ABSTRACT
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.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Papillomavirus Infections , Carcinoma, Squamous Cell/pathology , Carrier Proteins , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , DNA Damage , DNA, Viral/genetics , Head and Neck Neoplasms/genetics , Humans , Papillomaviridae/genetics , Signal Transduction , Squamous Cell Carcinoma of Head and Neck/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Specific Peptidase 7/metabolismABSTRACT
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.
Subject(s)
Cell Cycle Checkpoints/drug effects , DNA Damage , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Cell Cycle Checkpoints/radiation effects , Cell Line, Tumor , Humans , Indazoles/pharmacology , Piperidines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidinones/pharmacology , Radiation Tolerance/drug effectsABSTRACT
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.
Subject(s)
Biomarkers, Tumor , Head and Neck Neoplasms/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/metabolism , Animals , Disease Models, Animal , Dose-Response Relationship, Radiation , Head and Neck Neoplasms/etiology , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/therapy , Histones/metabolism , Homologous Recombination , Humans , Mice , Papillomavirus Infections/complications , Papillomavirus Infections/virology , Prognosis , Radiation Tolerance/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Ubiquitination , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
B7-H1 Antigen/genetics , Carcinoma, Squamous Cell/radiotherapy , Head and Neck Neoplasms/radiotherapy , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Aged , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/radiation effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Lymphocytes, Tumor-Infiltrating , Male , Middle Aged , Papillomaviridae/pathogenicity , Proteomics , RNA, Messenger/radiation effects , Radiation Tolerance/genetics , Signal Transduction/radiation effects , Axl Receptor Tyrosine KinaseABSTRACT
Platinum-based (Pt) chemotherapy is broadly utilized in the treatment of cancer. Development of more effective, personalized treatment strategies require identification of novel biomarkers of treatment response. Since Pt compounds are inactivated through cellular metabolic activity, we hypothesized that metabolic interrogation can predict the effectiveness of Pt chemotherapy in a pre-clinical model of head and neck squamous cell carcinoma (HNSCC).We tested the effects of cisplatin (CDDP) and carboplatin (CBP) on DNA damage, activation of cellular death cascades and tumor cell metabolism, specifically lactate production. Pt compounds induced an acute dose-dependent, transient drop in lactate generation in vitro, which correlated with effects on DNA damage and cell death. Neutralization of free radical stress abrogated these effects. The magnitude of this effect on lactate production correlated with the differential sensitivity of HNSCC cells to Pt compounds (CDDP vs CBP) and p53-driven Pt chemotherapy resistance. Using dual flank xenograft tumors, we demonstrated that Pt-driven effects on lactate levels correlate with effects on tumor growth delay in a dose-dependent manner and that lactate levels can define the temporal profile of Pt chemotherapy-induced metabolic stress. Lactate interrogation also predicted doxorubicin effects on cell death in both solid tumor (HNSCC) and acute myelogenous leukemia (AML) cell lines.Real-time metabolic interrogation of acute changes in cell and tumor lactate levels reflects chemotherapy effects on DNA damage, cell death and tumor growth delay. We have identified a real-time biomarker of chemotherapy effectiveness which can be used to develop adaptive, iterative and personalized treatment regimens against a variety of solid and hematopoietic malignancies.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/metabolism , Drug Resistance, Neoplasm/physiology , Head and Neck Neoplasms/metabolism , Lactic Acid/analysis , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Female , Humans , Lactic Acid/metabolism , Mice , Mice, Nude , Platinum Compounds/pharmacology , Squamous Cell Carcinoma of Head and Neck , Xenograft Model Antitumor AssaysABSTRACT
Radiotherapy is commonly used to treat a variety of solid tumors but improvements in the therapeutic ratio are sorely needed. The aim of this study was to assess the Chk1 kinase inhibitor, MK-8776, for its ability to radiosensitize human tumor cells. Cells derived from NSCLC and HNSCC cancers were tested for radiosensitization by MK-8776. The ability of MK-8776 to abrogate the radiation-induced G2 block was determined using flow cytometry. Effects on repair of radiation-induced DNA double strand breaks (DSBs) were determined on the basis of rad51, γ-H2AX and 53BP1 foci. Clonogenic survival analyses indicated that MK-8776 radiosensitized p53-defective tumor cells but not lines with wild-type p53. Abrogation of the G2 block was evident in both p53-defective cells and p53 wild-type lines indicating no correlation with radiosensitization. However, only p53-defective cells entered mitosis harboring unrepaired DSBs. MK-8776 appeared to inhibit repair of radiation-induced DSBs at early times after irradiation. A comparison of MK-8776 to the wee1 inhibitor, MK-1775, suggested both similarities and differences in their activities. In conclusion, MK-8776 radiosensitizes tumor cells by mechanisms that include abrogation of the G2 block and inhibition of DSB repair. Our findings support the clinical evaluation of MK-8776 in combination with radiation.
