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1.
Oncogene ; 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34773074

RESUMO

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

2.
Mol Cancer Ther ; 20(9): 1663-1671, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34158348

RESUMO

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

3.
Mol Ther ; 29(5): 1668-1682, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33845199

RESUMO

Cancer gene therapies are usually designed either to express wild-type copies of tumor suppressor genes or to exploit tumor-associated phenotypic changes to endow selective cytotoxicity. However, these approaches become less relevant to cancers that contain many independent mutations, and the situation is made more complex by our increased understanding of clonal evolution of tumors, meaning that different metastases and even regions of the same tumor mass have distinct mutational and phenotypic profiles. In contrast, the relatively genetically stable tumor microenvironment (TME) therefore provides an appealing therapeutic target, particularly since it plays an essential role in promoting cancer growth, immune tolerance, and acquired resistance to many therapies. Recently, a variety of different TME-targeted gene therapy and armed oncolytic strategies have been explored, with particular success observed in strategies targeting the cancer stroma, reducing tumor vasculature, and repolarizing the immunosuppressive microenvironment. Herein, we review the progress of these TME-targeting approaches and try to highlight those showing the greatest promise.

4.
Nature ; 592(7853): 209-213, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33828318

RESUMO

The trapped-ion quantum charge-coupled device (QCCD) proposal1,2 lays out a blueprint for a universal quantum computer that uses mobile ions as qubits. Analogous to a charge-coupled device (CCD) camera, which stores and processes imaging information as movable electrical charges in coupled pixels, a QCCD computer stores quantum information in the internal state of electrically charged ions that are transported between different processing zones using dynamic electric fields. The promise of the QCCD architecture is to maintain the low error rates demonstrated in small trapped-ion experiments3-5 by limiting the quantum interactions to multiple small ion crystals, then physically splitting and rearranging the constituent ions of these crystals into new crystals, where further interactions occur. This approach leverages transport timescales that are fast relative to the coherence times of the qubits, the insensitivity of the qubit states of the ion to the electric fields used for transport, and the low crosstalk afforded by spatially separated crystals. However, engineering a machine capable of executing these operations across multiple interaction zones with low error introduces many difficulties, which have slowed progress in scaling this architecture to larger qubit numbers. Here we use a cryogenic surface trap to integrate all necessary elements of the QCCD architecture-a scalable trap design, parallel interaction zones and fast ion transport-into a programmable trapped-ion quantum computer that has a system performance consistent with the low error rates achieved in the individual ion crystals. We apply this approach to realize a teleported CNOT gate using mid-circuit measurement6, negligible crosstalk error and a quantum volume7 of 26 = 64. These results demonstrate that the QCCD architecture provides a viable path towards high-performance quantum computers.

5.
Br J Cancer ; 124(11): 1809-1819, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33742147

RESUMO

BACKGROUND: The radiosensitising effect of the poly(ADP-ribose) polymerase inhibitor olaparib on tumours has been reported. However, its effect on normal tissues in combination with radiation has not been well studied. Herein, we investigated the therapeutic index of olaparib combined with hemithoracic radiation in a urethane-induced mouse lung cancer model. METHODS: To assess tolerability, A/J mice were treated with olaparib plus whole thorax radiation (13 Gy), body weight changes were monitored and normal tissue effects were assessed by histology. In anti-tumour (intervention) studies, A/J mice were injected with urethane to induce lung tumours, and were then treated with olaparib alone, left thorax radiation alone or the combination of olaparib plus left thorax radiation at 8 weeks (early intervention) or 18 weeks (late intervention) after urethane injection. Anti-tumour efficacy and normal tissue effects were assessed by visual inspection, magnetic resonance imaging and histology. RESULTS: Enhanced body weight loss and oesophageal toxicity were observed when olaparib was combined with whole thorax but not hemithorax radiation. In both the early and late intervention studies, olaparib increased the anti-tumour effects of hemithoracic irradiation without increasing lung toxicity. CONCLUSIONS: The addition of olaparib increased the therapeutic index of hemithoracic radiation in a mouse model of lung cancer.

