Effect of upregulation of ANGPT2 by DARPP-32 on angioenesis in gastric cancer.
34 Background: Gastric cancer is the second most frequent cause of cancer-related death worldwide. We have previously shown that Dopamine and cAMP regulated phosphoprotein MW 32 kDa (DARPP-32) and its truncated form (t-DARPP) are overexpressed in two-thirds of gastric adenocarcinomas. Angiopoietin 2 (ANGPT2) -TIE2 signaling is a secreted protein that acts as a key regulator of adult vascular homeostasis and blood vessel formation. Methods: The expression of DARPP-32 in the multi-step carcinogenesis cascade was examined using IHC analysis on 533 samples. ANGPT2 mRNA level was detected by real-time quantitative polymerase chain reaction (PCR) in 30 gastric cancer tissue samples and 30 normal gastric tissues. DARPP-32 and t-DARPP were over expressed using stable and transient expression in AGS and MKN-28 gastric cancer cell lines, lacking endogenous DARPP-32 to investigate the induction of ANGPT2 by DARPP-32 and t-DARPP. Results: We found that ANGPT2 was higher expressed in cancer samples than normal tissues from RT-PCR. We also found gastric cancer tissue samples expressed higher DARPP-32 and t-DARPP mRNA than normal gastric tissues. Over expression of DARPP-32 and t-DARPP led to a significant increase of the mRNA and protein levels of ANGPT2 as compared to empty vector control. Consistent with these findings, the condition media from DARPP-32 and t-DARRP expressing cells showed high levels of secreted ANGPT-2. TNF-α treatment induced the levels of ANGPT2 further in DARPP-32 and t-DARPP expressing cells as compared to control. Of note, this increase in NF-κB activity was significantly higher in DARPP-32 and t-DARPP expressing cells as compared to control. To confirm the angiogenic potential, we used condition media from DARPP-32 and t-DARPP expressing AGS cells and demonstrated its ability to stimulate tube formation on human umbilical vein endothelial cells (HUVEC) models than the condition medium from control cells. Conclusions: Our results suggest that DARPP-32 and t-DARPP over expression may participate in the angiogenesis of gastric cancer. The in vitro studies indicate that DARPP-32 and t-DARPP play a role in up regulation of ANGPT2 in gastric cancer cells by enhancing the TNF-α induced activation of NF-κB signaling pathway.