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1.
Cancers (Basel) ; 16(8)2024 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-38672668

RESUMO

The curative treatment of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC), utilizes radiation. The outcomes for HPV/p16-negative HNSCC are significantly worse than HPV/p16-positive tumors, with increased radiation resistance leading to worse locoregional recurrence (LRR) and ultimately death. This study analyzed the relationship between immune function and outcomes following radiation in HPV/p16-negative tumors to identify mechanisms of radiation resistance and prognostic immune biomarkers. A discovery cohort of 94 patients with HNSCC treated uniformly with surgery and adjuvant radiation and a validation cohort of 97 similarly treated patients were utilized. Tumor immune infiltrates were derived from RNAseq gene expression. The immune cell types significantly associated with outcomes in the discovery cohort were examined in the independent validation cohort. A positive association between high Th2 infiltration and LRR was identified in the discovery cohort and validated in the validation cohort. Tumor mutations in CREBBP/EP300 and CASP8 were significantly associated with Th2 infiltration. A pathway analysis of genes correlated with Th2 cells revealed the potential repression of the antitumor immune response and the activation of BRCA1-associated DNA damage repair in multiple cohorts. The Th2 infiltrates were enriched in the HPV/p16-negative HNSCC tumors and associated with LRR and mutations in CASP8, CREBBP/EP300, and pathways previously shown to impact the response to radiation.

2.
Clin Cancer Res ; 30(1): 187-197, 2024 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-37819945

RESUMO

PURPOSE: Radiation and platinum-based chemotherapy form the backbone of therapy in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). We have correlated focal adhesion kinase (FAK/PTK2) expression with radioresistance and worse outcomes in these patients. However, the importance of FAK in driving radioresistance and its effects on chemoresistance in these patients remains unclear. EXPERIMENTAL DESIGN: We performed an in vivo shRNA screen using targetable libraries to identify novel therapeutic sensitizers for radiation and chemotherapy. RESULTS: We identified FAK as an excellent target for both radio- and chemosensitization. Because TP53 is mutated in over 80% of HPV-negative HNSCC, we hypothesized that mutant TP53 may facilitate FAK-mediated therapy resistance. FAK inhibitor increased sensitivity to radiation, increased DNA damage, and repressed homologous recombination and nonhomologous end joining repair in mutant, but not wild-type, TP53 HPV-negative HNSCC cell lines. The mutant TP53 cisplatin-resistant cell line had increased FAK phosphorylation compared with wild-type, and FAK inhibition partially reversed cisplatin resistance. To validate these findings, we utilized an HNSCC cohort to show that FAK copy number and gene expression were associated with worse disease-free survival in mutant TP53, but not wild-type TP53, HPV-negative HNSCC tumors. CONCLUSIONS: FAK may represent a targetable therapeutic sensitizer linked to a known genomic marker of resistance.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/tratamento farmacológico , Infecções por Papillomavirus/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral
3.
Clin Cancer Res ; 29(23): 4958-4972, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37733794

RESUMO

PURPOSE: Ataxia-telangiectasia mutated (ATM) is the most frequently mutated DNA damage repair gene in non-small cell lung cancer (NSCLC). However, the molecular correlates of ATM mutations and their clinical implications have not been fully elucidated. EXPERIMENTAL DESIGN: Clinicopathologic and genomic data from 26,587 patients with NSCLC from MD Anderson, public databases, and a de-identified nationwide (US-based) NSCLC clinicogenomic database (CGDB) were used to assess the co-mutation landscape, protein expression, and mutational processes in ATM-mutant tumors. We used the CGDB to evaluate ATM-associated outcomes in patients treated with immune checkpoint inhibitors (ICI) with or without chemotherapy, and assessed the effect of ATM loss on STING signaling and chemotherapy sensitivity in preclinical models. RESULTS: Nonsynonymous mutations in ATM were observed in 11.2% of samples (2,980/26,587) and were significantly associated with mutations in KRAS, but mutually exclusive with EGFR (q < 0.1). KRAS mutational status constrained the ATM co-mutation landscape, with strong mutual exclusivity with TP53 and KEAP1 within KRAS-mutated samples. Those ATM mutations that co-occurred with TP53 were more likely to be missense mutations and associate with high mutational burden, suggestive of non-functional passenger mutations. In the CGDB cohort, dysfunctional ATM mutations associated with improved OS only in patients treated with ICI-chemotherapy, and not ICI alone. In vitro analyses demonstrated enhanced upregulation of STING signaling in ATM knockout cells with the addition of chemotherapy. CONCLUSIONS: ATM mutations define a distinct subset of NSCLC associated with KRAS mutations, increased TMB, decreased TP53 and EGFR co-occurrence, and potential increased sensitivity to ICIs in the context of DNA-damaging chemotherapy.


