RESUMEN

Gastroenteropancreatic neuroendocrine neoplasms (GEP­NEN) are a group of rare tumors whose specific pathogenetic mechanisms of resistance to therapies have not been completely revealed yet. Chemotherapy is the main therapeutic approach in patients with GEP­NEN, however, novel combination regimens and targeted therapy are continuously explored. In the present study, the anticancer effect of a novel Ruthenium (Ru)(II)­Bisdemethoxycurcumin (Ru­bdcurc) compound was evaluated in BON­1 cell line, one of the few cell lines derived from GEP­NEN, largely used in experimental research of this type of tumors. The experimental data revealed that the Ru­bdcurc compound induced cell death in a dose­dependent manner, in vitro. Biochemical studies demonstrated that, in response to the lower dose of treatment, BON­1 cells activated the nuclear factor erythroid 2­related factor 2 (NRF2) pathway with induction of some of its targets including catalase and p62 as well as of the antiapoptotic marker Bcl2, all acting as chemoresistance mechanisms. NRF2 induction associated also with increased expression of endogenous p53 which is reported to be dysfunctional in BON­1 cells and to inhibit apoptosis. Genetic or pharmacologic targeting of NRF2 inhibited the activation of the NRF2 pathway, as well as of endogenous dysfunctional p53, in response to the lower dose of Ru­bdcurc, increasing the cell death. To assess the interplay between NRF2 and dysfunctional p53, genetic targeting of p53 showed reduced activation of the NRF2 pathway in response to the lower dose of Ru­bdcurc, increasing the cell death. These findings identified for the first time a possible dysfunctional p53/NRF2 interplay in BON­1 cell line that can be a novel key determinant in cell resistance to cytotoxic agents to be evaluated also in GEP­NEN.

Asunto(s)

Antineoplásicos , Carcinoma Neuroendocrino , Curcumina , Tumores Neuroendocrinos , Rutenio , Humanos , Curcumina/farmacología , Proyectos Piloto , Factor 2 Relacionado con NF-E2 , Proteína p53 Supresora de Tumor/genética , Antineoplásicos/farmacología , Tumores Neuroendocrinos/tratamiento farmacológico

RESUMEN

Homeodomain-interacting protein kinase 2 (HIPK2) is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer. The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in colorectal cancer. We evaluated a retrospective cohort of colorectal cancer samples by IHC for HIPK2 expression and by next-generation sequencing (NGS) for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2-positive cells increases with tumor progression, significantly correlates with tumor-node-metastasis (TNM) staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer-related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated colorectal cancer cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in patients with colorectal cancer and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway. IMPLICATIONS: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated colorectal cancer.

Asunto(s)

Neoplasias Colorrectales , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Neoplasias Colorrectales/patología , Humanos , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Estudios Retrospectivos , Transducción de Señal/genética

RESUMEN

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

Asunto(s)

Empalme Alternativo/genética , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Citocinesis/genética , Intrones , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Portadoras/genética , Codón de Terminación , Exones , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Espastina/metabolismo , Transfección

RESUMEN

Centrosomal p53 has been described for three decades but its role is still unclear. We previously reported that, in proliferating human cells, p53 transiently moves to centrosomes at each mitosis. Such p53 mitotic centrosome localization (p53-MCL) occurs independently from DNA damage but requires ATM-mediated p53Ser15 phosphorylation (p53Ser15P) on discrete cytoplasmic p53 foci that, through MT dynamics, move to centrosomes during the mitotic spindle formation. Here, we show that inhibition of p53-MCL, obtained by p53 depletion or selective impairment of p53 centrosomal localization, induces centrosome fragmentation in human nontransformed cells. In contrast, tumor cells or mouse cells tolerate p53 depletion, as expected, and p53-MCL inhibition. Such tumor- and species-specific behavior of centrosomal p53 resembles that of the recently identified sensor of centrosome-loss, whose activation triggers the mitotic surveillance pathway in human nontransformed cells but not in tumor cells or mouse cells. The mitotic surveillance pathway prevents the growth of human cells with increased chance of making mitotic errors and accumulating numeral chromosome defects. Thus, we evaluated whether p53-MCL could work as a centrosome-loss sensor and contribute to the activation of the mitotic surveillance pathway. We provide evidence that centrosome-loss triggered by PLK4 inhibition makes p53 orphan of its mitotic dock and promotes accumulation of discrete p53Ser15P foci. These p53 foci are required for the recruitment of 53BP1, a key effector of the mitotic surveillance pathway. Consistently, cells from patients with constitutive impairment of p53-MCL, such as ATM- and PCNT-mutant carriers, accumulate numeral chromosome defects. These findings indicate that, in nontransformed human cells, centrosomal p53 contributes to safeguard genome integrity by working as sensor for the mitotic surveillance pathway.

