RESUMEN
Members of the p53 family of transcription factors-p53, p63, and p73-share a high degree of homology; however, members can be activated in response to different stimuli, perform distinct (sometimes opposing) roles and are expressed in different tissues. The level of complexity is increased further by the transcription of multiple isoforms of each homolog, which may interact or interfere with each other and can impact cellular outcome. Proteins perform their functions through interacting with other proteins (and/or with nucleic acids). Therefore, identification of the interactors of a protein and how they interact in 3D is essential to fully comprehend their roles. By utilizing an in silico protein-protein interaction prediction method-HMI-PRED-we predicted interaction partners of p53 family members and modeled 3D structures of these protein interaction complexes. This method recovered experimentally known interactions while identifying many novel candidate partners. We analyzed the similarities and differences observed among the interaction partners to elucidate distinct functions of p53 family members and provide examples of how this information may yield mechanistic insight to explain their overlapping versus distinct/opposing outcomes in certain contexts. While some interaction partners are common to p53, p63, and p73, the majority are unique to each member. Nevertheless, most of the enriched pathways associated with these partners are common to all members, indicating that the members target the same biological pathways but through unique mediators. p63 and p73 have more common enriched pathways compared to p53, supporting their similar developmental roles in different tissues.
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Factores de Transcripción , Proteína p53 Supresora de Tumor , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Overexpression of ΔNp63α, a member of the p53/p63/p73 family of transcription factors, is a molecular attribute of human squamous cancers of the head and neck, lung and skin. The TP63 gene plays important roles in epidermal morphogenesis and homeostasis, regulating diverse biological processes including epidermal fate decisions and keratinocyte proliferation and survival. When overexpressed experimentally in primary mouse keratinocytes, ΔNp63α maintains a basal cell phenotype including the loss of normal calcium-mediated growth arrest, at least in part through the activation and enhanced nuclear accumulation of the c-rel subunit of NF-κB (Nuclear Factor-kappa B). Initially identified for its role in the immune system and hematopoietic cancers, c-Rel has increasingly been associated with solid tumors and other pathologies. ΔNp63α and c-Rel have been shown to be associated in the nuclei of ΔNp63α overexpressing human squamous carcinoma cells. Together, these transcription factors cooperate in the transcription of genes regulating intrinsic keratinocyte functions, as well as the elaboration of factors that influence the tumor microenvironment (TME). This review provides an overview of the roles of ΔNp63α and c-Rel in normal epidermal homeostasis and elaborates on how these pathways may intersect in pathological conditions such as cancer and the associated TME.
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Carcinoma de Células Escamosas/metabolismo , Células Epiteliales/metabolismo , Homeostasis/fisiología , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinoma de Células Escamosas/patología , Células Epiteliales/patología , Humanos , Microambiente Tumoral/fisiologíaRESUMEN
The p63 gene is a member of the p53/p63/p73 family of transcription factors and plays a critical role in development and homeostasis of squamous epithelium. p63 is transcribed as multiple isoforms; ΔNp63α, the predominant p63 isoform in stratified squamous epithelium, is localized to the basal cells and is overexpressed in squamous cell cancers of multiple organ sites, including skin, head and neck, and lung. Further, p63 is considered a stem cell marker, and within the epidermis, ΔNp63α directs lineage commitment. ΔNp63α has been implicated in numerous processes of skin biology that impact normal epidermal homeostasis and can contribute to squamous cancer pathogenesis by supporting proliferation and survival with roles in blocking terminal differentiation, apoptosis, and senescence, and influencing adhesion and migration. ΔNp63α overexpression may also influence the tissue microenvironment through remodeling of the extracellular matrix and vasculature, as well as by enhancing cytokine and chemokine secretion to recruit pro-inflammatory infiltrate. This review focuses on the role of ΔNp63α in normal epidermal biology and how dysregulation can contribute to cutaneous squamous cancer development, drawing from knowledge also gained by squamous cancers from other organ sites that share p63 overexpression as a defining feature.
