ABSTRACT
Anaplastic thyroid cancer (ATC) is a rare, aggressive form of undifferentiated thyroid cancer, which exhibits rapid progression and is almost universally fatal. At least a subset of ATC is thought to arise from pre-existing well-differentiated thyroid cancer, most frequently papillary thyroid cancer (PTC). While PIK3CA mutations are rare in PTC, they are common in ATC and tend to co-occur with BRAF mutations. This provided the rationale for our study to identify the potential role of PIK3CA mutations in the progression from well-differentiated to undifferentiated thyroid cancer. We introduced PIK3CAE545K into the LAM1 PTC cell line, which carries a BRAFV600E mutation. In culture, the engineered cell line (LAM1:PIK3CAE545K) proliferated faster and demonstrated increased clonogenic potential relative to the parental line carrying an empty vector (LAM1EV). Both the LAM1EV and LAM1:PIK3CAE545K edited lines were implanted into hind flanks of athymic nude mice for in vivo determination of disease progression. While tumour weights and volumes were not significantly higher in LAM1:PIK3CAE545K mice, there was a decrease in expression of thyroid differentiation markers TTF-1, thyroglobulin, PAX8 and B-catenin, suggesting that introduction of PIK3CAE545K led to dedifferentiation in vivo. Collectively, this study provides evidence of a role for PIK3CAE545K in driving disease progression from a well-differentiated to an undifferentiated thyroid cancer; however, over-expression was not a determinant of an accelerated growth phenotype in ATC.
Subject(s)
Class I Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins B-raf , Thyroid Neoplasms , Animals , Cell Line , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/genetics , Humans , Mice , Mice, Nude , Mutation , Phenotype , Proto-Oncogene Proteins B-raf/genetics , Thyroid Cancer, Papillary/genetics , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathologyABSTRACT
BACKGROUND: Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs). METHODS: Five unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance. RESULTS: Prolonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20-35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib. CONCLUSIONS: We have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/drug therapy , MAP Kinase Signaling System , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Thiazoles/pharmacology , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Movement , Cell Proliferation , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Mice , Prognosis , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
Anaplastic thyroid cancer (ATC) is a rare, but nearly uniformly fatal disease that is typically resistant to chemotherapy and radiation. Alternative strategies to target this cancer at a molecular level are necessary in order to improve dismal outcomes for ATC patients. We examined the effects of flavopiridol, a CDK inhibitor, in a panel of ATC cell lines. When cell lines were treated over a ten-point concentration range, CAL62, KMH2 and BHT-101 cell lines had a sub micromolar half-maximal inhibitory concentration, while no effect was seen in the non-cancerous cell line IMR-90. Flavopiridol treatment resulted in decreased levels of the cell cycle proteins CDK9 and MCL1, and induced cell cycle arrest. Flavopiridol also decreased the in vitro ability of ATC cells to form colonies and impeded migration using a transwell migration assay. In vivo, flavopiridol decreased tumor weight and tumor volume over time in a patient-derived xenograft model of ATC. Given the observed in vitro and in vivo activity, flavopiridol warrants further investigation for treatment of ATC.
Subject(s)
Cell Cycle Checkpoints , Flavonoids/therapeutic use , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Thyroid Carcinoma, Anaplastic/drug therapy , Thyroid Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , Flavonoids/pharmacology , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Neoplasms/pathology , Transplantation, HeterologousABSTRACT
OBJECTIVES: Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. MATERIALS AND METHODS: SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. RESULTS: Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p < 0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. CONCLUSIONS: HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.
Subject(s)
Head and Neck Neoplasms/etiology , Papillomavirus Infections/complications , Syk Kinase/genetics , Adult , Aged , Animals , Biomarkers, Tumor , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Disease Susceptibility , Female , Gene Editing , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Immunohistochemistry , Male , Mice , Middle Aged , Neoplasm Grading , Neoplasm Staging , Papillomavirus Infections/virology , RNA Interference , RNA, Messenger/genetics , RNA, Small Interfering , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: The genomic landscape of head and neck cancer has been reported through The Cancer Genome Atlas project. We attempt to determine if high-risk human papillomavirus (HPV) or frequently mutated genes are correlated with survival in an oral cancer cohort. METHODS: Patient demographic data along with data from final pathology was collected. Tumor DNA was analyzed using a custom Illumina targeted sequencing panel. Five high-risk HPV types were tested by qPCR. Statistical analyses were used to identify associations between patient outcome and mutational status. RESULTS: High-risk HPV types were identified in 7% of cases; HPV status was not associated with survival. Mutations were identified in TP53, TERT promoter, & PIK3CA. Mutations in TP53 were significantly associated with poorer overall survival on multi-variate analysis (p = 0.03). CONCLUSIONS: Mutations in TP53 were associated with poor patient survival. Expanding our sample size may identify further predictors of outcome to direct customized cancer care.
