RESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Glioma/metabolismo , Glioma/radioterapia , Receptor IGF Tipo 1/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Daño del ADN , Glioma/genética , Glioma/patología , Humanos , Inmunohistoquímica , Clasificación del Tumor , Pirazoles/farmacología , Tolerancia a Radiación/efectos de los fármacos , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor IGF Tipo 1/genética , Transducción de Señal/efectos de los fármacos , Análisis de Matrices Tisulares , Triazinas/farmacologíaRESUMEN
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 patients with LSCC 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: Type 1 insulin-like growth factor receptor 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.
Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Factor de Crecimiento Epidérmico/metabolismo , Neoplasias Laríngeas/radioterapia , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/fisiología , Somatomedinas/metabolismo , Anciano , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Estudios de Cohortes , Femenino , Humanos , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patología , Laringectomía , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Tolerancia a RadiaciónRESUMEN
Internalization of ligand-activated type I IGF receptor (IGF1R) is followed by recycling to the plasma membrane, degradation or nuclear translocation. Nuclear IGF1R reportedly associates with clinical response to IGF1R inhibitory drugs, yet its role in the nucleus is poorly characterized. Here, we investigated the significance of nuclear IGF1R in clinical cancers and cell line models. In prostate cancers, IGF1R was predominantly membrane localized in benign glands, while malignant epithelium contained prominent internalized (nuclear/cytoplasmic) IGF1R, and nuclear IGF1R associated significantly with advanced tumor stage. Using ChIP-seq to assess global chromatin occupancy, we identified IGF1R-binding sites at or near transcription start sites of genes including JUN and FAM21, most sites coinciding with occupancy by RNA polymerase II (RNAPol2) and histone marks of active enhancers/promoters. IGF1R was inducibly recruited to chromatin, directly binding DNA and interacting with RNAPol2 to upregulate expression of JUN and FAM21, shown to mediate tumor cell survival and IGF-induced migration. IGF1 also enriched RNAPol2 on promoters containing IGF1R-binding sites. These functions were inhibited by IGF1/II-neutralizing antibody xentuzumab (BI 836845), or by blocking receptor internalization. We detected IGF1R on JUN and FAM21 promoters in fresh prostate cancers that contained abundant nuclear IGF1R, with evidence of correlation between nuclear IGF1R content and JUN expression in malignant prostatic epithelium. Taken together, these data reveal previously unrecognized molecular mechanisms through which IGFs promote tumorigenesis, with implications for therapeutic evaluation of anti-IGF drugs.Significance: These findings reveal a noncanonical nuclear role for IGF1R in tumorigenesis, with implications for therapeutic evaluation of IGF inhibitory drugs. Cancer Res; 78(13); 3497-509. ©2018 AACR.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-jun/genética , ARN Polimerasa II/metabolismo , Receptores de Somatomedina/metabolismo , Anciano , Línea Celular Tumoral , Movimiento Celular/genética , Núcleo Celular/patología , Supervivencia Celular/genética , Cromatina/genética , Cromatina/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Regiones Promotoras Genéticas/genética , Prostatectomía , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor IGF Tipo 1 , Transducción de Señal/genética , Sitio de Iniciación de la Transcripción , Regulación hacia ArribaRESUMEN
BACKGROUND: Activated type 1 insulin-like growth factor receptors (IGF-1Rs) undergo internalisation and nuclear translocation, promoting cell survival. We previously reported that IGF-1R inhibition delays DNA damage repair, sensitising prostate cancer cells to ionising radiation. Here we tested the clinical relevance of these findings. METHODS: We assessed associations between IGF-1R and clinical outcomes by immunohistochemistry in diagnostic biopsies of 136 men treated with 55-70 Gy external beam radiotherapy for prostate cancer, comparing results with publicly available transcriptional data in surgically treated patients. RESULTS: Following radiotherapy, overall recurrence-free survival was shorter in patients whose tumours contained high total, cytoplasmic and internalised (nuclear/cytoplasmic) IGF-1R. High total IGF-1R associated with high primary Gleason grade and risk of metastasis, and cytoplasmic and internalised IGF-1R with biochemical recurrence, which includes patients experiencing local recurrence within the radiation field indicating radioresistance. In multivariate analysis, cytoplasmic, internalised and total IGF-1R were independently associated with risk of overall recurrence, and cytoplasmic IGF-1R was an independent predictor of biochemical recurrence post radiotherapy. Insulin-like growth factor receptors expression did not associate with biochemical recurrence after radical prostatectomy. CONCLUSIONS: These data reveal increased risk of post-radiotherapy recurrence in men whose prostate cancers contain high levels of total or cytoplasmic IGF-1R.
