Pharmacological inhibition of Sox2 transcription factor-mediated tamoxifen resistance in breast cancer

Los capítulos IV y V están sujetos a confidencialidad por el autor. 137 p.El tamoxifeno, un antagonista del receptor de estrógeno (ER), es el fármaco más utilizado en pacientescon cáncer de mama ER-positivo. Sin embargo, el 30% de los casos desarrollan resistencia. Nuestrolaboratorio ha demostrado que el aumento del contenido de células madre cancerosas (CSCs) es mediadopor el incremento de la expresión del factor de transcripción Sox2 durante el desarrollo de resistencia altamoxifeno. En esta tesis, examinamos el potencial efecto inhibidor de los polioxometalatos (POMs)sobre Sox2 en células resistentes al tamoxifeno. En primer lugar, demostramos que diferentes POMsbloquean la actividad de unión al ADN de Sox2 in vitro. El derivado K6[P2W18O62] (PW) es el POM máseficaz en la inhibición del crecimiento celular induciendo una parada de ciclo celular y la muerte porapoptosis. Además, mostramos que PW bloquea específicamente la regulación del marcador de latransición epitelio-mesénquima (EMT) SNAI2 mediada por Sox2, que inhibe las capacidades demigración e invasión de células resistentes al tamoxifeno. Además, los ensayos in vivo en la membranacorioalantoidea de embriones de pollo confirman que la inhibición de Sox2 mediada por PW reduce elcontenido de la población de CSCs y restaura la sensibilidad al tamoxifeno. Molecularmente, lacorrelación inversa entre los niveles de expresión de Sox2 y ER se revierte con el tratamiento de PW encélulas resistentes al tamoxifeno. La regulación directa de Sox2 sobre el promotor del gen de ER se veafectada por el tratamiento con PW, lo que conlleva a la activación parcial de la vía de señalización deER y la restauración de la sensibilidad al tamoxifeno. Finalmente, la relevancia terapéutica de atacar a lasCSCs dependientes de Sox2 destaca el potencial uso clínico del derivado K6[P2W18O62] como inhibidorde Sox2 para tratar un subconjunto específico de pacientes con cáncer de mama resistente al tamoxifeno.CICbioGUN

Pharmacological inhibition of Sox2 transcription factor-mediated tamoxifen resistance in breast cancer

Los capítulos IV y V están sujetos a confidencialidad por el autor. 137 p.El tamoxifeno, un antagonista del receptor de estrógeno (ER), es el fármaco más utilizado en pacientescon cáncer de mama ER-positivo. Sin embargo, el 30% de los casos desarrollan resistencia. Nuestrolaboratorio ha demostrado que el aumento del contenido de células madre cancerosas (CSCs) es mediadopor el incremento de la expresión del factor de transcripción Sox2 durante el desarrollo de resistencia altamoxifeno. En esta tesis, examinamos el potencial efecto inhibidor de los polioxometalatos (POMs)sobre Sox2 en células resistentes al tamoxifeno. En primer lugar, demostramos que diferentes POMsbloquean la actividad de unión al ADN de Sox2 in vitro. El derivado K6[P2W18O62] (PW) es el POM máseficaz en la inhibición del crecimiento celular induciendo una parada de ciclo celular y la muerte porapoptosis. Además, mostramos que PW bloquea específicamente la regulación del marcador de latransición epitelio-mesénquima (EMT) SNAI2 mediada por Sox2, que inhibe las capacidades demigración e invasión de células resistentes al tamoxifeno. Además, los ensayos in vivo en la membranacorioalantoidea de embriones de pollo confirman que la inhibición de Sox2 mediada por PW reduce elcontenido de la población de CSCs y restaura la sensibilidad al tamoxifeno. Molecularmente, lacorrelación inversa entre los niveles de expresión de Sox2 y ER se revierte con el tratamiento de PW encélulas resistentes al tamoxifeno. La regulación directa de Sox2 sobre el promotor del gen de ER se veafectada por el tratamiento con PW, lo que conlleva a la activación parcial de la vía de señalización deER y la restauración de la sensibilidad al tamoxifeno. Finalmente, la relevancia terapéutica de atacar a lasCSCs dependientes de Sox2 destaca el potencial uso clínico del derivado K6[P2W18O62] como inhibidorde Sox2 para tratar un subconjunto específico de pacientes con cáncer de mama resistente al tamoxifeno.CICbioGUN

Dual Mechanisms of LYN Kinase Dysregulation Drive Aggressive Behavior in Breast Cancer Cells

The SRC-family kinase LYN is highly expressed in triple-negative/basal-like breast cancer (TNBC) and in the cell of origin of these tumors, c-KIT-positive luminal progenitors. Here, we demonstrate LYN is a downstream effector of c-KIT in normal mammary cells and protective of apoptosis upon genotoxic stress. LYN activity is modulated by PIN1, a prolyl isomerase, and in BRCA1 mutant TNBC PIN1 upregulation activates LYN independently of c-KIT. Furthermore, the full-length LYN splice isoform (as opposed to the Δaa25-45 variant) drives migration and invasion of aggressive TNBC cells, while the ratio of splice variants is informative for breast cancer-specific survival across all breast cancers. Thus, dual mechanisms-uncoupling from upstream signals and splice isoform ratios-drive the activity of LYN in aggressive breast cancers

Polyoxometalate inhibition of SOX2-mediated tamoxifen resistance in breast cancer

Background Increased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells. Methods Various POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis. Results POMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6− (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen. Conclusions Together, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.The authors thank grant support from the Basque Government (IT1722-22 and Elkartek KK-2022/00045) and Spanish Ministry of Science, Innovation and Universities (MICINN, grant PID2022-139530NB-I00) (JMGZ). This research was also funded by Elkartek (KK-2022/00045) by the Basque Government (MdMV), and by the Spanish Ministry of Science and Innovation MCIN/AEI/https://doi.org/10.13039/501100011033 (to MdMV and RK: CEX2021-001136-S; PRE2018-087073 to IG; PID2020-118464RB-I00 to MdMV, and PID2020-117649RB-100 to RK)

Laser Surface Microstructuring of a Bio-Resorbable Polymer to Anchor Stem Cells, Control Adipocyte Morphology, and Promote Osteogenesis

New strategies in regenerative medicine include the implantation of stem cells cultured in bio-resorbable polymeric scaffolds to restore the tissue function and be absorbed by the body after wound healing. This requires the development of appropriate micro-technologies for manufacturing of functional scaffolds with controlled surface properties to induce a specific cell behavior. The present report focuses on the effect of substrate topography on the behavior of human mesenchymal stem cells (MSCs) before and after co-differentiation into adipocytes and osteoblasts. Picosecond laser micromachining technology (PLM) was applied on poly (L-lactide) (PLLA), to generate different microstructures (microgrooves and microcavities) for investigating cell shape, orientation, and MSCs co-differentiation. Under certain surface topographical conditions, MSCs modify their shape to anchor at specific groove locations. Upon MSCs differentiation, adipocytes respond to changes in substrate height and depth by adapting the intracellular distribution of their lipid vacuoles to the imposed physical constraints. In addition, topography alone seems to produce a modest, but significant, increase of stem cell differentiation to osteoblasts. These findings show that PLM can be applied as a high-efficient technology to directly and precisely manufacture 3D microstructures that guide cell shape, control adipocyte morphology, and induce osteogenesis without the need of specific biochemical functionalization

A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer