Improving access to cancer clinical research in Brazil: recent advances and new opportunities. Expert opinions from the 4th CURA meeting, São Paulo, 2023.

Clinical research is the cornerstone of improvements in cancer care. However, it has been conducted predominantly in high-income countries with few clinical trials available in Brazil and other low-and-middle-income countries (LMIC). Of note, less than one-third of registered clinical trials addressing some of the most commonly diagnosed cancers (breast, lung and cervical) recruited patients from LMIC in the last years. The Institute Project CURA promoted the fourth CURA meeting, discussing barriers to cancer clinical research and proposing potential solutions. A meeting was held in São Paulo, Brazil, in June 2023 with representatives from different sectors: Brazilian Health Regulatory Agency (Anvisa), National Commission of Ethics in Research (CONEP), non-governmental organisations, such as the Latin American Cooperative Oncology Group, the Brazilian Society of Clinical Oncology (SBOC), Contract Research Organisations, pharmaceutical companies and investigators. A total of 16 experts pointed out achievements as shortening the time of regulatory processes involving Anvisa and CONEP, development of staff training programs, maintenance of the National Program of Oncological Attention (PRONON), and the foundation of qualified centres in North and Northeast Brazilian regions. Participants also highlighted the need to be more competitive in the field, which requires optimising ongoing policies and implementing new strategies as decentralisation of clinical research centres, public awareness campaigns, community-centered approaches, collaborations and partnerships, expansion of physicians-directed policies, exploring the role of the steering committee. Active and consistent reporting of the initiatives might help to propagate ongoing advances, increasing Brazilian participation in clinical cancer research. Engagement of all players is crucial to maintain continuous progress with further improvements in critical points including regulatory timelines and increments in qualified human resources which aligned with new educational initiatives focused on physicians and the general population will expand access to cancer clinical trials in Brazil.

Managing oncology clinical trials during COVID-19 pandemic.

São Paulo city is the epicenter of the Brazilian COVID-19 pandemic. The Instituto do Cancer do Estado de São Paulo is currently conducting 161 multinational sponsored trials plus 116 in house studies in the oncologic population. There are 242 currently active participants and 180 patients in follow-up. The management of the tightly controlled environment of clinical research becomes a challenge, and the Food and Drug Administration set of priority recommendations for patient safety while maintaining study integrity. Fast adaptations are necessary, and actions coalesce to participant protection from COVID-19. We pointed out critical processes for adjustments, and we believe that our experience may help other academic health centers.

Serum levels of VEGF and MCSF in HER2+ / HER2- breast cancer patients with metronomic neoadjuvant chemotherapy.

Metronomic therapy has been gaining importance in the neoadjuvant setting of breast cancer treatment. Its clinical benefits may involve antiangiogenic machinery. Cancer cells induce angiogenesis to support tumor growth by secreting factors, such as vascular endothelial growth factor (VEGF). In breast cancer, Trastuzumab (TZM) based treatment is of key importance and is believed to reduce diameter and volume of blood vessels as well as vascular permeability. Here in we investigated serum levels of angiogenic factors VEGF and MCSF in patients receiving metronomic neoadjuvant therapy with or without TZM. We observed in HER2+ cohort stable levels of MCSF through treatment, whereas VEGF trend was of decreasing levels. In HER2- cohort we observed increasing levels of MCSF and VEGF trend. Overall, HER2+ patients had better pathological response to treatment. These findings suggest that angiogenic pathway may be involved in TZM anti-tumoral effect in the neoadjuvant setting.

S-nitrosoglutathione and endothelial nitric oxide synthase-derived nitric oxide regulate compartmentalized ras S-nitrosylation and stimulate cell proliferation.

AIMS: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS: Ras signaling pathway stimulation by 50 and/or 100 µM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 µM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.

Rare tumors research in emerging countries.

