RESUMO
Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol consumption that induce a "precancerous field," with phosphoinositide 3-kinase (PI3K) signaling being a common driver. However, the preclinical effectiveness of PI3K inhibitors has not necessarily translated to remarkable benefit in HNSCC patients. Thus, we sought to determine how precancerous keratinocytes influence HNSCC proliferation, cancer stem cell (CSC) maintenance, and response to PI3K inhibitors. We used the NOK keratinocyte cell line as a model of preneoplastic keratinocytes because it harbors two frequent genetic events in HNSCC, CDKN2A promoter methylation and TP53 mutation, but does not form tumors. NOK cell coculture or NOK cell-conditioned media promoted HNSCC proliferation, PI3K inhibitor resistance, and CSC phenotypes. SOMAscan-targeted proteomics determined the relative levels of >1300 analytes in the media conditioned by NOK cells and HNSCC cells ± PI3K inhibitor. These results demonstrated that NOK cells release abundant levels of ligands that activate epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), two receptor tyrosine kinases with oncogenic activity. Inhibition of EGFR, but not FGFR, blunted PI3K inhibitor resistance and CSC phenotypes induced by NOK cells. Our results demonstrate that precancerous keratinocytes can directly support neighboring HNSCC by activating EGFR. Importantly, PI3K inhibitor sensitivity was not necessarily a cancer cell-intrinsic property, and the tumor microenvironment impacts therapeutic response and supports CSCs. Additionally, combined inhibition of EGFR with PI3K inhibitor diminished EGFR activation induced by PI3K inhibitor and potently inhibited cancer cell proliferation and CSC maintenance.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Lesões Pré-Cancerosas , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Queratinócitos/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptores de Fatores de Crescimento de Fibroblastos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Microambiente TumoralRESUMO
Reports regarding the frequency of SMAD4 loss in human head and neck squamous cell carcinoma (HNSCC) vary significantly. We have shown that SMAD4 deletion contributes to HNSCC initiation and progression. Therefore, accurately detecting genetic SMAD4 loss is critical to determine prognosis and therapeutic interventions in personalized medicine. We developed a SMAD4 fluorescence in situ hybridization (FISH) assay to identify chromosomal SMAD4 loss at the single cell level of primary HNSCC specimens and patient derived xenograft (PDX) tumors derived from HNSCCs. SMAD4 heterozygous loss was detected in 35% of primary HNSCCs and 41.3% of PDX tumors. Additionally, 4.3% of PDX tumors had SMAD4 homozygous loss. These frequencies of SMAD4 loss were similar to those in The Cancer Genome Atlas (TCGA). However, we identified significant heterogeneities of SMAD4 loss (partial or complete) among cells within each tumor. We also found that aneuploidy (monosomy and polysomy) contributed greatly to how to define chromosomal SMAD4 deletion. Furthermore, in cultured PDX tumors, SMAD4 mutant cells outcompeted SMAD4 wildtype cells, resulting in establishing homogenous SMAD4 mutant HNSCC cell lines with partial or complete genomic SMAD4 loss, suggesting a survival advantage of SMAD4 mutant cells. Taken together, our study reveals inter- and intra-tumor heterogeneities of SMAD4 chromosomal loss in HNSCCs. Further, SMAD4 FISH assay provides a platform for future clinical diagnosis of SMAD4 chromosomal loss that potentially serves as a molecular marker for prognosis and therapeutic intervention in cancer patients.
Assuntos
Carcinoma de Células Escamosas/genética , Aberrações Cromossômicas , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Neoplasias de Cabeça e Pescoço/genética , Proteína Smad4/genética , Animais , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hibridização in Situ Fluorescente , Camundongos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (eg, WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.
