RESUMO
The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.
Assuntos
Epigênese Genética , Histonas , Estresse Oxidativo , Proteínas Serina-Treonina Quinases , Humanos , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteoma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Dano ao DNA , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromatina/genética , Linhagem Celular Tumoral , Acetilação , Processamento de Proteína Pós-TraducionalRESUMO
Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.
Assuntos
Complexo Repressor Polycomb 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células K562 , Modelos Moleculares , Estrutura Molecular , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Ubiquitinação/efeitos dos fármacosRESUMO
The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucócitos/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Antineoplásicos/síntese química , Sítios de Ligação , Inibidores Enzimáticos/síntese química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Cinética , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , Leucócitos/enzimologia , Leucócitos/patologia , Modelos Moleculares , Proteína Meis1/genética , Proteína Meis1/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer, leading to improved overall survival. However, acquired resistance inevitably occurs. We aimed to identify, quantify, and assess the mechanisms of acquired resistance to trastuzumab. We established an acquired trastuzumab-resistant model in vitro from BT-474, a trastuzumab-sensitive, HER2-amplified breast-cancer cell line. A multi-omic strategy was implemented to obtain gene, proteome, and phosphoproteome signatures associated with acquired resistance to trastuzumab in HER2-positive breast cancer, followed by validation in human clinical samples. YAP1 dephosphorylation and TEAD2 overexpression were detected as significant alterations in the Hippo pathway in trastuzumab-resistant breast cancer. Because of the emerging role of these proteins as mediators of normal growth and tumorigenesis, we assessed the exogenous modulation of their activity, either by in vitro gene silencing or by pharmacological inhibition of the YAP1/TEAD complexes, both in vitro and in vivo. Moreover, we identified increased signaling through the Hippo pathway in human samples after progression following trastuzumab treatment. Finally, YAP1/TAZ nuclear accumulation in malignant cells in HER2 breast tumor was significantly associated with worse progression-free and overall survival in metastatic HER2-positive breast-cancer patients. Our results suggest the involvement of Hippo signaling in acquired trastuzumab resistance in breast cancer. Additionally, we provide novel evidence for a potential breast-cancer treatment strategy based on dual targeting of HER2 and Hippo pathway effectors, which may improve the antitumor activity of trastuzumab and help overcome resistance.
RESUMO
HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Quimiocina CCL5/metabolismo , Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Comunicação Autócrina , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Quimiocina CCL5/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Receptor ErbB-2/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The potential involvement of type 2 diabetes mellitus (T2DM) as a risk factor for colon cancer (CC) has been previously reported. While several clinical studies show a higher incidence of CC and a lower survival rate in diabetics, others report no association. Our own experience indicates that diabetes does not seem to worsen the prognosis once the tumor is present. Despite this controversy, there are no wide-spectrum molecular studies that delve into the impact of T2DM-related mechanisms in colon carcinogenesis. Here, we present a transcriptomic and proteomic profiling of paired tumor and normal colon mucosa samples in a cohort of 42 CC patients, 23 of which have T2DM. We used gene set enrichment and network approaches to extract relevant pathways in diabetics, referenced them to current knowledge, and tested them using in vitro techniques. Through our transcriptomics approach, we identified an unexpected overlap of pathways overrepresented in diabetics compared to nondiabetics, in both tumor and normal mucosa, including diabetes-related metabolic and signaling processes. Proteomic approaches highlighted several cancer-related signaling routes in diabetics found only in normal mucosa, not in tumors. An integration of the transcriptome and proteome analyses suggested the deregulation of key pathways related to colon carcinogenesis which converged on tumor initiation axis TEAD/YAP-TAZ as a potential initiator of the process. In vitro studies confirmed upregulation of this pathway in nontumor colon cells under high-glucose conditions. In conclusion, T2DM associates with deregulation of cancer-related processes in normal colon mucosa adjacent to tissue which has undergone a malignant transformation. These data support that in diabetic patients, the local microenvironment in normal colon mucosa may be a factor driving field cancerization promoting carcinogenesis. Our results set a new framework to study links between diabetes and colon cancer, including a new role of the TEAD/YAP-TAZ complex as a potential driver.
Assuntos
Neoplasias do Colo/etiologia , Neoplasias do Colo/genética , Diabetes Mellitus Tipo 2/complicações , Animais , Carcinogênese/genética , Carcinogênese/patologia , Estudos de Coortes , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Glucose/metabolismo , Humanos , Hiperglicemia/complicações , Mucosa Intestinal/patologia , Masculino , Camundongos Nus , Transdução de Sinais/genética , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy. OBJECTIVE: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer. CONCLUSION: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/uso terapêutico , Animais , Antineoplásicos Imunológicos/imunologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Trastuzumab/imunologiaRESUMO
Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006-19. ©2017 AACR.