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Carcinoma de Células Escamosas/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias Pulmonares/genética , Neoplasias Cutáneas/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Adhesión Celular , Linaje de la Célula/genética , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Epidermis/metabolismo , Epidermis/patología , Células Epiteliales/patología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
While overexpression of the p63 isoform, ΔNp63α, has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed ΔNp63α aberrantly maintains proliferation of primary mouse keratinocytes under conditions that normally induce growth arrest and differentiation. To identify genes downstream of dysregulated ΔNp63α that may contribute to squamous cancer development and progression, we performed microarray analyses using primary mouse keratinocytes. Herein we report that elevated ΔNp63α differentially regulates genes involved in a variety of cellular functions. Of note, multiple protease inhibitor mRNAs were downregulated including: maspin (serpinB5); plasminogen activator inhibitor-2 (PAI-2; serpinB2); and tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted TIMP-3 and PAI-2 protein declined in the presence of dysregulated ΔNp63α, however secreted maspin remained stable. Intracellular maspin protein expression decreased in response to overexpressed ΔNp63α, as did PAI-2. In contrast, TIMP-3 protein was not detected intracellularly, supporting a solely extracellular function. Electrophoretic mobility shift assays (EMSAs) using a maspin promoter p53/p63 consensus sequence revealed endogenous transcription factor(s) binding to this sequence in keratinocytes that was disrupted by overexpressed ΔNp63α. This was confirmed by ChIP assays. This binding was interrupted by the addition of antibodies recognizing p73, but not p53 or p63, and significantly diminished in EMSA reactions from p73(-/-) keratinocytes, confirming p73 as a constituent. Physical association between p73/ΔNp63α was observed in control ß-gal overexpressing keratinocytes and was enhanced in the presence of overexpressed ΔNp63α These findings underscore the importance of properly balanced p53 homologs for tissue homeostasis.
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Proteínas de Unión al ADN/fisiología , Queratinocitos/metabolismo , Proteínas Nucleares/fisiología , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas/genética , Serpinas/genética , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/fisiología , Animales , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Queratinocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genética , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Introduction: Amplification of human chromosome 3q26-29, which encodes oncoprotein ΔNp63 among other isoforms of the p63 family, is a feature common to squamous cell carcinomas (SCCs) of multiple tissue origins. Along with overexpression of ΔNp63, activation of the protooncogene, RAS, whether by overexpression or oncogenic mutation, is frequently observed in many cancers. In this study, analysis of transcriptome data from The Cancer Genome Atlas (TCGA) demonstrated that expression of TP63 mRNA, particularly ΔNp63 isoforms, and HRAS are significantly elevated in advanced squamous cell carcinomas of the head and neck (HNSCCs), suggesting pathological significance. However, how co-overexpressed ΔNp63 and HRAS affect the immunosuppressive tumor microenvironment (TME) is incompletely understood. Methods: Here, we established and characterized an immune competent mouse model using primary keratinocytes with retroviral-mediated overexpression of ΔNp63α and constitutively activated HRAS (v-rasHa G12R) to evaluate the role of these oncogenes in the immune TME. Results: In this model, orthotopic grafting of wildtype syngeneic keratinocytes expressing both v-rasHa and elevated levels of ΔNp63α consistently yield carcinomas in syngeneic hosts, while cells expressing v-rasHa alone yield predominantly papillomas. We found that polymorphonuclear (PMN) myeloid cells, experimentally validated to be immunosuppressive and thus representing myeloid-derived suppressor cells (PMN-MDSCs), were significantly recruited into the TME of carcinomas arising early following orthotopic grafting of ΔNp63α/v-rasHa-expressing keratinocytes. ΔNp63α/v-rasHa-driven carcinomas expressed higher levels of chemokines implicated in recruitment of MDSCs compared to v-rasHa-initiated tumors, providing a heretofore undescribed link between ΔNp63α/HRAS-driven carcinomas and the development of an immunosuppressive TME. Conclusion: These results support the utilization of a genetic carcinogenesis model harboring specific genomic drivers of malignancy to study mechanisms underlying the development of local immunosuppression.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Células Supresoras de Origen Mieloide , Humanos , Animales , Ratones , Carcinoma de Células Escamosas/genética , Inmunosupresores , Carcinoma de Células Escamosas de Cabeza y Cuello , Modelos Animales de Enfermedad , Microambiente Tumoral/genéticaRESUMEN
On December 16, 2020, the FDA granted regular approval to margetuximab-cmkb (MARGENZA), in combination with chemotherapy, for the treatment of adult patients with HER2-positive (HER2+) metastatic breast cancer who have received two or more prior anti-HER2 regimens, at least one of which was for metastatic disease. Approval was based on data from SOPHIA, a multicenter, randomized, open-label, active controlled study comparing margetuximab with trastuzumab, in combination with chemotherapy. The primary efficacy endpoint was progression-free survival (PFS) by blinded independent central review. SOPHIA demonstrated a 0.9-month difference in median PFS between the two treatment arms [5.8 vs. 4.9 months, respectively; stratified HR, 0.76 (95% confidence interval: 0.59-0.98; P = 0.0334)]. Overall survival (OS) was immature at the data cut-off date of September 10, 2019. Infusion-related reactions (IRR) are an important safety signal associated with margetuximab plus chemotherapy. In SOPHIA, 13% of patients treated with margetuximab plus chemotherapy reported IRRs, of which 1.5% were grade 3. The most commonly reported adverse drug reactions (>10%) with margetuximab in combination with chemotherapy were fatigue/asthenia, nausea, diarrhea, vomiting, constipation, headache, pyrexia, alopecia, abdominal pain, peripheral neuropathy, arthralgia/myalgia, cough, decreased appetite, dyspnea, IRR, palmar-plantar erythrodysesthesia, and extremity pain. Overall, the favorable risk-benefit profile for margetuximab when added to chemotherapy supported its approval for the intended indication.
Asunto(s)
Neoplasias de la Mama , Adulto , Anticuerpos Monoclonales/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/patología , Aprobación de Drogas , Femenino , Humanos , Receptor ErbB-2/uso terapéutico , Trastuzumab/efectos adversosRESUMEN
Transcriptional regulation by p53 is critical for p53-mediated tumour suppression; however, p53-mediated transactivation has been dissociated from p53-mediated biological processes including apoptosis, DNA repair, and differentiation. We compared the effects of a mutant allele, p53(QS - val135), containing a double mutation in the amino-terminus abrogating transactivation activity and a modification at amino acid 135 partially affecting DNA binding, to complete loss of p53. We applied in vitro endpoints correlated with epithelial tumourigenesis and an in vivo assay of tumour phenotype to assess whether loss of p53-mediated transcriptional regulation underlies the malignant phenotype of p53(-/-)/v-ras(Ha)-overexpressing keratinocytes. Transactivation deficiency of p53QS-val135 was confirmed by reporter gene assays in fibroblasts and differentiating keratinocytes. Ras oncogene-induced senescence was lost in both p53(QS - val135/QS - val135) and p53(-/-) keratinocytes. Similarly, p53(QS - val135/QS - val135), like p53(-/-), cooperated with v-ras(Ha) to enhance malignant conversion. The tumours arising in p53(QS - val135/QS - val135) keratinocytes displayed strong nuclear p53 expression; thus, the p53(QS - val135) allele was maintained and the deficient transactivation function of the expressed p53QS mutant protein was supported by absence of p21(waf1) in these tumours. The p53(QS - val135) allele did not confer a dominant-negative phenotype, as p53(+/QS - val135) keratinocytes senesced normally in response to v-ras(Ha) expression and formed benign tumours. While p53(-/-) keratinocytes displayed diminished response to TGF-beta, p53(QS - val135/QS - val135) and p53(+/+) keratinocytes responded equivalently, indicating that the requirement for p53 in maximizing TGF-beta-mediated growth regulation is independent of its transactivation domain and that the ability of keratinocytes to respond to TGF-beta is insufficient to suppress the malignant phenotype in this model. Furthermore, TGF-beta enhances p53QS-induced activation of a dual p53-TGF-beta responsive reporter in a keratinocyte cell line. These findings support an essential role for p53-mediated transcriptional regulation in suppressing malignancies arising from ras-induced skin tumours, consistent with previous findings in spontaneous carcinogenesis in other organs, and highlight the potential importance of senescence for tumour suppression in vivo.
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Carcinoma de Células Escamosas/prevención & control , Transformación Celular Neoplásica/genética , Genes p53/genética , Neoplasias Cutáneas/prevención & control , Factor de Crecimiento Transformador beta/fisiología , Animales , Carcinoma de Células Escamosas/genética , Células Cultivadas , Senescencia Celular/genética , Genotipo , Queratinocitos/trasplante , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Neoplasias Cutáneas/genética , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.