Assuntos
Ataxia Telangiectasia , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator 2 Relacionado a NF-E2/genética , Mutação , Receptores ErbB/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
4.
Cancer Cell ; 41(7): 1363-1380.e7, 2023 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-37327788

RESUMO

Inactivating STK11/LKB1 mutations are genomic drivers of primary resistance to immunotherapy in KRAS-mutated lung adenocarcinoma (LUAD), although the underlying mechanisms remain unelucidated. We find that LKB1 loss results in enhanced lactate production and secretion via the MCT4 transporter. Single-cell RNA profiling of murine models indicates that LKB1-deficient tumors have increased M2 macrophage polarization and hypofunctional T cells, effects that could be recapitulated by the addition of exogenous lactate and abrogated by MCT4 knockdown or therapeutic blockade of the lactate receptor GPR81 expressed on immune cells. Furthermore, MCT4 knockout reverses the resistance to PD-1 blockade induced by LKB1 loss in syngeneic murine models. Finally, tumors from STK11/LKB1 mutant LUAD patients demonstrate a similar phenotype of enhanced M2-macrophages polarization and hypofunctional T cells. These data provide evidence that lactate suppresses antitumor immunity and therapeutic targeting of this pathway is a promising strategy to reversing immunotherapy resistance in STK11/LKB1 mutant LUAD.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Animais , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/terapia , Adenocarcinoma de Pulmão/metabolismo , Lactatos/metabolismo , Lactatos/farmacologia , Lactatos/uso terapêutico , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/tratamento farmacológico , Macrófagos , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
5.
Cancer Res ; 82(5): 916-928, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34965932

RESUMO

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to "BRCAness" in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC. SIGNIFICANCE: In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA , DNA Viral/genética , Neoplasias de Cabeça e Pescoço/genética , Humanos , Papillomaviridae/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
6.
Nat Commun ; 12(1): 6340, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732714

RESUMO

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment.


Assuntos
Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Mutação com Ganho de Função , Histona Acetiltransferases/metabolismo , Recombinação Homóloga , Acetilação , Animais , Apoptose , Proteína BRCA1/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Masculino , Camundongos Nus , Mutação , Neoplasias/genética , Neoplasias/terapia , Domínios Proteicos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Int J Radiat Biol ; 97(8): 1121-1128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32073931

RESUMO

PURPOSE: Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however, normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status. MATERIALS AND METHODS: PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(-) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(-) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(-) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(-) cells. RESULTS: In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(-) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(-) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(-) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(-). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(-) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(-) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition. CONCLUSIONS: Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(-). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Dano ao DNA , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Indazóis/farmacologia , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Tolerância a Radiação/efeitos dos fármacos
8.
Head Neck ; 41(1): 46-55, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30561022

RESUMO

BACKGROUND: Photon (X-ray) radiotherapy (XRT) kills cells via DNA damage, however, how proton radiotherapy (PRT) causes cell death in head and neck squamous cell carcinoma (HNSCC) is unclear. We investigated mechanisms of HNSCC cell death after XRT versus PRT. METHODS: We assessed type of death in 2 human papillomavirus (HPV)-positive and two HPV-negative cell lines: necrosis and apoptosis (Annexin-V fluorescein isothiocyanate [FITC]); senescence (ß-galactosidase); and mitotic catastrophe (γ-tubulin and diamidino-phenylindole [DAPI]). RESULTS: The XRT-induced or PRT-induced cellular senescence and mitotic catastrophe in all cell lines studied suggested that PRT caused cell death to a greater extent than XRT. After PRT, mitotic catastrophe peaked in HPV-negative and HPV-positive cells at 48 and 72 hours, respectively. No obvious differences were noted in the extent of cell necrosis or apoptosis after XRT versus PRT. CONCLUSION: Under the conditions and in the cell lines reported here, mitotic catastrophe and senescence were the major types of cell death induced by XRT and PRT, and PRT may be more effective.


Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Fótons , Terapia com Prótons , Radioterapia/métodos , Apoptose/efeitos da radiação , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/virologia , Linhagem Celular Tumoral/efeitos da radiação , Senescência Celular/efeitos da radiação , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Mitose/efeitos da radiação , Necrose , Papillomaviridae/patogenicidade , Transdução de Sinais/efeitos da radiação
9.
Clin Cancer Res ; 24(3): 600-607, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29113987

RESUMO

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC).Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)-negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients.Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy.Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600-7. ©2017 AACR.


Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Histonas/metabolismo , Recombinação Homóloga , Humanos , Camundongos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Prognóstico , Tolerância a Radiação/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Clin Cancer Res ; 23(11): 2713-2722, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28476872

RESUMO

Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1-low expression group (P = 0.01, 1.9 × 10-3, and 9 × 10-4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10-4 and P = 4 × 10-4, respectively).Conclusions: Taken together, our data point to a targetable Axl-PI3 kinase-PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713-22. ©2017 AACR.


Assuntos
Antígeno B7-H1/genética , Carcinoma de Células Escamosas/radioterapia , Neoplasias de Cabeça e Pescoço/radioterapia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Idoso , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfócitos do Interstício Tumoral , Masculino , Pessoa de Meia-Idade , Papillomaviridae/patogenicidade , Proteômica , RNA Mensageiro/efeitos da radiação , Tolerância a Radiação/genética , Transdução de Sinais/efeitos da radiação , Receptor Tirosina Quinase Axl
11.
Head Neck ; 39(4): 708-715, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28039958

RESUMO

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal carcinomas response better to X-ray therapy (XRT) than HPV-negative disease. Whether HPV status influences the sensitivity of head and neck cancer cells to proton therapy or the relative biological effectiveness (RBE) of protons versus XRT is unknown. METHODS: Clonogenic survival was used to calculate the RBE; immunocytochemical analysis and neutral comet assay were used to evaluate unrepaired DNA double-strand breaks. RESULTS: HPV-positive cells were more sensitive to protons and the unrepaired double-strand breaks were more numerous in HPV-positive cells than in HPV-negative cells (p < .001). Protons killed more cells than did XRT at all fraction sizes (all RBEs > 1.06). Cell line type and radiation fraction size influenced the RBE. CONCLUSION: HPV-positive cells were more sensitive to protons than HPV-negative cells maybe through the effects of HPV on DNA damage and repair. The RBE for protons depends more on cell type and fraction size than on HPV status. © 2016 Wiley Periodicals, Inc. Head Neck 39: 708-715, 2017.


Assuntos
Neoplasias Orofaríngeas/radioterapia , Papillomaviridae/isolamento & purificação , Terapia com Prótons/métodos , Eficiência Biológica Relativa , Células Tumorais Cultivadas/efeitos da radiação , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/virologia , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Tolerância a Radiação , Radioterapia/métodos , Valores de Referência , Carcinoma de Células Escamosas de Cabeça e Pescoço
12.
Bioelectromagnetics ; 37(7): 471-80, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27434783

RESUMO

Devices that combine magnetic resonance imaging with linear accelerators (MRL) represent a novel tool for MR-guided radiotherapy. However, whether magnetic fields (MFs) generated by these devices affect the radiosensitivity of tumors is unknown. We investigated the influence of a 1.5-T MF on cell viability and radioresponse of human solid tumors. Human head/neck cancer and lung cancer cells were exposed to single or fractionated 6-MV X-ray radiation; effects of the MF on cell viability were determined by cell plating efficiency and on radioresponsiveness by clonogenic cell survival. Doses needed to reduce the fraction of surviving cells to 37% of the initial value (D0s) were calculated for multiple exposures to MF and radiation. Results were analyzed using Student's t-tests. Cell viability was no different after single or multiple exposures to MRL than after exposure to a conventional linear accelerator (Linac, without MR-generated MF) in 12 of 15 experiments (all P > 0.05). Single or multiple exposures to MF had no influence on cell radioresponse (all P > 0.05). Cells treated up to four times with an MRL or a Linac further showed no changes in D0s with MF versus without MF (all P > 0.05). In conclusion, MF within the MRL does not seem to affect in vitro tumor radioresponsiveness as compared with a conventional Linac. Bioelectromagnetics. 37:471-480, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Imageamento por Ressonância Magnética/instrumentação , Aceleradores de Partículas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Humanos , Tolerância a Radiação , Radiometria , Raios X
13.
Clin Cancer Res ; 22(18): 4643-50, 2016 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-27036135

