Immunogenic Cell Death Role in Urothelial Cancer Therapy.

PURPOSE: Bladder cancer is the 13th most common cause of cancer death with the highest lifetime cost for treatment of all cancers. This scoping review clarifies the available evidence on the role of a novel therapeutic approach called immunogenic cell death (ICD) in urothelial cancer of the bladder. METHODS: In accordance with the recommendations of the Joanna Briggs Institute, we searched MEDLINE (Ovid), EMBASE, CENTRAL databases, and supplemented with manual searches through the conferences, Google scholar, and clinicaltrials.gov for published studies up to April 2022. We included literature that studied molecular mechanisms of ICD and the role of certain danger-associated molecular patterns (DAMPs) in generating ICD, safety and efficacy of different ICD inducers, and their contributions in combination with other urothelial cancer treatments. RESULTS: Oncolytic viruses, radiotherapy, certain chemo/chemo radiation therapy combinations, photodynamic therapy, and novel agents were studied as ICD-inducing treatment modalities in the included studies. ICD was observed in vitro (murine or human urothelial carcinoma) in ten studies, eight studies were performed on mouse models (orthotopic or subcutaneous), and five clinical trials assessed patient response to ICD inducing agents. The most common studied DAMPs were Calreticulin, HMGB1, ATP, and Heat Shock Proteins (HSP) 70 and 90, which were either expressed on the cancer cells or released. CONCLUSION: ICD inducers were able to generate lasting antitumor immune responses with memory formation in animal studies (vaccination effect). In clinical trials these agents generally had low side effects, except for one trial, and could be used alone or in combination with other cancer treatment strategies in urothelial cancer patients.

Novo papel da proteína XPC na regulação dos complexos da cadeia de transporte de elétrons e desequilíbrio redox / New role of XPC protein in regulating the electron transport chain complexes and redox unbalance

Espécies reativas de oxigênio (EROs) são normalmente e continuamente geradas em mitocôndrias, majoritariamente na cadeia de transporte de elétrons (CTE). Harman (1956, 1972 e 1992) teorizou que os radicais livres gerados nas mitocôndrias seriam a principal causa do envelhecimento. De fato, durante o envelhecimento é observado um desequilíbrio entre formação e remoção de EROs, que resulta em estresse redox. Essa condição favorece a formação de lesões oxidadas no DNA, acarretando em mutagênese ou morte celular. Diversos mecanismos moleculares cooperam para o reparo de DNA. Duas vias de reparo de DNA lidam com a maioria das lesões: o reparo por excisão de base (BER) e o reparo por excisão de nucleotídeos (NER). A via BER corrige pequenas modificações de bases que surgem de reações de desaminação, alquilação e oxidação. A via NER é mais versátil, reconhecendo lesões que distorcem a dupla hélice de DNA, como danos induzidos por luz UV e adutos volumos. Pacientes xeroderma pigmentoso (XP-A a XP-G) herdam mutações em um de sete genes que codificam proteínas envolvidas na via NER, ou em um gene que codifica uma polimerase translesão (XP-V). A doença é caracterizada por fotosensibilidade e incidência elevada de neoplasias cutâneas. A proteína XPC atua na etapa de reconhecimento da lesão de DNA na subvia de reparo global do genoma (GG-NER), e sua mutação dá origem aos sintomas clássicos de XP. Novas funções de XPC foram recentemente descritas: i) atuando como cofator na via BER auxiliando as DNA glicosilases OGG1, TDG e SMUG; ii) atuando como cofator transcricional de elementos responsivos a Oct4/Sox2, RXR e PPARα; e iii) na adaptação metabólica na transformação de queratinócitos. Então, propusemo-nos a investigar as relações entre XPC e a manutenção da integridade do DNA mitocondrial, a sensibilidade celular a estresse redox mitocondrial e possíveis alterações bioenergéticas e redox. Para tal, padronizamos um ensaio in vitro de cinética de incisão em DNA plasmidial a fim de investigarmos o possível papel de XPC no reparo de lesões oxidadas em mtDNA. Porém, nossos dados revelaram que XPC não se encontra em mitocôndrias. Apesar disso, células XP-C são mais sensíveis ao tratamento com azul de metileno (AM), antimicina A (AA) e rotenona (ROT), que geram estresse redox mitocondrial. A sensibilidade à AA foi completamente revertida em células corrigidas. Células XP-C apresentaram alterações quanto ao uso dos complexos mitocondriais, com diminuição da taxa de consumo de oxigênio (OCR) via complexo I e um aumento da OCR via complexo II, dependente da presença de XPC. Ademais, a linhagem XP-C apresentou um desequilíbrio redox mitocondrial com maior produção de EROs e menor atividade de GPx. O DNA mitocondrial de células XP-C apresentou níveis elevados de lesão e deleção, que no entanto não retornaram aos níveis encontrados em células selvagens na linhagem XP-C corrigida. Observamos uma acentuada diminuição da expressão de PPARGC1A, um importante regulador de biogênese mitocondrial. Contudo, não foi possível determinar o mecanismo de supressão da expressão de PPARGC1A. Por fim, identificamos que o tipo de mutação em XPC pode estar associado a expressão de PPARGC1A. Esse estudo abre novas possibilidade na investigação do papel de proteína XPC, à parte da instabilidade genômica, na adaptação metabólica e desequilíbrio redox em direção da progressão tumoral

