Radiation and Stemness Phenotype May Influence Individual Breast Cancer Outcomes: The Crucial Role of MMPs and Microenvironment

Breast cancer is the most common cancer in women. Radiotherapy (RT) is one of the mainstay treatments for cancer but in some cases is not effective. Cancer stem cells (CSCs) within the tumor can be responsible for recurrence and metastasis after RT. Matrix metalloproteases (MMPs), regulated mainly by tissue inhibitors of metalloproteinases (TIMPs) and histone deacetylases (HDACs), may also contribute to tumor development by modifying its activity after RT. The aim of this work was to study the effects of RT on the expression of MMPs, TIMPs and HDACs on different cell subpopulations in MCF-7, MDA-MB-231 and SK-BR-3 cell lines. We assessed the in vitro expression of these genes in different 3D culture models and induced tumors in female NSG mice by orthotopic xenotransplants. Our results showed that gene expression is related to the cell subpopulation studied, the culture model used and the single radiation dose administered. Moreover, the crucial role played by the microenvironment in terms of cell interactions and CSC plasticity in tumor growth and RT outcome is also shown, supporting the use of higher doses (6 Gy) to achieve better control of tumor developmentThis research was funded by the FUNDACIÓN PROGRESO Y SALUD, Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI-730), the INSTITUTO DE SALUD CARLOS III, Ministerio de Ciencia, Innovación y Universidades (PIE16-00045) and by the Chair “Doctors Galera-Requena in cancer stem cell research” (CMC-CTS963)

Matrix metalloproteases and TIMPs as prognostic biomarkers in breast cancer patients treated with radiotherapy: A pilot study

Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E‐cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (‐1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐10, ‐12 and ‐13) and TIMPs (‐1 to ‐4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP‐1 and TIMP‐3 levels. Significant correlations were found between MMP‐3 and TIMP‐4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP‐9 and TIMP‐3 levels could be predictive of RT toxicity. For this reason, MMP‐3, MMP‐9, TIMP‐3 and TIMP‐4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT.FUNDACIÓN PROGRESO Y SALUD, Grant/Award Number: PI‐730; Instituto de Salud Carlos III, Grant/Award Number: PIE16‐00045; Oncología Básica y Clínica, Grant/Award Number: CTS‐20

miRNAs as radio-response biomarkers for breast cancer stem cells

In breast cancer (BC), the presence of cancer stem cells (CSCs) has been related to relapse, metastasis, and radioresistance. Radiotherapy (RT) is an extended BC treatment, but is not always effective. CSCs have several mechanisms of radioresistance in place, and some miRNAs are involved in the cellular response to ionizing radiation (IR). Here, we studied how IR affects the expression of miRNAs related to stemness in different molecular BC subtypes. Exposition of BC cells to radiation doses of 2, 4, or 6 Gy affected their phenotype, functional characteristics, pluripotency gene expression, and in vivo tumorigenic capacity. This held true for various molecular subtypes of BC cells (classified by ER, PR and HER-2 status), and for BC cells either plated in monolayer, or being in suspension as mammospheres. However, the effect of IR on the expression of eight stemness- and radioresistance-related miRNAs (miR-210, miR-10b, miR-182, miR-142, miR-221, miR-21, miR-93, miR-15b) varied, depending on cell line subpopulation and clinicopathological features of BC patients. Therefore, clinicopathological features and, potentially also, chemotherapy regimen should be both taken into consideration, for determining a potential miRNA signature by liquid biopsy in BC patients treated with RT. Personalized and precision RT dosage regimes could improve the prognosis, treatment, and survival of BC patients.This work has been partially funded by the Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía and European Regional Development Fund (ERDF), ref. SOMM17/6109/ UGR, and with grants from the Ministry of Economy and Competitiveness (FEDER funds, projects no. PIE16/00045) and from the Chair ‘Doctors Galera- Requena in cancer stem cell research’ (CMC-CTS963)

