Assuntos
Proteínas de Membrana/antagonistas & inibidores , Microtúbulos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Membrana/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Invasividade Neoplásica , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Tumoral de Célula-Tronco , UbiquitinaçãoRESUMO
Several observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset in atherosclerosis and restenosis. We characterized the phenotypic features of SMC subsets in cultures derived from human carotid endarterectomy specimens. Specimens comprised an undiseased portion (thin intimal thickening with the underlying media) and a diseased portion (atherosclerotic plaque with the underlying media). From plaque tissues of the diseased portion, only macrophage-derived foam cells were retrieved. From medial tissues, two SMC phenotypes were isolated: large SMCs (flat with a monolayered growth pattern, from the undiseased portion) and small SMCs (fusiform and growing in multilayers, from the undiseased and diseased portions after co-culture with macrophage-derived foam cells). Small SMCs displayed higher proliferative and migratory activities and were less differentiated than large SMCs. Proteomic analysis showed that calmodulin was predominant in small SMCs. Co-culture of large SMCs with macrophage-derived foam cells induced a transition to the small phenotype with increased calmodulin expression. The calmodulin inhibitor W-7 decreased the proliferation of small SMCs and prevented the large to small phenotypic transition. In vivo, calmodulin was markedly expressed in SMCs of atherosclerotic plaques and was barely detectable in the media. Macrophage-derived foam cells promote selective migration from the media of atheroma-prone SMCs characterized by calmodulin overexpression. Further studies of small SMCs could be instrumental in understanding atherosclerosis pathogenesis and in planning therapeutic strategies.
Assuntos
Calmodulina/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Placa Aterosclerótica/patologia , Becaplermina , Calmodulina/antagonistas & inibidores , Calmodulina/genética , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Proliferação de Células/efeitos dos fármacos , Separação Celular , Tamanho Celular , Meios de Cultivo Condicionados/farmacologia , Endarterectomia das Carótidas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fenótipo , Placa Aterosclerótica/genética , Proteômica , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologiaRESUMO
The tumour microenvironment (TME) is a complex and constantly evolving collection of cells and extracellular components. Cancer cells and the surrounding environment influence each other through different types of processes. Characteristics of the TME include abnormal vasculature, altered extracellular matrix, cancer-associated fibroblast and macrophages, immune cells, and secreted factors. Within these components, several molecules and pathways are altered and take part in the support of the tumour. Epigenetic regulation, kinases, phosphatases, metabolic regulators, and hormones are some of the players that influence and contribute to shaping the tumour and the TME. All these characteristics contribute significantly to cancer progression, metastasis, and immune escape, and may be the target for new approaches for cancer treatment.
RESUMO
Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. PDOs derived from two patients with TP53 mutations were tested with two different HDAC inhibitors (SAHA and NKL54). Cell death induction, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and the downregulation of highly expressed genes. A similar differential effect is observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death, which is characterized by the upregulation of BH3-only genes BIK and BMF. Up-regulation of BIK is associated with increased accessibility in an intronic region that has enhancer properties. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In summary, our exploratory study indicates a mechanism of action for SAHA and demonstrate the low efficacy of NKL54 as a single agent for apoptosis induction, using two PDOs. These observations need to be validated in a larger cohort of PDOs.
