Abstract
The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.
Highlights
The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure
We sought to know whether MIF had any role in regulating mitochondrial structural dynamics per se, because the loss of mitochondrial functionality and metabolic crisis are often linked with aberrant mitochondrial structure [19, 20]
To check whether this enhanced mitochondrial fission could be attributed to any specific cell cycle alteration, flow cytometry– based AGS cell cycle analysis was done, which revealed a radical increase (Ϸ24% in siMIF-treated cells compared with 3% in control) in the sub-G0 population upon MIF knockdown (Fig. 2B)
Summary
MIF knockdown in gastric adenocarcinoma, hepatocellular carcinoma, cervical adenocarcinoma, and colorectal carcinoma cells destabilized mitochondria, increased mitochondrial fission, and reduced cell viability. Confocal micrographs of live cells and corresponding scatter plots revealed significant retention of filamentous mitochondria in the MIF-depleted cells supplemented with exogenous MIF compared with siMIF-treated only cells (Fig. 6B) Together, these data put forward evidence supporting the specific role of MIF on mitochondrial structure and cell survival. The knockdown of NF-B neutralized the rescue effect of MIF supplementation in MIF KD cells as revealed by the flow cytometric data, where significant apoptosis was detected in the MIF-supplemented cells with MIF–NF-B double knockdown (Fig. 9F) In this context, to check whether the restoration of the cellular NF-B level in MIF-silenced cells ameliorated mitochondrial and cellular pathology, NF-B was overexpressed by transfecting the AGS cells with NF-B p65 plasmid (pNF–B p65), and the parameters of the mitochondrial structure and function along with cell viability were followed (Fig. 10). The data indicated that selective knockdown of CD74 induced pronounced deterioration of mitochondrial function and morphological integrity to trigger apoptosis
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