing a transgenic GHR antagonist (GHA mice) to generate groups of animals with genotypes Ghrflox/flox;Rosa-Cre-ERT2;C3(1)/TAg+/0, Ghrflox/flox;C3(1)/TAg+/0, Ghrflox/flox;Pb-Cre+/0;C3(1)/TAg+/0, and GHA+/0;C3(1)/TAg+/0. Mice with all four of these genotypes had a high incidence of off-target tumors in the head and neck salivary glands resulting in premature death or lethargy requiring euthanasia (Figure 1) that were not described in the original characterization of the C3(1)/TAg model’s survivability (26). However, these tumors have previously been identified in a separate study of mammary tumors in this model (31). Due to early mortality, further analysis focused on a 6 month timepoint and five groups of mice with genotype and treatment as follows: Ghrflox/flox;Rosa-Cre-ERT2;C3(1)/TAg+/0 with vehicle treatment (vehicle control), Ghrflox/flox;C3(1)/TAg+/0 with tamoxifen treatment (tamoxifen control), Ghrflox/flox;Rosa-Cre-ERT2;C3(1)/TAg+/0 with tamoxifen treatment (global Ghr deletion), Ghrflox/flox;Pb-Cre+/0;C3(1)/TAg+/0 with no treatment (prostate-specific Ghr deletion), and GHA+/0;C3(1)/TAg+/0 with no treatment (GHA). Global Ghr deletion in the C3(1)/TAg prostate cancer model reduced Ghr RNA expression relative to a housekeeping gene, Tbp, in the mouse DLP (Figure 2A) and liver (Figure 2B) and resulted in decreased serum IGF-1 (Figure 2C) and testosterone (Figure 2D) compared to vehicle- and tamoxifen-control tissue and serum. Prostate-specific Ghr recombination in the C3(1)/TAg prostate cancer model reduced Ghr RNA expression relative to a housekeeping gene, Tbp, in the mouse DLP (Figure 2A) but not the liver (Figure 2B) and resulted in no statistically significant change in serum IGF-1 compared to control (Figure 2C). Transgenic GH antagonist expression in the C3(1)/TAg prostate cancer model induced statistically significant downregulation of Ghr in the mouse liver (Figure 2B) but not the DLP (Figure 2A) and resulted in decreased serum IGF-1 compared to control tissue or serum (Figure 2C). There were no statistically significant differences in mouse body weight or prostate wet weight among groups (data not shown).

Figure 1.

Figure 1.

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A Kaplan-Meier curve is shown for genotypes Ghrflox/flox;Rosa-Cre-ERT2;C3(1)/TAg+/0 (n=36), Ghrflox/flox;C3(1)/TAg+/0 (n=18), Ghrflox/flox;Pb-Cre+/0;C3(1)/TAg+/0 (n=15), and GHA+/0;C3(1)/TAg+/0 (n=20). A Mantel-Cox log-rank text reveals no significant difference in survival curves between groups.

Figure 2.

Figure 2.

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RT-qPCR was used to measure Ghr expression in the DLP (A) and livers (B) of mice. Expression is presented relative to the expression of the housekeeping gene Tbp. Serum IGF-1 levels were measured by ELISA (C). Serum testosterone was quantified by LC-MS/MS (D). N = 3–7 per group. Statistically significant differences from vehicle control determined by ANOVA with Tukey’s multiple comparison’s test are indicated; *p<0.05, **p<0.01.

Histopathological assessment revealed that at 6 months PIN lesions did not develop as extensively in the prostates of C3(1)/TAg mice in our study relative to previous characterization of this model (26). Only one mouse developed identifiable invasive carcinoma in the prostate (tamoxifen control group) while two developed PIN1 (one each in vehicle and tamoxifen control groups). Varying instances of focal tufting without atypia and focal crowding were also observed (Supplementary Table S1). However, extensive epithelial stratification was identified and abundant throughout all groups. All three forms of GH signaling disruption including prostate epithelium specific Ghr deletion reduced the extent of prostate epithelial cell stratification in the prostate (Figure 3A–E, K) and reduced epithelial proliferation as determined by Ki67 labeling index (Figure 3F–J, L).

Figure 3.

Figure 3.

