Caracterización de MYBBP como un nuevo gen supresor de tumores

El microambiente tumoral es un entorno heterogéneo y dinámico que se caracteriza por las constantes interacciones entre las células tumorales y las células del estroma y los continuos cambios en la distribución de nutrientes esenciales para el desarrollo del tumor. Frecuentemente se producen deficiencias locales y/o temporales en la vascularización del tumor que provocan la ausencia de glucosa, de oxígeno o de otros nutrientes esenciales para el desarrollo del mismo, generando situaciones con altos niveles de estrés celular. La caracterización de los mecanismos moleculares que favorecen la adaptación de las células tumorales a entornos críticos permitiría identificar nuevas dianas con potencial antitumoral. Por este motivo, realizamos un rastreo genético de pérdida de función en ausencia de glucosa en el que identificamos el gen MYB binding protein 1A (MYBBP1A). MYBBP1A es un regulador de factores de transcripción implicado en la regulación de varios procesos biológicos esenciales. En este trabajo hemos analizado muestras de tumores humanos, confirmando que la pérdida de MYBBP1A es un evento genético que se produce en tumores humanos, especialmente en tumores renales. Hemos analizado la expresión de MYBBP1A en una cohorte de 97 pacientes de cáncer renal, en la que hemos observado que la pérdida o reducción de la expresión de MYBBP1A se produce en el 8% de los tumores. Esta pérdida estaba asociada a la aparición de metástasis y peor pronóstico en términos de supervivencia libre de enfermedad y supervivencia global. En base a esta observación y a que se ha descrito que la degradación de MYBBP1A está regulada por pVHL, que se pierde frecuentemente en tumores renales, hemos utilizado líneas celulares de carcinoma renal para estudiar la posible función de MYBBP1A como supresor tumoral. Para estudiar el mecanismo molecular a través del cual MYBBP1A participaría en el proceso de tumorigénesis hemos analizado el efecto de la sobreexpresión y del silenciamiento de MYBBP1A en líneas celulares de carcinoma renal. La sobreexpresión de MYBBP1A suprime el crecimiento celular en todas las líneas celulares utilizadas independientemente del contexto molecular. Por otro lado, el silenciamiento de MYBBP1A induce el aumento de algunas propiedades tumorales en las líneas celulares que expresan c-MYB y no expresan pVHL. En estas líneas la reducción de la expresión de MYBBP1A induce la activación de c-MYB, conduciendo a la activación de la transcripción de sus genes diana CD34 y CXCR4 y al aumento del fenotipo equivalente a célula madre tumoral. Además de inducir el fenotipo de célula madre, hemos encontrado que c-MYB activa la transcripción de PGC-1α. Así mismo, la reducción de MYBBP1A activa a PGC-1α directa e indirectamente. De forma directa, la reducción de MYBBP1A permite la actividad de PGC-1α y de forma indirecta aumenta los niveles de PGC-1α a través de la activación de c-MYB. Finalmente, la activación de PGC-1α conduce al cambio metabólico de glucólisis a OXPHOS, el cual es más eficiente para producir ATP en condiciones de limitación de glucosa. Por tanto, la combinación de estos dos efectos como resultado de la disminución de la expresión de MYBBP1A proporciona una ventaja selectiva a las células tumorales. Además, hemos analizado la expresión de MYBBP1A y su correlación con la expresión de genes de vías de señalización implicadas en los procesos de iniciación y desarrollo tumoral. Hemos observado que la expresión de MYBBP1A correlaciona negativamente con la expresión de genes involucrados en el fenotipo de célula madre, identificando un subgrupo de carcinomas de células renales de células claras con un patrón de expresión característico. También hemos encontrado un conjunto de genes regulados transcripcionalmente por c-MYB cuya expresión correlaciona negativamente con MYBBP1A y positivamente con PGC-1α. Además, hemos identificado un subgrupo de tumores, en torno al 8% del total de las muestras, con un patrón de expresión característico: bajos niveles de expresión de MYBBP1A y altos niveles de expresión de PGC-1α y de los genes diana de c-MYB. Por último, hemos observado que la expresión de MYBBP1A correlaciona negativamente con la expresión de genes del ciclo de los ácidos tricarboxílicos. También hemos encontrado que el 9% de las muestras tumorales presentan bajos niveles de expresión de MYBBP1A y altos niveles de expresión de los genes del ciclo de los ácidos tricarboxílicos. Por tanto, estos análisis bioinformáticos confirman nuestros datos experimentales.Tumor microenvironment is heterogeneous and dynamic due to constant tumor and stromal cells interactions and continuous changes in nutrient distribution that are essential for tumor development. Frequently, there is a local and/or temporal lack of efficient vasculature that leads to glucose, oxygen or other nutrients depletion, increasing the stress levels of tumor cells. Characterization of molecular mechanisms that enable tumor cells to adapt to host microenvironments would allow identifying new targets for cancer therapy. To this end, we have performed a genetic loss of function screen in absence of glucose, identifying MYB binding protein 1A (MYBBP1A). MYBBP1A is a regulator of transcription factors involved in the control of several relevant biological processes. In this work we have analyzed the expression of MYBBP1A in human tumors, confirming that MYBBP1A loss is a genetic event found in renal tumors. In a cohort of 97 patients of renal cancer the MYBBP1A expression is lost or reduced in 8% of tumors. This loss was associated with metastasis and poor prognosis, measured by disease free survival and overall survival. Based on this observation and published data that MYBBP1A is a target of pVHL, which is frequently lost in renal tumors, we have used renal carcinoma cell lines to study the possible role of MYBBP1A as a tumor suppressor. In order to study the molecular mechanism through MYBBP1A would take part in tumorigenesis, we analyzed the effect of MYBBP1A overexpression and downregulation in renal carcinoma cell lines. MYBBP1A overexpression suppresses cell growth in all cell lines used regardless of the molecular context. On the other hand, downregulation of MYBBP1A increases some tumor properties in cell lines that express c-MYB and do not express pVHL. In these cell lines downregulation of MYBBP1A induces c-MYB activation, inducing transcriptional activation of its target genes CD34 and CXCR4, which lead to an increase in cancer stem cell-like phenotype. In addition, we have shown that c-MYB induce the transcription of PGC-1α. Hence, MYBBP1A dowrnregulaion activates PGC-1α direct and indirectly. In a direct manner, reduction of the MYBBP1A expression des-represses PGC-1α activity and indirectly increases PGC-1α mRNA levels through c-MYB activation. Finally, PGC-1α activation leads to a metabolic shift from glycolysis to OXPHOS, which is more efficient to produce ATP under glucose limitations. Therefore, combined effects of MYBBP1A downregulation provide selective advantage over other tumor cells. Furthermore, we analyzed MYBBP1A expression and its correlation to the expression of genes of signaling pathways involved in tumor initiation and development. We observed that MYBBP1A correlated negatively with the expression of genes involved in stem cell phenotype, detecting a subgroup of clear cell renal cell carcinomas with a characteristic expression pattern. We have also found some genes regulated transcriptionally by c-MYB whose expression correlated negatively with MYBBP1A and positively with PGC-1α expression. In addition, we identified a subgroup of tumors, 8% of all tumor samples, with a characteristic expression pattern: low expression of MYBBP1A and high expression of PGC-1α and c-MYB target genes. Finally, we observed that MYBBP1A expression correlated negatively with the expression of tricarboxylic acid cycle genes. We also detected that 9% of tumor samples showed low expression of MYBBP1A and high expression of tricarboxylic acid cycle genes. This bioinformatic analysis supports our experimental conclusions