ABSTRACT
Phosphoinositide 3-kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival and proliferation. Phosphorylation of Akt Thr308 by PI3K-PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K-activated Akt signaling is well established in HNSCC, the significance of mTORC2-driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2-specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.
Subject(s)
Head and Neck Neoplasms/drug therapy , Mechanistic Target of Rapamycin Complex 2/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Squamous Cell Carcinoma of Head and Neck/drug therapy , Antineoplastic Agents/pharmacology , CRISPR-Cas Systems , Cell Line, Tumor , Cisplatin/pharmacology , Erlotinib Hydrochloride/pharmacology , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Mechanistic Target of Rapamycin Complex 2/genetics , Phosphatidylinositol 3-Kinases/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolismABSTRACT
Smoking has historically been recognized as a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). This study aimed to assess the mutational differences between heavy smokers (>20 pack years) and never smokers among the HNSCC patients within The Cancer Genome Atlas (TCGA). Single nucleotide variation and copy number aberration differences between heavy smokers and never smokers were compared within human papillomavirus-positive (HPV-positive) (n = 67) and HPV-negative (n = 431) TCGA cohorts with HNSCC, and the impact of these mutations on survival were assessed. No genes were differentially mutated between smoking and never-smoking patients with HPV-positive tumors. By contrast, in HPV-negative tumors, NSD1 and COL1A11 were found to be more frequently mutated in heavy smokers, while CASP8 was more frequently altered in never smokers. HPV-negative patients with NSD1 mutations experienced significantly improved overall survival compared with NSD1 WT patients. This improved prognosis was validated in an independent cohort of 77 oral cavity cancer patients and a meta-analysis that included 2 additional data sets (688 total patients, hazard ratio for death 0.44, 95% CI, 0.30-0.65). NSD1 mutations are more common in HPV-negative heavy smokers, define a cohort with favorable prognosis, and may represent a clinically useful biomarker to guide treatment deintensification for HPV-negative patients.
ABSTRACT
Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K/AKT/mTOR signaling axis, particularly in the PIK3CA gene. PI3K-targeted agents have therefore gained considerable preclinical and clinical interest as emerging therapies for HNSCC. Identification of predictive biomarkers of response would advance the clinical application of PI3K-targeted drugs for patients, in order to achieve maximal benefit. To date, studies of drug biomarkers have largely focused on screening cell lines, with much more limited in vivo testing, usually only as validation. This approach has rarely enabled accurate predictions of clinical efficacy. Recently, clinical trials of PDX models (PDX clinical trials) have been introduced as a preclinical approach to interrogate interpatient response heterogeneity. Already, PDX clinical trial responses have been demonstrated to correlate closely with patient outcomes. Here, using both an HNSCC specific, 28-cell line panel and a PDX clinical trial of 80 xenografts derived from 20 unique HNSCC tumors, we systematically examine patterns of response to PI3K inhibition in HNSCC. We find EGFR, AKT1 and CSMD1 copy number aberrations, but not PIK3CA mutations, to be associated with responsiveness to PI3K-targeted drugs. Further, we reveal PI3Kα inhibition to be almost globally tumoristatic in HNSCC xenografts regardless of PIK3CA mutational status, emphasizing its potential as a stabilizing neoadjuvant therapy for HNSCC patients.
Subject(s)
Carcinoma, Squamous Cell/prevention & control , Cetuximab/pharmacology , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Head and Neck Neoplasms/prevention & control , Xenograft Model Antitumor Assays/methods , Adult , Aged , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Male , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Middle Aged , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
Anaplastic thyroid cancer (ATC) is a rare and lethal human malignancy with no known effective therapies in the majority of cases. Despite the use of conventional treatments such as chemotherapy, radiation and surgical resection, this disease remains almost universally fatal. In the present study, we identified the JAK2 inhibitor Lestaurtinib as a potent compound when testing against 13 ATC cell lines. Lestaurtinib demonstrated a potent antiproliferative effect in vitro at nanomolar concentrations. Furthermore, Lestaurtinib impeded cell migration and the ability to form colonies from single cells using scratch-wound and colony formation assays, respectively. Flow cytometry was used for cell cycle analysis following drug treatment and demonstrated arrest at the G2/M phase of the cell cycle, indicative of a cytostatic effect. In vivo studies using the chick chorioallantoic membrane xenograft models demonstrated that treatment with Lestaurtinib resulted in a significant decrease in endpoint tumor volume and vascularity using power Doppler ultrasound imaging. Overall, this study provides evidence that Lestaurtinib is a potent antiproliferative agent with potential antiangiogenic activity that warrants further investigation as a targeted therapy for ATC.