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Representative images of H&E-stained (A-E) and Ki67-stained (F-J) sections are shown for vehicle control (A, F), tamoxifen control (B, G), global Ghr deletion (C, H) prostate-specific Ghr deletion (D, I) and GHA (E, J) mouse DLPs. Scale bar for each image = 50 μm. Dotted borders in A-E highlight examples of areas identified as epithelial stratification for quantification purposes. Arrows in F-J point to examples of Ki67 positive cells (brown stain). (K) Area of stratification was assessed by an observer blinded to group and taken as a percentage of total prostatic area with a multi-layered epithelium (n=3–6 per group). (L) Ki67 labeling index was determined by counting Ki67 positive cells and total cells based in nuclear hematoxylin stain by an observer blinded to group, (n=3–5 per group). Statistically significant differences determined by ANOVA and Tukey’s multiple comparisons test are indicated, *p<0.05, **p<0.01.

RNA sequencing was performed comparing tamoxifen control DLPs to global Ghr deletion DLPs. Blocking GH signaling changed the expression of 75 and 42 genes in C3(1)/TAg and WT DLPs, respectively. Volcano plots of differentially regulated genes’ log2 fold change vs. p-value are shown in Figure 4. Genes with log2 foldchange greater than 1 are shown in red, and genes with log2 foldchange less than −1 are shown in cyan. Ghr deletion in C3(1)/TAg mice resulted in significant expression changes for 75 genes of which 63 were downregulated (Figure 4A). In WT mice, global Ghr deletion significantly changed the expression of 42 genes, 28 of which were downregulated (Figure 4B). Gene set enrichment analysis of canonical gene sets (C2) identified differentially enriched gene sets in C3(1)/TAg and WT mice in Tables 1 and 2, respectively.

Figure 4.

Figure 4.

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A volcano plot of differentially expressed genes in the DLP of C3(1)/TAg (A) and WT (B) mice with tamoxifen-induced Ghr deletion compared tamoxifen treated controls with log2 fold change on the x-axis and the negative log10 of the adjusted p-value on the y-axis. Thresholds for significance are −log10p-value > 2 and log2FoldChange > 1 or < −1. Genes upregulated in response to Ghr deletion are colored red, and genes downregulated in response to Ghr deletion are colored cyan.

Table 1.

Differentially enriched C2 genesin C3(1)/TAg mouse DLPs upon Ghr deletion.

Gene Set name log2FoldChange Corrected p-value
REACTOME_INTERLEUKIN_1_FAMILY_SIGNALING 1.7413 2.914×10−6
KRIGE_RESPONSE_TO_TOSEDOSTAT_24HR_DN 1.7289 3.384×10−6
THUM_SYSTOLIC_HEART_FAILURE_DN 1.7121 4.073×10−6
LOCKWOOD_AMPLIFIED_IN_LUNG_CANCER 1.7090 4.318×10−6
GRESHOCK_CANCER_COPY_NUMBER_UP 1.7090 4.316×10−6
REACTOME_SIGNALING_BY_INTERLEUKINS 1.6783 6.083×10−6
AMBROSINI_FLAVOPIRIDOL_TREATMENT_TP53 1.6697 6.761×10−6
RICKMAN_TUMOR_DIFFERENTIATED_WELL_VS_MODERATELY_UP 1.6692 6.759×10−6
JOSEPH_RESPONSE_TO_SODIUM_BUTYRATE_DN 1.6617 7.423×10−6
JAEGER_METASTASIS_DN 1.6567 8.002×10−6
REACTOME_CYTOCHROME_P450_ARRANGED_BY_SUBSTRATE_TYPE 1.6418 9.414×10−6
SILIGAN_BOUND_BY_EWS_FLT1_FUSION 1.6418 9.411×10−6
REACTOME_REGULATION_OF_LIPID_METABOLISM_BY_PPARALPHA 1.6418 9.408×10−6
WANG_LMO4_TARGETS_UP 1.5653 2.3004×10−6
SABATES_COLORECTAL_ADENOMA_UP 1.5647 2.914×10−5
SERVITJA_LIVER_HNF1A_TARGETS_DN 1.5612 2.464×10−5
SENESE_HDAC1_AND_HDAC2_TARGETS_UP 1.5224 3.946×10−5
IWANAGA_CARCINOGENESIS_BY_KRAS_PTEN_DN 1.5019 4.511×10−5
HAN_SATB1_TARGETS_UP 1.5008 4.589×10−5
RICKMAN_HEAD_AND_NECK_CANCER_A 1.4944 4.887×10−5
HOLLEMAN_VINCRISTINE_RESISTANCE_ALL_DN 1.4567 7.782×10−5
ENK_UV_RESPONSE_EPIDERMIS_UP 1.4008 1.360×10−4
REACTOME_METABOLISM_OF_LIPIDS 1.3783 1.807×10−4
REACTOME_BIOLOGICAL_OXIDATIONS 1.3665 1.893×10−4
REACTOME_PHASE_I_FUNCTIONALIZATION_OF_COMPOUNDS 1.3665 1.888×10−4
HOELZEL_NF1_TARGETS_DN 1.3498 2.384×10−4
TURASHVILI_BREAST_LOBULAR_CARCINOMA_VS_LOBULAR_NORMAL_UP 1.3439 2.445×10−4
JAATINEN_HEMATOPOIETIC_STEM_CELL_DN 1.3406 2.483×10−4
FARMER_BREAST_CANCER_APOCRINE_VS_LUMINAL 1.3296 2.761×10−4
ACEVEDO_LIVER_CANCER_DN 1.3181 3.214×10−4
MARTORIATI_MDM4_TARGETS_FETAL_LIVER_DN 1.3010 3.676×10−4
ENK_UV_RESPONSE_EPIDERMIS_DN 1.2851 4.262×10−4
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Table 2.

