The PI-3-kinase/Akt signaling pathway is critical for cancer cell growth and survival (1
) and a number of inhibitors of PI-3-kinase or Akt have been, or will soon be introduced into clinical trial as antitumor agents (13
). Determining which patients respond to these drugs will play a role in how, and at what pace they move through clinical development (35
). Our in vivo
antitumor studies in a panel of 13 molecularly characterized human tumor cell line-derived xenografts found that tumors with mutant PIK3CA or PTEN null, but without mutant Ras were sensitive to PX-866. The three most sensitive lines, which displayed a cytostatic or regression response had activating mutations in PI-3-kinase. It is noteworthy that an activating mutation in Raf in the HT-29 derived xenograft together with a PIK3CA activating mutation was insufficient to reverse the cytostatic effects of PX-866. The BxPC3 pancreatic cancer cell line with no reported mutations in the PI-3-kinase/Akt pathway also showed a cytostatic response to PX-866. This cell line has been characterized as having an inactivating mutation in Smad 4, which has recently been shown to allow these cells to downregulate PTEN and thus activate PI-3-kinase signaling (36
). This illustrates that while individual mutations or deletions in the PI-3-kinase/Akt/PTEN signaling pathway may be sufficient to predict response to PX-866, they are not be necessary for response as multiple inputs can influence this pathway. Most importantly we found that mutant oncogenic Ras is a negative predictor of response to the PI-3-kinase inhibitor PX-866 in xenografts, even those with concurrent activating mutations in PI-3-kinase, thus PI-3-Kinase mutation cannot be used as individual marker for sensitivity.
RPPA technology was used to measure protein levels and activation in the cell lines in vivo
. Several proteins known to be directly involved in PI-3-kinase/Akt signaling were studied, including PIK3CA, Akt, and GSK. Neither total nor phosphoprotein levels were significantly different between sensitive and resistant lines nor was there a significant association with the in vivo
antitumor response. Thus, the expression level and activation of these PI-3-kinase/Akt pathway proteins in vitro
under basal conditions does not translate into in vivo
tumor sensitivity. Although this finding excludes the level of Akt Thr308
phosphorylation as a predictive biomarker, AKT phosphorylation remains a surrogate endpoint for measuring the efficacy of target inhibition as phosphorylation of Akt was inhibited by PX-866 in vivo
, independent of the sensitivity or resistance of the tumor to PX-866 in terms of its growth. Cyclin B and c-Myc were shown to be significantly overexpressed in cell lines forming PX-866 resistant xenografts compared to cell lines showing sensitivity to PX-866. In previous studies, oncogenic Ras has been shown to have the ability to upregulate total c-Myc levels both through an increase in mRNA levels (31
) and increases in protein stability (32
). Cyclin B was increased in cells resistant to PX-866 treatment and showed a significant negative association with antitumor response. Cyclin B has been shown to be upregulated during Ras induced transformation and is associated with an increased mitotic rate. Whether cyclin B and c-Myc act are factors contributing to the resistance of mutant Ras tumors to PX-866, or only serve as markers of mutant Ras is not known. Additionally, the data derived from the H-Ras lines possessing the ability to activate specific downstream pathways showed that all three pathways studied are capable of activating cyclin B and c-Myc. Therefore it is possible that the robust expression of these proteins in the mutant Ras lines in the RPPA may come from a cooperative effect between the pathways.
