REST Inactivation and Coexpression of ASCL1 and POU3F4 Are Necessary for the Complete Transformation of RB1/TP53-Inactivated Lung Adenocarcinoma into Neuroendocrine Carcinoma.

Although recent reports have revealed the importance of the inactivation of both RB1 and TP53 in the transformation from lung adenocarcinoma into neuroendocrine carcinoma (NEC), the requirements for complete transformation into NEC have not been elucidated. To investigate alterations in the characteristics associated with the inactivation of RB1/TP53 and define the requirements for transformation into NEC cells, RB1/TP53 double-knockout A549 lung adenocarcinoma cells were established, and additional knockout of REST and transfection of ASCL1 and POU class 3 homeobox transcription factors (TFs) was conducted. More than 60 genes that are abundantly expressed in neural cells and several genes associated with epithelial-to-mesenchymal transition were up-regulated in RB1/TP53 double-knockout A549 cells. Although the expression of chromogranin A and synaptophysin was induced by additional knockout of REST (which mimics the status of most NECs), the expression of another neuroendocrine marker, CD56, and proneural TFs was not induced. However, coexpression of ASCL1 and POU3F4 in RB1/TP53/REST triple-knockout A549 cells induced the expression of not only CD56 but also other proneural TFs (NEUROD1 and insulinoma-associated 1) and induced NEC-like morphology. These findings suggest that the inactivation of RB1 and TP53 induces a state necessary for the transformation of lung adenocarcinoma into NEC and that further inactivation of REST and coexpression of ASCL1 and POU3F4 are the triggers for complete transformation into NEC.

Endocrine secretory granule production is caused by a lack of REST and intragranular secretory content and accelerated by PROX1.

Endocrine secretory granules (ESGs) are morphological characteristics of endocrine/neuroendocrine cells and store peptide hormones/neurotransmitters. ESGs contain prohormones and ESG-related molecules, mainly chromogranin/secretogranin family proteins. However, the precise mechanism of ESG formation has not been elucidated. In this study, we experimentally induced ESGs in the non-neuroendocrine lung cancer cell line H1299. Since repressive element 1 silencing transcription factor (REST) and prospero homeobox 1 (PROX1) are closely associated with the expression of ESG-related molecules, we edited the REST gene and/or transfected PROX1 and then performed molecular biology, immunocytochemistry, and electron and immunoelectron microscopy assays to determine whether ESG-related molecules and ESGs were induced in H1299 cells. Although chromogranin/secretogranin family proteins were induced in H1299 cells by knockout of REST and the induction was accelerated by the PROX1 transgene, the ESGs could not be defined by electron microscopy. However, a small number of ESGs were detected in the H1299 cells lacking REST and expressing pro-opiomelanocortin (POMC) by electron microscopy. Furthermore, many ESGs were produced in the REST-lacking and PROX1- and POMC-expressing H1299 cells. These findings suggest that a lack of REST and the expression of genes related to ESG content are indispensable for ESG production and that PROX1 accelerates ESG production.Trial registration: Not applicable.