Estudio de la relevancia de la interacción entre SPN Y PP1 en tumorigénesis

El gen SPN es un supresor de tumores importante en la progresión y malignidad tumoral en muchos tipos de tumores, entre ellos el cáncer de mama. Esta característica parece ser dependiente, al menos parcialmente, de su asociación a la fosfatasa PP1 y de la capacidad de esta holoenzima de desfosforilar a pRB. Mediante un análisis mutagénico se han descrito 122 mutaciones en la proteína SPN en tumores humanos, con una frecuencia aproximada del 0,2%. Estas mutaciones se encuentran a lo largo de toda la secuencia de la proteína, siendo de especial interés aquellas que pudieran afectar a la interacción de SPN con PP1 y pRB. Hemos caracterizado 4 mutaciones de SPN en dicha región y hemos comprobado que las mutaciones de SPN afectan de distinta manera a la interacción con PP1, tanto con la isoforma PP1α como con la isoforma PP1γ. Sin embargo, ni SPN endógena ni las proteínas SPN mutantes interaccionan con PP1β. Hemos observado que la holoenzima PP1-SPN interacciona con todas las proteínas de la familia de pRB (pRB, p107 y p130), al menos en sus formas fosforiladas, y que las mutaciones de SPN no afectan a esta interacción pero sí a la capacidad de dicha holoenzima de desfosforilar a las proteínas de la familia de pRB. Además, las mutaciones de SPN parecen afectar a la localización celular de la holoenzima, colocalizando con PP1 en el citoplasma. El efecto de cada una de las mutaciones en las líneas celulares de cáncer de mama T47D y MDA-MB-468 es diferente y solo A566V ha demostrado un aumento claro en las propiedades tumorales de las células y en el fenotipo de célula madre tumoral en ambas líneas celulares. De hecho, esta mutación aparece en distintos tipos de tumores humanos y ha sido encontrada en dos análisis mutacionales independientes. Las células que sobreexpresan la mutación A566V de SPN tienen mayores niveles de pRB y p107 fosforilada durante la transición G0/G1, así como mayores niveles de pRB fosforilada y, parcialmente, de p130 fosforilada al final de G1, probablemente debido a una deficiente desfosforilación por parte de la holoenzima PP1α/γ-SPN. Esto haría que la fase G1 sea más corta y que, por tanto, las células proliferen más rápido. Por tanto, la holoenzima PP1α/γ-SPN parece estar implicada en la regulación de la desfosforilación de las proteínas de la familia de pRB en la transición G0/G1 y, parcialmente, al final de G1, pero no a la salida de mitosis, fase en la que PP1β tiene un papel más relevante. Además, las células con la sobreexpresión de la mutación SPN-A566V presentan mayores niveles de expresión de genes relacionados con la pluripotencia de las células madre, como OCT4 y SOX2, por lo que podría existir una conexión entre la autorrenovación de las células y el ciclo celular a través de las proteínas de la familia de pRB y la holoenzima PP1-SPN. El efecto de las distintas mutaciones en las propiedades tumorales de las células parece depender del contexto molecular, ya que es más claro y consistente en la línea T47D que en la línea MDA-MB-468. Esto puede deberse a que la línea MDA-MB-468 no expresa pRB, dado que las mutaciones de SPN afectan a la interacción con PP1 y pRB y aunque esta línea sí exprese p107 y p130, sus funciones no son totalmente redundantes a las de pRB. Adicionalmente, se ha analizando el efecto de la mutación SPN-A566V en una línea inmortalizada no tumorigénica, MCF10A, que expresa p53 silvestre. Hemos comprobado que solo las células que sobreexpresaban tanto la mutación SPN-A566V como la mutación de p53 vieron aumentadas las propiedades tumorales de las células, así como las propiedades relacionadas con el fenotipo de célula madre tumoral. Por tanto, esto parecía indicar que la mutación A566V de SPN sería un evento que promueve la progresión tumoral una vez que esta se ha iniciado, principalmente a causa de mutaciones en p53. Al igual que otras mutaciones oncogénicas, la actividad de las mutaciones de SPN depende del contexto molecular en el que se produzcan, favoreciéndose la actividad protumoral cuando también se produce la inactivación de genes esenciales como p53. Estos datos confirman previas observaciones in vivo en ratones knock-out de Spn, en las que la ausencia de Spn por sí sola producía un aumento en la proliferación celular de algunos tejidos, necesitando también de la ausencia de p53 para permitir el desarrollo de tumores

