As the most frequently diagnosed cancer in North American men, prostate cancer can progress to the androgen independent stage after initial response to androgen ablation therapy. The molecular mechanisms involved in the hormonal progression of prostate cancer are not completely understood. Here, we analyze changes in the transcriptome of prostate cancer cells at different stages of progression to reveal potential mechanisms. Applying Affymetrix GeneChip technology, we identified the transcriptomes in response to stimulation of androgen and PKA pathways in human prostate cancer cells. In addition to PSA, other common target genes were identified. Genes differentially expressed in response to androgen and stimulation of the PKA pathway in vitro were also differentially expressed during hormonal progression in vivo. Upon androgen stimulation, androgen receptor binds to a functional androgen response element within the promoter region of SESN1, a p53 targeted gene, and represses its expression. The expression of SESN1 was induced by castration in LNCaP xenografts, but the expression was eventually suppressed again in the androgen independent stage of prostate cancer. Knockdown of SESN1 promoted the proliferation of prostate cancer cells. Expression patterns of androgen-regulated genes in androgen independent tumours were revealed to be more similar to that from before castration than to the tumors under androgen ablation. The β-catenin, a potent coactivator of the androgen receptor, and Wnt pathway was deregulated in androgen-independent tumours. There was increased nuclear colocalization and interaction of androgen receptor and β-catenin with hormonal progression of prostate cancer. This study provides insight into hormonal effects on prostate cancer and possible pathways involved in the development of androgen independent disease, as well as potential therapeutic targets. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Lithium is given to millions of bipolar disorder or post-traumatic disorder patients. The recent studies also support a role for lithium in treating neurodegenerative disease such as Parkinson's disease and stroke. Lithium treatment leads to lithium nephropathy, which includes lithium-induced nephrogenic diabetic insipidus (NDI), lithium-induced renal cell proliferation leading to the formation of microcysts in the kidney, and lithium-induced renal fibrosis. However, there is still a gap in understanding the mechanisms and signaling pathways involved in regulating lithium-induced nephropathy. mTOR pathway activation and primary cilia are known to be associated with the abnormal renal cell proliferation and the formation of renal cysts in polycystic kidney disease, a renal disease model similar to our lithium model. The activation of hedgehog pathway is associated with the renal fibrosis observed in the unilateral ureteral obstruction and unilateral ischemia reperfusion injury models of chronic renal injury. Thus, I hypothesize that mTOR signaling pathway, primary cilia and hedgehog pathway may all contribute to lithium-induced nephropathy. To address the hypothesis that the mTOR signaling pathway may be responsible for lithium-induced renal collecting duct proliferation, mTOR pathway activation was assessed in lithium-treated mice and lithium-treated mouse inner medullary collecting duct (mIMCD3) cells. Lithium activated mTOR signaling pathway in renal collecting duct cells both in vivo and in vitro. Rapamycin, an inhibitor of mTOR, blocked lithium-induced renal cell proliferation in renal cortex and medulla in vivo and in renal collecting duct cells in vitro, supporting the hypothesis. However, rapamycin did not improve lithium-induced reduction of urine osmolality, suggesting mTOR signaling pathway may not contribute to lithium-induced NDI. To address the hypothesis that primary cilia may be necessary for lithium-induced mTOR activation and renal cell proliferation, primary cilia deficient cells were used to assess mTOR pathway activation and cell proliferation in response to lithium treatment. The absence of primary cilia abolished lithium-induced activation of mTOR pathway and cell proliferation, which supports the hypothesis. To address the hypothesis that lithium elongates primary cilia length, which is mediated by mTOR signaling pathway, primary cilia length alternation was assessed in the kidney and in mIMCD3 cells in response to lithium treatment. Lithium increased primary cilia length in renal collecting duct cells of cortex, outer medulla, and inner medulla kidney regions in vivo and in mIMCD3 cells in vitro. Rapamycin reversed lithium-induced elongation of primary cilia in renal cortical and outer medullary collecting duct cells in vivo, and blocked the increase of primary cilia length in mIMCD3 cells in vitro, which support the hypothesis. To address the hypothesis that lithium activates the hedgehog pathway in a Smoothened (smo, a key regulator of the hedgehog pathway)-dependent manner in renal collecting duct cells, mIMCD3 cells were treated with lithium or lithium/Smo inhibitor or lithium/Smo activator. Hedgehog signaling pathway is activated by lithium in mIMCD3 cells, which is partially Smo-dependent. However, the role of hedgehog signaling pathway in regulating lithium-induced fibrosis was not assessed in the study. Future studies are required to determine the role of the hedgehog pathway in the lithium model.

