RESUMEN
Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49f(high)/ALDH1A1(high)/H3K4/K27me3(low) subpopulation (CD49f+) of tumor cells. A strikingly similar CD49f(high)/H3K27me3(low) subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49f(high)/ALDH(high), label retaining cells (LRC) proliferated immediately in vivo, with time the CD49f(low)/ALDH(low), non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49f(high)/ALDH(high), label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2 phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f- cells can "reprogram" and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a "moving target" and their eradication might require more persistent strategies.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Células Madre Neoplásicas/patología , Animales , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Embrión de Pollo , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Células Madre Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
The stress-activated kinase p38 plays key roles in tumor suppression and induction of tumor cell dormancy. However, the mechanisms behind these functions remain poorly understood. Using computational tools, we identified a transcription factor (TF) network regulated by p38alpha/beta and required for human squamous carcinoma cell quiescence in vivo. We found that p38 transcriptionally regulates a core network of 46 genes that includes 16 TFs. Activation of p38 induced the expression of the TFs p53 and BHLHB3, while inhibiting c-Jun and FoxM1 expression. Furthermore, induction of p53 by p38 was dependent on c-Jun down-regulation. Accordingly, RNAi down-regulation of BHLHB3 or p53 interrupted tumor cell quiescence, while down-regulation of c-Jun or FoxM1 or overexpression of BHLHB3 in malignant cells mimicked the onset of quiescence. Our results identify components of the regulatory mechanisms driving p38-induced cancer cell quiescence. These may regulate dormancy of residual disease that usually precedes the onset of metastasis in many cancers.
Asunto(s)
Neoplasias/patología , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Embrión de Pollo , Biología Computacional/métodos , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Humanos , Imidazoles/farmacología , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Piridinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Pancreatic endoplasmic reticulum kinase (PERK)-eIF2 alpha signaling, a component of the endoplasmic reticulum (ER) stress response, has been proposed as a therapeutic target due to its importance to cell survival in hypoxic tumors. In this study, we show that in addition to promoting survival, PERK can also suppress tumor growth of advanced carcinomas. Our results show that in squamous carcinoma T-HEp3 cells, which display low PERK-eIF2 alpha signaling, inducible activation of an Fv2E-PERK fusion protein results in a strong G(0)-G(1) arrest in vitro. Most importantly, Fv2E-PERK activation, in addition to promoting survival in vitro, inhibits T-HEp3 and SW620 colon carcinoma growth in vivo. Increased PERK activation is linked to enhanced p-eIF2 alpha levels, translational repression, and a decrease in Ki67, pH 3, and cycD1/D3 levels, but not to changes in angiogenesis or apoptosis. Experimental reduction of PERK activity, or overexpression of GADD34 in a spontaneously arising in vivo quiescent variant of HEp3 cells that displays strong basal PERK-eIF2 alpha activation, reverts their quiescent phenotype. We conclude that the growth-inhibitory function of PERK is preserved in tumors and upon proper reactivation can severely inhibit tumor growth through induction of quiescence. This is an important consideration in the development of PERK-based therapies, as its inhibition may facilitate the proliferation of slow-cycling or dormant tumor cells.
Asunto(s)
Carcinoma de Células Escamosas/patología , Proliferación Celular , eIF-2 Quinasa/fisiología , Animales , Apoptosis/genética , Carcinoma de Células Escamosas/genética , Supervivencia Celular/genética , Embrión de Pollo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Transducción de Señal/fisiología , Células Tumorales Cultivadas , eIF-2 Quinasa/genéticaRESUMEN
Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2alpha. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2alpha and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-DeltaC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKDeltaC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.