Subject(s)
Checkpoint Kinase 1/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Radiation-Sensitizing Agents/pharmacology , Tumor Suppressor Protein p53/physiology , Cell Line, Tumor , DNA Breaks, Double-Stranded , G2 Phase/radiation effects , Histones/analysis , Humans , Pyrimidinones , Tumor Suppressor Protein p53/geneticsABSTRACT
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.
Subject(s)
Focal Adhesion Kinase 1/metabolism , Head and Neck Neoplasms/metabolism , Proteome , Proteomics , Radiation Tolerance , Biomarkers, Tumor , Cell Line, Tumor , DNA Damage , Focal Adhesion Kinase 1/genetics , Gene Dosage , Gene Expression , Head and Neck Neoplasms/etiology , Head and Neck Neoplasms/radiotherapy , Humans , Proteomics/methods , Radiation Tolerance/genetics , Treatment OutcomeABSTRACT
Repurposing agents approved for other indications to radiosensitize tumors may be advantageous. The study by Hill and colleagues utilizes nelfinavir, an HIV protease inhibitor (PI), in combination with radiotherapy in rectal cancer in a prospective study. This combination may improve tumor perfusion and regression compared with radiotherapy alone.
Subject(s)
Nelfinavir/therapeutic use , Radiation-Sensitizing Agents/therapeutic use , Rectal Neoplasms/drug therapy , Rectal Neoplasms/radiotherapy , Combined Modality Therapy , Humans , Magnetic Resonance Imaging , Radiation Dose Hypofractionation , Rectal Neoplasms/diagnosis , Tomography, X-Ray Computed , Treatment OutcomeABSTRACT
Ionizing radiation is the primary nonsurgical treatment modality for solid tumors. Its effectiveness is impacted by temporal constraints such as fractionation, hypoxia, and development of radioresistant clones. Biomarkers of acute radiation response are essential to developing more effective clinical algorithms. We hypothesized that acute perturbations in tumor lactate levels act as a surrogate marker of radiation response. In vitro experiments were carried out using validated human-derived cell lines from three histologies: anaplastic thyroid carcinoma (ATC), head and neck squamous cell carcinoma (HNSCC), and papillary thyroid carcinoma (PTC). Cellular metabolic activity was measured using standard biochemical assays. In vivo validation was performed using both an orthotopic and a flank derivative of a previously established ATC xenograft murine model. Irradiation of cells and tumors triggered a rapid, dose-dependent, transient decrease in lactate levels that was reversed by free radical scavengers. Acute lactate perturbations following irradiation could identify hypoxic conditions and correlated with hypoxia-induced radioresistance. Mutant TP53 cells and cells in which p53 activity was abrogated (shRNA) demonstrated a blunted lactate response to irradiation, consistent with a radioresistant phenotype. Lactate measurements therefore rapidly detected both induced (i.e., hypoxia) and intrinsic (i.e., mutTP53-driven) radioresistance. We conclude that lactate is a quantitative biomarker of acute genotoxic stress, with a temporal resolution that can inform clinical decision making. Combined with the spatial resolution of newly developed metabolic imaging platforms, this biomarker could lead to the development of truly individualized treatment strategies.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/metabolism , Carcinoma/metabolism , Head and Neck Neoplasms/metabolism , Lactic Acid/metabolism , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Neoplasms/metabolism , Animals , Carcinoma/genetics , Carcinoma/radiotherapy , Carcinoma, Papillary , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/radiotherapy , Cell Line, Tumor , DNA Damage/radiation effects , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/radiotherapy , Humans , Mice , Radiation Tolerance/genetics , Radiation, Ionizing , Squamous Cell Carcinoma of Head and Neck , Thyroid Cancer, Papillary , Thyroid Carcinoma, Anaplastic/genetics , Thyroid Carcinoma, Anaplastic/radiotherapy , Thyroid Neoplasms/genetics , Thyroid Neoplasms/radiotherapy , Tumor Suppressor Protein p53/geneticsABSTRACT
