STAT3 protein is an important transcription factor and a central mediator of many signaling pathways. Its constitutive activation drives oncogenic functions and contributes to the progression and the maintenance of many human tumors. In addition, STAT3 is an important regulator of immune system and it is implicated in the cancer-related inflammation. Currently, STAT3 is considered a promising anti-cancer target in several contexts so it would be interesting to study the role of this protein in multiple aspects of pathological conditions. The first part of my research was focused in the involvement of STAT3 in microglia activation under amyloid stimulation. Microglia are the unique resident immune cells of the CNS acting as primary mediators of inflammation. They are responsible of the release of pro-inflammatory cytokines and the related neurodegenerative disorders such as Alzheimer’s disease. The JAK/STAT3 system was proven to be involved in the transcriptional regulation of many inflammatory factors. Particularly, in the neuronal compartment the JAK/STAT3 signaling cooperates in the neurotoxicity, induced by beta amyloid exposure, and it is engaged in the chronic microglia activation by several stimuli. However, molecular and kinetic details of STAT3 involvement in the microglia-mediated neuroinflammation are still unknown. In order to understand the complex signaling leading to early and late responsiveness of microglia to amyloid, we analyzed STAT3 signaling and its post-translational modifications (PTMs) by Western Blotting. PTMs are known to influence the protein activities and to trigger diverse cellular events [59]. The second part of my research was focused in studying the role of STAT3 in Prostate Cancer (PCa). PCa is characterized by an evident clinical, histological and biological heterogeneity. Clinically PCa goes from “latent”, to an “aggressive” form. The “latent” form, a slow-growing tumor, appears histological differentiated, and indolent, without any symptoms. On the contrary, the aggressive form is a fast-growing tumor with a lethal progression. Identify the PCa type is an important step for the optimal treatment and the consequent decrease of the morbidity and mortality incidence. Recently, the early stage detection is based mostly on evaluation of the serum PSA (Prostate Specific Antigen) levels in patients. However, the most important limitation of PSA is in its specificity: it can be elevated, also, in non-malignant conditions, such as prostatitis and benign prostatic hypertrophy. Actually, all the parameters used in urology, the PSA levels, Gleason Score classification and the TNM system, are not sufficient to predict the tumor progression types. Therefore, the clinicians and researchers are forced to find more precise and sensitive biomarker suitable for PCa diagnostics as well as prognostics and therapy. In this scenario many data suggest a crucial role of STAT3 in prostate cancer progression when it was found constitutively activated. Thanks to the collaboration with pathologist of “Policlinico Umberto I” we analyzed STAT3 PTMs in FFPE (Formalin Fixed and Paraffin Embedded) tissues, came from radical prostatectomy, by immunoblotting and immunohistochemistry assays. The goal was to investigate STAT3 activity in early and advanced tumor stages. In particular, we were interested finding a correlation between PTMs and different PCa clinical stages. In addition, we looked at the presence of specific STAT3 interactors, ERp57, Ref-1 and p300, known to be involved in particular cellular conditions like inflammation or oxidative stress. Finally, the next goal was to understand the influences of cell signaling in STAT3 gene expression profile in vivo and in vitro. The last part of my PhD program started in the Memorial Sloan-Kettering Cancer Center, working on HER2+ Brest Cancer (BC) in the laboratory of doctor Jacquelin F. Bromberg. In the adjuvant scenario, for patients, with HER-2 over-expressing Breast Cancer, treatment with Trastuzumab is the standard of care and Lapatinib is currently approved for metastatic disease. However, despite these agents have been extremely effective in treating HER2+ BC, de novo or acquired drug resistance is common and the cure for metastatic disease is still an utopia. This problem highlights the importance of developing new therapeutic strategies and simultaneous or sequential targeting of multiple pathways could thwart the development of drug resistance. Preclinical studies showed that the interleukin-6/JAK2/STAT3 pathway is preferentially activated in BC cells. The aim of this study was to demonstrate that JAK2 inhibition restores the sensitivity to the anti-HER2 drug, Lapatinib, a tyrosine Kinase inhibitor (TKI), in BC cell lines. We analyzed the synergistic effects of drugs combination (AZD1478 and Lapatinib) by MTT assay and Compusyn software. In addition, we looked also at the influences of those two drugs in HER family proteins and the corresponding downstream signaling pathways expression and activation. It would be interesting to extend the experiment in Lapatinib resistant BC cell lines and compare the behavior of the acquired resistant cells with the primary resistant ones treated with JAK2 inhibitor alone and in combination with Lapatinib.
Stat3, trasduttore di segnale e fattore di trascrizione, è una proteina nota per essere coinvolta nello sviluppo e nella progressione di molti tipi di tumori. Dati di letteratura dimostrano la sua attivazione costitutiva legata all' espressione di geni che regolano importanti processi cellulari: sopravvivenza, infiammazione, ciclo cellulare, angiongenesi e metastasi. La prima parte della mia ricerca si è focalizzata sul coinvolgimento della STAT3 nell' attivazione microgliale sotto stimolazione delle beta amiloide. La microglia rappresentano le cellule immunitarie del sistema nervoso centrale, mediatori dell' infiammazione, essi sono responsabili del rilascio di citochine pro-infiammatorie. Tuttavia, il ruolo difensivo di queste cellule può trasformarsi in uno altrettanto dannoso, scatenando una infiammazione cronica responsabile di alcune patologie neurodegenerative, tra cui l'Alzheimer. Il segnale JAK/STAT3 è noto per essere coinvolto nella regolazione trascrizionale di diversi fattori dell'infiammazione, inoltre è stato dimostrato il suo coinvolgimento nella neurotossicità indotta dalla beta-amiloide e nell'attivazione cronica microgliale. In questo studio è stata analizzata la STAT3 e le sue modificazioni post-traduzionale (PTMs), in cellule BV2 e di microglia primarie allo scopo di mettere alla luce i processi cinetici e molecolari della proteina durante il processo di infiammazione indotta dalla microglia.la seconda parte della ricerca è stata rivolta ad indagare il ruolo della STAT3, delle sue PTMs e dei suoi interattori in tessuti FFPE di tumore alla prostata con diversi gradi di Gleason. Inoltre è stato effettuato anche una correlazione tra le PTMs e la diversa espressione genica. L'obiettivo è di poter fornire nuovi biomarker in grado di dare una più precisa informazione sulla stadiazione tumorale al momento della diagnosi nonchè gettare nuove basi per una ricerca terapeutica più mirata. infine, l'ultima parte del progetto è stato svolto al Memorial Sloan-Kettering Cancer Center, per la durata di un anno, nel laboratorio della Dott. Jacquelin F. Bromberg dove mi sono focalizzata nello studio del tumore alla mammella sovraesprimenti il recettore HER2. In particolare ho analizzato in vitro gli effetti della combinazione di farmaci (Laparinib e AZD1478) come possibile forma terapeutica contro la resistenza primaria e secondaria sviluppata nei pazienti sotto trattamento con farmaci anti HER2.
Stat3-Mediated Signaling Pathway in Physio-Phatological Processes / Romaniello, Donatella. - STAMPA. - (2015).