Assuntos
Carcinoma de Células Escamosas/patologia , Comunicação Celular , Neoplasias de Cabeça e Pescoço/patologia , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral , Via de Sinalização Wnt , Animais , Apoptose , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this report, we describe in detail the evolving procedures to optimize humanized mouse cohort generation, including optimal conditioning, choice of lineage for engraftment, threshold for successful engraftment, HNSCC tumor implantation, and immune and stroma cell analyses. We developed a dual infusion protocol of human hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stem cells (MSCs), leading to incremental human bone marrow engraftment, and exponential increase in mature peripheral human immune cells, and intratumor homing that includes a more complete lineage reconstitution. Additionally, we have identified practical rules to predict successful HSPC/MSC expansion, and a peripheral human cell threshold associated with bone marrow engraftment, both of which will optimize cohort generation and management. The tremendous advances in immune therapy in cancer have made the need for appropriate and standardized models more acute than ever, and therefore, we anticipate that this manuscript will have an immediate impact in cancer-related research. The need for more representative tools to investigate the human tumor microenvironment (TME) has led to the development of humanized mouse models. However, the difficulty of immune system engraftment and minimal human immune cell infiltration into implanted xenografts are major challenges. We have developed an improved method for generating mismatched humanized mice (mHM), using a dual infusion of human HSPCs and MSCs, isolated from cord blood and expanded in vitro. Engraftment with both HSPCs and MSCs produces mice with almost twice the percentage of human immune cells in their bone marrow, compared to mice engrafted with HSPCs alone, and yields 9- to 38-fold higher levels of mature peripheral human immune cells. We identified a peripheral mHM blood human B cell threshold that predicts an optimal degree of mouse bone marrow humanization. When head and neck squamous cell carcinoma (HNSCC) tumors are implanted on the flanks of HSPC-MSC engrafted mice, human T cells, B cells, and macrophages infiltrate the stroma of these tumors at 2- to 8-fold higher ratios. In dually HSPC-MSC engrafted mice we also more frequently observed additional types of immune cells, including regulatory T cells, cytotoxic T cells, and MDSCs. Higher humanization was associated with in vivo response to immune-directed therapy. The complex immune environment arising in tumors from dually HSPC-MSC engrafted mice better resembles that of the originating patient's tumor, suggesting an enhanced capability to accurately recapitulate a human TME.
Assuntos
Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Biomarcadores , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Transplante HeterólogoRESUMO
BACKGROUND: The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. METHODS: We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. RESULTS: PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. CONCLUSIONS: Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.
Assuntos
Antígeno B7-H1 , Neoplasias de Cabeça e Pescoço , Animais , Humanos , Camundongos , Neoplasias de Cabeça e Pescoço/genética , Camundongos Endogâmicos NOD , Receptor de Morte Celular Programada 1 , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
The epidermal growth factor receptor (EGFR) inhibitor cetuximab is the only FDA-approved oncogene-targeting therapy for head and neck squamous cell carcinoma (HNSCC). Despite variable treatment response, no biomarkers exist to stratify patients for cetuximab therapy in HNSCC. Here, we applied unbiased hierarchical clustering to reverse-phase protein array molecular profiles from patient-derived xenograft (PDX) tumors and revealed 2 PDX clusters defined by protein networks associated with EGFR inhibitor resistance. In vivo validation revealed unbiased clustering to classify PDX tumors according to cetuximab response with 88% accuracy. Next, a support vector machine classifier algorithm identified a minimalist biomarker signature consisting of 8 proteins - caveolin-1, Sox-2, AXL, STING, Brd4, claudin-7, connexin-43, and fibronectin - with expression that strongly predicted cetuximab response in PDXs using either protein or mRNA. A combination of caveolin-1 and Sox-2 protein levels was sufficient to maintain high predictive accuracy, which we validated in tumor samples from patients with HNSCC with known clinical response to cetuximab. These results support further investigation into the combined use of caveolin-1 and Sox-2 as predictive biomarkers for cetuximab response in the clinic.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Caveolina 1/metabolismo , Cetuximab/uso terapêutico , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Fatores de Transcrição SOXB1/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Cetuximab/farmacologia , Neoplasias de Cabeça e Pescoço/fisiopatologia , Humanos , CamundongosRESUMO
Resistance to immunotherapy is a significant challenge, and the scarcity of human models hinders the identification of the underlying mechanisms. To address this limitation, we constructed an autologous humanized mouse (aHM) model with hematopoietic stem and progenitor cells (HSPC) and tumors from 2 melanoma patients progressing to immunotherapy. Unlike mismatched humanized mouse (mHM) models, generated from cord blood-derived HSPCs and tumors from different donors, the aHM recapitulates a patient-specific tumor microenvironment (TME). When patient tumors were implanted on aHM, mHM, and NOD/SCID/IL2rg-/- (NSG) cohorts, tumors appeared earlier and grew faster on NSG and mHM cohorts. We observed that immune cells differentiating in the aHM were relatively more capable of circulating peripherally, invading into tumors and interacting with the TME. A heterologous, human leukocyte antigen (HLA-A) matched cohort also yielded slower growing tumors than non-HLA-matched mHM, indicating that a less permissive immune environment inhibits tumor progression. When the aHM, mHM, and NSG cohorts were treated with immunotherapies mirroring what the originating patients received, tumor growth in the aHM accelerated, similar to the progression observed in the patients. This rapid growth was associated with decreased immune cell infiltration, reduced interferon gamma (IFNγ)-related gene expression, and a reduction in STAT3 phosphorylation, events that were replicated in vitro using tumor-derived cell lines. IMPLICATIONS: Engrafted adult HSPCs give rise to more tumor infiltrative immune cells, increased HLA matching leads to slower tumor initiation and growth, and continuing immunotherapy past progression can paradoxically lead to increased growth.
Assuntos
Imunoterapia/métodos , Melanoma/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.
Assuntos
Neoplasias de Cabeça e Pescoço/patologia , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Macrófagos Associados a Tumor/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/metabolismo , Masculino , Camundongos Endogâmicos NOD , Monócitos/metabolismo , Monócitos/patologia , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Cancer stem cells (CSC) drive growth, therapy resistance, and recurrence in head and neck squamous cell carcinoma (HNSCC). Regulation of protein translation is crucial for normal stem cells and CSCs; its inhibition could disrupt stemness properties, but translation inhibitors are limited clinically due to toxicity. SVC112 is a synthetic derivative of bouvardin, a plant-derived translation elongation inhibitor. SVC112 had greater antiproliferative effects on HNSCC cells compared with the FDA-approved translation inhibitor omacetaxine mepesuccinate (HHT). SVC112 preferentially inhibited cancer cells compared with patient-matched cancer-associated fibroblasts, whereas HHT was equally toxic to both. SVC112 reduced sphere formation by cell lines and CSCs. SVC112 alone inhibited the growth of patient-derived xenografts (PDX), and SVC112 combined with radiation resulted in tumor regression in HPV-positive and HPV-negative HNSCC PDXs. Notably, CSC depletion after SVC112 correlated with tumor response. SVC112 preferentially impeded ribosomal processing of mRNAs critical for stress response and decreased CSC-related proteins including Myc and Sox2. SVC112 increased cell-cycle progression delay and slowed DNA repair following radiation, enhancing colony and sphere formation radiation effects. In summary, these data demonstrate that SVC112 suppresses CSC-related proteins, enhances the effects of radiation, and blocks growth of HNSCC PDXs by inhibiting CSCs. SIGNIFICANCE: Inhibiting protein elongation with SVC112 reduces tumor growth in head and neck squamous cell carcinoma and increases the effects of radiation by targeting the cancer stem cell pool.
Assuntos
Neoplasias de Cabeça e Pescoço/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas/efeitos da radiação , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Peptídeos Cíclicos/química , Inibidores da Síntese de Proteínas/uso terapêutico , Dosagem Radioterapêutica , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The flow cytometry mutation assay (FCMA) uses hybrid CHO A(L) cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T(pot)) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59(-) cells sorted on the peak day of expression was roughly half that of CD59(+) cells. Cloned EMS-treated CD59(-) cells had a doubling time of 16.7 vs. 14.1 h for CD59(+) cells. Triple mutants (CD59(-)/CD44(-)/CD90(-)) were preferentially lost from the population over time, while the proportion of CD59(-)/CD90(-) mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth.
Assuntos
Antígenos CD59/genética , Cromossomos Humanos Par 11/genética , Análise Mutacional de DNA/métodos , Citometria de Fluxo/métodos , Testes de Mutagenicidade , Alquilantes/farmacologia , Amianto/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos Par 11/efeitos dos fármacos , Cromossomos Humanos Par 11/efeitos da radiação , Metanossulfonato de Etila/farmacologia , Raios gama , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Mutagênicos/farmacologia , Mutação , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismoRESUMO
The CHO A(L) cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59(-) regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59(-) clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.