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Neoplasias de la Próstata/radioterapia , Receptor IGF Tipo 1/fisiología , Anciano , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Receptor IGF Tipo 1/análisisRESUMEN
Chordomas are rare primary malignant bone tumors arising from embryonal notochord remnants of the axial skeleton. Chordomas commonly recur following surgery and radiotherapy, and there is no effective systemic therapy. Previous studies implicated receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R), in chordoma biology. We report an adult female patient who presented in 2003 with spinal chordoma, treated with surgery and radiotherapy. She underwent further surgery for recurrent chordoma in 2008, with subsequent progression in pelvic deposits. In June 2009, she was recruited onto the Phase I OSI-906-103 trial of EGFR inhibitor erlotinib with linsitinib, a novel inhibitor of IGF-1R/insulin receptor (INSR). Treatment with 100 mg QD erlotinib and 50 mg QD linsitinib was well-tolerated, and after 18 months a partial response was achieved by RECIST criteria. From 43 months, a protocol modification allowed intra-patient linsitinib dose escalation to 50 mg BID. The patient remained stable on trial treatment for a total of 5 years, discontinuing treatment in August 2014. She subsequently experienced further disease progression for which she underwent pelvic surgery in April 2015. Analysis of DNA extracted from 2008 (pre-trial) tissue showed that the tumor harbored wild-type EGFR, and a PIK3CA mutation was detected in plasma, but not tumor DNA. The 2015 (post-trial) tumor harbored a mutation of uncertain significance in ATM, with no detectable mutations in other components of a 50 gene panel, including EGFR, PIK3CA, and TP53. By immunohistochemistry, the tumor was positive for brachyury, the molecular hallmark of chordoma, and showed weak-moderate membrane and cytoplasmic EGFR. IGF-1R was detected in the plasma membrane and cytoplasm and was expressed more strongly in recurrent tumor than the primary. We also noted heterogeneous nuclear IGF-1R, which has been linked with sensitivity to IGF-1R inhibition. Similar variation in IGF-1R expression and subcellular localization was noted in 15 further cases of chordoma. In summary, this exceptionally durable response suggests that there may be merit in evaluating combined IGF-1R/INSR and EGFR inhibition in patients with chordomas that recur following failure of local treatment.
RESUMEN
Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ â IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma/tratamiento farmacológico , Receptor IGF Tipo 1/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Esquema de Medicación , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacología , Melanoma/genética , Melanoma/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Pirazinas/administración & dosificación , Pirazinas/farmacología , Receptor IGF Tipo 1/metabolismo , Análisis de Supervivencia , Temozolomida , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Head and neck squamous cell carcinomas (HNSCC) are treated with surgery, radiotherapy and cisplatin-based chemotherapy, but survival from locally-advanced disease remains poor, particularly in patients whose tumors are negative for Human papillomavirus (HPV). Type 1 IGF receptor (IGF-1R) is known to promote tumorigenesis and resistance to cancer therapeutics. Here, we assessed IGF-1R immunohistochemistry on tissue microarrays containing 852 cores from 346 HNSCC patients with primary tumors in the oropharynx (n = 231), larynx (85), hypopharynx (28), oral cavity (2). Of these, 236 (68%) were HPV-negative, 110 (32%) positive. IGF-1R was detected in the cell membrane of 36% and cytoplasm of 92% of HNSCCs; in 64 cases with matched normal tonsillar epithelium, IGF-1R was overexpressed in the HNSCCs (P < 0.001). Overall survival (OS) and disease-specific survival (DSS) were reduced in patients whose tumors contained high membrane IGF-1R [OS: hazard ratio (HR) = 1.63, P = 0.006; DSS: HR = 1.63, P = 0.016], cytoplasmic IGF-1R (OS: HR = 1.58, P = 0.009; DSS: HR = 1.58, P = 0.024) and total IGF-1R (OS: HR = 2.02, P < 0.001; DSS: HR = 2.2, P < 0.001). High tumor IGF-1R showed significant association with high-tumor T-stage (P < 0.001) and HPV-negativity (P < 0.001), and was associated with shorter OS when considering patients with HPV-positive (P = 0.01) and negative (P = 0.006) tumors separately. IGF-1R was independently associated with survival in multivariate analysis including HPV, but not when lymphovascular invasion, perineural spread and T-stage were included. Of these factors, only IGF-1R can be manipulated; the association of IGF-1R with aggressive disease supports experimental incorporation of anti-IGF-1R agents into multimodality treatment programs for HPV-negative and high IGF-1R HPV-positive HNSCC.