Asunto(s)
eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis , Mama/enzimología , Ciclo Celular , Línea Celular , Retículo Endoplásmico/enzimología , Células Epiteliales/citología , Células Epiteliales/enzimología , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Ratones , Fosforilación , ARN Mensajero/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Regulación hacia ArribaRESUMEN
In this chapter, we present an approach using genomic and ribonomic profiling to investigate functional gene programs in a tumor growth model. To reach this goal, ribonomic profiling was combined with RNA interference in a tumor dormancy model. Strategies merging functional genomic technologies are outlined for the identification of novel posttranscriptionally regulated targets of p38 to show that they are functionally linked to the induction or interruption of cellular growth in cancer. In the first section of this chapter, we describe a method for the detection of mRNA subsets associated with RNA-binding proteins such as hnRNP A1 using (1) immunopurification of mRNA-protein complexes, from either whole cell lysates or subcellular fractions and (2) gene expression arrays to find those mRNAs bound to hnRNP A1. In the second section, short hairpin RNA technology was used to create a library of shRNAs that target p38 induced mRNAs expression libraries are utilized to "knockdown" the genes identified in the first section. Finally, this library of gene candidates is evaluated in vivo to address their functional role in the induction or maintenance of dormancy.
Asunto(s)
División Celular/genética , Silenciador del Gen , MicroARNs , Neoplasias/genética , Complejo Silenciador Inducido por ARN/fisiología , Regulación hacia Abajo , Perfilación de la Expresión Génica , Biblioteca de Genes , Humanos , MicroARNs/fisiología , Neoplasias/patología , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Cancer dormancy is a poorly understood stage of cancer progression. However, the ability to control this step of the disease offers novel therapeutic opportunities. Here we summarize recent findings that implicate the extracellular matrix and adhesion receptor signaling in the escape or induction of tumor dormancy. We further review evidence suggesting that imbalances in the activity ratio of ERK to p38 signaling may determine the fate (i.e., tumorigenicity vs. dormancy) of different carcinoma cells. Special attention is placed on the mechanisms that p38 signaling regulates during the induction of dormancy and how modulation of these pathways may offer a therapeutic opportunity. We also review evidence for a novel drug-resistance mechanism in dormant tumor cells that when blocked may enable killing of dormant tumor cells. Finally, we explore the notion that dormancy of tumor cells may be the result of a selective adaptive response that allows disseminated tumor cells to pause their growth and cope with stress signaling imposed by dissemination and/or treatment until growth can be restored.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proliferación Celular , Sistema de Señalización de MAP Quinasas , Células Madre Neoplásicas/metabolismo , Estrés Fisiológico/metabolismo , Animales , Carcinoma de Células Escamosas/patología , Supervivencia Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Modelos Animales , Metástasis de la Neoplasia , Neoplasia Residual , Células Madre Neoplásicas/patología , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The mechanisms that determine whether a tumor cell that has disseminated to a secondary site will resume growth immediately, die or enter a state of dormancy are poorly understood. Although tumor dormancy represents a common clinical finding, studying the mechanisms behind this stage of tumor progression has been challenging. Furthermore, it is thought that dormant tumor cells are refractory to chemotherapy due to their lack of proliferation. However, whether this is the only reason for their chemo-resistance remains to be proven. In this review we summarize recent findings that provide a mechanistic explanation about how stress signaling through the p38(SAPK) pathway and ER-stress signaling may coordinate the induction of growth arrest and drug-resistance in a model of squamous carcinoma dormancy. We further discuss how dormant tumor cells may enter this stage to adapt to strenuous conditions that do not favor immediate growth after dissemination. Finally, we propose that this response may recapitulate an evolutionarily conserved program of life-span extension through adaptation and tolerance to stress.
