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1.
Comput Math Methods Med ; 2022: 2402567, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35535229

RESUMO

Objective: To study the expression and prognostic value of CDK6 in stomach cancer and the function of CDK4/6 inhibitor PD-0332991 on the proliferation of stomach cancer cells. Methods: Immunohistochemistry was used to detect the expression of CDK6 in stomach cancer tissues and adjacent normal tissues and to analyze the effect of CDK6 on clinicopathological parameters of stomach cancer patients. Kaplan-Meier plotter was employed to study the relationship between CDK6 and overall survival in stomach cancer. Western blot and RT-PCR were used to detect protein and gene expression of CDK6 in different cells. The effects of CDK4/6 inhibitor PD-0332991 on apoptosis and aging of stomach cancer cells were detected by flow cytometry and ß-galactosidase aging staining assay. The effects of CDK4/6 inhibitor PD-0332991 on the invasion and migration of stomach cancer cells were explored by the wound healing experiment and the Transwell experiment. The supernatant of stomach cancer cells was collected, and the effect of CDK4/6 inhibitor PD-0332991 on tumor markers of stomach cancer cells was detected by biochemical immunoassay. Results: (1) CDK6 was highly expressed in stomach cancer tissues and cells. (2) Abnormally elevated CDK6 expression results in shorter survival in stomach cancer patients. (3) CDK4/6 inhibitor PD-0332991 could block the proliferation of stomach cancer cells, but not stomach epithelial proliferation. PD-0332991 could inhibit the secretion of pro-GRP by MGC 823. (4) PD-0332991 could advance the development of the apoptosis and senescence of stomach cancer cells and suppressed the invasion and migration of stomach cancer cells. Conclusion: CDK6 expression is elevated in gastric cancer, and the CDK4/6 inhibitor PD-0332991 can remarkably promote apoptosis and senescence of stomach cancer cells and effectively inhibit the migration and invasion of stomach cancer cells.


Assuntos
Quinase 6 Dependente de Ciclina , Neoplasias Gástricas , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Piperazinas/farmacologia , Piridinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo
2.
Drug Des Devel Ther ; 16: 1083-1097, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35431540

RESUMO

Purpose: To enhance the cytotoxicities of our obtained CDK4 inhibitors and get CDK4/HDACs inhibitors with potent enzymatic inhibitory and anti-proliferative activities. Methods: A series of novel CDK4/HDACs inhibitors were designed and synthesized by incorporating the HDAC pharmacophores (hydroxylamine or o-diaminoaniline) into the basic structure of our newly obtained 2-anilino-4-triazolpyrimidine based CDK4 inhibitors. The enzymatic inhibitory (HDAC1, CDK2, CDK4, and CDK6) activities and cytotoxicities of these compounds were evaluated. Moreover, HDAC isoforms inhibitory activity, cell cycle arrest assay, cell apoptosis analysis, cell migration, and cell colony formation assay were performed for the representative compound 11k. Results: Most of these compounds showed excellent HDAC1 inhibitory activities (IC50s: 0.68~244.5 nM) and anti-proliferative activities against cancer cell lines. Some compounds displayed potent CDK4 inhibitory activities and a certain selectivity towards CDK2 and CDK6. Compound 11k exhibited potent enzymatic (CDK4: IC50=23.59 nM; HDAC1: IC50=61.11 nM; HDAC2: IC50=80.62 nM; HDAC6: IC50=45.33 nM) and anti-proliferative activities against H460, MDA-MB-468, HCT116, and HepG2 cell lines with IC50 values 1.20, 1.34, 2.07, and 2.66 µM, respectively. Further mechanistic studies revealed that compound 11k could arrest the cell cycle in G0/G1 phase and induce apoptosis in HCT116 and MDA-MB-468 cells. In addition, compound 11k significantly inhibited the migration and cell colony formation of H460 and HCT116 cells. Conclusion: This study suggested that the incorporation of the HDAC pharmacophore into CDK4 inhibitor scaffold to design CDK/HDAC inhibitors might be a tractable strategy to enhance the antitumor potency of compounds.


Assuntos
Antineoplásicos , Desenho de Fármacos , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
3.
Nat Rev Urol ; 19(5): 305-320, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35264774

RESUMO

The treatment of advanced and metastatic kidney cancer has entered a golden era with the addition of more therapeutic options, improved survival and new targeted therapies. Tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors and immune checkpoint blockade have all been shown to be promising strategies in the treatment of renal cell carcinoma (RCC). However, little is known about the best therapeutic approach for individual patients with RCC and how to combat therapeutic resistance. Cancers, including RCC, rely on sustained replicative potential. The cyclin-dependent kinases CDK4 and CDK6 are involved in cell-cycle regulation with additional roles in metabolism, immunogenicity and antitumour immune response. Inhibitors of CDK4 and CDK6 are now commonly used as approved and investigative treatments in breast cancer, as well as several other tumours. Furthermore, CDK4/6 inhibitors have been shown to work synergistically with other kinase inhibitors, including mTOR inhibitors, as well as with immune checkpoint inhibitors in preclinical cancer models. The effect of CDK4/6 inhibitors in kidney cancer is relatively understudied compared with other cancers, but the preclinical studies available are promising. Collectively, growing evidence suggests that targeting CDK4 and CDK6 in kidney cancer, alone and in combination with current therapeutics including mTOR and immune checkpoint inhibitors, might have therapeutic benefit and should be further explored.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/tratamento farmacológico , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/uso terapêutico , Feminino , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Renais/tratamento farmacológico , Masculino , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/uso terapêutico
4.
Pathol Res Pract ; 232: 153839, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35303521

RESUMO

Our main goal was to investigate the potential utility of MDM2/CDK4 immunohistochemistry to act as surrogate for FISH to identify a subset of lipoma-like ALT/WDL that might, otherwise, be underdiagnosed on initial screening. Lack of cytologic atypia in lipomatous tumors with negative expression for MDM2/CDK4 IHC does not fully exclude the possibility of underlying MDM2/CDK4 amplification. Present study identified that isolated CDK4/p16 positive expression, in the absence of MDM2 expression, may have potential utility during the initial screening of these tumors but the proportion of conventional lipomas, which may also exhibit low levels of CDK4/p16 expression remains uncertain.


Assuntos
Lipoma , Lipossarcoma , Biomarcadores Tumorais/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Humanos , Imuno-Histoquímica , Lipoma/diagnóstico , Lipoma/genética , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Lipossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo
5.
Oncogene ; 41(18): 2540-2554, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35301407

RESUMO

Low-risk gestational trophoblastic neoplasia including choriocarcinoma is often effectively treated with Methotrexate (MTX) as a first line therapy. However, MTX resistance (MTX-R) occurs in at least ≈33% of cases. This can sometimes be salvaged with actinomycin-D but often requires more toxic combination chemotherapy. Moreover, additional therapy may be needed and, for high-risk patients, 5% still die from the multidrug-resistant disease. Consequently, new treatments that are less toxic and could reverse MTX-R are needed. Here, we compared the proteome/phosphoproteome of MTX-resistant and sensitive choriocarcinoma cells using quantitative mass-spectrometry to identify therapeutically actionable molecular changes associated with MTX-R. Bioinformatics analysis of the proteomic data identified cell cycle and DNA damage repair as major pathways associated with MTX-R. MTX-R choriocarcinoma cells undergo cell cycle delay in G1 phase that enables them to repair DNA damage more efficiently through non-homologous end joining in an ATR-dependent manner. Increased expression of cyclin-dependent kinase 4 (CDK4) and loss of p16Ink4a in resistant cells suggested that CDK4 inhibition may be a strategy to treat MTX-R choriocarcinoma. Indeed, inhibition of CDK4/6 using genetic silencing or the clinically relevant inhibitor, Palbociclib, induced growth inhibition both in vitro and in an orthotopic in vivo mouse model. Finally, targeting the ATR pathway, genetically or pharmacologically, re-sensitised resistant cells to MTX in vitro and potently prevented the growth of MTX-R tumours in vivo. In short, we identified two novel therapeutic strategies to tackle MTX-R choriocarcinoma that could rapidly be translated into the clinic.


Assuntos
Coriocarcinoma , Quinase 6 Dependente de Ciclina/metabolismo , Metotrexato , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Coriocarcinoma/tratamento farmacológico , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Dactinomicina , Feminino , Humanos , Metotrexato/farmacologia , Camundongos , Gravidez , Proteômica
6.
Cell Death Dis ; 13(2): 163, 2022 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-35184131

RESUMO

During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.


Assuntos
Neoplasias da Mama , Quinase 6 Dependente de Ciclina , Fator IX , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Senescência Celular/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Fator IX/genética , Feminino , Humanos , Células MCF-7
7.
Exp Cell Res ; 414(1): 113053, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35149086

RESUMO

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called "oncogene-induced senescence (OIS)", prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called "HBEC3KT"), immortalized with hTERT ("T") and Cdk4 ("K"), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated ß-galactosidase staining (SA-ßG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.


Assuntos
Telomerase , Brônquios/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Senescência Celular/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Telomerase/genética , Telomerase/metabolismo
8.
JCO Precis Oncol ; 6: e2100211, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35108033

RESUMO

PURPOSE: Soft tissue and bone sarcomas are rare malignancies that exhibit significant pathologic and molecular heterogeneity. Deregulation of the CDKN2A-CCND-CDK4/6-retinoblastoma 1 (Rb) pathway is frequently observed in about 25% of unselected sarcomas and is pathognomonic for specific sarcoma subtypes. This genomic specificity has fueled the clinical evaluation of selective CDK4/6 inhibitors in sarcomas. Here, we highlight successes, opportunities, and future challenges for using CDK4/6 inhibitors to treat sarcoma. MATERIALS AND METHODS: This review summarizes the current evidence for the use of CDK4/6 inhibitors in sarcoma while identifying molecular rationale and predictive biomarkers that provide the foundation for targeting the CDK4/6 pathway in sarcoma. A systematic review was performed of articles indexed in the PubMed database and the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov). For each sarcoma subtype, we discuss the preclinical rationale, case reports, and available clinical trials data. RESULTS: Despite promising clinical outcomes in a subset of sarcomas, resistance to CDK4/6 inhibitors results in highly heterogeneous clinical outcomes. Current clinical data support the use of CDK4/6 inhibitors in subsets of sarcoma primarily driven by CDK4/6 deregulation. When dysregulation of the Rb pathway is a secondary driver of sarcoma, combination therapy with CDK4/6 inhibition may be an option. Developing strategies to identify responders and the mechanisms that drive resistance is important to maximize the clinical utility of these drugs in patients with sarcoma. Potential biomarkers that indicate CDK4/6 inhibitor sensitivity in sarcoma include CDK4, CCND, CCNE, RB1, E2F1, and CDKN2A. CONCLUSION: CDK4/6 inhibitors represent a major breakthrough for targeted cancer treatment. CDK4/6 inhibitor use in sarcoma has led to limited, but significant, early clinical success. Targeted future clinical research will be key to unlocking the potential of CDK4/6 inhibition in sarcoma.


Assuntos
Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Sarcoma , Neoplasias de Tecidos Moles , Ensaios Clínicos como Assunto , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Genômica/métodos , Humanos , Sarcoma/tratamento farmacológico , Sarcoma/enzimologia , Neoplasias de Tecidos Moles/enzimologia , Estados Unidos
9.
Science ; 375(6577): eabc1495, 2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35025636

RESUMO

Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin D­CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor­positive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin D­CDK4/6 inhibitors in cancer treatment.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinogênese , Proliferação de Células/efeitos dos fármacos , Senescência Celular , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
10.
Cell Biol Int ; 46(5): 737-746, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35032143

RESUMO

Hair follicles regenerate periodically by spontaneously undergoing cycles of growth, regression, and relative quiescence. During the hair cycle, follicle stem cells residing in a specialized niche remain quiescent, and they are stimulated to proliferate throughout the growth phase of the hair follicle. Although cell cycle regulators play a prominent role during the activation of hair follicle stem cells, the identity and the role of these regulators have not been confirmed. Herein, we reported that stem cells located in the bulge region of the HF (BuSCs) express high levels of cyclin-dependent kinase 4 (CDK4) through the quiescent phase of the hair cycle. Using gain- and loss-of-function studies, we have determined that the CDK4 protein level affects the number of BuSCs. Transgenic expression of CDK4 in the bulge region of the hair follicles reduces the number of BuSCs, whereas CDK4 ablation resulted in an increasing number of BuSCs. These results suggest that deregulation of CDK4 protein levels contributes to distorting the self-renewal/proliferation balance and, in turn, altering the number of BuSCs.


Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Folículo Piloso , Células-Tronco , Animais , Folículo Piloso/metabolismo , Queratinócitos , Camundongos
11.
Biochem Biophys Res Commun ; 595: 62-68, 2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-35093641

RESUMO

BACKGROUND: P16ink4a can accumulate in senescent cells and can be induced by different oncogenic stimulations. These functions make p16ink4a a biomarker of senescence and cancer. However, the exact role of p16ink4a remains unclear in cardiovascular disease. This study was aimed to investigate the role of p16ink4a in cardiac remodeling after myocardial infarction (MI). METHODS: In vivo, gain and loss of function experiments using p16ink4a overexpression and knockdown adenovirus were induced to determine the effect of p16ink4a on cardiac structure and function after MI. The in vitro effects of p16ink4a were evaluated by overexpression and knockdown adenovirus of p16ink4a on isolated neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). RESULTS: Expression level of p16ink4a was increased after MI and enriched in the infarction area. In vivo, overexpression of p16ink4a protected, while knockdown of p16ink4a worsened cardiac function. In vitro, p16ink4a did not influence the hypertrophy of NMCMs. Overexpression of p16ink4a inhibited the proliferation and migration of NMCFs and reduced the level of collagen I and α-SMA. Consistently, knockdown of p16ink4a in vitro displayed the opposite effects. Further mechanism studies revealed that p16ink4a affected the expression level of cyclin-dependent kinase 4 (CDK4) and phosphorylation of retinoblastoma (pRb), which could be a potential pathway in regulating cardiac remodeling after MI. CONCLUSION: Overexpression of 16ink4a in cardiac fibroblasts can ameliorate cardiac dysfunction and attenuate pathological cardiac remodeling in mice after MI by regulating the p16ink4a/CDK4/pRb pathway.


Assuntos
Quinase 4 Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Expressão Gênica , Infarto do Miocárdio/genética , Proteína do Retinoblastoma/genética , Remodelação Ventricular/genética , Animais , Animais Recém-Nascidos , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Quinase 4 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Masculino , Camundongos Endogâmicos ICR , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosforilação , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/genética
12.
Sci Rep ; 12(1): 1275, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35075231

RESUMO

Understanding human genome alterations is necessary to optimize genome-based cancer therapeutics. However, some newly discovered mutations remain as variants of unknown significance (VUS). Here, the mutation c.1403A > G in exon 10 of the platelet-derived growth factor receptor-alpha (PDGFRA) gene, a VUS found in adult glioblastoma multiforme (GBM), was introduced in human embryonal kidney 293 T (HEK293T) cells using genome editing to investigate its potential oncogenic functions. Genome editing was performed using CRISPR/Cas9; the proliferation, drug sensitivity, and carcinogenic potential of genome-edited cells were investigated. We also investigated the mechanism underlying the observed phenotypes. Three GBM patients carrying the c.1403A > G mutation were studied to validate the in vitro results. The c.1403A > G mutation led to a splice variant (p.K455_N468delinsN) because of the generation of a 3'-acceptor splice site in exon 10. PDGFRA-mutated HEK293T cells exhibited a higher proliferative activity via PDGFRα and the cyclin-dependent kinase (CDK)4/CDK6-cyclin D1 signaling pathway in a ligand-independent manner. They showed higher sensitivity to multi-kinase, receptor tyrosine kinase, and CDK4/CDK6 inhibitors. Of the three GBM patients studied, two harbored the p.K455_N468delinsN splice variant. The splicing mutation c.1403A > G in PDGFRA is oncogenic in nature. Kinase inhibitors targeting PDGFRα and CDK4/CDK6 signaling should be evaluated for treating GBM patients harboring this mutation.


Assuntos
Glioblastoma/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Edição de Genes , Glioblastoma/tratamento farmacológico , Células HEK293 , Humanos , Terapia de Alvo Molecular , Sítios de Splice de RNA , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais
13.
Cancer Chemother Pharmacol ; 89(1): 31-40, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34655298

RESUMO

BACKGROUND: Osteosarcoma is a prevalent type of bone tumor in children and adolescents, with limited treatment and poor prognosis. Abemaciclib, an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), is approved for the treatment of advanced breast cancer as single agent therapy and is currently under investigation in clinical trials for the treatment of several solid tumors. METHODS: The efficacy of abemaciclib was determined using osteosarcoma cellular assays and xenograft mouse model. The combination studies were performed based on the Chou-Talalay method. Immunoblotting analysis was performed to determine the underlying mechanisms of abemaciclib in osteosarcoma cell lines. RESULTS: Abemaciclib potently inhibits growth, anchorage-independent colony formation and survival of a panel of osteosarcoma cell lines, with IC50 range from 90 nM to >20 µM. In addition, the combination of abemaciclib and doxorubicin is synergistic and antagonistic in abemaciclib-sensitive (IC50 <1 µM) and abemaciclib-resistant (IC50 >1 µM), respectively. Abemaciclib inhibits tumor formation and growth in a dose-dependent manner without causing significant drug toxicity in mice. The combination of abemaciclib and doxorubicin results in much greater efficacy than doxorubicin alone in inhibiting tumor growth throughout the whole treatment duration. Abemaciclib acts on osteosarcoma via suppressing CDK4/6-Cyclin D-Rb pathway. CONCLUSIONS: Our pre-clinical evidence provides a rationale of initializing clinical trial of investigating the efficacy of abemaciclib in combination with doxorubicin in osteosarcoma patients. Our work also highlights the therapeutic value of CDK4/6 inhibition in osteosarcoma with proper function of Rb.


Assuntos
Aminopiridinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Aminopiridinas/administração & dosagem , Animais , Benzimidazóis/administração & dosagem , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclina D/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos SCID , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Eur J Med Chem ; 228: 114024, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34875521

RESUMO

Multiple myeloma (MM) ranks second in malignant hematopoietic cancers, and the most common anti-MM drugs easily generate resistance. CDK4/6 have been validated to play determinant roles in MM, but no remarkable progress has been obtained from clinical trials of CDK4/6 inhibitors for MM. To discover novel CDK6 inhibitors with better potency and high druggability, structure-based virtual screening was conducted to identify compound 10. Further chemical optimization afforded a better derivative, compound 32, which exhibited strong inhibition of CDK4/6 and showed high selectivity over 360+ kinases, including homologous CDKs. The in vivo evaluation demonstrated that compound 32 possessed low toxicity (LD50 > 10,000 mg/kg), favorable bioavailability (F% = 51%), high metabolic stability (t1/2 > 24 h) and strong anti-MM potency. In summary, we discovered a novel CDK4/6 inhibitor bearing favorable drug-like properties and offered a great candidate for MM preclinical studies.


Assuntos
Antineoplásicos/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Descoberta de Drogas , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
15.
Cancer Sci ; 113(2): 802-807, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34866279

RESUMO

Extramammary Paget's disease (EMPD) is a rare adnexal neoplasm commonly seen in the genital areas among the senior population. The prognosis of advanced EMPD is not favorable; thus, the development of potential treatments has long been sought. Cyclin-dependent kinase (CDK) 4/6 inhibitors such as abemaciclib and palbociclib have been proven effective against metastatic breast cancer; however, no studies have addressed CDK4/6 inhibitors as an EMPD treatment. We herein examine the efficacy of CDK4/6 inhibitors against an EMPD patient-derived xenograft (PDX) model. Abemaciclib (50 mg/kg/day) or palbociclib (120 mg/kg/day) was given orally to tumor-bearing NOD/Scid mice over a 3-week period. We also investigated the protein expression levels of CDK4/6 and cyclin D1 through immunohistochemical staining using EMPD clinical samples. Treatment with abemaciclib or palbociclib as a single agent was found to significantly suppress tumor growth in EMPD-PDX. The Ki-67-positive ratio of the treated EMPD-PDX tumors was significantly lower than that of the nontreated tumors. Clinically, the expression levels of CDK4 and cyclin D1 were significantly higher in the EMPD tumor cells than in the normal epidermis. Our results suggest that CDK4/6 inhibitors could be novel and potent therapeutics for the treatment of EMPD.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Doença de Paget Extramamária/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Animais , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Doença de Paget Extramamária/metabolismo , Doença de Paget Extramamária/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pele/metabolismo , Pele/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Arthritis Rheumatol ; 74(5): 860-870, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34882985

RESUMO

OBJECTIVE: One of the histologic characteristics of systemic sclerosis (SSc) is an increased number of dermal myofibroblasts, and transforming growth factor ß (TGFß) plays a crucial role in the promotion of myofibroblast differentiation from fibroblasts, leading to dermal fibrosis. This study was undertaken to 1) examine whether inhibition of the cell cycle with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor suppresses the proliferation of fibroblasts and their differentiation into myofibroblasts, and 2) assess the therapeutic effects of a CDK4/6 inhibitor, administered as monotherapy or in combination with a TGFß receptor (TGFßR) inhibitor, on dermal fibrosis in murine models of SSc. METHODS: Fibroblasts obtained from the skin of patients with SSc were cultured in the presence or absence of TGFß. The effects of palbociclib, a CDK4/6 inhibitor, on fibroblast proliferation and TGFß-induced differentiation into myofibroblasts were examined using bromodeoxyuridine uptake assays as well as immunofluorescence and immunoblotting analyses. Murine models of HOCl- and bleomycin-induced dermal fibrosis were used to study the effect of a CDK4/6 inhibitor on dermal fibrosis, with the CDK4/6 inhibitor treatment administered as monotherapy or in combination with galunisertib, a TGFßR inhibitor. RESULTS: Addition of a CDK4/6 inhibitor to the cell cultures suppressed the proliferation of human dermal SSc fibroblasts and their TGFß-induced differentiation into myofibroblasts, without inhibiting canonical and noncanonical TGFß signals. In murine models of dermal fibrosis, treatment of mice with a CDK4/6 inhibitor decreased dermal thickness and collagen content, as well as dermal fibroblast proliferation and the numbers of myofibroblasts. Combination therapy with the CDK4/6 inhibitor and TGFßR inhibitor resulted in additive antifibrotic effects. Mechanistically, the CDK4/6 inhibitor suppressed the expression of cellular communication network 2 and cadherin-11, which are proteins that have important roles in the development and progression of fibrosis. CONCLUSION: Results of this study demonstrate the therapeutic effect of a CDK4/6 inhibitor on dermal fibrosis when administered as monotherapy or in combination with a TGFßR inhibitor. CDK4/6 inhibitors, including palbociclib used in the present study, may represent novel agents for the treatment of SSc, which, if used in combination with a TGFßR inhibitor, might result in increased efficacy.


Assuntos
Escleroderma Sistêmico , Animais , Células Cultivadas , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/farmacologia , Quinase 4 Dependente de Ciclina/uso terapêutico , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Escleroderma Sistêmico/patologia , Pele/patologia , Fator de Crescimento Transformador beta/metabolismo
17.
Cancer Res ; 82(4): 648-664, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34853070

RESUMO

The invasive leading edge represents a potential gateway for tumor metastasis. The role of fibroblasts from the tumor edge in promoting cancer invasion and metastasis has not been comprehensively elucidated. We hypothesize that cross-talk between tumor and stromal cells within the tumor microenvironment results in activation of key biological pathways depending on their position in the tumor (edge vs. core). Here we highlight phenotypic differences between tumor-adjacent-fibroblasts (TAF) from the invasive edge and tumor core fibroblasts from the tumor core, established from human lung adenocarcinomas. A multiomics approach that includes genomics, proteomics, and O-glycoproteomics was used to characterize cross-talk between TAFs and cancer cells. These analyses showed that O-glycosylation, an essential posttranslational modification resulting from sugar metabolism, alters key biological pathways including the cyclin-dependent kinase 4 (CDK4) and phosphorylated retinoblastoma protein axis in the stroma and indirectly modulates proinvasive features of cancer cells. In summary, the O-glycoproteome represents a new consideration for important biological processes involved in tumor-stroma cross-talk and a potential avenue to improve the anticancer efficacy of CDK4 inhibitors. SIGNIFICANCE: A multiomics analysis of spatially distinct fibroblasts establishes the importance of the stromal O-glycoproteome in tumor-stroma interactions at the leading edge and provides potential strategies to improve cancer treatment. See related commentary by De Wever, p. 537.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Quinase 4 Dependente de Ciclina/genética , Genômica/métodos , Neoplasias/genética , Proteômica/métodos , Proteína do Retinoblastoma/genética , Células Estromais/metabolismo , Células A549 , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética
18.
Eur J Med Chem ; 229: 114009, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-34839996

RESUMO

Bruton's tyrosine kinase (BTK) regulates multiple important signaling pathways and plays a key role in the proliferation, survival, and differentiation of B-lineage cells and myeloid cells. BTK is a promising target for the treatment of hematologic malignancies. Ibrutinib, the first-generation BTK inhibitor, was approved to treat several B-cell malignancies. Despite the remarkable potency and efficacy of ibrutinib against various lymphomas and leukemias in the clinics, there are also some clinical limitations, such as off-target toxicities and primary/acquired drug resistance. As strategies to overcome these challenges, second- and third-generation BTK inhibitors, BTK-PROTACs, as well as combination therapies have been explored. In this review, we summarize clinical developments of the first-, second- and third-generation BTK inhibitors, as well as recent advances in BTK-PROTACs and ibrutinib-based combination therapies.


Assuntos
Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Piperidinas/química , Inibidores de Proteínas Quinases/química , Adenina/química , Adenina/metabolismo , Adenina/uso terapêutico , Tirosina Quinase da Agamaglobulinemia/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico
19.
Life Sci ; 288: 120176, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34848192

RESUMO

AIMS: Glioblastoma (GBM) is a highly malignant brain tumor. After treatment with the first-line drug temozolomide, only 50% of patients are responsive. Recent literature shows that the difficulty in treating GBM is mainly due to the heterogeneity of its four major cellular states, which are characterized by differences in EGFR, PDGFRA, CDK4, and NF1. Therefore, development of a multitarget drug is a potential strategy for treating heterogeneous GBM. MAIN METHODS: In this study, the antitumor ability of a potent heat shock protein 90 inhibitor, NVP-AUY922 (AUY922), was evaluated in GBM cell lines (U-87 MG and T98G cells) and patient-derived GBM cell lines [P#5 and P#5 temozolomide-resistant (TMZ-R) cells]. KEY FINDINGS: We found that AUY922 significantly reduced cell viability and colony formation in four GBM cell lines. AUY922 also significantly induced apoptosis by increasing PARP1 cleavage and the number of annexin V-positive cells. The autophagy indicators as MAP1LC3B cleavage and MAP1LC3B puncta were increased after AUY922 treatment. AUY922-induced cell death could be partially reversed by pharmacological inhibition of either apoptotic inhibitor or autophagy inhibitor. Moreover, AUY922 reduced the mRNA and protein expressions of EGFR, PDGFRA, CDK4, and NF1, which contribute to the four cellular state subtypes in GBM cells. In addition, the downstream signaling proteins of these four proteins, AKT/p-AKT, MAPK/p-MAPK, and BRAF, were downregulated after AUY922 treatment. SIGNIFICANCE: Taken together, AUY922 led to GBM cell death via apoptosis and autophagy, and reduced the mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1in heterogeneous GBM cells.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/farmacologia , Neurofibromina 1/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Resorcinóis/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Tumorais Cultivadas
20.
Lab Invest ; 102(4): 391-400, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34921235

RESUMO

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.


Assuntos
Neoplasias Ósseas , Células-Tronco Mesenquimais , Osteossarcoma , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteossarcoma/tratamento farmacológico , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética
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