The cargo protein MAP17 (PDZK1IP1) regulates the cancer stem cell pool activating the Notch pathway by abducting NUMB

Purpose: Cancer stem cells (CSC) are self-renewing tumor cells, with the ability to generate diverse differentiated tumor cell subpopulations. They differ from normal stem cells in the deregulation of the mechanisms that normally control stem cell physiology. CSCs are the origin of metastasis and highly resistant to therapy. Therefore, the understanding of the CSC origin and deregulated pathways is important for tumor control. Experimental Design: We have included experiments in vitro, in cell lines and tumors of different origins. We have used patient-derived xenografts (PDX) and public transcriptomic databases of human tumors. Results: MAP17 (PDZKIP1), a small cargo protein overexpressed in tumors, interacts with NUMB through the PDZ-binding domain activating the Notch pathway, leading to an increase in stem cell factors and cancer-initiating–like cells. Identical behavior was mimicked by inhibiting NUMB. Conversely, MAP17 downregulation in a tumor cell line constitutively expressing this gene led to Notch pathway inactivation and a marked reduction of stemness. In PDX models, MAP17 levels directly correlated with tumorsphere formation capability. Finally, in human colon, breast, or lung there is a strong correlation of MAP17 expression with a signature of Notch and stem cell genes. Conclusions: MAP17 overexpression activates Notch pathway by sequestering NUMB. High levels of MAP17 correlated with tumorsphere formation and Notch and Stem gene transcription. Its direct modification causes direct alteration of tumorsphere number and Notch and Stem pathway transcription. This defines a new mechanism of Notch pathway activation and Stem cell pool increase that may be active in a large percentage of tumors.Ministerio de Economía y Competitividad PI15/00045, CTS-1848Junta de Andalucía PI-00-96-2014, PI-0306-201

Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy

NumbL, or Numb-like, is a close homologue of Numb, and is part of an evolutionary conserved protein family implicated in some important cellular processes. Numb is a protein involved in cell development, in cell adhesion and migration, in asymmetric cell division, and in targeting proteins for endocytosis and ubiquitination. NumbL exhibits some overlapping functions with Numb, but its role in tumorigenesis is not fully known. Here we showed that the downregulation of NumbL alone is sufficient to increase NICD nuclear translocation and induce Notch pathway activation. Furthermore, NumbL downregulation increases epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related gene transcripts and CSC-like phenotypes, including an increase in the CSC-like pool. These data suggest that NumbL can act independently as a tumor suppressor gene. Furthermore, an absence of NumbL induces chemoresistance in tumor cells. An analysis of human tumors indicates that NumbL is downregulated in a variable percentage of human tumors, with lower levels of this gene correlated with worse prognosis in colon, breast and lung tumors. Therefore, NumbL can act as an independent tumor suppressor inhibiting the Notch pathway and regulating the cancer stem cell pool.España, Ministerio de Economía y Competitividad PI12/00137, PI15/00045España, Consejería de Ciencia e Innovación CTS-6844España, Consejería de Ciencia e Innovación CTS-1848España, Junta de Andalucía, Consejería de Salud PI-0306-2012España, Junta de Andalucía, Consejería de Salud PI-0096-201

CDH18 is a fetal epicardial biomarker regulating differentiation towards vascular smooth muscle cells

