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2.
PLoS One ; 11(11): e0166185, 2016.
Article in English | MEDLINE | ID: mdl-27855189

ABSTRACT

BACKGROUND: Hyperactivation of STAT3 via constitutive phosphorylation of tyrosine 705 (Y705) is common in most human cancers, including head and neck squamous carcinoma (HNSCC). STAT3 is rarely mutated in cancer and the (epi)genetic alterations that lead to STAT3 activation are incompletely understood. Here we used an unbiased approach to identify genomic and epigenomic changes associated with pSTAT3(Y705) expression using data generated by The Cancer Genome Atlas (TCGA). METHODS AND FINDINGS: Mutation, mRNA expression, promoter methylation, and copy number alteration data were extracted from TCGA and examined in the context of pSTAT3(Y705) protein expression. mRNA expression levels of 1279 genes were found to be associated with pSTAT3(705) expression. Association of pSTAT3(Y705) expression with caspase-8 mRNA expression was validated by immunoblot analysis in HNSCC cells. Mutation, promoter hypermethylation, and copy number alteration of any gene were not significantly associated with increased pSTAT3(Y705) protein expression. CONCLUSIONS: These cumulative results suggest that unbiased approaches may be useful in identifying the molecular underpinnings of oncogenic signaling, including STAT3 activation, in HNSCC. Larger datasets will likely be necessary to elucidate signaling consequences of infrequent alterations.


Subject(s)
Carcinoma, Squamous Cell/genetics , Genomics , Head and Neck Neoplasms/genetics , STAT3 Transcription Factor/genetics , Transcriptome/genetics , Caspase 8/metabolism , Cell Line, Tumor , DNA Copy Number Variations/genetics , DNA Methylation/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Mutation Rate , Phosphorylation , Promoter Regions, Genetic , Protein Array Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , STAT3 Transcription Factor/metabolism , Squamous Cell Carcinoma of Head and Neck
3.
Oncotarget ; 7(17): 23300-11, 2016 Apr 26.
Article in English | MEDLINE | ID: mdl-27004400

ABSTRACT

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.


Subject(s)
Amphiregulin/metabolism , Carcinoma, Squamous Cell/drug therapy , Erlotinib Hydrochloride/pharmacology , Head and Neck Neoplasms/drug therapy , Mitogen-Activated Protein Kinase 1/genetics , Mutation , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Proliferation/drug effects , Female , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Mice , Mice, Nude , Signal Transduction/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
JAMA Oncol ; 1(2): 238-44, 2015 May.
Article in English | MEDLINE | ID: mdl-26181029

ABSTRACT

IMPORTANCE: Randomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response. OBJECTIVE: To determine a mechanism of exceptional response to erlotinib therapy in HNSCC. DESIGN, SETTING, AND PARTICIPANTS: Single patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response. INTERVENTION: A brief course of erlotinib monotherapy followed by surgical resection. MAIN OUTCOMES AND MEASURES: Identification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants. RESULTS: No EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR-mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells. CONCLUSIONS AND RELEVANCE: Selective erlotinib use in HNSCC may be informed by precision oncology approaches.


Subject(s)
Antineoplastic Agents/administration & dosage , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Erlotinib Hydrochloride/administration & dosage , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Mitogen-Activated Protein Kinase 1/genetics , Protein Kinase Inhibitors/administration & dosage , Tongue Neoplasms/drug therapy , Tongue Neoplasms/genetics , Adult , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Biopsy , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/pathology , Chemotherapy, Adjuvant , DNA Mutational Analysis , Drug Administration Schedule , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Genetic Predisposition to Disease , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/pathology , Humans , Male , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Targeted Therapy , Mutation , Neoadjuvant Therapy , Neoplasm Staging , Phenotype , Phosphorylation , Predictive Value of Tests , Randomized Controlled Trials as Topic , Remission Induction , Squamous Cell Carcinoma of Head and Neck , Time Factors , Tongue Neoplasms/enzymology , Tongue Neoplasms/pathology , Treatment Outcome
5.
Proc Natl Acad Sci U S A ; 111(3): 1114-9, 2014 Jan 21.
Article in English | MEDLINE | ID: mdl-24395800

ABSTRACT

The underpinnings of STAT3 hyperphosphorylation resulting in enhanced signaling and cancer progression are incompletely understood. Loss-of-function mutations of enzymes that dephosphorylate STAT3, such as receptor protein tyrosine phosphatases, which are encoded by the PTPR gene family, represent a plausible mechanism of STAT3 hyperactivation. We analyzed whole exome sequencing (n = 374) and reverse-phase protein array data (n = 212) from head and neck squamous cell carcinomas (HNSCCs). PTPR mutations are most common and are associated with significantly increased phospho-STAT3 expression in HNSCC tumors. Expression of receptor-like protein tyrosine phosphatase T (PTPRT) mutant proteins induces STAT3 phosphorylation and cell survival, consistent with a "driver" phenotype. Computational modeling reveals functional consequences of PTPRT mutations on phospho-tyrosine-substrate interactions. A high mutation rate (30%) of PTPRs was found in HNSCC and 14 other solid tumors, suggesting that PTPR alterations, in particular PTPRT mutations, may define a subset of patients where STAT3 pathway inhibitors hold particular promise as effective therapeutic agents.


