ABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) are major players in tumor-stroma communication, and participate in several cancer hallmarks to drive tumor progression and metastatic dissemination. This study investigates the driving effects of tumor-secreted factors on CAF biology, with the ultimate goal of identifying effective therapeutic targets/strategies for head and neck squamous cell carcinomas (HNSCC). METHODS: Functionally, conditioned media (CM) from different HNSCC-derived cell lines and normal keratinocytes (Kc) were tested on the growth and invasion of populations of primary CAFs and normal fibroblasts (NFs) using 3D invasion assays in collagen matrices. The changes in MMPs expression were evaluated by RT-qPCR and kinase enrichment was analyzed using mass spectrometry phosphoproteomics. RESULTS: Our results consistently demonstrate that HNSCC-secreted factors (but not Kc CM) specifically and robustly promoted pro-invasive properties in both CAFs and NFs, thereby reflecting the plasticity of fibroblast subtypes. Concomitantly, HNSCC-secreted factors massively increased metalloproteinases levels in CAFs and NFs. By contrast, HNSCC CM and Kc CM exhibited comparable growth-promoting effects on stromal fibroblasts. Mechanistically, phosphoproteomic analysis predominantly revealed phosphorylation changes in fibroblasts upon treatment with HNSCC CM, and various promising kinases were identified: MKK7, MKK4, ASK1, RAF1, BRAF, ARAF, COT, PDK1, RSK2 and AKT1. Interestingly, pharmacologic inhibition of RAF1/BRAF using sorafenib emerged as the most effective drug to block tumor-promoted fibroblast invasion without affecting fibroblast viability CONCLUSIONS: Our findings demonstrate that HNSCC-secreted factors specifically fine tune the invasive potential of stromal fibroblasts, thereby generating tumor-driven pro-invasive niches, which in turn to ultimately facilitate cancer cell dissemination. Furthermore, the RAF/BRAF inhibitor sorafenib was identified as a promising candidate to effectively target the onset of pro-invasive clusters of stromal fibroblasts in the HNSCC microenvironment.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Humans , Squamous Cell Carcinoma of Head and Neck/pathology , Carcinoma, Squamous Cell/pathology , Sorafenib/therapeutic use , Proto-Oncogene Proteins B-raf/metabolism , Secretome , Cell Line, Tumor , Head and Neck Neoplasms/pathology , Fibroblasts/metabolism , Tumor Microenvironment/physiologyABSTRACT
In order to assess Stathmin as an immunohistochemical (IHC) indicator of phosphatidylinositol 3-kinase (PI3K) pathway activity in HPV-negative head & neck squamous cell carcinoma (HNSCC), we compared Stathmin IHC to expression of other pathway components. We also evaluated the relationship between Stathmin IHC and the mutational status of four key pathway genes. Finally, we ascertained whether Stathmin IHC correlates with tumor grade or primary site. Correlation exists between high Stathmin expression and high pAKT1 expression, indicating a role for Stathmin IHC as a marker of pathway activity. Our analysis did not show correlation between Stathmin IHC and mutation of the four genes evaluated. We also observed an association between high Stathmin expression and oropharyngeal primary site. Our results suggest utility of Stathmin IHC as an indicator of PI3K pathway activity, and thereby demonstrate potential relevance of Stathmin IHC in the context of HNSCC.
ABSTRACT
This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4+, CD8+ and FOXP3+ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4+ and CD8+ TILs are significantly associated with smoking and alcohol habits. CD4+ and CD8+ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3+ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4+ and CD8+ TILs and a high tumoral CD8+/FOXP3+ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3+ TILs and a low stromal CD8+/FOXP3+ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8+/FOXP3+ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8+/FOXP3+ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.
ABSTRACT
INTRODUCCIÓN Y OBJETIVOS: La disfunción del complejo E-cadherina/catenina está relacionada directamente con la carcinogénesis y el desarrollo de metástasis. El objetivo de este trabajo es investigar el significado pronóstico de la expresión de E-cadherina y beta-catenina en carcinomas de células escamosas de laringe e hipofaringe tratados quirúrgicamente. MATERIAL Y MÉTODOS: Se obtuvieron muestras de tejido tumoral de 133 pacientes consecutivos con carcinomas escamosos de cabeza y cuello (68 de laringe y 65 carcinomas de hipofaringe), que fueron sometidos a tratamiento quirúrgico en nuestro hospital entre 2000 y 2005. La expresión de E-cadherina y beta-catenina se analizó mediante inmunohistoquímica, cuantificando el porcentaje de células teñidas y la intensidad de la tinción. RESULTADOS: La expresión de E-cadherina y beta-catenina fue evaluable en 59 muestras de carcinomas de laringe y en 58 de hipofaringe. En tumores de laringe se observó una asociación significativa entre la baja expresión de beta-catenina de membrana y tumores avanzados T4 y la recidiva tumoral. A nivel de hipofaringe se encontró una asociación significativa de la expresión positiva de Beta-catenina nuclear con pobre diferenciación histológica (p = 0,02). En el análisis multivariante solo la presencia de metástasis ganglionares era factor predictor independiente de disminución de la supervivencia específica para enfermedad en carcinoma de células escamosas de laringe. CONCLUSIONES: La expresión de E-cadherina y beta-catenina no parece tener utilidad pronóstica superior al TNM en los carcinomas epidermoides de laringe e hipofaringe
INTRODUCTION AND OBJECTIVES: Dysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development. The aim of this paper is to investigate the prognostic significance of E-cadherin and Beta-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas. MATERIAL AND METHODS: Tumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005. E-cadherin and beta-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining. RESULTS: E-cadherin and beta-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas. In the laryngeal tumours, a significant association was found between the low expression of membrane Beta-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear beta-catenin and poor histological differentiation (P = .02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas. CONCLUSIONS: The expression of E-cadherin and beta-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Laryngeal Neoplasms/pathology , Hypopharyngeal Neoplasms/pathology , Carcinoma, Squamous Cell/pathology , Cadherins/analysis , beta Catenin/analysis , Retrospective Studies , Immunohistochemistry , Tissue Array Analysis , Human papillomavirus 16/isolation & purification , Laryngeal Neoplasms/mortality , Hypopharyngeal Neoplasms/mortality , Kaplan-Meier Estimate , Neoplasm Staging , Neoplasm Grading , Risk FactorsABSTRACT
INTRODUCTION AND OBJECTIVES: Dysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development. The aim of this paper is to investigate the prognostic significance of E-cadherin and ß-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas. MATERIAL AND METHODS: Tumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005. E-cadherin and ß-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining. RESULTS: E-cadherin and ß-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas. In the laryngeal tumours, a significant association was found between the low expression of membrane ß-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear ß-catenin and poor histological differentiation (P=.02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas. CONCLUSIONS: The expression of E-cadherin and ß-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification.
ABSTRACT
Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cells (CSC). The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Here, we show that SOX2 depletion dramatically reduced the ability of undifferentiated pleomorphic sarcoma (UPS) cells to form tumorspheres and to initiate tumor growth. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. Moreover, using a reporter system (SORE6) which allows to monitor viable cells expressing SOX2 and/or OCT4, we found that SORE6+ cells were significantly more tumorigenic than the SORE6- subpopulation. In agreement with this findings, SOX2 expression in sarcoma patients was associated to tumor grade, differentiation, invasive potential and lower patient survival. Finally, we studied the effect of a panel of anti-tumor drugs on the SORE6+ cells of the UPS model and patient-derived chondrosarcoma lines. We found that the mithramycin analogue EC-8042 was the most efficient in reducing SORE6+ cells in vitro and in vivo. Overall, this study demonstrates that SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations.
ABSTRACT
Melatonin mitigates cancer initiation, progression and metastasis through inhibition of both the synthesis of estrogens and the transcriptional activity of the estradiol-ER (Estrogen receptor) complex in the estrogen-dependent breast cancer cell line MCF-7. Moreover, melatonin improves the sensitivity of MCF-7 to chemotherapeutic agents and protects against their side effects. It has been described that melatonin potentiates the anti-proliferative effects of doxorubicin; however, the molecular changes involving gene expression and the activation/inhibition of intracellular signaling pathways remain largely unknown. Here we found that melatonin enhanced the anti-proliferative effect of doxorubicin in MCF-7 but not in MDA-MB-231 cells. Strikingly, doxorubicin treatment induced cell migration and invasion, and melatonin effectively counteracted these effects in MCF-7 but not in estrogen-independent MDA-MB-231 cells. Importantly, we describe for the first time the ability of melatonin to downregulate TWIST1 (Twist-related protein 1) in estrogen-dependent but not in estrogen-independent breast cancer cells. Combined with doxorubicin, melatonin inhibited the activation of p70S6K and modulated the expression of breast cancer, angiogenesis and clock genes. Moreover, melatonin regulates the levels of TWIST1-related microRNAs, such as miR-10a, miR-10b and miR-34a. Since TWIST1 plays a pivotal role in the epithelial to mesenchymal transition, acquisition of metastatic phenotype and angiogenesis, our results suggest that inhibition of TWIST1 by melatonin might be a crucial mechanism of overcoming resistance and improving the oncostatic potential of doxorubicin in estrogen-dependent breast cancer cells.
ABSTRACT
Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Cell Proliferation/genetics , Dasatinib/pharmacology , Lung Neoplasms/genetics , Proto-Oncogene Proteins c-yes/genetics , A549 Cells , Animals , Antineoplastic Agents/therapeutic use , CRISPR-Cas Systems , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Dasatinib/therapeutic use , Gene Amplification , Gene Knockdown Techniques , Gene Knockout Techniques , Humans , Lung Neoplasms/drug therapy , Mice , Prognosis , Proto-Oncogene Mas , Proto-Oncogene Proteins c-yes/antagonists & inhibitors , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Focal adhesion kinase (FAK) and cortactin overexpression is frequently detected in a variety of cancers, and has been associated with poor clinical outcome. However, there are no data in cutaneous squamous cell carcinoma (cSCC). OBJECTIVE: To investigate the relationship of FAK and cortactin expression with the clinicopathologic features and the impact on the prognosis of cSCC patients. METHODS: FAK and cortactin expression was analyzed by immunohistochemistry on paraffin-embedded tissue samples from 100 patients with cSCC, and correlated with the clinical data. RESULTS: FAK overexpression was a significant risk factor for nodal metastasis with crude and adjusted ratios (HRs) of 2.04, (95% CI [1.08-3.86], [P = 0.029]) and 2.23 (95% CI [1.01-4.91], [P = 0.047]), respectively. Cortactin expression was not a significant risk factor for nodal metastasis. CONCLUSION: These findings demonstrate that FAK overexpression is an independent predictor of nodal metastasis that might be helpful for risk stratification and management of patients with cSCC.
Subject(s)
Biomarkers, Tumor/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Lymph Nodes/pathology , Skin Neoplasms/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , Adult , Aged , Biopsy, Needle , Cohort Studies , Disease-Free Survival , Female , Humans , Immunohistochemistry , Lymphatic Metastasis/pathology , Male , Middle Aged , Predictive Value of Tests , Prognosis , Retrospective Studies , Skin Neoplasms/epidemiology , Skin Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/epidemiology , Squamous Cell Carcinoma of Head and Neck/pathology , Survival AnalysisABSTRACT
Early diagnosis of laryngeal squamous cell carcinoma (LSCC) at the stage of dysplasia could greatly improve the outcome of affected patients. For the first time we compared the mutational landscape of non-progressing dysplasia (NPD; n = 42) with progressing dysplasia (PD; n = 24), along with patient-matched LSCC biopsies; a total of 90 samples. Using targeted next-generation sequencing identified non-synonymous mutations in six genes (PIK3CA, FGFR3, TP53, JAK3, MET, FBXW7), and mutations were validated by Sanger sequencing and/or qPCR. Analysis was extended in silico to 530 head and neck (HNSCC) cases using TCGA data. Mutations in PIK3CA and FGFR3 were detected in PD and LSCC cases, as well as other HNSCC cases, but absent in NPD cases. In contrast, mutations in JAK3, MET and FBXW7 were found in NPD cases but not PD, LSCC or other HNSCC cases. TP53 was the most frequently mutated gene in both PD and NPD cases. With the exception of R248W, mutations were mutually exclusive. Moreover, five of seven PD mutations were located in motif H2 of p53, whereas none of the NPD mutations were. In summary, we propose that the mutational profile of laryngeal dysplasia has utility for the early detection of patients at risk of progression.
