Increased EZH2 expression in prostate cancer is associated with metastatic recurrence following external beam radiotherapy

Background Enhancer of zeste 2 (EZH2) promotes prostate cancer progression. We hypothesized that increased EZH2 expression is associated with postradiotherapy metastatic disease recurrence, and may promote radioresistance. Methods EZH2 expression was investigated using immunohistochemistry in diagnostic prostate biopsies of 113 prostate cancer patients treated with radiotherapy with curative intent. Associations between EZH2 expression in malignant and benign tissue in prostate biopsy cores and outcomes were investigated using univariate and multivariate Cox regression analyses. LNCaP and PC3 cell radiosensitivity was investigated using colony formation and γH2AX assays following UNC1999 chemical probe‐mediated EZH2 inhibition. Results While there was no significant association between EZH2 expression and biochemical recurrence following radiotherapy, univariate analysis revealed that prostate cancer cytoplasmic and total EZH2 expression were significantly associated with metastasis development postradiotherapy (P = 0.034 and P = 0.003, respectively). On multivariate analysis, the prostate cancer total EZH2 expression score remained statistically significant (P = 0.003), while cytoplasmic EZH2 expression did not reach statistical significance (P = 0.053). No association was observed between normal adjacent prostate EZH2 expression and biochemical recurrence or metastasis. LNCaP and PC3 cell treatment with UNC1999 reduced histone H3 lysine 27 tri‐methylation levels. Irradiation of LNCaP or PC3 cells with a single 2 Gy fraction with UNC1999‐mediated EZH2 inhibition resulted in a statistically significant, though modest, reduction in cell colony number for both cell lines. Increased γH2AX foci were observed 24 hours after ionizing irradiation in LNCaP cells, but not in PC3, following UNC1999‐mediated EZH2 inhibition vs controls. Conclusions Taken together, these results reveal that high pretreatment EZH2 expression in prostate cancer in diagnostic biopsies is associated with an increased risk of postradiotherapy metastatic disease recurrence, but EZH2 function may only at most play a modest role in promoting prostate cancer cell radioresistance

IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits

Mature type 1 insulin-like growth factor receptors (IGF-1Rs) are heterotetrameric structures comprising two extracellular α-subunits disulphide-bonded to two transmembrane β-subunits with tyrosine kinase activity. IGF-1R is a well-known cell surface mediator of malignant growth, with an incompletely understood role upon nuclear import as a transcriptional regulator. Previous characterisation of nuclear IGF-1R focused on IGF-1Rβ. Here, we aimed to clarify the source of nuclear IGF-1R and investigate whether α-subunits contribute to nuclear IGF-1R function. Using prostate cancer cell lines DU145 and 22Rv1 we detected nuclear α- and β-subunits, with increase in nuclear signal upon IGF-treatment and reduction in response to IGF-1R inhibitor BMS-754807. Following biotinylation of cell surface proteins, biotinylated α- and β-subunits were detected in nuclear extracts of both cell lines. Furthermore, α- and β-subunits reciprocally co-precipitated from nuclear extract. Finally, we detected recruitment of both subunits to regulatory regions of chromatin, including the promoter of the oncogene JUN, that we previously identified in ChIP-seq as sites of IGF-1Rβ enrichment. These data confirm the cell surface origin of nuclear IGF-1R, suggest the presence of nuclear αβ complexes and reveal that both IGF-1Rα- and β-subunits contribute to pro-tumorigenic functions of nuclear IGF-1R

Bad to the bone: The role of the insulin-like growth factor axis in osseous metastasis

Bone metastases are a frequent complication of cancer that are associated with considerable morbidity. Current treatments may temporarily palliate the symptoms of bone metastases but often fail to delay their progression. Bones provide a permissive environment because they are characterized by dynamic turnover, secreting factors required for bone maintenance but also stimulating the establishment and growth of metastases. Insulin-like growth factors (IGF) are the most abundant growth factors in bone and are required for normal skeletal development and function. Via activation of the IGF-1 receptors (IGF-1R) and variant insulin receptors, IGFs promote cancer progression, aggressiveness, and treatment resistance. Of specific relevance to bone biology, IGFs contribute to the homing, dormancy, colonization, and expansion of bone metastases. Furthermore, preclinical evidence suggests that tumor cells can be primed to metastasize to bone by a high IGF-1 environment in the primary tumor, suggesting that bone metastases may reflect IGF dependency. Therapeutic targeting of the IGF axis may therefore provide an effective method for treating bone metastases. Indeed, anti-IGF-1R antibodies, IGF-1R tyrosine kinase inhibitors, and anti-IGF-1/2 antibodies have demonstrated antitumor activity in preclinical models of prostate and breast cancer metastases, either alone or in combination with other agents. Several studies suggest that such treatments can inhibit bone metastases without affecting growth of the primary tumor. Although previous trials of anti-IGF-1R drugs have generated negative results in unselected patients, these considerations suggest that future clinical trials of IGF-targeted agents may be warranted in patients with bone metastases

