• 2019-10
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  • br di erent mechanisms of which one of


    different mechanisms, of which one of the most important mechanisms may be aerobic glycolysis [28,29]. Although the correlation between HK2 and tumorigenesis has been confirmed by mouse models [30], the specific mechanism by which HK2 mediates the development of colon cancer remains unclear.
    Currently, some studies suggest that there may be interactions be-tween AKT2 and HK2. It has been reported that miR-29b negatively regulates AKT2/AKT3 expression, resulting in the down-regulation of HK2/PKM2, which leads to a decrease in the Warburg effect and a slowing of ovarian cancer progression [31]. Moreover, Zhuo et al. de-monstrated that the elevation of HK2 in osteosarcoma induced by ac-tivated PI3K/Akt signaling exerts anti-apoptotic and proliferative ef-fects by regulating the Warburg effect [32]. In addition, double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of HK2 mitochondria translocation [33]. However, how AKT2 and HK2 function together in colon cancer remains poorly understood to date.
    In the present study, an interaction between AKT2 and HK2 but not AKT1 or AKT3, in colon cancer cells was identified by im-munoprecipitation assays. Since AKT is known to be closely related to insulin signaling and HK2 is a key rate-limiting enzyme in glycolysis, is a major subtype in insulin-sensitive tissue and is upregulated in tumors, one of the consequences of this interaction may be to alter the aerobic glycolysis metabolism of tumor cells. In addition, HK2 up-regulation results in increased glycolysis rates. In this study, we reported that the overexpression of AKT2 can simultaneously increase the expression level and phosphorylation level of HK2, enhanced the activity of cel-lular hexokinase, and promoted aerobic glycolysis in HCT-116 and HT-29 cells, while producing large amounts of lactic acid. A knockout of the hk2 gene following the overexpression of AKT2 can significantly reduce the hexokinase activity of the cells and the amount of lactic Norfloxacin pro-duced. Rescuing the stable overexpression of HK2 in OV AKT2 hk2 cells significantly increased the hexokinase activity and amount of  Cellular Signalling 58 (2019) 99–110
    lactic acid, but the mutant HK2T473A did not. These results indicate that AKT2 contributes to increased levels of aerobic glycolysis through HK2 in colon cancer cells, can more efficiently obtain the energy required by these cells, and obtain a large number of intermediate products ne-cessary for cellular proliferation, further promoting tumor develop-ment. This is similar to a previous report that c-Src can mediate HK2 to increase hexokinase activity and lactic acid production in tumor cells [34].
    In addition, recent research shows that HK2 contributes to the in-hibition of apoptosis through the suppression of the formation of mi-tochondrial permeability transition pores in association with voltage-dependent anion channel (VDAC) protein [11]. We also found that AKT2 mediates HK2 resistance to apoptosis induced by serum starva-tion. Since HK2 is closely related to the nutritional metabolism of tumor cells, we only tested serum starvation-induced apoptosis in the present study. In a future experiment, we will examine whether AKT2 mediates HK2 resistance to apoptosis induced by other factors.
    AKT has been well-characterized as a serine/threonine protein ki-nase that phosphorylates the RXRXX(S/T) signature sequence [24], and HK2 exhibits the typical AKT phosphorylation sequence, QHRAR-QKT473. Moreover, a study has confirmed that the total AKT can phosphorylate Thr-473 HK2 and increase the binding of mitochondria and HK2 to protect cardiomyocytes [25]; however, the authors did not determine which subtype of AKT plays a role in this process. In addi-tion, in colon cancer cells, no studies to date have assessed whether AKT2 can phosphorylate Thr-473 of HK2. Indeed, although wild type HK2 was found to be phosphorylated by AKT2, the phosphorylation did not occur when threonine 473 in HK2 was mutated.
    Our further studies indicate that AKT2/HK2 mediates the promotion of colon cancer cell invasion, xenograft tumors, and metastasis that can be triggered by EGF-stimulated PI3K activation. PI3K acts as an up-stream kinase for AKT2 and HK2. In addition, AKT2 has been reported to be a molecule located upstream from mTOR. Although AKT2 acts as an upstream molecule of HK2 and can directly interact and phosphor-ylate HK2, rapamycin, an inhibitor of mTOR, was also found to reduce the expression of HK2 in this study. This finding should be studied further to explain the specific associated molecular mechanisms in fu-ture research.
    We also investigated whether AKT2 affects p-STAT3, NF-κB, Bcl-2, Bcl-XL, HIF1α, MMP2, and MMP9 via the phosphorylation of the T473 site of HK2, which are important molecules closely related to cancer cell invasion, apoptosis, and metastasis. We found that AKT2 regulates NF-κB, HIF1α, MMP2, Norfloxacin and MMP9 via the phosphorylation of the HK2 T473 site. A previous study showed that HK2 is a downstream target of HIF1α [30]. However, in our study, we found that HK2 is the upstream molecule of HIF1α. This inconsistency may indicate that there are in-teractions in complex cellular signaling pathways in different cancer cells. We also found that AKT2 does not regulate the level of p-STAT3, Bcl-2, and Bcl-XL protein expression via HK2. In addition, to gain in-sight into the specific role of the AKT2-HK2-NF-κB/HIF1α/MMP2/ MMP9 axis in colon cancer, there is a need to investigate how HK2 could modulate the expression of MMP2/9, HIF-1a and NF-κB in the future.