Although cisplatin has played a role in "standard-of-care" multimodality therapy for patients with advanced squamous cell carcinoma of the head and neck (HNSCC), the rate of treatment failure remains particularly high for patients receiving cisplatin whose tumors have mutations in the TP53 gene. We found that cisplatin treatment of HNSCC cells with mutant TP53 leads to arrest of cells in the G2 phase of the cell cycle, leading us to hypothesize that the wee-1 kinase inhibitor MK-1775 would abrogate the cisplatin-induced G2 block and thereby sensitize isogenic HNSCC cells with mutant TP53 or lacking p53 expression to cisplatin. We tested this hypothesis using clonogenic survival assays, flow cytometry, and in vivo tumor growth delay experiments with an orthotopic nude mouse model of oral tongue cancer. We also used a novel TP53 mutation classification scheme to identify which TP53 mutations are associated with limited tumor responses to cisplatin treatment. Clonogenic survival analyses indicate that nanomolar concentration of MK-1775 sensitizes HNSCC cells with high-risk mutant p53 to cisplatin. Consistent with its ability to chemosensitize, MK-1775 abrogated the cisplatin-induced G2 block in p53-defective cells leading to mitotic arrest associated with a senescence-like phenotype. Furthermore, MK-1775 enhanced the efficacy of cisplatin in vivo in tumors harboring TP53 mutations. These results indicate that HNSCC cells expressing high-risk p53 mutations are significantly sensitized to cisplatin therapy by the selective wee-1 kinase inhibitor, supporting the clinical evaluation of MK-1775 in combination with cisplatin for the treatment of patients with TP53 mutant HNSCC.
Subject(s)
Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Head and Neck Neoplasms/genetics , Mutation/genetics , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cellular Senescence/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Damage , Drug Synergism , Humans , Mice, Nude , Mitosis/drug effects , Nuclear Proteins/metabolism , Phenotype , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Pyrimidinones , Reactive Oxygen Species/metabolismABSTRACT
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.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , ErbB Receptors/metabolism , Head and Neck Neoplasms/metabolism , Receptor, IGF Type 1/metabolism , Signal Transduction/drug effects , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cetuximab , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , Disease Models, Animal , ErbB Receptors/genetics , Gene Expression , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/therapy , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Protein Multimerization/drug effects , Radiation , Radiation Tolerance/drug effects , Radiotherapy , Receptor, IGF Type 1/genetics , Tumor Burden/drug effects , Tumor Burden/radiation effects , Tumor Suppressor p53-Binding Protein 1 , Xenograft Model Antitumor AssaysABSTRACT
The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H2AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H2O2 and converted H2O2-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.
Subject(s)
Indazoles/pharmacology , Piperidines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Radiation-Sensitizing Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , DNA Breaks, Single-Stranded/drug effects , DNA Breaks, Single-Stranded/radiation effects , DNA Repair/drug effects , DNA Repair/radiation effects , Female , Histones/metabolism , Humans , Hydrogen Peroxide/pharmacology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Microscopy, Fluorescence , Oxidants/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Rad51 Recombinase/metabolism , Tumor Stem Cell Assay , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.
Subject(s)
Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/therapy , MicroRNAs/metabolism , Radiation-Sensitizing Agents/metabolism , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Nude , MicroRNAs/genetics , Neoplasm Transplantation , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Signal Transduction/radiation effectsABSTRACT
PURPOSE: To evaluate the feasibility of administering sunitinib in combination with androgen deprivation therapy and external-beam intensity modulated radiation therapy (XRT) in patients with localized high-risk prostate cancer. METHODS AND MATERIALS: Seventeen men with localized adenocarcinoma of the prostate with cT2c-cT4 or Gleason 8-10 or prostate-specific antigen >20 ng/mL received initial androgen deprivation (leuprolide 22.5 mg every 12 weeks plus oral bicalutamide 50 mg daily) for 4-8 weeks before oral sunitinib 12.5, 25, or 37.5 mg daily for 4 weeks as lead-in, then concurrently with and 4 weeks after XRT (75.6 Gy in 42 fractions to prostate and seminal vesicles). A 3+3 sequential dose-escalation design was used to assess the frequency of dose-limiting toxicity (DLT) and establish a maximal tolerated dose of sunitinib. RESULTS: Sunitinib at 12.5- and 25-mg dose levels was well tolerated. The first 4 patients enrolled at 37.5 mg experienced a DLT during