ABSTRACT
Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.
ABSTRACT
Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.
Subject(s)
Carcinoma, Squamous Cell , Fanconi Anemia , Head and Neck Neoplasms , Mouth Neoplasms , Animals , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Disease Models, Animal , Fanconi Anemia/complications , Fanconi Anemia/genetics , Fanconi Anemia/pathology , Keratins , Mice , Mice, Knockout , Mouth Neoplasms/genetics , Squamous Cell Carcinoma of Head and NeckABSTRACT
Background: Filamentous plant virus-derived nanoparticles are biodegradable and noninfectious to humans. Their structure is also amenable to chemical modifications. They constitute an appealing material for biomedical applications including imaging and drug delivery. We had previously used turnip mosaic virus-derived nanoparticles (TuMV-NPs) to increase antibody-sensing in vivo, to prevent biofilm formation and to build biological nanoscaffolds. Materials & methods: We analyzed TuMV-NP biodistribution and tumor homing using in vivo imaging. We studied in vitro the interaction with human cancer cell lines and the antiproliferative effect of epigallocatechin gallate-functionalized TuMV-NPs. Results & conclusion: TuMV-NPs are efficiently internalized by human cells and show good tumor homing. The antiproliferative effect of epigallocatechin gallate-TuMV-NPs suggests that they could offer a potential anticancer therapy.
Cancer is the second leading cause of death worldwide, just behind cardiovascular disease. It accounts for nearly 10 million deaths annually, and new strategies to improve early detection and drug delivery are urgently needed. Nanoparticles are small structures within the nanometer range (1 billionth of a meter) that can be used to deliver either an imaging probe (tracer) to allow the detection of a tumor or drugs to kill tumor cells. There are many types of nanoparticles; those based on plant viruses are especially appealing for biomedical purposes because they are biodegradable and noninfectious to humans. Also, their physicochemical properties, such as symmetry, uniformity and loading capacity, make them excellent nanocarriers. We report here for the first time the ability of nanoparticles derived from the turnip mosaic virus (TuMV), a well-known virus naturally infecting cruciferous plants (e.g., broccoli, turnip, radish, cabbage) but not humans, to deliver a fluorescent imaging probe that allows tumor detection in vivo. Moreover, TuMV nanoparticles were used to deliver a natural chemotherapeutic agent of plant origin to different types of tumor cells (lung, colorectal, breast, and head and neck), showing increased antiproliferative capacity compared to the nonvehiculized drug.
Subject(s)
Nanoparticles , Potyvirus , Humans , Polyphenols/pharmacology , Tissue DistributionABSTRACT
Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes-WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO-revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.
ABSTRACT
Fanconi anemia (FA) patients have an exacerbated risk of head and neck squamous cell carcinoma (HNSCC). Treatment is challenging as FA patients display enhanced toxicity to standard treatments, including radio/chemotherapy. Therefore, better therapies as well as new disease models are urgently needed. We have used CRISPR/Cas9 editing tools in order to interrupt the human FANCA gene by the generation of insertions/deletions (indels) in exon 4 in two cancer cell lines from sporadic HNSCC having no mutation in FA-genes: CAL27 and CAL33 cells. Our approach allowed efficient editing, subsequent purification of single-cell clones, and Sanger sequencing validation at the edited locus. Clones having frameshift indels in homozygosis did not express FANCA protein and were selected for further analysis. When compared with parental CAL27 and CAL33, FANCA-mutant cell clones displayed a FA-phenotype as they (i) are highly sensitive to DNA interstrand crosslink (ICL) agents such as mitomycin C (MMC) or cisplatin, (ii) do not monoubiquitinate FANCD2 upon MMC treatment and therefore (iii) do not form FANCD2 nuclear foci, and (iv) they display increased chromosome fragility and G2 arrest after diepoxybutane (DEB) treatment. These FANCA-mutant clones display similar growth rates as their parental cells. Interestingly, mutant cells acquire phenotypes associated with more aggressive disease, such as increased migration in wound healing assays. Therefore, CAL27 and CAL33 cells with FANCA mutations are phenocopies of FA-HNSCC cells.
