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1.
J Med Virol ; 95(5): e28796, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37218551

RESUMO

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the read out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Tripsina , Internalização do Vírus , SARS-CoV-2/metabolismo , Peptídeo Hidrolases
2.
Semin Cancer Biol ; 68: 21-30, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562955

RESUMO

Quinacrine, also known as mepacrine, has originally been used as an antimalarial drug for close to a century, but was recently rediscovered as an anticancer agent. The mechanisms of anticancer effects of quinacrine are not well understood. The anticancer potential of quinacrine was discovered in a screen for small molecule activators of p53, and was specifically shown to inhibit NFκB suppression of p53. However, quinacrine can cause cell death in cells that lack p53 or have p53 mutations, which is a common occurrence in many malignant tumors including high grade serous ovarian cancer. Recent reports suggest quinacrine may inhibit cancer cell growth through multiple mechanisms including regulating autophagy, FACT (facilitates chromatin transcription) chromatin trapping, and the DNA repair process. Additional reports also suggest quinacrine is effective against chemoresistant gynecologic cancer. In this review, we discuss anticancer effects of quinacrine and potential mechanisms of action with a specific focus on gynecologic and breast cancer where treatment-refractory tumors are associated with increased mortality rates. Repurposing quinacrine as an anticancer agent appears to be a promising strategy based on its ability to target multiple pathways, its selectivity against cancer cells, and the synergistic cytotoxicity when combined with other anticancer agents with limited side effects and good tolerability profile.


Assuntos
Antimaláricos/uso terapêutico , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Reposicionamento de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinacrina/uso terapêutico , Animais , Humanos
3.
Biochem Biophys Res Commun ; 571: 26-31, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34303192

RESUMO

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.


Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Amidas/farmacologia , Antivirais/química , Benzimidazóis/farmacologia , COVID-19/virologia , Carbamatos/farmacologia , Domínio Catalítico , Simulação por Computador , RNA-Polimerase RNA-Dependente de Coronavírus/química , Ciclopropanos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Fluorenos/farmacologia , Humanos , Lactamas Macrocíclicas/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pandemias , Prolina/análogos & derivados , Prolina/farmacologia , Conformação Proteica , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia
4.
J Med Virol ; 93(3): 1296-1303, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32964419

RESUMO

The recent outbreak of the novel coronavirus, SARS-CoV-2, has emerged to be highly pathogenic in nature. Although lungs are considered as the primary infected organs by SARS-CoV-2, some of the other organs, including the brain, have also been found to be affected. Here, we have discussed how SARS-CoV-2 might infect the brain. The infection of the respiratory center in the brainstem could be hypothesized to be responsible for the respiratory failure in many COVID-19 patients. The virus might gain entry through the olfactory bulb and invade various parts of the brain, including the brainstem. Alternatively, the entry might also occur from peripheral circulation into the central nervous system by compromising the blood-brain barrier. Finally, yet another possible entry route could be its dispersal from the lungs into the vagus nerve via the pulmonary stretch receptors, eventually reaching the brainstem. Therefore, screening neurological symptoms in COVID-19 patients, especially toward the breakdown of the respiratory center in the brainstem, might help us better understand this disease.


Assuntos
Encéfalo/virologia , COVID-19/fisiopatologia , COVID-19/virologia , Vias Neurais/virologia , Centro Respiratório/virologia , SARS-CoV-2/patogenicidade , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , COVID-19/patologia , Citocinas/metabolismo , Humanos , Inflamação , Vias Neurais/fisiopatologia , Neurônios/virologia , Centro Respiratório/patologia , Centro Respiratório/fisiopatologia , Insuficiência Respiratória , Tropismo Viral
5.
Cell Physiol Biochem ; 41(2): 795-805, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28214851

RESUMO

BACKGROUND/AIMS: Epithelial-to-mesenchymal transition (EMT) plays an essential role in the transition from early to invasive phenotype, however the underlying mechanisms still remain elusive. Herein, we propose a mechanism through which the class-III deacetylase SIRT1 regulates EMT in ovarian cancer (OC) cells. METHODS: Expression analysis was performed using Q-PCR, western blot, immunofluorescence and fluorescence-IHC study. Matrigel invasion assay was used for the invasion study. Morphological alterations were observed by phalloidin-staining. Co-immunoprecipitation study was performed to analyze protein-protein interaction. RESULTS: Overexpression of SIRT1-WT as well as Resveratrol-mediated SIRT1 activation antagonized the invasion of OC cells by suppressing EMT. SIRT1 deacetylates HIF1α, to inactivate its transcriptional activity. To further validate HIF1α inactivation, its target gene, i.e. ZEB1, an EMT-inducing factor was found to attenuate upon SIRT1 activation. To uncover the regulatory factor governing SIRT1 expression, lysophosphatidic acid (LPA), a highly enriched oncolipid in ascites/serum of OC patients, was found to down-regulate SIRT1 expression. Importantly, LPA was found to induce the mesenchymal switch in OC cells through suppression of SIRT1. Decreased level of SIRT1 was further validated in ovarian tissue samples of OC patients. CONCLUSION: We have identified a mechanism that relates SIRT1 down-regulation to LPA-induced EMT in OC cells and may open new arenas on developing novel anti-cancer therapeutics.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Sirtuína 1/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microscopia de Fluorescência , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética
6.
Mol Cancer ; 14: 162, 2015 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-26298390

RESUMO

BACKGROUND: Most ovarian cancers are highly invasive in nature and the high burden of metastatic disease make them a leading cause of mortality among all gynaecological malignancies. The homeodomain transcription factor, PITX2 is associated with cancer in different tissues. Our previous studies demonstrated increased PITX2 expression in human ovarian tumours. Growing evidence linking activation of TGF-ß pathway by homeodomain proteins prompted us to look for the possible involvement of this signalling pathway in PITX2-mediated progression of ovarian cancer. METHODS: The status of TGF-ß signalling in human ovarian tissues was assessed by immunohistochemistry. The expression level of TGFB/INHBA and other invasion-associated genes was measured by quantitative-PCR (Q-PCR) and Western Blot after transfection/treatments with clones/reagents in normal/cancer cells. The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay. The PITX2- and activin-induced epithelial-mesenchymal transition (EMT) was evaluated by Q-PCR of respective markers and confocal/phase-contrast imaging of cells. RESULTS: Human ovarian tumours showed enhanced TGF-ß signalling. Our study uncovers the PITX2-induced expression of TGFB1/2/3 as well as INHBA genes (p < 0.01) followed by SMAD2/3-dependent TGF-ß signalling pathway. PITX2-induced TGF-ß pathway regulated the expression of invasion-associated genes, SNAI1, CDH1 and MMP9 (p < 0.01) that accounted for enhanced motility/invasion of ovarian cancers. Snail and MMP9 acted as important mediators of PITX2-induced invasiveness of ovarian cancer cells. PITX2 over-expression resulted in loss of epithelial markers (p < 0.01) and gain of mesenchymal markers (p < 0.01) that contributed significantly to ovarian oncogenesis. PITX2-induced INHBA expression (p < 0.01) contributed to EMT in both normal and ovarian cancer cells. CONCLUSIONS: Overall, our findings suggest a significant contributory role of PITX2 in promoting invasive behaviour of ovarian cancer cells through up-regulation of TGFB/INHBA. We have also identified the previously unknown involvement of activin-A in promoting EMT. Our work provides novel mechanistic insights into the invasive behavior of ovarian cancer cells. The extension of this study have the potential for therapeutic applications in future.


Assuntos
Proteínas de Homeodomínio/genética , Subunidades beta de Inibinas/genética , Neoplasias Ovarianas/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Subunidades beta de Inibinas/biossíntese , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Ovarianas/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Proteína Homeobox PITX2
7.
J Virol ; 88(8): 4319-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24478427

RESUMO

UNLABELLED: Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a ß-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting ß-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La ß-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein. IMPORTANCE: Hepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented the development of a small-animal model, which is important for development of new antiviral drugs. Although a number of human-specific proteins are responsible for this species selectivity and some of these proteins--mostly entry factors--have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence allows the mouse La to behave like its human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.


Assuntos
Hepacivirus/fisiologia , Hepatite C/metabolismo , Especificidade de Hospedeiro , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Hepacivirus/genética , Hepatite C/genética , Hepatite C/virologia , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/genética , RNA Viral/metabolismo , Alinhamento de Sequência , Replicação Viral
8.
J Virol ; 87(12): 6713-26, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23552417