RESUMO

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC. EXPERIMENTAL DESIGN: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC. RESULTS: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2-M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas. CONCLUSIONS: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643-50. ©2016 AACR.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteoma , Proteômica , Tolerância a Radiação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Dano ao DNA , Quinase 1 de Adesão Focal/genética , Dosagem de Genes , Expressão Gênica , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Proteômica/métodos , Tolerância a Radiação/genética , Resultado do Tratamento
14.
Cancer Med ; 4(1): 65-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25355701

RESUMO

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Modelos Animais de Doenças , Receptores ErbB/genética , Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Multimerização Proteica/efeitos dos fármacos , Radiação , Tolerância a Radiação/efeitos dos fármacos , Radioterapia , Receptor IGF Tipo 1/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Anticancer Res ; 34(12): 6981-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25503124

RESUMO

BACKGROUND: Positive transcription elongation factor-b (P-TEFb) is a complex containing CDK9 and a cyclin (T1, T2 or K). The effect of inhibition of P-TEFb by 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB) on cell radiosensitivity and the underlying mechanisms were investigated. MATERIALS AND METHODS: Six human cancer cell lines were subjected to (3)H-uridine incorporation, cell viability and clonogenic cell survival assays; cell-cycle redistribution and apoptosis assay; western blots and nuclear 53BP1 foci analysis after exposing the cells to DRB with/without γ-radiation. RESULTS: DRB suppressed colony formation and enhanced radiosensitivity of all cell lines. DRB caused a further increase in radiation-induced apoptosis and cell-cycle redistribution depending on p53 status. DRB prolonged the presence of radiation-induced nuclear p53 binding protein-1 (53BP1) foci and suppressed the expression of sirtuin-1 (SIRT1) and casein kinase 2-alpha (CK2α), suggesting an inhibition of DNA repair processes. CONCLUSION: Our findings indicate that DRB has the potential to increase the efficacy of radiotherapy and warrants further investigation using in vivo tumor models.


Assuntos
Caseína Quinase II/antagonistas & inibidores , Diclororribofuranosilbenzimidazol/farmacologia , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Sirtuína 1/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Caseína Quinase II/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos da radiação , Sirtuína 1/biossíntese , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
16.
Radiother Oncol ; 105(2): 241-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23010482

RESUMO

BACKGROUND AND PURPOSE: Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect. MATERIALS AND METHODS: Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells. RESULTS: All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity. CONCLUSIONS: Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.


Assuntos
Carcinoma de Células Escamosas/radioterapia , Núcleo Celular/metabolismo , Reparo do DNA/efeitos dos fármacos , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Tiazóis/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteína Tirosina Quinase CSK , Ciclo Celular , Dasatinibe , Receptores ErbB/análise , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Quinases da Família src/análise , Quinases da Família src/metabolismo
17.
J Thorac Oncol ; 7(8): 1211-7, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22617250

RESUMO

INTRODUCTION: The radiation doses used to treat unresectable lung cancer are often limited by the proximity of normal tissues. Overexpression of c-Met, a receptor tyrosine kinase, occurs in about half of non-small-cell lung cancers (NSCLCs) and has been associated with resistance to radiation therapy and poor patient survival. We hypothesized that inhibiting c-Met would increase the sensitivity of NSCLC cells to radiation, enhancing the therapeutic ratio, which may potentially translate into improved local control. METHODS: We tested the radiosensitivity of two high-c-Met-expressing NSCLC lines, EBC-1 and H1993, and two low-c-Met-expressing lines, A549 and H460, with and without the small-molecule c-Met inhibitor MK-8033. Proliferation and protein expression were measured with clonogenic survival assays and Western blotting, respectively. γ-H2AX levels were evaluated by immunofluorescence staining. RESULTS: MK-8033 radiosensitized the high-c-Met-expressing EBC-1 and H1993 cells but not the low-c-Met-expressing cell lines A549 and H460. However, irradiation of A549 and H460 cells increased the expression of c-Met protein at 30 minutes after the irradiation. Subsequent targeting of this up-regulated c-Met by using MK-8033 followed by a second radiation dose reduced the clonogenic survival of both A549 and H460 cells. MK-8033 reduced the levels of radiation-induced phosphorylated (activated) c-Met in A549 cells. CONCLUSIONS: These results suggest that inhibition of c-Met could be an effective strategy to radiosensitize NSCLC tumors with high basal c-Met expression or tumors that acquired resistance to radiation because of up-regulation of c-Met.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Neoplasias Pulmonares/radioterapia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/uso terapêutico , Apoptose/efeitos da radiação , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Radioisótopos de Césio , Imunofluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaio Tumoral de Célula-Tronco
18.
Anticancer Res ; 31(6): 2141-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21737634