Mitochondria continuously produce reactive oxygen species (ROS), mainly at the electron transport chain. Harman (1956, 1972 e 1992) proposed that normal aging is driven by increased mitochondrially generated free radicals. Indeed, during the course of aging there is an increased imbalance between formation and removal of ROS, leading to redox stress. This condition favours the formation of oxidized DNA lesions, given rise to mutations and cell death. Several molecular mechanisms cooperates to repair the DNA. Two DNA repair pathways deal with the majority of lesions: base excision repair (BER) and nucleotide excision repair (NER). The BER pathway corrects small base modifications that arise from deamination, alkylation and oxidation reactions. The NER pathway is more versitile, recognizing helix-distorting lesions, such as UV-induced damage and bulky adducts. Xeroderma pigmentosum (XP-A to XP-G) patients inherit mutations in one of seven protein-coding genes involved in NER pathway, or in a gene coding a translesion DNA polymerase (XP-V). Photosensitivity and a thousand-fold increased in the risk of developing cutaneous neoplasms are the main clinical features of XP. XPC protein functions in the recognition step of global genome NER (GG-NER) sub-pathway, and mutations in this gene lead to classical XP symptoms. Recently, it has been described that XPC acts: i) as a cofactor in BER pathway through functional interaction with DNA glycosylases OGG1, TDG and SMUG1; ii) as coactivator in transcription at Oct4/Sox2, RXR and PPARα responsive elements; iii) in metabolic shift during keratinocytes transformation. Thus, we sought to investigate a possible role for XPC in the maintenance of mtDNA integrity, cellular sensitivity to mitochondrial redox stress and eventual bioenergetic and redox changes. For this purpose, we established an in vitro plasmid incision assay to investigate the possible role of XPC in the repair of oxidized lesions in mitochondrial DNA. However, our data revealed that XPC did not localized in mitochondria. Nonetheless, XP-C cells are more sensitive to methylene blue, antimycin A (AA) and rotenone treatment, which induce mitochondrial redox stress. The XP-C sensitivity to AA was completely reverted in XPC-corrected cells. XP-C cells presented altered usage of mitochondrial complexes, with decreased oxygen consumption rate (OCR) via complex I and increased OCR through complex II, an XPC-dependent phenomenon. Furthermore, the XP-C cell line showed mitochondrial redox imbalance with increased ROS production and decrease GPx activity. MtDNA from XP-C cells accumulate lesions and deletions, which, however, were found at similar levels in the corrected cell line. We identified a sharp decrease in the expression of PPARGC1A, a master regulator of mitochondrial biogenesis. Nevertheless, it was not possible to determine the mechanism of suppression of PPARGC1A expression. Finally, our results suggest a possible link between the type of XPC mutation and PPARGC1A expression. This study unfolds new possible roles for XPC, aside from its established roles in genomic instability, in metabolic adaptation and redox imbalance towards tumour progression

