Throughout all studies it was of interest that despite
Throughout all studies, it was of interest that despite marked alterations in the top and middle bands by either proteasomal or lysosomal inhibitors, the effect on the bottom band was not significant nor consistent. Considering this, it could be suggested that the bottom band may not be result of subsequent processing after the lysosomal step, rather it could be result of a separate mechanism that results in cleavage of the top band (Fig. 11A). Such separate processing is consistent with previous studies where cleavage was indicated to be responsible for the bottom NDRG1 band in cancer cells. In fact, this cleavage event was consistent with pseudotrypsin digestion at the N-terminus between residues Cys49-Gly50, potentially leading to the NDRG1 41-kDa protein . The role of both the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by immunofluorescence studies using confocal microscopy. However, it should be noted that the fluorescent signal obtained by confocal microscopy represents a combination of all three NDRG1 bands and it does not distinguish between the top, middle and bottom NDRG1 isoforms. There was a slight increase in co-localization of NDRG1 with proteasomal marker (PSMD9) after incubation with MG132 or Baf A1. On the other hand, there was a marked increase in co-localization of NDRG1 with PSMD9 upon incubation MG132 and Baf A1. These findings are in accordance with our proposed model of NDRG1 processing, where processing by the proteasome then proceeds to autophagic metabolism (Fig. 11A). Thus, inhibition of both processes were required to observe a marked accumulation of NDRG1 in proteasomes. There was increased co-localization of NDRG1 with LC3 stained autophagosomes after inhibition of proteasomal and/or autophagic processing, which could be due to the downstream processing role of autophagy in NDRG1 metabolism. These findings substantiate that both proteasomal and autophagic pathways are intricately involved in NDRG1 processing. Hypoxia is a well-known, physiologically-relevant, NDRG1-inducing factor in tumor Calcipotriol  and in the current investigation was able to increase levels of the middle NDRG1 band (Fig. 4A). Under hypoxic conditions, the proteasomal inhibitor resulted in a marked increase in the levels of the top band, with a significant decrease in the middle NDRG1 band. In terms of the development of NDRG1 as a therapeutic target, novel thiosemicarbazone-based anti-cancer agents (e.g., Dp44mT) can markedly and significantly increase the top and middle bands of NDRG1, and its phosphorylation at Ser330 and Thr346, which was correlated with its anti-cancer activity [15,28,39,45]. Furthermore, the anti-metastatic effects of these agents have been reported to be dependent on NDRG1 expression . In contrast to hypoxia, which only increased the middle NDRG1 band, Dp44mT up-regulated both the top and middle bands indicating differential processing by different induction stimuli. NDRG1 is a known to be an important metastasis suppressor, which regulates plethora of cellular pathways involved in cancer progression, such as EGFR, ROCK-pMLC, FAK-paxillin, Src, β-catenin, etc. [3,4,, , ,54]. In future studies, it will be interesting to assess the role of the diverse investigative conditions employed in this current study (i.e., proteasomal or lysosomal inhibition or both) on the downstream NDRG1 signaling. In this investigation, rather than focusing on other possible post-translational processes (e.g., SUMOlyation; ), we performed detailed mechanistic studies examining NDRG1 phosphorylation and upstream kinases involved in this process. The effect of proteasomal and lysosomal inhibitors on the Dp44mT-induced top and middle NDRG1 bands directly correlated with the phosphorylated forms of this protein at Ser330 and Thr346. However, while the bottom NDRG1 band at 41-kDa was also detected after probing for phosphorylated NDRG1, its levels were not directly correlated with the bottom band of total NDRG1. Collectively, these observations suggest the top and middle NDRG1 bands are predominantly the phosphorylated forms of the protein, while the bottom band could be a cleavage product, as reported previously . This remains consistent with the hypothesis described above and illustrated in the Schematic in Fig. 11A.