RESUMEN
Recently, cancer stem cells (CSCs) have been identified as the major cause of both chemotherapy and radiotherapy resistance. Evidence from experimental studies applying both in vitro and in vivo preclinical models suggests that CSCs survive after conventional therapy protocols. Several mechanisms are proposed to be involved in CSC resistance to radiotherapy. Among them, stimulated DNA double-strand break (DSB) repair capacity in association with aldehyde dehydrogenase (ALDH) activity seems to be the most prominent mechanism. However, thus far, the pathway through which ALDH activity stimulates DSB repair is not known. Therefore, in the present study, we investigated the underlying signaling pathway by which ALDH activity stimulates DSB repair and can lead to radioresistance of breast cancer cell lines in vitro. When compared with ALDH-negative cells, ALDH-positive cells presented significantly enhanced cell survival after radiation exposure. This enhanced cell survival was associated with stimulated Nanog, BMI1 and Notch1 protein expression, as well as stimulated Akt activity. By applying overexpression and knockdown approaches, we clearly demonstrated that Nanog expression is associated with enhanced ALDH activity and cellular radioresistance, as well as stimulated DSB repair. Akt and Notch1 targeting abrogated the Nanog-mediated radioresistance and stimulated ALDH activity. Overall, we demonstrate that Nanog signaling induces tumor cell radioresistance and stimulates ALDH activity, most likely through activation of the Notch1 and Akt pathways.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Neoplasias de la Mama/metabolismo , Tolerancia a Radiación , Transducción de Señal , Neoplasias de la Mama/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Humanos , Células MCF-7 , Proteína Homeótica Nanog/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/efectos de la radiaciónRESUMEN
Ionizing radiation (IR) and epidermal growth factor (EGF) stimulate Y-box binding protein-1 (YB-1) phosphorylation at Ser-102 in KRAS wild-type (KRASwt) cells, whereas in KRAS mutated (KRASmut) cells, YB-1 is constitutively phosphorylated, independent of IR or EGF. YB-1 activity stimulates the repair of IR-induced DNA double-strand breaks (DSBs) in the nucleus. Thus far, the YB-1 nuclear translocation pattern after cell exposure to various cellular stressors is not clear. In the present study, we investigated the pattern of YB-1 phosphorylation and its possible translocation to the nucleus in KRASwt cells after exposure to IR, EGF treatment, and conditional expression of mutated KRAS(G12V). IR, EGF, and conditional KRAS(G12V) expression induced YB-1 phosphorylation in both the cytoplasmic and nuclear fractions of KRASwt cells. None of the stimuli induced YB-1 nuclear translocation, while p90 ribosomal s6 kinase (RSK) translocation was enhanced in KRASwt cells after any of the stimuli. EGF-induced RSK translocation to the nucleus and nuclear YB-1 phosphorylation were completely blocked by the EGF receptor kinase inhibitor erlotinib. Likewise, RSK inhibition blocked RSK nuclear translocation and nuclear YB-1 phosphorylation after irradiation and KRAS(G12V) overexpression. In summary, acute stimulation of YB-1 phosphorylation does not lead to YB-1 translocation from the cytoplasm to the nucleus. Rather, irradiation, EGF treatment, or KRAS(G12V) overexpression induces RSK activation, leading to its translocation to the nucleus, where it activates already-existing nuclear YB-1. Our novel finding illuminates the signaling pathways involved in nuclear YB-1 phosphorylation and provides a rationale for designing appropriate targeting strategies to block YB-1 in oncology as well as in radiation oncology.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Transporte Activo de Núcleo Celular/efectos de la radiación , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Fosforilación/efectos de la radiación , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras)/genética , Estrés Fisiológico/efectos de la radiación , Regulación hacia ArribaRESUMEN
Transforming growth factor beta-3 (TGF-ß3) has been shown to decrease scar formation after scheduled topical applications to the cutaneous wounds. This study aimed to continuously deliver TGF-ß3, during the early phase of wound healing, by engineering a dermal equivalent (DE) using TGF-ß3 expressing bone marrow stromal cells (BM-SCs) and human dehydrated amniotic membrane (hDAM). To engineer a DE, rat BM-SCs were seeded on the hDAM and TGF-ß3 was transiently transfected into the BM-SCs using a plasmid vector. Pieces of the dermal equivalent were transplanted onto the full-thickness excisional skin wounds in rats. The process of wound healing was assessed by image analysis, Manchester Scar Scale (MSS), and histopathological studies 7, 14, 21, and 85 days after the excision. The results confirmed accurate construction of recombinant pcDNA3.1-TGF-ß3 expression system and showed that the transfected BM-SCs seeded on hDAM expressed TGF-ß3 mRNA and protein from day 3 through day 7 after transfection. After implantation of the DE, contraction of the wounds was measured from day 7 through 21 and analyzed by linear regression, which revealed that the rate of wound contraction in all experimental groups was similar. Histologic evaluation demonstrated that transfected BM-SCs decreased retention and recruitment of the cells during the early stage of wound healing, decreased the formation of vascular structures and led to formation of uniformly parallel collagen bundles. MSS scores showed that TGF-ß3 secreting cells significantly improved the cosmetic appearance of the healed skin and decreased the scar formation. From these results, it could be concluded that transient secretion of TGF-ß3, during the early phase of healing, by BM-SCs seeded on hDAM can improve the cosmetic appearance of the scar in cutaneous wounds without negatively affecting the process of wound repair.