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Blocking the mitogen-activated protein kinase (MAPK) pathway with the MEK1/2 inhibitor trametinib has produced promising results in patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that trametinib treatment leads to overexpression and activation of the epidermal growth factor receptor (EGFR) in HNSCC cell lines and patient-derived xenografts. Knockdown of EGFR improved trametinib treatment efficacy both in vitro and in vivo. Mechanistically, we demonstrated that trametinib-induced EGFR overexpression hyperactivates the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. In vitro, blocking the PI3K pathway with GDC-0941 (pictilisib), or BYL719 (alpelisib), prevented AKT pathway hyperactivation and enhanced the efficacy of trametinib in a synergistic manner. In vivo, a combination of trametinib and BYL719 showed superior antitumor efficacy vs. the single agents, leading to tumor growth arrest. We confirmed our findings in a syngeneic murine head and neck cancer cell line in vitro and in vivo. Taken together, our findings show that trametinib treatment induces hyperactivation of EGFR/PI3K/AKT; thus, blocking of the EGFR/PI3K pathway is required to improve trametinib efficacy in HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço , Fosfatidilinositol 3-Quinase , Humanos , Animais , Camundongos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Receptores ErbB/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Ovarian cancer (OC) is known for exhibiting low response rates to immune checkpoint inhibitors that activate T cells. However, immunotherapies that activate B cells have not yet been extensively explored and may be a potential target, as B cells that secrete immunoglobulins have been associated with better outcomes in OC. Although the secretion of immunoglobulins is often mediated by the microbiome, it is still unclear what role they play in limiting the progression of OC. METHODS: We conducted an in-vivo CRISPR screen of immunodeficient (NSG) and immune-intact wild type (WT) C57/BL6 mice to identify tumor-derived immune-escape mechanisms in a BRAC1- and TP53-deficient murine ID8 OC cell line (designated ITB1). To confirm gene expression and signaling pathway activation in ITB1 cells, we employed western blot, qPCR, immunofluorescent staining, and flow cytometry. Flow cytometry was also used to identify immune cell populations in the peritoneum of ITB1-bearing mice. To determine the presence of IgA-coated bacteria in the peritoneum of ITB1-bearing mice and the ascites of OC patients, we employed 16S sequencing. Testing for differences was done by using Deseq2 test and two-way ANOVA test. Sequence variants (ASVs) were produced in Qiime2 and analyzed by microeco and phyloseq R packages. RESULTS: We identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a tumor-derived immune suppressive mediator in ITB1 cells. Knockout of TRAF3 (TRAF3KO) activated the type-I interferon pathway and increased MHC-I expression. TRAF3KO tumors exhibited a growth delay in WT mice vs. NSG mice, which was correlated with increased B cell infiltration and activation compared to ITB1 tumors. B cells were found to be involved in the progression of TRAF3KO tumors, and B-cell surface-bound and secreted IgA levels were significantly higher in the ascites of TRAF3KO tumors compared to ITB1. The presence of commensal microbiota was necessary for B-cell activation and for delaying the progression of TRAF3KO tumors in WT mice. Lastly, we observed unique profiles of IgA-coated bacteria in the ascites of OC-bearing mice or the ascites of OC patients. CONCLUSIONS: TRAF3 is a tumor-derived immune-suppressive modulator that influences B-cell infiltration and activation, making it a potential target for enhancing anti-tumor B-cell responses in OC.
Assuntos
Neoplasias Ovarianas , Fator 3 Associado a Receptor de TNF , Humanos , Feminino , Camundongos , Animais , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Ascite , Camundongos Knockout , Neoplasias Ovarianas/patologia , Imunoglobulina A/metabolismo , Linhagem Celular TumoralRESUMO
The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression. SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.