Subject(s)
Fanconi Anemia Complementation Group A Protein/deficiency , Fanconi Anemia/pathology , Gene Editing , Head and Neck Neoplasms/pathology , Mutation , Phenotype , Squamous Cell Carcinoma of Head and Neck/pathology , Cell Movement , Cell Proliferation , DNA Damage , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/genetics , Head and Neck Neoplasms/genetics , Humans , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Tumor Cells, Cultured , Wound HealingABSTRACT
Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.
Subject(s)
Cell Proliferation , Head and Neck Neoplasms/metabolism , Proto-Oncogene Proteins c-vav/genetics , Proto-Oncogene Proteins c-vav/metabolism , Squamous Cell Carcinoma of Head and Neck/metabolism , Animals , Cell Differentiation , Disease Models, Animal , Epidermis/metabolism , Epidermis/pathology , GTP Phosphohydrolases , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Hyperplasia/pathology , Keratinocytes/pathology , Mice , Mice, Knockout , Mucous Membrane/metabolism , Prognosis , RNA, Messenger/metabolism , Signal Transduction , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , TranscriptomeABSTRACT
Epidermal stem cells are responsible for normal tissue homeostasis and contribute to tissue regeneration during injury. Several assays measuring stem cell frequency and function can be used to assess epidermal stem cell potential. However, the ultimate assay that accounts for stemness is the capacity to sustain in vivo long-term tissue regeneration and maintenance. We can use this type of analysis to interrogate whether a specific genetic alteration (e.g., activation or inactivation of any gene thought to be involved in stem cell quiescence or proliferation) confers increased or decreased stem cell potential.
Subject(s)
Keratinocytes/cytology , Keratinocytes/transplantation , Stem Cells/cytology , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Epidermis/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Keratinocytes/metabolism , Mice , RegenerationABSTRACT
Head and neck cancer affects the upper aerodigestive tract and is the sixth leading cancer worldwide by incidence and the seventh by cause of death. Despite significant advances in surgery and chemotherapy, molecularly targeted therapeutic options for this type of cancer are scarce and long term survival rates remain low. Recently, comprehensive genomic studies have highlighted the most commonly altered genes and signaling pathways in this cancer. The Hippo-YAP pathway has been identified as a key oncogenic pathway in multiple tumors. Expression of genes controlled by the Hippo downstream transcriptional coactivators YAP (Yes-associated protein 1) and TAZ (WWTR1, WW domain containing transcription regulator 1) is widely deregulated in human cancer including head and neck squamous cell carcinoma (HNSCC). Interestingly, YAP/TAZ signaling might not be as essential for the normal homeostasis of adult tissues as for oncogenic growth, altogether making the pathway an amenable therapeutic target in cancer. Recent advances in the role of Hippo-YAP pathway in HNSCC have provided evidence that genetic alterations frequent in this type of cancer such as PIK3CA (phosphatidylinositide 3-kinase catalytic subunit alpha) overexpression or FAT1 (FAT atypical cadherin 1) functional loss can result in YAP activation. We discuss current therapeutic options targeting this pathway which are currently in use for other tumor types.
ABSTRACT
Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.
Subject(s)
Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Animals , Cell Line, Tumor , Cisplatin/therapeutic use , Enhancer of Zeste Homolog 2 Protein/physiology , Female , Histocompatibility Antigens , Humans , Mice , Urinary Bladder Neoplasms/immunology , Urinary Bladder Neoplasms/pathologyABSTRACT
PURPOSE: Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP). EXPERIMENTAL DESIGN: Bladder cancer cell lines were tested for in vitro sensitivity to CDK4/6 inhibition. A novel metastatic bladder cancer mouse model was developed and used to test its in vivo activity. RESULTS: Cell lines tested were sensitive to CDK4/6 inhibition, independent on RB1 gene status. Transcriptome analyses and knockdown experiments revealed a major role for FOXM1 in this response. CDK4/6 inhibition resulted in reduced FOXM1 phosphorylation in vitro and in vivo and showed synergy with CDDP, allowing a significant tumor regression. FOXM1 exerted important oncogenic roles in bladder cancer. CONCLUSIONS: CDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.
