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    • To determine whether the inhibition of RSK and

    2018-10-20

    To determine whether the inhibition of RSK and TTK inhibits neovascularization in vivo, we gave mice LL/2 tumor grafts. Both BI-D1870 and AZ3146, used at doses determined to be non-toxic to animals, significantly improved survival, inhibited tumor growth, and decreased vascular density. In contrast, SU5416 had no effect on survival. In agreement with a recent study (Ogawara et al., 2014), SU5416 did not affect LL/2 tumor graft growth nor significantly decreased vessel density. In Fmoc-O-Phospho-Tyr-OH to Ogawara\'s and our studies, there is a previous report showing that SU5416 significantly inhibited tumor angiogenesis and metastasis of an LL/2 model (Cuneo et al., 2007). This discrepancy may be due to the fact that Cuneo et al. (2007) began treatment with SU5416 either immediately or 1 hr after injecting LL/2 cells, whereas in our study and that of Ogawara et al. (2014), we began treatment once tumors reached a minimal volume of 100 mm3. Therefore, in the LL/2 model SU5416 may be effective in inhibiting host vessels from infiltrating the tumor, but ineffective once the tumor is well vascularized. Ogawara et al. (2014) showed that SU5416 significantly reduced tumor growth in B16 and C-26 with no effect on LL/2 tumor grafts. This was attributed to high levels of VEGF within B16 and C-26 tumors, compared with LL/2 tumors, suggesting that VEGF does not play a major role in the angiogenesis of LL/2 tumors; instead, other proangiogenic factors, such as bFGF, are responsible for angiogenesis/tumor growth in LL/2. Other studies showed that inhibition of VEGF in tumors can lead to upregulation of bFGF and other proangiogenic factors (Lu and Bergers, 2013) to overcome VEGF inhibition. Interestingly, RSK is a downstream target of bFGF (Czaplinska et al., 2014) and VEGF (Seko et al., 1998), which constitute two major proangiogenic pathways involved in tumor growth (Lu and Bergers, 2013). Although many of the signaling events involved in developmental angiogenesis are also involved in tumor angiogenesis, there are distinct differences between these two processes, which lead to dysfunctionality of the tumor vasculature. In the case of tumors, tissue disorganization, high enzymatic activity, overproduction of growth factors and extracellular matrix components, and changes in pH and oxygen in the tumor environment lead to detachment of pericytes, leakiness of vessels, and loss of vascular integrity (Jin and Jakobsson, 2012). This could explain why AZ3146 and BI-D1870 resulted in a significant decrease in vessel density in the tumor but had no effect on the vessel density in normal host tissue. This, along with the lack of change in body weight and behavior, indicates that these drugs at the doses used were not toxic. We also investigated the effect of AZ3146 and BI-D1870 on the downstream phosphorylation of targets in tumors. AZ3146 significantly decreased SMAD2 phosphorylation in tumors. SMAD2 can positively regulate VEGF release in various tumor cell lines (Seystahl et al., 2015) and plays a role in angiogenesis (Assis et al., 2015; Pen et al., 2008). BI-D1870 had no effect on LKB1 phosphorylation in tumors, unlike EBs. This finding is not surprising, as LKB1 has been shown to promote physiological angiogenesis (Londesborough et al., 2008), whereas in cancer cells it acts as a tumor suppressor and inhibits angiogenesis (Zhuang et al., 2006). However, treatment with BI-D1870 significantly decreased RPS6 phosphorylation in tumors. Interestingly, decreased phosphorylation of RPS6 has been correlated with decreased tumor angiogenesis (Hayashi et al., 2005).
    Experimental Procedures
    Acknowledgments We thank Alex Manno, Kamal Garcha, Neil Adams, Lauren Beck, Divya Santhanam, Nayasta Kusdaya, Yeji An, Jennifer Du, Leanne Studley, Gessica Raponi, Jodi Garner, and Milan Ganguly for advice and/or technical assistance. We thank the Ontario Institute for Cancer Research and Dr. Gordon Keller for reagents. This study was supported by a Terry Fox New Frontiers Program Project grant from the Canadian Institutes for Health Research and by the Ontario Institute for Cancer Research. L.H. was supported by an Ontario Institute for Regenerative Medicine postdoctoral research award.