Algunas evidencias de aplicación

Libro temático especializadoLa sustentabilidad también debe aplicarse al sistema de producción, buscando impulsar transformaciones graduales de los estilos y modelos productivos tradicionales a unas de mayor eficiencia. Y donde se incorpore la dimensión ambiental y geográfico-espacial, para crear estructuras productivas más progresivas y equitativas en las sociedades. Todo esto, como alternativa para revertir las tendencias de escasez y agotamiento de los recursos naturales, así como de los desequilibrios globales, cuyos costos permean todos los tejidos humanos. De esta manera, la “sustentabilidad productiva” se concibe como la generación de bienes y servicios con ciertos estándares de calidad, bajo un esquema de eficiencia, rendimiento y de organización inclusiva e integrada, con baja presión al ambiente y uso racional de los recursos, garantizando la estadía y permanencia de los insumos y materiales en el tiempo. Desde esta perspectiva, la producción sustentable y el crecimiento de largo plazo pueden ser explicados por la capacidad que tienen las economías para generar e incorporar conocimientos y tecnologías. De ahí que, la educación y las cualificaciones del capital humano, los cambios en la organización de la producción y la calidad institucional, sean elementos nodales para avanzar en la consolidación de este ambiente productivo

Chemical, Physical and Biological Approaches to Prevent Ochratoxin Induced Toxicoses in Humans and Animals

Ochratoxins are polyketide derived fungal secondary metabolites with nephrotoxic, immunosuppressive, teratogenic, and carcinogenic properties. Ochratoxin-producing fungi may contaminate agricultural products in the field (preharvest spoilage), during storage (postharvest spoilage), or during processing. Ochratoxin contamination of foods and feeds poses a serious health hazard to animals and humans. Several strategies have been investigated for lowering the ochratoxin content in agricultural products. These strategies can be classified into three main categories: prevention of ochratoxin contamination, decontamination or detoxification of foods contaminated with ochratoxins, and inhibition of the absorption of consumed ochratoxins in the gastrointestinal tract. This paper gives an overview of the strategies that are promising with regard to lowering the ochratoxin burden of animals and humans

Importancia de las células madre tumorales (CSCS) y de las metaloproteasas (MMPS) en respuesta a la radiación en cáncer de mama