Radioresistant laryngeal cancers upregulate type 1 IGF receptor and exhibit increased cellular dependence on IGF and EGF signalling

Objectives Patients failing radiotherapy for laryngeal squamous cell carcinoma (LSCC) often require salvage total laryngectomy which has major functional consequences, highlighting a need for biomarkers of radiotherapy resistance. In other tumour types, radioresistance has been linked to epidermal growth factor receptor (EGFR) and type 1 insulin‐like growth factor receptor (IGF‐1R). Here, we evaluated IGF‐1R and EGFR as predictors and mediators of LSCC radioresistance. Design We compared IGF‐1R and EGFR immunohistochemical scores in LSCC patients achieving long‐term remission post‐radiotherapy (n=23), patients treated with primary laryngectomy (n=22) or salvage laryngectomy following radiotherapy recurrence (n=18). To model radioresistanceandnbsp;in vitro, two LSCC cell lines underwent clinically‐relevant irradiation to 55 Gy in 2.75 Gy fractions. Results IGF‐1R expression was higher in pre‐treatment biopsies of radiotherapy‐failures compared with those in long‐term remission, and was upregulated post‐radiotherapy. Patients undergoing primary laryngectomy had more advanced T/N stage and greater tumour IGF‐1R content than those achieving long‐term remission. Pre‐treatment EGFR did not associate with radiotherapy outcomes but showed a trend to upregulation post‐irradiation.andnbsp;In vitro, radiosensitivity was enhanced by inhibition of EGFR but not IGF. Repeated irradiation upregulated IGF‐1R in BICR18 and SQ20B cells and EGFR in SQ20B, and enhanced SQ20B radioresistance. Repeatedly‐irradiated SQ20B_55 cells were not radiosensitised by inhibition of IGF and/or EGFR, but IGF‐1R:EGFR co‐inhibition suppressed baseline cell survival more effectively than blockade of either pathway alone, and more effectively than in parental cells. Conclusions Radiation upregulates IGF‐1R and may enhance IGF/EGFR dependence, suggesting that IGF/EGFR blockade may have activity in LSCCs that recur post‐radiotherapy.</p

IGF-1R nuclear import and recruitment to chromatin involves both alpha and beta subunits

Mature type 1 insulin-like growth factor receptors (IGF-1Rs) are heterotetrameric structures comprising two extracellular α-subunits disulphide-bonded to two transmembrane β-subunits with tyrosine kinase activity. IGF-1R is a well-known cell surface mediator of malignant growth, with an incompletely understood role upon nuclear import as a transcriptional regulator. Previous characterisation of nuclear IGF-1R focused on IGF-1Rβ. Here, we aimed to clarify the source of nuclear IGF-1R and investigate whether α-subunits contribute to nuclear IGF-1R function. Using prostate cancer cell lines DU145 and 22Rv1 we detected nuclear α- and β-subunits, with increase in nuclear signal upon IGF-treatment and reduction in response to IGF-1R inhibitor BMS-754807. Following biotinylation of cell surface proteins, biotinylated α- and β-subunits were detected in nuclear extracts of both cell lines. Furthermore, α- and β-subunits reciprocally co-precipitated from nuclear extract. Finally, we detected recruitment of both subunits to regulatory regions of chromatin, including the promoter of the oncogene JUN, that we previously identified in ChIP-seq as sites of IGF-1Rβ enrichment. These data confirm the cell surface origin of nuclear IGF-1R, suggest the presence of nuclear αβ complexes and reveal that both IGF-1Rα- and β-subunits contribute to pro-tumorigenic functions of nuclear IGF-1R

Radioresistant laryngeal cancers upregulate type 1 IGF receptor and exhibit increased cellular dependence on IGF and EGF signalling

