Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.

Rab5C enhances resistance to ionizing radiation in rectal cancer.

Rectal cancer represents one third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment reducing the risk of local recurrence. However, a pathologic complete response is only achieved in 10-40% of cases and the mechanisms associated with resistance are poorly understood. To identify potential targets for preventing therapy resistance, a proteomic analysis of biopsy specimens collected from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment was performed and compared with residual tumor tissues removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation. To better understand the role of these proteins in therapy resistance, a rectal adenocarcinoma cell line was irradiated to generate a radiotherapy-resistant lineage. These cells overexpressed the same three proteins identified in the tissue samples. Furthermore, radiotherapy resistance in this in vitro model was found to involve modulation of epidermal growth factor receptor (EGFR) internalization by Rab5C in response to irradiation, affecting expression of the DNA repair proteins, Ku70 and Ku80, and cell resistance. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells and they should be further investigated. KEY MESSAGES: • Rab5C orchestrates a mechanism of radioresistance in rectal adenocarcinoma cell. • Rab5C modulates EGFR internalization and its relocalization to the nucleus. • In the nucleus, EGFR can modulate the expression of the DNA repair proteins, Ku70 and Ku80. • Rab5C, Ku70, and Ku80 are overexpressed in tumor tissues that contain tumor cells that are resistant to chemoradiation treatment.

Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells.

Expression of aquaporin 2 following facial nerve crush in rats.

CONCLUSION: We demonstrated an early increase in aquaporin 2 (AQP2) expression in a motor nerve (extratemporal facial nerve, FN) following acute peripheral compression (crush), concomitant to effective development of motor dysfunction (facial palsy). The early increase in AQP2 expression that occurred concomitantly with the appearance of a deficit in a peripheral motor nerve suggests that this protein is involved in the physiological events associated with post-injury edema, similar to the already demonstrated behavior of AQP4 in the central nervous system (CNS). OBJECTIVE: The aim of this study was to assess the expression of AQP2 in the FN of rats up to 7 days after crush. METHODS: The extratemporal trunk of the right FN of rats was subjected to mechanical crush, and the expression of AQP2 in the affected (right) and non-affected (left) FN was measured by means of western blotting at days 1, 3, and 7 after injury. Behavioral analysis of the development of facial palsy was also performed over the same time period. RESULTS: Increased expression of AQP2 was shown in the affected FN compared with its corresponding control at day 1 after compression, simultaneously with the appearance of facial palsy.

Regulação da atividade de STAT3 e o papel da proteína STI1/Hop secretada em glioblastoma / Regulation of STAT3 activity and the role of secreted STI1/Hop in glioblastoma

O Glioblastoma (GBM) é o tumor de células gliais mais comum e agressivo dentre os tumores cerebrais primários. Caracterizar mecanismos moleculares associados com a progressão desse tumor pode auxiliar no desenvolvimento de novas estratégias para seu tratamento e garantir a maior sobrevida de pacientes. A proteína STAT3 (Proteína transdutora de sinal e ativadora de transcrição 3) é um fator de transcrição ativado por fosforilação e conhecido por seu importante papel na gliomagênese. Com o uso de microarranjos de tecidos (TMAs) avaliamos a expressão total e a localização nuclear de STAT3 e de suas formas fosforiladas pSTAT3 (Y705) e pSTAT3(S727) em astrocitomas e tecido cerebral não tumoral. STAT3 possui uma localização nuclear aumentada em GBMs humanos, quando comparada com astrocitomas de menor grau ou tecido cerebral não tumoral. Interessantemente, o aumento da localização nuclear de STAT3 nos GBMs não está associado com o incremento de suas formas fosforiladas. Além disso, altos níveis de STAT3 nuclear em GBM estão correlacionados com o menor tempo livre de recidiva e a uma menor sobrevida nesses pacientes. Esse perfil não foi visto para as formas fosforiladas, indicando que outros mecanismos de ativação de STAT3, que não a via canônica de fosforilação, podem estar presentes nos GBMs. A identificação dessas modificações pode representar uma nova estratégia terapêutica para a abordagem desses tumores uma vez que as drogas disponíveis atualmente têm como alvo os domínios fosforilados de STAT3. Uma modificação pós-traducional que poderia contribuir para a translocação nuclear de STAT3 é a SUMOilação. A proteína PIAS1 (Proteína inibidora da atividade de STAT1) está envolvida neste mecanismo por mediar a adição de SUMO (Pequena proteína modificadora relacionada à ubiquitina) às proteínas-alvo. Nossos dados mostraram que PIAS1 apresenta maior localização nuclear em GBM quando comparado ao tecido não tumoral...

Glioblastoma is the most common and aggressive primary brain tumor. This work was conducted to characterize molecular mechanisms associated with GBM progression that could assist in developing of new strategies for its treatment and ensure better overall survival of these patients. STAT3 (Signal Transducer and Activator of Transcription 3) is a transcription factor activated by phosphorylation and known for its important role in gliomagenesis. Using tissue microarrays (TMAs) we evaluated the total expression and nuclear localization of STAT3 and its phosphorylated forms, pSTAT3 (Y705) and pSTAT3 (S727) in astrocytomas and non-tumor brain tissue. GBMs showed higher levels of nuclear STAT3 compared to lower grade astrocitomas or non-tumor brain tissue. Interestingly, increased nuclear STAT3 in GBMs is not associated with the improvement of its phosphorylated forms. Moreover, high levels of nuclear STAT3 in GBMs correlate with lower free-recurrence survival and overall survival of these patients. This profile followed by its phosphorylated forms, indicating that other activation mechanisms besides than the canonical STAT3 phosphorylation is present in GBM. The identification of these post-translation modifications may represent new therapeutic strategies for the treatment of these tumors since the currently available drugs target only the phosphorylation sites of STAT3. A posttranslational modification that could contribute to nuclear translocation of STAT3 is the SUMOylation although we were unable to see this interaction in cultured cells. On the other hand, GBMs have a higher nuclear PIAS1 compared to non-tumor brain tissue. PIAS1 protein (Protein Inhibitor of Activity of STAT1) is involved in protein SUMOylation by mediating the addition of SUMO to target proteins. Moreover, PIAS1 promotes nuclear retention of the co-chaperone STI1/Hop (Stress inducible protein 1/Hsp70- Hsp90 organizing protein) in astrocytes treated with gamma radiation, which may...