Desialylation of Atg5 by sialidase (Neu2) enhances autophagosome formation to induce anchorage-dependent cell death in ovarian cancer cells

Increased sialylation is one of the hallmarks of ovarian cancer (OC) but its relation with programmed cell death is not known. Here we explored the molecular interplay between autophagy, apoptosis/anoikis, and aberrant-expression of the PI3K-Akt/mTOR pathway in the context of sialidase. OC is accompanied by low expression of cytosolic sialidase (Neu2) and similar to 10-fold more alpha 2,6- than alpha 2,3-linked sialic acids found through qPCR, western blot, and flow cytometry. Interestingly, Neu2 overexpression cleaved alpha 2,6- and alpha 2,3-linked sialic acids and reduced cell viability. Several autophagy-related molecules like LC3B/Atg3/Atg5/Atg7/Atg12/Atg16L1/Beclin1 were upregulated upon Neu2 overexpression. Atg5, a crucial protein for autophagosome formation, was desialylated by overexpressed Neu2. Desialylated Atg5 now showed enhanced association both with Atg12 and Atg16L1 leading to more autophagosome formation. Neu2-overexpressing cells exhibited extrinsic pathway-mediated apoptosis as reflected the in activation of Fas/FasL/FADD/Bid/caspase 8/caspase 6/caspase 3/PARP cleavage. There was also increased Bax, reduced Bcl2, and several cell-cycle molecules (CDK2/CDK4/CDK6/cyclin-B1/cyclin-E). Inhibition of autophagy using bafilomycin A1 or Beclin1 siRNA leads to reversal of Neu2-induced apoptosis suggesting their possible relationship. Additionally, overexpressed Neu2 inhibited growth factor-mediated signaling molecules involved in the PI3K/Akt-mTOR pathway probably through their desialylation. Furthermore, overexpressed Neu2 inhibited epithelial (ZO-1/Claudin1), mesenchymal (snail/slug), and cell-adhesion (integrin-beta 3/focal-adhesion kinase) molecules suggesting anchorage-dependent cell death (anoikis). Such changes were absent in the presence of bafilomycin A1 indicating the involvement of autophagy in Neu2-induced anoikis. The physiological relevance of our in vitro observations was further confirmed in the OC xenograft model. Taken together, it is the first report demonstrating that Atg5 is a sialoglycoprotein having alpha 2,6- and alpha 2,3-linked sialic acids and its desialylation by overexpressed Neu2 leads to its activation for autophagosome formation, which induced apoptosis/anoikis in OC.

Automated summary

This study reveals the molecular interplay between autophagy, apoptosis, and the PI3K-Akt/mTOR pathway in the context of sialidase, shedding light on the relationship between increased sialylation and ovarian cancer. Overexpression of Neu2 activates autophagy and apoptosis pathways, while inhibiting growth factor signaling molecules, ultimately leading to cell death.