CDH18は胎児期の心外膜細胞の指標であり胎児心外膜から平滑筋細胞の分化を制御している. 京都大学プレスリリース. 2022-02-09.Using stem cells to regenerate the heart. 京都大学プレスリリース. 2022-02-09.The epicardium is a mesothelial layer covering the myocardium serving as a progenitor source during cardiac development. The epicardium reactivates upon cardiac injury supporting cardiac repair and regeneration. Fine-tuned balanced signaling regulates cell plasticity and cell-fate decisions of epicardial-derived cells (EPCDs) via epicardial-to-mesenchymal transition (EMT). However, powerful tools to investigate epicardial function, including markers with pivotal roles in developmental signaling, are still lacking. Here, we recapitulated epicardiogenesis using human induced pluripotent stem cells (hiPSCs) and identified type II classical cadherin CDH18 as a biomarker defining lineage specification in human active epicardium. The loss of CDH18 led to the onset of EMT and specific differentiation towards cardiac smooth muscle cells. Furthermore, GATA4 regulated epicardial CDH18 expression. These results highlight the importance of tracing CDH18 expression in hiPSC-derived epicardial cells, providing a model for investigating epicardial function in human development and disease and enabling new possibilities for regenerative medicine

Coordinated downregulation of Spinophilin and the catalytic subunits of PP1, PPP1CA/B/C, contributes to a worse prognosis in lung cancer

The scaffold protein Spinophilin (Spinophilin, PPP1R9B) is one of the regulatory subunits of phosphatase-1 (PP1), directing it to distinct subcellular locations and targets. The loss of Spinophilin reduces PP1 targeting to pRb, thereby maintaining higher levels of phosphorylated pRb. Spinophilin is absent or reduced in approximately 40% of human lung tumors, correlating with the malignant grade. However, little is known about the relevance of the coordinated activity or presence of Spinophilin and its reported catalytic partners in the prognosis of lung cancer. In the present work, we show that the downregulation of Spinophilin, either by protein or mRNA, is related to a worse prognosis in lung tumors. This effect is more relevant in squamous cell carcinoma, SCC, than in adenocarcinoma. Downregulation of Spinophilin is related to a decrease in the levels of its partners PPP1CA/B/C, the catalytic subunits of PP1. A decrease in these subunits is also related to prognosis in SCC and, in combination with a decrease in Spinophilin, are markers of a poor prognosis in these tumors. The analysis of the genes that correlate to Spinophilin in lung tumors showed clear enrichment in ATP biosynthesis and protein degradation GO pathways. The analysis of the response to several common and pathway-related drugs indicates a direct correlation between the Spinophilin/PPP1Cs ratio and the response to oxaliplatin and bortezomib. This finding indicates that this ratio may be a good predictive biomarker for the activity of the drugs in these tumors with a poor prognosis.España, Mineco Plan Estatal de I+D+I 2013-2016España, ISCIII Fis: PI15/00045CIBER de Cáncer CB16/12/00275, CB16/12/00443, CB16/12/00442España, Junta de Andalucía, Consejeria de Ciencia e Innovacion CTS-1848España, Junta de Andalucía, Consejeria de Salud PI-0096-201

New markers for human ovarian cancer that link platinum resistance to the cancer stem cell phenotype and define new therapeutic combinations and diagnostic tools

BACKGROUND: Ovarian cancer is the leading cause of gynecologic cancer-related death, due in part to a late diagnosis and a high rate of recurrence. Primary and acquired platinum resistance is related to a low response probability to subsequent lines of treatment and to a poor survival. Therefore, a comprehensive understanding of the mechanisms that drive platinum resistance is urgently needed. METHODS: We used bioinformatics analysis of public databases and RT-qPCR to quantitate the relative gene expression profiles of ovarian tumors. Many of the dysregulated genes were cancer stem cell (CSC) factors, and we analyzed its relation to therapeutic resistance in human primary tumors. We also performed clustering and in vitro analyses of therapy cytotoxicity in tumorspheres. RESULTS: Using bioinformatics analysis, we identified transcriptional targets that are common endpoints of genetic alterations linked to platinum resistance in ovarian tumors. Most of these genes are grouped into 4 main clusters related to the CSC phenotype, including the DNA damage, Notch and C-KIT/MAPK/MEK pathways. The relative expression of these genes, either alone or in combination, is related to prognosis and provide a connection between platinum resistance and the CSC phenotype. However, the expression of the CSC-related markers was heterogeneous in the resistant tumors, most likely because there were different CSC pools. Furthermore, our in vitro results showed that the inhibition of the CSC-related targets lying at the intersection of the DNA damage, Notch and C-KIT/MAPK/MEK pathways sensitize CSC-enriched tumorspheres to platinum therapies, suggesting a new option for the treatment of patients with platinum-resistant ovarian cancer. CONCLUSIONS: The current study presents a new approach to target the physiology of resistant ovarian tumor cells through the identification of core biomarkers. We hypothesize that the identified mutations confer platinum resistance by converging to activate a few pathways and to induce the expression of a few common, measurable and targetable essential genes. These pathways include the DNA damage, Notch and C-KIT/MAPK/MEK pathways. Finally, the combined inhibition of one of these pathways with platinum treatment increases the sensitivity of CSC-enriched tumorspheres to low doses of platinum, suggesting a new treatment for ovarian cancerSpanish Ministry of Education FPU12/01380Spanish Ministry of Economy and Competitivity, Plan Estatal de I + D + I 2013–2016Spanish Ministry of Science, Innovation and Universities (RTI2018–097455-B-I00)CIBER de Cáncer (CD16/12/00275)Spanish Consejería de Salud of the Junta de Andalucia (PI-0397-2017