Assuntos
Neoplasias da Mama/genética , Ciclina B1/deficiência , Resistencia a Medicamentos Antineoplásicos/genética , Maitansina/análogos & derivados , Receptor ErbB-2/genética , Trastuzumab/farmacologia , Ado-Trastuzumab Emtansina , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Ciclina B1/metabolismo , Modelos Animais de Doenças , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Maitansina/farmacologia , Camundongos , Ligação Proteica , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A subset of HER2+ breast cancer patients manifest clinical resistance to trastuzumab. Recently, miR-26a and miR-30b have been identified as trastuzumab response regulators, and their target gene CCNE2 seems to play an important role in resistance to trastuzumab therapy. Cell viability was evaluated in trastuzumab treated HER2+ BT474 wt (sensitive), BT474r (acquired resistance), HCC1954 (innate resistance), and MDA-MB-231 (HER2-) cell lines, and the expression of miR-26a, miR-30b, and their target genes was measured. BT474 wt cell viability decreased by 60% and miR-26a and miR-30b were significantly overexpressed (~3-fold, p = 0.003 and p = 0.002, respectively) after trastuzumab treatment, but no differences were observed in resistant and control cell lines. Overexpression of miR-30b sensitized BT474r cells to trastuzumab (p = 0.01) and CCNE2, was significantly overexpressed after trastuzumab treatment in BT474r cells (p = 0.032), but no significant changes were observed in sensitive cell line. When CCNE2 was silenced BT474r cell sensitivity to trastuzumab increased (p = 0.03). Thus, the molecular mechanism of trastuzumab action in BT474 cell line may be regulated by miR-26a and miR-30b and CCNE2 overexpression might play an important role in acquired trastuzumab resistance in HER2+ breast cancer given that resistance was diminished when CCNE2 was silenced.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Ciclinas/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ciclinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Trastuzumab/farmacologiaRESUMO
The tumor suppressor microRNA-199b (miR-199b) is a negative SET regulator associated with poor outcome in some human cancers. However, its expression levels as well as potential biological and clinical significance in colorectal cancer (CRC) remain completely unexplored. The PP2A inhibitor SET has shown promising therapeutic and clinical implications in metastatic CRC (mCRC) but the molecular mechanisms underlying SET deregulation are currently unknown. We show here miR-199b downregulation in 4 out of 5 CRC SET-overexpressing cell lines and its inverse correlation with SET overexpression in CRC patients. Moreover, miR-199b led to PP2A activation through a direct SET inhibition, impaired cell viability and enhanced oxaliplatin sensitivity in CRC cells. MiR-199b was found downregulated in 25% of cases, and associated with lymph metastasis (p = 0.049), presence of synchronous metastasis at diagnosis (p = 0.026) and SET overexpression (p < 0.001). Furthermore, low miR-199b levels determined shorter overall (p < 0.001), progression-free survival (p = 0.003) and predicted clinical benefit to oxaliplatin treatment. The miR-199b prognostic impact was particularly evident in both younger and KRAS wild-type subgroups. Multivariate analyses confirmed its independent prognostic impact. Altogether, our results show that miR-199b is a tumor suppressor whose downregulation independently determines worse outcome and emerges as a potential contributing mechanism to inhibit PP2A via SET overexpression in a subgroup of mCRC patients.
Assuntos
Neoplasias Colorretais/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Chaperonas de Histonas/genética , MicroRNAs/genética , Proteína Fosfatase 2/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA , Feminino , Células HT29 , Chaperonas de Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Proteína Fosfatase 2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Trastuzumab became the therapy of choice for patients with HER2-positive breast cancer in 1998, and it has provided clinical benefit ever since. However, a significant percentage of patients show primary resistance to trastuzumab at diagnosis, and most patients with metastatic disease that initially respond to trastuzumab eventually progress (acquired resistance). Consequently, there is an urgent need to improve our knowledge of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. We generated new cell lines derived from BCCL through extended exposure to trastuzumab. Drug-conditioned populations were authenticated for their molecular profile and their resistance rate was determined. Heterogeneous HER2 amplification was observed across most of the BCCLs, ranging from cells without HER2 amplification to elevated HER2 gene copy numbers in others. Using a phospho-antibody array we analyzed the status of kinase receptors and effectors from different cellular pathways. This revealed that HER2, AKT, and S6RP presented high phosphorylation levels with specific variations between sensitive and resistant populations. In addition, differences in phosphorylation levels for several of those pathways targets were found between sensitive and resistant lines. Furthermore, a biochemical study characterized patterns of molecular alterations similar to those commonly described in breast cancer. Finally, a subcutaneous xenograft murine model confirmed the resistance to trastuzumab of the established cell line. We conclude that these resistant BCCLs can be a valuable tool to gain insight into the mechanisms of acquisition of trastuzumab resistance.