No description available.

Maternal immune activation (MIA) can affect fetal brain development and thus behavior of young and adult offspring. Reports have shown that increased Interleukin-6 (IL-6) in the maternal serum plays a key role in altering fetal brain development, and may impair social behaviors in the offspring. Interestingly, these effects could be attenuated by blocking IL-6. The current study investigated the effects of luteolin, a citrus bioflavonoid, and its structural analog, diosmin, on IL-6 induced JAK2/STAT3 (Janus tyrosine kinase-2/signal transducer and activator of transcription-3) phosphorylation and signaling as well as behavioral phenotypes of MIA offspring. Luteolin and diosmin inhibited neuronal JAK2/STAT3 phosphorylation both in vitro and in vivo following IL-6 challenge as well as significantly diminishing behavioral deficits in social interaction. Importantly, our results showed that diosmin (10 mg/kg day) was able to block the STAT3 signal pathway; significantly opposing MIA-induced abnormal behavior and neuropathological abnormalities in MIA/adult offspring. Diosmin's molecular inhibition of JAK2/STAT3 pathway may underlie the attenuation of abnormal social interaction in IL-6/MIA adult offspring.

Indiana University-Purdue University Indianapolis (IUPUI) / Hearing loss and vestibular dysfunction are inner ear disease states that arise from an array of diverse etiologies that interfere with mechanosensory hair cell function, including: congenital syndromes, noise-induced trauma, ototoxic drugs, and aging. The investigation of normal inner ear development and the pathological aberrations that cause inner ear disease has been previously advanced through formation of an easily generated, scalable, accurate in vitro model system that readily facilitates experimental applications. This model utilizes a 3D floating cell culture protocol which guides differentiation of stem cell aggregates into inner ear organoids, which are vesicles containing a sensory epithelium with functioning mechanosensory hair cells. Inner ear organoid formation enables studying the effects of modulating the signaling pathways that guide developing inner ear structure and function. The Notch signaling pathway heavily influences the formation of the inner ear through two major mechanisms: lateral induction of sensory progenitor cells and lateral inhibition to determine which of those progenitors differentiate into mechanosensory hair cells. The effects of inhibiting Notch signaling within the inner ear organoid system were explored through application of the ɣ-secretase inhibitor MDL28170 (MDL) at a concentration of 25μM on day 8 of organoid culture. Aggregates were harvested on day 32, fixed, sectioned, and stained according to a standard immunohistochemistry protocol. Sections were stained for the mechanosensory hair cell markers Myosin7a (Myo7a) and Sox2. MDL-treated aggregates demonstrated statistically significant reductions in the total number of vesicles and the number of vesicles containing hair cells compared to control aggregates. In contrast to control aggregates which demonstrated two distinct organoid variants (protruding and embedded), MDL-treated aggregates only formed the embedded variant. Differences in the expression pattern of Sox2, which is also a marker of stemness and neural progenitor cells were also noted between the two conditions. MDL-treated aggregates demonstrated regions of ‘ectopic’ Sox2 expression whereas Sox2 expression in control aggregates was consistently expressed within Myo7a+ regions.