Role of the holoenzyme PP1-SPN in the dephosphorylation of the rb family of tumor suppressors during cell cycle

Cell cycle progression is highly regulated by modulating the phosphorylation status of the retinoblastoma protein (pRB) and the other two members of the RB family, p107 and p130. This process is controlled by a balance in the action of kinases, such as the complexes formed by cyclindependent kinases (CDKs) and cyclins, and phosphatases, mainly the protein phosphatase 1 (PP1). However, while the phosphorylation of the RB family has been largely studied, its dephosphorylation is less known. Phosphatases are holoenzymes formed by a catalytic subunit and a regulatory protein with substrate specificity. Recently, the PP1-Spinophilin (SPN) holoenzyme has been described as the main phosphatase responsible for the dephosphorylation of RB proteins during the G0/G1 transition and at the end of G1. Moreover, SPN has been described as a tumor suppressor dependent on PP1 in lung and breast tumors, where it promotes tumorigenesis by increasing the cancer stem cell pool. Therefore, a connection between the cell cycle and stem cell biology has also been proposed via SPN/PP1/RB proteins

A new treatment for sarcoma extracted from combination of miRNA deregulation and gene association rules

Ministerio de Ciencia e Innovación ID2021-122629OB-I00Centro de Investigación Biomédica en Red (CIBER) CB16/12/00275Junta de Andalucía PI-0397- 2017, P18-RT-2501Asociación Española contra el Cáncer GC16173720CAR

A Six-Gene Prognostic and Predictive Radiotherapy-Based Signature for Early and Locally Advanced Stages in Non-Small-Cell Lung Cancer

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, generating an enormous economic and social impact that has not stopped growing in recent years. Cancer treatment for this neoplasm usually includes surgery, chemotherapy, molecular targeted treatments, and ionizing radiation. The prognosis in terms of overall survival (OS) and the disparate therapeutic responses among patients can be explained, to a great extent, by the existence of widely heterogeneous molecular profiles. The main objective of this study was to identify prognostic and predictive gene signatures of response to cancer treatment involving radiotherapy, which could help in making therapeutic decisions in patients with NSCLC. To achieve this, we took as a reference the differential gene expression pattern among commercial cell lines, differentiated by their response profile to ionizing radiation (radiosensitive versus radioresistant lines), and extrapolated these results to a cohort of 107 patients with NSCLC who had received radiotherapy (among other therapies). We obtained a six-gene signature (APOBEC3B, GOLM1, FAM117A, KCNQ1OT1, PCDHB2, and USP43) with the ability to predict overall survival and progression-free survival (PFS), which could translate into a prediction of the response to the cancer treatment received. Patients who had an unfavorable prognostic signature had a median OS of 24.13 months versus 71.47 months for those with a favorable signature, and the median PFS was 12.65 months versus 47.11 months, respectively. We also carried out a univariate analysis of multiple clinical and pathological variables and a bivariate analysis by Cox regression without any factors that substantially modified the HR value of the proposed gene signature