RESUMO
MAPK phosphatase-1 (MKP-1) is overexpressed during malignant transformation of the breast in many patients, and it is usually associated with chemoresistance through interference with JNK-driven apoptotic pathways. Although the molecular settings of the mechanism have been documented, details about the contribution of MKP-1 to the failure of chemotherapeutic interventions are unclear. Transient overexpression of MKP-1 and treatment with JNK-modulating agents in breast carcinoma cells confirmed the mediation of MKP-1 in the resistance to taxanes and anthracyclines in breast cancer, through the inactivation of JNK1/2. We next assessed MKP-1 expression and JNK1/2 phosphorylation status in a large cohort of samples from 350 early breast cancer patients treated with adjuvant anthracycline-based chemotherapy. We detected that MKP-1 overexpression is a recurrent event predominantly linked to dephosphorylation of JNK1/2 with an adverse impact on relapse of the tumor and overall and disease-free survival. Moreover, MKP-1 and p-JNK1/2 determinations in 64 locally advanced breast cancer patients treated with neoadjuvant taxane-based chemotherapy showed an inverse correlation between MKP-1 overexpression (together with JNK1/2 inhibition) and the pathologic response of the tumors. Our results emphasize the importance of MKP-1 as a potential predictive biomarker for a subset of breast cancer patients with worse outcome and less susceptibility to treatment. Mol Cancer Ther; 15(11); 2780-90. ©2016 AACR.
Assuntos
Antraciclinas/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Taxoides/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Fosfatase 1 de Especificidade Dupla/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Gradação de Tumores , Prognóstico , Modelos de Riscos Proporcionais , RecidivaRESUMO
Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision has emerged as the standard treatment for locally advanced rectal cancer (LARC) patients. However, many cases do not respond to neoadjuvant CRT, suffering unnecessary toxicities and surgery delays. Thus, identification of predictive biomarkers for neoadjuvant CRT is a current clinical need. In the present study, microRNA-31 expression was measured in formalin-fixed paraffin-embedded (FFPE) biopsies from 78 patients diagnosed with LARC who were treated with neoadjuvant CRT. Then, the obtained results were correlated with clinical and pathological characteristics and outcome. High microRNA-31 (miR-31) levels were found overexpressed in 34.2% of cases. Its overexpression significantly predicted poor pathological response (p = 0.018) and worse overall survival (OS) (p = 0.008). The odds ratio for no pathological response among patients with miR-31 overexpression was 0.18 (Confidence Interval = 0.06 to 0.57; p = 0.003). Multivariate analysis corroborated the clinical impact of miR-31 in determining pathological response to neoadjuvant CRT as well as OS. Altogether, miR-31 quantification emerges as a novel valuable clinical tool to predict both pathological response and outcome in LARC patients.
Assuntos
MicroRNAs/genética , Neoplasias Retais/diagnóstico , Neoplasias Retais/genética , Adulto , Idoso , Biomarcadores , Quimiorradioterapia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Curva ROC , Neoplasias Retais/mortalidade , Neoplasias Retais/terapia , Resultado do TratamentoRESUMO
FTY720 (Fingolimod, Gilenya () ) is an FDA-approved immunosuppressant currently used in the treatment of multiple sclerosis. However, a large number of studies over the last few years have shown that FTY720 shows potent antitumor properties that suggest its potential usefulness as a novel anticancer agent. Interestingly, the restoration of protein phosphatase 2A (PP2A) activity mediated by FTY720 could play a key role in its antitumor effects. Taking into account that PP2A inactivation is a common event that determines poor outcome in several tumor types, FTY720 could serve as an alternative therapeutic strategy for cancer patients with such alterations.
Assuntos
Cloridrato de Fingolimode/farmacologia , Imunossupressores/farmacologia , Neoplasias/tratamento farmacológico , Proteína Fosfatase 2/efeitos dos fármacos , Humanos , Imunossupressores/uso terapêutico , Esclerose Múltipla/tratamento farmacológicoRESUMO
Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.
Assuntos
Leiomiossarcoma/genética , Mutação , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Neoplasias Retroperitoneais/genética , Análise Mutacional de DNA/economia , Análise Mutacional de DNA/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leiomiossarcoma/diagnóstico , Masculino , Mutação Puntual , Neoplasias Retroperitoneais/diagnóstico , Espaço Retroperitoneal/patologiaRESUMO
Protein phosphatase 2A (PP2A) is a well-known tumor suppressor frequently inhibited in human cancer. Alterations affecting PP2A subunits together with the deregulation of endogenous PP2A inhibitors such as CIP2A and SET have been described as contributing mechanisms to inactivate PP2A in prostate cancer. Moreover, recent findings highlight that functional inactivation of PP2A could represent a key event in the acquisition of castration-resistant phenotype and a novel molecular target with high impact at both clinical and therapeutic levels in prostate cancer.
Assuntos
Autoantígenos/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Proteína Fosfatase 2/genética , Fatores de Transcrição/metabolismo , Apoptose/genética , Autoantígenos/genética , Autoantígenos/uso terapêutico , Proliferação de Células/genética , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Chaperonas de Histonas/genética , Chaperonas de Histonas/uso terapêutico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/uso terapêutico , Terapia de Alvo Molecular , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/terapia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/uso terapêutico , Receptores Androgênicos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/uso terapêuticoRESUMO
Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients.