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1.
Cancer ; 126(4): 894-907, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31714594

RESUMO

BACKGROUND: Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors exhibit promising activity against ovarian cancers, but their efficacy can be limited by acquired drug resistance. This study explores the role of autophagy in regulating the sensitivity of ovarian cancer cells to PARP inhibitors. METHODS: Induction of autophagy was detected by punctate LC3 fluorescence staining, LC3I to LC3II conversion on Western blot analysis, and electron microscopy. Enhanced growth inhibition and apoptosis were observed when PARP inhibitors were used with hydroxychloroquine, chloroquine (CQ), or LYS05 to block the hydrolysis of proteins and lipids in autophagosomes or with small interfering RNA against ATG5 or ATG7 to prevent the formation of autophagosomes. The preclinical efficacy of the combination of CQ and olaparib was evaluated with a patient-derived xenograft (PDX) and the OVCAR8 human ovarian cancer cell line. RESULTS: Four PARP inhibitors (olaparib, niraparib, rucaparib, and talazoparib) induced autophagy in a panel of ovarian cancer cells. Inhibition of autophagy with CQ enhanced the sensitivity of ovarian cancer cells to PARP inhibitors. In vivo, olaparib and CQ produced additive growth inhibition in OVCAR8 xenografts and a PDX. Olaparib inhibited PARP activity, and this led to increased reactive oxygen species (ROS) and an accumulation of γ-H2AX. Inhibition of autophagy also increased ROS and γ-H2AX and enhanced the effect of olaparib on both entities. Treatment with olaparib increased phosphorylation of ATM and PTEN while decreasing the phosphorylation of AKT and mTOR and inducing autophagy. CONCLUSIONS: PARP inhibitor-induced autophagy provides an adaptive mechanism of resistance to PARP inhibitors in cancer cells with wild-type BRCA, and a combination of PARP inhibitors with CQ or other autophagy inhibitors could improve outcomes for patients with ovarian cancer.


Assuntos
Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Indazóis/farmacologia , Camundongos Nus , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
2.
Cancer ; 126(15): 3579-3592, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32484926

RESUMO

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.


Assuntos
Quinase do Linfoma Anaplásico/genética , Neoplasias Ovarianas/tratamento farmacológico , Fator de Transcrição STAT3/genética , Proteínas rho de Ligação ao GTP/genética , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/genética , Sobrevivência Celular/genética , Crizotinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos
3.
Clin Genet ; 95(5): 558-568, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30614523

RESUMO

Breast Cancer (BC) is the most commonly diagnosed cancer and is the leading cause of cancer deaths in women. BC is a heterogeneous disease with different clinical and genetic features. According to immunohistochemical markers, BC is subdivided into four main subtypes: luminal A, luminal B, ERBB2 positive and triple negative. Long non-coding RNAs (lncRNAs) are transcripts with more than 200 nucleotides and deregulated lncRNAs are associated with human diseases, including BC. In order to improve BC molecular classification, non-coding RNAs (ncRNAs), including lncRNAs, have been used. In this review, we focus on lncRNAs with differential expression in BC subtypes and how these RNAs may act to contribute to BC heterogeneity. We also emphasize the potential of these lncRNAs as biomarkers.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Estrogênios/metabolismo , Feminino , Humanos , RNA Longo não Codificante/metabolismo , Receptor ErbB-2/metabolismo
4.
J Clin Invest ; 132(11)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642638

RESUMO

Poly(ADP-ribose) polymerase inhibitors (PARP inhibitors) have had an increasing role in the treatment of ovarian and breast cancers. PARP inhibitors are selectively active in cells with homologous recombination DNA repair deficiency caused by mutations in BRCA1/2 and other DNA repair pathway genes. Cancers with homologous recombination DNA repair proficiency respond poorly to PARP inhibitors. Cancers that initially respond to PARP inhibitors eventually develop drug resistance. We have identified salt-inducible kinase 2 (SIK2) inhibitors, ARN3236 and ARN3261, which decreased DNA double-strand break (DSB) repair functions and produced synthetic lethality with multiple PARP inhibitors in both homologous recombination DNA repair deficiency and proficiency cancer cells. SIK2 is required for centrosome splitting and PI3K activation and regulates cancer cell proliferation, metastasis, and sensitivity to chemotherapy. Here, we showed that SIK2 inhibitors sensitized ovarian and triple-negative breast cancer (TNBC) cells and xenografts to PARP inhibitors. SIK2 inhibitors decreased PARP enzyme activity and phosphorylation of class-IIa histone deacetylases (HDAC4/5/7). Furthermore, SIK2 inhibitors abolished class-IIa HDAC4/5/7-associated transcriptional activity of myocyte enhancer factor-2D (MEF2D), decreasing MEF2D binding to regulatory regions with high chromatin accessibility in FANCD2, EXO1, and XRCC4 genes, resulting in repression of their functions in the DNA DSB repair pathway. The combination of PARP inhibitors and SIK2 inhibitors provides a therapeutic strategy to enhance PARP inhibitor sensitivity for ovarian cancer and TNBC.


Assuntos
Antineoplásicos , Neoplasias Ovarianas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas , Antineoplásicos/uso terapêutico , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética
5.
Cancers (Basel) ; 13(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503955

RESUMO

Salt-induced kinase 2 (SIK2) is a serine-threonine kinase that regulates centrosome splitting, activation of PI3 kinase and phosphorylation of class IIa HDACs, affecting gene expression. Previously, we found that inhibition of SIK2 enhanced sensitivity of ovarian cancer cells to paclitaxel. Carboplatin and paclitaxel constitute first-line therapy for most patients with ovarian carcinoma, producing a 70% clinical response rate, but curing <20% of patients with advanced disease. We have asked whether inhibition of SIK2 with ARN-3261 enhances sensitivity to carboplatin in ovarian cancer cell lines and xenograft models. ARN-3261-induced DNA damage and apoptosis were measured with γ-H2AX accumulation, comet assays, and annexin V. ARN-3261 inhibited growth of eight ovarian cancer cell lines at an IC50 of 0.8 to 3.5 µM. ARN-3261 significantly enhanced sensitivity to carboplatin in seven of eight ovarian cancer cell lines and a carboplatin-resistant cell line tested. Furthermore, ARN-3261 in combination with carboplatin produced greater inhibition of tumor growth than carboplatin alone in SKOv3 and OVCAR8 ovarian cancer xenograft models. ARN-3261 enhanced DNA damage and apoptosis by downregulating expression of survivin. Thus, a SIK2 kinase inhibitor enhanced carboplatin-induced therapy in preclinical models of ovarian cancer and deserves further evaluation in clinical trials.

6.
Chem Sci ; 12(10): 3526-3543, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-34163626

RESUMO

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

7.
Cancers (Basel) ; 11(4)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003488

RESUMO

Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during "second look" operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15-33) and the switch II region of DIRAS3 (residues 93-107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.

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