RESUMO
Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.
Assuntos
Neoplasias Cerebelares/metabolismo , Receptores ErbB/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Fatores de Transcrição/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Células Cultivadas , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Cicloexilaminas/farmacologia , Derme/citologia , Derme/efeitos dos fármacos , Derme/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/genética , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Patched , Receptor Patched-1 , Fosforilação/efeitos dos fármacos , Análise Serial de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiofenos/farmacologia , Fatores de Transcrição/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138(neg)) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138(neg) cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
