ABSTRACT
Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.
Subject(s)
Carrier Proteins/genetics , Gene Expression Regulation, Neoplastic/genetics , Lung Neoplasms/genetics , Signal Transduction/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line, Tumor , Cohort Studies , Computational Biology , DNA Mutational Analysis , Disease Progression , Epithelial-Mesenchymal Transition/genetics , Female , HEK293 Cells , Humans , Lung/pathology , Lung Neoplasms/pathology , Magnetic Resonance Spectroscopy , Male , Mice , Phosphorylation , Polymorphism, Single Nucleotide , Proline/genetics , Protein Binding/genetics , Protein Domains/genetics , Proteolysis , Threonine/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Placental human growth hormone-variant (hGH-V) and pituitary human growth hormone-N (hGH-N) are of identical size (22 kDa) but differ in 13 residues scattered throughout the protein. Several isoforms of GH are produced by the hGH-N and hGH-V genes including a 20-kDa hGH-V resulting from a 45-bp deletion caused by the use of an alternative acceptor site within exon 3. To date, the biological properties of the 20-kDa GH-V have not been characterized in vivo. Using young male Wistar rats fed either chow or a high-fat (HF) diet for 4 wk postweaning, we investigated the effect of 7 days treatment with either 22-kDa hGH-N, 20-kDa hGH-V (5 ug x g(-1) x day(-1) sc), or vehicle on body composition and endocrine and metabolic profiles. Total body growth (absolute weight gain and linear growth trajectory) in the 20-kDa hGH-V-treated animals was intermediary between that of control and hGH-N-treated animals. Both 22-kDa hGH-N and 20-kDa hGH-V significantly reduced total body fat mass compared with control animals, and there were no differences between the GH isoforms in anti-lipogenic activity in animals fed the HF diet. Fasting plasma insulin and C peptide were significantly increased in animals on the HF diet and further increased by hGH-N but were unchanged in 20-kDa hGH-V-treated animals compared with saline-treated controls. Plasma volume as assessed by hematocrit was increased in hGH-N-treated animals but was unchanged in 20-kDa hGH-V-treated animals compared with controls. Furthermore, 20-kDa hGH-V had reduced lactogenic (prolactin receptor mediated) activity characteristic of hGH-N as tested in vitro compared with the 20-kDa hGH-N and 22-kDa hGH-N variants. In summary, placental 20-kDa hGH-V retains some of the growth-promoting and all antilipogenic activities of pituitary 22-kDa hGH-N but has diminished diabetogenic and lactogenic properties compared with the native 22-kDa hGH-N.