Assuntos
Neoplasias do Colo , Inibidores de Histona Desacetilases , Humanos , Inibidores de Histona Desacetilases/farmacologia , Cromatina/genética , Ácidos Hidroxâmicos/farmacologia , Apoptose/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/genéticaRESUMO
Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across various TMZ-resistant GBM cell models, patient-derived GBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Proteômica , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular TumoralRESUMO
Dopamine (DA) therapy of non-functioning pituitary adenomas (NFA) can result in tumor stabilization and shrinkage. However, the mechanism of action is still unknown. Previous evidence showed that DA can inhibit pituitary vascular endothelial growth factor expression (VEGF), that may be involved in pituitary tumor growth. The aim of our study was to clarify whether VEGF secretion modulation might mediate the effects of DA agonists on cell proliferation in human NFA. We assessed DA receptor subtype 2 (DR2) expression in 20 NFA primary cultures, where we also investigated the effects of a selective DR2 agonist, cabergoline (Cab), on VEGF secretion and on cell viability. All NFA samples expressed α-subunit and DR2 was expressed in 11 samples. In DR2 expressing tumors, Cab significantly reduced cell viability (-25%; P < 0.05) and VEGF secretion (-20%; P < 0.05). These effects were counteracted by treatment with the DA antagonist sulpiride. Cab antiproliferative effects were blocked by VEGF. Our data demonstrate that Cab, via DR2, inhibits cell viability also by reducing VEGF secretion in a selected group of NFA, supporting that DA agonists can be useful in the medical therapy of DR2 expressing NFA.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Ergolinas/farmacologia , Neoplasias Hipofisárias/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Cabergolina , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Receptores Dopaminérgicos/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Tumor heterogeneity affects diagnosis, prognosis and response to therapy. Heterogeneity is found in both normal and neoplastic human mammary gland. Indeed, luminal ER-negative cells can give rise to various phenotypes, including ER-negative and ER-positive mammary tumors. As a result, the tumor phenotype does not necessarily reflects the cell of origin of cancer. With regard to the ER status, heterogeneity can challenge endocrine therapies, where the elimination of responsive clones could lead to reduced treatment efficacy and tumor relapse through the expansion of the resistant clones. The aim of this study was to investigate breast tumor heterogeneity and its role in endocrine resistance onset. For this purpose, we used ER+ (T47D, CAMA1) and triple-negative breast cancer cell lines (TNBC; MDA-MB-231, HCC70), co-cultures using 2D and 3D models. Our results showed that ER status is modulated when ER+ cells are cultured in the presence of TNBC cells, leading to a different response to endocrine therapy, demonstrating that the response to treatment can be affected by the influence that different breast cancer cell types exert on each other. In addition, ER+ positive cells doubling time was modified after exposure to TNBC cell co-culturing. Further experiments are required to fully elucidate the molecular mechanism of these observations.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/genética , Prognóstico , Neoplasias Mamárias Animais/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Cancer cells must adapt to the hostile conditions of the microenvironment in terms of nutrition, space, and immune system attack. Mutations of DNA are the drivers of the tumorigenic process, but mutations must be able to hijack cellular functions to sustain the spread of mutant genomes. Transcriptional control is a key function in this context and is controlled by the rearrangement of the epigenome. Unlike genomic mutations, the epigenome of cancer cells can in principle be reversed. The discovery of the first epigenetic drugs triggered a contaminating enthusiasm. Unfortunately, the complexity of the epigenetic machinery has frustrated this enthusiasm. To develop efficient patient-oriented epigenetic therapies, we need to better understand the nature of this complexity. In this review, we will discuss recent advances in understanding the contribution of HDACs to the maintenance of the transformed state and the rational for their selective targeting.
Assuntos
Metilação de DNA , Epigenômica , Carcinogênese , Epigênese Genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Microambiente TumoralRESUMO
Despite cancer having been usually considered the result of genetic mutations, it is now well established that epigenetic dysregulations play pivotal roles in cancer onset and progression. Hence, inactivation of tumour suppressor genes can be gained not only by genetic mutations, but also by epigenetic mechanisms such as DNA methylation and histone modifications. To occur, epigenetic events need to be triggered by genetic alterations of the epigenetic regulators, or they can be mediated by intracellular and extracellular stimuli. In this last setting, the tumour microenvironment (TME) plays a fundamental role. Therefore, to decipher how epigenetic changes are associated with TME is a challenge still open. The complex signalling between tumour cells and stroma is currently under intensive investigation, and most of the molecules and pathways involved still need to be identified. Neoplastic initiation and development are likely to involve a back-and-forth crosstalk among cancer and stroma cells. An increasing number of studies have highlighted that the cancer epigenome can be influenced by tumour microenvironment and vice versa. Here, we discuss about the recent literature on tumour-stroma interactions that focus on epigenetic mechanisms and the reciprocal regulation between cancer and TME cells.
RESUMO
Understanding how an epigenetic regulator drives different cellular responses can be a tricky task. Very often, their activities are modulated by large multiprotein complexes, the composition of which is context- and time-dependent. As a consequence, experiments aimed to unveil the functions of an epigenetic regulator can provide different outcomes and conclusions, depending on the circumstances. HDAC9 (histone deacetylase), an epigenetic regulator that influences different differentiating and adaptive responses, makes no exception. Since its discovery, different phenotypes and/or dysfunctions have been observed after the artificial manipulation of its expression. The cells and the microenvironment use multiple strategies to control and monitor HDAC9 activities. To date, some of the genes under HDAC9 control have been identified. However, the exact mechanisms through which HDAC9 can achieve all the different tasks so far described, remain mysterious. Whether it can assemble into different multiprotein complexes and how the cells modulate these complexes is not clearly defined. In summary, despite several cellular responses are known to be affected by HDAC9, many aspects of its network of interactions still remain to be defined.
