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  • (±)-Baclofen The multiple NDRG bands observed

    2019-07-08

    The multiple NDRG1 bands observed in immunoblots are probably the result of post-translational modifications such as phosphorylation [16], small ubiquitin-like modifier (SUMO) modification [17] and/or cleavage of NDRG1 [18]. Proteasome-mediated NDRG1 degradation was previously reported and NDRG1 phosphorylation was suggested to signal this degradation pathway [19,20]. Phosphorylation of NDRG1 at Ser330 and Thr346 by serum- and glucocorticoid-regulated kinase 1 (SGK1) was shown to be crucial to inhibit NF-ĸB signaling and CXC cytokine synthesis [21]. Additionally, NDRG1 cleavage was identified in prostate cancer cells, but not normal prostate epithelial cells, suggesting this process could influence the anti-oncogenic function of NDRG1 [18]. Our previous studies have demonstrated that NDRG1 is up-regulated by cellular iron depletion and hypoxia through hypoxia-inducible factor-1α (HIF-1α)-dependent and -independent mechanisms at protein and mRNA levels [[22], [23], [24]]. Furthermore, due to its well-characterized roles as a metastasis suppressor, and since metastasis kills 90% of cancer patients [25], NDRG1 has been identified as a molecular target of a new group of anti-cancer agents of the di-2-pyridylketone thiosemicarbazone class that potently inhibit tumor growth and metastasis [6,22,[26], [27], [28], [29], [30], [31], [32]]. The first lead agent of this class of agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), leads to marked NDRG1 up-regulation in many tumor cell-types [15,22,28,33], with an analogue of this agent entering Phase I clinical trials [34]. In fact, relative to control-treated cells, Dp44mT induced up-regulation of the 46- and 47-kDa NDRG1 bands, as well as increasing the p-NDRG1 (Ser330 and Thr346) bands at 46–47-kDa [15].
    Materials and methods
    Results
    Discussion NDRG1 is an intriguing metastasis suppressor that has been demonstrated to play a role inhibiting the progression and metastasis of a variety of solid tumors, including pancreatic cancer [2]. NDRG1 has been shown to inhibit a number of critical oncogenic cellular signaling pathways (e.g., EGFR pathway, ROCK-pMLC, FAK-paxillin, Src, β-catenin, etc.) in cancer (±)-Baclofen [3,4,[8], [9], [10],54]. Previous studies using immunoblotting have reported the presence of three different NDRG1 isoforms that could be the product of post-translational modifications, such as phosphorylation or truncation [15,18,21,28]. To understand the mechanism(s) relating to the generation of NDRG1 isoforms, the current investigation dissected the roles of two established protein processing pathways, namely that mediated by the proteasome and lysosome. Surprisingly, both these latter protein-processing machineries affected different NDRG1 isoforms (Fig. 11A). First, the levels of the top NDRG1 band (47-kDa band) were increased by two well-characterized proteasomal inhibitors, namely MG132 and Epoxomycin [47]. Each of these inhibitors have their own specific profile of inhibiting different proteolytic activities [47], nonetheless, both increased the top NDRG1 band, demonstrating the role of the proteasome in NDRG1 processing (Fig. 11A). Second, the middle NDRG1 band (46-kDa) was increased by the lysosomal acidification inhibitor, Baf A1, that inhibits both chaperone-mediated and macroautophagy and also 3-MA that inhibits only the latter process [38]. This suggested the middle band was being processed and potentially degraded via the lysosome (Fig. 11A). Of note, these well-established classical inhibitors are known to affect cellular protein degradation machinery, i.e., proteasomal (e.g., MG132; [47]) or autophagy (e.g., Baf A1; [38]), rather than transcription/translation. In the presence of proteasomal inhibitors, lysosomal inhibitors were unable to increase the middle NDRG1 band (Fig. 2). This suggests that a sequential processing of NDRG1 occurs with the functional proteasome being required for subsequent lysosome-mediated metabolism. These effects were observed in three different cell-types, suggesting a more general mechanism. Of interest, other proteins also have been reported to be processed by coordinated proteasomal and lysosomal metabolism, such as the EGFR [62]. Such processing of NDRG1 may be critical in terms of different aspects of its biological activities. For instance, it has been demonstrated that NDRG1 plays a role in suppressing metastasis via inhibiting multiple oncogenic pathways and this could, at least in part, be mediated through these different isoforms.