Akt is known to play key roles in tumor malignancy, including in tumor cell survival, invasion, and metastasis (11
). Regulation of Akt has been extensively studied, and it is well established that Akt activity is under the control of PI-3K (11
). More than 15 physiological substrates of Akt have been reported, including GSK-3, BAD, caspase 9, forkhead, Mdm-2, and p21WAF1 (4
), and the roles of Akt during cell proliferation, cell survival, and insulin response of cells have been relatively well characterized by investigation of the effects of phosphorylation of these substrates. The mechanism of Akt involvement in tumor metastasis, however, has remained unclear.
Metastasis and invasion are complex biological processes that include cell detachment, cell migration, tumor cell invasion of blood and/or lymphatic vessels, attachment to the vessel wall, and extravasation and proliferation at the target site (3
). The invasion activity of tumor cells is an important characteristic of metastatic tumors. The results of the present study showed induction of a dramatic increase in invasion activity by ARK5 overexpression in human pancreatic cancer cell line PANC-1 and human colon cancer cell line DLD-1, in which endogenous ARK5 expression is very weak, and the ARK5-induced invasion activity was found to be controlled by Akt. At the present time, five factors including ARK5 have been identified as members of the AMPK catalytic subunit family, and our group has reported that AMPK-α1 and AMPK-α2 are involved in tumor growth and survival (31
). However, only ARK5 of the AMPK catalytic subunit family members was regulated by Akt and showed extensive invasion activity; overexpression of α1, α2, SNARK, or MELK did not promote invasion activity (our unpublished data). In the present study, an extensive induction of invasion activity was triggered by CA-Akt1. Interestingly, the increased invasion activity induced by CA-Akt1 was not suppressed by LY294002. LY294002 suppresses Akt activation through blockage of the PI-3K-PDK-1 pathway (61
), and CA-Akt1 is the membrane-targeted version (9
). In the present study, IGF-1 induced both Akt and ARK5 activation, and LY294002 suppressed this activation completely. Therefore, it seems interesting that LY294002 does not suppress invasion activity stimulated by CA-Akt1 in the Matrigel assay. Recently, some reports suggested that PI-3K-PDK-1 pathway-independent phosphorylation of Akt and CA-Akt1 activation were not suppressed by LY294002 (38
). CA-Akt1 may be activated by a LY294002-insensitive pathway in the present setting. Since the increase in activity was completely reversed by the elimination of ARK5 by the RNAi technique and by functional suppression of ARK5 by transfection of DN-ARK5, even in the presence of constitutively active Akt, the results strongly suggest that Akt-induced invasion activity is mediated by ARK5. Human colon cancer cell line SW480, in which ARK5 is expressed at the same level as in the P/ARK and D/ARK cell lines, exhibited almost the same level of Akt/ARK5-dependent invasion activity. Moreover, the PANC-1 cell line showed weak invasion activity, and ARK5 overexpression increased it. These findings suggest that ARK5-induced invasion activity is not an artificial phenomenon.