In colony formation assays the HCT-116 H-Ras line with specific activation of PI-3-Kinase was found to be the only line sensitive to the effects of PI-3-kinase inhibition, indicating this line had been made more dependent on PI-3-Kinase signaling. Cells with the wild type H-Ras showed greater amounts of apoptosis than cells with mutant K-Ras, which may reflect differences between the two Ras isoforms in their utilization of downstream signaling (37
), or the ability of parental K-Ras to utilize less characterized pathways downstream of Ras. This also suggests the slowing of growth seen in mutant Ras tumors in vivo
following PX-866 treatment may be a result increased apoptosis. The highest apoptosis was seen in the H-Ras cell line selectively activating PI-3-K, as it lacked the resistance provided by parallel signaling pathways. Of interest, HCT-116 K-Ras null cells which retain a mutant PI-3-Kinase were not sensitized to PX-866 suggesting that in cell lines arising from a Ras mutation, in contrast to tumors with exclusive PI-3-Kinase mutations, the input from Ras may be essential for PI-3-Kinase signaling to be utilized in tumorigenic processes. This observation agrees with previous studies that found that despite this PI-3-kinase heterozygous mutation, to sensitize HCT-116 to the effects of PI-3-Kinase inhibitors in colony formation and growth inhibition assays, a homozygous PIK3CA variant need to be created and the assays performed in low serum conditions, additionally HCT-116 cells retaining mutant K-Ras but with only a homozygous wild type PI-3-kinase were able to form tumors (38
). In contrast, K-Ras null HCT-116 cells have been shown to lack the ability to form tumors (22
When injected into mice the oncogenic H-Ras and selective H-Ras cells all retained the ability to form tumors. Despite both having increased Akt activity compared with the parental HCT-116 line, the cells with an active H-Ras responded similarly to PX-866 as the parental HCT-116, while PI-3-kinase activated H-Ras cells showed a 23% greater response to PX-866 than control cells. H-Ras cells displaying low levels of Akt activity than the other constructs showed activity similar to the parental HCT-116 or HCT-116 H-Ras lines. The H-Ras line specific for RalGDS signaling showed an intermediate response, with a 17% increase in activity of PX-866 against the tumor. Together these results show that the magnitude of Akt activation does not determine response to PI-3-kinase inhibition but that activation of pathways downstream of oncogenic Ras, parallel or compensatory for active PI-3-kinase, can rescue tumors from inhibition.
Mutant active Ras has recently been described to predict resistance to small molecule inhibitors and antibodies against the EGF receptor (39
) presumably due to the down-stream location of Ras in EGF receptor signaling (41
). Additionally, Ras-driven cell lines exhibit a modest response to MEK inhibitors, showing a growth delay when grown as xenografts, while B-Raf driven xenografts display a cytostatic response (42
). This is similar to our observation with PX-866 in the context of PI-3-kinase signaling and derive from the ability of Raf and PI-3-kinase to converge on redundant downstream mediators, “funnel factors”, of translation, such as eiF4e (43
), survival factors such as Bad (44
), and cyclin D for cell cycle progression (42
). Ultimately, treatment of Ras-driven tumors may lie in the direct inhibition of the active Ras protein itself, or the combination of PI-3-kinase inhibitors, including PX-866, with other agents targeting endpoints of the Ras pathway.
The relationships of the signaling pathways we have studied are shown in . Oncogenic Ras effectively negates the effects of other potential predictors such as mutant PIK3CA which has been proposed to be a positive marker of response to inhibitors of PI-3-kinase/Akt signaling and decreases the reliance of the tumor on PI3-kinase signaling, in favor of other Ras dependent signaling pathways. Thus, it may be necessary to know the mutational status of K-Ras, PIK3CA and PTEN as markers for predicting response to PI-3-kinase inhibition. This information may have considerable significance in tumor types, such as ovarian, endometrial, and colon where mutations in PIK3CA or PTEN and Ras have been found to coexist at relevant rates (45
Signaling in resistant and sensitive lines
In summary, we have studied the activity of the PI-3-kinase inhibitor PX-866 in a panel of tumor cell line-derived xenografts and shown mutant oncogenic Ras to be a negative predictor of response to PX-866, both independently and in the presence of positive prognostic indictors such as PI3lCA and loss of PTEN activity. Known effects of Ras-induced transformation such as increased cyclin B and c-Myc were also negatively associated with antitumor response to PX-866 The level of activation of PI-3-kinase signaling measured with phospho-Akt was not sufficient to predict in vivo antitumor response to PX-866. Ras constructs modified to activate specific components of the Ras signaling pathway showed that multiple pathways are utilized for growth and survival both in vitro and in vivo in Ras dependent signaling.