lead-in, and a drug interaction between sunitinib and bicalutamide was suspected. The protocol was revised and concurrent bicalutamide omitted. Of the next 3 patients enrolled at 37.5 mg, 2 of 3 receiving concurrent therapy experienced DLTs during radiation: grade 3 diarrhea and grade 3 proctitis, respectively. Only 1 of 7 patients completed sunitinib at 37.5 mg daily, whereas 3 of 3 patients (25 mg as starting dose) and 3 of 4 patients (25 mg as reduced dose) completed therapy. CONCLUSIONS: The feasibility of combined vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor (PDGFR) inhibitor therapy, androgen deprivation, and radiation therapy for prostate cancer was established. Using a daily dosing regimen with lead-in, concurrent, and post-XRT therapy, the recommended phase 2 dose of sunitinib is 25 mg daily.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/radiotherapy , Androgen Antagonists/administration & dosage , Antineoplastic Agents/administration & dosage , Indoles/administration & dosage , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/radiotherapy , Pyrroles/administration & dosage , Adenocarcinoma/pathology , Aged , Aged, 80 and over , Androgen Antagonists/adverse effects , Anilides/administration & dosage , Anilides/adverse effects , Antineoplastic Agents/adverse effects , Combined Modality Therapy/methods , Drug Interactions , Feasibility Studies , Goserelin/administration & dosage , Humans , Indoles/adverse effects , Leuprolide/therapeutic use , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm Grading , Neoplasm Staging , Nitriles/administration & dosage , Nitriles/adverse effects , Prostate/radiation effects , Prostate-Specific Antigen/blood , Prostatic Neoplasms/pathology , Pyrroles/adverse effects , Radiotherapy, Intensity-Modulated/adverse effects , Radiotherapy, Intensity-Modulated/methods , Seminal Vesicles/radiation effects , Sunitinib , Tosyl Compounds/administration & dosage , Tosyl Compounds/adverse effectsABSTRACT
BACKGROUND: Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. METHODS: The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib's ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. RESULTS: Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. CONCLUSIONS: We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro. The fact that tumor growth delay was enhanced when sunitinib was given after radiotherapy was completed suggests that sunitinib may be acting on the irradiated tumor stroma and suppressing its ability to sustain regrowth of the irradiated tumor. Based on these preclinical findings, we suggest that the combination of sunitinib and radiation for the treatment of prostate cancer deserves further development.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Indoles/pharmacology , Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Radiation-Sensitizing Agents/pharmacology , Xenograft Model Antitumor Assays , Animals , Blotting, Western , Fluorescent Antibody Technique , Humans , Male , Mice , Mice, Nude , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Proto-Oncogene Proteins c-kit/metabolism , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/metabolism , Sunitinib , Tumor Cells, Cultured , Tumor Stem Cell Assay , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
PURPOSE: Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a classification of TP53 mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and radiation sensitivity. EXPERIMENTAL DESIGN: Seventy-four patients with HNSCC treated with surgery and postoperative radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and the in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using short hairpin RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and reactive oxygen species (ROS) assays were carried out. The effect of the drug metformin on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an orthotopic xenograft model. RESULTS: Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly predicted LRR (P = 0.03). Cell lines with disruptive mutations were significantly more radioresistant (P < 0.05). Expression of disruptive TP53 mutations significantly decreased radiation-induced senescence, as measured by SA-ß-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence. Examination of our patient cohort showed that LRR was decreased in patients taking metformin. CONCLUSIONS: Disruptive TP53 mutations in HNSCC tumors predicts for LRR, because of increased radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC with disruptive TP53, presaging the possibility of personalizing HNSCC treatment.
