RESUMO
OBJECTIVE: Dedifferentiated endometrial carcinoma (DDEC) characterized by SWItch/Sucrose Non-Fermentable (SWI/SNF) complex inactivation is a highly aggressive type of endometrial cancer without effective systemic therapy options. Its uncommon nature and aggressive disease trajectory pose significant challenges for therapeutic progress. To address this obstacle, we focused on developing preclinical models tailored to this tumor type and established patient tumor-derived three-dimensional (3D) spheroid models of DDEC. METHODS: High-throughput drug repurposing screens were performed on in vitro 3D spheroid models of DDEC cell lines (SMARCA4-inactivated DDEC-1 and ARID1A/ARID1B co-inactivated DDEC-2). The dose-response relationships of the identified candidate drugs were evaluated in vitro, followed by in vivo evaluation using xenograft models of DDEC-1 and DDEC-2. RESULTS: Drug screen in 3D models identified multiple cardiac glycosides including digoxin and digitoxin as candidate drugs in both DDEC-1 and DDEC-2. Subsequent in vitro dose-response analyses confirmed the inhibitory activity of digoxin and digitoxin with both drugs showing lower IC50 in DDEC cells compared to non-DDEC endometrial cancer cells. In in vivo xenograft models, digoxin significantly suppressed the growth of DDEC tumors at clinically relevant serum concentrations. CONCLUSION: Using biologically precise preclinical models of DDEC derived from patient tumor samples, our study identified digoxin as an effective drug in suppressing DDEC tumor growth. These findings provide compelling preclinical evidence for the use of digoxin as systemic therapy for SWI/SNF-inactivated DDEC, which may also be applicable to other SWI/SNF-inactivated tumor types.
Assuntos
Digoxina , Neoplasias do Endométrio , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Digoxina/farmacologia , Digoxina/uso terapêutico , Humanos , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Animais , Linhagem Celular Tumoral , Camundongos , Esferoides Celulares/efeitos dos fármacos , Reposicionamento de Medicamentos , Digitoxina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga EscalaRESUMO
Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
Assuntos
Homeostase , Ferro , Neoplasias , Humanos , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Ferroptose , Ferro/metabolismo , Proteína 1 Reguladora do Ferro , Neoplasias/metabolismo , Neoplasias/genética , Ligação Proteica , Receptores da Transferrina/metabolismo , Receptores da Transferrina/genéticaRESUMO
Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.
Assuntos
Adenocarcinoma , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Mutação/genética , GenômicaRESUMO
Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.
Assuntos
Ribonucleoproteína Nuclear Heterogênea A1 , Esclerose Múltipla , Humanos , Processamento Alternativo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Esclerose Múltipla/genética , RNA , Splicing de RNA/genéticaRESUMO
OBJECTIVE: Inhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. METHODS: A recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. RESULTS: Loss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. CONCLUSIONS: NF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.
Assuntos
Carcinoma Endometrioide , Cistadenocarcinoma Seroso , Neoplasias Ovarianas , Feminino , Humanos , Proteína BRCA1 , Neurofibromina 1/genética , Imuno-Histoquímica , Proteína BRCA2 , Neoplasias Ovarianas/patologia , Carcinoma Endometrioide/patologia , Cistadenocarcinoma Seroso/patologia , Carcinoma Epitelial do OvárioRESUMO
Eph receptors and their ephrin ligands are viewed as promising targets for cancer treatment; however, targeting them is hindered by their context-dependent functionalities. To circumvent this, we explore molecular landscapes underlying their pro- and anti-malignant activities. Using unbiased bioinformatics approaches, we construct a cancer-related network of genetic interactions (GIs) of all Ephs and ephrins to assist in their therapeutic manipulation. We also apply genetic screening and BioID proteomics and integrate them with machine learning approaches to select the most relevant GIs of one Eph receptor, EPHB6. This identifies a crosstalk between EPHB6 and EGFR, and further experiments confirm the ability of EPHB6 to modulate EGFR signaling, enhancing the proliferation of cancer cells and tumor development. Taken together, our observations show EPHB6 involvement in EGFR action, suggesting its targeting might be beneficial in EGFR-dependent tumors, and confirm that the Eph family genetic interactome presented here can be effectively exploited in developing cancer treatment approaches.