Microglial stress inducible protein 1 promotes proliferation and migration in human glioblastoma cells.

Microglial activation is a key event in the progression and infiltration of tumors. We have previously demonstrated that the co-chaperone stress inducible protein 1 (STI1), a cellular prion protein (PrP(C)) ligand, promotes glioblastoma (GBM) proliferation. In the present study, we examined the influence of microglial STI1 in the growth and invasion of the human glioblastoma cell line GBM95. We demonstrated that soluble factors secreted by microglia into the culture medium (microglia conditioned medium; MG CM) caused a two-fold increase in the proliferation of GBM95 cells. This effect was reversed when STI1 was removed from the MG CM. In this context, we have shown that microglial cells synthesize and secrete STI1. Interestingly, no difference was observed in proliferation rates when GBM cells were maintained in MG CM or MG CM containing an anti-PrP(C) neutralizing antibody. Moreover, rec STI1 and rec STI1(Δ230-245), which lack the PrP(C) binding site, both promoted similar levels of GBM95 proliferation. In the migration assays, MG CM favored the migration of GBM95 cells, but migration failed when STI1 was removed from the MG CM. We detected metalloproteinase 9 (MMP-9) activity in the MG CM, and when cultured microglia were treated with an anti-STI1 antibody, MMP-9 activity decreased. Our results suggest that STI1 is secreted by microglia and favors tumor growth and invasion through the participation of MMP-9 in a PrP(C)-independent manner.

STI1 promotes glioma proliferation through MAPK and PI3K pathways.

Gliomas are tumors derived from glia or their precursors within the central nervous system. Clinically, gliomas are divided into four grades and the glioblastoma multiforme (GBM), also referred as grade IV astrocytoma, is the most aggressive and the most common glioma in humans. The prognosis for patients with GBM remains dismal, with a median survival of 9-12 months. Despite their striking heterogeneity, common alterations in specific cellular signal transduction pathways occur within most GBMs. Previous work from our group identified the co-chaperone stress-inducible protein 1 (STI1) as a cell surface ligand for cellular prion (PrP(C)), which leads to the activation of several signal transduction pathways, some of which modulate cell survival. In the present work, we used thymidine incorporation assays to investigate the effect of STI1 upon proliferation of the human glioblastoma-derived cell line A172. Here we report that STI1 is secreted by and induces proliferation in tumor cells, an effect that is modulated by the Erk and PI3K pathways, and that, in contrast to glioma cells, STI1 does not induce proliferation of normal glia. In addition, our data suggest the involvement of PrP(C) in STI1-induced proliferation of A172 cells. These results provide initial evidence of a new functional role for STI1 on the physiology of human gliomas, and may lead to the identification of new therapeutic targets in these tumors.

Heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications.

