Papel do Receptor Lgr5 no Prognóstico e na Biologia de Sarcomas de Partes Moles / Role of the Lgr5 Receptor in the Prognosis and Biology of Soft Tissue Sarcomas

INTRODUÇÃO: Os sarcomas de partes moles (SPM) de alto grau são neoplasias heterogêneas, de prognóstico ruim e que apresentam poucas alternativas de tratamento. A identificação de marcadores de resposta tumoral ao tratamento, prognóstico, e até ao desenvolvimento de novas drogas, é uma busca incessante para um melhor tratamento dos sarcomas. Neste aspecto, o receptor Lgr5 tem um grande potencial em ser um novo alvo molecular, sendo um marcador de células-tronco das criptas intestinais e glândulas mamárias que também atua como um modulador negativo da sinalização da via Wnt/ß-catenina, uma das mais importantes na biologia de sarcomas emerge como um promissor candidato para estudos pré-clínicos, uma vez que já foi demonstrada sua importância em tumores do trato gastrointestinal. Para isso, os modelos de tumor de xenoenxerto derivado do paciente (PDX) representam uma plataforma valiosa para identificar novos biomarcadores e novos alvos, assim como o Lgr5 para avaliar a resposta à terapia e os mecanismos de resistência. OBJETIVO: Este estudo teve como objetivo estabelecer, caracterizar e testar a proteína Lgr5 através de ensaios in vitro e in vivo para desvendar a importância de Lgr5 na biologia de SPM e estabelecer uma estrutura integrada, convergente e translacional para o estudo deste tipo de tumor. MATERIAIS E MÉTODOS: Para a determinação da expressão de Lgr5 foi estabelecida duas coortes de estudo, uma retrospectiva oriunda do Registro Institucional de Sarcomas e uma prospectiva, onde foram convidados pacientes operados na Instituição com o intuito de gerar Patient-derived xenografts (PDX), um modelo-pré-clínico que possui a capacidade de manter as características moleculares dos tumores dos pacientes. Para isso, foram utilizados fragmentos implantados em camundongos imunossuprimidos para gerar esses modelos tumorais derivados de pacientes, além dos estudos funcionais in vitro utilizando linhagens de SPM para análise de perfil de expressão da proteína Lgr5 através de ensaios com imunofluorescência para verificar a capacidade de expressão de Lgr5, citometria de fluxo para verificar o padrão e quantidade de proteína nas amostras analisadas e western blotting para obter um padrão de marcação da proteína Lgr5. Além dos ensaios funcionais para avaliar a participação da proteína na proliferação, se a expressão da proteína interfere no poder migratório das células e tumores de SPM e capacidade de auto renovação, bem como sua associação com os dados clínicos e dados de sobrevida. RESULTADOS: O Registro Institucional retrospectivo conta com mais de 300 pacientes, já o Registro prospectivo com 70 pacientes que derivaram a geração de 33 PDX. Foi observado que pacientes com H-score superior a 20 apresentaram sobrevida global menor em 5 anos em comparação com o H-score de pacientes com valores inferiores a 20. Agora na outra análise feita, o H-score de pacientes com valores superiores a 25 é pior em comparação com os que apresentaram valores inferiores a 25 nos dados de sobrevida livre de doença. Além disso, células que superexpressam a proteína Lgr5 tem maior capacidade migratória (p= 0.02) e uma tendência de aumento na proliferação e auto renovação. Realizamos o teste de implante dessas populações positivas e negativas de Lgr5, separadas previamente por cell sorting. Para isso foram utilizados animais Balb/c Nude. Sugerindo que a expressão da proteína transduzida pode ser modulada por mecanismos compensatórios que precisam ser explorados. CONCLUSÃO: A construção do Registro Institucional de SPM é um grande passo para o melhor compreendimento da biologia dos Sarcomas, além da possibilidade de estudar novos alvos terapêuticos desse tumor raro, uma vez que os estudos e artigos científicos ainda são muito escassos. A geração dos modelos PDX também foi uma estratégia implantada muito bem executada com a geração de 33 PDX de diversos subtipos histológicos. Além da proteína Lgr5 induzir a migração celular a sua expressão está relacionada a um pior prognóstico, uma vez que, quanto maior a expressão de Lgr5 menor é a sobrevida global do paciente.

INTRODUCTION: High-grade soft tissue sarcomas (STS) are heterogeneous neoplasms with a poor prognosis and few treatment alternatives. The identification of tumor response markers to treatment, prognosis, and even the development of new drugs, is an incessant search for a better treatment of sarcomas. In this aspect, the Lgr5 receptor has great potential to be a new molecular target, being a marker of stem cells of the intestinal crypts and mammary glands that also acts as a negative modulator of the signaling of the Wnt/ß-catenin pathway, one of the most important in the biology of sarcomas emerges as a promising candidate for preclinical studies, since its importance in tumors of the gastrointestinal tract has already been demonstrated. To that end, patient-derived xenograft (PDX) tumor models represent a valuable platform to identify new biomarkers and new targets, as does Lgr5 to assess therapy response and resistance mechanisms. OBJECTIVE: This study aimed to establish, characterize, and test the Lgr5 protein through in vitro and in vivo assays to unravel the importance of Lgr5 in the biology of PMS and to establish an integrated, convergent and translational framework for the study of this type of tumor. MATERIALS AND METHODS: To determine the expression of Lgr5, two study cohorts were established, a retrospective one from the Institutional Registry of Sarcomas and a prospective one, in which patients operated on at the Institution were invited to generate Patient-derived xenografts (PDX), a pre-model -clinical that has the ability to maintain the molecular characteristics of patients' tumors. For this, fragments implanted in immunosuppressed mice were used to generate these tumor models derived from patients, in addition to in vitro functional studies using SPM strains to analyze the expression profile of the Lgr5 protein through immunofluorescence assays to verify the ability to express Lgr5, flow cytometry to verify the pattern and amount of protein in the analyzed samples and western blotting to obtain a pattern of labeling of the Lgr5 protein. In addition to functional assays to assess the protein's participation in proliferation, whether protein expression interferes with the migratory power of SPM cells and tumors and self-renewal capacity, as well as its association with clinical data and survival data. RESULTS: The Institutional Retrospective Registry has more than 300 patients, while the Prospective Registry has 70 patients who derived the generation of 33 PDX. It was observed that patients with an H-score greater than 20 had a lower overall survival at 5 years compared to the H-score of patients with values below 20. Now in the other analysis performed, the H-score of patients with values greater than 25 it is worse compared to those who had values less than 25 in the disease-free survival data. Furthermore, cells that overexpress the Lgr5 protein have greater migratory capacity (p=0.02) and a tendency to increase proliferation and self-renewal. We performed the implant test of these positive and negative populations of Lgr5, previously separated by cell sorting. For this, Balb/c Nude animals were used. Suggesting that the expression of the transduced protein can be modulated by compensatory mechanisms that need to be explored. CONCLUSION: The construction of the Institutional Registry of PMS is a big step towards a better understanding of the biology of Sarcomas, in addition to the possibility of studying new therapeutic targets for this rare tumor, since studies and scientific articles are still very scarce. The generation of PDX models was also an implemented strategy very well executed with the generation of 33 PDX of several histological subtypes. In addition to the Lgr5 protein inducing cell migration, its expression is related to a worse prognosis, since the higher the Lgr5 expression, the lower the overall survival of the patient

The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology.

BACKGROUND: The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. RESULTS: We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. CONCLUSION: We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology

Abstract Background The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. Results We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. Conclusion We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

Expression of stem cell factors in the adult sea cucumber digestive tube.

Homeostatic cell turnover has been extensively characterized in mammals. In their adult tissues, lost or aging differentiated cells are replenished by a self-renewing cohort of stem cells. The stem cells have been particularly well studied in the intestine and are clearly identified by the expression of marker genes including Lgr5 and Bmi1. It is, however, unknown if the established principles of tissue renewal learned from mammals would be operating in non-mammalian systems. Here, we study homeostatic cell turnover in the sea cucumber digestive tube, the organ with high tissue plasticity even in adult animals. Both the luminal epithelium and mesothelium express orthologs of mammalian Lgr5 and Bmi1. However, unlike in mammals, there is no segregation of these positively labeled cells to specific regions in the luminal epithelium, where most of the cell proliferation would take place. In the mesothelium, the cells expressing the stem cell markers are tentatively identified as peritoneocytes. There are significant differences among the five anatomical gut regions in cell renewal dynamics and stem factor expression. The cloaca differs from the rest of the digestive tube as the region with the highest expression of the Lgr5 ortholog, lowest level of Bmi1 and the longest retention of BrdU-labeled cells.