MMP-2 and MMP-9 are well known to play key roles in tumor metastasis (37
), and ARK5 overexpression has been found to be associated with increased activation of MMP-2 and MMP-9. MT1-MMP is well known to be the activator of these MMPs (48
), and the results of our study demonstrated that ARK5 greatly stimulates MT1-MMP production. The ARK5-induced invasion is probably related to this increased MT1-MMP production. It has recently been reported that MT1-MMP production is regulated at the translational level by Akt during tumor cell invasion induced by IGF-1, which activates mTOR (mammalian target of rapamycin) via Akt activation (63
). Although overexpressed ARK5 induced MT1-MMP production, there was no change in expression of MT1-MMP mRNA in P/ARK and PANC-1 cell lines. The results of the present study demonstrated that the increase in production of MT1-MMP was suppressed by rapamycin and ARK5i. IGF-1-induced mTOR phosphorylation was suppressed by DN-Akt1, DN-ARK5, and ARK5i in the present study. Although our present results suggested that mTOR phosphorylation induced by IGF-1 is mediated by ARK5 downstream of Akt, the phosphorylation site of mTOR (Ser2448) did not match the AMPK family member phosphorylation motif (22
). Therefore, it seems reasonable that ARK5 modulates mTOR phosphorylation downstream of Akt but does not phosphorylate it directly. It is also interesting that phosphorylation of FKHRL1 was not affected by antisense ARK5 or its RNAi. Because both mTOR and FKHRL1 are reported to be phosphorylated by Akt directly (5
), it seems likely that some factor(s) regulating mTOR phosphorylation is phosphorylated by ARK5 to permit mTOR phosphorylation. It is also possible that ARK5 might phosphorylate mTOR directly at a site other than Ser2448 to cause a conformational change allowing Ser2448 phosphorylation. These possibilities should be carefully examined in the future because mTOR regulation is more complex than previously thought and is increasingly important in cancer research (49
When the P/ARK cell line was transplanted into nude mice, an increase in tumor volume was observed in the mice compared to the PANC-1 cell line. One remarkable feature of the P/ARK tumors was the absence of massive necrosis within the tumor. The necrotic areas in the P/ARK tumors were scattered and resembled not archipelagos but islands, a feature we refer to as piecemeal necrosis. Every area of piecemeal necrosis was significantly smaller than the area of necrosis in the PANC-1 tumors, and the total area of necrosis in the P/ARK tumors was also significantly smaller than in the PANC-1 tumors. Tumors derived from both PANC-1 and P/ARK cell lines contained high levels of phosphorylated Akt; however, P/ARK- but not PANC-1-derived tumors showed a significant decrease in the ratio of necrotic cell death and a marked induction of metastasis. The survival and growth of cells in tumors are largely dependent on the nutrient and oxygen supply (10
). ARK5 induces tolerance to nutrient starvation in vitro (55
), and such tolerance has been found to be involved in tumorigenesis in nude mice (31
). Tumorigenesis is also well known to be dependent on angiogenesis (15
); however, no significant changes in the numbers of microvessels were detected with anti-mouse CD31 antibody in the present study. Nor did angiogenesis-related gene expression profiling by DNA microarray technology show any significant changes (our unpublished data). Since our previous work showed that ARK5 overexpression conferred tolerance to the cell death caused by glucose starvation (55
), our present findings are highly consistent with our previous findings. These observations suggest that the accelerated tumorigenesis of P/ARK tumors is independent of angiogenesis, indicating austerity, i.e., stronger tolerance of P/ARK to oxygen and nutrient starvation (14
). It is highly probable that the austerity caused by ARK5 is one of the mechanisms of accelerated tumorigenesis without massive necrosis.
The results of this study demonstrated that both tumorigenesis and invasion are stimulated by ARK5. It is noteworthy that ARK5 is a member of the AMPK catalytic subunit family, which is activated by exposure to various stresses causing elevation of the intracellular 5′-AMP concentration. Actually overexpression of ARK5 confers tolerance to glucose starvation (55
), which is a stress that leads to a decrease in ATP and an increase in AMP. Therefore, it is reasonable to postulate that an insufficient blood supply to tumor tissue and tumor hypoxia cause activation of ARK5 through both Akt activation and increased AMP, resulting in tolerance to nutrient starvation and up-regulation of MT1-MMP production, leading to MMP-2 and MMP-9 activation and stimulating tumor invasion and metastasis. These observations are highly consistent with the notion that tumor hypoxia is the driving force of tumor malignancy (28
). Moreover, ARK5 activation is directly regulated by Akt, and Akt is overexpressed or amplified in pancreatic adenocarcinoma (29
). The PANC-1 cell line has been widely used as a nonliver metastatic pancreatic cancer cell line (59
). It has also been reported that Akt is overexpressed and activated in clinical pancreatic cancers (47
). The present study showed activation of Akt in PANC-1-derived tumors, but invasion and metastasis were strongly induced only in the presence of overexpressed ARK5. This suggests that ARK5 is a key factor during the tumorigenesis of colorectal and pancreatic cancer. ARK5 and related biological processes may serve as new targets of cancer therapy.