CONCLUSIONES 1. La expresión de los marcadores de CSCs de mama (ALDH1, CD44+ y CD24-/low) varía con la dosis de radiación administrada. En la subpoblación positiva (CSCs), a dosis altas (6 Gy) aumenta el CD44+, el cual se asocia con la EMT y un mal pronóstico en cáncer de mama. 2. En los estudios in vitro de cultivo 3D y 3D+lrECM, la expresión de MMPs, TIMPs y HDACs varía en función de la línea tumoral (MCF-7, MDA-MB-231 y SK-BR-3), la dosis de radiación (0, 2 y 6 Gy), la subpoblación celular (general, positiva o CSCs, y negativa o no-CSCs) y el modelo de cultivo 3D (esferas en suspensión o embebidas en Matrigel). 3. Tras la irradiación celular y en ambos modelos de cultivo 3D, la MMP-1 aumenta su expresión en la subpoblación positiva (CSCs) de la línea triple negativa MDA-MB-231, la cual es la más radioresistente. Este hecho sugiere la importancia de MMP-1 en el proceso de invasión y metástasis tras el tratamiento con radiación. 4. El microambiente tumoral también se altera tras el tratamiento con radiación, afectando a la expresión de MMPs, TIMPs y HDACs. 5. En el estudio in vitro de co-cultivo (líneas celulares tumorales con fibroblastos), en general, la mayoría de genes estudiados aumentan su expresión a dosis altas de radiación. Este comportamiento apoya la importancia de los componentes del microambiente tumoral (entre ellos, fibroblastos) en la progresión de la enfermedad. 6. En el estudio in vivo, los tumores formados a partir de células MDA-MB-231 irradiadas a 2 Gy previo a la inoculación han sido de mayor volumen con respecto al control en las subpoblaciones general y positiva. Este hecho pone de manifiesto que dosis bajas de radiación serían insuficientes para erradicar las CSCs debido a su radioresistencia, contribuyendo al crecimiento tumoral. 7. En el desarrollo tumoral in vivo, los tumores formados a partir de células MDA-MB-231 irradiadas a 6 Gy previo a la inoculación han sido los más pequeños dentro de cada subpoblación celular. Este resultado sugiere que a dosis altas de radiación (6 Gy) se reduce la velocidad de crecimiento tumoral y el volumen final del mismo. 8. El estudio piloto realizado en pacientes con cáncer de mama pone de manifiesto que la RT induce alteraciones en los niveles séricos de MMPs y TIMPs. 9. Las pacientes de cáncer de mama con recidiva tumoral muestran un aumento en los niveles séricos de todas las MMPs analizadas y una disminución en los niveles de TIMP-1. Este hecho apoya la teoría de que TIMP-1 es el principal inhibidor de las MMPs y el más afín a ellas. Es importante considerar que el tamaño muestral es pequeño y se requieren estudios adicionales para corroborar estos hallazgos. 10. Finalmente, los resultados obtenidos en este trabajo confirman la importancia del microambiente y sus componentes, y de las CSCs en el desarrollo tumoral y en la respuesta al tratamiento con radiación.CONCLUSIONS 1. The expression of the BCSCs markers (ALDH1, CD44+ and CD24-/low) varies with the dose of radiation administered. In the positive subpopulation (CSCs), high doses of radiation (6 Gy) increases CD44+, which is associated with EMT and a poor prognosis in BC. 2. In the in vitro 3D and 3D+lrECM studies, the expression of MMPs, TIMPs and HDACs varies depending on the tumor line (MCF-7, MDA-MB-231 and SK-BR-3), the radiation dose (0, 2 and 6 Gy), the cell subpopulation (general, positive or CSCs, and negative or non-CSCs) and the 3D culture model (spheres suspended or embedded in Matrigel). 3. After cell irradiation and in both 3D culture models, MMP-1 increases its expression in the positive subpopulation (CSCs) of the MDA-MB-231 triple negative line, which is the most resistant. This fact suggests the importance of MMP-1 in the process of invasion and metástasis after radiation treatment. 4. The tumor microenvironment is also altered after radiation treatment, affecting the expression of MMPs, TIMPs and HDACs. 5. In the in vitro co-culture study (tumor lines with fibroblasts), in general, most of the genes studied increase their expression at high doses of radiation. This behavior supports the importance of the components of the tumor’s microenvironment (including fibroblasts) in the progression of the disease. 6. In the in vivo study, tumors formed from MDA-MB-231 cells irradiated at 2 Gy prior to inoculation have been of higher volume with respect to the control in the general and positive subpopulations. This fact shows that low doses of radiation would be insufficient to eradicate CSCs due to their radioresistance, contributing to tumor growth. 7. In tumor development in vivo, tumors formed from MDA-MB-231 cells irradiated at 6 Gy prior to inoculation dose have been the smallest within each cell subpopulation. This result suggests that at high doses of radiation (6 Gy) the tumor growth rate and the final volume of the tumor are reduced. 8. The pilot study carried out in BC patients shows that RT induces alterations in the serum levels of MMPs and TIMPs. 9. BC patients with tumor recurrence show an increase in serum levels of all MMPs analyzed and a decrease in TIMP-1 levels. This fact supports the theory that TIMP-1 is the main inhibitor of MMPs and the most related to them. The sample size is small and additional studies are required. 10. Finally, the results obtained in this work confirm the importance of the microenvironment and its components, and of the CSCs in tumor development and in the response to radiation treatment.Tesis Univ. Granada