Assuntos
Efrinas , Neoplasias , Efrinas/genética , Proteômica , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Transdução de Sinais , Receptores ErbB/genética , Neoplasias/genéticaRESUMO
PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Cetuximab/farmacologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Large-scale genetic screens are becoming increasingly used as powerful tools to query the genome to identify therapeutic targets in cancer. The advent of the CRISPR technology has revolutionized the effectiveness of these screens and has made it possible to carry out loss-of-function screens to identify cancer-specific genetic interactions. Such loss-of-function screens can be performed in silico, in vitro, and in vivo, depending on the scale of the screen, as well as research questions to be answered. Performing screens in vivo has its challenges but also advantages, providing opportunities to study the tumor microenvironment and cancer immunity. In this chapter, we present a procedural framework and associated notes for conducting in vivo CRISPR knockout screens in cancer models to study cancer biology, anti-tumor immune responses, tumor microenvironment, and predicting treatment responses.
Assuntos
Sistemas CRISPR-Cas , Neoplasias , Humanos , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma , Neoplasias/genética , Microambiente Tumoral/genéticaRESUMO
The lack of targeted therapies for triple-negative breast cancer (TNBC) contributes to their high mortality rates and high risk of relapse compared to other subtypes of breast cancer. Most TNBCs (75%) have downregulated the expression of CREB3L1 (cAMP-responsive element binding protein 3 like 1), a transcription factor and metastasis suppressor that represses genes that promote cancer progression and metastasis. In this report, we screened an FDA-approved drug library and identified four drugs that were highly cytotoxic towards HCC1806 CREB3L1-deficient TNBC cells. These four drugs were: (1) palbociclib isethionate, a CDK4/6 inhibitor, (2) lanatocide C (also named isolanid), a Na+/K+-ATPase inhibitor, (3) cladribine, a nucleoside analog, and (4) homoharringtonine (also named omacetaxine mepesuccinate), a protein translation inhibitor. Homoharringtonine consistently showed the most cytotoxicity towards an additional six TNBC cell lines (BT549, HCC1395, HCC38, Hs578T, MDA-MB-157, MDA-MB-436), and several luminal A breast cancer cell lines (HCC1428, MCF7, T47D, ZR-75-1). All four drugs were then separately evaluated for possible synergy with the chemotherapy agents, doxorubicin (an anthracycline) and paclitaxel (a microtubule stabilizing agent). A strong synergy was observed using the combination of homoharringtonine and paclitaxel, with high cytotoxicity towards TNBC cells at lower concentrations than when each was used separately.
Assuntos
Antineoplásicos , Neoplasias de Mama Triplo Negativas , Adenosina Trifosfatases , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cladribina/uso terapêutico , Doxorrubicina/uso terapêutico , Excipientes , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Nucleosídeos/uso terapêutico , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.
RESUMO
Neuroendocrine prostate cancer (NEPC) represents a highly aggressive form of prostate tumors. NEPC results from trans-differentiated castration-resistant prostate cancer (CRPC) with increasing evidence indicating that the incidence of NEPC often results from the adaptive response to androgen deprivation therapy. Recent studies have shown that a subset of NEPC exhibits overexpression of the MYCN oncogene along with the loss of tumor suppressing TP53 and RB1 activities. N-MYC is structurally disordered with no binding pockets available on its surface and so far, no clinically approved drug is available. We adopted a drug-repurposing strategy, screened ~1800 drug molecules, and identified fludarabine phosphate to preferentially inhibit the proliferation of N-MYC overexpressing NEPC cells by inducing reactive oxygen species (ROS). We also show that fludarabine phosphate affects N-MYC protein levels and N-MYC transcriptional targets in NEPC cells. Moreover, enhanced ROS production destabilizes N-MYC protein by inhibiting AKT signaling and is responsible for the reduced survival of NEPC cells and tumors. Our results indicate that increasing ROS production by the administration of fludarabine phosphate may represent an effective treatment option for patients with N-MYC overexpressing NEPC tumors.