Heat shock proteins (Hsps) are overexpressed in a wide range of human cancers and are implicated in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. We review the current status of the role of Hsp expression in cancer with special emphasis on the clinical setting. Although Hsp levels are not informative at the diagnostic level, they are useful biomarkers for carcinogenesis in some tissues and signal the degree of differentiation and the aggressiveness of some cancers. In addition, the circulating levels of Hsp and anti-Hsp antibodies in cancer patients may be useful in tumor diagnosis. Furthermore, several Hsp are implicated with the prognosis of specific cancers, most notably Hsp27, whose expression is associated with poor prognosis in gastric, liver, and prostate carcinoma, and osteosarcomas, and Hsp70, which is correlated with poor prognosis in breast, endometrial, uterine cervical, and bladder carcinomas. Increased Hsp expression may also predict the response to some anticancer treatments. For example, Hsp27 and Hsp70 are implicated in resistance to chemotherapy in breast cancer, Hsp27 predicts a poor response to chemotherapy in leukemia patients, whereas Hsp70 expression predicts a better response to chemotherapy in osteosarcomas. Implication of Hsp in tumor progression and response to therapy has led to its successful targeting in therapy by 2 main strategies, including: (1) pharmacological modification of Hsp expression or molecular chaperone activity and (2) use of Hsps in anticancer vaccines, exploiting their ability to act as immunological adjuvants. In conclusion, the present times are of importance for the field of Hsps in cancer, with great contributions to both basic and clinical cancer research.

Effect of heat-shock on rhesus monkey lymphocytes and IL-2-dependent cells.

Treatment of Rhesus monkey peripheral blood lymphocytes and IL-2 dependent cell lines with heat prior to incubation with mitogens or IL-2, respectively, induces significant cell changes at the nuclear level, detected by DNA staining with Vindelov's propidium iodide and the simultaneous measurement of its red fluorescence and 90 degrees light scatter. These changes are an increase in their nuclear granularity and in apparently fragmented DNA which shows less fluorescence intensity than DNA from nuclei in the G0G1 phase, a phenomenon suggestive of apoptosis. Treated cells also show an increased number of nuclei in G1 or early S phase, with a reduction in those reaching the G2 or M phases. After heat-shock treatment, CTLL-2 cells show an increase in their response to low doses of recombinant IL-2 and an impaired ability to proliferate at higher IL-2 concentrations. These results provide further evidence for the regulatory role of stress-induced events.

Effect of heat-shock on rhesus monkey lymphocytes and IL-2-dependent cells

Treatment of Rhesus monkey peripheral blood lymphocytes and IL-2 dependent cell lines with heat prior to incubation with mitogens or IL-2, respectively, induces significant cell changes at the nuclear level, detected by DNA staining with Vindelov's propidium iodide and the simultaneous measurement of its red fluorescence and 90 degrees light scatter. These changes are an increase in their nuclear granularity and in apparently fragmented DNA which shows less fluorescence intensity than DNA from nuclei in the G0G1 phase, a phenomenon suggestive of apoptosis. Treated cells also show an increased number of nuclei in G1 or early S phase, with a reduction in those reaching the G2 or M phases. After heat-shock treatment, CTLL-2 cells show an increase in their response to low doses of recombinant IL-2 and an impaired ability to proliferate at higher IL-2 concentrations. These results provide further evidence for the regulatory role of stress-induced events

Modulation for antigen presentation in tuberculosis by using synthetic peptides.

Competition assay technology has been a very useful tool in the study of parasite antigens and has been inferred but never proven that this approach can be applied to select T-cell epitopes by using another microorganisms. In this study, HLA-restricted T-cell clones specific to synthetic peptides derived from the 65 kDa mycobacterial protein were used to investigate whether these peptides are able to compete with each other at the level of MHC-binding sites in tuberculosis. Fixed APCs were pulsed with suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptide. The results showed that two peptides from this protein were able to compete with each other inducing a significant inhibition in the proliferation assays while there was no competition by using a control peptide. The amount of cross-reactivity was influenced by the peptide concentrations. More important was the observation that these peptides were able to bind to the same HLA-class II molecules therefore blocking the binding of each other. The fact that these peptides have not an identical amino acid sequence support the idea that the MHC-peptide interaction must have a broad specificity to be able to bind a large number of peptides. These results demonstrate that it is possible to modulate the antigen presentation by blocking the peptide MHC-class II interaction in tuberculosis and support the idea that this approach facilitates the selection of appropriate T-cell epitopes to be incorporated in a vaccine.