Bruna R. Rodrigues

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.

A Comparison between Manual and Automated Evaluations of Tissue Microarray Patterns of Protein Expression

Tissue microarray technology enables us to evaluate the pattern of protein expression in large numbers of samples. However, manual data acquisition and analysis still represent a challenge because they are subjective and time-consuming. Automated analysis may thus increase the speed and reproducibility of evaluation. However, the reliability of automated analysis systems should be independently evaluated. Herein, the expression of phosphorylated AKT and mTOR was determined by ScanScope XT (Aperio; Vista, CA) and ACIS III (Dako; Glostrup, Denmark) and compared with the manual analysis by two observers. The percentage of labeled pixels or nuclei analysis had a good correlation between human observers and automated systems (κ = 0.855 and 0.879 for ScanScope vs. observers and κ = 0.765 and 0.793 for ACIS III vs. observers). The intensity of labeling determined by ScanScope was also correlated with that found by the human observers (correlation index of 0.946 and 0.851 for pAKT and 0.851 and 0.875 for pmTOR). However, the correlation between ACIS III and human observation varied for labeling intensity and was considered poor in some cases (correlation index of 0.718 and 0.680 for pAKT and 0.223 and 0.225 for pmTOR). Thus, the percentage of positive pixels or nuclei determination was satisfactorily performed by both systems; however, labeling intensity was better identified by ScanScope XT.

Disruption of prion protein–HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival

Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)–Hsp90-organizing protein (HOP) with cellular prion protein (PrPC) triggers a large number of trophic effects in the nervous system. We found that both PrPC and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I–III. High levels of PrPC and HOP were associated with greater GBM proliferation and lower patient survival. HOP–PrPC binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrPC binding site (HOP230–245) abrogates this effect. PrPC knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230–245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230–245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrPC–HOP engagement is a promising approach for GBM therapy.

Nuclear unphosphorylated STAT3 correlates with a worse prognosis in human glioblastoma

Glioblastoma (GBM) is currently the most aggressive form of brain tumor identified, and STAT3 is known to play an important role in gliomagenesis. Moreover, while several studies have used pharmacological approaches to modulate STAT3 activity, the results have been contradictory. In this study, expressions of STAT3, pSTAT3 (Y705), and pSTAT3 (S727) were evaluated using immunohistochemistry assays of tissue microarrays containing non-neoplastic tissue (NN, n=12), grade II astrocytomas (n=33), grade III astrocytomas (n=12), and GBM (n=85) specimens. In GBM specimens, STAT3 was overexpressed and exhibited greater nuclear localization compared with lower grade astrocytomas and NN. Conversely, nuclear localization of pSTAT3 (Y705) and pSTAT3 (S727) exhibited a similar phenotype in both GBMs and NNs. MET was also detected as a non-canonical pathway marker for STAT3. For tumors with higher levels of STAT3 nuclear localization, and not pSTAT3 (Y705) and pSTAT3 (S727), these specimens exhibited increased levels of MET expression. Thus, a non-canonical pathway may mediate a proportion of the STAT3 that translocates to the nucleus. Moreover, tumors which exhibited greater nuclear localization of STAT3 corresponded with patients that presented with lower rates of recurrence-free survival and overall survival. In contrast, the phosphorylated forms of STAT3 did not correlate with patient survival. These findings suggest that phosphorylation-independent mechanisms may mediate the nuclear translocation and activation of STAT3. Further studies are needed to identify the mechanisms involved, especially those that provide targets to achieve efficient inhibition and control of GBM progression.

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.

Abstract B45: Rab5C enhances resistance to ionizing radiation in rectal cancer

Rectal cancer is currently the second most common cancer in the large intestine, and represents one-third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment and it reduces the risk of local recurrence. However, a pathologic complete response is only achieved in 10–40% of cases and the mechanisms associated with chemoradiation 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. These results were then compared with a proteomic analysis of residual rectal adenocarcinoma tissues that were removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation treatment. To better understand the possible 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 that were 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, and this affected expression of the DNA repair proteins, Ku70 and Ku80. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells to neoadjuvant treatment and they should be further investigated. Supported by FAPESP - Fundacao de Amparo a Pesquisa do Estado de Sao Paulo. Note: This abstract was not presented at the conference. Citation Format: Antuani Rafael Baptistella, Marcos Vinicios Salles Dias, Fernanda Giudice, Bruna Roz Rodrigues, Petrus Paulo Combas Eufrazio Silva, Edson Kuatelela Cassinela, Tonielli Lacerda, Fabio Marchi, Adriana Franco Paes Leme, Maria Dirlei Begnami, Samuel Aguiar Junior, Vilma Regina Martins. Rab5C enhances resistance to ionizing radiation in rectal cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B45.

