EXAMINING MECHANISTIC UNDERPINNINGS OF CHEMORESISTANCE IN TRIPLE NEGATIVE BREAST CANCER

Background: Triple negative breast cancer (TNBC) is not only the most aggressive subtype of breast cancer, but it does also not have many targeted therapeutic options due to the lack of hormone receptor expression and enrichment of HER2. TNBCs are also more prone to the development of chemoresistance and metastatic progression, which are the main obstacles to reducing TNBC-related mortality. Question: The objective of this study is to identify targetable key node (s) that contribute to the development of chemoresistance in TNBC. Method: Differentially expressed genes in TNBC patients with or without relapse were analyzed to select functionally important genes in chemoresistant TNBC. TRIM29 was selected based on survival analysis. Carboplatin-resistant TNBC cells were established to explore the phenotypic and molecular differences. Various growth and migration assays were used to explore the phenotype of chemoresistant TNBC cells. Expression of TRIM29 and related pathways were assessed by immunoblotting and immunofluorescence analysis. Cells with TRIM29-knockout (KO) were established by the CRISPR system. Result: Chemoresistant TNBC cells overexpress TRIM29. Exhibiting the functional importance, overexpression of TRIM29 in MDAMB231 confers resistance to carboplatin. A stable knockout of TRIM29 in carboplatin-resistant cells results in improved response to carboplatin. Mechanistically, an enhanced expression of β-catenin and YAP1 is observed in chemoresistant as well as TRIM29-overxpressing cells. Both β-catenin and YAP1 exhibit nuclear colocalization in TRIM29-overexpressing cells. TRIM29-KO in carboplatin-resistant HCC1806 results in a drastic decrease in β-catenin level, but, paradoxically an increased level of YAP1 is observed indicating a feedback loop demanding further investigation. Conclusion:Our findings implicate TRIM29 enrichment as an important node in chemoresistant TNBC that may concomitantly modulate YAP1 and β-catenin as downstream oncogenic effectors

Mechanisms of liver regeneration in a clinical model for preoperative induction of liver hypertrophy prior to major liver resection

The liver is a central organ for homeostasis and has the unusual and remarkable property of regeneration with rapid restoration of its volume and function. Liver regeneration (LR) is a very enigmatic and complex process involving numerous intra and extrahepatic signals and pathways. Interestingly, genetic knockdown studies have often demonstrated delays in the course of regeneration, but no single signal has been identified to be both sufficient and necessary for LR. Furthermore, LR can be modified by multiple patient, liver or trauma-related factors. As a consequence, diverse clinical applications have been developed and used extensively including hepatic resections to remove liver tumours, split liver transplantation from donors, portal vein occlusion, artificial support in acute liver insufficiency or cellular therapy. Recently, a surgical technique named associated liver partition with portal vein ligation for staged hepatectomy (ALPPS) has shown an unprecedented property of accelerating LR. This procedure results in a greater speed of cell hypertrophy in the future liver remnant when compared with the gold standard portal vein embolization (PVE). These techniques are used for preoperative optimization of a small future liver remnant, avoid postoperative liver failure and, in turn, improve morbidity and mortality. Unfortunately, tumoral progression has been observed after PVE precluding the potential of curative hepatectomy. The ALPPS procedure may speed this hepatic resection and increase resectability rates. However, due to its associated high morbidity and mortality rates, diverse groups have developed modifications of the ALPPS technique (ALPPS variants). This includes radiofrequency assisted liver partition ablation with portal vein ligation for staged hepatectomy (RALPPS). By associating a line of necrosis with radiofrequency ablation following portal vein ligation, this two-stage technique allows a safe and rapid LR without an increased morbidity or mortality associated with the procedure. Herein, a general review of the process of LR is presented as well as further research of the intrinsic mechanisms of LR induced by PVE and the ALPPS variant, RALPPS.Open Acces

A Comprehensive Model and Modulation of Cellular Signaling Involved in Early Mammary Development and Aggressive Cancer Using a Novel Recombinant Protein of the G3 Domain of Laminin-5