Differentially enriched C2 genesin WT mouse DLPs upon Ghr deletion.

Gene Set name log2FoldChange Corrected p-value
REACTOME_CYTOKINE_SIGNALING_IN_IMMUNE_SYSTEM 1.405405405 1.962×10−4
ACEVEDO_LIVER_TUMOR_VS_NORMAL_ADJACENT_TISSUE_UP 1.399361967 2.080×10−4
REACTOME_TRANSLATION 1.383204633 2.474×10−4
REACTOME_EUKARYOTIC_TRANSLATION_INITIATION 1.383204633 2.475×10−4
RHEIN_ALL_GLUCOCORTICOID_THERAPY_DN 1.375625626 2.692×10−4
YAMASHITA_LIVER_CANCER_WITH_EPCAM_UP 1.375625626 2.693×10−4
REACTOME_SELENOAMINO_ACID_METABOLISM 1.375625626 2.693×10−4
GRAESSMANN_APOPTOSIS_BY_SERUM_DEPRIVATION_UP 1.349249249 3.466×10−4
GRAESSMANN_RESPONSE_TO_MC_AND_SERUM_DEPRIVATION_UP 1.349249249 3.470×10−4
RAY_TUMORIGENESIS_BY_ERBB2_CDC25A_DN 1.349249249 3.475×10−4
PUJANA_BRCA1_PCC_NETWORK 1.330343222 4.253×10−4
ACEVEDO_NORMAL_TISSUE_ADJACENT_TO_LIVER_TUMOR_DN 1.313738739 4.913×10−4
GRAESSMANN_APOPTOSIS_BY_DOXORUBICIN_UP 1.313563564 4.989×10−4
RAY_TUMORIGENESIS_BY_ERBB2_CDC25A_UP −1.32307833 4.655×10−4
PEDRIOLI_MIR31_TARGETS_DN −1.329615792 4.242×10−4
OSWALD_HEMATOPOIETIC_STEM_CELL_IN_COLLAGEN_GEL_UP −1.417455808 1.733×10−4
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Using the UALCAN data portal (32) to interrogate data from the TCGA database (33), we identified statistically significant increases in GHR expression relative to normal prostates in prostate cancers with ERG-fusion genes or ETV1-fusion genes that represent about 50% of prostate cancer cases (Figure 5A). On average, there is increased expression of GHR in human prostate cancers with ERG-fusions (Figure 5A) as well as a statistically significant positive correlation between GHR expression and ERG expression in prostate tumors (Figure 5B).

Figure 5.

Figure 5.

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TCGA project data for GHR expression in human prostate cancers separated by molecular signature are shown (A) (data visualization and statistics via the UALCAN data portal, p<1×10−12 for normal vs ERG-fusion, p=1.16×10−4 for normal vs ETV1-fusion). The UALCAN data portal was used to compare GHR (vertical axis) and ERG (horizontal axis) expression in TCGA prostate tumors (B). Pearson Correlation coefficient: 0.58.