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Carcinoma de Células Escamosas/mortalidad , Neoplasias de Cabeza y Cuello/mortalidad , Infecciones por Papillomavirus/complicaciones , Receptor IGF Tipo 1/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virología , Transformación Celular Neoplásica/genética , Terapia Combinada , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/virología , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Papillomaviridae , Carcinoma de Células Escamosas de Cabeza y Cuello , Adulto JovenRESUMEN
Inhibition of type 1 IGF receptor (IGF-1R) sensitizes to DNA-damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non-homologous end-joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF-1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF-1R-inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time-dependent accumulation of γH2AX foci in IGF-1R -inhibited or -depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild-type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2(-/-) colorectal cancer cells, compared with isogenic BRCA2(+/-) cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO-3306, which impairs HR by inhibiting CDK1-mediated BRCA1 phosphorylation. R0-3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF-1R inhibition, with 2.4-fold reduction in AZ12253801 GI50 and 13-fold reduction in GI80. These data suggest that responses to IGF-1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild-type, BRCA mutant) that may be intrinsically sensitive to IGF-1R inhibitory drugs.
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Roturas del ADN de Doble Cadena , Reparación del ADN , Recombinación Homóloga/genética , Receptor IGF Tipo 1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Western Blotting , Compuestos de Boro/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Histonas/metabolismo , Recombinación Homóloga/efectos de los fármacos , Humanos , Isoxazoles/farmacología , Masculino , Microscopía Fluorescente , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Pirimidinas/farmacología , Quinolinas/farmacología , Interferencia de ARN , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Receptor IGF Tipo 1/antagonistas & inhibidores , Tiazoles/farmacologíaRESUMEN
Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Resistencia a Antineoplásicos , Factor I del Crecimiento Similar a la Insulina/fisiología , Fosfoproteínas/fisiología , Receptor IGF Tipo 1/antagonistas & inhibidores , Proteínas ras/metabolismo , Animales , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proteínas Dishevelled , Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Concentración 50 Inhibidora , Isoxazoles/farmacología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Pirimidinas/farmacología , Receptor IGF Tipo 1/metabolismo , Proteínas Wnt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
IGF-1R inhibitors arrived in the clinic accompanied by optimism based on preclinical activity of IGF-1R targeting, and recognition that low IGF bioactivity protects from cancer. This was tempered by concerns about toxicity to normal tissue IGF-1R and cross-reactivity with insulin receptor (InsR). In fact, toxicity is not a show-stopper; the key issue is efficacy. While IGF-1R inhibition induces responses as monotherapy in sarcomas and with chemotherapy or targeted agents in common cancers, negative Phase 2/3 trials in unselected patients prompted the cessation of several Pharma programs. Here, we review completed and on-going trials of IGF-1R antibodies, kinase inhibitors and ligand antibodies. We assess candidate biomarkers for patient selection, highlighting the potential predictive value of circulating IGFs/IGFBPs, the need for standardized assays for IGF-1R, and preclinical evidence that variant InsRs mediate resistance to IGF-1R antibodies. We review hypothesis-led and unbiased approaches to evaluate IGF-1R inhibitors with other agents, and stress the need to consider sequencing with chemotherapy. The last few years were a tough time for IGF-1R therapeutics, but also brought progress in understanding IGF biology. Even failed studies include patients who derived benefit; they should be investigated to identify features distinguishing the tumors and host environment of responders from non-responders. We emphasize the importance of incorporating biospecimen collection into trial design, and wording patient consents to allow post hoc analysis of trial material as new data become available. Such information represents the key to unlocking the potential of this approach, to inform the next generation of trials of IGF signalling inhibitors.