Assuntos
Antígenos CD59/genética , Citometria de Fluxo/métodos , Mutação Puntual , Animais , Células CHO , Linhagem Celular , Cromossomos Humanos Par 11 , Células Clonais , Cricetinae , Cricetulus , Metanossulfonato de Etila/toxicidade , Humanos , Testes de Mutagenicidade , TransfecçãoRESUMO
Radiation therapy (RT) is a cornerstone of treatment in the management of head and neck squamous cell carcinomas (HNSCC), yet treatment failure and disease recurrence are common. The p38/MK2 pathway is activated in response to cellular stressors, including radiation, and promotes tumor inflammation in a variety of cancers. We investigated MK2 pathway activation in HNSCC and the interaction of MK2 and RT in vitro and in vivo. We used a combination of an oropharyngeal SCC tissue microarray, HNSCC cell lines, and patient-derived xenograft (PDX) tumor models to study the effect of RT on MK2 pathway activation and to determine how inhibition of MK2 by pharmacologic (PF-3644022) and genetic (siRNA) methods impacts tumor growth. We show that high phosphorylated MK2 (p-MK2) levels are associated with worsened disease-specific survival in p16-negative HNSCC patients. RT increased p-MK2 in both p16-positive, HPV-positive and p16-negative, HPV-negative HNSCC cell lines. Pharmacologic inhibition or gene silencing of MK2 in vitro abrogated RT-induced increases in p-MK2; inflammatory cytokine expression and expression of the downstream MK2 target, heat shock protein 27 (HSP27); and markers of epithelial-to-mesenchymal transition. Mouse PDX models treated with a combination of RT and MK2 inhibitor experienced decreased tumor growth and increased survival. Our results suggest that MK2 is a potential prognostic biomarker for head and neck cancer and that MK2 pathway activation can mediate radiation resistance in HNSCC.
Assuntos
Citocinas/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Infecções por Papillomavirus/complicações , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Radioterapia/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/virologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Nus , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Fosforilação , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: To describe differences in cancer stem cell (CSC) presence and behavior associated with their intratumor compartment of origin using a patient-derived xenograft (PDX) model of oral cavity squamous cell carcinoma (OCSCC). MATERIALS AND METHODS: Four HPV-negative OCSCC PDX cases were selected (CUHN004, CUHN013, CUHN096, CUHN111) and the percentage of CSCs (ALDH+CD44high) was measured in the tumor Leading Edge (LE) and Core compartments of each PDX tumor case via fluorescence activated cell sorting (FACS). The fraction of cells in the proliferative phase was measured by Ki-67 labelling index of paraffin embedded tissue. The proliferation and invasion of LE versus Core CSCs were compared using sphere and Matrigel invasion assays, respectively. RESULTS: Both CUHN111 and CUHN004 demonstrate CSC enrichment in their LE compartments while CUHN013 and CUHN096 show no intratumor difference. Cases with LE CSC enrichment demonstrate greater Ki-67 labelling at the LE. CSC proliferative potential, assessed by sphere formation, reveals greater sphere formation in CUHN111 LE CSCs, but no difference between CUHN013 LE and Core CSCs. CUHN111 CSCs do not demonstrate an intratumor difference in invasiveness while CUHN013 LE CSCs are more invasive than Core CSCs. CONCLUSION: A discrete intratumor CSC niche is present in a subset of OCSCC PDX tumors. The CSC functional phenotype with regard to proliferation and invasion is associated with the intratumor compartment of origin of the CSC: LE or Core. These individual functional characteristics appear to be modulated independently of one another and independently of the presence of an intratumor CSC niche.
Assuntos
Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/etiologia , Neoplasias Bucais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nicho de Células-Tronco , Idoso , Animais , Biomarcadores , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/patologiaRESUMO
Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB-MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3-ETV6 and MYB-NFIB)Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935-43. ©2018 AACR.
Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Mutação , Recidiva , Neoplasias das Glândulas Salivares/metabolismo , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A(L)) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A(L) cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis.
Assuntos
Antígenos CD/genética , Cromossomos Humanos Par 11/genética , Marcadores Genéticos , Glicosilfosfatidilinositóis/genética , Mutação , Animais , Antígenos CD59/genética , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo , Proteína-1 Reguladora de Fusão/genética , Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Células Híbridas , Reação em Cadeia da Polimerase , Tetraspanina 24 , Antígenos Thy-1/genéticaRESUMO
Human patient-derived xenograft (PDX) tumors, propagated in immunodeficient mice, are rapidly growing in use as a model for cancer research. Horizontal transfer between mice, without in vitro cell culture, allows these tumors to retain many of their unique characteristics from their individual patient of origin. However, the immunodeficient mouse strains used to grow these tumors are susceptible to numerous opportunistic pathogens, including Corynebacterium bovis. At our institution, 2 in vivo tumor banks of PDX tumors had been maintained within nude mouse colonies enzootically infected with C. bovis. Elimination of C. bovis from these colonies required the aseptic harvest and horizontal transfer of tumor tissue between infected and naïve recipient mice without cross-contamination. Out of necessity, we developed a standard operating procedure using enhancements to traditional aseptic surgical technique with concurrent application of both procedural and physical barriers to prevent C. bovis transmission. By using these methods, all 61 unique PDX tumor models were successfully harvested from C. bovis-infected mice and transferred into recipient mice without transmission of infection. Our data demonstrate that, in situations where C. bovis-free colonies can be established and maintained, this procedure can successfully be used to eliminate C. bovis from an in vivo tumor bank of valuable PDX tumors.
Assuntos
Infecções por Corynebacterium/prevenção & controle , Corynebacterium/classificação , Xenoenxertos/microbiologia , Neoplasias Experimentais/microbiologia , Animais , Infecções por Corynebacterium/microbiologia , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/patologiaRESUMO
Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion-positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551-63. ©2017 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Proteínas de Fusão Oncogênica/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Fusão Oncogênica/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinase/genética , RNA Mensageiro/análise , Fatores de Transcrição SOXB1/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antígenos CD , Antineoplásicos/farmacologia , Caderinas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Divisão Celular , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Papillomaviridae/isolamento & purificação , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Retinal Desidrogenase , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Células Tumorais CultivadasRESUMO
Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A(L)). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59- and CD59+ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.
Assuntos
Análise Mutacional de DNA/métodos , Citometria de Fluxo/métodos , Mutagênicos/toxicidade , Animais , Antígenos CD59/metabolismo , Células CHO , Cromossomos Humanos Par 11 , Cricetinae , Reagentes de Ligações Cruzadas/toxicidade , Metanossulfonato de Etila/toxicidade , Humanos , Mitomicina/toxicidade , Compostos Organometálicos/toxicidade , Radiação Ionizante , Fatores de TempoRESUMO
BACKGROUND: This phase 1, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), antitumor activity, and molecular correlates of IPI-926, a Hedgehog pathway (HhP) inhibitor, combined with cetuximab in patients with relapsed/metastatic squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Cetuximab was given with a 400mg/m(2) loading dose followed by 250mg/m(2) weekly. IPI-926 was given daily starting two weeks after cetuximab initiation. A "3+3" study design was used. Prior therapy with cetuximab was allowed. Tumor biopsies occurred prior to cetuximab initiation, prior to IPI-926 initiation, and after treatment with both drugs. RESULTS: Nine patients were enrolled. The RP2D was 160mg, the same as the single-agent IPI-926 MTD. Among 9 treated, 8 evaluable patients, the best responses were 1 partial response (12.5%), 4 stable disease (50%), and 3 disease progressions (37.5%). The median progression free survival was 77days (95% confidence interval 39-156). Decreases in tumor size were seen in both cetuximab-naïve patients (one HPV-positive, one HPV-negative). The most frequent treatment-emergent adverse events were fatigue, muscle cramps, and rash. No DLTs were observed. Tumor shrinkage and progression free survival were associated with intra-tumoral ErbB and HhP gene expression down-regulation during therapy, supporting the preclinical hypothesis. CONCLUSION: Treatment with IPI-926 and cetuximab yielded expected toxicities with signs of anti-tumor activity. Serial tumor biopsies were feasible and revealed proof-of-concept biomarkers.