Identificación de TRIM29 localizado en la región cromosómica 11q23.3 como biomarcador de resistencia a Doxorubicina y proliferación celular en cáncer de mama triple negativo

El cáncer de mama es una enfermedad compleja y heterogénea en la que los pacientes pueden presentar síntomas similares y padecer la misma enfermedad por razones genéticas completamente diferentes. Los factores que contribuyen a esta heterogeneidad incluyen la variación en el genoma de cada paciente, las diferencias en el origen y la naturaleza de la célula tumoral y los eventos genéticos que contribuyen a la progresión del tumor. La recaída en cáncer de mama es una de las mayores causas de morbilidad en pacientes que desarrollan la enfermedad y, entre ellas, entre un 15-20% poseerán tumores de mama triple negativo (TN). La mortalidad de pacientes con cáncer de mama TN es considerablemente mayor si se compara con otros tumores mamarios, siendo complicado predecir respuestas individuales al tratamiento. Estos datos resaltan la necesidad de nuevas estrategias que integren información tanto clínica, como genética y molecular para poder predecir respuestas individuales a fármacos o la progresión de la enfermedad. En la presente tesis doctoral se ha realizado el estudio del estado de la región 11q23.3 y se ha identificado el biomarcador TRIM29, presente en dicha región. Se ha estudiado el estado del gen, la función de TRIM29 en líneas celulares de cáncer de mama, su relación en la proliferación, migración y la resistencia a tratamientos mediante Doxorubicina. Para llevar a cabo los objetivos propuestos realizamos diferentes experimentos basados en técnicas de biología molecular, celular y técnicas bioinformáticas. Para la hibridación mediante Hibridación Fluorescente in situ (FISH), se elaboraron sondas no comerciales a partir del marcaje de Cromosomas Artificiales Bacterianos (BACs), con la finalidad de visualizar el estado de la región genómica de interés. De un stock de 59 líneas celulares, obtuvimos DNA, RNA, y proteína, con el fin de estudiar el estado de la región génica y la expresión de TRIM29 a nivel de mensajero y proteína. Se seleccionaron las lineas celulares con diferente expresión de TRIM29 y se modificó su expresión con la finalidad de estudiar los efectos tanto a nivel funcional como para observar si variaba la co-expresion con genes relacionados positivamente. Por último, a nivel celular, estudiamos las diferencias en migración, proliferación y resistencia frente a los tratamientos, entre las líneas control y las modificadas. Además, se elaboraron Tissue Microarray (TMAs) de parafinas de 97 tumores de cáncer de mama para comprobar la expresión de expresión de TRIM29 en los diferentes subtipos grácias a los marcadores de clasificación estudiados en pacientes.Breast cancer is a complex and heterogeneous disease where the patitets present the same syntomphs and suffer the same disease, but due to different genetic reasons. Some factors that contribute to this heterogeneity include patient genome variation, differences between the origin and the nature of the tumoral cell, and tumor progression due to genomic changes. Breast cancer recurrence is one of the biggest morbility causes in patients that suffer the disease, and 15-20% of them will develop triple negative breast cancer tumors (TN). Comparing it with other tumor subtypes, the mortality of patients harbouring TN tumors is higher, in part, due to the difficulty of getting personalized drugs. These data show the need to look for new drug strategy, where clinical, genetics and molecular data could be integred and compared. The main objetive in this Thesis is to analyze the genomic status of 11q23.3 chromosomal region and the gene TRIM29 that mapped at this region as a candidate biomarker of breast cancer proliferation and agressivenes but also as a of therapeutic response both in breast cancer cell lines and patients. To accomplish the objectives proposed, we will perform different experiments based in molecular biology, celular and bioinformatic techniques. In order to perform the fluorescence in situ hybridization (FISH), non-commercial probes where done by using labelled BACs. Starting from samples of DNA, RNA and protein of 59 cell lines, we will study the status of TRIM29 region and TRIM29 mRNA and protein expression level. Moreover, we have 600 DNA breast cancer patients and controls collection, where we will be able to study alterations in TRIM29 region, and we will try to relate the gain or the deletion with the different subtypes of breast cancer. Besides, Tissue Microarray (TMA) of 97 breast cancer patients will be used to observe the expression of TRIM29 in the different subtypes according to the inmunohistochemycal subtype classification markers. Finally, to observe the biological implications of the biomarker, we will modify the expression of TRIM29 in TN breast cancer cell lines, who do not express the gene, but also in those who overexpress it. Once we get modified cell lines, we will study the differences in TRIM29 and related genes expression, migration, proliferation and drug treatment resistance between control and modified cell lines

Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer

Breast cancer; HeterodimerizationCáncer de mama; HeterodimerizaciónCàncer de mama; HeterodimeritzacióAnti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer.A.A.-A., E.J.A., and F.B.-M. were supported by Asociación Española contra el Cáncer AECC (PRDVA18013LLUC to A.A.-A., POSTD211413AREN to E.J.A., and AECC_Postdoctoral17-1062 to F.B.-M.). A.M.-S. was funded by the Spanish Government (PFIS FI20/00188). J.Ar. is supported by Breast Cancer Research Foundation (BCRF-20-08), Instituto de Salud Carlos III Project reference number AC15/00062, and the EC under the framework of the ERA-NET TRANSCAN-2 initiative cofinanced by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociación Española Contra el Cáncer (AECC). A.P. was supported by Instituto de Salud Carlos III—PI19/01846, Breast Cancer Now—2018NOVPCC1294. P.E. and A.L. were funded by Instituto de Salud Carlos III and cofinanced by FEDER (PI18/01219 to P.E. and CB16/12/00481 to A.L.). J.M.C. was funded by Sociedad Española de Oncología Médica (Rio Hortega-SEOM) and Compromiso ADAMED

Circadian PERformance in breast cancer: a germline and somatic genetic study of PER3VNTR polymorphisms and gene co-expression.

Polymorphisms in the PER3 gene have been associated with several human disease phenotypes, including sleep disorders and cancer. In particular, the long allele of a variable number of tandem repeat (VNTR) polymorphism has been previously linked to an increased risk of breast cancer. Here we carried out a combined germline and somatic genetic analysis of the role of the PER3VNRT polymorphism in breast cancer. The combined data from 8284 individuals showed a non-significant trend towards increased breast cancer risk in the 5-repeat allele homozygous carriers (OR = 1.17, 95% CI: 0.97-1.42). We observed allelic imbalance at the PER3 locus in matched blood and tumor DNA samples, showing a significant retention of the long variant (risk) allele in tumor samples, and a preferential loss of the short repetition allele (p = 0.0005). Gene co-expression analysis in healthy and tumoral breast tissue samples uncovered significant associations between PER3 expression levels with those from genes which belong to several cancer-associated pathways. Finally, relapse-free survival (RFS) analysis showed that low expression levels of PER3 were linked to a significant lower RSF in luminal A (p = 3 × 10-12) but not in the rest of breast cancer subtypes

Targeting HER2-AXL heterodimerization to overcome resistance to HER2 blockade in breast cancer

Anti-HER2 therapies have markedly improved prognosis of HER2-positive breast cancer. However, different mechanisms play a role in treatment resistance. Here, we identified AXL overexpression as an essential mechanism of trastuzumab resistance. AXL orchestrates epithelial-to-mesenchymal transition and heterodimerizes with HER2, leading to activation of PI3K/AKT and MAPK pathways in a ligand-independent manner. Genetic depletion and pharmacological inhibition of AXL restored trastuzumab response in vitro and in vivo. AXL inhibitor plus trastuzumab achieved complete regression in trastuzumab-resistant patient-derived xenograft models. Moreover, AXL expression in HER2-positive primary tumors was able to predict prognosis. Data from the PAMELA trial showed a change in AXL expression during neoadjuvant dual HER2 blockade, supporting its role in resistance. Therefore, our study highlights the importance of targeting AXL in combination with anti-HER2 drugs across HER2-amplified breast cancer patients with high AXL expression. Furthermore, it unveils the potential value of AXL as a druggable prognostic biomarker in HER2-positive breast cancer. AXL is a prognostic biomarker and a potential therapeutic target to restore trastuzumab response in HER2 + breast cancer