Assuntos
Neoplasias de Cabeça e Pescoço , Proteômica , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Neoplasias de Cabeça e Pescoço/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
BACKGROUND: Although the mitogen-activated protein kinases (MAPK) pathway is hyperactive in head and neck cancer (HNC), inhibition of MEK1/2 in HNC patients has not shown clinically meaningful activity. Therefore, we aimed to characterize the effect of MEK1/2 inhibition on the tumor microenvironment (TME) of MAPK-driven HNC, elucidate tumor-host interaction mechanisms facilitating immune escape on treatment, and apply rationale-based therapy combination immunotherapy and MEK1/2 inhibitor to induce tumor clearance. METHODS: Mouse syngeneic tumors and xenografts experiments were used to analyze tumor growth in vivo. Single-cell cytometry by time of flight, flow cytometry, and tissue stainings were used to profile the TME in response to trametinib (MEK1/2 inhibitor). Co-culture of myeloid-derived suppressor cells (MDSC) with CD8+ T cells was used to measure immune suppression. Overexpression of colony-stimulating factor-1 (CSF-1) in tumor cells was used to show the effect of tumor-derived CSF-1 on sensitivity to trametinib and anti-programmed death- 1 (αPD-1) in mice. In HNC patients, the ratio between CSF-1 and CD8A was measured to test the association with clinical benefit to αPD-1 and αPD-L1 treatment. RESULTS: Using preclinical HNC models, we demonstrated that treatment with trametinib delays HNC initiation and progression by reducing tumor cell proliferation and enhancing the antitumor immunity of CD8+ T cells. Activation of CD8+ T cells by supplementation with αPD-1 antibody eliminated tumors and induced an immune memory in the cured mice. Mechanistically, an early response to trametinib treatment sensitized tumors to αPD-1-supplementation by attenuating the expression of tumor-derived CSF-1, which reduced the abundance of two CSF-1R+CD11c+ MDSC populations in the TME. In contrast, prolonged treatment with trametinib abolished the antitumor activity of αPD-1, because tumor cells undergoing the epithelial to mesenchymal transition in response to trametinib restored CSF-1 expression and recreated an immune-suppressive TME. CONCLUSION: Our findings provide the rationale for testing the trametinib/αPD-1 combination in HNC and highlight the importance of sensitizing tumors to αPD-1 by using MEK1/2 to interfere with the tumor-host interaction. Moreover, we describe the concept that treatment of cancer with a targeted therapy transiently induces an immune-active microenvironment, and supplementation of immunotherapy during this time further activates the antitumor machinery to cause tumor elimination.
Assuntos
Neoplasias de Cabeça e Pescoço , Microambiente Tumoral , Animais , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Imunoterapia , CamundongosRESUMO
We hypothesize that the biomechanical properties of cells can predict their viability, with Young's modulus representing the former and cell sensitivity to ultrasound representing the latter. Using atomic force microscopy, we show that the Young's modulus stiffness measure is significantly lower for superficial cancer cells (squamous cell carcinomas and melanoma) compared with noncancerous keratinocyte cells. In vitro findings reveal a significant difference between cancerous and noncancerous cell viability at the four ultrasound energy levels evaluated, with different cell lines exhibiting different sensitivities to the same ultrasound intensity. Young's modulus correlates with cell viability (R 2 = 0.93), indicating that this single biomechanical property can predict cell sensitivity to ultrasound treatment. In mice, repeated ultrasound treatment inhibits tumor growth without damaging healthy skin tissue. Histopathological tumor analysis indicates ultrasound-induced focal necrosis at the treatment site. Our findings provide a strong rationale for developing ultrasound as a noninvasive selective treatment for superficial cancers.
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Uncontrolled growth of ovarian cancer cells is the fifth leading cause of female cancer deaths since most ovarian cancer patients are diagnosed at an advanced stage of metastatic disease. Here, we report on the sensor for monitoring the cancer treatment efficiency in real-time. We measure the optical interaction between the evanescent fields of microfiber and ovarian cancer inter-cellular medium at different treatment stages. Spectral absorption signatures are correlated with optical micrographs and western blot tests. We found that the treatment of tumor cells with induces both cells growth arrest and alter the spectral lines in a dose-dependent manner. These observations are mediated by surface roughness out of silica glass material, form an essential step toward the development of early detection of response to cancer therapy.
Assuntos
Técnicas Biossensoriais , Proliferação de Células/efeitos dos fármacos , Nanofibras/química , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/patologia , Dióxido de Silício/químicaRESUMO
Most head and neck cancer (HNC) patients are resistant to cetuximab, an antibody against the epidermal growth factor receptor. Such therapy resistance is known to be mediated, in part, by stromal cells surrounding the tumor cells; however, the mechanisms underlying such a resistance phenotype remain unclear. To identify the mechanisms of cetuximab resistance in an unbiased manner, RNA-sequencing (RNA-seq) of HNC patient-derived xenografts (PDXs) was performed. Comparing the gene expression of HNC-PDXs before and after treatment with cetuximab indicated that the transforming growth factor-beta (TGF-beta) signaling pathway was upregulated in the stromal cells of PDXs that progressed on cetuximab treatment (CetuximabProg-PDX). However, in PDXs that were extremely sensitive to cetuximab (CetuximabSen-PDX), the TGF-beta pathway was downregulated in the stromal compartment. Histopathological analysis of PDXs showed that TGF-beta-activation was detected in cancer-associated fibroblasts (CAFs) of CetuximabProg-PDX. These TGF-beta-activated CAFs were sufficient to limit cetuximab efficacy in vitro and in vivo. Moreover, blocking the TGF-beta pathway using the SMAD3 inhibitor, SIS3, enhanced cetuximab efficacy and prevented the progression of CetuximabProg-PDX. Altogether, our findings indicate that TGF-beta-activated CAFs play a role in limiting cetuximab efficacy in HNC.