Subject(s)
Genetic Predisposition to Disease , Laryngeal Neoplasms/genetics , Laryngeal Neoplasms/pathology , Mutation , Precancerous Conditions/genetics , Precancerous Conditions/pathology , Adult , Aged , Aged, 80 and over , Alleles , Amino Acid Substitution , Biomarkers, Tumor , Computational Biology/methods , DNA Mutational Analysis , Disease Progression , Female , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Neoplasm Grading , Neoplasm Invasiveness , Neoplasm Staging , Reproducibility of Results , Risk Assessment , Risk FactorsABSTRACT
Tumor growth and progression is the result of a complex process controlled not only by malignant cancer cells but also by the surrounding tumor microenvironment (TME). Cancer associated fibroblasts (CAFs), the most abundant cellular component of TME, play an active role in tumor invasion and metastasis by promoting cancer cell invasion through cell-cell interactions and secretion of pro-invasive factors such as extracellular matrix (ECM)-degrading proteases. Due to their tumor-promoting activities, there is an emerging interest in investigating CAFs biology and its potential as drug targets for cancer therapies. Here we describe an easy and highly reproducible quantitative method to analyze CAF invasive activity by forming multicellular spheroids embedded into a three-dimensional (3D) matrix that mimics in vivo ECM. Subsequently, invasion is monitored over time using a time-lapse microscope. We also provide an automated image analysis system that enables the rapid quantification of the spheroid area increase (invasive area) over time. The use of a 96-well plate format with one CAF spheroid per well and the automated analysis provides a method suitable for drug screening test, such as protease inhibitors.
Subject(s)
Cancer-Associated Fibroblasts/pathology , Cell Culture Techniques/methods , Endopeptidases/metabolism , Protease Inhibitors/pharmacology , Spheroids, Cellular/metabolism , Cancer-Associated Fibroblasts/metabolism , Cell Communication/drug effects , Cell Culture Techniques/instrumentation , Cell Line, Tumor , Collagen/metabolism , Dipeptides/pharmacology , Drug Screening Assays, Antitumor/methods , Extracellular Matrix/metabolism , Head and Neck Neoplasms/pathology , Humans , Neoplasm Invasiveness/pathology , Neoplasm Invasiveness/prevention & control , Neoplasms/drug therapy , Neoplasms/pathology , Primary Cell Culture , Spheroids, Cellular/drug effects , Squamous Cell Carcinoma of Head and Neck/pathology , Tumor Microenvironment/drug effectsABSTRACT
Deregulated SRC/FAK signaling leads to enhanced migration and invasion in many types of tumors. In myxoid and round cell liposarcoma (MRCLS), an adipocytic tumor characterized by the expression of the fusion oncogene FUS-CHOP, SRC have been found as one of the most activated kinases. Here we used a cell-of-origin model of MRCLS and an MRCLS cell line to thoroughly characterize the mechanisms of cell invasion induced by FUS-CHOP using in vitro (3D spheroid invasion assays) and in vivo (chicken chorioallantoic membrane model) approaches. FUS-CHOP expression activated SRC-FAK signaling and increased the invasive ability of MRCLS cells. In addition, FAK expression was found to significantly correlate with tumor aggressiveness in sarcoma patient samples. The involvement of SRC/FAK activation in FUS-CHOP-mediated invasion was further confirmed using the SRC inhibitor dasatinib, the specific FAK inhibitor PF-573228, and FAK siRNA. Notably, dasatinib and PF573228 could also efficiently block the invasion of cancer stem cell subpopulations. Downstream of SRC/FAK signaling, we found that FUS-CHOP expression increases the levels of the RHO/ROCK downstream effector phospho-MLC2 (T18/S19) and that this activation was prevented by dasatinib or PF573228. Moreover, the ROCK inhibitor RKI-1447 was able to completely abolish invasion in FUS-CHOP-expressing cells. These data uncover the involvement of SRC/FAK/RHO/ROCK signaling axis in FUS-CHOP-mediated invasion, thus providing a rationale for testing inhibitors of this pathway as potential novel antimetastatic agents for MRCLS treatment.
