RESUMEN
RNA interference has boosted the field of functional genomics, by making it possible to carry out 'loss-of-function' screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes-RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23-were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.
Asunto(s)
Neoplasias de la Mama/genética , División Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 8 , ARN Interferente Pequeño/genética , Secuencia de Bases , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Cartilla de ADN , Proteínas de Unión al ADN/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Nucleares/genética , Nucleoproteínas/genética , Fosfoproteínas/genética , Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The pituitary adenylate cyclase-activating polypeptide (PACAP) is a trophic factor that promotes neuronal survival and neurite outgrowth. However, the signaling pathways and the transcriptional mechanisms involved are not completely elucidated. Our previous studies aimed at characterizing the transcriptome of PACAP-differentiated PC12 cells revealed an increase in the expression of nuclear factor κB2 (NF-κB2) gene coding for p100/p52 subunit of NF-κB transcription factor. Here, we examined the role of the NF-κB pathway in neuronal differentiation promoted by PACAP. We first showed that PACAP-driven survival and neuritic extension in PC12 cells are inhibited following NF-κB pathway blockade. PACAP stimulated both c-Rel and p52 NF-κB subunit gene expression and nuclear translocation, whereas c-Rel down-regulation inhibited cell survival and neuritogenesis elicited by the neuropeptide. PACAP-induced c-Rel nuclear translocation was inhibited by ERK1/2 and Ca(2+) blockers. Furthermore, the neuropeptide stimulated NF-κB p100 subunit processing into p52, indicative of activation of the NF-κB alternative pathway. Taken together, our data show that PACAP promotes both survival and neuritogenesis in PC12 cells by activating NF-κB pathway, most likely via classical and alternative signaling cascades involving ERK1/2 kinases, Ca(2+), and c-Rel/p52 dimers.
Asunto(s)
Señalización del Calcio/fisiología , Núcleo Celular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Neuritas/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Núcleo Celular/genética , Supervivencia Celular/fisiología , Proteína Quinasa 3 Activada por Mitógenos/genética , Subunidad p52 de NF-kappa B/genética , Células PC12 , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Proteínas Proto-Oncogénicas c-rel/genética , RatasRESUMEN
Triple-negative breast cancers (TNBCs) account for a large proportion of breast cancer deaths, due to the high rate of recurrence from residual, resistant tumor cells. New treatments are needed, to bypass chemoresistance and improve survival. The WNT pathway, which is activated in TNBCs, has been identified as an attractive pathway for treatment targeting. We analyzed expression of the WNT coreceptors LRP5 and LRP6 in human breast cancer samples. As previously described, LRP6 was overexpressed in TNBCs. However, we also showed, for the first time, that LRP5 was overexpressed in TNBCs too. The knockdown of LRP5 or LRP6 decreased tumorigenesis in vitro and in vivo, identifying both receptors as potential treatment targets in TNBC. The apoptotic effect of LRP5 knockdown was more robust than that of LRP6 depletion. We analyzed and compared the transcriptomes of cells depleted of LRP5 or LRP6, to identify genes specifically deregulated by LRP5 potentially implicated in cell death. We identified serine/threonine kinase 40 (STK40) as one of two genes specifically downregulated soon after LRP5 depletion. STK40 was found to be overexpressed in TNBCs, relative to other breast cancer subtypes, and in various other tumor types. STK40 depletion decreased cell viability and colony formation, and induced the apoptosis of TNBC cells. In addition, STK40 knockdown impaired growth in an anchorage-independent manner in vitro and slowed tumor growth in vivo. These findings identify the largely uncharacterized putative protein kinase STK40 as a novel candidate treatment target for TNBC.