Asunto(s)
Apoptosis/fisiología , Canales de Cloruro/metabolismo , Daño del ADN , Proteínas Proto-Oncogénicas c-bcl-2 , Proteína p53 Supresora de Tumor/metabolismo , Animales , Sitios de Unión/genética , Células Cultivadas , Canales de Cloruro/genética , Expresión Génica , Genes p53 , Queratinocitos/citología , Queratinocitos/metabolismo , Potenciales de la Membrana , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transfección , Proteína X Asociada a bcl-2RESUMEN
deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.
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Diferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Queratinocitos/fisiología , Fosfoproteínas , Transactivadores , Factores de Transcripción/metabolismo , Adenoviridae/genética , Clorometilcetonas de Aminoácidos/farmacología , Animales , Sitios de Unión , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Calcio/metabolismo , Calcio/farmacología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Proteínas Filagrina , Genes Reporteros , Genes Supresores de Tumor , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Osteosarcoma/genética , Osteosarcoma/patología , Isoformas de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de TumorRESUMEN
On February 22, 2013, the FDA licensed ado-trastuzumab emtansine (Kadcyla; Genentech, Inc.) for use as a single agent for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC) who previously received trastuzumab and a taxane, separately or in combination. The clinical basis for licensure was a phase III trial in 991 patients with HER2-positive MBC that randomly allocated patients to receive ado-trastuzumab emtansine (n=495) or lapatinib in combination with capecitabine (n=496). The coprimary endpoints were progression-free survival (PFS) based on tumor assessments by an independent review committee and overall survival (OS). Statistically significant improvements in PFS and OS were observed in patients receiving ado-trastuzumab emtansine compared with patients receiving lapatinib plus capecitabine [difference in PFS medians of 3.2 months, HR, 0.65 (95% confidence interval, CI, 0.55-0.77), P<0.0001 and difference in OS medians of 5.8 months, HR, 0.68 (95% CI, 0.55-0.85), P=0.0006]. The most common adverse reactions in patients receiving ado-trastuzumab emtansine were fatigue, nausea, musculoskeletal pain, thrombocytopenia, headache, increased aminotransferase levels, and constipation. Other significant adverse reactions included hepatobiliary disorders and left ventricular dysfunction. Given the PFS and OS results, the benefit-risk profile was considered favorable.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Receptor ErbB-2/genética , Ado-Trastuzumab Emtansina , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/efectos adversos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Aprobación de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Humanos , Maitansina/administración & dosificación , Maitansina/efectos adversos , Persona de Mediana Edad , Trastuzumab , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Mouse models have informed us that p63 is critical for normal epidermal development and homeostasis. The p53/p63/p73 family is expressed as multiple protein isoforms due to a combination of alternative promoter usage and C-terminal alternative splicing. These isoforms can mimic or interfere with one another, and their balance ultimately determines biological outcome in a context-dependent manner. While not frequently mutated, p63, and in particular the ΔNp63 subclass, is commonly overexpressed in human squamous cell cancers. In vitro keratinocytes and murine transgenic and transplantation models have been invaluable in elucidating the contribution of altered p63 levels to cancer development, and studies have identified the roles for ΔNp63 isoforms in keratinocyte survival and malignant progression, likely due in part to their transcriptional regulatory function. These findings can be extended to human cancers; for example, the novel recognition of NF κ B/c-Rel as a downstream effector of p63 has identified a role for NF κ B/c-Rel in human squamous cell cancers. These models will be critical in enhancing the understanding of the specific molecular mechanisms of cancer development and progression.