Subject(s)
Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/metabolism , Mutation , Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics , STAT3 Transcription Factor/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Survival , Computer Simulation , HEK293 Cells , Humans , Immunohistochemistry , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Structure, Tertiary , Proteome , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Transfection
6.
Article in English | MEDLINE | ID: mdl-24273416

ABSTRACT

Recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. Treatment is limited to chemotherapeutic approaches. Cisplatin is an established and effective treatment for R/M HNSCC, and many studies have investigated cisplatin treatment in combination with other agents. Even when being treated with first line therapy (cisplatin + 5-fluorouracil + cetuximab), overall survival is only 10 months, indicating the need for novel chemotherapeutics and treatment regimens. Current research is focused on molecular targeting therapies inhibiting epidermal growth factor receptor, phosphoinositide-3-kinase/Akt/mammalian target of rapamycin, and vascular endothelial growth factor pathways. A variety of clinical trials have been completed and are currently underway with encouraging results. Finally, future directions of cisplatin-based R/M HNSCC treatment may include targeting specific pathways known to induce cisplatin resistance, such as nucleotide excision repair and inhibition of apoptosis, in hopes to enhance response to cisplatin therapy.

7.
Oncotarget ; 4(6): 936-47, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23801058

ABSTRACT

The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Oxadiazoles/chemistry , Oxadiazoles/pharmacokinetics , Proto-Oncogene Proteins c-myc/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Binding Sites , HEK293 Cells , Humans , Mass Spectrometry , Protein Binding , Protein Multimerization , Proto-Oncogene Proteins c-myc/genetics , Structure-Activity Relationship
8.
Cancer Discov ; 3(7): 761-9, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23619167

ABSTRACT

Genomic findings underscore the heterogeneity of head and neck squamous cell carcinoma (HNSCC). Identification of mutations that predict therapeutic response would be a major advance. We determined the mutationally altered, targetable mitogenic pathways in a large HNSCC cohort. Analysis of whole-exome sequencing data from 151 tumors revealed the phosphoinositide 3-kinase (PI3K) pathway to be the most frequently mutated oncogenic pathway (30.5%). PI3K pathway-mutated HNSCC tumors harbored a significantly higher rate of mutations in known cancer genes. In a subset of human papillomavirus-positive tumors, PIK3CA or PIK3R1 was the only mutated cancer gene. Strikingly, all tumors with concurrent mutation of multiple PI3K pathway genes were advanced (stage IV), implicating concerted PI3K pathway aberrations in HNSCC progression. Patient-derived tumorgrafts with canonical and noncanonical PIK3CA mutations were sensitive to an mTOR/PI3K inhibitor (BEZ-235), in contrast to PIK3CA-wild-type tumorgrafts. These results suggest that PI3K pathway mutations may serve as predictive biomarkers for treatment selection.


Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , Phosphatidylinositol 3-Kinases/genetics , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Head and Neck Neoplasms/pathology , High-Throughput Nucleotide Sequencing , Humans , Imidazoles/administration & dosage , Mutation , Neoplasm Staging , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Quinolines/administration & dosage , Squamous Cell Carcinoma of Head and Neck
9.
PLoS One ; 7(3): e32500, 2012.
Article in English | MEDLINE | ID: mdl-22403666

ABSTRACT

BACKGROUND: A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system. PRINCIPAL FINDINGS: Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. ß-galactosidase production was used to examine translation from mRNA containing the bzpR 5' UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no ß-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5' UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated. CONCLUSIONS: The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5' UTR.


Subject(s)
Dictyostelium/growth & development , Dictyostelium/metabolism , Protozoan Proteins/metabolism , eIF-2 Kinase/metabolism , 5' Untranslated Regions/genetics , Base Sequence , Dictyostelium/enzymology , Dictyostelium/genetics , Gene Deletion , Molecular Sequence Data , Open Reading Frames/genetics , Phosphorylation , Protozoan Proteins/biosynthesis , Protozoan Proteins/genetics
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