The full text of the thesis is currently restricted. / The full text will be available at the end of the embargo period: 1st Nov 2021

La protéine O-fucosyltransférase 1 (POFUT1) réticulaire, dont le gène est localisé dans la région chromosomique 20q11.21 chez l’Homme, catalyse le transfert d’un fucose qui sera O-lié sur la sérine ou la thréonine présente dans la séquence consensus (C2X4S/TC3), portée par un domaine EGF-like d’une glycoprotéine membranaire ou sécrétée. Le knockdown de Pofut1 (Po -) dans la lignée myoblastique murines C2C12 conduit à la formation de myotubes allongés et minces, à faible nombre de noyaux ainsi qu’à une sous-expression du marqueur myogénique tardif Myf6, suggérant des défauts significatifs dans la fusion secondaire. La signalisation NFATc2/IL-4 est décrite comme la voie principale associée à cette étape. Nous montrons que la moindre expression de Nfatc2 dans les myotubes Po - est corrélée à une baisse de l'IL-4 sécrétée et à une faible quantité de son récepteur (IL-4Rα) présent chez les cellules de réserve qui doivent participer à la fusion avec les myotubes naissants. La neutralisation de l’IL-4Rα sur les C2C12 sauvages provoque des défauts d'accrétion myonucléaire, semblables à ceux observés pour les Po -. Ainsi, POFUT1 pourrait être un nouveau médiateur de la croissance des myotubes au cours du processus myogénique, notamment par la signalisation NFATc2/IL-4. La glycoprotéine WIF1, cible potentielle de POFUT1, est un antagoniste de la signalisation WNT via sa fixation aux protéines WNT. Cette voie est connue pour être impliquée dans la prolifération et la différenciation des myoblastes. Néanmoins, aucune donnée ne concerne le rôle de WIF1 dans le processus myogénique. Par un apport exogène de WIF1, nous avons montré l’augmentation de la prolifération et l’altération de la différenciation myoblastique des C2C12. Lors de la prolifération, une augmentation de l’expression de Myf5 et une diminution de MyoG sont observées. A 7 jours de différenciation, les myotubes Po - ont un diamètre plus petit que les myotubes sauvages et ils sont plus nombreux à avoir un faible nombre de noyaux, traduisant des défauts de fusion. Nous démontrons pour la première fois, l’implication de la protéine WIF1 dans le processus myogénique. Récemment, POFUT1 a aussi été proposé comme nouveau biomarqueur pour certains cancers, mais pas évalué dans le cancer colorectal (CCR). Nous avons donc collecté des données issues de 626 tumeurs et 51 tissus adjacents non tumoraux disponibles dans FireBrowse, celles de lignées cellulaires cancéreuses colorectales et de prélèvements tumoraux provenant du Centre de Ressources Biologiques du CHU de Limoges. Une surexpression de POFUT1 est observée dès le stade I, majoritairement due à une amplification de la région 20q11.21. Elle est significativement associée aux adénocarcinomes non mucineux et à une localisation rectale. De plus, l’expression de POFUT1 est corrélée à celles des récepteurs NOTCH ainsi qu’au processus métastatique, probablement par activation de la voie NOTCH. A ce titre, POFUT1 pourrait être considéré comme un nouveau biomarqueur pour le diagnostic du CCR. / The ER protein O-fucosyltransferase 1 (POFUT1), whose gene is located at the 20q11.21 chromosomic region in humans, catalyzes O-linked fucose addition to serine or threonine present in the consensus sequence (C2X4S/TC3) carried by EGF-like domain of membrane or secreted glycoprotein. Pofut1 knockdown (Po -) in murine myoblast C2C12 cell line leads to formation of elongated and thin myotubes, with a low number of nuclei and to downexpression of the late myogenic marker Myf6, suggesting significant defects in secondary fusion. NFATc2/IL-4 signaling is described as the main pathway associated to this step. We showed that the slightest expression of Nfatc2 in Po - myotubes is correlated with a decrease in IL-4 secretion and a lower quantity of IL 4Rα in reserve cells, which had to fuse with nascent myotubes. IL-4Rα neutralization on wild-type C2C12 causes myonuclear accretion defects, similar to those observed in Po -. Then, POFUT1 could be a new mediator of myotube growth during myogenic process, particularly through NFATc2/IL-4 signaling. The glycoprotein WIF1, potential POFUT1 target, is an antagonist of WNT signaling via its binding to WNT proteins. This pathway is involved in proliferation and differentiation of myoblasts. However, no data are available on WIF1 role in the myogenic process. Through exogenous WIF1 treatment, we showed a proliferation increase and a myoblast differentiation impairment in C2C12. During proliferation, increase in Myf5 and decrease in MyoG expressions are observed. At 7 days of differentiation, Po - myotubes have a smaller diameter than wild-type ones and are more numerous to have a small number of nuclei, reflecting fusion defects. For the first time, we demonstrate the involvement of WIF1 in the myogenic process. Recently, POFUT1 was proposed to be a new biomarker for several cancers, but not evaluated in colorectal cancer (CRC). We used data from 626 tumors and 51 adjacent non-tumor tissues available at FireBrowse, colorectal cancer cell lines and tumor samples from the Biological Resource Centre of Limoges hospital. A POFUT1 overexpression is observed from stage I, mainly due to amplification of 20q11.21 region. It is significantly associated to non-mucinous adenocarcinoma and to rectum location. Moreover, POFUT1 expression is correlated with those of NOTCH receptors as well as the metastatic process, probably by activation of the NOTCH pathway. POFUT1 could therefore be considered as a new biomarker for CRC diagnosis.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Atypical teratoid rhabdoid tumor (ATRT) is a highly malignant pediatric central nervous system tumor. The prognosis is often poor, with a 2‐year survival rate estimated at 15%. This dismal prognosis highlights the need to develop new treatment modalities for this devastating pediatric tumor. Recently, a tumor suppressing signaling pathway known as Hippo has emerged as a possible cancer treatment target. The Hippo signaling pathway is involved in organ growth and maintenance, and is dysregulated in many diverse cancers. We used quantitative real‐time PCR to evaluate the mRNA expression profile of Hippo pathway genes. We then used determined the protein expression of various Hippo components using Western blots. The results of this study suggest that Hippo plays a definite role in atypical teratoid rhabdoid tumor.

Aconitase is an enzyme of the Krebs cycle that catalyzes the isomerization of citrate to isocitrate. In addition to its enzymatic activity, Aco1 has been reported to bind to mitochondrial DNA (mtDNA) and mediate its maintenance in the budding yeast S. cerevisiae. In the absence of Aco1, cells rapidly lose mtDNA and become “petite” mutants. The purpose of this study is to uncover the mechanism behind mtDNA loss due to an aco1 deletion mutation. We found that an aco1 mutation activates the mitochondria-to-nucleus retrograde (RTG) signaling pathway, resulting in increased expression of citrate synthases (CIT) through the activation of two transcription factors Rtg1 and Rtg3. Increased activity of CIT leads to increased iron accumulation in cells, which is known to raise reactive oxygen species (ROS). By deleting RTG1, RTG3, genes encoding citrate synthases, orMRS3 and MRS4, encoding two irontransporters in the mitochondrial inner membranes, mtDNA loss can be prevented in aco1 deletion mutant cells. We further show that the loss of SOD1, encoding the cytoplasmic isoform of superoxide dismutase, but not SOD2, encoding the mitochondrial isoform of superoxide dismutase, prevents mtDNA loss in aco1 mutant cells. Altogether, our data suggest that mtDNA loss in aco1 mutant cells is caused by the activation of the RTG pathway and subsequent iron accumulation and toxicity in the mitochondria.

A leucemia linfoide aguda (LLA) é uma neoplasia hematológica agressiva, caracterizada pela expansão clonal de progenitores linfoides e ativação exacerbada de vias de sinalização. A via de sinalização de IGF1R/IRS1 inicia-se pela ligação do ligante IGF1 ao seu receptor transmembrana IGF1R, e subsequente ativação de seu substrato IRS1, que transmite sinais mitogênicos e antiapoptóticos, principalmente através da modulação das vias de sinalização PI3K/AKT/mTOR e MAPK . Estas vias de sinalização desempenham uma importante função na proliferação, sobrevivência e migração de células de leucêmicas. Dessa forma, o objetivo do nosso trabalho foi investigar a participação da via de sinalização IGF1R/IRS1 na LLA. Linhagens celulares Jurkat, MOLT-4, Namalwa e Raji foram tratadas ou não com inibidor de IGF1R/IRS1-2, NT157, ou com inibidor de IGF1R/IR, OSI-906, e submetidas à avaliação da viabilidade celular, apoptose, proliferação, ciclo celular, migração e expressão/ativação gênica e proteica. Células mononucleares de pacientes com LLA e de doadores saudáveis foram submetidas aos ensaios de viabilidade e apoptose, após tratamento com NT157 e OSI-906. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células CEM em camundongos NSG. O estudo foi aprovado pelo Comitê de Ética em Pesquisa e Comitê de Ética no Uso dos Animais da Instituição. A análise estatística foi realizada pelo teste ANOVA e teste t de Student. O tratamento com NT157 reduziu a viabilidade e a proliferação, induziu apoptose e modulou o ciclo celular em todas as linhagens testadas (p<0,05). Similarmente, OSI- 906 reduziu a viabilidade e a proliferação, modulou o ciclo celular, porém não foi capaz de induzir apoptose nas linhagens de LLA (p<0,05). Os tratamentos com NT157 e com OSI-906 diminuíram significativamente a migração de Jurkat em fibronectina, porém não modularam a migração de Namalwa. Em um contexto molecular, a exposição ao NT157 resultou em inibição da fosforilação de proteínas da via de sinalização PI3K/AKT/mTOR e modulou a expressão de 25 genes relacionados com a via de sinalização MAPK, dentre eles CDKN1A (p21), FOS e JUN (p<0,05). OSI-906modulou a ativação das proteínas da via de sinalização PI3K/AKT/mTOR e a expressão gênica de p21, FOS e JUN, porém de uma forma diferente da modulação encontrada pelo tratamento com NT157 (p<0,05). Em células mononucleares de pacientes com LLA, NT157 induziu uma resposta heterogênea na viabilidade e induziu apoptose, e OSI-906 reduziu a viabilidade, porém não foi capaz de induzir apoptose nestes pacientes (p<0,05). Os tratamentos com NT157 e OSI-906 não apresentaram citotoxicidade em células de doadores saudáveis. Adicionalmente, o tratamento in vivo com veículo ou NT157 na dose de 50mg/kg/dia, via intraperitoneal, em modelos de xenotransplante com células CEM em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IGF1R/IRS1-2, por NT157, e de IGF1R/IR, por OSI-906, apresentaram efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com LLA. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Nossos resultados revelaram que NT157 exerce um efeito citotóxico nas células de LLA, enquanto que OSI-906 tem um efeito predominantemente citostático. Estes dados indicam que o inibidor de IGF1R/IRS1-2, NT157, obteve resultados antineoplásicos mais atrativos e a inibição direta de IRS1 pode ser um potencial alvo terapêutico em LLA. / Acute lymphoid leukemia (ALL) is an aggressive hematological neoplasm, characterized by clonal expansion of lymphoid progenitors and exacerbated activation of signaling pathways. The IGF1R/IRS1 signaling pathway initiated through binding of the ligand IGF1 to its transmembrane receptor IGF1R, and the subsequent activation of its substrate IRS1, which transmit mitogenic and antiapoptotic signals, mainly through the modulation of the PI3K/AKT/mTOR and MAPK signaling pathways. These signaling pathways play an important function in cell proliferation, survival and migration of leukemia cells. Therefore, the objective of our study was to investigate the participation of the IGF1R/IRS1 signaling pathway in ALL. Jurkat, MOLT-4, Namalwa and Raji cell lines were treated or not with IGF1R/IRS1-2 inhibitor, NT157, or with IGF1R/IR inhibitor, OSI-906, and evaluated for cell viability, apoptosis, proliferation, cell cycle, migration, gene and protein expression/activation. Mononuclear cells from patients with ALL and from healthy donors were submitted to the viability and apoptosis assays, after treatment with NT157 and OSI-906. The NT157 effect in vivo was evaluated using CEM cell line xenograft model in NSG mice. The study was approved by the Research Ethics Committee and the Ethics Committee on the Use of Animals of the Institution. Statistical analysis was performed by ANOVA and Student t test. Treatment with NT157 reduced viability and proliferation, induced apoptosis, and modulated the cell cycle in all cell lines tested (p<0.05). Similarly, OSI-906 reduced viability and proliferation, modulated the cell cycle, but was not able to induce apoptosis in ALL cell lines (p<0.05). Treatments with NT157 and OSI-906 significantly decreased the migration of Jurkat in fibronectin, but did not modulate the Namalwa migration. In a molecular context, the NT157 exposure resulted in inhibition of the PI3K/AKT/mTOR signaling pathway protein phosphorylation and modulated the expression of 25 genes related to the MAPK signaling pathway, including CDKN1A (p21), FOS and JUN (p<0,05). OSI-906 modulated the activation of the PI3K/AKT/mTOR signaling pathway proteins and the p21, FOS and JUN geneexpression, but in a different way from the modulation found by treatment with NT157 (p<0.05). In mononuclear cells of patients with ALL, NT157 induced a heterogeneous response in viability and induced apoptosis, and OSI-906 reduced viability, but was not able to induce apoptosis in these patients (p<0.05). Treatments with NT157 and OSI- 906 did not show cytotoxicity in healthy donor cells. Additionally, in vivo treatment with vehicle or NT157 at the dose of 50 mg/kg/day, intraperitoneally, in xenotransplantation models with CEM cells in NSG mice (n=5 for each group) showed no antineoplastic effects. In conclusion, the pharmacological inhibition of IGF1R/IRS1- 2, by NT157, and IGF1R/IR, by OSI-906, showed significant antineoplastic effects in cell line models and in primary samples of patients with ALL. The results of in vivo studies in xenotransplantation models indicate the need for pharmacokinetic and pharmacodynamic studies for NT157. In conclusions, our results revealed that NT157 exerts a cytotoxic effect on ALL cell lines and primary ALL cells, whereas OSI-906 has a predominantly cytostatic effect. These data indicate that the IGF1R/IRS1-2 inhibitor, NT157, obtained more attractive antineoplastic results and the direct inhibition of IRS1 may be a potential therapeutic to target in ALL.