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Antineoplásicos/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Ensayos Clínicos como Asunto , Humanos , Valor Predictivo de las Pruebas , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismoRESUMEN
The type 1 insulin-like growth factor receptor (IGF-1R) is a transmembrane glycoprotein composed of two extracellular alpha subunits and two beta subunits with tyrosine kinase activity. The IGF-1R is frequently upregulated in cancers and signals from the cell surface to promote proliferation and cell survival. Recent attention has focused on the IGF-1R as a target for cancer treatment. Here, we report that the nuclei of human tumor cells contain IGF-1R, detectable using multiple antibodies to alpha- and beta-subunit domains. Cell-surface IGF-1R translocates to the nucleus following clathrin-mediated endocytosis, regulated by IGF levels. The IGF-1R is unusual among transmembrane receptors that undergo nuclear import, in that both alpha and beta subunits traffic to the nucleus. Nuclear IGF-1R is phosphorylated in response to ligand and undergoes IGF-induced interaction with chromatin, suggesting direct engagement in transcriptional regulation. The IGF dependence of these phenomena indicates a requirement for the receptor kinase, and indeed, IGF-1R nuclear import and chromatin binding can be blocked by a novel IGF-1R kinase inhibitor. Nuclear IGF-1R is detectable in primary renal cancer cells, formalin-fixed tumors, preinvasive lesions in the breast, and nonmalignant tissues characterized by a high proliferation rate. In clear cell renal cancer, nuclear IGF-1R is associated with adverse prognosis. Our findings suggest that IGF-1R nuclear import has biological significance, may contribute directly to IGF-1R function, and may influence the efficacy of IGF-1R inhibitory drugs.
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Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Neoplasias/metabolismo , Receptor IGF Tipo 1/metabolismo , Células 3T3 , Animales , Neoplasias de la Mama/metabolismo , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Neoplasias Renales/metabolismo , Masculino , Ratones , Neoplasias de la Próstata/metabolismo , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Notch signalling is essential for the development and maintenance of the colonic epithelium. Its inhibition induces a differentiation phenotype in vivo and reduces adenomas in APCmin mice. Whether Notch signals are also required in colorectal cancer (CRC) has remained elusive. Therefore, 64 CRC cell lines were analysed for the occurrence of proteolytically processed, active Notch. RESULTS: 63 CRC lines contained a fragment with approximately the size of the Notch1 intracellular domain (NICD), which is required for signalling. Subsequent analyses with an antibody that specifically recognises the free Val1744 residue generated by gamma-secretase-mediated cleavage of Notch1 showed that a subset of CRC cells lacks this specific Val1744-NICD. Surprisingly, inhibition of Val1744-NICD signalling with different gamma-secretase inhibitors (GSI) did not lead to substantial effects on CRC cell line growth or survival. However, transient activation of Erk upon GSI treatment was detected. Since cisplatin relies on Erk activation for bioactivity in some cells, platinum compounds were tested together with GSI and enhanced cell killing in a subset of Val1744-NICD-positive CRC cell lines was detected. Erk inhibition ablated this combination effect. CONCLUSION: We conclude that gamma-secretase inhibition results in activation of the MAP kinases Erk1/2 and, when used in conjunction, enhances cell death induced by platinum compounds in a large subset of colorectal cancer cell lines.Furthermore the activation of Erk appears to be of particular importance in mediating the enhanced effect seen, as its inhibition abrogates the observed phenomenon. These findings do not only highlight the importance of signalling pathway crosstalk but they may also suggest a new avenue of combination therapy for some colorectal cancers.
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Proliferación Celular , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neoplasias Pancreáticas/metabolismo , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Adenilil Ciclasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Humanos , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Interferencia de ARN , Complejo Shelterina , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND/AIM: The effects of rapamycin (RAPA) were examined in active Heymann nephritis (HN), an experimental model of human membranous nephropathy (MN). Current opinion on the therapy of MN is controversial, and medications used for its treatment have not yielded the expected results. METHODS: In a two-part study, we examined the effects of RAPA (1.5 mg/kg/day) during the induction phase of HN and on the evolving disease. In both parts, control groups of immunized rats not treated with RAPA and control groups of unimmunized rats were observed and sacrificed concurrently with the treated groups. RESULTS: During the induction phase no significant changes in proteinuria were observed in the group treated with RAPA, in comparison to those in the untreated group (p < 0.001). During the evolving disease RAPA significantly lowered proteinuria (p < 0.001). The characteristic pathohistologic changes and IgG depositions along the glomerular basement membrane were considerably diminished, and infiltration of CD8+ cells completely prevented. CONCLUSION: RAPA demonstrated beneficial effects on disease progression, given either in the induction phase or during evolving HN. It would be desirable to investigate the effect of RAPA on patients with MN.