Abstract 3942: Cell death resistance promoted by radiotherapy in a colorectal cell line

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Colorectal adenocarcinoma is the fourth more frequent cause of death by cancer in the world. Despite some similarities, the rectal cancer has characteristics that differentiate it from the colon cancer, resulting in different treatments and outcomes. The rectal cancer in stages II and III is treated with neoadjuvant chemoradiation followed by surgery, and the response to neoadjuvant treatment in patients is variable and unpredictable. Up to now, surgery intervention is required to assess the pathologic response to therapy. The identification of predictive biomarkers for response to therapy is of great importance, in order to identify the patients that will benefit from such treatment. To better understand the molecular alterations in cells that resist to the radiotherapy, we started our research using WiDr, a colon tumor cell line. Cell cycle synchronized WiDr cells were submitted to two cycles of irradiation (4Gy in each cycle and 2 week of interval between the cycles) and resistant cells were isolated. Irradiated WiDr cells (RxTWiDr) shown a better capacity to form colonies, in colony growth assays, when compared to parental WiDr cells. This characteristic was maintained when they were submitted to a new round of irradiation. In addition, WiDrRxT cells treated with Staurosporine presented a higher viability in MTT assays than WiDr cells. We also collected the conditioned medium (CM) from RxTWiDr and WiDr cells, and treated the parental WiDr cells. The parental cells treated with CM from RxTWiDr cells for 15 minutes shown an increase in the ERK1/2 activation when compared to cells treated CM from WiDr cells. These results demonstrated that radio resistant cells are more resilient than parental cells and that their secreted factors activated specific signaling pathways via ERK1/2 phosphorylation. The nature of these factors is under investigation using proteomics. Supported by Sao Paulo State Foundation (FAPESP) Citation Format: Antuani R. Baptistella, Bruna Rodrigues, Marcos Dias, Fernanda Giudice, Tonielli Lacerda, Tiago Santos, Felipe Silva, Petrus Paulo Silva, Samuel Aguiar, Vilma Martins. Cell death resistance promoted by radiotherapy in a colorectal cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3942. doi:10.1158/1538-7445.AM2014-3942

Abstract 4880: Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Glioblastoma multiforme (GBM) is the most malignant form of astrocytomas and they release a heterogeneous population of microvesicles (MVEs) to the extracellular space. These MVEs are involved in intercellular communication by carrying oncogenes, and miRNAs that contribute with the tumor aggressiveness. However, the mechanisms of MVEs biogenesis and secretion in GBM are poorly understood. Material and Methods: Using five glioblastoma cell lines (LN-18, LN-229, U87MG, A-172 and U118MG) the vesicular biogenesis was characterize analyzing the expression of markers involved in steps of this process by western blotting. MVEs were also quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®). Immunofluorescence was performed in these cell lines to characterize the cellular distribution of Rab27b, which is responsible for driving the multivesicular bodies to the plasma membrane. Immunohistochemistry (IHC) measured Rab27b expression in a tissue microarray with 60 GBMs samples and 13 non-neoplastic human brain tissues used as controls. IHC intensity was scored from 1 to 3 (1 weak; 2, moderate; 3, strong) and positive cells, from 0 to 3 (0, 0%; 1, 1-10%+; 2, 11-50%+; 3, ≥ 51%+). The final IHC score was represented by intensity + positivity. Students t-test was performed to compare Rab27b expression in GBM and non neoplastic tissues. Kaplan-Meier and log-rank tests were used to correlate overall survival with Rab27b expression. Results: The cell lines showed a heterogeneous expression of markers involved in vesicle biogenesis. LN-18 cells presented higher levels of Cavelin-1, Flotillin -1, Alix and VPS4, p<0.01, but lower levels of Rab27b, p<0.0001. On the other hand, the U87MG cells standed out by higher levels of Rab27b, compared to the other cell lines studied, p<0.0001. Interesting, our results pointed that MVEs secretion is directed correlated to the expression pattern of Rab27b in GBM cells. Indeed, LN-18 cells showed lower levels of MVEs secretion (p<0.001) while U87MG cells presented higher MVEs secretion, p<0.001. In addition, Rab27b and CD63 (MVEs marker) were retained in the perinuclear region of LN -18 cells while in U87MG cells both proteins were distributed throughout the cytoplasm. Finally, Rab27b is overexpressed in GBM samples when compared to non-neoplastic tissue and patients presenting high expression of Rab27b showed lower survival outcomes. Conclusions: Our findings suggest that Rab27b may be crucial to the secretion of MVEs process in GBM. Furthermore, the overexpression of Rab27b in glioblastoma patients coincides with a poor prognostic phenotype. Supported by Sao Paulo State Foundation (FAPESP). Citation Format: Bruna R. Rodrigues, Fernanda S. Giudice, Marcos S. Dias, Antuani R. Baptistella, Rafael R. Malagoli, Paulo I. Sanematsu, Sergio Suzuki, Mariana Reis, Vilma R. Martins. Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4880. doi:10.1158/1538-7445.AM2014-4880