RESUMEN
On June 8, 2012, the U.S. Food and Drug Administration (FDA) approved pertuzumab (Perjeta, Genentech) for use in combination with trastuzumab (Herceptin, Genentech) and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer (MBC) who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Approval was based on the results of a randomized, double-blind, placebo-controlled trial conducted in 808 patients with HER2-positive MBC. Patients were randomized (1:1) to receive pertuzumab (n = 402) or placebo (n = 406) in combination with trastuzumab and docetaxel. The primary endpoint was progression-free survival (PFS) and a key secondary endpoint was overall survival (OS). A statistically significant improvement in PFS (difference in medians of 6.1 months) was observed in patients receiving pertuzumab [HR, 0.62; 95% confidence interval (CI), 0.51-0.75; P < 0.0001]. A planned interim analysis suggested an improvement in OS (HR, 0.64; 95% CI, 0.47-0.88; P = 0.0053) but the HR and P value did not cross the stopping boundary. Common adverse reactions (>30%) observed in patients on the pertuzumab arm included diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy. No additive cardiac toxicity was observed. Significant manufacturing issues were identified during the review. On the basis of substantial evidence of efficacy for pertuzumab in MBC and the compelling public health need, FDA did not delay availability to patients pending final resolution of all manufacturing concerns. Therefore, FDA approved pertuzumab but limited its approval to lots not affected by manufacturing problems. The applicant agreed to multiple manufacturing and testing postmarketing commitments under third-party oversight to resolve manufacturing issues.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aprobación de Drogas , Metástasis de la Neoplasia/tratamiento farmacológico , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/administración & dosificación , Docetaxel , Femenino , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptor ErbB-2/metabolismo , Taxoides/administración & dosificación , Trastuzumab , Resultado del Tratamiento , Estados Unidos , United States Food and Drug AdministrationRESUMEN
p63 is critical for squamous epithelial development, and elevated levels of the ΔNp63α isoform are seen in squamous cell cancers of various organ sites. However, significant controversy exists regarding the role of p63 isoforms as oncoproteins or tumor suppressors. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in primary keratinocytes. Elevated levels of ΔNp63α in vitro promote long-term survival and block both replicative and oncogene-induced senescence in primary keratinocytes, as evidenced by the expression of SA-ß-gal and the presence of nuclear foci of heterochromatin protein 1γ. The contribution of ΔNp63α to cancer development was assessed using an in vivo grafting model of experimental skin tumorigenesis that allows distinction between benign and malignant tumors. Grafted lenti-ΔNp63α keratinocytes do not form tumors, whereas lenti-GFP/v-ras(Ha) keratinocytes develop well-differentiated papillomas. Lenti-ΔNp63α/v-ras(Ha) keratinocytes form undifferentiated carcinomas. The average volume of lenti-ΔNp63α/v-ras(Ha) tumors was significantly higher than those in the lenti-GFP/v-ras(Ha) group, consistent with increased BrdU incorporation detected by immunohistochemistry. The block in oncogene-induced senescence corresponds to sustained levels of E2F1 and phosphorylated AKT, and is associated with loss of induction of p16(ink4a)/p19(arf). The relevance of p16(ink4a)/p19(arf) loss was demonstrated in grafting studies of p19(arf)-null keratinocytes, which develop malignant carcinomas in the presence of v-ras(Ha) similar to those arising in wildtype keratinocytes that express lenti-ΔNp63α and v-ras(Ha). Our findings establish that ΔNp63α has oncogenic activity and its overexpression in human squamous cell carcinomas contributes to the malignant phenotype, and implicate its ability to regulate p16(ink4a)/p19(arf) in the process.
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Transformación Celular Neoplásica/metabolismo , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/antagonistas & inhibidores , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Regulación de la Expresión Génica , Queratinocitos/patología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Supervivencia Celular , Transformación Celular Neoplásica/patología , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/metabolismo , Expresión Génica , Células HEK293 , Humanos , Queratinocitos/metabolismo , Lentivirus/genética , Ratones , Fenotipo , Factores de Tiempo , Regulación hacia Arriba/genética , Proteínas ras/metabolismoRESUMEN
Inflammation-induced activation of proto-oncogenic NF-κB/REL and dysfunction of tumor suppressor TP53/p63/p73 family transcription factors are key events in cancer progression. How inflammatory signaling coordinates dysregulation of these two transcription factor families during oncogenesis remains incompletely understood. Here, we observed that oncoprotein c-REL and tumor suppressor TAp73 are coexpressed and complex with ΔNp63α in the nucleus of a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with mutant (mt)TP53. TNF-α, a proinflammatory cytokine, promoted c-REL nuclear translocation, c-REL/ΔNp63α interaction, and dissociation of TAp73 from ΔNp63α and the nucleus to the cytoplasm, whereas c-REL siRNA knockdown attenuated this effect. Overexpression of c-REL or a c-REL κB-site DNA-binding mutant enhanced protein interaction with ΔNp63α and TAp73 dissociation, implicating c-REL/ΔNp63α-specific interactions in these effects. We discovered that TNF-α or genetic alteration of c-REL expression inversely modulates ΔNp63α/TAp73 interactions on distinct p63 DNA-binding sites, including those for key growth arrest and apoptotic genes p21WAF1, NOXA, and PUMA. Functionally, c-REL repressed these genes and the antiproliferative effects of TNF-α or TAp73. Conversely, c-REL siRNA depletion enhanced TAp73 promoter interaction and expression of genes mediating growth arrest and apoptosis. Similar to TNF-α-treated HNSCC lines, human HNSCC tumors and hyperplastic squamous epithelia of transgenic mice overexpressing ΔNp63α that exhibit inflammation also show increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 localization. These findings unveil a novel and reversible dynamic mechanism whereby proinflammatory cytokine TNF-α-induced c-REL/ΔNp63α interactions inactivate tumor suppressor TAp73 function, promoting TNF-α resistance and cell survival in cancers with mtTP53.