Rare tumors, when considered as a group, represent a significant burden to society as they may account for up to 25% of the mortality by cancer in nations like the United States. In contrast with the current scenario in highly incident cancer types, little progress has been achieved in the treatment of the most rare cancers. The reasons for this apparent stagnation are mostly intrinsic to logistical difficulties in performing large clinical trials in rare diseases and will be addressed further in this article. Because both cancer incidence and clinical research are booming in emerging nations, we also aim to address the current and future role of these countries in research and the drug development process in rare tumor types.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases.

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.

The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

Protein tyrosine phosphorylation and protein tyrosine nitration in redox signaling.

Reversible phosphorylation of protein tyrosine residues by polypeptide growth factor-receptor protein tyrosine kinases is implicated in the control of fundamental cellular processes including the cell cycle, cell adhesion, and cell survival, as well as cell proliferation and differentiation. During the last decade, it has become apparent that receptor protein tyrosine kinases and the signaling pathways they activate belong to a large signaling network. Such a network can be regulated by various extracellular cues, which include cell adhesion, agonists of G protein-coupled receptors, and oxidants. It is well documented that signaling initiated by receptor protein tyrosine kinases is directly dependent on the intracellular production of oxidants, including reactive oxygen and nitrogen species. Accumulated evidence indicates that the intracellular redox environment plays a major role in the mechanisms underlying the actions of growth factors. Oxidation of cysteine thiols and nitration of tyrosine residues on signaling proteins are described as posttranslational modifications that regulate, positively or negatively, protein tyrosine phosphorylation (PTP). Early observations described the inhibition of PTP activities by oxidants, resulting in increased levels of proteins phosphorylated on tyrosine. Therefore, a redox circuitry involving the increasing production of intracellular oxidants associated with growth-factor stimulation/cell adhesion, oxidative reversible inhibition of protein tyrosine phosphatases, and the activation of protein tyrosine kinases can be delineated.

Regulation of p21Waf1 expression and TNFalpha biosynthesis by glutathione modulators in PMA induced-THP1 differentiation: involvement of JNK and ERK pathways.

Oxidative modifications of proteins are fundamental biochemical events that regulate cellular signaling, protein expression, and function. The redox status is balanced by reductants in which GSH plays a major role. This study investigated whether or not p21Waf1 expression and TNFalpha biosynthesis in macrophage differentiation/activation were regulated by GSH modulators and whether or not the JNK and ERK pathway were involved. We observed an increase of p21Waf1 expression and TNFalpha biosynthesis in the THP1 monocyte/macrophage cell line treated with PMA. Treatment of THP1 cultures with NAC prior to adding PMA abrogates the expression of p21Waf1 mRNA and decreases the level of TNFalpha whereas GSH depletion by BSO enhances the levels of TNFalpha with minor effects on p21Waf1 expression. To assess whether or not ERK and JNK were involved in the redox mechanism of p21Waf1 and TNFalpha, we used pharmacological inhibitors for JNK and ERK. Both PD98095 and dicoumarol were capable of blocking TNFalpha production but had only a small effect on p21Waf1 expression. We next observed that activation of JNK was significantly inhibited in cells pretreated with NAC with no effect on ERK. Taken together, our findings suggest that the modulation of GSH regulate the magnitude the cell response to PMA in which JNK and ERK have a particular role in redox signaling.

Nitric oxide induces thioredoxin-1 nuclear translocation: possible association with the p21Ras survival pathway.

One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21(RasC118S)). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway.

Tyrosine phosphorylation in nitric oxide-mediated signaling events.

In this chapter, we provide an overview of nitric oxide (NO)-tyrosine phosphorylation signal transduction pathways, integrating them with the cyclic guanosine monophosphate (cGMP) and S-nitrosylation-mediated pathways that are triggered by NO. The second half of this chapter includes a description of the methods that our laboratory has used extensively to characterize the mechanisms involved in signaling events mediated by this pathway. These include assays for detecting protein tyrosine phosphorylation, tyrosine phosphorylation of the epidermal growth factor (EGF) receptor, phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases, transfection of cells with modified forms of p21Ras, and an assay of p21Ras.

Nitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cells.

The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.