RESUMO

Human La protein has been implicated in facilitating the internal initiation of translation as well as replication of hepatitis C virus (HCV) RNA. Previously, we demonstrated that La interacts with the HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG within stem-loop IV by its RNA recognition motif (RRM) (residues 112 to 184) and influences HCV translation. In this study, we have deciphered the role of this interaction in HCV replication in a hepatocellular carcinoma cell culture system. We incorporated mutation of the GCAC motif in an HCV monocistronic subgenomic replicon and a pJFH1 construct which altered the binding of La and checked HCV RNA replication by reverse transcriptase PCR (RT-PCR). The mutation drastically affected HCV replication. Furthermore, to address whether the decrease in replication is a consequence of translation inhibition or not, we incorporated the same mutation into a bicistronic replicon and observed a substantial decrease in HCV RNA levels. Interestingly, La overexpression rescued this inhibition of replication. More importantly, we observed that the mutation reduced the association between La and NS5B. The effect of the GCAC mutation on the translation-to-replication switch, which is regulated by the interplay between NS3 and La, was further investigated. Additionally, our analyses of point mutations in the GCAC motif revealed distinct roles of each nucleotide in HCV replication and translation. Finally, we showed that a specific interaction of the GCAC motif with human La protein is crucial for linking 5' and 3' ends of the HCV genome. Taken together, our results demonstrate the mechanism of regulation of HCV replication by interaction of the cis-acting element GCAC within the HCV IRES with human La protein.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Motivos de Aminoácidos/genética , Hepacivirus/genética , Fosfoproteínas/metabolismo , RNA Viral/metabolismo , Replicação Viral , Motivos de Aminoácidos/efeitos da radiação , Linhagem Celular Tumoral , Códon de Iniciação , Elementos Facilitadores Genéticos , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Hepatócitos/virologia , Humanos , Mutação Puntual , Biossíntese de Proteínas , RNA Viral/genética , Replicon/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo
9.
J Virol ; 87(18): 10105-13, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23843634

RESUMO

BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease.


Assuntos
Vírus BK/genética , Vírus BK/fisiologia , Tropismo Viral , Internalização do Vírus , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus BK/classificação , DNA Viral/química , DNA Viral/genética , Feminino , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência de DNA , Sorotipagem , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
10.
Mol Ther ; 21(1): 57-67, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22910295

RESUMO

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.


Assuntos
Peptídeos/farmacologia , Ribossomos/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Humanos , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
11.
Transl Oncol ; 41: 101879, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38262110

RESUMO

Fluctuations in the number of regulatory molecules and differences in timings of molecular events can generate variation in gene expression among genetically identical cells in the same environmental condition. This variation, termed as expression noise, can create differences in metabolic state and cellular functions, leading to phenotypic heterogeneity. Expression noise and phenotypic heterogeneity have been recognized as important contributors to intra-tumor heterogeneity, and have been associated with cancer growth, progression, and therapy resistance. However, how expression noise changes with cancer progression in actual cancer patients has remained poorly explored. Such an analysis, through identification of genes with increasing expression noise, can provide valuable insights into generation of intra-tumor heterogeneity, and could have important implications for understanding immune-suppression, drug tolerance and therapy resistance. In this work, we performed a genome-wide identification of changes in gene expression noise with cancer progression using single-cell RNA-seq data of lung adenocarcinoma patients at different stages of cancer. We identified 37 genes in epithelial cells that showed an increasing noise trend with cancer progression, many of which were also associated with cancer growth, EMT and therapy resistance. We found that expression of several of these genes was positively associated with expression of mitochondrial genes, suggesting an important role of mitochondria in generation of heterogeneity. In addition, we uncovered substantial differences in sample-specific noise profiles which could have implications for personalized prognosis and treatment.

12.
Pharmaceuticals (Basel) ; 16(4)2023 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-37111346

RESUMO

Multiple myeloma is a malignancy of immunoglobulin-secreting plasma cells that is now often treated in the newly diagnosed and relapsed and/or refractory settings with monoclonal antibodies targeting lineage-specific markers used either alone or in rationally designed combination regimens. Among these are the anti-CD38 antibodies daratumumab and isatuximab, and the anti-Signaling lymphocytic activation molecule family member 7 antibody elotuzumab, all of which are used in their unconjugated formats. Single-chain variable fragments from antibodies also form a key element of the chimeric antigen receptors (CARs) in the B-cell maturation antigen (BCMA)-targeted CAR T-cell products idecabtagene vicleucel and ciltacabtagene autoleucel, which are approved in the advanced setting. Most recently, the bispecific anti-BCMA and T-cell-engaging antibody teclistamab has become available, again for patients with relapsed/refractory disease. Another format into which antibodies can be converted to exert anti-tumor efficacy is as antibody-drug conjugates (ADCs), and belantamab mafodotin, which also targets BCMA, represented the first such agent that gained a foothold in myeloma. Negative results from a recent Phase III study have prompted the initiation of a process for withdrawal of its marketing authorization. However, belantamab remains a drug with some promise, and many other ADCs targeting either BCMA or other plasma cell surface markers are in development and showing potential. This contribution will provide an overview of some of the current data supporting the possibility that ADCs will remain a part of our chemotherapeutic armamentarium against myeloma moving forward, and also highlight areas for future development.

13.
Oncogene ; 42(37): 2725-2736, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37550562

RESUMO

PG545 (Pixatimod) is a highly sulfated small molecule known for its ability to inhibit heparanase and disrupt signaling mediated by heparan-binding-growth factors (HB-GF). Previous studies indicated that PG545 inhibits growth factor-mediated signaling in ovarian cancer (OC) to enhance response to chemotherapy. Here we investigated the previously unidentified mechanisms by which PG545 induces DNA damage in OC cells and found that PG545 induces DNA single- and double-strand breaks, reduces RAD51 expression in an autophagy-dependent manner and inhibits homologous recombination repair (HRR). These changes accompanied the ability of PG545 to inhibit endocytosis of the heparan-sulfate proteoglycan interacting DNA repair protein, DEK, leading to DEK sequestration in the tumor microenvironment (TME) and loss of nuclear DEK needed for HRR. As a result, PG545 synergized with poly (ADP-ribose) polymerase inhibitors (PARPis) in OC cell lines in vitro and in 55% of primary cultures of patient-derived ascites samples ex vivo. Moreover, PG545/PARPi synergy was observed in OC cells exhibiting either de novo or acquired resistance to PARPi monotherapy. PG545 in combination with rucaparib also generated increased DNA damage, increased antitumor effects and increased survival of mice bearing HRR proficient OVCAR5 xenografts compared to monotherapy treatment in vivo. Synergistic antitumor activity of the PG545/rucaparib combination was likewise observed in an immunocompetent syngeneic ID8F3 OC model. Collectively, these results suggest that targeting DEK-HSPG interactions in the TME through the use of PG545 may be a novel method of inhibiting DNA repair and sensitizing cells to PARPis.


Assuntos
Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Saponinas , Animais , Feminino , Humanos , Camundongos , Inibidores da Angiogênese/farmacologia , Linhagem Celular Tumoral , Reparo do DNA , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Microambiente Tumoral , Saponinas/farmacologia , Saponinas/uso terapêutico
15.
FEBS Open Bio ; 12(9): 1657-1676, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35686673

RESUMO

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a "metabolic disorder," is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1-SIRT7) belong to the group of NAD+ deacetylase and ADP-ribosylation enzymes that function as NAD+ sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non-malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2-driven activatory phosphorylation of DRP1 at serine-616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA-mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.


Assuntos
Dinâmica Mitocondrial , Neoplasias Ovarianas , Sirtuínas , Feminino , Glicólise , Humanos , NAD/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , Sirtuínas/metabolismo
16.
Cancer Res ; 82(9): 1675-1681, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35260879

RESUMO

Abundant fibrotic stroma is a typical feature of most solid tumors, and stromal activation promotes oncogenesis, therapy resistance, and metastatic dissemination of cancer cells. Therefore, targeting the tumor stroma in combination with standard-of-care therapies has become a promising therapeutic strategy in recent years. The leucine-rich repeat-containing protein 15 (LRRC15) is involved in cell-cell and cell-matrix interactions and came into focus as a promising anticancer target owing to its overexpression in mesenchymal-derived tumors such as sarcoma, glioblastoma, and melanoma and in cancer-associated fibroblasts in the microenvironment of breast, head and neck, lung, and pancreatic tumors. Effective targeting of LRRC15 using specific antibody-drug conjugates (ADC) has the potential to improve the outcome of patients with LRRC15-positive (LRRC15+) cancers of mesenchymal origin or stromal desmoplasia. Moreover, LRRC15 expression may serve as a predictive biomarker that could be utilized in the preclinical assessment of cancer patients to support personalized clinical outcomes. This review focuses on the role of LRRC15 in cancer, including clinical trials involving LRRC15-targeted therapies, such as the ABBV-085 ADC for patients with LRRC15+ tumors. This review spans perceived knowledge gaps and highlights the clinical avenues that need to be explored to provide better therapeutic outcomes in patients.


Assuntos
Fibroblastos Associados a Câncer , Glioblastoma , Imunoconjugados , Sarcoma , Fibroblastos Associados a Câncer/metabolismo , Glioblastoma/metabolismo , Humanos , Imunoconjugados/farmacologia , Proteínas de Membrana/metabolismo , Sarcoma/tratamento farmacológico , Microambiente Tumoral
17.
Oncogene ; 41(33): 4003-4017, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804016

RESUMO

PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.


Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Linhagem Celular Tumoral , Proliferação de Células , Glicólise , Via de Sinalização Hippo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Fosfofrutoquinase-2/metabolismo , Piridinas , Quinolinas , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Microambiente Tumoral
18.
NAR Cancer ; 4(3): zcac022, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35875052

RESUMO

A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

19.
Cancer Res ; 82(6): 1038-1054, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34654724

RESUMO

Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with ß1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases.Significance: This study identifies that LRRC15 activates ß1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody-drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.


Assuntos
Imunoconjugados , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Adesão Celular , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Integrinas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia
20.
Cancers (Basel) ; 13(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919392

RESUMO

We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

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