Assuntos
Carcinoma Neuroendócrino , Neoplasias da Próstata , Antagonistas de Androgênios/uso terapêutico , Carcinoma Neuroendócrino/patologia , Linhagem Celular Tumoral , Reposicionamento de Medicamentos , Humanos , Masculino , Proteína Proto-Oncogênica N-Myc/metabolismo , Neoplasias da Próstata/patologia , Espécies Reativas de Oxigênio/uso terapêutico , Fosfato de Vidarabina/análogos & derivadosRESUMO
The human hepatocyte nuclear factor 1 homeobox A (HNF1A) gene loci express the protein-coding HNF1A transcript and a long non-coding RNA in the anti-sense (HNF1A-AS1) direction. HNF1A-AS1 is expressed in numerous types of cancers and poor clinical outcomes such as higher mortality rates, greater metastatic capacity, and poor prognosis of the disease are the results of this expression. In this study, we determined the epigenetic features of the HNF1A gene loci, and expression and cellular localization of HNF1A-AS1 RNA, HNF1A RNA, and HNF1A protein in colorectal cancer (HT-29, HTC116, RKO, and SW480) and normal colon epithelial (CCD841) cells. The HT-29 HNF1A gene had active histone marks (H3K4me3, H3K27ac) and DNase 1 accessible sites at the promoter regions of the HNF1A and HNF1A-AS1 genes. These epigenetic marks were not observed in the other colorectal cancer cells or in the normal colon epithelial cells. Consistent with the active gene epigenetic signature of the HNF1A gene in HT-29 cells, HNF1A protein, and HNF1A/HNF1A-AS1 transcripts were detected in HT-29 cells but poorly, if at all observed, in the other cell types. In HT-29 cells, HNF1A-AS1 localized to the nucleus and was found to bind to the enhancer of zeste homolog 2 (EZH2, a member of PRC2 complex) and potentially form RNA-DNA triplexes with DNase 1 accessible sites in the HT-29 genome. These activities of HNF1A-AS1 may contribute to the oncogenic properties of this long non-coding RNA.
Assuntos
Neoplasias do Colo , RNA Longo não Codificante , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/genética , Desoxirribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
DEAD/H-box helicases are implicated in virtually every aspect of RNA metabolism, including transcription, pre-mRNA splicing, ribosomes biogenesis, nuclear export, translation initiation, RNA degradation, and mRNA editing. Most of these helicases are upregulated in various cancers and mutations in some of them are associated with several malignancies. Lately, synthetic lethality (SL) and synthetic dosage lethality (SDL) approaches, where genetic interactions of cancer-related genes are exploited as therapeutic targets, are emerging as a leading area of cancer research. Several DEAD/H-box helicases, including DDX3, DDX9 (Dbp9), DDX10 (Dbp4), DDX11 (ChlR1), and DDX41 (Sacy-1), have been subjected to SL analyses in humans and different model organisms. It remains to be explored whether SDL can be utilized to identity druggable targets in DEAD/H-box helicase overexpressing cancers. In this review, we analyze gene expression data of a subset of DEAD/H-box helicases in multiple cancer types and discuss how their SL/SDL interactions can be used for therapeutic purposes. We also summarize the latest developments in clinical applications, apart from discussing some of the challenges in drug discovery in the context of targeting DEAD/H-box helicases.
RESUMO
The progression of prostate cancer (PC) into neuroendocrine prostate cancer (NEPC) is a major challenge in treating PC. In NEPC, the PC cells undergo neuroendocrine differentiation (NED); however, the exact molecular mechanism that triggers NED is unknown. Peripheral nerves are recently shown to promote PC. However, their contribution to NEPC was not studied well. In this study, we explored whether sympathetic neurosignaling contributes to NED. We found that human prostate tumors from patients that later developed metastases and castration-resistant prostate cancer (CRPC), a stage preceding to NEPC, have high sympathetic innervations. Our work revealed that high concentrations of the sympathetic neurotransmitter norepinephrine (NE) induces NED-like changes in PC cells in vitro, evident by their characteristic cellular and molecular changes. The NE-mediated NED was effectively inhibited by the Adrß2 blocker propranolol. Strikingly, propranolol along with castration also significantly inhibited the development and progression of NEPC in vivo in an orthotopic NEPC model. Altogether, our results indicate that the NE-Adrß2 axis is a potential therapeutic intervention point for NEPC.