MAP17 (PDZK1IP1) and pH2AX are potential predictive biomarkers for rectal cancer treatment efficacy

Rectal cancer represents approximately 10% of cancers worldwide. Preoperative chemoradiotherapy increases complete pathologic response and local control, although it offers a poor advantage in survivorship and sphincter saving compared with that of radiotherapy alone. After preoperative chemoradiotherapy, approximately 20% of patients with rectal cancer achieve a pathologic complete response to the removed surgical specimen; this response may be related to a better prognosis and an improvement in disease-free survival. However, better biomarkers to predict response and new targets are needed to stratify patients and obtain better response rates. MAP17 (PDZK1IP1) is a small, 17 kDa non-glycosylated membrane protein located in the plasma membrane and Golgi apparatus and is overexpressed in a wide variety of human carcinomas. MAP17 has been proposed as a predictive biomarker for reactive oxygen species, ROS, inducing treatments in cervical tumors or laryngeal carcinoma. Due to the increase in ROS, MAP17 is also associated with the marker of DNA damage, phosphoH2AX (pH2AX). In the present manuscript, we examined the values of MAP17 and pH2AX as surrogate biomarkers of the response in rectal tumors. MAP17 expression after preoperative chemoradiotherapy is able to predict the response to chemoradiotherapy, similar to the increase in pH2AX. Furthermore, we explored whether we can identify molecular targeted therapies that could help improve the response of these tumors to radiotherapy. In this sense, we found that the inhibition of DNA damage with olaparib increased the response to radio- and chemotherapy, specifically in tumors with high levels of pH2AX and MAP17.Spanish Ministry of Economy and Competitivity, Plan Estatal de I+D+I 2013–2016, ISCIII (Fis: PI15/00045) and CIBER de Cáncer (CB16/12/00275)co-funded by FEDER from Regional Development European Funds (European Union), Consejería de Ciencia e Innovación (CTS-1848)Consejería de Salud of the Junta de Andalucía (PI-0096–2014)