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Apoptosis/genética , Carcinoma de Células Escamosas/patología , Proteínas de Unión al ADN/metabolismo , Neoplasias de Cabeza y Cuello/patología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Sitios de Unión , Carcinoma de Células Escamosas/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Núcleo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN de Neoplasias/metabolismo , Resistencia a Medicamentos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes p53 , Genes rel , Neoplasias de Cabeza y Cuello/genética , Humanos , Complejos Multiproteicos , Regiones Promotoras Genéticas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-rel , ARN Interferente Pequeño/farmacologíaRESUMEN
The p53 homologue DeltaNp63alpha is overexpressed and inhibits apoptosis in a subset of human squamous cell carcinomas (SCC). Here, we report that in normal keratinocytes overexpressing DeltaNp63alpha and in human squamous carcinoma cells, DeltaNp63alpha physically associates with phosphorylated, transcriptionally active nuclear c-Rel, a nuclear factor-kappaB family member, resulting in increased c-Rel nuclear accumulation. This accumulation and the associated enhanced proliferation driven by elevated DeltaNp63alpha are attenuated by c-Rel small interfering RNA or overexpression of mutant IkappaBalphaM, indicating that c-Rel-containing complex formation is critical to the ability of elevated DeltaNp63alpha to maintain proliferation in the presence of growth arresting signals. Consistent with a role in growth regulation, DeltaNp63alpha-c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human squamous carcinoma cells and normal keratinocytes overexpressing DeltaNp63alpha. The relationship between DeltaNp63alpha and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of primary head and neck SCC. This is the first report indicating that high levels of DeltaNp63alpha interact with activated c-Rel in keratinocytes and SCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers.
Asunto(s)
Proliferación Celular , Células Epiteliales/fisiología , FN-kappa B/fisiología , Transducción de Señal/fisiología , Transactivadores/metabolismo , Transactivadores/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Animales , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patología , Células Cultivadas , Células Epiteliales/metabolismo , Neoplasias de Cabeza y Cuello/etiología , Neoplasias de Cabeza y Cuello/patología , Humanos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-rel/metabolismo , Proteínas Proto-Oncogénicas c-rel/fisiología , Homología de Secuencia , Transactivadores/genética , Factores de Transcripción , Transfección , Proteína p53 Supresora de Tumor/química , Proteínas Supresoras de Tumor/genéticaRESUMEN
p63 is a member of a gene family also including the p53 tumor suppressor and p73. In contrast to p53, p63 is rarely mutated in human cancers. Rather, gene amplification and dysregulated expression of p63 protein have been observed, particularly in squamous cell carcinomas. p63 is essential for development of stratified squamous epithelium, including the epidermis. The p63 gene is expressed as multiple protein isoforms with different functional capacities, and the balance of these isoforms, along with the presence or absence of the other family members, p53 and p73, can impact biological outcome. Both gene silencing and overexpression approaches have been utilized to elucidate the contributions of specific p63 isoforms to normal epidermal morphogenesis and tissue maintenance. While numerous studies have established the essential nature of p63 in the epidermis, the basis of this requirement, and the unique, as well as, overlapping functions of the individual isoforms, remain controversial. In this review, we summarize the current understanding of roles played by specific p63 isoforms within the context of epidermal morphogenesis and homeostasis of the established epidermis, and the potential impact of p63 dysregulation on cancer development.