6.
Cancer Res ; 81(7): 1667-1680, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33558336

RESUMO

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/metabolismo , Carcinogênese/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Polimorfismo de Nucleotídeo Único/fisiologia , Prognóstico , Fatores de Risco , Transdução de Sinais/genética , Resultado do Tratamento
7.
Cancer Res ; 81(8): 2128-2141, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33509941

RESUMO

Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR-CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. SIGNIFICANCE: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2128/F1.large.jpg.


Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Replicação do DNA , Receptor IGF Tipo 1/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleotídeo Redutases/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Desoxirribonucleosídeos/metabolismo , Regulação para Baixo , Fibroblastos , Xenoenxertos , Histonas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Receptores Nucleares Órfãos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptor IGF Tipo 1/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Esferoides Celulares
8.
Cell Death Differ ; 28(4): 1333-1346, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33168956

RESUMO

T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

9.
Clin Transl Radiat Oncol ; 25: 61-66, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33072895

RESUMO

Lung cancer is the leading cause of cancer mortality worldwide and most patients are unsuitable for 'gold standard' treatment, which is concurrent chemoradiotherapy. CONCORDE is a platform study seeking to establish the toxicity profiles of multiple novel radiosensitisers targeting DNA repair proteins in patients treated with sequential chemoradiotherapy. Time-to-event continual reassessment will facilitate efficient dose-finding.

10.
Int J Cancer ; 147(5): 1474-1484, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32159854

RESUMO

Replication stress is a common feature of cancer cells. Ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signalling, a DNA damage repair (DDR) pathway, is activated by regions of single-stranded DNA (ssDNA) that can arise during replication stress. ATR delays cell cycle progression and prevents DNA replication fork collapse, which prohibits cell death and promotes proliferation. Several ATR inhibitors have been developed in order to restrain this protective mechanism in tumours. It is known, however, that despite other effective anticancer chemotherapy treatments targeting DDR pathways, resistance occurs. This begets the need to identify combination treatments to overcome resistance and prevent tumour cell growth. We conducted a drug screen to identify potential synergistic combination treatments by screening an ATR inhibitor (VE822) together with compounds from a bioactive small molecule library. The screen identified adefovir dipivoxil, a reverse transcriptase inhibitor and nucleoside analogue, as a compound that has increased cytotoxicity in the presence of ATR, but not ATM or DNA-dependant protein kinase (DNA-PK) inhibition. Here we demonstrate that adefovir dipivoxil induces DNA replication stress, activates ATR signalling and stalls cells in S phase. This simultaneous induction of replication stress and inhibition of ATR signalling lead to a marked increase in pan-nuclear γH2AX-positive cells, ssDNA accumulation and cell death, indicative of replication catastrophe.


Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Replicação do DNA/efeitos dos fármacos , Organofosfonatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Adenina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos
11.
J Clin Invest ; 130(1): 258-271, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581151

RESUMO

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.


Assuntos
Carcinoma Hepatocelular , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Neoplasias Hepáticas Experimentais , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Células MCF-7 , Camundongos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Biol Med ; 16(2): 234-246, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31516745

RESUMO

Objective: Patients with BRAF-mutant colorectal cancer (CRC) have a poor prognosis. Molecular status is not currently used to select which drug to use in combination with radiotherapy. Our aim was to identify drugs that radiosensitise CRC cells with known BRAF status. Methods: We screened 298 oncological drugs with and without ionising radiation in colorectal cancer cells isogenic for BRAF. Hits from rank product analysis were validated in a 16-cell line panel of human CRC cell lines, using clonogenic survival assays and xenograft models in vivo. Results: Most consistently identified hits were drugs targeting cell growth/proliferation or DNA damage repair. The most effective class of drugs that radiosensitised wild-type and mutant cell lines was PARP inhibitors. In clonogenic survival assays, talazoparib produced a radiation enhancement ratio of 1.9 in DLD1 (BRAF-wildtype) cells and 1.8 in RKO (BRAF V600E) cells. In DLD1 xenografts, talazoparib significantly increased the inhibitory effect of radiation on tumour growth (P ≤ 0.01). Conclusions: Our method for screening large drug libraries for radiosensitisation has identified PARP inhibitors as promising radiosensitisers of colorectal cancer cells with wild-type and mutant BRAF backgrounds.