Subject(s)
Antineoplastic Agents/pharmacology , Carbazoles/pharmacology , Thyroid Carcinoma, Anaplastic/drug therapy , Thyroid Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Chick Embryo , Dose-Response Relationship, Drug , Furans , Humans , Janus Kinase 2/metabolism , Thyroid Carcinoma, Anaplastic/blood supply , Thyroid Carcinoma, Anaplastic/diagnostic imaging , Thyroid Neoplasms/blood supply , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVES: The PI3K/AKT/mTOR pathway is frequently altered in head and neck squamous cell cancer (HNSCC), making this pathway a logical therapeutic target. However, PI3K targeting is not universally effective. Biomarkers of response are needed to stratify patients likely to derive benefit and exclude those unlikely to respond. MATERIALS AND METHODS: We examined the sensitivity of cell lines with constitutively-active (G12V mutant) HRAS and wild-type HRAS to PI3K inhibition using flow cytometry and cell viability assays. We then overexpressed and silenced HRAS and measured sensitivity to the PI3K inhibitor BYL719. Immunoblotting was used to determine activation of the PI3K pathway. MEK and mTOR inhibitors were then tested in HRAS mutant cells to determine their efficacy. RESULTS: HRAS mutant cell lines were non-responsive to PI3K inhibition. Overexpression of HRAS led to reduced susceptibility to PI3K inhibition, while knockdown improved sensitivity. Immunoblotting revealed suppressed AKT phosphorylation upon PI3K inhibition in both wild-type and HRAS mutant cell lines, however mutant lines maintained phosphorylation of S6, downstream of mTOR. Targeting mTOR effectively reduced viability of HRAS mutant cells and we subsequently examined the ERK-TSC2-mTOR cascade as a mediator of resistance to PI3K inhibition. CONCLUSIONS: HRAS mutant cells are resistant to PI3K inhibition and our findings suggest the involvement of a signalling intersection of the MAPK and PI3K pathways at the level of ERK-TSC2, leading to persistent mTOR activity. mTOR inhibition alone or in combination with MAPK pathway inhibition may be a promising therapeutic strategy for this subset of HNSCC tumors.
Subject(s)
Drug Resistance, Neoplasm/physiology , Head and Neck Neoplasms/drug therapy , MAP Kinase Signaling System , Neoplasm Proteins/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras)/physiology , Squamous Cell Carcinoma of Head and Neck/drug therapy , Thiazoles/pharmacology , Tuberous Sclerosis Complex 2 Protein/physiology , Cell Cycle/drug effects , Cell Line, Tumor , Genes, ras , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Head and Neck Neoplasms/physiopathology , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Molecular Targeted Therapy , Mutation, Missense , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , RNA Interference , Ribosomal Protein S6 Kinases/metabolism , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , Squamous Cell Carcinoma of Head and Neck/physiopathology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/physiologyABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is a common cancer diagnosis worldwide. Despite advances in treatment, HNSCC has very poor survival outcomes, emphasizing an ongoing need for development of improved therapeutic options. The distinct tumor characteristics of human papillomavirus (HPV)-positive vs. HPV-negative disease necessitate development of treatment strategies tailored to tumor HPV-status. High-throughput robotic screening of 1,433 biologically and pharmacologically relevant compounds at a single dose (4 µM) was carried out against 6 HPV-positive and 20 HPV-negative HNSCC cell lines for preliminary identification of therapeutically relevant compounds. Statistical analysis was further carried out to differentiate compounds with preferential activity against cell lines stratified by the HPV-status. These analyses yielded 57 compounds with higher activity in HPV-negative cell lines, and 34 with higher-activity in HPV-positive ones. Multi-point dose-response curves were generated for six of these compounds (Ryuvidine, MK-1775, SNS-032, Flavopiridol, AZD-7762 and ARP-101), confirming Ryuvidine to have preferential potency against HPV-negative cell lines, and MK-1775 to have preferential potency against HPV-positive cell lines. These data comprise a valuable resource for further investigation of compounds with therapeutic potential in the HNSCC.
ABSTRACT
Albendazole is an anti-helminthic drug that has been shown to exhibit anti-cancer properties, however its activity in head and neck squamous cell cancer (HNSCC) was unknown. Using a series of in vitro assays, we assessed the ability of albendazole to inhibit proliferation in 20 HNSCC cell lines across a range of albendazole doses (1 nM-10 µM). Cell lines that responded to treatment were further examined for cell death, inhibition of migration and cell cycle arrest. Thirteen of fourteen human papillomavirus-negative HNSCC cell lines responded to albendazole, with an average IC50 of 152 nM. In contrast, only 3 of 6 human papillomavirus-positive HNSCC cell lines responded. Albendazole treatment resulted in apoptosis, inhibition of cell migration, cell cycle arrest in the G2/M phase and altered tubulin distribution. Normal control cells were not measurably affected by any dose tested. This study indicates that albendazole acts to inhibit the proliferation of human papillomavirus-negative HNSCC cell lines and thus warrants further study as a potential chemotherapeutic agent for patients suffering from head and neck cancer.