Asunto(s)

Centrosoma/metabolismo , Mitosis , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Cromosomas Humanos , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética

RESUMEN

Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A- and B-type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1kof/kof ) mice exhibit telomeric defects and that Ft1kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1kof/kof ; p53ko/ko and Ft1kof/kof ; p53+/ko ) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53-dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model.

Asunto(s)

Progeria/genética , Proteínas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Proteínas Reguladoras de la Apoptosis , Células Cultivadas , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Mutación , Progeria/metabolismo , Progeria/patología , Proteínas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

HIPK2 is a Y-regulated S/T kinase involved in various cellular processes, including cell-fate decision during development and DNA damage response. Cis-autophosphorylation in the activation-loop and trans-autophosphorylation at several S/T sites along the protein are required for HIPK2 activation, subcellular localization, and subsequent posttranslational modifications. The specific function of a few of these autophosphorylations has been recently clarified; however, most of the sites found phosphorylated by mass spectrometry in human and/or mouse HIPK2 are still uncharacterized. In the process of studying HIPK2 in human colorectal cancers, we identified a mutation (T566P) in a site we previously found autophosphorylated in mouse Hipk2. Biochemical and functional characterization of this site showed that compared to wild type (wt) HIPK2, HIPK2-T566P maintains nuclear-speckle localization and has only a mild reduction in kinase and growth arresting activities upon overexpression. Next, we assessed cell response following UV-irradiation or treatment with doxorubicin, two well-known HIPK2 activators, by evaluating cell number and viability, p53-Ser46 phosphorylation, p21 induction, and caspase cleavage. Interestingly, cells expressing HIPK2-T566P mutant did not respond to UV-irradiation, while behaved similarly to wt HIPK2 upon doxorubicin-treatment. Evaluation of HIPK2-T566 phosphorylation status by a T566-phospho-specific antibody showed constitutive phosphorylation in unstressed cells, which was maintained after doxorubicin-treatment but inhibited by UV-irradiation. Taken together, these data show that HIPK2-T566 phosphorylation contributes to UV-induced HIPK2 activity but it is dispensable for doxorubicin response.

Asunto(s)

Neoplasias Óseas/tratamiento farmacológico , Proteínas Portadoras/metabolismo , Doxorrubicina/farmacología , Osteosarcoma/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias Óseas/enzimología , Neoplasias Óseas/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Humanos , Ratones , Osteosarcoma/enzimología , Osteosarcoma/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transfección , Rayos Ultravioleta

RESUMEN

Small interfering RNAs (siRNAs) are widely used to study gene function and extensively exploited for their potential therapeutic applications. HIPK2 is an evolutionary conserved kinase that binds and phosphorylates several proteins directly or indirectly related to apoptosis. Recently, an alternatively spliced isoform skipping 81 nucleotides of exon 8 (Hipk2-Δe8) has been described. Selective depletion of Hipk2 full-length (Hipk2-FL) with a specific siRNA that spares the Hipk2-Δe8 isoform has been shown to strongly induce apoptosis, suggesting an unpredicted dominant-negative effect of Hipk2-FL over the Δe8 isoform. From this observation, we sought to take advantage and assessed the therapeutic potential of generating Hipk2 isoform unbalance in tumor-initiating cells derived from colorectal cancer patients. Strong reduction of cell viability was induced in vitro and in vivo by the originally described exon 8-specific siRNA, supporting a potential therapeutic application. However, validation analyses performed with additional exon8-specific siRNAs with different stabilities showed that all exon8-targeting siRNAs can induce comparable Hipk2 isoform unbalance but only the originally reported e8-siRNA promotes cell death. These data show that loss of viability does not depend on the prevalence of Hipk2-Δe8 isoform but it is rather due to microRNA-like off-target effects.

Asunto(s)

Apoptosis/genética , Proteínas Portadoras/genética , Eliminación de Gen , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Empalme Alternativo , Animales , Western Blotting , Línea Celular Transformada , Línea Celular Tumoral , Supervivencia Celular/genética , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Exones/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones Desnudos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/metabolismo , Tratamiento con ARN de Interferencia/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de Xenoinjerto