Objectives Patients failing radiotherapy for laryngeal squamous cell carcinoma (LSCC) often require salvage total laryngectomy which has major functional consequences, highlighting a need for biomarkers of radiotherapy resistance. In other tumour types, radioresistance has been linked to epidermal growth factor receptor (EGFR) and type 1 insulin‐like growth factor receptor (IGF‐1R). Here, we evaluated IGF‐1R and EGFR as predictors and mediators of LSCC radioresistance. Design We compared IGF‐1R and EGFR immunohistochemical scores in LSCC patients achieving long‐term remission post‐radiotherapy (n=23), patients treated with primary laryngectomy (n=22) or salvage laryngectomy following radiotherapy recurrence (n=18). To model radioresistance in vitro, two LSCC cell lines underwent clinically‐relevant irradiation to 55 Gy in 2.75 Gy fractions. Results IGF‐1R expression was higher in pre‐treatment biopsies of radiotherapy‐failures compared with those in long‐term remission, and was upregulated post‐radiotherapy. Patients undergoing primary laryngectomy had more advanced T/N stage and greater tumour IGF‐1R content than those achieving long‐term remission. Pre‐treatment EGFR did not associate with radiotherapy outcomes but showed a trend to upregulation post‐irradiation. In vitro, radiosensitivity was enhanced by inhibition of EGFR but not IGF. Repeated irradiation upregulated IGF‐1R in BICR18 and SQ20B cells and EGFR in SQ20B, and enhanced SQ20B radioresistance. Repeatedly‐irradiated SQ20B_55 cells were not radiosensitised by inhibition of IGF and/or EGFR, but IGF‐1R:EGFR co‐inhibition suppressed baseline cell survival more effectively than blockade of either pathway alone, and more effectively than in parental cells. Conclusions Radiation upregulates IGF‐1R and may enhance IGF/EGFR dependence, suggesting that IGF/EGFR blockade may have activity in LSCCs that recur post‐radiotherapy.</p

Bad to the bone: The role of the insulin-like growth factor axis in osseous metastasis

Bone metastases are a frequent complication of cancer that are associated with considerable morbidity. Current treatments may temporarily palliate the symptoms of bone metastases but often fail to delay their progression. Bones provide a permissive environment because they are characterized by dynamic turnover, secreting factors required for bone maintenance but also stimulating the establishment and growth of metastases. Insulin-like growth factors (IGF) are the most abundant growth factors in bone and are required for normal skeletal development and function. Via activation of the IGF-1 receptors (IGF-1R) and variant insulin receptors, IGFs promote cancer progression, aggressiveness, and treatment resistance. Of specific relevance to bone biology, IGFs contribute to the homing, dormancy, colonization, and expansion of bone metastases. Furthermore, preclinical evidence suggests that tumor cells can be primed to metastasize to bone by a high IGF-1 environment in the primary tumor, suggesting that bone metastases may reflect IGF dependency. Therapeutic targeting of the IGF axis may therefore provide an effective method for treating bone metastases. Indeed, anti-IGF-1R antibodies, IGF-1R tyrosine kinase inhibitors, and anti-IGF-1/2 antibodies have demonstrated antitumor activity in preclinical models of prostate and breast cancer metastases, either alone or in combination with other agents. Several studies suggest that such treatments can inhibit bone metastases without affecting growth of the primary tumor. Although previous trials of anti-IGF-1R drugs have generated negative results in unselected patients, these considerations suggest that future clinical trials of IGF-targeted agents may be warranted in patients with bone metastases

The ERANOS Code and data system for fast reactor neutronic analyses

International audienceThe main modelling options of ERANOS 2.0, the latest version of the ERANOS fast reactor analysis code and data system, are described. These include the ECCO cell and lattice code (Collision Probability Method in many groups using the subgroup method), the BISTRO 2-D S n transport code, the TGV/VARIANT 3-D Nodal Variational Transport code, and a special procedure for creating equivalent homogeneous cross sections for control rods. The recommended "reference" core calculation route is presented. A summary of the ERANOS extensive validation is provided. It is concluded that ERANOS 2.0 can predict the main characteristics of conventional as well as advanced liquid-metal-cooled fast reactors with an excellent accuracy, and that it can also be used for modelling advanced fast reactor cores, source-driven sub-critical media and gas-cooled fast reactors

Type 1 IGF receptor associates with adverse outcome and cellular radioresistance in paediatric high-grade glioma.

High-grade glioma (HGG) is highly resistant to therapy, prompting us to investigate the contribution of insulin-like growth factor receptor (IGF-1R), linked with radioresistance in other cancers. IGF-1R immunohistochemistry in 305 adult HGG (aHGG) and 103 paediatric/young adult HGG (pHGG) cases revealed significant association with adverse survival in pHGG, with median survival of 13.5 vs 29 months for pHGGs with moderate/strong vs negative/weak IGF-1R (p = 0.011). Secondly, we tested IGF-1R inhibitor BMS-754807 in HGG cells, finding minimal radiosensitisation of 2/3 aHGG cell lines (dose enhancement ratios DERs < 1.60 at 2-8 Gy), and greater radiosensitisation of 2/2 pHGG cell lines (DERs ≤ 4.16). BMS-754807 did not influence radiation-induced apoptosis but perturbed the DNA damage response with altered induction/resolution of γH2AX, 53BP1 and RAD51 foci. These data indicate that IGF-1R promotes radioresistance in pHGG, potentially contributing to the association of IGF-1R with adverse outcome and suggesting IGF-1R as a candidate treatment target in pHGG

Targeting IGF perturbs global replication through ribonucleotide reductase dysfunction

Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR–CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers