In order to evaluate the potential of TORC2 inhibition as a strategy to impair tumor cell proliferation, we have analyzed the consequences of Rictor depletion in two distinct tumor cell lines MCF7 and PC3. Inhibition of TORC2 activity prevented proliferation and anchorage independent growth of both MCF7 and PC3 cells, although MCF7 were more sensitive to reduced TORC2 activity. Analysis of cell cycle profiles revealed a clear enrichment of cells in G1 phase in TORC2-inhibited cells, which coincided with a strong downregulation of Cyclin D1. These findings suggest that loss of HM site phosphorylation may be sufficient to mimic the effects of a more global inhibition of AKT activity in this context, although we cannot rule out the possibility of AKT-independent effects. In sum, our study shows that proliferation and anchorage-independent growth of tumor cells can be efficiently prevented by inhibiting TORC2, which therefore could be an effective strategy for treatment of cancers that depend on high AKT activity.
Which AKT functions are dependent on TORC2-mediated HM phosphorylation? Several studies provide evidence that two downstream effectors of AKT, FOXO1/3a and TORC1, respond differently to loss of AKT HM phosphorylation. In both Drosophila and mammalian systems, loss of TORC2 activity reduces phosphorylation-mediated inhibition of FOXO, whereas TORC1 activity is unaffected by loss of AKT HM phosphorylation [
4,
6‐
8]. This difference is reflected in the Drosophila TORC2 loss-of-function phenotypes. While loss of AKT, Rheb or TOR cause a clear reduction of cell size [
14‐
16], inhibition of TORC2, and consequently AKT HM phosphorylation, does not (VH & SC, unpublished observation). FOXO1/3a, on the other hand, has been shown to be important for regulation of resistance to oxidative stress and other apoptotic stimuli, and indeed mammalian cells lacking TORC2 or AKT HM phosphorylation are hypersensitive to apoptotic stimuli induced by H
2O
2 [
7], indole-3-carbinol [
17] or etoposide [
5]. The effect of TORC2 on cell proliferation has been studied in fibroblasts derived from knock-out mouse models of essential TORC2 components Rictor and Sin1 with controversial results. Shiota and coworkers analyzed proliferation of Rictor-deficient MEFs and showed that the cells displayed modestly slower proliferation compared to wild type [
18], whereas Jacinto and coworkers analyzed sin1-deficient MEFs, and found that they proliferated similarly to wild type cells [
7]. In the two tumor cell lines analyzed here, acute loss of TORC2 activity clearly slowed proliferation by slowing or blocking cells in G1 phase of the cell cycle. Interestingly, a recent study showed that many gliomas overexpress Rictor and have elevated TORC2 activity that contributes to their tumorigenity [
19]. Results obtained in our study suggest that the potential of preventing tumor growth by inhibition of TORC2 is not only limited to gliomas, but can be used to inhibit growth of other types of tumors as well.
Sarbassov and coworkers discovered that prolonged treatments with rapamycin or its derivatives can also inhibit TOR complex 2, in addition to the well-established inhibition of TORC1 [
17]. Therefore, rapamycin derivatives might be useful in preventing growth of tumors dependent on AKT HM phosphorylation. However, strong inhibition of TORC1 may have undesirable consequences. Therefore development of small-molecular inhibitors specific to TORC2 is necessary.