br AKT can be activated by upstream kinases In
AKT can be activated by upstream kinases. In particular, it has been
Fig. 4. HK2 activity is required for AKT2-stimulated tumor invasion, tumorigenesis and metastasis.
A and B, HCT-116 cell migration assay; C and D, HT-29 cell migration assay; E, HK2 activity is required for AKT2-stimulated xenograft tumor growth. Tumors were collected and examined six weeks after inoculation of HT-29 cells, respectively. Pictures of isolated tumors were taken. F, Tumor weights were determined. G and H, Tumors shown in E were measured for HK activity and lactate production. I, Lung metastatic burden in BALB/c nude mice 45 days after tail vein injection of HT-29 cells, as determined by counting the number of micrometastases per section (arrows point to metastatic areas). J, The lung metastatic nodule numbers in BALB/c nude mice 45 days after tail vein injection of HT-29 cells. K, Representative HE staining of metastatic foci in the lung tissues. L, Similar changes in the lung metastatic nodule numbers were also detected in the ratio of metastatic tumor area/total lung area. Data are represented as mean ± SD (n = 6). * indicates p < .05, ** indicates p < .01 vs. control, ns refers to no significant diﬀerence.
Fig. 5. Potential upstream regulators and downstream eﬀector molecules associated with AKT2- and/or HK2-mediated promotion of colon cancer cell progression. A, HCT-116 and HT-29 K 252a treated with EGF (100 ng/mL for 24 h); B, HCT-116 and HT-29 cells treated with the P13K/AKT inhibitor LY294002 (10 nM for 24 h). C, HCT-116 and HT-29 cells treated with the mTOR inhibitor rapamycin (100 nM for 24 h). Western-blot for p-STAT3, NF-κB p65 (nuclear), Bcl-2, Bcl-XL, HIF1α, MMP2 and MMP9 expression in (D) HCT-116 cells and (E) HT-29 cells. H2B Histone was used as the nuclear loading control whereas β-actin served as a total protein loading control. All assays were performed in triplicate and showed representative blots. Quantification of blots and statistical analysis were showed in supplemental fig. S1 and S2.
well-established that epidermal growth factor (EGF) activates the AKT signaling pathway. Therefore, we use EGF to stimulate colon cancer cells and increase the activity of AKT2. As shown Fig. 5A, following the treatment of HCT-116 and HT-29 cells with 100 ng/mL of EGF for 24 h, the level of p-PI3K, p-AKT2, and HK2 expression was significantly in-creased. There was no significant change in the level of total PI3K and AKT2 expression. These results indicate that EGF can eﬀectively acti-vate AKT and promote HK2 expression.
After treating HCT-116 and HT-29 cells with 10 nM of the PI3K inhibitor, LY294002, for 24 h, the expression of p-PI3K and p-AKT2 protein could not be detected and the expression of HK2 protein was significantly reduced. There was also no significant down-regulation of the total level of PI3K and AKT2 expression (Fig. 5B). These results indicate that the inhibitor of PI3K, LY294002, is able to inhibit HK2 expression. Taken together, the above data confirmed that PI3K is the upstream kinase of AKT2, and HK2 is a downstream molecule of AKT2. The level of HK2 expression in colon cancer cells is regulated by
activated PI3K and AKT2.
3.11. Other downstream AKT2/HK2 molecules involved in colon cancer progression
mTOR has been shown to be one of the downstream molecules of AKT kinase. As shown in Fig. 5C, after treating HCT-116 and HT-29 cells for 24 h with 100 nM rapamycin, an mTOR inhibitor, the level of both mTOR and p-mTOR expression were decreased. The level of HK2 expression was also significantly reduced. These results indicate that the inhibition of mTOR was able to reduce the expression of HK2, which may be a downstream molecular target of mTOR.
We next examined other potential downstream molecules involved in the AKT2/HK2-mediated progression of colon cancer. As shown in Fig. 5D and E, the level of expression of the transcription factor, phosphorylated signal transducers and activators of transcription 3 (p-STAT3), was significantly increased in OV AKT2 cells compared to
normal cells. However, knocking out the hk2 gene in OV AKT2 cells did
not reduce the level of p-STAT3 expression. In addition, the stable rescue of HK2 or HK2T473A mutant overexpression in OV AKT2 hk2
cells did not alter the level of p-STAT3 expression. These results in-dicate that although the overexpression of AKT2 can increase HK2 ex-pression, the up-regulation of p-STAT3 expression is mediated by AKT2, not through HK2.
We also tested whether the transcription factor, nuclear factor-κB (NF-κB), is a downstream molecular target of AKT2 or HK2. It has been well-established that the nuclear translocation of NF-κB p65 is an ef-fective active form. The overexpression of AKT2 can significantly in-crease the level of NF-κB p65 expression. The knockout of the hk2 gene after overexpressing AKT2 has significantly reduced the level of NF-κB p65 expression. Moreover, rescuing the stable overexpression of HK2 in OV AKT2 hk2 cells significantly increased the expression of NF-κB p65, rather than the HK2T473A mutant. Therefore, we hypothesized that HK2 can directly regulate the expression of NF-κB p65, and AKT2 can aﬀect NF-κB p65 expression through HK2.