Subject(s)
Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 6/genetics , Forkhead Box Protein M1/genetics , Urinary Bladder Neoplasms/drug therapy , Aged , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Female , Heterografts , Humans , Male , Mice , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Phosphorylation/drug effects , Progression-Free Survival , Protein Kinase Inhibitors/pharmacology , Retinoblastoma Binding Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
The Hippo-YAP (Yes-associated protein) pathway is a key regulator of tissue growth, organ size and stem cell function. More recently, a fundamental role for this pathway has emerged in stem cell function and tumorigenesis. Activation of the transcriptional co-activator YAP promotes cell-contact independent proliferation, epithelial to mesenchymal transition (EMT), cancer stem cell features and drug resistance. In this review, we describe the main components of the pathway, the microenvironment and the cell-intrinsic cues governing its activation, the downstream players of the pathway and the biological implications of their activation in the context of cancer. We will focus on the existing knowledge of this pathway in head and neck squamous carcinoma (HNSCC), its clinical value in this type of cancer as a marker of poor prognosis and resistance to therapy, as well as the most encouraging therapeutic strategies targeting the pathway.
Subject(s)
Cell Cycle Proteins/physiology , Head and Neck Neoplasms/physiopathology , Squamous Cell Carcinoma of Head and Neck/physiopathology , Transcription Factors/physiology , Biomarkers, Tumor/metabolism , Carcinogenesis/genetics , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/physiology , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Hippo Signaling Pathway , Humans , Prognosis , Protein Serine-Threonine Kinases/physiology , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathologyABSTRACT
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and although new therapeutic approaches have been recently evaluated, overall patient survival is still poor. Thus, new effective and selective clinical treatments are urgently needed. An analysis of data from large-scale, high-throughput drug screening cell line projects identified Bosutinib, a Src/Abl inhibitor that is currently used for the treatment of chronic myelogenous leukemia, as a candidate drug to treat HNSCC. Using a panel of HNSCC-derived cell lines, we found that treatment with Bosutinib reduced cell proliferation and induced apoptosis of sensitive cell lines. The drug rapidly inhibited Src and EGFR (epidermal growth factor receptor) phosphorylation, and sensitivity to Bosutinib was correlated with the activation status of EGFR. Similar findings were observed in in vivo xenograft assays using HNSCC derived cells. Moreover, in the presence of mutations in PIK3CA, the combination of Bosutinib with the PI3Kα inhibitor Alpelisib showed a synergistic effect. These results suggest that Bosutinib could be a new effective drug for the treatment of HNSCC, particularly in tumors with high EGFR activity. Its combination with Alpelisib could especially benefit patients bearing activating mutations of PIK3CA.
Subject(s)
Aniline Compounds/pharmacology , Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/drug therapy , Nitriles/pharmacology , Quinolines/pharmacology , Thiazoles/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Drug Combinations , Drug Synergism , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Injections, Subcutaneous , Mice , Mice, Nude , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Using Sos1 knockout (Sos1-KO), Sos2-KO, and Sos1/2 double-knockout (Sos1/2-DKO) mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis, including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds, as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth, and prevented malignant progression of papillomas in a DMBA (7,12-dimethylbenz[α]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation. Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair, as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors.
Subject(s)
SOS1 Protein/metabolism , Skin Neoplasms/etiology , Skin/metabolism , Son of Sevenless Proteins/metabolism , Animals , Carcinogenesis , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Homeostasis , Mice , Mice, Knockout , Neovascularization, Physiologic , Papilloma/metabolism , Papilloma/pathology , SOS1 Protein/deficiency , SOS1 Protein/genetics , Skin/blood supply , Skin/cytology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Son of Sevenless Proteins/deficiency , Son of Sevenless Proteins/genetics , Wound HealingABSTRACT
OBJECTIVES: Phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is commonly altered in many human tumors, leading to the activation of p110α enzymatic activity that stimulates growth factor-independent cell growth. PIK3CA alterations such as mutation, gene amplification and overexpression are common in head and neck squamous cell carcinoma (HNSCC) and. We aim to explore how these alterations and clinical outcome are associated, as well as the molecular mechanisms involved. MATERIAL AND METHODS: Mutation and copy-number variation in PIK3CA, and whole-genome expression profiles, were analyzed in primary HNSCC tumors from The Cancer Genome Atlas (TCGA) cohort (nâ¯=â¯243). The results were validated in an independent cohort form the University Hospital of A Coruña (UHAC, nâ¯=â¯62). Expression of the PIK3CA gene protein product (PI3K p110α) and nuclear YAP were assessed in tissue microarrays in a cohort from the University Hospital 12 de Octubre (UH12O, nâ¯=â¯91). RESULTS: Only high expression of the PIK3CA gene was associated with poor clinical outcome. The study of gene expression, transcription factor and protein signatures suggested that the activation of the Hippo-YAP pathway, involved in organ size, stem cell maintenance and tumorigenesis, could underlie tumor progression in PI3KCA overexpressing tumors. Tissue arrays showed that PI3K p110α levels correlated with YAP nuclear localization in HNSCC tumors. CONCLUSIONS: High expression of PIK3CA in HNSCC primary tumors identifies patients at high risk for recurrence. In these tumors, progression could rely on the Hippo-YAP pathway instead of the canonical Akt/mTOR pathway. This observation could have important implications in the therapeutic options for patients.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/genetics , Class I Phosphatidylinositol 3-Kinases/genetics , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , Transcription Factors/metabolism , Adult , Aged , Aged, 80 and over , Disease Progression , Female , Humans , Male , Middle Aged , Prognosis , YAP-Signaling Proteins , Young AdultABSTRACT
Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy.
