RESUMEN
Triple-negative breast cancer (TNBC), which constitutes 10-20 percent of all breast cancers, is aggressive, has high metastatic potential, and carries a poor prognosis due to limited treatment options. LT-IIc, a member of the type II subfamily of ADP-ribosylating-heat-labile enterotoxins that bind to a distinctive set of cell-surface ganglioside receptors-is cytotoxic toward TNBC cell lines, but has no cytotoxic activity for non-transformed breast epithelial cells. Here, primary TNBC cells, isolated from resected human tumors, showed an enhanced cytotoxic response specifically toward LT-IIc, in contrast to other enterotoxins that were tested. MDA-MB-231 cells, a model for TNBC, were used to evaluate potential mechanisms of cytotoxicity by LT-IIc, which induced elevated intracellular cAMP and stimulated the cAMP response element-binding protein (CREB) signaling pathway. To dissect the role of ADP-ribosylation, cAMP induction, and ganglioside ligation in the cytotoxic response, MDA-MB-231 cells were exposed to wild-type LT-IIc, the recombinant B-pentamer of LT-IIc that lacks the ADP-ribosylating A polypeptide, or mutants of LT-IIc with an enzymatically inactivated A1-domain. These experiments revealed that the ADP-ribosyltransferase activity of LT-IIc was nonessential for inducing the lethality of MDA-MB-231 cells. In contrast, a mutant LT-IIc with an altered ganglioside binding activity failed to trigger a cytotoxic response in MDA-MB-231 cells. Furthermore, the pharmacological inhibition of ganglioside expression protected MDA-MB-231 cells from the cytotoxic effects of LT-IIc. These data establish that ganglioside ligation, but not the induction of cAMP production nor ADP-ribosyltransferase activity, is essential to initiating the LT-IIc-dependent cell death of MDA-MB-231 cells. These experiments unveiled previously unknown properties of LT-IIc and gangliosides in signal transduction, offering the potential for the targeted treatment of TNBC, an option that is desperately needed.
Asunto(s)
Enterotoxinas , Gangliósidos , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Gangliósidos/metabolismo , Enterotoxinas/toxicidad , Femenino , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacosRESUMEN
Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galß1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc-host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galß1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Toxinas Bacterianas/metabolismo , Ceramidas/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Gangliósidos/metabolismo , Adyuvantes Inmunológicos/química , Animales , Toxinas Bacterianas/química , Sitios de Unión , Secuencia de Carbohidratos , Células Cultivadas , Ceramidas/química , Enterotoxinas/química , Proteínas de Escherichia coli/química , Gangliósidos/química , Macrófagos/metabolismo , Ratones , Especificidad por SustratoRESUMEN
PURPOSE: Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin. METHODS: Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively. RESULTS: Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype. CONCLUSIONS: Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties.
Asunto(s)
Células Madre Embrionarias/metabolismo , Neuronas/metabolismo , Retinoblastoma/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Genes Relacionados con las Neoplasias , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Proteína Homeótica Nanog , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/inmunología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Transporte de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína Reelina , Retina/metabolismo , Retina/patología , Retinoblastoma/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genéticaRESUMEN
Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIb(T13I), and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 µg when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa's activity in humans.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos Bacterianos/administración & dosificación , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli/inmunología , Inmunoglobulina G/sangre , Ricina/inmunología , Vacunas/inmunología , Adyuvantes Inmunológicos/química , Administración Intranasal , Animales , Anticuerpos Neutralizantes/sangre , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/administración & dosificación , Escherichia coli Enterotoxigénica/química , Enterotoxinas/administración & dosificación , Proteínas de Escherichia coli/administración & dosificación , Inyecciones Intradérmicas , Ratones , Ricina/química , Vacunas/administración & dosificaciónRESUMEN
Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/administración & dosificación , Enterotoxinas/administración & dosificación , Proteínas de Escherichia coli/administración & dosificación , Inflamación/prevención & control , Piel/inmunología , Vacunas de Subunidad/uso terapéutico , Vacunas Sintéticas/uso terapéutico , Vacunas/administración & dosificación , Adyuvantes Inmunológicos , Administración Intranasal , Animales , Anticuerpos Neutralizantes/uso terapéutico , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Sinergismo Farmacológico , Enterotoxinas/inmunología , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/inmunología , Proteínas de Escherichia coli/metabolismo , Femenino , Inmunidad Mucosa , Inmunización , Inflamación/inmunología , Inflamación/metabolismo , Ratones , Ratones Endogámicos BALB C , Piel/metabolismo , Vacunas/inmunología , Vacunas/metabolismoRESUMEN
A plethora of human pathogens invade and/or colonize mucosal surfaces. Elaboration of strong, protective immune responses against those pathogens by mucosal vaccination, however, is hampered by endogenous regulatory systems in the mucosae that dampen responses to foreign antigens (Ags). To overcome those natural barriers, mucosal adjuvants must be employed. Using a mouse mucosal immunization model and AgI/II, a weak immunogen from Streptococcus mutans, LT-IIc, a new member of the type II subgroup of the heat-labile enterotoxin family, was shown to have potent mucosal adjuvant properties. In comparison to mice intranasally immunized only with AgI/II, co-administration of AgI/II with LT-IIc enhanced production of Ag-specific IgA antibodies in the saliva and vaginal fluids and Ag-specific IgA and IgG in the serum. Secretion of IL-2, IL-6, IL-17, IFN-γ, and TNF-α was enhanced in cultures of AgI/II-stimulated splenic cells isolated from mice that had received LT-IIc as a mucosal adjuvant. In contrast, secretion of IL-10 was suppressed in those cells. This pattern of cytokine secretion suggested that LT-IIc stimulates both Th1 and Th2 immune responses. In contrast to LT-IIa and LT-IIb, the original members of the type II subgroup that also are mucosal adjuvants, LT-IIc dramatically enhanced secretion of IL-1α and IL-1ß in peritoneal macrophages that had been co-cultured with LPS. Furthermore, the B pentameric subunit of LT-IIc augmented uptake of Ag by bone marrow-derived dendritic cells to levels that exceeded those attained by use of LPS or by the B pentamers of LT-IIa or LT-IIb. These data confirmed that LT-IIc is a strong mucosal adjuvant with immunomodulatory properties that are distinguishable from those of LT-IIa and LT-IIb and which has immunomodulatory properties that may be exploitable in vaccine development.