RESUMO
In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment.
Assuntos
Neoplasias da Mama/patologia , Carcinogênese/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Feminino , Humanos , Proteínas de Membrana/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX®) and enzalutamide (XTANDI®). However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8-19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide.
Assuntos
Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Processamento Alternativo , Androgênios/farmacologia , Animais , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores Androgênicos/genética , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , TransfecçãoRESUMO
Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.
RESUMO
As there is growing evidence for the tumor microenvironment's role in tumorigenesis, we investigated the role of fibroblast-expressed kinases in triple-negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that led to upregulation of NR4A1 in TNBC cells, where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease-free survival, highlighting it as a therapeutic target for TNBC.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/biossíntese , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias de Mama Triplo Negativas/enzimologia , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Fibroblastos/patologia , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Pancreatic Neuroendocrine Neoplasms (pNEN) are rare tumors which treatment still represent an important clinical problem, due to the paucity of medical treatments. Due to tumor complexity, techniques as 3D cultures are important to study drug activity in a more realistic model. This study aims to compare three different 3D culture methods in order to understand which one can be considered the best option in terms of experimental easiness and reproducibility in studying the efficacy of a target drug on pNEN. The BON1 cell line was used as a pNEN model and the well-known Receptor Tyrosine Kinase inhibitor Sunitinib was used in order to better investigate the different features of each method. The investigated methods are: (1) 96-well hanging drop plates (HD plates), (2) 24-well plates with a cell-repellent surface, and (3) ultra-low attachment 96-well plates with clear round bottom (ULA plates). The evaluated parameters during the study were: cell seeding, easiness in spheroids formation, morphology, culture maintenance, medium change, spheroids monitoring, picture quality, spheroid perimeter measurement reproducibility error, possibility to perform assays into the seeding plate, overall time of the experiment. Moreover, we investigated how culture methods can influence experimental outcomes evaluating perimeter changes, cell viability and immunohistochemistry of spheroids treated with different Sunitinib concentrations. Results showed that each method has weak and strong points but, considering the easiness of spheroids maintenance and reproducibility results, ULA plates method appears to be the best approach to culture BON1 spheroids and, therefore, to study pNEN.
RESUMO
Broncho-pulmonary neuroendocrine neoplasms (BP-NENs) are neoplasms orphan of an efficient therapy. Available medical treatments derived from clinical trials are not specific for the management of this malignancy. Sunitinib is a multi-receptor tyrosine-kinases (RTKs) inhibitor that has already shown its efficacy in NENs, but there are no available data about its action in BP-NENs. Therefore, our aim was to understand the effects of RTKs inhibition promoted by sunitinib in order to evaluate new putative targets useful in malignancy treatment. Since our results underlined a role for EGFR and IGF1R in modulating sunitinib antiproliferative action, we investigated the effects of erlotinib, an EGFR inhibitor, and linsitinib, an IGF1R inhibitor, in order to understand their function in regulating cells behaviour. Cell viability and caspase activation were evaluated on two immortalised human BP-NEN cell lines and primary cultures. Our results showed that after treatment with sunitinib and/or IGF1, EGF and VEGF, the antiproliferative effect of sunitinib was counteracted by EGF and IGF1 but not by VEGF. Therefore, we evaluated with AlphaScreen technology the phosphorylated EGFR and IGF1R levels in primary cultures treated with sunitinib and/or EGF and IGF1. Results showed a decrease of p-IGF1R after treatment with sunitinib and an increase after co-treatment with IGF1. Then, we assessed cell viability and caspase activation on BP-NEN cell lines after treatment with linsitinib and/or erlotinib. Results demonstrate that these two agents have a stronger antiproliferative effect compared to sunitinib. In conclusion, our results suggest that IGF1R and EGF1R could represent putative molecular targets in BP-NENs treatment.
RESUMO
Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and post-chemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.