Characterization of an activating R1353H insulin-like growth factor 1 receptor variant in a male with extreme tall height.

OBJECTIVE: The insulin-like growth factor1 receptor (IGF1R) is important in growth and development, and inactivating IGF1R mutations cause short stature and relatively high levels of serum IGF-I. We identified an unclassified IGF1RR1353H variant in a male with extreme tall height, very low levels of serum IGF-I and delayed and prolonged growth spurt. The index case's mother and three sons all carried the variant, but so far only the eldest son (age 18 years) presented with tall height. We hypothesized that the variant could constitute an activating mutation. DESIGN: The IGF1RR1353H variant was investigated in Igf1r-/- mouse embryonic fibroblasts (R-cells) by cell cycle, colony formation and transcriptome analyses. RESULTS: The IGF1RR1353H (R-1353) exhibited significantly increased cell proliferation, G1-S progression and colony formation in soft agar. RNA sequencing identified 195 differentially expressed genes between R-WT and R-1353 (adjusted P < 1E-100). Most genes were upregulated in R-1353, including the gene encoding the androgen receptor (AR). Gene expression profiling showed the most significant enrichment in extracellular matrix organization (P = 2.76E-7), collagen biosynthesis (P = 1.21E-5) and cell adhesion (P = 7.38E-5). Retrospective biochemical analysis of the index case revealed decreased testosterone and sex hormone-binding globulin levels, whereas LH and FSH were within normal ranges. This profile suggests an increased sensitivity to androgen, which is compatible with the enhanced expression of Ar in R-1353 cells. CONCLUSIONS: Our findings suggest that R1353H constitutes an activating IGF1R variant. The possible deregulation of collagen turnover and increased androgen sensitivity implicates an association to tall phenotype in male carriers.

Telomerase promoter mutations and copy number alterations in solitary fibrous tumours.

AIMS: Solitary fibrous tumour (SFT) is an infrequently metastasising mesenchymal tumour defined by the NAB2-STAT6 fusion gene. Activating mutations in the telomerase reverse transcriptase (hTERT) gene promoter has been reported to associate with adverse patient outcome in SFTs. METHODS: We analysed the hTERT gene for promoter mutations and copy number alterations in 43 primary extrameningeal SFTs (9 malignant and 34 benign tumours according to WHO 2013 criteria), six local recurrences and three metastatic lesions. RESULTS: Activating -124 C>T (n=12) or -148 C>T (n=2) mutations were found in 33% of the tumours and associated with older age (P=0.006), necrosis (P=0.009), higher mitotic rate (P=0.003), nuclear atypia (P=0.002), malignant histological diagnosis (P=0.04) and worse progression-free survival (P=0.023). We also observed frequent (24%) hTERT promoter mutations in histologically benign tumours without metastasis (mean follow-up >9 years), and in 14%-18% of low-risk SFTs as determined by three risk-stratification models. Mutations were seen in 2/6 metastatic tumours and metastatic lesions. hTERT copy number gain was seen in 11/28 hTERT promoter wild-type cases. CONCLUSIONS: Activating hTERT promoter mutations associate with aggressive histopathological features, indicating a role in tumour progression. Given the comparatively high prevalence of hTERT promoter mutations in low-risk and non-metastasising lesions, further studies are required to clarify the prognostic value of hTERT promoter analysis before implementing the analysis in clinical diagnostics.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Loss of miR-514a-3p regulation of PEG3 activates the NF-kappa B pathway in human testicular germ cell tumors.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.

SUMO-modified insulin-like growth factor 1 receptor (IGF-1R) increases cell cycle progression and cell proliferation.

Increasing number of studies have shown nuclear localization of the insulin-like growth factor 1 receptor (nIGF-1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF-1R have, however, still not been disclosed. Previously, we reported that IGF-1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple-SUMO-site-mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R-). Cell clones (R-WT and R-TSM) expressing equal amounts of IGF-1R were selected for experiments. Phosphorylation of IGF-1R, Akt, and Erk upon IGF-1 stimulation was equal in R-WT and R-TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R-WT proliferated substantially faster than R-TSM, which did not differ significantly from the empty vector control. Upon IGF-1 stimulation G1-S-phase progression of R-WT increased from 12 to 38%, compared to 13 to 20% of R-TSM. The G1-S progression of R-WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO-IGF-1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO-IGF-1R dependent mechanisms seem important.

Nuclearly translocated insulin-like growth factor 1 receptor phosphorylates histone H3 at tyrosine 41 and induces SNAI2 expression via Brg1 chromatin remodeling protein.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase that has crucial roles in cell proliferation and protection from apoptosis. It is therefore not surprising that IGF-1R is often found overexpressed in many types of tumors. This has made IGF-1R a prominent target molecule for pharmacological companies to develop new anti-cancer agents. However, several clinical trials during the last 5 years using IGF-1R specific antibodies have shown disappointing results. We have previously shown that upon IGF-1 stimulation, the receptor becomes SUMOylated and translocates into the nucleus of cancer cells to act as a transcription co-factor. Soon after our original study, several others have reported nuclear IGF-1R (nIGF-1R) as well, and some of them have demonstrated a prognostic value of nIGF-1R expression in cancer. In the current study we demonstrate that nIGF-1R binds to and phosphorylates histone H3 at tyrosine 41 (H3Y41) in HeLa cells. Furthermore, our results suggest that phosphorylation of H3Y41 by nIGF-1R, stabilizes the binding of Brg1 chromatin remodeling protein to Histone H3. Our findings suggest that phosphorylated nIGF-1R, rather than total nIGF-1R, plays a superior role in these contexts. We identified SNAI2 oncogene as a target gene for nIGF-1R and its expression was decreased upon mutation of H3Y41 or by Brg1 knockdown. Furthermore, we demonstrate that both IGF-1R and Brg1 binds to the SNAI2 promoter. As SNAI2 protein is implicated in e.g. cancer invasion and metastasis, the nIGF-1R-mediated effects shown in this study may influence such important tumor phenotypic actions.

The Nucleus-Localized Epidermal Growth Factor Receptor Is SUMOylated.

The epidermal growth factor receptor (EGFR) plays important roles in normal and cancer cell growth. The EGFR has principally two different signaling pathways: the canonical kinase route induced at the plasma membrane resulting in an intracellular phosphorylation cascade via MAPKs and PI3K and the more recently discovered pathway by which the receptor functions as a transcriptional co-activator inside the cell nucleus. Full length EGFR translocates to the inner nuclear membrane, via the endoplasmic reticulum, through association with the sec61ß translocon. The c-myc (MYC) and cyclin D1 (CNND1) genes represent two target genes for nuclear EGFR (nEGFR). Here we show that EGFR is SUMOylated and that the SUMO-1-modified receptors are almost unexceptionally nuclear. Co-immunoprecipitation experiments suggest that EGFR is multi-SUMOylated. Using two mass spectrometry-based strategies (matrix-assisted laser desorption ionization time of flight and electrospray ionization liquid chromatography with tandem mass spectrometry), lysine 37 was identified as a SUMO-1-modified residue by both methods. A lysine 37 site mutant (K37R) was transfected into EGFR deficient cells. Total SUMOylation of EGFR was not altered in the K37R-transfected cells, confirming the presence of other SUMOylation sites. To gain preliminary insight into the possible functional role of EGFR SUMOylation, we compared the effect of expression of the wild-type EGFR with the K37R mutant on promoter activity and expression of CMYC and CNND1. Our results indicate that SUMO-1 modification may affect the transcriptional activity of EGFR, which might have additional impact on, e.g., cancer progression.

Nuclear IGF1R is a transcriptional co-activator of LEF1/TCF.

Recent findings suggest that nuclear IGF1R binds to enhancer regions and functions as a transcriptional cofactor. However, the downstream transcriptional regulators of this pathway remain to be defined. Here, we show that nuclear IGF1R associates with the transcription factor LEF1 and increases promoter activity of LEF1 downstream target genes cyclin D1 and axin2. Furthermore, nuclear IGF1R augments protein levels of cyclin D1 and axin2. Our findings suggest a novel function for IGF1R, thus further emphasizing the important role of this receptor in cancer biology.