Subject(s)
Carcinogenesis/genetics , Carcinoma, Squamous Cell/genetics , Cyclin E/genetics , Histones/genetics , Skin Neoplasms/genetics , Carcinoma, Squamous Cell/chemically induced , Carcinoma, Squamous Cell/pathology , Cell Differentiation/drug effects , Cell Differentiation/genetics , DNA Damage/drug effects , Doxorubicin/administration & dosage , Doxorubicin/adverse effects , Gene Expression Regulation, Neoplastic/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , Humans , Keratinocytes/drug effects , Keratinocytes/pathology , Mitosis/drug effects , Mitosis/genetics , Polyploidy , Primary Cell Culture , Skin Neoplasms/chemically induced , Skin Neoplasms/pathologyABSTRACT
IKKß (encoded by IKBKB) is a protein kinase that regulates the activity of numerous proteins important in several signaling pathways, such as the NF-κB pathway. IKKß exerts a protumorigenic role in several animal models of lung, hepatic, intestinal, and oral cancer. In addition, genomic and proteomic studies of human tumors also indicate that IKBKB gene is amplified or overexpressed in multiple tumor types. Here, the relevance of IKKß in skin cancer was determined by performing carcinogenesis studies in animal models overexpressing IKKß in the basal skin layer. IKKß overexpression resulted in a striking resistance to skin cancer development and an increased expression of several tumor suppressor proteins, such as p53, p16, and p19. Mechanistically, this skin tumor-protective role of IKKß is independent of p53, but dependent on the activity of the Ink4a/Arf locus. Interestingly, in the absence of p16 and p19, IKKß-increased expression favors the appearance of cutaneous spindle cell-like squamous cell carcinomas, which are highly aggressive tumors. These results reveal that IKKß activity prevents skin tumor development, and shed light on the complex nature of IKKß effects on cancer progression, as IKKß can both promote and prevent carcinogenesis depending on the cell type or molecular context.Implications: The ability of IKKß to promote or prevent carcinogenesis suggests the need for further evaluation when targeting this protein. Mol Cancer Res; 15(9); 1255-64. ©2017 AACR.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p16/metabolism , I-kappa B Kinase/biosynthesis , Skin Neoplasms/metabolism , Tumor Suppressor Protein p14ARF/metabolism , Animals , Cyclin-Dependent Kinase Inhibitor p16/genetics , Epigenesis, Genetic , I-kappa B Kinase/genetics , Mice , Mice, Transgenic , Mutation , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis.