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

MAP17 and SGLT1 Protein Expression Levels as Prognostic Markers for Cervical Tumor Patient Survival

MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types. © 2013 Perez et al.This work was supported by grants from the Spanish Ministry of Science and Innovation and FEDER funds (SAF2009-08605), Consejeria de Ciencia e Innovacion and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142) and FIS (PI12/00137). AC’s laboratory is also funded by a fellowship from the Fundacion Oncologica FERO, supported by Fundació Josep Botet.Peer Reviewe

Spinophilin loss correlates with poor patient prognosis in advanced stages of colon carcinoma

[Purpose+ The genomic region 17q21 is frequently associated with microsatellite instability and LOH in cancer, including gastric and colorectal carcinomas. This region contains several putative tumor suppressor genes, including Brca1, NM23, prohibitin, and spinophilin (Spn, PPP1R9B, neurabin II). The scaffold protein Spn is one of the regulatory subunits of phosphatase-1 (PP1) that targets PP1 to distinct subcellular locations and couples PP1 to its target. Thus, Spn may alter cell-cycle progression via the regulation of the phosphorylation status of the retinoblastoma protein, a direct target of PP1. Therefore, we analyzed whether Spn levels were reduced in colorectal carcinomas and whether Spn levels correlated with prognosis or response to therapy.[Experimental Design] By means of immunohistochemistry or quantitative PCR, we studied the levels of Spn in stages II, III, and IV colorectal carcinoma tumors and correlated to other clinicopathologic features as well as prognosis or response to therapy.[Results] Spn was lost in a percentage of human gastric, small intestine, and colorectal carcinomas. In patients with colorectal carcinoma, tumoral Spn downregulation correlated with a more aggressive histologic phenotype (poorer tumor differentiation and higher proliferative Ki67 index). Consistent with this observation, lower Spn protein expression levels were associated with faster relapse and poorer survival in patients with stage III colorectal carcinoma, particularly among those receiving adjuvant fluoropyrimidine therapy. We validated this result in an independent cohort of patients with metastatic colorectal carcinoma treated with standard chemotherapy. Although patients that achieved an objective tumor response exhibited Spn levels similar to nontumoral tissue, nonresponding patients showed a significant reduction in Spn mRNA levels.[Conclusions] Our data suggest that Spn downregulation contributes to a more aggressive biologic behavior, induces chemoresistance, and is associated with a poorer survival in patients with advanced stages of colorectal carcinoma. © 2013 American Association for Cancer Research.This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2009-08605), Fondo de Investigacion Sanitaria (PI12/00137), Consejeria de Ciencia e Innovacion, and Consejeria de Salud of the Junta de Andalucia (CTS-6844 and PI-0142). A. Carnero's laboratory is also funded by a fellowship from Fundacion Oncologica FERO. P. Estevez-Garcia and I. Lopez-Calderero are supported by Rio Ortega Fellowships and S. Molina-Pinelo is supported by a Sara Borrell fellowship. R. Garcia-Carbonero is funded by the Instituto de Salud Carlos III, Ministerio de Sanidad, Spain (PI 10.02164).Peer Reviewe

The Tumor Suppressor Roles of MYBBP1A, a Major Contributor to Metabolism Plasticity and Stemness

The MYB binding protein 1A (MYBBP1A, also known as p160) acts as a co-repressor of multiple transcription factors involved in many physiological processes. Therefore, MYBBP1A acts as a tumor suppressor in multiple aspects related to cell physiology, most of them very relevant for tumorigenesis. We explored the different roles of MYBBP1A in different aspects of cancer, such as mitosis, cellular senescence, epigenetic regulation, cell cycle, metabolism plasticity and stemness. We especially reviewed the relationships between MYBBP1A, the inhibitory role it plays by binding and inactivating c-MYB and its regulation of PGC-1α, leading to an increase in the stemness and the tumor stem cell population. In addition, MYBBP1A causes the activation of PGC-1α directly and indirectly through c-MYB, inducing the metabolic change from glycolysis to oxidative phosphorylation (OXPHOS). Therefore, the combination of these two effects caused by the decreased expression of MYBBP1A provides a selective advantage to tumor cells. Interestingly, this only occurs in cells lacking pVHL. Finally, the loss of MYBBP1A occurs in 8%–9% of renal tumors. tumors, and this subpopulation could be studied as a possible target of therapies using inhibitors of mitochondrial respiration.This research was funded by grants from the Spanish Ministry of Economy and Competitivity, Plan Estatal de I+D+I 2013-2016 and Grants from the Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, a la Agencia Estatal de Investigación (AEI) y al Fondo Europeo de Desarrollo Regional (FEDER): RTI2018-097455-B-I00 (MCIU/AEI/FEDER, UE); CIBER de Cáncer (CB16/12/00275), co-funded by FEDER from Regional Development European Funds (European Union). Especial thanks to the Fundacion AECC for supporting this work. BFA was funded by Spanish Ministry of Education (FPU12/01380)