13.
EMBO Mol Med ; 11(7): e9982, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31273933

RESUMO

Due to compromised homologous recombination (HR) repair, BRCA1- and BRCA2-mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA2-deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia (CLL). We establish that chlorambucil is specifically toxic to BRCA1/2-deficient cells, including olaparib-resistant and cisplatin-resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA2-deficient xenografts and inhibits growth of olaparib-resistant patient-derived tumour xenografts (PDTXs). We demonstrate that chlorambucil inflicts replication-associated DNA double-strand breaks (DSBs), similarly to cisplatin, and we identify ATR, FANCD2 and the SNM1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA2-compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA-deficient tumours.


Assuntos
Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Clorambucila/farmacologia , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores Ativados por Proliferador de Peroxissomo/antagonistas & inibidores , Ftalazinas/farmacologia , Piperazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cricetinae , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos SCID , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
EMBO J ; 38(13): e100532, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268606

RESUMO

Lung cancer remains the leading cause of cancer-related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4-hydroxylase alpha-2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem-like programming and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF-1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.


Assuntos
Adenocarcinoma de Pulmão/patologia , Metilação de DNA , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Prolil Hidroxilases/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
15.
PLoS One ; 14(2): e0212172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753240

RESUMO

The identification and measurement of tumours is a key requirement in the study of tumour development in mouse models of human cancer. Disease burden in autochthonous tumours, such as those arising in the lung, can be seen with non-invasive imaging, but cannot be accurately measured using standard tools such as callipers. Lung imaging is further complicated in the mouse due to instabilities arising from the rapid but cyclic cardio-respiratory motions, and the desire to use free-breathing animals. Female A/JOlaHsd mice were either injected (i.p.) with PBS 0.1ml/10g body weight (n = 6), or 10% urethane/PBS 0.1ml/10g body weight (n = 12) to induce autochthonous lung tumours. Cardio-respiratory synchronised bSSFP MRI, at 200 µm isotropic resolution was performed at 8, 13 and 18 weeks post induction. Images from the same mouse at different time points were aligned using threshold-based segmented masks of the lungs (ITK-SNAP and MATLAB) and tumour volumes were determined via threshold-based segmentation (ITK-SNAP).Scan times were routinely below 10 minutes and tumours were readily identifiable. Image registration allowed serial measurement of tumour volumes as small as 0.056 mm3. Repetitive imaging did not lead to mouse welfare issues. We have developed a motion desensitised scan that enables high sensitivity MRI to be performed with high throughput capability of greater than 4 mice/hour. Image segmentation and registration allows serial measurement of individual, small tumours. This allows fast and highly efficient volumetric lung tumour monitoring in cohorts of 30 mice per imaging time point. As a result, adaptive trial study designs can be achieved, optimizing experimental and welfare outcomes.


Assuntos
Neoplasias Pulmonares , Pulmão , Imageamento por Ressonância Magnética , Movimento (Física) , Neoplasias Experimentais , Carga Tumoral , Animais , Feminino , Pulmão/diagnóstico por imagem , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Camundongos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/patologia
16.
BMC Cancer ; 19(1): 102, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678647