Subject(s)
Down-Regulation , Epidermis/metabolism , Skin Neoplasms/genetics , Transcription Factors/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Animals , Cell Line , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Keratinocytes/metabolism , Mice , Microscopy, Fluorescence , Mutation , Neoplasm Metastasis , Phosphoproteins/genetics , Point Mutation , Skin/pathology , Skin Neoplasms/metabolism , Stem Cells/cytology , Trans-Activators/geneticsABSTRACT
Hair follicle stem cells (HF-SCs) alternate between periods of quiescence and proliferation, to finally differentiate into all the cell types that constitute the hair follicle. Also, they have been recently identified as cells of origin in skin cancer. HF-SCs localize in a precise region of the hair follicle, the bulge, and molecular markers for this population have been established. Thus, HF-SCs are good model to study the potential role of oncogenic activations on SC physiology. Expression of a permanently active form of Akt (myrAkt) in basal cells leads to Akt hyperactivation specifically in the CD34(+)Itga6(H) population. This activation causes bulge stem cells to exit from quiescence increasing their response to proliferative stimuli and affecting some functions such as cell migration. HF-SC identity upon Akt activation is preserved; in this sense, increased proliferation does not result in stem cell exhaustion with age suggesting that Akt activation does not affect self-renewal an important aspect for normal tissue maintenance and cancer development. Genome-wide transcriptome analysis of HF-SC isolated from myrAkt and wild-type epidermis underscores changes in metabolic pathways characteristic of cancer cells. These differences manifest during a two-step carcinogenesis protocol in which Akt activation in HF-SCs results in increased tumor development and malignant transformation.
Subject(s)
Carcinogenesis/metabolism , Epidermis/enzymology , Skin Neoplasms/enzymology , Stem Cells/enzymology , Animals , Cell Proliferation , Cells, Cultured , Enzyme Activation , Epidermis/pathology , Keratinocytes/enzymology , Mice, Transgenic , Proto-Oncogene Proteins c-akt , Re-Epithelialization , Signal TransductionABSTRACT
The retinoblastoma gene product (pRb) controls proliferation and differentiation processes in stratified epithelia. Importantly, and in contrast to other tissues, Rb deficiency does not lead to spontaneous skin tumor formation. As the cyclin-dependent kinase inhibitor p21 regulates proliferation and differentiation in the absence of pRb, we analyzed the consequences of deleting p21 in pRb-ablated stratified epithelia (hereafter pRb(ΔEpi);p21-/-). These mice display an enhancement of the phenotypic abnormalities observed in pRb(ΔEpi) animals, indicating that p21 partially compensates pRb absence. Remarkably, pRb(ΔEpi);p21-/- mice show an acute skin inflammatory phenotype and develop spontaneous epithelial tumors, particularly affecting tongue and oral tissues. Biochemical analyses and transcriptome studies reveal changes affecting multiple pathways, including DNA damage and p53-dependent signaling responses. Comparative metagenomic analyses, together with the histopathological profiles, indicate that these mice constitute a faithful model for human head and neck squamous cell carcinomas. Collectively, our findings demonstrate that p21, in conjunction with pRb, has a central role in regulating multiple epithelial processes and orchestrating specific tumor suppressor functions.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Epithelium/metabolism , Gene Expression Regulation, Neoplastic , Inflammation , Retinoblastoma Protein/metabolism , Animals , Carcinoma, Squamous Cell/metabolism , Cell Proliferation , DNA Damage , Female , Gene Expression Profiling , Head and Neck Neoplasms/metabolism , Humans , Keratinocytes/cytology , Mice , Mice, SCID , Phenotype , Signal Transduction , Skin/pathology , Transcriptome , Tumor Suppressor Protein p53/metabolismABSTRACT
The specific ablation of Rb1 gene in stratified epithelia (Rb(F/F);K14cre) promotes proliferation and altered differentiation but is insufficient to produce spontaneous tumors. The pRb relative, p107, compensates some of the functions of pRb in these tissues; however, Rb(F/F);K14cre;p107(-/-) mice die postnatally. Here we show, using an inducible mouse model (Rb(F/F);K14creER(TM)), that p107 exerts specific tumor suppressor functions in the absence of pRb in stratified epithelia. The simultaneous absence of pRb and p107 produces impaired p53 transcriptional functions and reduction of Pten expression, allowing spontaneous squamous carcinoma development. These tumors display significant overlap with human squamous carcinomas, supporting that Rb(F/F);K14creER(TM);p107(-/-) mice might constitute a new model for these malignancies. Remarkably tumor development in vivo is partially alleviated by mTOR inhibition. These data demonstrate the existence of a previously unreported functional connection between pRb, Pten and p53 tumor suppressors, through p107, of a particular relevance in squamous tumor development.