Stat3-Mediated Signaling Pathway in Physio-Phatological Processes
ROMANIELLO, DONATELLA
01/01/2015
Abstract
STAT3 protein is an important transcription factor and a central mediator of many signaling pathways. Its constitutive activation drives oncogenic functions and contributes to the progression and the maintenance of many human tumors. In addition, STAT3 is an important regulator of immune system and it is implicated in the cancer-related inflammation. Currently, STAT3 is considered a promising anti-cancer target in several contexts so it would be interesting to study the role of this protein in multiple aspects of pathological conditions. The first part of my research was focused in the involvement of STAT3 in microglia activation under amyloid stimulation. Microglia are the unique resident immune cells of the CNS acting as primary mediators of inflammation. They are responsible of the release of pro-inflammatory cytokines and the related neurodegenerative disorders such as Alzheimer’s disease. The JAK/STAT3 system was proven to be involved in the transcriptional regulation of many inflammatory factors. Particularly, in the neuronal compartment the JAK/STAT3 signaling cooperates in the neurotoxicity, induced by beta amyloid exposure, and it is engaged in the chronic microglia activation by several stimuli. However, molecular and kinetic details of STAT3 involvement in the microglia-mediated neuroinflammation are still unknown. In order to understand the complex signaling leading to early and late responsiveness of microglia to amyloid, we analyzed STAT3 signaling and its post-translational modifications (PTMs) by Western Blotting. PTMs are known to influence the protein activities and to trigger diverse cellular events [59]. The second part of my research was focused in studying the role of STAT3 in Prostate Cancer (PCa). PCa is characterized by an evident clinical, histological and biological heterogeneity. Clinically PCa goes from “latent”, to an “aggressive” form. The “latent” form, a slow-growing tumor, appears histological differentiated, and indolent, without any symptoms. On the contrary, the aggressive form is a fast-growing tumor with a lethal progression. Identify the PCa type is an important step for the optimal treatment and the consequent decrease of the morbidity and mortality incidence. Recently, the early stage detection is based mostly on evaluation of the serum PSA (Prostate Specific Antigen) levels in patients. However, the most important limitation of PSA is in its specificity: it can be elevated, also, in non-malignant conditions, such as prostatitis and benign prostatic hypertrophy. Actually, all the parameters used in urology, the PSA levels, Gleason Score classification and the TNM system, are not sufficient to predict the tumor progression types. Therefore, the clinicians and researchers are forced to find more precise and sensitive biomarker suitable for PCa diagnostics as well as prognostics and therapy. In this scenario many data suggest a crucial role of STAT3 in prostate cancer progression when it was found constitutively activated. Thanks to the collaboration with pathologist of “Policlinico Umberto I” we analyzed STAT3 PTMs in FFPE (Formalin Fixed and Paraffin Embedded) tissues, came from radical prostatectomy, by immunoblotting and immunohistochemistry assays. The goal was to investigate STAT3 activity in early and advanced tumor stages. In particular, we were interested finding a correlation between PTMs and different PCa clinical stages. In addition, we looked at the presence of specific STAT3 interactors, ERp57, Ref-1 and p300, known to be involved in particular cellular conditions like inflammation or oxidative stress. Finally, the next goal was to understand the influences of cell signaling in STAT3 gene expression profile in vivo and in vitro. The last part of my PhD program started in the Memorial Sloan-Kettering Cancer Center, working on HER2+ Brest Cancer (BC) in the laboratory of doctor Jacquelin F. Bromberg. In the adjuvant scenario, for patients, with HER-2 over-expressing Breast Cancer, treatment with Trastuzumab is the standard of care and Lapatinib is currently approved for metastatic disease. However, despite these agents have been extremely effective in treating HER2+ BC, de novo or acquired drug resistance is common and the cure for metastatic disease is still an utopia. This problem highlights the importance of developing new therapeutic strategies and simultaneous or sequential targeting of multiple pathways could thwart the development of drug resistance. Preclinical studies showed that the interleukin-6/JAK2/STAT3 pathway is preferentially activated in BC cells. The aim of this study was to demonstrate that JAK2 inhibition restores the sensitivity to the anti-HER2 drug, Lapatinib, a tyrosine Kinase inhibitor (TKI), in BC cell lines. We analyzed the synergistic effects of drugs combination (AZD1478 and Lapatinib) by MTT assay and Compusyn software. In addition, we looked also at the influences of those two drugs in HER family proteins and the corresponding downstream signaling pathways expression and activation. It would be interesting to extend the experiment in Lapatinib resistant BC cell lines and compare the behavior of the acquired resistant cells with the primary resistant ones treated with JAK2 inhibitor alone and in combination with Lapatinib.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