RESUMO

BACKGROUND: Due to the lack of effective therapies and poor prognosis in TNBC (triple-negative breast cancer) patients, there is a strong need to develop effective novel targeted therapies for this subtype of breast cancer. Inhibition of heat shock protein 90 (HSP90), a conserved molecular chaperone that is involved in the regulation of oncogenic client proteins, has shown to be a promising therapeutic approach for TNBC. However, both intrinsic and acquired resistance to HSP90 inhibitors (HSP90i) limits their effectiveness in cancer patients. METHODS: We developed models of acquired resistance to HSP90i by prolonged exposure of TNBC cells to HSP90i (ganetespib) in vitro. Whole transcriptome profiling and a 328-compound bioactive small molecule screen were performed on these cells to identify the molecular basis of acquired resistance to HSP90i and potential therapeutic approaches to overcome resistance. RESULTS: Among a panel of seven TNBC cell lines, the most sensitive cell line (Hs578T) to HSP90i was selected as an in vitro model to investigate acquired resistance to HSP90i. Two independent HSP90i-resistant clones were successfully isolated which both showed absence of client proteins degradation, apoptosis induction and G2/M cell cycle arrest after treatment with HSP90i. Gene expression profiling and pathway enrichment analysis demonstrate significant activation of the survival JAK-STAT signalling pathway in both HSP90i-resistant clones, possibly through IL6 autocrine signalling. A bioactive small molecule screen also demonstrated that the HSP90i-resistant clones showed selective sensitivity to JAK2 inhibition. Inhibition of JAK and HSP90 caused higher induction of apoptosis, despite prior acquired resistance to HSP90i. CONCLUSIONS: Acquired resistance to HSP90i in TNBC cells is associated with an upregulated JAK-STAT signalling pathway. A combined inhibition of the JAK-STAT signalling pathway and HSP90 could overcome this resistance. The benefits of the combined therapy could be explored further for the development of effective targeted therapy in TNBC patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-6/metabolismo , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
17.
Oncotarget ; 9(17): 13666-13681, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568385

RESUMO

Cancer-associated fibroblasts (CAFs) are an emerging target for cancer therapy as they promote tumour growth and metastatic potential. However, CAF targeting is complicated by the lack of knowledge-based strategies aiming to selectively eliminate these cells. There is a growing body of evidence suggesting that a pro-inflammatory microenvironment (e.g. ROS and cytokines) promotes CAF formation during tumorigenesis, although the exact mechanisms involved remain unclear. In this study, we reveal that a prolonged pro-inflammatory stimulation causes a de facto deficiency in base excision repair, generating unrepaired DNA strand breaks and thereby triggering an ATF4-dependent reprogramming of normal fibroblasts into CAF-like cells. Based on the phenotype of in vitro-generated CAFs, we demonstrate that midostaurin, a clinically relevant compound, selectively eliminates CAF-like cells deficient in base excision repair and prevents their stimulatory role in cancer cell growth and migration.

18.
Int J Radiat Oncol Biol Phys ; 100(3): 767-775, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29413287

RESUMO

PURPOSE: Poly (ADP-ribose) polymerase (PARP) inhibitors have been shown to enhance the radiosensitivity of cancer cells in vitro in a replication-dependent manner. Their in vivo radiosensitizing effects have also been demonstrated in preclinical tumor models. However, whether PARP inhibition can enhance the response to radiation therapy in normal tissues has been largely neglected. We hypothesized that PARP inhibition might also potentiate the response of replicating normal tissues to radiation therapy. In this study, we examined the normal tissue response in mice treated with PARP inhibitors (BMN673 or AZD2281) in combination with thoracic irradiation. METHODS AND MATERIALS: The antitumor effects of fractionated irradiation (5 Gy × 4) in combination with BMN673 were evaluated in nude mice bearing established Calu-6 human lung cancer xenografts. The normal tissue response was evaluated in C57BL6 mice that were treated with BMN673 or AZD2281 combined with fractionated irradiation, 5 Gy × 4, delivered to the whole thorax. Body weight and histology of the esophagus and skin in the field of irradiation were examined. The DNA damage response in the esophagus and skin was assessed by γH2AX immunohistochemistry. RESULTS: While PARP inhibition enhanced irradiation-induced tumor growth inhibition in nude mice, it was also associated with significant body weight loss and increased damage to the esophagus and skin within the field of irradiation in C57BL6 mice. PARP inhibition compromised the repair of irradiation-induced DNA damage in the esophagus and skin. CONCLUSIONS: Although PARP inhibition enhanced the antitumor response to fractionated irradiation, it also enhanced the irradiation response in replicating normal tissues. Therefore, our study suggests that additional caution may be warranted in the clinical development of combination therapies using PARP inhibitors and radiation therapy, in particular where the field of irradiation includes the esophagus.


