The DLK1/MEG3 Locus is Related to the Differentiation of Pituitary Neuroendocrine Tumors

Background: Pituitary neuroendocrine tumors (PitNETs) represent the neoplastic proliferation of the anterior pituitary gland. Transcription factors play a key role in the differentiation of PitNETs according to the 2017 WHO classification of tumors of endocrine organs. However, for a substantial proportion of PitNETs, the etiology is poorly understood.Methods: To analyze the diversity genes and pathways based on transcriptomic data of 172 patients by transcriptome sequencing, difference, correlation, and enrichment analysis. The transcriptomic data and clinical characteristics are applied to find the clinical significance of key genes by correlation analysis and ROC curve. Series functional experiments demonstrated the role of DLK1/MEG3 locus through antibody blocking and RNA interference in GH3, MMQ and ATT20 cell line.Results: In this study, we found the imprinting disorders of the 14q32.2 region and DLK1/MEG3 locus associated with the differentiation of PitNETs. As being specific feature change in somatotroph adenomas compared with other subtype adenomas, DLK1/MEG3 locus promoted somatotroph differentiation and inhibited tumor proliferation in PIT1(+) PitNET patients, furthermore, the level of DLK1 played a critical role in the trend of somatotroph differentiation or lactotroph differentiation in PIT1(+) PitNET patients. Anti-DLK1 monoclonal antibody blockade or siMEG3 both indicated that the DLK1/MEG3 domain significantly induced the synthesis and secretion of growth hormone/insulin-like growth factor-1 (GH/IGF-1) and inhibited cell growth and colony formation and that the loss of DLK1 activated the mTOR signaling pathway in high DLK1-expressing and PIT1(+) GH3 cell lines. There was a mild effect in the low DLK1-expressing and PIT1(+) MMQ cell lines and no effect in the PIT1(-) ATT20 cell line, regardless of whether anti-DLK1 monoclonal antibody blockade or siMEG3 was used. Conclusions: These findings emphasize that expression at the DLK1/MEG3 locus plays a key role in the differentiation of PitNETs in patients depending on the level of PIT1. In addition, these findings provide potential molecular target data for patient stratification and treatment in the future.

Sorting of PC2 to the regulated secretory pathway in AtT20 cells

PC2 and PC3 are neuroendocrine specific members of the eukaryotic subtilisin-like proprotein convertase (PC) family. Both are sorted via the regulated secretory pathway into secretory granules. In order to identify sequences in PC2 which are involved in targeting to the regulated secretory pathway we expressed a series of PC2 cDNAs containing mutations in the C terminal or propeptide domains in the mouse corticotrophic AtT20 cell line. Sorting of endogenous PC3 was used as a control. PC2 and PC3 were secreted with similar kinetics and sorted to secretory granules with similar efficiencies. Deletions of up to 50 amino acids from the C-terminus of proPC2 had no effect on secretion or sorting, but larger deletions completely prevented maturation or secretion. Two large deletions within the propeptide also prevented secretion. Smaller deletions between the primary and secondary cleavage sites, or of the primary cleavage site, reduced the amount of protein secreted but did not affect sorting to secretory granules. Replacement of the propeptide of PC2 with that of the endogenous PC3 also had no effect on secretion or sorting. The results indicate that targeting of proPC2 to the regulated secretory pathway is dependent on more than one region within the proPC2 molecule.

Tubular early endosomal networks in AtT20 and other cells.

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.

An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage.

We have raised a rabbit antiserum against a synthetic peptide corresponding to the cleavage site between beta-lipotropic hormone and the ACTH moieties of murine pro-opiomelanocortin (POMC). After affinity purification, the anti-cleavage site antibody immunoprecipitates POMC from extracts of AtT20 cells but it does not immunoprecipitate the ACTH in such extracts or any of the other products of cleavage of POMC. By contrast, an antiserum raised against pure swine ACTH immunoprecipitates both POMC and ACTH from AtT20 cell extracts. Using the anti-cleavage site antibody we have shown that all the POMC synthesized during a 15-min pulse-labeling with [35S]methionine is cleaved at this site within 1 h. By immunoelectron microscopy we show that approximately 25-30% of peripheral secretory granules in AtT20 cells can be labeled with the anti-cleavage site antibody while anti-ACTH antiserum labels all these granules. This establishes that at least some POMC is packaged into secretory granules before its proteolytic cleavage.