Abstract 4444: The role of small GTPase Rab7 in the secretion of extracellular microvesicles by head and neck squamous cells carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant tumor that shows its highest incidence in the upper aerodigestive tract and is considered one of the six most common malignancies worldwide. Among the processes involved in the poor prognosis of HNSCC, local invasion and subsequent metastasis are the most important events. Recent evidences have shown that metastasis can be the result of extracellular microvesicles (MVEs) being released from the tumor cells to the blood or lymphatic system. The molecular mechanisms involved in MVEs biogenesis and secretion are, however, poorly understood. Herein, we have performed a screen targeting for the components of MVEs biogenesis in three HNSCC cell lines (SCC-9, Cal27 and FaDu) through western blotting. MVEs were quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®) and the lysosomal compartment was followed in the cell lines through immunofluorescence. Comparing the three cell lines, FaDu cells presented higher expression of proteins responsible for MVEs formation (p Supported by Sao Paulo Research Foundation (FAPESP) Citation Format: Fernanda S. Giudice, Bruna R. Rodrigues, Marcos V. S. Dias, Antuani R. Baptistella, Tonielli Cristina S. Lacerda, Luiz P. Kowalski, Vilma R. Martins. The role of small GTPase Rab7 in the secretion of extracellular microvesicles by head and neck squamous cells carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2014-4444

Abstract 4400: The levels of Prion protein and its ligand HOP modulate glioblastoma proliferation and predict a lower survival outcome.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A main concern in glioblastoma (GBM) therapy is the resistance of these tumors to current treatment protocols. The identification of molecules that can be target to treat these tumors is of major interest. The prion protein (PrPC) is a glycosylphosphatidylinositol-anchored protein very abundant in neurons and astrocytes and its interaction with a diverse number of ligands modulates cell survival and differentiation. One of these ligands is the secreted co-chaperone Hsp70-Hsp90 organizing protein (HOP), which reaches the extracellular space in exosome-like microvesicles. HOP is secreted by a number of cancer cells and seems to be a relevant biomarker in ovarian tumors. However, the role of PrPC-HOP complex in tumor biology is largely unknown. Herein, 185 cases o gliomas (33 grade I, 46 grade II, 15 grade II and 91 GBM) arranged in tissue microarrays were evaluated for the expression of PrPC and its ligand HOP. Both proteins were highly expressed in GBM when compared to tumors of grade I, II or III and also when compared to non-tumor samples (p<0.05 for all comparisons). High expression levels of both proteins in GBMs were direct correlated with a higher tumor proliferation, measured by the number of cells positive for Ki-67. Patients with high levels of both proteins presented a lower survival than those with high levels of HOP but lower levels of PrPC (p=0.017). The treatment of a human GBM cell line U87MG, which presents high expression of PrPC, with soluble HOP increases proliferation by three times however any effect was observed when cells were treated with a soluble HOP lacking the PrPC binding site (HOPΔ230-245). Both HOP or HOPΔ230-245 have no effect in U87MG cells whose PrPC expression was knocked down by 98%. In accordance, the intra-tumor infusion of HOP, but not HOPΔ230-245, increased tumor growth in nude mice bearing U87MG orthotopic xenografts. Remarkably, U87MG cells in which PrPC expression was knocked down were unable to growth tumors in nude mice. Together these results demonstrated that PrPC and HOP are significantly expressed in GBMs and higher levels of these proteins are associated to the patient group presenting a lower survival. The manipulation of PrPC and HOP interaction in cell cultures or in vivo affects tumor growth indicating that the interference with their engagement may represent a therapeutic target in GBMs. Supported by The State of Sao Paulo Foundation. Citation Format: Marilene H. Lopes, Nicole Gilda Queiroz, Tiago Goss Santos, Bruna R. Rodrigues, Isabela W. Cunha, Bruno Costa Silva, Vilma R. Martins. The levels of Prion protein and its ligand HOP modulate glioblastoma proliferation and predict a lower survival outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2013-4400