In our work, we have performed a functional protein signal pathway activation mapping and gene expression analysis of prostate tumor xenografts treated with a MEK inhibitor and found that MAP kinase inhibition caused hyperactivation of the upstream components of the canonical MAPK pathway as well as upregulation of other signaling events regulating cell growth and survival. This demonstrates the problem in basing a therapy on a simple catalogue of activated proteins in cancer cells, because an aberrantly activated protein could be part of a feedback or feed-forward control system. Without knowing its network role, inhibiting this protein could result in contributing to disease progression rather than curative therapy.
Previous studies have highlighted the complexity of selecting a targeted therapeutic agent based on the activation of a single network component (26
). Elevated mTOR activity has been observed in multiple cancers and mTOR inhibitors have shown robust activity in model systems. However, the clinical trial results with mTOR inhibitors have been more modest than predicted (27
). Studies have shown that while inhibiting mTOR activity in lung, breast, colon, and prostate cancer cells effectively suppressed the phosphorylation of downstream effectors such as p70S6K and 4EBP1, it increased the phosphorylation of AKT (26
). This increase in AKT activity attenuated the effect of mTOR inhibition and facilitated cancer cell growth and survival. Only upon discovery of this mTOR – AKT feedback control system could effective combinatorial treatments be determined; inhibition of IGFR-1 in breast and prostate cancer cell lines, and of PI3K in lung cancer cell lines, sensitized cells to mTOR inhibition (26
We utilized global analysis of protein activation and gene expression to identify compensatory events and facilitate the design of effective drug combinations. The usefulness of global analysis for identifying drug combinations was recently demonstrated using the KrasG12V/Lkb1−/− mouse model for NSCLC (35
). Analysis of gene expression and phosphoproteome profiles between primary KrasG12V tumors, primary KrasG12V/Lkb1−/−tumors and metastatic KrasG12V/Lkb1−/− tumors showed an increase in genes associated with the FAK/Src and PI3K/AKT pathways. Targeting the PI3K/AKT, MAPK, and Src pathways in combination significantly reduced tumor burden in the KrasG12V/Lkb1−/− mice compared to targeting either Src alone or PI3K/AKT and MAPK together. These experiments conceptually overlap with our own results showing that identification of compensatory signaling pathways can be used to rationally develop drug combinations.
When we combined inhibitors of IKK (NFκB pathway), or mTOR (Phosphatidyl inositol pathway) with MEK inhibition we observed synergistic cytotoxicity in CWR22Rv1 cells and we observed additivity when we combined MEK and Hedgehog inhibition according to Bliss Independence (17
). Not yet determined is the precise mechanism of synergy with these drug combinations. An increase in NFκB signaling has been associated with prostate cancer (36
). Moreover, a recent study has found that inflammatory infiltration and activation of IKK-alpha in tumor cells is associated with prostate cancer progression (28
). The activation of IKK-alpha in tumor cells following castration was dependent upon IKK-beta in infiltrating immune cells and the release of lymphotoxin. Inhibition of any component of this signaling resulted in a significant delay in the appearance of castration-resistant prostate cancer. Inhibition of MEK may trigger up regulation of NFκB signaling since NFκB activation can lead to an increase in Bcl-X(L) in some systems (37
). Such an up regulation could blunt the effectiveness of therapies by facilitating cell survival and castration-resistance.
mTOR is a protein kinase downstream of PTEN/PI3K/Akt signaling that regulates protein translation, cell growth, and apoptosis (38
). The implication of inhibiting mTOR in isolation is described above. Our data suggest that inhibiting MEK in vivo
leads to an increase in Akt and mTOR activity. This observation is consistent with previous work demonstrating that blockade of EGFR to MAPK signaling conferred a decrease in IRS-1 serine phosphorylation thereby promoting IGFR to Akt signaling (39
). MAPK signaling can affect IRS-1 serine phosphorylation either through direct phosphorylation by ERK or through the ability of ERK to transactivate p70S6K (39
). The inhibition of MEK in prostate xenografts appears to trigger a similar response and the combination of MEK and mTOR inhibition may counteract the effect of MEK inhibition on IRS-1 phosphorylation.
Hedgehog signaling is a major regulator of cellular differentiation and proliferation that is elevated in prostate cancer (29
). Previous studies have suggested cross talk between Hedgehog and MAPK signaling; specifically ERK involvement in Gli regulation (41
). In pancreatic cancer Gli is required for KRas mediated tumorigenesis (42
). Recently, direct evidence for ERK and JNK binding and phosphorylation of Gli transcription factors was reported (45
). Loss of ERK signaling in prostate cancer may trigger an increase in canonical Hedgehog signaling. The combination of MEK and Hedgehog inhibition then leads to additive growth inhibition.
One implication of these observations is that a combination therapy targeting MEK along with inhibiting IKK, mTOR, or Hedgehog may be efficacious for the treatment of prostate cancer, although further work is necessary testing these combinations in preclinical models. Previously we showed that in vivo Ras blockade could restore androgen sensitivity to a castration resistant prostate cancer xenograft, C4-2 cells (7
). This suggests that combining MEK inhibition with IKK, mTOR, or Hedgehog inhibition may be effective with androgen ablation. Also, since multiple signaling pathways are elevated in response to MEK inhibition it may be more efficacious in the clinic to use a cocktail of drugs targeting the compensatory pathways. One fundamental question remaining is if the compensatory pathways elevated in response to MEK inhibition observed in this study will be observed clinically. In our hands, CWR22Rv1 cells are the only AR positive prostate cancer cell line with active MAPK in vitro. We did not observe any additive or synergistic effect on cell cytotoxicity when testing the above combinations on LNCaP, C4-2, and LAPC4 cells. This is likely due to the lack of active MAPK in vitro, however, it is possible that the compensatory effects and subsequent effective derived drug combinations may be unique to a given cell line or individual. The broader implication of the data presented herein suggests that the conceptual paradigm of a global analysis to identify the compensatory signal transduction pathways in response to a molecular targeted agent can be used to determine effective drug combinations for the treatment of cancer, especially in the context of personalized medicine.