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Epidermis/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/fisiología , Fosfoproteínas/fisiología , Neoplasias Cutáneas/metabolismo , Transactivadores/fisiología , Animales , Células Epidérmicas , Homeostasis , Humanos , Ratones , Morfogénesis , Neoplasias Cutáneas/patologíaRESUMEN
In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.
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Cisplatino/toxicidad , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Aductos de ADN/metabolismo , Dosificación de Gen/efectos de los fármacos , Dosificación de Gen/fisiología , Ratones , Ratones Noqueados , Factores de TiempoRESUMEN
Ectodermal dysplasias (EDs) are a group of human pathological conditions characterized by anomalies in organs derived from epithelial-mesenchymal interactions during development. Dlx3 and p63 act as part of the transcriptional regulatory pathways relevant in ectoderm derivatives, and autosomal mutations in either of these genes are associated with human EDs. However, the functional relationship between both proteins is unknown. Here, we demonstrate that Dlx3 is a downstream target of p63. Moreover, we show that transcription of Dlx3 is abrogated by mutations in the sterile alpha-motif (SAM) domain of p63 that are associated with ankyloblepharon-ectodermal dysplasia-clefting (AEC) dysplasias, but not by mutations found in ectrodactylyectodermal dysplasia-cleft lip/palate (EEC), Limb-mammary syndrome (LMS) and split hand-foot malformation (SHFM) dysplasias. Our results unravel aspects of the transcriptional cascade of events that contribute to ectoderm development and pathogenesis associated with p63 mutations.
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Displasia Ectodérmica/etiología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Fosfoproteínas/genética , Transactivadores/genética , Factores de Transcripción/genética , Animales , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Displasia Ectodérmica/genética , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Masculino , Ratones , Mutación , Osteosarcoma/patología , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Piel/citología , Transcripción GenéticaRESUMEN
An increasing number of monoclonal antibodies for cancer diagnosis and treatment are in clinical use and in the development pipeline, with more expected as new molecular targets are identified. As with all drugs, product quality, an appropriate pre-clinical pharmacology-toxicology testing program, and well-designed clinical trials are essential for a successful drug development program. However, protein products such as monoclonal antibodies present unique regulatory concerns. The derivation from biological sources as well as the constantly evolving technologies utilized to develop these products demands continuous appraisal of safety concerns, even while the accumulated experience with these protein products has facilitated their safety evaluations. Because of the complex nature of these products and their inherent heterogeneity, a mechanistic understanding of the mode of action along with careful attention to product design and manufacture are critical to assuring a safe, effective and consistent product. Protein products may be highly species specific, thus pharmacologically relevant animal models are an important component in accurately assessing pre-clinical safety and establishing initial dosing. Furthermore, the immunogenicity of protein products can impact its safety profile, dose exposure, and efficacy. Mechanistic insight should form the basis of biological assays used for monitoring efficacy, safety, lot-to-lot consistency and manufacturing changes. The inherent uniqueness of each product necessitates a flexible case-by-case approach for biologics review that is based on a strong scientific understanding of relative risks. This review will provide an overview of approaches used in the development of antibody-based cancer therapeutics and the scientific basis of regulatory reviews.
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Anticuerpos Monoclonales , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Evaluación Preclínica de Medicamentos , Control de Medicamentos y Narcóticos , HumanosRESUMEN
As a broad-acting cyclin-dependent kinase inhibitor, p21(WAF1) occupies a central position in the cell cycle regulation of self-renewing tissues such as oral mucosa and skin. In addition to regulating normal cell cycle progression decisions, p21(WAF1) integrates genotoxic insults into growth arrest and apoptotic signaling pathways that ultimately determine cell fate. As a result of its complex interactions with cell cycle machinery and response to mutagenic agents, p21(WAF1) also has stage-specific roles in epithelial carcinogenesis. Finally, a view is emerging of p21(WAF1) as not merely a cyclin-dependent kinase inhibitor, but also as a direct participant in regulating genes involved in growth arrest, senescence, and aging, thus providing an additional layer of control over matters of the cell cycle. This review discusses these various roles played by p21(WAF1) in cell cycle control, and attempts to relate these to epithelial cell biology, with special emphasis on keratinocytes.