GH-dependent growth of experimentally induced carcinomas in vivo.

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.

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

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.

Mammary Tumor Growth and Proliferation Are Dependent on Growth Hormone in Female SV40 C3(1) T-Antigen Mice.

Female SV40 C3(1) T-antigen (C3(1)/TAg) transgenic mice develop mammary tumors that are molecularly similar to human basal-like breast cancers with 100% incidence at 16 weeks of age. To determine the requirement for growth hormone (GH) signaling in these tumors, genetic crosses were used to create cohorts of female mice that were homozygous for a floxed growth hormone receptor (Ghr) gene and carried one copy each of the Rosa-Cre-ERT2 transgene and the C3(1)/TAg transgene (Ghrflox/flox; Rosa-Cre-ERT2; C3(1)/TAg+/0 mice). When the largest mammary tumor reached 200 mm3, mice were treated with tamoxifen to delete Ghr or with vehicle as a control. An additional group of Ghrflox/flox; C3(1)/TAg+/0 mice were also treated with tamoxifen when the largest mammary tumor reached 200 mm3 as a control for the effects of tamoxifen. After 3 weeks, tumors in mice in which Ghr was deleted began to shrink while vehicle and tamoxifen treatment control mouse tumors continued to grow. Pathological analysis of tumors revealed similar growth patterns and varying levels of necrosis throughout all groups. A decrease in cancer cell proliferation in Ghr-/- tumors relative to controls was observed as measured by Ki67 immunohistochemistry labeling index. These data suggest that even established C3(1)/TAg mammary tumors are dependent on the GH/IGF-1 axis.

GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression.

Previous studies investigating the effects of blocking the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis in prostate cancer found no effects of the growth hormone receptor (GHR) antagonist, pegvisomant, on the growth of grafted human prostate cancer cells in vivo. However, human GHR is not activated by mouse GH, so direct actions of GH on prostate cancer cells were not evaluated in this context. The present study addresses the species specificity of GH-GHR activity by investigating GH actions in prostate cancer cell lines derived from a mouse Pten-deletion model. In vitro cell growth was stimulated by GH and reduced by pegvisomant. These in vitro GH effects were mediated at least in part by the activation of JAK2 and STAT5. When Pten-mutant cells were grown as xenografts in mice, pegvisomant treatment dramatically reduced xenograft size, and this was accompanied by decreased proliferation and increased apoptosis. RNA sequencing of xenografts identified 1765 genes upregulated and 953 genes downregulated in response to pegvisomant, including many genes previously implicated as cancer drivers. Further evaluation of a selected subset of these genes via quantitative reverse transcription-polymerase chain reaction determined that some genes exhibited similar regulation by pegvisomant in prostate cancer cells whether treatment was in vivo or in vitro, indicating direct regulation by GH via GHR activation in prostate cancer cells, whereas other genes responded to pegvisomant only in vivo, suggesting indirect regulation by pegvisomant effects on the host endocrine environment. Similar results were observed for a prostate cancer cell line derived from the mouse transgenic adenocarcinoma of the mouse prostate (TRAMP) model.

Oligo(Lactic Acid)8-Docetaxel Prodrug-Loaded PEG-b-PLA Micelles for Prostate Cancer.

Docetaxel (DTX) is among the most frequently prescribed chemotherapy drugs and has recently been shown to extend survival in advanced prostate cancer patients. However, the poor water solubility of DTX prevents full exploitation of this potent anticancer drug. The current marketed formulation, Taxotere®, contains a toxic co-solvent that induces adverse reactions following intravenous injection. Nano-sized polymeric micelles have been proposed to create safer, water-soluble carriers for DTX, but many have failed to reach the clinic due to poor carrier stability in vivo. In this study, we aimed to improve micelle stability by synthesizing an ester prodrug of DTX, oligo(lactic acid)8-docetaxel (o(LA)8-DTX), for augmented compatibility with the core of poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) micelles. Due to the enhancement of drug-carrier compatibility, we were able to load 50% (w/w) prodrug within the micelle, solubilize 20 mg/mL o(LA)8-DTX (~12 mg/mL DTX-equivalent) in aqueous media, and delay payload release. While the micelle core prohibited premature degradation, o(LA)8-DTX was rapidly converted to parent drug DTX through intramolecular backbiting (t1/2 = 6.3 h) or esterase-mediated degradation (t1/2 = 2.5 h) following release. Most importantly, o(LA)8-DTX micelles proved to be as efficacious but less toxic than Taxotere® in a preclinical mouse model of prostate cancer.

Mammary Tumors Growing in the Absence of Growth Hormone Are More Sensitive to Doxorubicin Than Wild-Type Tumors.

Previously, we reported that N-methyl-N-nitrosourea (MNU)-induced mammary tumors could be established in mutant spontaneous dwarf rats (SDRs), which lack endogenous growth hormone (GH) by supplementing with exogenous GH, and almost all such tumors regressed upon GH withdrawal. When the highly inbred SDR line was outcrossed to wild-type (WT) Sprague-Dawley rats, MNU-induced mammary tumors could still be established in resulting outbred SDRs by supplementing with exogenous GH. However, unlike tumors in inbred SDRs, 65% of mammary tumors established in outbred SDRs continued growth after GH withdrawal. We further tested whether these tumors were more sensitive to doxorubicin than their WT counterparts. To accomplish this, MNU-induced mammary tumors were established in WT rats and in SDRs supplemented with exogenous GH. Once mammary tumors reached 1 cm3 in size, exogenous GH was withdrawn from SDRs, and the subset that harbored tumors that continued or resumed growth in the absence of GH were selected for doxorubicin treatment. Doxorubicin was then administered in 6 injections over 2 weeks at 2.5 mg/kg or 1.25 mg/kg for both the WT and SDR groups. The SDR mammary tumors that had been growing in the absence of GH regressed at both doxorubicin doses while WT tumors continued to grow robustly. The regression of SDR mammary tumors treated with 1.25 mg/kg doxorubicin was accompanied by reduced proliferation and dramatically higher apoptosis relative to the WT mammary tumors treated with 1.25 mg/kg doxorubicin. These data suggest that downregulating GH signaling may decrease the doxorubicin dose necessary to effectively treat breast cancer.

Prostate epithelial-specific expression of activated PI3K drives stromal collagen production and accumulation.

We genetically engineered expression of an activated form of P110 alpha, the catalytic subunit of PI3K, in mouse prostate epithelium to create a mouse model of direct PI3K activation (Pbsn-cre4Prb;PI3KGOF/+ ). We hypothesized that direct activation would cause rapid neoplasia and cancer progression. Pbsn-cre4Prb;PI3KGOF/+ mice developed widespread prostate intraepithelial hyperplasia, but stromal invasion was limited and overall progression was slower than anticipated. However, the model produced profound and progressive stromal remodeling prior to explicit epithelial neoplasia. Increased stromal cellularity and inflammatory infiltrate were evident as early as 4 months of age and progressively increased through 12 months of age, the terminal endpoint of this study. Prostatic collagen density and phosphorylated SMAD2-positive prostatic stromal cells were expansive and accumulated with age, consistent with pro-fibrotic TGF-ß pathway activation. Few reported mouse models accumulate prostate-specific collagen to the degree observed in Pbsn-cre4Prb;PI3KGOF/+ . Our results indicate a signaling process beginning with prostatic epithelial PI3K and TGF-ß signaling that drives prostatic stromal hypertrophy and collagen accumulation. These mice afford a unique opportunity to explore molecular mechanisms of prostatic collagen accumulation that is relevant to cancer progression, metastasis, inflammation and urinary dysfunction. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.