Expression of metalloproteases in breast cancer stem cells after irradiation

INTRODUCTION: The CSCs constitute a small sub-population of cells in the tumor. They are characterized by their capacity for self-renewal, differentiation, metastasis, quiescence, tumorigenesis, survival in the blood stream, expression of surface markers, and resistance to chemotherapy and radiotherapy. Ionizing radiation treatment causes senescence, genomic and epigenetic instability, metabolism and cell cycle changes, DNA damage, modified DNA repair capacity, and CSC death. CSCs surviving radiation treatment generate survival mechanisms that enhance their invasion and metastasis processes. Different types of metalloproteinase (MMP) intervene in these processes. MMPs are family of endopeptidases secreted as zymogens by cells in the tumor and microenvironment. They are also implicated in other aspects of carcinogenesis such as EMT, tumor growth, apoptosis evasion, angiogenesis, inflammatory response, and pre-metastatic niche formation. These enzymes are regulated by TIMPs and, epigenetically, by HDACs. OBJECTIVE: To establish the proportion of CSCs in two human breast cancer cell lines after different radiation doses, and to determine CSC expression of different enzymes related to the tumor microenvironment (MMPs, TIMPs and HDACs) in the positive and negative CSC populations. MATERIAL AND METHODS: Two breast cancer cell lines (MCF-7 and MDA-MB-231) were maintained in monolayer culture and then cultured in sphere medium. At 24 h after seeding in monolayer, cells were irradiated at 2, 4 or 6 Gy, maintaining a non-irradiated control. Cells were then cultured for 7 days to form colonies, followed by flow cytometry studies to measure stem cell markers (ALDH1, CD24 and CD44). From the general population of cells cultured in sphere media, positive and negative CSCs were separated with flow cytometry. The two cell sub-populations were irradiated at doses of 0, 2, 4, or 6 Gy. After 24 hours of irradiation, RT-PCR was used for RNA extraction and quantification and cDNA synthesis, and qPCR for gene expression measurement. The genes measured in both positive and negative sub-populations were: MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, HDAC-1, HDAC-2, HDAC-4, TIMP-1 and TIMP-2. RESULTS: After ionizing radiation treatment, the proportion of CSCs markers varied according to the dosage and cell type. ALDH positivity was increased at 4 Gy in MCF-7 cells, while ALDH1 and CD44 were increased at 6 Gy in the MDA-MB-231 line. More genes were expressed in the MDA-MB-231 versus MCF-7 line. MMP-1, MMP-2, HDAC-4, and TIMP-1 genes were expressed in both cell lines. MMP-3, MMP-9, MMP-13, HDAC-2 and TIMP-2 were also expressed In MDA-MB-231 cells. In general, genes were more expressed in the negative population than in the positive, highlighting the positive CSCs expression of MMP-2 and MMP-9, which are relarelated to the formation of secondary tumors. CONCLUSIONS: Radiation enhances the population of CSCs in different ways according to the dose and cell line. Post-radiation, a larger number and higher proportion of genes were expressed in the MDA-MB-231 cell line, which is more radioresistant in comparison to MCF-7. The increased MMP-2 and MMP-9 expression after radiation would contribute to modifying the cell survival capacity and differentiation status of MDA-MB-231 cells and may correspond to a phenotype linked to carcinogenesis. The increase in this expression with higher radiation dose in the positive CSC population may play a role in the development of secondary tumors, facilitating ECM degradation by the cells that survive radiation. Administration of inhibitors of these MMPs to patients undergoing radiotherapy may be useful to avoid the radiation-induced development of a more aggressive phenotype that promotes tumor progression. Both MMP-2 and MMP-9 may be considered novel therapeutic targets in cancer treatment.INTRODUCCIÓN: Las CSCs constituyen una pequeña subpoblación de células dentro del tumor. Estas células se caracterizan por la capacidad de auto-renovación, diferenciación, metástasis, quiescencia, tumorigénesis, supervivencia al torrente sanguíneo, expresión de marcadores de superficie y resistencia a la quimioterapia y radioterapia. El tratamiento con radiación ionizante provoca en las CSCs senescencia, inestabilidad genómica y epigenética, alteración del metabolismo y del ciclo celular, daño en el ADN y modificación de su reparación, y muerte celular. Las CSCs que sobreviven al tratamiento con radiación generan mecanismos de supervivencia que les permite favorecer los procesos de invasión y metástasis, en los que también intervienen distintos tipos de metaloproteasas (MMP s). Las MMPs son una familia de endopeptidasas, secretadas como zimógenos tanto por las células tumorales como por las células del microambiente. Están además implicadas en otros procesos de la carcinogénesis como la TEM, el crecimiento tumoral, la evasión de la apoptosis, la angiogénesis, la inflamación, y la formación de nichos pre-metastásicos. Estas enzimas estám reguladas por los TIMPs y, epigenéticamente, por las HDACs. OBJETIVO: Establecer la proporción de CSCs en dos líneas celulares tumorales de cáncer de mama humano para distintas dosis de radiación y, determinar, en caso de que exista, la expresión de distintas enzimas asociadas al microambiente tumoral (MMPs, TIMPs y HDACs) en las poblaciones CSCs positiva y negativa. RESULTADOS: Tras el tratamiento con radiación ionizante, el porcentaje de marcadores de CSCs varía en función de la dosis y del tipo celular. En MCF-7 se observa un incremento de ALDH1 a los 4 Gy, mientras que en MDA-MB-231, aumentan los marcadores ALDH1 y CD44 a los 6 Gy. Se han expresado mayor número de genes en la línea MDA-MB-231 que en la MC F-7. Los genes expresados en ambas líneas han sido: MMP-1, MMP-2, HDAC-4 y TIMP-1. En MDA-MB-231, además, se han expresado la MMP-3, MMP-9, MMP-13, HDAC-2 y TIMP-2. De forma general, los genes han sido más expresados en la población negativa que en la positiva, destacando la expresión en las CSCs positivas de la MMP-2 y la MMP-9, las cuales están relacionadas con la formación de tumores secundarios. CONCLUSIONES: La radiación favorece la selección de CSCs de distinta manera dependiendo de la dosis y de la línea celular irradiada. Además, después de este tratamiento, mayor número de genes y en mayor proporción han sido expresados en la línea celular MDA-MB-231, la cual es más radio-resistente que la MCF-7. La expresión aumentada de MMP-2 y MMP-9 tras el tratamiento con radiación estaría contribuyendo, por tanto, a que las células MDA-MB-231 modifiquen su capacidad de supervivencia celular, su estado de diferenciación y a que reflejen un fenotipo asociado al proceso de carcinogénesis. El hecho de que esta expresión aumente con la dosis de radiación en la población CSC+, podría justificar el desarrollo de tumores secundarios, dado que aquellas células que no han sido eliminadas con radiación degradarían la MEC con mayor facilidad. En este sentido, sería interesante utilizar, junto a la radioterapia, inhibidores de estas MMPs para evitar que, tras el empleo de radiación, las células desarrollaran un fenotipo más agresivo que favorezca la progresión tumoral. De esta manera, tanto MMP-2 como MMP-9 podrían ser consideradas como nuevas dianas terapéuticas en el tratamiento del cáncer.Universidad de Granada. Máster en Avances en Radiología Diagnóstica y Terapéutica y Medicina Física. Curso Académico 2013-201