Subject(s)
Acute-Phase Proteins/metabolism , Focal Adhesion Kinase 1/metabolism , Liposarcoma, Myxoid/genetics , Liposarcoma, Myxoid/metabolism , Oncogene Proteins, Fusion/genetics , RNA-Binding Protein FUS/genetics , Signal Transduction , Transcription Factor CHOP/genetics , rho-Associated Kinases/metabolism , src-Family Kinases/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Liposarcoma, Myxoid/pathology , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/metabolism , RNA, Small Interfering/genetics , RNA-Binding Protein FUS/metabolism , Transcription Factor CHOP/metabolismABSTRACT
BACKGROUND: Binding of tumor-expressed programmed cell death ligand 1 (PD-L1) to the programmed cell death 1 (PD-1) surface receptor blocks T-cell activation thereby leading to immune evasion. Tumor PD-L1 expression has been associated with poor outcome in a wide variety of cancers; however, data in cutaneous squamous cell carcinoma (cSCC) are scarce and conflicting. OBJECTIVE: To investigate the relationship of tumor PD-L1 expression with the clinicopathologic features and prognosis of cSCC. METHODS: PD-L1 expression was analyzed by immunohistochemistry on paraffin-embedded tissue samples from 100 patients with cSCC. Cumulative/dynamic receiver operating characteristic curve was used to determine the optimal PD-L1 threshold. Kaplan-Meier estimators and Cox proportional hazards regression models were also used. RESULTS: On the basis of cumulative/dynamic receiver operating characteristic curves, we defined the cut-off score for PD-L1 expression as ≥25% of tumor cells positively stained. PD-L1 expression was a significant risk factor for nodal metastasis with crude and adjusted hazard ratios of 3.39 (1.71-6.65) and 6.54 (2.28-18.78), respectively. LIMITATIONS: This is a retrospective study limited to cSCC of the head and neck. CONCLUSION: These findings indicate that tumor PD-L1 expression predicts increased risk for nodal metastasis in patients with cSCC.
Subject(s)
B7-H1 Antigen/biosynthesis , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/secondary , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Aged , Aged, 80 and over , Female , Humans , Lymphatic Metastasis , Male , Retrospective StudiesABSTRACT
Trabectedin has been approved for second-line treatment of soft tissue sarcomas. However, its efficacy to target sarcoma initiating cells has not been addressed yet. Here, we used pioneer models of myxoid/round cell liposarcoma (MRCLS) and undifferentiated pleomorphic sarcoma (UPS) developed from transformed human mesenchymal stromal/stem cells (MSCs) to evaluate the effect of trabectedin in the cell type responsible for initiating sarcomagenesis and their derived cancer stem cells (CSC) subpopulations. We found that low nanomolar concentrations of trabectedin efficiently inhibited the growth of sarcoma-initiating cells, induced cell cycle arrest, DNA damage and apoptosis. Interestingly, trabectedin treatment repressed the expression of multiple genes responsible for the development of the CSC phenotype, including pluripotency factors, CSC markers and related signaling pathways. Accordingly, trabectedin induced apoptosis and reduced the survival of CSC-enriched tumorsphere cultures with the same efficiency that inhibits the growth of bulk tumor population. In vivo, trabectedin significantly reduced the mitotic index of MRCLS xenografts and inhibited tumor growth at a similar extent to that observed in doxorubicin-treated tumors. Combination of trabectedin with campthotecin (CPT), a chemotherapeutic drug that shows a robust anti-tumor activity when combined with alkylating agents, resulted in a very strong synergistic inhibition of tumor cell growth and highly increased DNA damage and apoptosis induction. Importantly, the enhanced anti-tumor activity of this combination was also observed in CSC subpopulations. These data suggest that trabectedin and CPT combination may constitute a novel strategy to effectively target both the cell-of-origin and CSC subpopulations in sarcoma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Camptothecin/administration & dosage , Dioxoles/administration & dosage , Liposarcoma, Myxoid/drug therapy , Liposarcoma/drug therapy , Neoplastic Stem Cells/drug effects , Tetrahydroisoquinolines/administration & dosage , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , DNA Damage/drug effects , Drug Synergism , Humans , Liposarcoma/pathology , Liposarcoma, Myxoid/pathology , Mice , Neoplastic Stem Cells/pathology , Signal Transduction/drug effects , Trabectedin , Xenograft Model Antitumor AssaysABSTRACT
The PI3K/AKT/mTOR signaling pathway has emerged as one of the most frequently deregulated in head and neck squamous cell carcinomas (HNSCC). Numerous alterations of various upstream and downstream components have been described; however, their prognostic significance and impact on HNSCC patient survival remains to be established. This was addressed using an unbiased cohort of 93 consecutive and homogeneous surgically treated HNSCC patients and results confirmed in 432 HNSCC patients. Our findings reveal the high prevalence of S6 phosphorylation, a surrogate marker of mTORC1 activation, in HNSCC specimens (>70%) and, more importantly, demonstrate its relevance on clinical outcome. Phosphorylation of ribosomal protein S6 on either Ser235/236 or Ser240/244 was consistently and significantly correlated with favorable prognosis, although with differences depending on the tumor site. Thus, p-S6 expression was significantly correlated with better disease-specific survival specifically in the subgroup of laryngeal carcinoma patients (P< 0.001). In addition, multivariate regression models revealed p-S6 to be an inverse and independent predictor of lymph-node metastasis (P= 0.004) and distant metastasis (P= 0.006). Taken together, this study unveils an unprecedented correlation of mTOR activation with improved clinical outcome in patients with laryngeal carcinomas and uncovers the potential of p-S6 expression as a good prognostic biomarker and an inverse predictor of lymph node and distant metastases. These results should be of broad interest as immunohistochemical detection of p-S6 may help to stratify patients and guide treatment decisions.