RESUMO
Over the past two decades, the concept of synthetic lethality (SL) that queries genetic relationships between gene pairs has gradually emerged as one of the best strategies to selectively eliminate cancer cells. Some of the most successful approaches to identify synthetic lethal interactions (SLIs) were largely dependent on pooled screening formats that require heavy validation in order to mitigate false positives. Here, we describe a high-throughput method to identify SLIs using CRISPR-based strategy that covers, high-throughput production of plasmid DNA preparations, lentiviral production, and subsequent cellular transduction using single guide RNAs (sgRNAs). This method could be adopted to query hundreds of SLIs. As an example, we describe the methods associated with building an interaction map for DNA damage and repair (DDR) genes. The use of multiwell plates and image-based quantification allows a comparative measurement of SLIs at a high-resolution on a one-by-one basis. Furthermore, this scalable, arrayed CRISPR screening method can be applied to multiple cancer cell types, and genes of interest, resulting in new functional discoveries that can be exploited therapeutically.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , RNA Guia de Cinetoplastídeos/genética , Mutações Sintéticas LetaisRESUMO
Despite the success of targeted therapies including immunotherapies in cancer treatments, tumor resistance to targeted therapies remains a fundamental challenge. Tumors can evolve resistance to a therapy that targets one gene by acquiring compensatory alterations in another gene, such compensatory interaction between two genes is referred to as synthetic rescue (SR) interactions. To identify SRs, here we describe an algorithm, INCISOR, that leverages tumor transcriptomics and clinical information from 10,000 patients as well as data from experimental screens. INCISOR can identify SRs that are common across several cancer-types in genome-wide fashion by sifting through half a billion possible gene-gene combinations and provide a framework to design therapies to tackle resistance.
Assuntos
Neoplasias , Algoritmos , Humanos , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/terapia , TranscriptomaRESUMO
Cancer is one of the leading causes of death and chromosomal instability (CIN) is a hallmark feature of cancer. CIN, a source of genetic variation in either altered chromosome number or structure contributes to tumor heterogeneity and has become a hot topic in recent years prominently for its role in therapeutic responses. Synthetic lethality and synthetic rescue based approaches, for example, advancing CRISPR-Cas9 platform, are emerging as a powerful strategy to identify new potential targets to selectively eradicate cancer cells. Unfortunately, only few of them are further explored therapeutically due to the difficulty in linking these targets to small molecules for pharmacological intervention. This, however, can be alleviated by the efforts to bring chemical, bioactivity, and genomic data together, as well as established computational approaches. In this chapter, we will discuss some of these advances, including established databases and in silico target-ligand prediction, with the aim to navigate through the synthetically available chemical space to the biologically targetable landscape, and eventually, to the chemical modeling of synthetic lethality and synthetic rescue interactions, that are of great clinical and pharmaceutical relevance and significance.
Assuntos
Mutações Sintéticas Letais , Instabilidade Cromossômica , Genômica , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia-induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5'-monophosphate-activated protein kinase activation and increased acetyl-coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.
Assuntos
Bicarbonatos , Neoplasias , Liases de Carbono-Enxofre , Anidrase Carbônica IX , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Hipóxia , Ferro , Neoplasias/genéticaRESUMO
BACKGROUND: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. METHODS: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments. RESULTS: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. CONCLUSIONS: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.
Assuntos
Apoptose/genética , Proteínas de Fluorescência Verde , Infecções por HIV , Macrófagos , RNA Interferente Pequeno , Linfócitos T CD4-Positivos/virologia , Estudo de Associação Genômica Ampla , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Linfócitos TRESUMO
Gastro-esophageal (GE) cancers are one of the major causes of cancer-related death in the world. There is a need for novel biomarkers in the management of GE cancers, to yield predictive response to the available therapies. Our study aims to identify leading genes that are differentially regulated in patients with these cancers. We explored the expression data for those genes whose protein products can be detected in the plasma using the Cancer Genome Atlas to identify leading genes that are differentially regulated in patients with GE cancers. Our work predicted several candidates as potential biomarkers for distinct stages of GE cancers, including previously identified CST1, INHBA, STMN1, whose expression correlated with cancer recurrence, or resistance to adjuvant therapies or surgery. To define the predictive accuracy of these genes as possible biomarkers, we constructed a co-expression network and performed complex network analysis to measure the importance of the genes in terms of a ratio of closeness centrality (RCC). Furthermore, to measure the significance of these differentially regulated genes, we constructed an SVM classifier using machine learning approach and verified these genes by using receiver operator characteristic (ROC) curve as an evaluation metric. The area under the curve measure was > 0.9 for both the overexpressed and downregulated genes suggesting the potential use and reliability of these candidates as biomarkers. In summary, we identified leading differentially expressed genes in GE cancers that can be detected in the plasma proteome. These genes have potential to become diagnostic and therapeutic biomarkers for early detection of cancer, recurrence following surgery and for development of targeted treatment.