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Here, we reported for the first time an increased expression of c-Met protein in primary cultures of human dermal and pulmonary fibroblasts of late passages. This suggests that c-Met could serve as an early marker of cellular senescence (CS). The levels of c-Met-related signaling proteins phospho-Akt and Stat3 were also increased in (pre)senescent fibroblasts. Considering the anti-apoptotic activity of Akt and the involvement of Stat3 in mediating the effects of proinflammatory cytokines, the findings of this study indicate that c-Met could contribute through its downstream targets or partners to at least two major phenotypical features of CS - resistance to apoptosis and senescence-associated secretory phenotype.
Assuntos
Senescência Celular/fisiologia , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-met , Biomarcadores , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
AXL overexpression is a common resistance mechanism to anti-cancer therapies, including the resistance to BYL719 (Alpelisib) - the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) - in esophagus and head and neck squamous cell carcinoma (ESCC, HNSCC respectively). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here we demonstrated that the AP-1 transcription factors, c-JUN and c-FOS, regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus positive (HPVPos) and negative (HPVNeg) tumor cells to BYL719 in vitro. Blocking of the c-JUN N-terminal kinase (JNK) using SP600125 in combination with BYL719 showed a synergistic anti-proliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719-SP600125 drug combination led to the arrest of tumor growth in cell line-derived and patient-derived xenograft models, and in syngeneic head and neck murine cancer models. Collectively, our data suggests that JNK inhibition in combination with anti-PI3K therapy is a new therapeutic strategy that should be tested in HPVPos and HPVNeg HNSCC and ESCC patients.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tiazóis/farmacologia , Fator de Transcrição AP-1/metabolismo , Animais , Antracenos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Sinergismo Farmacológico , Neoplasias Esofágicas , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Serina-Treonina Quinases TOR/metabolismo , Tiazóis/uso terapêutico , Língua/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Despite of remarkable progress made in the head and neck cancer (HNC) therapy, the survival rate of this metastatic disease remain low. Tailoring the appropriate therapy to patients is a major challenge and highlights the unmet need to have a good preclinical model that will predict clinical response. Hence, we developed an accurate and time efficient drug screening method of tumor ex vivo analysis (TEVA) system, which can predict patient-specific drug responses. In this study, we generated six patient derived xenografts (PDXs) which were utilized for TEVA. Briefly, PDXs were cut into 2 × 2 × 2 mm3 explants and treated with clinically relevant drugs for 24 h. Tumor cell proliferation and death were evaluated by immunohistochemistry and TEVA score was calculated. Ex vivo and in vivo drug efficacy studies were performed on four PDXs and three drugs side-by-side to explore correlation between TEVA and PDX treatment in vivo. Efficacy of drug combinations was also ventured. Optimization of the culture timings dictated 24 h to be the time frame to detect drug responses and drug penetrates 2 × 2 × 2 mm3 explants as signaling pathways were significantly altered. Tumor responses to drugs in TEVA, significantly corresponds with the drug efficacy in mice. Overall, this low cost, robust, relatively simple and efficient 3D tissue-based method, employing material from one PDX, can bypass the necessity of drug validation in immune-incompetent PDX-bearing mice. Our data provides a potential rationale for utilizing TEVA to predict tumor response to targeted and chemo therapies when multiple targets are proposed.
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An understanding of the mechanisms underlying acquired resistance to cetuximab is urgently needed to improve cetuximab efficacy in patients with head and neck squamous cell carcinoma (HNSCC). Here, we present a clinical observation that MET pathway activation constitutes the mechanism of acquired resistance to cetuximab in a patient with HNSCC. Specifically, RNA sequencing and mass spectrometry analysis of cetuximab-sensitive (CetuxSen ) and cetuximab-resistant (CetuxRes ) tumors indicated MET amplification and overexpression in the CetuxRes tumor compared to the CetuxSen lesion. Stimulation of MET in HNSCC cell lines was sufficient to reactivate the MAPK pathway and to confer resistance to cetuximab in vitro and in vivo. In addition to the direct role of MET in reactivation of the MAPK pathway, MET stimulation abrogates the well-known cetuximab-induced compensatory feedback loop of HER2/HER3 expression. Mechanistically, we showed that the overexpression of HER2 and HER3 following cetuximab treatment is mediated by the ETS homologous transcription factor (EHF), and is suppressed by MET/MAPK pathway activation. Collectively, our findings indicate that evaluation of MET and HER2/HER3 in response to cetuximab in HNSCC patients can provide the rationale of successive line of treatment.