Hub genes are connected to many upstream regulators so their occurrence in a hypothesis network is less surprising.
Many upstream regulators and downstream effectors of these small GTPases have been isolated, and their modes of activation and action have gradually been elucidated.
Many upstream regulators inhibited by metronomic cyclophosphamide, including hypoxia-inducible factors and MAP kinases, have glioma-promoting activity; their inhibition may contribute to the therapeutic effectiveness of the six-day repeating metronomic cyclophosphamide schedule.
We therefore construct many possible upstream regulators and networks serving as hypotheses for the actual biological mechanism underlying the data, and then score those regulators by their statistical significance.
When all mouse genes showing concordance in both tumor models at the time when CPA-induced tumor regression is well underway (i.e., treatment day 18 in U251 tumors and day 24 in 9L tumors) were considered, many more upstream regulators were identified (Table 5; Additional file 1: Table S7).
AKT/mTOR and MAPK pathways are the main upstream regulators of autophagy (He and Klionsky, 2009).
MTORC1 is a key negative regulator of autophagy, and many upstream stimuli regulate autophagy through the MTORC1 pathway.
Many other activated upstream regulators identified here (Tables 3 and 5) are also associated with anti-tumor immune responses.
Many upstream activators and regulators of IKK activity have been identified, including the NF- κB-inducing kinase (NIK) (Nakano et al, 1998), protein kinase C ζ (PKC ζ) (Lallena et al, 1999), transforming growth factor β-activated kinase (TAK-1) (Ninomiya-Tsuji et al, 1999), MEK kinase (MEKK) 1, 2 and 3 (Zhao and Lee, 1999), and S6 kinase (Schouten et al, 1997).
Further, many of the inhibited upstream regulators identified (Tables 3 and 5) have strong pro-tumor activities (e.g., TGFB1, which increases glioma malignancy [ 49]), consistent with the overall strong anti-tumor responses that we have seen in the metronomic CPA-treated gliomas.
Many co-acting genes are upstream regulators of well-documented regulators of the TRAIL signaling machinery, such as NF-κB, p53, or AKT.