Metalloproteinases 1 and 3 as Potential Biomarkers in Breast Cancer Development

Breast cancer continues to be one of the main causes of morbidity and mortality globally and was the leading cause of cancer death in women in Spain in 2020. Early diagnosis is one of the most effective methods to lower the incidence and mortality rates of breast cancer. The human metalloproteinases (MMP) mainly function as proteolytic enzymes degrading the extracellular matrix and plays important roles in most steps of breast tumorigenesis. This retrospective cohort study shows the immunohistochemical expression levels of MMP-1, MMP-2, MMP-3, and MMP-9 in 154 women with breast cancer and 42 women without tumor disease. The samples of breast tissue are assessed using several tissue matrices (TMA). The percentages of staining (≤50%–>50%) and intensity levels of staining (weak, moderate, or intense) are considered. The immunohistochemical expression of the MMP-1-intensity (p = 0.043) and MMP-3 percentage (p = 0.018) and intensity, (p = 0.025) present statistically significant associations with the variable group (control–case); therefore, expression in the tumor tissue samples of these MMPs may be related to the development of breast cancer. The relationships between these MMPs and some clinicopathological factors in breast cancer are also evaluated but no correlation is found. These results suggest the use of MMP-1 and MMP-3 as potential biomarkers of breast cancer diagnosis.Fundación Progreso Salud, grant number PI-0730-2013, and by ISCIII, grant number PIE16/00045

CSC Radioresistance: A Therapeutic Challenge to Improve Radiotherapy Effectiveness in Cancer

Radiotherapy (RT) is a modality of oncologic treatment that can be used to treat approximately 50% of all cancer patients either alone or in combination with other treatment modalities such as surgery, chemotherapy, immunotherapy, and therapeutic targeting. Despite the technological advances in RT, which allow a more precise delivery of radiation while progressively minimizing the impact on normal tissues, issues like radioresistance and tumor recurrence remain important challenges. Tumor heterogeneity is responsible for the variation in the radiation response of the different tumor subpopulations. A main factor related to radioresistance is the presence of cancer stem cells (CSC) inside tumors, which are responsible for metastases, relapses, RT failure, and a poor prognosis in cancer patients. The plasticity of CSCs, a process highly dependent on the epithelial–mesenchymal transition (EMT) and associated to cell dedifferentiation, complicates the identification and eradication of CSCs and it might be involved in disease relapse and progression after irradiation. The tumor microenvironment and the interactions of CSCs with their niches also play an important role in the response to RT. This review provides a deep insight into the characteristics and radioresistance mechanisms of CSCs and into the role of CSCs and tumor microenvironment in both the primary tumor and metastasis in response to radiation, and the radiobiological principles related to the CSC response to RT. Finally, we summarize the major advances and clinical trials on the development of CSC-based therapies combined with RT to overcome radioresistance. A better understanding of the potential therapeutic targets for CSC radiosensitization will provide safer and more efficient combination strategies, which in turn will improve the live expectancy and curability of cancer patients