Subject(s)
Aging/radiation effects , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Mutation/genetics , Neoplasm Recurrence, Local/diagnosis , Radiation Tolerance/genetics , Tumor Suppressor Protein p53/genetics , Aging/genetics , Animals , Apoptosis/genetics , Apoptosis/radiation effects , Blotting, Western , Carcinoma, Squamous Cell/mortality , Carcinoma, Squamous Cell/radiotherapy , Fluorescent Antibody Technique , Head and Neck Neoplasms/mortality , Head and Neck Neoplasms/radiotherapy , Humans , Immunoenzyme Techniques , Mice , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/mortality , Reactive Oxygen Species/metabolism , Survival Rate , Treatment Failure , Tumor Cells, CulturedABSTRACT
PURPOSE: Radiotherapy is commonly used to treat a variety of solid tumors. However, improvements in the therapeutic ratio for several disease sites are sorely needed, leading us to assess molecularly targeted therapeutics as radiosensitizers. The aim of this study was to assess the wee1 kinase inhibitor, MK-1775, for its ability to radiosensitize human tumor cells. EXPERIMENTAL DESIGN: Human tumor cells derived from lung, breast, and prostate cancers were tested for radiosensitization by MK-1775 using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of MK-1775 to abrogate the radiation-induced G2 block, thereby allowing cells harboring DNA lesions to prematurely progress into mitosis, was determined using flow cytometry and detection of γ-H2AX foci. The in vivo efficacy of the combination of MK-1775 and radiation was assessed by tumor growth delay experiments using a human lung cancer cell line growing as a xenograft tumor in nude mice. RESULTS: Clonogenic survival analyses indicated that nanomolar concentrations of MK-1775 radiosensitized p53-defective human lung, breast, and prostate cancer cells but not similar lines with wild-type p53. Consistent with its ability to radiosensitize, MK-1775 abrogated the radiation-induced G2 block in p53-defective cells but not in p53 wild-type lines. MK-1775 also significantly enhanced the antitumor efficacy of radiation in vivo as shown in tumor growth delay studies, again for p53-defective tumors. CONCLUSIONS: These results indicate that p53-defective human tumor cells are significantly radiosensitized by the potent and selective wee1 kinase inhibitor, MK-1775, in both the in vitro and in vivo settings. Taken together, our findings strongly support the clinical evaluation of MK-1775 in combination with radiation.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Neoplasms , Nuclear Proteins/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Radiation-Sensitizing Agents/pharmacology , Tumor Suppressor Protein p53/deficiency , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/radiotherapy , Cell Line, Tumor , Combined Modality Therapy , Female , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/radiation effects , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/radiotherapy , Male , Mice , Mice, Nude , Neoplasms/drug therapy , Neoplasms/pathology , Neoplasms/radiotherapy , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/radiotherapy , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyrimidinones , Transplantation, Heterologous , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor AssaysABSTRACT
Radiotherapy plays a crucial role in the treatment of many malignancies; however, locoregional disease progression remains a critical problem. This has stimulated laboratory research into understanding the basis for tumor cell resistance to radiation and the development of strategies for overcoming such resistance. We know that some cell signaling pathways that respond to normal growth factors are abnormally activated in human cancer and that these pathways also invoke cell survival mechanisms that lead to resistance to radiation. For example, abnormal activation of the epidermal growth factor receptor (EGFR) promotes unregulated growth and is believed to contribute to clinical radiation resistance. Molecular blockade of EGFR signaling is an attractive strategy for enhancing the cytotoxic effects of radiotherapy and, as shown in numerous reports, the radiosensitizing effects of EGFR antagonists correlate with a suppression of the ability of the cells to repair radiation-induced DNA double strand breaks (DSBs). The molecular connection between the EGFR and its governance of DNA repair capacity appears to be mediated by one or more signaling pathways downstream of this receptor. The purpose of this review is to highlight what is currently known regarding EGFR signaling and the processes responsible for repairing radiation-induced DNA lesions that would explain the radiosensitizing effects of EGFR antagonists.
Subject(s)
DNA Repair/physiology , ErbB Receptors/radiation effects , Radiobiology , Signal Transduction/radiation effects , Animals , Cell Line, Tumor/radiation effects , DNA Breaks, Double-Stranded/radiation effects , DNA Damage/radiation effects , DNA Repair/radiation effects , ErbB Receptors/metabolism , Humans , Neoplasms/genetics , Neoplasms/radiotherapy , Radiation Injuries/prevention & control , Radiation ToleranceSubject(s)
Cell Cycle Proteins/metabolism , DNA Repair/genetics , Genomic Instability/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Humans , PTEN Phosphohydrolase/genetics , Signal Transduction/physiologyABSTRACT
PURPOSE: This study assessed the role of oxidative stress and loss of glutathione in ABT-737-induced apoptosis. METHODS: Jurkat human acute lymphocytic leukemia cells and HeLa cells transfected with a tet-regulated Bcl-2 expression system were treated with ABT-737 or its less active stereoisomer. GSH concentrations, intracellular reactive oxygen species (ROS), caspase activation and apoptotic DNA fragmentation were measured. RESULTS: ABT-737 induced oxidative stress through decreased GSH and increased intracellular hydrogen peroxide and superoxide levels. Apoptotic DNA fragmentation and caspase activation were the consequences of this oxidative stress. Combining ABT-737 with ROS-inducing agents such as adaphostin or etoposide enhanced cell death. CONCLUSIONS: These results demonstrate that inhibition of Bcl-2 causes a loss of GSH, an increase in ROS, caspase activation and subsequent apoptosis. Clinically, redox alterations as a consequence of Bcl-2 inhibition by ABT-737 should be considered in devising combination therapies with this novel agent or its derivatives.