RESUMO

AIM: To assess radiosensitzing potential of huachansu (HCS) and delineate the underlying mechanisms. MATERIALS AND METHODS: Lung cancer cell lines were exposed to HCS, radiation or both and subjected to survival assays, Western blots, apoptosis assay and immunocytochemical analysis. RESULTS: HCS suppressed the viability of all three lung lines tested and enhanced radiosensitivity of H460 and A549 (wild-type p53) only with no effect on H1299 (p53 null) cells. HCS prolonged the presence of radiation-induced γH2AX foci and increased radiation-induced apoptosis. Western blots showed that HCS increased cleaved caspase-3 and cleaved poly-(ADP-ribose) polymerase (PARP) levels, as well as reducing BCL-2 and p53 protein levels in H460 cells. CONCLUSION: HCS-enhanced radiosensitivity of human lung cancer lines appeared to be p53-dependent. Inhibition of DNA repair and increase in radiation-induced apoptosis may have served as underlying mechanisms. These data suggest that HCS may have potential to improve the efficacy of radiotherapy.


Assuntos
Venenos de Anfíbios/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Radiossensibilizantes/farmacologia , Venenos de Anfíbios/química , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/farmacologia , Linhagem Celular Tumoral , Terapia Combinada , Dano ao DNA , DNA de Neoplasias/efeitos da radiação , Relação Dose-Resposta a Droga , Histonas/biossíntese , Histonas/genética , Humanos , Neoplasias Pulmonares/genética
19.
Clin Cancer Res ; 17(7): 2035-43, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21349997

RESUMO

PURPOSE: Radiotherapy plays an integral role in the treatment of head and neck squamous cell carcinoma (HNSCC). Although proteins involved in DNA repair may predict HNSCC response to radiotherapy, none has been validated in this context. We examined whether differential expression of double-strand DNA break (DSB) repair proteins in HNSCC, the chief mediators of DNA repair following irradiation, predict for treatment outcomes. EXPERIMENTAL DESIGN: Archival HNSCC tumor specimens (n = 89) were assembled onto a tissue microarray and stained with antibodies raised against 38 biomarkers. The biomarker set was enriched for proteins involved in DSB repair, in addition to established mechanistic markers of radioresistance. Staining was correlated with treatment response and survival alongside established clinical and pathologic covariates. Results were validated in an independent intramural cohort (n = 34). RESULTS: Ku80, a key mediator of DSB repair, correlated most closely with clinical outcomes. Ku80 was overexpressed in half of all tumors, and its expression was independent of all other covariates examined. Ku80 overexpression was an independent predictor for both locoregional failure and mortality following radiotherapy (P < 0.01). The predictive power of Ku80 overexpression was confined largely to HPV-negative HNSCC, where it conferred a nine-fold greater risk of death at two years. CONCLUSIONS: Ku80 overexpression is a common feature of HNSCC, and is a candidate DNA repair-related biomarker for radiation treatment failure and death, particularly in patients with high-risk HPV-negative disease. It is a promising, mechanistically rational biomarker to select individual HPV-negative HNSCC patients for strategies to intensify treatment.


Assuntos
Carcinoma de Células Escamosas/radioterapia , DNA Helicases/metabolismo , Reparo do DNA , Neoplasias de Cabeça e Pescoço/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alphapapillomavirus , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/virologia , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Autoantígeno Ku , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Análise Serial de Proteínas , Células Tumorais Cultivadas
20.
Clin Cancer Res ; 17(7): 1815-27, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21350000

RESUMO

PURPOSE: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data. RESULTS: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo. CONCLUSION: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/patologia , Piperidinas/farmacologia , Quinazolinas/farmacologia , Radiossensibilizantes/farmacologia , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Tolerância a Radiação/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
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