Assuntos
Esôfago/efeitos dos fármacos , Esôfago/efeitos da radiação , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Radiossensibilizantes/farmacologia , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Animais , Terapia Combinada/efeitos adversos , Dano ao DNA , Feminino , Xenoenxertos , Histonas/análise , Humanos , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Órgãos em Risco/efeitos da radiação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Lesões Experimentais por Radiação/patologia , Perda de Peso
19.
EMBO Mol Med ; 9(10): 1398-1414, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28729482

RESUMO

Maintenance of genome integrity requires the functional interplay between Fanconi anemia (FA) and homologous recombination (HR) repair pathways. Endogenous acetaldehyde, a product of cellular metabolism, is a potent source of DNA damage, particularly toxic to cells and mice lacking the FA protein FANCD2. Here, we investigate whether HR-compromised cells are sensitive to acetaldehyde, similarly to FANCD2-deficient cells. We demonstrate that inactivation of HR factors BRCA1, BRCA2, or RAD51 hypersensitizes cells to acetaldehyde treatment, in spite of the FA pathway being functional. Aldehyde dehydrogenases (ALDHs) play key roles in endogenous acetaldehyde detoxification, and their chemical inhibition leads to cellular acetaldehyde accumulation. We find that disulfiram (Antabuse), an ALDH2 inhibitor in widespread clinical use for the treatment of alcoholism, selectively eliminates BRCA1/2-deficient cells. Consistently, Aldh2 gene inactivation suppresses proliferation of HR-deficient mouse embryonic fibroblasts (MEFs) and human fibroblasts. Hypersensitivity of cells lacking BRCA2 to acetaldehyde stems from accumulation of toxic replication-associated DNA damage, leading to checkpoint activation, G2/M arrest, and cell death. Acetaldehyde-arrested replication forks require BRCA2 and FANCD2 for protection against MRE11-dependent degradation. Importantly, acetaldehyde specifically inhibits in vivo the growth of BRCA1/2-deficient tumors and ex vivo in patient-derived tumor xenograft cells (PDTCs), including those that are resistant to poly (ADP-ribose) polymerase (PARP) inhibitors. The work presented here therefore identifies acetaldehyde metabolism as a potential therapeutic target for the selective elimination of BRCA1/2-deficient cells and tumors.


Assuntos
Acetaldeído/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Fibroblastos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Rad51 Recombinase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Br J Cancer ; 117(4): 503-512, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28677687

RESUMO

BACKGROUND: Tumour-specific radiosensitising treatments may enhance the efficacy of radiotherapy without exacerbating side effects. In this study we determined the radiation response following depletion or inhibition of TOPK, a mitogen-activated protein kinase kinase family Ser/Thr protein kinase that is upregulated in many cancers. METHODS: Radiation response was studied in a wide range of cancer cell lines and normal cells using colony formation assays. The effect on cell cycle progression was assessed and the relationship between TOPK expression and therapeutic efficacy was studied in a cohort of 128 prostate cancer patients treated with radical radiotherapy. RESULTS: TOPK knockdown did not alter radiation response in normal tissues, but significantly enhanced radiosensitivity in cancer cells. This result was recapitulated in TOPK knockout cells and with the TOPK inhibitor, OTS964. TOPK depletion altered the G1/S transition and G2/M arrest in response to radiation. Furthermore, TOPK depletion increased chromosomal aberrations, multinucleation and apoptotic cell death after irradiation. These results suggest a possible role for TOPK in the radiation-induced DNA damage checkpoints. These findings have clinical relevance, as elevated TOPK protein expression was associated with poorer clinical outcomes in prostate cancer patients treated with radical radiotherapy. CONCLUSIONS: This study demonstrates that TOPK disruption may cause tumour-specific radiosensitisation in multiple different tumour types.


Assuntos
Pontos de Checagem do Ciclo Celular , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Recidiva Local de Neoplasia/metabolismo , Neoplasias da Próstata/radioterapia , Tolerância a Radiação , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Técnicas de Silenciamento de Genes , Células HCT116 , Células HeLa , Humanos , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Neoplasias da Próstata/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Taxa de Sobrevida
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