Asunto(s)
Retículo Endoplásmico/enzimología , Neoplasias/enzimología , Neoplasias/patología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Resistencia a Antineoplásicos , Factor 2 Eucariótico de Iniciación/agonistas , Humanos , Metástasis de la Neoplasia , Transducción de Señal , eIF-2 Quinasa/metabolismoRESUMEN
It has been proposed that occult, disseminated metastatic cells are refractory to chemotherapy due to lack of proliferation. We have shown that p38 activation induces dormancy of squamous carcinoma cells. We now show that p38 signaling in these cells activates a prosurvival mechanism via the up-regulation of the endoplasmic reticulum (ER) chaperone BiP and increased activation of the ER stress-activated eukaryotic translation initiator factor 2alpha kinase RNA-dependent protein kinase-like ER kinase (PERK) allowing dormant tumor cells to resist drug toxicity. RNA interference and dominant-negative expression studies revealed that both BiP and PERK signaling promote survival and drug resistance of dormant cells, and that BiP up-regulation prevents Bax activation. We propose that stress-dependent activation of p38 via BiP up-regulation and PERK activation protects dormant tumor cells from stress insults, such as chemotherapy.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/enzimología , Proteínas de Choque Térmico/biosíntesis , Chaperonas Moleculares/biosíntesis , eIF-2 Quinasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/fisiología , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Etopósido/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Chaperonas Moleculares/antagonistas & inhibidores , Fosforilación , Proteómica , Interferencia de ARN , Regulación hacia Arriba , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismo , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
Nonsense-mediated mRNA decay (NMD) in mammalian cells targets cap-binding protein 80 (CBP80)-bound mRNA during or after a pioneer round of translation. It is unknown whether eukaryotic translation initiation factor 4G (eIF4G) functions in the pioneer round. We show that baculovirus-produced CBP80 and CBP20 independently interact with eIF4GI. The interactions between eIF4G and the heterodimer CBP80/20 suggest that eIF4G has a function in the pioneer initiation complex rather than merely a presence during remodeling to the steady-state complex. First, NMD is inhibited upon eIF4G cleavage by HIV-2 or poliovirus 2A protease. Second, eIF4GI coimmunopurifies with pre-mRNA, indicating that it associates with transcripts before the pioneer round. Third, eIF4G immunopurifies with Upf NMD factors and eIF4AIII, which are constituents of the pioneer translation initiation complex. We propose a model in which eIF4G serves to connect CBP80/20 with other initiation factors during the pioneer round of translation.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Unión Proteica , ARN Mensajero/metabolismoRESUMEN
The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2alpha coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2alpha in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2alpha, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.
Asunto(s)
Proteínas Portadoras/metabolismo , Codón sin Sentido/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Células COS , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Chlorocebus aethiops , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Humanos , Luciferasas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Fosfoproteínas/genética , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Pruebas de Precipitina , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Elevated manganese superoxide dismutase (Sod2) levels have been reported to be associated with an increased frequency of tumor invasion and metastasis in certain cancers, and the aim of this study is to examine the molecular mechanisms by which this occurs. EXPERIMENTAL DESIGN: Sod2 and catalase overexpressing HT-1080 fibrosarcoma cell lines were used to evaluate the H(2)O(2)-dependent regulation of matrix metalloproteinase (MMP)-1 promoter activity, mitogen-activated protein (MAP) kinase signaling, DNA-binding activity, and MMP mRNA levels. The invasive and metastatic potential of Sod2 overexpressing cells was characterized using subrenal capsular implantation or tail vein injection of tumor cells into nude mice, respectively. RESULTS: Our data reveal that Sod2 overexpression increases the DNA-binding activity of transcription factors critical for MMP expression but also enhances MMP-1 promoter activity via the Ras//MAP/extracellular signal-regulated kinase (MEK) signaling cascade. A single nucleotide polymorphism that creates an Ets site at position -1607 bp confers Sod2-dependent MMP-1 promoter activity. Sod2 overexpression also increases the mRNA levels of MMPs-2, -3, -7, -10, -9, -11 and enhances the metastatic potential of fibrosarcoma cells when implanted in immunodeficient mice. The Sod2-dependent increases in AP-1 and SP-1 DNA-binding activity, MMP-1 promoter activity, general MMP expression, and collagen degradation can be reversed by the hydrogen peroxide-detoxifying enzyme, catalase. CONCLUSION: MMPs play a critical role in the process of stromal invasion and metastasis, and these findings suggest that the association between increased Sod2 and poor prognosis in certain cancers may be attributed to elevated MMP production.