Identifying molecular mechanisms that determine resistance to CDK4/6 inhibitors in HER2-enriched advanced luminal breast cancer
[eng] Breast Cancer (BCa) is the most prevalent cancer among women worldwide. It is a very heterogeneous disease at the histological, molecular and clinical level. Consequently, different classifications are used for stratifying patients. The histopathological classification is used in patient clini...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2021 |
| País: | España |
| Recursos: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/185269 |
| Acesso em linha: | https://hdl.handle.net/2445/185269 http://hdl.handle.net/10803/674158 |
| Access Level: | acceso abierto |
| Palavra-chave: | Biologia molecular Citologia Genètica mèdica Càncer de mama Metàstasi Molecular biology Cytology Medical genetics Breast cancer Metastasis |
| Resumo: | [eng] Breast Cancer (BCa) is the most prevalent cancer among women worldwide. It is a very heterogeneous disease at the histological, molecular and clinical level. Consequently, different classifications are used for stratifying patients. The histopathological classification is used in patient clinical management. It is based on the expression of three different markers: Estrogen receptor (ER) and progesterone receptor, both hormone receptors, and the human epidermal growth factor receptor 2 (HER2). Recent technical advances have developed a novel classification based on the gene expression profile of the tumors. Four intrinsic molecular subtypes are defined by the expression of 50 specific genes, this gene signature is called PAM50. Accordingly, BCa is classified in Luminal A, Luminal B, HER2-enriched (HER2-E) and Basal –like. These subtypes have critical differences in incidence, survival and response to treatment and have a predictive and prognostic value at the treatment level. The histopathological classification and the molecular classification are independent. In February 2015, the FDA approved a new drug for postmenopausal women with metastatic ER+/HER2-negative BCa, CDK4/6 inhibitors (CDK4/6i). CDK4/6 are serine-threonine kinases that have a key role in cell cycle regulation by allowing S phase progression. These inhibitors have provided clinical improvement in terms of progression-free survival and overall survival compared with endocrine therapy alone. CDK4/6i are now standard of care for treating metastatic ER+ BCa patients. PAM50 has been used to predict benefit from CDK4/6i in retrospective analysis of clinical trials. According to these retrospective analyses, CDK4/6i are only beneficial in luminal A and B. Thus, HER2-E tumors, which were a big proportion of treated tumors, did not benefit from CDK4/6i. Understanding mechanisms responsible for primary or secondary resistance to CDK4/6i in advanced ER+ BCa is crucial to improve patient outcome. We aim to identify new candidate genes responsible for resistance to palbociclib (CDK4/6i) treatment in the HER2-E intrinsic subtype of ER+/HER2- BCa by combining an unbiased CRISPR-Cas9-based genome-wide screening approach and the analysis of clinical data. To approach this question, we characterized CDK4/6i response in ER+ BCa cell lines. We determined ER+ BCa cell lines that are palbo-resistant or palbo-sensitive based on palbociclib IC50. Next, we subjected the palbo-resistant cell lines to CRISPR-cas9 sgRNA pooled genome-wide screening. We identified different genes that confer resistance to palbociclib in vitro. In parallel to the unbiased CRISPR/Cas9 knock-out screening, we interrogated which genes were associated with worse resp onse to CDK4/6i using two different patient cohorts (CDK patient cohort and CORALEEN data set). We integrated all 11 the data and we identified eleven common genes out of the three analyses. Nine out of the eleven genes were related to cell cycle progression machinery such as E2F1 and CCNE1, whereas the other two genes were membrane molecules associated with signal transduction. We selected one of these membrane molecules for further validation based on its tight association with HER2-E molecular subtype. High expression of this gene was associated with worst progression-free survival and overall survival in the CDK patient cohort. The patients that had progression disease presented a higher mRNA level of this resistance driver. Similar observations were found in samples from the CORALEEN clinical trial. Patients that did not respond to CDK4/6i treatment showed an increased expression of this gene. At the cellular level, different in vitro strategies were followed to determine the effect of the expression of this gene in palbociclib response. We developed acquired resistant cell lines from palbo-sensitive MCF7 cells and used them to validate our candidate. Moreover, loss- and gain-of-function approaches confirmed the association of this gene with an increased resistance to palbociclib treatment. Finally, we study how mechanistically this gene drives resistance to palbociclib. To this end, we interrogated alterations in gene expression of different genes after silencing this membrane protein in different cell lines. We detected reduced CCNE1 expression levels. CCNE1 gene was identified through the CRISPR/Cas9 screen and it was associated with no response and progression disease in both CDK and CORALEEN patient cohorts. Importantly, when we silenced CCNE1 in ZR75 cells we successfully increased CDK4/6i sensitivity. In addition, we optimized a proximity-dependent labeling approach, BioID, in order to describe the interactome of our gene of interest in parental and acquired resistant MCF7 cells. We found interesting downstream signaling transducers to further analyzed as a mechanism of action. In conclusion, we succeed identifying a new driver of CDK4/6i resistance in ER+/HER2-negative advanced BCa classified as HER2-E molecular subtype. |
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