Stable transfected derivatives of PTEN-P2 and TRAMP-C2 cell lines were created with expression constructs containing either mouse Ghr (PTEN-P2_mGhr and TRAMP-C2_mGhr) or an empty vector control (PTEN-P2_EV and TRAMP-C2_EV). Compared to empty vector controls, the resulting PTEN-P2_mGhr and TRAMP-C2_mGhr overexpressing cells had 2.9- and 2.5-fold increases in mouse Ghr expression, respectively (Supplementary Figure S1A). Compared to empty vector controls, PTEN-P2_mGhr and TRAMP-C2_mGhr cells grew faster in vitro under standard culture conditions (Supplementary Figure S1B). These four stably transfected cell line derivatives were surgically grafted under the kidney capsules of 10-week-old male Balb/C nu/nu mice for 5 weeks. In both PTEN-P2 and TRAMP-C2 cells, the upregulation of Ghr resulted in increased xenograft size and weight (Figure 6). Similarly, Ghr upregulation resulted in increased proliferation as measured by immunohistochemical staining for proliferation marker Ki67 (Figure 7).

Figure 6.

Figure 6.

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PTEN-P2 or TRAMP-C2 cells stably transfected with a plasmid containing either mouse Ghr (_mGhr) or an empty vector control (_EV) suspended in rat collagen pellets (3.5 × 105 cells per graft) were surgically grafted under the kidney capsules of 10-week-old male Balb/C nu/nu mice. Xenografts were grown for 5 weeks. Examples of resultant xenografts are shown for PTEN-P2_EV (A), PTEN-P2_mGhr (B), TRAMP-C2_EV (C) and TRAMP-C2_mGhr (D). Photos in A-D show kidneys (dark red) with xenografts (lighter areas on kidney surface inside the dashed lines) and a ruler for scale. (E) Graft masses are shown for empty vector controls (black bars, n=5 per group) and Ghr upregulated (grey bars, n=4 per group). Statistically significant differences from control are indicated *p<0.05; Student’s t-test).

Figure 7.

Figure 7.

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Representative images of Ki67-stained sections are shown for PTEN-P2_EV (A), PTEN-P2_mGhr (B), TRAMP-C2_EV (C) and TRAMP-C2_mGhr (D). Scale bar for each image = 50 μm. Arrows point to examples of Ki67 positive cells (brown stain). (E) Ki67 labeling index was determined by counting Ki67 positive cells and total cells based in nuclear hematoxylin stain by an observer blinded to group, n=4 per group. Statistically significant differences determined by ANOVA are indicated, *p<0.05, **p<0.01.

RNA was collected from mouse DLPs for Ghr deletion and tamoxifen control groups for both C3(1)/TAg and WT background mice. Six genes previously identified to be differentially regulated by GH signaling disruption (28)were found to be down-regulated upon Ghr deletion by RNA-seq for both the C3(1)/TAg and WT backgrounds here and were confirmed by quantitative RT-PCR (Figure 8A). RNA was also collected from Pten mutant and TRAMP cell line xenografts overexpressing Ghr or EV controls. Some of the six genes down-regulated by Ghr deletion were regulated in the opposite direction compared to Ghr deletion in Ghr-overexpressing xenografts with some differences between the Pten mutant and TRAMP cell lines (Figure 8B). The expression of four genes previously found to be down-regulated in prostate cancer xenografts by GHR antagonist pegvisomant (Ccnd1, Ccnd2, Cdk4, and Grem1) and two genes previously found to be up-regulated in prostate cancer xenografts by pegvisomant (Ar, Cdh1) (28) was also evaluated for Ghr overexpressing and EV control xenografts and expression of all six genes was oppositely regulated upon Ghr overexpression (Figure 8C) compared to the previously observed pattern for pegvisomant-treated xenografts (28).

Figure 8.

Figure 8.

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RNA extracted from DLPs of global Ghr deletion or tamoxifen groups in C3(1)/TAg and WT mice were used to confirm expression changes via RT-qPCR in genes first identified by RNA-seq (A). Gene expression as measured by qPCR in PTEN-P2 and TRAMP-C2 grafts with overexpression of Ghr (mGhr) compared to empty vector (EV) controls for genes identified as Ghr regulated in this study (B) and genes previously implicated by disruption of the GH/IGF-1 axis with pegvisomant treatment (28) (C). Gene expression is shown relative to the expression of the housekeeping gene, Tbp, and normalized to control = 1. Statistically significant differences determined by ANOVA with Tukey’s multiple comparison’s test are indicated, *p<0.05, **p<0.01; n = 3–4 per group.

DISCUSSION

Humans with Laron syndrome appear to be protected from certain diseases, including cancer and diabetes (34). None of the 169 subjects with Laron syndrome in one study developed cancer while their non-Laron relatives developed cancer at expected rates of ~20% (34). Mouse models of Laron syndrome also have lower rates of cancer (35) and are protected from experimentally induced cancer (9). The C3(1)/TAg model of mouse prostate cancer failed to develop PIN as expected when crossed with the mouse model of Laron syndrome (GHR null mice), reflecting an outcome similar to the protection seen in human Laron syndrome subjects (9). However, an important limitation of these epidemiological and experimental studies is that both human Laron patients and the Laron mouse model possess disrupted GH/IGF-1 axes from conception throughout their entire lives, which has limited clinical relevance (21,22). Because of this limitation, these studies fail to address the potential necessity for continuous GH signaling in prostate cancer. To model the potential therapeutic capacity of blocking GH signaling after prostate cancers develop, we used a standardized tamoxifen inducible Cre-lox system (Rosa-Cre-ER) that provided temporal control of Ghr expression. Likewise, spatial control of Ghr expression (PbCre) provided the opportunity to identify direct action of GH signaling in prostate epithelial cells. Lastly, a transgenically expressed GH antagonist (GHA) gave the ability to mimic chronic GH antagonism and determine its effect on prostate cancer in the C3(1)/TAg model.

GH/IGF-1 axis activity was downregulated in all three models of disruption (Figure 2A–C). Compared to vehicle and tamoxifen control groups, global Ghr deletion, prostate-specific Ghr deletion, and GHA mice exhibited varying levels of axis disruption. At the RNA level, DLP Ghr expression was reduced in both global and prostate-specific deletion groups (Figure 2A). In the liver, Ghr RNA was reduced in the global deletion and GHA groups (Figure 2B). GHA-induced downregulation of Ghr is consistent with previous investigations of gene regulation in this model (36). Considering genetic changes, serum IGF-1 was lowered in mice with global Ghr and GHA as expected due to blocking GHR function, but not prostate-specific Ghr recombination for these mice had an intact axis outside of the prostate epithelium (Figure 2C). Additionally, tamoxifen-induced Ghr deletion had reduced serum testosterone (Figure 2D) which is consistent with studies of Laron patients with increased serum testosterone following treatment with IGF-1 (37).

C3(1)/TAg expression transforms cells through mechanisms that are functionally similar to those that occur in human prostate carcinogenesis (26,38). Original characterization of the model identified low-grade PIN in the ventral and dorsolateral lobes of the mouse prostate at two months of age, and high-grade PIN was found in both lobes by five months. Invasive carcinomas were reported in the model as early as seven months. After histopathological assessment by a pathologist, it was revealed that at six months PIN lesions did not develop as extensively in the prostate of C3(1)/TAg mice as originally described. Only one mouse developed identifiable invasive carcinoma in the prostate and two mice displayed instances of PIN1. The lesion penetrance discrepancy between the original characterization and the current study can potentially be due to the mixed background of mice that was required for this study as has been identified in studies of the PTEN mouse prostate cancer model with mixed backgrounds, for example (39). However, extensive epithelial stratification was identified and abundant throughout all groups. Considering these differences in pathology between our model and the original characterization, we found that all three groups with disrupted GH/IGF-1 axes showed reduced prostate duct stratification compared to controls (Figure 3A–E, K). Additionally, proliferation of prostate cells was reduced in GHR-disrupted C3(1)/TAg mice (Figure 3F–J, L).

