Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 1. GHR expression in normal and cancerous lung tissue. (a) GHR levels in clinical samples representing 18 SCC and 40 lung adenocarcinoma analysed from microarray data (GSE10245) retrieved from Gene Expression Omnibus (GEO).24 A significant correlation was determined at P = 0.003. Gene expression analysis of four genes (b) GHR, (c) EGFR, (d) SOCS2 and (e) SOCS3 in 60 clinical samples of normal lung tissue and NSCLC from a non-smoking female cohort in accordance with microarray data (GSE19804).25 A significant difference was observed (Po0.0001). The expression levels of other genes (SOCS1, CISH and GH2) did not differ significantly (Supplementary Figure 1).

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Expressing, Microarray, Gene Expression

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 4. GHRP495T impairs SOCS2 binding to GHR. (a) No difference in SOCS2 transcript induction between WT GHR and GHRP495T. HEK293 cells transduced with WT GHR and GHRP495T were maintained in serum-starved media with sustained GH (2.3 nM) then RNA was extracted at indicated time points. SOCS2 levels were determined by real-time PCR and normalised to GAPDH reference gene. Data presented as mean ± s.e.m. analysed by one-way ANOVA (****Po0.0001, ***Po0.001). Representative of three independent experiments confirmed in three separate lines generated by independent transductions. (b) HEK293 cells stably expressing WT GHR or GHRP495T, and parental cells transfected with SOCS2 expression plasmid for 24 h and serum-starved overnight before 2.3 nM GH (+) or vehicle (−) stimulation for 15 min. Lysates were harvested and co-IP with SOCS2 antibody as described in Materials and methods. Protein complex from the immunoprecipitates were immunoblotted (IB) using anti-HA antibody for GHR (mature (m) receptor and precursor (p) receptor) and anti-SOCS2 (input). As control, parental cell line with no transduced GHR was used and a small volume of total cell lysates used for co-IP was probed for GHR levels (HA-tag) to indicate GHR levels. (c) Graph represents the signal intensity of total GHR (HA-tag) pull down in GHRP495T relative to WT GHR relative to SOCS2 input, corrected for endogenous GHR expression. Data presented as mean ± s.e.m. analysed by Student’s t-test (***Po0.001) and representative of nine independent experiments confirmed in three independently transduced cell lines. (d) CISH protein does not interact directly with GHR as compared with SOCS2. HEK293 cells stably expressing WT GHR co-transfected with CISH or SOCS2 expression plasmids for 24 h and serum-starved overnight before 2.3 nM GH (+) or vehicle (-) stimulation for 15 min. Lysates were harvested and co-IP with SOCS2 and CISH antibodies simultaneously. Protein complex from the immunoprecipitates was immunoblotted using anti-HA antibody (for GHR) and anti-SOCS2 and anti-CISH antibodies. As a control, parental cell line with no transduced GHR, but transfected with CISH was used and a small volume of total cell lysates used for co-IP was probed for GHR levels (HA-tag) to indicate endogenous GHR levels (see Supplementary Figure 2).

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Binding Assay, Transduction, Real-time Polymerase Chain Reaction, Generated, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Control

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 5. GHRP495T generates structural changes in the receptor intracellular domain. (a) Clustal Omega multiple sequence alignment of the GHR polypeptide in close proximity to Pro 495 (red). The Tyr residue, a known active STAT5 binding site is coloured blue. Symbols below indicates (*) identical residues, (:) conservation of strongly similar properties and (.) conservation of weakly similar properties. (b) Importance of residues surrounding Pro495 to SOCS2 binding. HEK293 cells stably transduced with WT GHR, GHRP495T, GHRP495K, GHRP495A and GHRT494A were subjected to co-IP analysis as described in Materials and methods. Protein complexes from the immunoprecipitates were immunoblotted for GHR (anti-HA-tag) (mature (m) receptor and precursor (p) receptor) and SOCS2. As control, a small volume of total cell lysates used for co-IP was probed to indicate endogenous levels of GHR, SOCS2, and GAPDH (loading control). (c) Graph represents the signal intensity of total GHR (HA-tag) pull down relative to SOCS2 input, corrected for endogenous GHR expression. Data presented as mean ± s.e.m. analysed by one-way ANOVA (***Po0.001) and representative of three independent experiments confirmed in three lines generated by independent transductions. (d) GHRP495T changes the structural ensemble in the GHR intracellular domain by nuclear magnetic resonance spectroscopy analysis. Secondary Cα-chemical shift (SCS) of GHR ICD455-588 WT (top panel). Consecutive positive SCS-values indicate transiently folded helices (TH) marked in grey boxes. The original TH5, as previously reported34 was re-evaluated as two interrupted transient helices as TH5.1 (E462-L475), TH5.2 (P478-S488), and are shown together with TH6 and TH7 (top panel). SCS of GHR ICD455-588 P495T (middle panel). P495T induces helicity around the mutation site and before in the C-terminal of TH5.2. Numbering includes the signal peptide. Model illustrating the change in structural ensemble around T495 (bottom panel) and the inter-conversion equilibrium between disordered and helical structures, where the helicity is increased in the GHR ICD455-588 P495T .

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Sequencing, Residue, Binding Assay, Stable Transfection, Transduction, Co-Immunoprecipitation Assay, Control, Expressing, Generated, Nuclear Magnetic Resonance, Spectroscopy, Mutagenesis

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 6. GHRP495T degradation is impaired. (a) Effect of BFA treatment on WT GHR and GHRP495T. Time course analysis on HEK293 cells transduced with WT GHR and GHRP495T subjected to 2.3 nM GH stimulation for 15 min in the presence of BFA and harvested at indicated time points. Immunoblot demonstrating GHR (HA-tag) levels (mature (m) receptor and precursor (p) receptor) and β-TUBULIN (loading control). (b) Graph indicating fold change in mature GHR levels normalised to β-TUBULIN relative to ‘0 min’ time point after GH removal. (c) Immunoblot indicating GHR (HA-tag) following BFA treatment, as above (a) in the presence of SOCS2. (d) Graph indicating fold change in mature GHR levels in the presence of SOCS2, normalised to β-TUBULIN relative to ‘0 min’ time point after GH removal. (b, d) Data presented as mean ± s.e.m. analysed by two-way ANOVA (**Po0.01, *Po0.05) and representative of at least three independent experiments confirmed in two independently transduced lines (see Supplementary Figure 3); (e) GHRP495T is less amenable to degradation owing to SOCS2 as evident from TIRF microscopy (allows detection of fluorescent proteins only at, or near the cell membrane) images of HEK293 cells transduced with WT GHR-GFP and GHRP495T-GFP transfected with SOCS2-mCherry in the absence of exogenous GH. Colocalisation (yellow) of SOCS2 (red) and receptor (green) was more pronounced in GHRP495T (See Supplementary Figure 4). Separate channel images shown below. (f) GHR levels on cell surface of HEK293 cells expressed as normalised fluorescent intensity in the presence or absence of SOCS2. Data presented as mean ± s.e.m. from 30 cells per condition across two independently transduced cell lines and analysed by one-way ANOVA (**Po0.01) relative to WT GHR-GFP no SOCS2. Student’s t-test (#Po0.05) between WT GHR-GFP + SOCS2 and GHRP495T-GFP + SOCS2. (g) SOCS2 levels at the cell surface expressed as normalised fluorescent intensity for the cells analysed above. Data presented as mean± s.e.m. from 30 cells per condition from three independent experiments across two independently transduced lines and analysed by Student’s t-test (ns, not significant). (h) HEK293 cells transduced with WT GHR and GHRP495T treated with proteasomal inhibitor MG-132 (10 μM) for 2 h before GH (2.3 nM) addition for 15 min. Immunoblot indicating GHR (HA-tag) levels with mature and precursor receptor and two remnant bands observed at ~ 60 and ~ 43 kDa only in WT GHR lysates and undetectable in GHRP495T (see Supplementary Figure 5). Blot representative of four independent experiments.

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Transduction, Western Blot, Control, Microscopy, Membrane, Transfection

Journal: Oncogene

Article Title: A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

doi: 10.1038/onc.2017.352

Figure Lengend Snippet: Figure 7. GHRP495T increases transcripts associated with tumour progression. BEAS-2B cells transduced with WT GHR and GHRP495T stimulated with GH (2.3 nM) for 15 min, then RNA was harvested at 60 (a) and 120 min (b) post-initial treatment. (a) Transcript levels of early GH-response genes c-FOS, EGR1, JUN at 60 min in WT GHR and GHRP495T cells normalised to B2M. (b) Transcript levels of genes associated with epithelial–mesenchymal transition (EMT) (TWIST1, SNAI2 and TGFB1), proliferation (CCND1, MYC), and elevated GHR signalling (GHR, EGFR, SOCS2 and SOCS3) at 120 min in WT GHR and GHRP495T cells normalised to B2M (see Supplementary Figure 6). Data presented as mean ± s.e.m. analysed by one-way ANOVA (***Po0.001, **Po0.01, *Po0.05) and representative of three independent experiments confirmed in three independently transduced lines.

Article Snippet: SOCS2 expression plasmid pCMV-SOCS2-Flag was purchased from Origene (Rockville, MD, USA) and CISH expression plasmid was a kind gift from Dr Patrick Lau (IMB, The University of Queensland).

Techniques: Transduction