MicroRNA regulating metabolic reprogramming in tumor cells: New tumor markers

Metabolic reprogramming is a feature of cancer cells that provides fast energy production and the abundance of precursors required to fuel uncontrolled proliferation. The Warburg effect, increase in glucose uptake and preference for glycolysis over oxidative phosphorylation (OXPHOS) as major source of energy even in the presence of oxygen, is the main metabolic adaptation of cancer cells but not the only one. Increased glutaminolysis is also observed in cancer cells, being another source of adenosine triphosphate production and supply of intermediates for macromolecule biosynthesis. The ability to shift from OXPHOS to glycolysis and vice versa, known as metabolic plasticity, allows cancer cells to adapt to continuous changes in the tumor microenvironment. Metabolic reprogramming is linked to the deregulation of pathways controlled by hypoxia-inducible factor 1 alpha, MYC, or p53, and microRNAs (miRNAs) have emerged as key regulators of these signaling pathways. miRNAs target metabolic enzymes, oncogenes, and tumor suppressors involved in metabolic reprogramming, becoming crucial elements in the cross talk of molecular pathways that promotes survival, proliferation, migration, and consequently, tumor progression and metastasis. Moreover, several miRNAs have been found downregulated in different human cancers. Due to this fact and their central role in metabolism regulation, miRNAs may be considered as biomarkers for cancer therapy

Mitophagy in Cancer: A Tale of Adaptation

In the past years, we have learnt that tumors co-evolve with their microenvironment, and that the active interaction between cancer cells and stromal cells plays a pivotal role in cancer initiation, progression and treatment response. Among the players involved, the pathways regulating mitochondrial functions have been shown to be crucial for both cancer and stromal cells. This is perhaps not surprising, considering that mitochondria in both cancerous and non-cancerous cells are decisive for vital metabolic and bioenergetic functions and to elicit cell death. The central part played by mitochondria also implies the existence of stringent mitochondrial quality control mechanisms, where a specialized autophagy pathway (mitophagy) ensures the selective removal of damaged or dysfunctional mitochondria. Although the molecular underpinnings of mitophagy regulation in mammalian cells remain incomplete, it is becoming clear that mitophagy pathways are intricately linked to the metabolic rewiring of cancer cells to support the high bioenergetic demand of the tumor. In this review, after a brief introduction of the main mitophagy regulators operating in mammalian cells, we discuss emerging cell autonomous roles of mitochondria quality control in cancer onset and progression. We also discuss the relevance of mitophagy in the cellular crosstalk with the tumor microenvironment and in anti-cancer therapy responses

Mitophagy in Cancer: A Tale of Adaptation

In the past years, we have learnt that tumors co-evolve with their microenvironment, and that the active interaction between cancer cells and stromal cells plays a pivotal role in cancer initiation, progression and treatment response. Among the players involved, the pathways regulating mitochondrial functions have been shown to be crucial for both cancer and stromal cells. This is perhaps not surprising, considering that mitochondria in both cancerous and non-cancerous cells are decisive for vital metabolic and bioenergetic functions and to elicit cell death. The central part played by mitochondria also implies the existence of stringent mitochondrial quality control mechanisms, where a specialized autophagy pathway (mitophagy) ensures the selective removal of damaged or dysfunctional mitochondria. Although the molecular underpinnings of mitophagy regulation in mammalian cells remain incomplete, it is becoming clear that mitophagy pathways are intricately linked to the metabolic rewiring of cancer cells to support the high bioenergetic demand of the tumor. In this review, after a brief introduction of the main mitophagy regulators operating in mammalian cells, we discuss emerging cell autonomous roles of mitochondria quality control in cancer onset and progression. We also discuss the relevance of mitophagy in the cellular crosstalk with the tumor microenvironment and in anti-cancer therapy responses