Decreases in stratification and proliferation in the C3(1)/TAg model were accompanied by changes in gene expression (Figure 4A). C3(1)/TAg prostates in mice with global Ghr deletion possessed upregulated gene sets involved in interleukin expression and anti-metastatic genetic mechanisms which hints at GH disruption’s contribution to pro-immune, anti-cancer effects (Table 1). These changes in gene expression in the C3(1)/TAg model were mirrored by gene expression changes in the WT mouse DLP, though less robustly (Figure 4B). Differentially enriched gene sets in WT mice with disrupted GH/IGF-1 axes similarly involved the immune system and downregulated genes involved in tumorigenesis, providing further evidence that deletion of Ghr, and therefore disruption of the entire GH/IGF1 axis, contributes to transcriptional opposition to carcinogenesis and immune system regulation of uncontrolled growth (Table 2). Given the duplicative representation of the immune system in the current gene and gene set analysis, further examination of the impact of the immune system and immune cell infiltration in the C3(1)/TAg model of cancer is warranted. Additionally, global GHR regulation of immune-related genes in the WT mouse DLP should be explored upstream of the lower urinary tract to determine gene regulation in the prostate. These data align with the translational dysregulation observed in prostate cancer cells treated with pegvisomant in a previous study, indicating that the genetic means of GH disruption outlined here mimic some of the effects of pharmacological GH disruption in terms of downstream transcriptional effects on cancer-related genes and gene sets (28).

Pegvisomant is an FDA-approved GHR antagonist used as a pharmaceutical treatment for acromegaly and has been investigated in models of prostate cancer in combination with enzalutamide resulting in decreased expression of cancerous matrix metalloproteinases and serum prostate specific antigen (40). In vitro and pre-clinical in vivo studies of pegvisomant’s effectiveness in prostate cancer is tantalizing. Previous studies of pegvisomant use in vitro and in vivo identified therapeutic potential as well as 2,718 differentially expressed genes in Pten mutant mouse prostate cancer cells (28). Of these genes, global deletion of Ghr similarly changed the expression of seven of them: Cfb, Gcom1, Rpl15-ps3, Arnt2, Cyp4a12a, Exph5, and Pbk. The downregulation of Gcom1, Rpl15-ps3, and Pbk infers GH disruption via genetic or pharmacological means has a greater effect on cellular signal transduction than simply blocking the activation of JAK or STAT. Conversely, the upregulation of Cfb, Arnt2, Cyp4a12a, and Exph5 reinforces gene set analysis that indicates GH disruption plays a role in the upregulation of immune system activity (Tables 1 and 2).

Investigation of differential expression of genes in C3(1)/TAg and WT mice identify a few genes of interest that are regulated in part by AR. Interestingly, while one study observed a tamoxifen-induced increase in testosterone levels in acromegaly patients (41), tamoxifen-induced deletion of Ghr resulted in decreased serum testosterone (Figure 2D). Conversely, another study observed an increase in testosterone in Laron patients supplemented with IGF-1 (37). These findings suggest that GH may have an indirect role in promoting androgen-regulated genes in prostate cancer.

Previous studies have identified that local expression and overexpression of GH occurs in advanced human prostate cancers and contributes to cancer progression (40). This study identified GHR as an additional component of the GH signaling pathway that is altered in prostate cancer. UALCAN data portal interrogation of TCGA data revealed an overexpression of GHR relative to normal prostate in human prostate cancers with ERG-fusion genes or ETV1-fusion genes (Figure 5A). These two subtypes represent about half of all prostate cancer cases (33). Additionally, a positive correlation links GHR and ERG expression in human prostate tumors (Figure 5B). We speculate that pathways downstream of GHR may be playing a cooperative role with ETS fusion genes which has similarly been reported in PI3K pathway activation (42). To further highlight this relationship between GHR and prostate tumors, we found that PTEN-P2 and TRAMP-C2 mouse prostate cancer cells that overexpress mouse Ghr grew more robustly than normal Ghr controls (Figure 6). Similarly, both PTEN-P2 and TRAMP-C2 Ghr overexpressing cells had a higher proliferation rate when grown in vivo than controls (Figure 7). PTEN-P2 cells’ transformation is driven by a heterozygous deletion of the tumor suppressor gene Pten while TRAMP-C2 is derived from a similar C3(1)/TAg-induced carcinoma used in genetic studies here (43,44). These data reveal a cooperation between Ghr overexpression and multiple oncogenic insults to drive tumor growth. While previous studies have identified the cancer driving potential of autocrine GH signaling (45), this study provides the first functional evidence that Ghr overexpression may drive prostate cancer progression. Additionally, overexpression of Ghr resulted in the opposite regulation of genes previously implicated in this study via tamoxifen-induced Ghr deletion (Figure 4) or with pegvisomant treatment ((28); Figure 8). For example, all commonly differentially regulated genes in C3(1)/TAg and WT mouse prostates were downregulated after Ghr deletion. Ghr overexpression, however, induced upregulation of some of those genes (Figure 8B). As well, pegvisomant treatment altered gene expression in PTEN-P2 and TRAMP-C2 cells in the opposite manner that Ghr overexpression did here ((28); Figure 8C). These data describing the overexpression of GHR in human prostate cancers with ERG- and ETV1-fusion genes provides rationale to develop a new transgenic model with prostate-specific Ghr overexpression. This model would provide a way to investigate the potential role of Ghr as a driver of prostate cancer initiation and progression and the relationship between the GH/IGF-1 axis and fusion genes involving the ETS family of transcription factors.