The mammary gland is a unique and specialized epidermal organ; mammary organogenesis begins in the embryo but is not fully complete until puberty. As such, formation of the mammary gland depends on temporally and spatially regulated developmental steps that require coordination of multiple biological and cell signaling processes; many of which have parallels with cancer development. Research describing the events that occur between birth and puberty is lacking and little is known about human breast development of youth. Since mammary gland development requires a coordinated balance between cell growth, proliferation, and apoptosis, it is critical to understand which signaling pathways are utilized to relay developmental signals, and how these pathways and their targets interact and cooperate with age. Additionally, interactions between integrin molecules and their laminin ligands, especially Laminin-5 (Ln-5; also known as Laminin-332), regulate multiple facets of both embryonic development and tumor growth, invasion, and metastasis. α6β4 integrin serves as a marker to detect distant metastases in the early stages of specific malignancies and β4 integrin overexpression has been found in basal-like breast cancers, correlating with aggressiveness to institute a prognostic β4 signature that increases with tumor grade. The mechanism α6β4 integrin utilizes to modulate oncogenic signaling through association with Ln-5 molecules in the ECM is the basis for the recombinant protein (rG3, the third of five G domains of Ln-5) produced for the work reported in this dissertation. Here, it is shown there are specific transcriptional differences and a unique interaction of a gene set over time that contributes to postnatal mammary gland development, and this model clearly shares similarities and signaling pathways with oncogenic development. Especially important are pathways of the adaptive and innate immunities, ECM remodeling and integrin interactions, and extrinsic and intrinsic TP53-mediated apoptosis, greater understanding of which could lead to early detection of potential tumorigenic growth and identification of potential treatment avenues. Presented is a comprehensive model of early mammary development along with several panels of biomarkers that possess a role in normal mammary development, are involved in aggressive cancers, and are affected by apoptosis induced by rG3 treatment. rG3 has proven to be a valuable tool to study apoptotic pathways and the crosstalk among those pathways

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

Colorectal Cancer

Colorectal cancer is one of the most common malignancies worldwide, and the pathogenesis of this neoplasm is probably one of the most studied. The knowledge obtained over time has led to the development of screening and early diagnosis systems, allowing a significant reduction in the incidence of this neoplasm. However, what is currently known probably represents only the tip of the iceberg of the biology of this tumor. It was recently shown that the gut microbiota may contribute to colorectal cancerogenesis. In addition, several novel targeted therapies are now applied to patients with colorectal carcinoma. Nonetheless, several questions are still unanswered. Could the modulation of the gut microbiota modify the risk of tumor progression or the efficacy of therapies? Are there any predictive biomarkers of the risk of tumor progression or the efficacy of target therapies? Is metastatic colorectal cancer one or more diseases? This book collects a series of scientific articles reflecting part of the state of the art regarding colorectal cancer, seeking to answer these questions

Úloha Trim15 a UCHL3 v regulaci buněčného cyklu pomocí ubikvitin signalizace.

(ČEŠTINA) Ubikvitinace patří k důležitým regulačním mechanismům buňky, které kontrolují různé biologické procesy mezi které patří diferenciace, transkripce a buněčné dělení. Buněčné dělení vyžaduje duplikaci celého genomu v průběhu S- fáze buněčného cyklu, která je následována rovnoměrným rozdělením genetické informace mezi dvě dceřiné buňky v průběhu mitózy. Nesprávná regulace buněčného dělení může vést k aneuploidii, tedy k abnormálnímu počtu chromozomů v buňce. Aneuploidie jsou známou příčinnou vzniku rakoviny. Systematická analýza genomu tisíců vzorků z rakovinných buněk ukázala, že většina nádorů má abnormální počet chromozomů. V mé dizertační práci jsem se zabývala studiem dvou proteinů, které jsou součástí ubikvitin- proteazomového systému, konkrétně deubikvitináza UCHL3 a ubikvitin ligáza Trim15. UCHL3 jsme identifikovali pomocí "high- throughput" testování, které bylo cíleno na rozpoznání dosud neznámých faktorů regulujících buněčné dělení. Předchozí studie ukazují, že zvýšenou expresi UCHL3 můžeme najít v buňkách některých nádorů a to především ve vzorcích rakoviny tlustého střeva. Můj výzkum ukázal, že UCHL3 reguluje správné seskupení chromozomů v metafázi a jejich následné rozdělení do dvou dceřiných buněk. Ztráta (delece) UCHL3 vede k nesprávnému uchycení chromozomů k dělícímu vřeténku...(ENGLISH) Ubiquitin signaling is a key regulatory mechanism for many important cellular processes such as transcription, differentiation and cell division. Cell division requires duplication of all genetic material during S-phase followed by its precise partitioning between two daughter cells during mitosis. Misregulation of the complex mitotic machinery may lead to aneuploidy and genomic instability, known drivers of tumorigenesis. Indeed, systematic genetic analysis of many cancer tissues over the last decades, indicates the presence of severe chromosome abnormalities in thousands of cancer tissue samples. In this work, I investigated the function of two components of ubiquitin signaling, the deubiquitinating enzyme UCHL3 and the E3 ubiquitin ligase TRIM15. The hypothesized role of E3 ligase TRIM15 in the cell cycle regulation could not be confirmed by our experiments, but I observed an effect on cell adhesion and motility instead. UCHL3 was identified using high-content visual siRNA screen, as a critical factor controlling genome segregation and integrity. Interestingly, it has been previously reported that UCHL3 levels are altered in various cancer types, especially colon cancer. My data demonstrate that UCHL3 drives proper alignment of chromosomes at the metaphase plate by facilitating...Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc