RESUMEN
Histone lysine methyltransferase 2D (KMT2D) is the most frequently mutated epigenetic modifier in head and neck squamous cell carcinoma (HNSCC). However, the role of KMT2D in HNSCC tumorigenesis and whether its mutations confer any therapeutic vulnerabilities remain unknown. Here we show that KMT2D deficiency promotes HNSCC growth through increasing glycolysis. Additionally, KMT2D loss decreases the expression of Fanconi Anemia (FA)/BRCA pathway genes under glycolytic inhibition. Mechanistically, glycolytic inhibition facilitates the occupancy of KMT2D to the promoter/enhancer regions of FA genes. KMT2D loss reprograms the epigenomic landscapes of FA genes by transiting their promoter/enhancer states from active to inactive under glycolytic inhibition. Therefore, combining the glycolysis inhibitor 2-DG with DNA crosslinking agents or poly (ADP-ribose) polymerase (PARP) inhibitors preferentially inhibits tumor growth of KMT2D-deficient mouse HNSCC and patient-derived xenografts (PDXs) harboring KMT2D-inactivating mutations. These findings provide an epigenomic basis for developing targeted therapies for HNSCC patients with KMT2D-inactivating mutations.
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Glucólisis , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Humanos , Ratones , Glucólisis/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/deficiencia , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Regulación Neoplásica de la Expresión Génica , Ensayos Antitumor por Modelo de Xenoinjerto , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Femenino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Regiones Promotoras Genéticas/genética , Proteína de la Leucemia Mieloide-LinfoideRESUMEN
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.
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Antígeno B7-H1 , Neoplasias de Cabeza y Cuello , Animales , Humanos , Ratones , Neoplasias de Cabeza y Cuello/genética , Ratones Endogámicos NOD , Receptor de Muerte Celular Programada 1 , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
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.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Lesiones Precancerosas , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Queratinocitos/metabolismo , Células Madre Neoplásicas/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores de Factores de Crecimiento de Fibroblastos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Microambiente TumoralRESUMEN
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.
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Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Caveolina 1/metabolismo , Cetuximab/uso terapéutico , Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Factores de Transcripción SOXB1/metabolismo , Animales , Antineoplásicos Inmunológicos/farmacología , Cetuximab/farmacología , Neoplasias de Cabeza y Cuello/fisiopatología , Humanos , RatonesRESUMEN
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.
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Inmunoterapia/métodos , Melanoma/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Neoplasias de Cabeza y Cuello/patología , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Macrófagos Asociados a Tumores/patología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Retroalimentación Fisiológica , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Ácido Hialurónico/metabolismo , Masculino , Ratones Endogámicos NOD , Monocitos/metabolismo , Monocitos/patología , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Microambiente Tumoral , Macrófagos Asociados a Tumores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
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.
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Neoplasias de Cabeza y Cuello/terapia , Células Madre Neoplásicas/efectos de los fármacos , Péptidos Cíclicos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Quimioradioterapia/métodos , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Relación Dosis-Respuesta en la Radiación , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Recurrencia Local de Neoplasia , Células Madre Neoplásicas/efectos de la radiación , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Péptidos Cíclicos/química , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Dosificación Radioterapéutica , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/etiología , Neoplasias de la Boca/metabolismo , Células Madre Neoplásicas/metabolismo , Nicho de Células Madre , Anciano , Animales , Biomarcadores , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Inmunohistoquímica , Inmunofenotipificación , Masculino , Ratones , Persona de Mediana Edad , Neoplasias de la Boca/patología , Estadificación de Neoplasias , Células Madre Neoplásicas/patologíaRESUMEN
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.
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Citocinas/metabolismo , Neoplasias de Cabeza y Cuello/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Infecciones por Papillomavirus/complicaciones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Radioterapia/métodos , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/virología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Desnudos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Fosforilación , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Carcinoma de Células Escamosas/genética , Aberraciones Cromosómicas , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Neoplasias de Cabeza y Cuello/genética , Proteína Smad4/genética , Animales , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Humanos , Hibridación Fluorescente in Situ , Ratones , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Carcinoma de Células Escamosas/patología , Comunicación Celular , Neoplasias de Cabeza y Cuello/patología , Células Madre Neoplásicas/patología , Microambiente Tumoral , Vía de Señalización Wnt , Animales , Apoptosis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Modelos Animales de Enfermedad , Neoplasias de Cabeza y Cuello/patología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Biomarcadores , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Trasplante HeterólogoRESUMEN
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.
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Biomarcadores de Tumor , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/metabolismo , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/patología , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunofenotipificación , Ratones , Mutación , Recurrencia , Neoplasias de las Glándulas Salivales/metabolismo , Secuenciación del Exoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Proteínas de Fusión Oncogénica/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Proteínas de Fusión Oncogénica/genética , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Infecciones por Corynebacterium/prevención & control , Corynebacterium/clasificación , Xenoinjertos/microbiología , Neoplasias Experimentales/microbiología , Animales , Infecciones por Corynebacterium/microbiología , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/patologíaRESUMEN
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.
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Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasa/genética , ARN Mensajero/análisis , Factores de Transcripción SOXB1/genética , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Antígenos CD , Antineoplásicos/farmacología , Cadherinas/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , División Celular , Proliferación Celular , Transformación Celular Neoplásica/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Papillomaviridae/aislamiento & purificación , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Retinal-Deshidrogenasa , Factores de Transcripción SOXB1/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Esferoides Celulares , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Células Tumorales CultivadasRESUMEN
Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas.
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Carcinoma de Células Escamosas/enzimología , Neoplasias de Cabeza y Cuello/enzimología , Proteínas de Neoplasias/fisiología , Receptor EphB4/fisiología , Animales , Apoptosis , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral/efectos de la radiación , Reparación del ADN , Puntos de Control de la Fase G2 del Ciclo Celular , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Queratinocitos/enzimología , Ratones , Terapia Molecular Dirigida , Proteínas de Neoplasias/deficiencia , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación , Receptor EphB4/deficiencia , Carga Tumoral , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dysregulation of the Notch1 receptor has been shown to facilitate the development and progression of colorectal cancer (CRC) and has been identified as an independent predictor of disease progression and worse survival. Although mutations in the NOTCH1 receptor have not been described in CRC, we have previously discovered a NOTCH1 gene copy number gain in a portion of CRC tumor samples. Here, we demonstrated that a NOTCH1 gene copy number gain is significantly associated with worse survival and a high percentage of gene duplication in a cohort of patients with advanced CRC. In our CRC patient-derived tumor xenograft (PDTX) model, tumors harboring a NOTCH1 gain exhibited significant elevation of the Notch1 receptor, JAG1 ligand and cleaved Notch1 activity. In addition, a significant association was identified between a gain in NOTCH1 gene copy number and sensitivity to a Notch1-targeting antibody. These findings suggest that patients with metastatic CRC that harbor a gain in NOTCH1 gene copy number have worse survival and that targeting this patient population with a Notch1 antibody may yield improved outcomes.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Variaciones en el Número de Copia de ADN , Dosificación de Gen , Receptor Notch1/genética , Animales , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores de Tumor , Proteínas de Unión al Calcio/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Duplicación de Gen , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-1 , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Metástasis de la Neoplasia , Pronóstico , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/metabolismo , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Cetuximab/uso terapéutico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Supervivencia sin Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Proyectos Piloto , Resultado del TratamientoRESUMEN
UNLABELLED: Oncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers. SIGNIFICANCE: TRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.