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Glomerulonefritis Membranosa/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Sirolimus/uso terapéutico , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Membrana Basal Glomerular/patología , Glomerulonefritis Membranosa/patología , Glomerulonefritis Membranosa/prevención & control , Masculino , Proteinuria/tratamiento farmacológico , Ratas , Ratas WistarRESUMEN
Activation of the inhibitor of NF-kappaB kinase/NF-kappaB (IKK/NF-kappaB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were generated to mediate acinar cell-specific expression of IKK2 alleles. Expression of dominant-negative IKK2 ameliorated cerulein-induced pancreatitis but did not affect activation of trypsin, an initial event in experimental pancreatitis. Notably, expression of constitutively active IKK2 was sufficient to induce acute pancreatitis. This acinar cell-specific phenotype included edema, cellular infiltrates, necrosis, and elevation of serum lipase levels as well as pancreatic fibrosis. IKK2 activation caused increased expression of known NF-kappaB target genes, including mediators of the inflammatory response such as TNF-alpha and ICAM-1. Indeed, inhibition of TNF-alpha activity identified this cytokine as an important effector of IKK2-induced pancreatitis. Our data identify the IKK/NF-kappaB pathway in acinar cells as being key to the development of experimental pancreatitis and the major factor in the inflammatory response typical of this disease.
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Quinasa I-kappa B/metabolismo , Páncreas/citología , Páncreas/enzimología , Pancreatitis/enzimología , Pancreatitis/etiología , Animales , Ceruletida/toxicidad , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Ratones , Ratones Transgénicos , RatasRESUMEN
BACKGROUND: Activation of the nuclear factor kappaB (NF-kappaB) system is a major event in acute and chronic inflammatory processes. NF-kappaB cascades are comprised of IkappaB kinases, IkappaBs and NF-kappaB dimers. Little is known of the individual roles of these proteins in organ specific inflammation. The aim of the present study was to analyse the consequences of ectopic IkappaB kinase-2 (IKK2) activation in the pancreas of mice. METHODS: Transgenic mice were generated using an inducible genetic system (tet system) to conditionally overexpress a gain of function mutant of IKK2 (tetO-IKK2-EE) in the pancreas. To achieve transgene expression in the pancreas, these animals were crossed with CMV-rtTA mice that are known to express the rtTA protein in the pancreas. RESULTS: In these double transgenic animals, doxycycline treatment induced expression of IKK2-EE (IKK2(CA)) in pancreatic acinar cells resulting in moderate activation of the IkappaB kinase complex, as measured by the immune complex kinase assay, and up to 200-fold activation of the transgene expression cassette, as detected by luciferase assay. IKK2(CA) expression in the pancreas had a mosaic appearance. Ectopic IKK2(CA) mostly activated the classical NF-kappaB pathway. The activation level of the NF-kappaB cascade induced by IKK2(CA) was considerably lower compared with that observed after supramaximal caerulein stimulation but still led to the formation of leucocyte infiltrates first observed after 4 weeks of doxycycline stimulation with a maximum after 8-12 weeks. The infiltrates were mainly composed of B lymphocytes and macrophages. Increased mRNA levels of tumour necrosis factor alpha and RANTES were detected in pancreatic acinar cells. However, only minor damage to pancreatic tissue was observed. A combination of supramaximal caerulein stimulation with induction of IKK2(CA) caused increased tissue damage compared with either IKK2(CA) or caerulein alone. CONCLUSIONS: Our observations suggest that the role of IKK2 activation in pancreatic acini is to induce leucocyte infiltration, but at a moderate level of activation it is not sufficient to induce pancreatic damage in mice. The IKK2(CA) induced infiltrations resemble those observed in autoimmune pancreatitis, indicating a role for IKK2/NF-kappaB in this disease. IKK2(CA) in pancreatic acinar cells increases tissue damage of secretagogue induced experimental pancreatitis underlining the proinflammatory role of the IKK/NF-kappaB pathway in this disease.