CONCLUSIONS

In this study, we investigated the effects of conditionally deleting Ghr in the C3(1)/TAg mouse model of prostate cancer. This study showed that GH/IGF-1 axis disruption through global Ghr deletion, prostate-specific Ghr deletion, or GHA expression reduced the extent of prostate epithelial stratification in the C3(1)/TAg model through decreased proliferation. By deleting Ghr specifically in the prostate epithelium, we’ve shown that GHR activity directly contributes to cellular transformation and proliferation. Further, gene regulation downstream of GHR and IGF-1 was significantly altered upon global deletion of Ghr. Overexpression of Ghr also increased tumor size of xenografted mouse prostate cancer cells. Together, these loss-of-function and gain-of-function models that altered GHR activity demonstrated a pro-proliferative role for GHR in prostate cancer cells and point to a potentially clinically relevant target for prostate cancer treatment.

Supplementary Material

fS1

Supplementary Figure S1. In vitro Ghr expression and cell viability is increased upon stable transfection of a plasmid containing mouse Ghr. (A) Gene expression as measured by RT-qPCR in PTEN-P2 and TRAMP-C2 cells reveals an upregulation of Ghr expression when treated with a plasmid containing mouse Ghr (mGhr) compared to an empty vector (EV). Gene expression is relative to the expression of the housekeeping gene, Tbp. (B) PTEN-P2 and TRAMP-C2 cells overexpressing Ghr have higher viability in vitro. Cell viability was determined utilizing reduced resorufin fluorescence and normalized to control = 1. Statistically significant differences determined by ANOVA with Tukey’s multiple comparison’s test are indicated, *p<0.05, **p<0.01; n = 3 per group.

tS1

Supplementary Table S1. Instances of a description by a histopathologist’s assessment of the pathology within mouse prostates by group.

ACKNOWLEDGEMENTS

This work was supported by the National Cancer Institute (R21CA238105). The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute. Special thanks to Sandra Splinter BonDurant and Molly Zeller at the University of Wisconsin-Madison Biotechnology Center DNA Sequencing Facility and Amita Kapoor and Cody Corbett at the Wisconsin National Primate Research Center.

Footnotes

DISCLOSURES

The authors have no conflicts of interest to disclose.

DATA AVAILABILITY

RNA-Seq data is available for download from NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/GEO) with accession numbers GSE197640 and GSE197683. Additional data are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

fS1

Supplementary Figure S1. In vitro Ghr expression and cell viability is increased upon stable transfection of a plasmid containing mouse Ghr. (A) Gene expression as measured by RT-qPCR in PTEN-P2 and TRAMP-C2 cells reveals an upregulation of Ghr expression when treated with a plasmid containing mouse Ghr (mGhr) compared to an empty vector (EV). Gene expression is relative to the expression of the housekeeping gene, Tbp. (B) PTEN-P2 and TRAMP-C2 cells overexpressing Ghr have higher viability in vitro. Cell viability was determined utilizing reduced resorufin fluorescence and normalized to control = 1. Statistically significant differences determined by ANOVA with Tukey’s multiple comparison’s test are indicated, *p<0.05, **p<0.01; n = 3 per group.

NIHMS1858851-supplement-fS1.tif (13.7MB, tif)
tS1

Supplementary Table S1. Instances of a description by a histopathologist’s assessment of the pathology within mouse prostates by group.

NIHMS1858851-supplement-tS1.docx (13.1KB, docx)

Data Availability Statement

RNA-Seq data is available for download from NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/GEO) with accession numbers GSE197640 and GSE197683. Additional data are available from the corresponding author upon reasonable request.

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