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/pathology , Ribosomal Protein S6 Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/mortality , Cohort Studies , Disease-Free Survival , Female , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/mortality , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Phosphatidylinositol 3-Kinases/metabolism , Prognosis , Proto-Oncogene Proteins c-akt/metabolism , Squamous Cell Carcinoma of Head and NeckABSTRACT
Podoplanin is involved in actin remodeling of the cytoskeleton of tumor cells and may promote tumor cell invasion by increasing cell motility and formation of filopodia-like membrane protrusions. Podoplanin is expressed in a variety of tumors, but its role in head and neck cancer, particularly in oral squamous cell carcinoma, remains unclear. We studied podoplanin expression by immunohistochemistry in 92 oral squamous cell carcinomas (OSCC) using a monoclonal antibody against an epitope of podoplanin (D2-40). In terms of the number of stained cells, 34 OSCC (38 %) had low podoplanin expression (less than 33 % of cells), 33 (36 %) showed moderate expression (between 34 and 66 % of cells), and 21 (22 %) showed high expression. The intensity of immunostaining was strong in 26 (28 %) cases, moderate in 36 (40 %), and weak or negative in the remaining 30 tumors (32 %). Immunohistochemical expression of podoplanin was associated with a tumor histological grade. A diffuse pattern of podoplanin expression significantly decreased in moderately differentiated (37 %) and poorly differentiated (20 %) carcinomas compared to well-differentiated (43 %) carcinomas. In addition, the focal expression of podoplanin in the invasion front of the tumor, without expression in the tumor center, was observed in 72 % of well-differentiated tumors, 27 % of moderate tumors, and 0 % of poorly differentiated tumors. Moreover, a trend was found toward an association of diffuse podoplanin staining with the development of second primary carcinomas (13 %), in contrast to its expression in the invasion front (3 %). No association was observed between podoplanin expression and nodal metastasis.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/pathology , Membrane Glycoproteins/biosynthesis , Mouth Neoplasms/pathology , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/mortality , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Membrane Glycoproteins/analysis , Middle Aged , Mouth Neoplasms/metabolism , Mouth Neoplasms/mortality , Prognosis , Proportional Hazards Models , Retrospective Studies , Young AdultABSTRACT
NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.
Subject(s)
Epithelium/metabolism , Homeodomain Proteins/metabolism , Animals , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Cell Line , Cell Line, Tumor , Cell Lineage , Cell Proliferation , Epithelium/enzymology , Esophagus/metabolism , Female , Homeodomain Proteins/genetics , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Male , Mice , Mice, Inbred C57BL , Mitosis , Nanog Homeobox Protein , Promoter Regions, Genetic , Species SpecificityABSTRACT
Metastasis remains a major cause of mortality in head and neck squamous cell carcinoma (HNSCC). Current clinicopathological features have shown limited predictability for the risk of distant metastasis in individual patients, and therefore more accurate and reliable markers are needed. The aim of this study was to investigate the ability of various molecular markers present in primary tumors to predict the risk of developing distant metastasis. Restrictive clinical criteria were applied for patient selection in order to carry out a case-control study with comparable clinical features on a group-wide basis and a similar risk of metastasis. All patients were surgically treated (with postoperative radiotherapy when appropriate) and classified as stage IV disease. Immunohistochemical analysis was performed for a panel of proteins known to participate in cellular processes relevant to metastatic dissemination (E-cadherin, annexin A2, cortactin, FAK, EGFR, p53, and p-AKT). Results showed that the loss of E-cadherin expression was significantly correlated with the risk of distant metastasis (P = 0.002; log-rank test), while the loss of annexin A2 expression was nearly statistically significant (P = 0.06). None of the other protein markers assessed were associated with the development of distant metastasis. Therefore, according to our data the loss of epithelial adhesion seems to play a central role in the development of metastasis in HNSCC, and more importantly, immunohistochemical assessment of key proteins involved in cell adhesion regulation, such as E-cadherin could represent a useful tool to evaluate easily and routinely the metastatic potential of these carcinomas.
Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Adhesion/genetics , Hypopharyngeal Neoplasms/pathology , Laryngeal Neoplasms/pathology , Neoplasm Metastasis/genetics , Annexin A2/genetics , Annexin A2/immunology , Biomarkers, Tumor/immunology , Cadherins/genetics , Cadherins/immunology , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/radiotherapy , Carcinoma, Squamous Cell/surgery , Combined Modality Therapy , Humans , Hypopharyngeal Neoplasms/drug therapy , Hypopharyngeal Neoplasms/genetics , Hypopharyngeal Neoplasms/radiotherapy , Hypopharyngeal Neoplasms/surgery , Immunohistochemistry , Laryngeal Neoplasms/drug therapy , Laryngeal Neoplasms/genetics , Laryngeal Neoplasms/radiotherapy , Laryngeal Neoplasms/surgery , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/pathology , Neoplasm Metastasis/radiotherapy , Neoplasm Staging , Tissue Array AnalysisABSTRACT
OBJECTIVES: Recent studies have identified podoplanin, a mucin-type transmembrane glycoprotein, as a biomarker for oral cancer risk in patients with oral leukoplakia (OPL). The aim of this study was to investigate the potential association between podoplanin and the risk of malignant transformation of OPL with epithelial dysplasia. MATERIALS AND METHODS: In this retrospective study, podoplanin immunoexpression was analyzed in 58 patients with oral leukoplakia that showed epithelial dysplasia. Lesions with podoplanin expression in the basal and suprabasal layers of oral epithelium at one area or showing suprabasal expression at two or more areas were considered as positive. Association between podoplanin expression and oral cancer development was analyzed. RESULTS: Twenty-two of the 58 lesions (38%) were classified as podoplanin-positive, and the remaining 36 (62%) lesions were considered podoplanin-negative. The expression of podoplanin was correlated with the grade of dysplasia (p<0.0005), and with the risk of progression to oral cancer (p<0.0005). In multivariate survival analysis, only premalignant oral lesions displaying positive podoplanin expression showed a significantly increased risk of developing an oral squamous cell carcinoma (hazard ratio=8.738, p=0.007). CONCLUSION: Podoplanin could be a valuable biomarker for risk assessment of malignant transformation in patients with OPL along with histological assessment.
Subject(s)
Carcinogenesis , Leukoplakia, Oral/metabolism , Membrane Glycoproteins/metabolism , Mouth Neoplasms/metabolism , Aged , Biomarkers, Tumor/metabolism , Female , Humans , Leukoplakia, Oral/pathology , Male , Middle Aged , Mouth Neoplasms/pathology , Retrospective StudiesABSTRACT
Compelling evidence indicates that the human ether-à-go-go voltage-gated potassium channels (hEAG1) may represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression of hEAG1 potassium channel subunit in both primary tumors and HNSCC-derived cell lines to ascertain its clinical and biological role in tumor progression. Our findings demonstrate that hEAG1 is frequently aberrantly expressed in a high percentage of primary tumors (83 %, 45/54 cases) and HNSCC-derived cell lines (83 %, 10/12 cell lines). hEAG1 expression increased during HNSCC progression and was more frequent in advanced tumors. Strikingly, hEAG1 expression was also detected in a notable proportion (39 %, 17/44 cases) of patient-matched normal adjacent mucosa, whereas no expression was detected in normal epithelia from non-oncologic patients without exposure to tobacco carcinogens. In an attempt to identify the underlying mechanisms of aberrant hEAG1 expression in HNSCC, we found that hEAG1 gene copy gain occurred at a low frequency (15 %, 13/88 cases) in primary tumors but was not observed in early stages of HNSCC tumorigenesis. Furthermore, this study provides original evidence supporting the involvement of histone acetylation (i.e., H3Ac and H4K16Ac activating marks) in the regulation of hEAG1 expression in HNSCC. In addition, functional studies in HNSCC cells further revealed that hEAG1 expression is a biologically relevant feature that promotes cell proliferation and invasion, although independently of its ion-conducting function. Our findings strongly support the notion that hEAG1 may represent a promising candidate as tumor marker and membrane therapeutic target for HNSCC treatment.
