The data presented herein describe the biochemical and biological effects of PI3K isotype selective small molecule inhibitors on a panel of breast cancer cell lines. We demonstrate that these are highly effective at inhibiting the PI3K pathway, at inhibiting cell number and inducing cell death and/or cell cycle arrest, in a cell type-dependent manner depending on distinct genetic characteristics.
The importance of increased PI3K signaling in breast cancer is highlighted by the finding that p110α is mutated at high frequency in this disease [9
]. In particular these have been shown to be activating mutations, making this isoform of great interest with regard to the usefulness of p110α selective small molecules [6
]. The p110α mutation status in the breast cancer cell lines studied here has recently been determined, both by the Sanger Center, (data on their website) and in [12
]. BT20, BT474, T47D and MCF7 contain p110α mutations, and BT549, Hs578t, MDA-MB-468, MDA-MB-231, SKBr3 express wild type p110. We show that while p110α inhibitors are generally effective at the inhibition of PI3K signaling both biochemically and biologically, there was no clear increase in sensitivity seen in cell lines expressing mutant p110α. Moreover, it was also not obvious that cells expressing mutated p110α showed increased basal phosphorylation of either PKB/Akt or S6. A possible explanation for this is that while four of the 10 cell lines showed p110α mutations, all of the cancer cell lines analyzed had mutations in either p110α, PTEN, Ras or amplification of ErbB2, all of which could cause elevated PI3K pathway activity. Another explanation attests to whether the presence of a particular genetic alteration automatically implies that the altered gene is a good target for therapy. Whilst this has proven true for oncogenes such as bcr-abl and kit [38
], current evidence suggests that mutations in EGFR may not predict response to EGFR inhibitors in lung cancer [39
], and B-Raf mutations in melanoma do not currently appear to predict response to sorafenib [41
], a compound which inhibits B-Raf activity.
It should be noted that MCF10A cells, which are karyotypically normal and are not transformed, were equally sensitive to p110α inhibitors, and were in fact the most sensitive to MEK inhibitors, as measured by flow cytometry. p110α inhibitors were also recently shown to inhibit glucose uptake in non-transformed adipocytes and myotubes [17
]. It remains unclear whether strong effects on `normal' cells in culture will translate into systemic toxicity in the clinic.
Loss of PTEN is well documented to be associated with a poor clinical outcome in breast cancer [42
]. The unique sensitivity of MDA-MB-468 and BT549 cells to the p110β selective inhibitors, TGX-286 and PIK-108 is therefore of particular interest. These are the only cell lines studied which respond to these compounds when examining PKB/Akt and S6 phosphorylation. In addition, these compounds also cause a G1 arrest in MDA-MB-468 cells. Therefore, while “normal” and WT PTEN expressing tumor cells are equally sensitive to p110α inhibitors (with the exception of cell lines expressing mutant Ras, discussed below), PTEN mutant tumor cells may become more dependent on additional isoforms such as p110β. This correlation was extended to an isogenic system in which knockdown of PTEN in MDA-MB-231 cells using siRNA was sufficient to confer responsiveness to p110β inhibitors. It is unclear why PTEN mutated/deleted cells show a particular isotype dependence, although cellular localization of PTEN and particular p110 isoforms could play a role. In this regard, it has been reported that both PTEN [44
] and p110β [45
] can be localized to the nucleus. Although somewhat preliminary, these results may suggest a window of therapeutic opportunity in tumors expressing mutant PTEN to p110β inhibition. Interestingly, a particular reliance of p110β in prostate tumor cells has been previously noted by other investigators [46
]. PC3 cells, which express a mutant form of PTEN, show a high basal level of PKB/Akt phosphorylation, which is reduced upon inhibition of p110β expression, but not p110α. p110β inhibition also resulted in inhibition of invasive growth through matrigel in these cells, in contrast to p110α inhibitors [46
]. While PTEN mutations are relatively rare in breast tumors, decreases in PTEN levels are more frequent and have been shown to have prognostic significance in this disease. Tumors that have low levels of PTEN without mutations may therefore also be candidates for p110β inhibition. In addition, preliminary experiments in prostate cancer (data not shown) and glioblastoma [47
] cell lines show a similar correlation between PTEN mutations and sensitivity to p110β inhibitors. These tumors display a greater incidence of PTEN mutations and may represent a wider arena for this class of drugs.
Distinct pharmacodymamic responses were observed among the inhibitors tested and the cell lines studied. Although we have not analyzed the stability of these compounds, their structures do not suggest any aqueous instability, and most are effective over a 72 hour time period in at least one or more cell lines. In addition, other cellular screens have shown phenotypes consistent with inhibition of their respective targets [17
]. One explanation for such heterogeneous pharmacodynamic responses could be that different cell lines metabolize drugs at differing rates. An alternative explanation could be that different cell types have the ability to switch their dependence for a particular PI3K isoform after one is inhibited. These hypotheses could be tested by re-adding either the same compounds, and/or different ones at later time points to see whether this is sufficient to reduce the restored PKB/Akt phosphorylation. Given that PI-103 also inhibits p110β, one might have expected this compound to also induce a G1 arrest in MDA-MB-468 cells. However, pharmacodynamic data from shows that in MDA-MB-468 cells, the effects of PI-103 on PI3K inhibition are considerably more transient when compared to TGX-286 and PIK-108 which could explain this apparent discrepancy. These data further illustrate the importance of pharmacodynamics in drug responses and highlight the need for reliable pharmacodynamic markers in treated patients.
The finding that Hs578t cells are relatively resistant in terms of cell proliferation to the drugs studied whereas PI3K signaling is inhibited indicates that these cells can dispense with PI3K signals for cell proliferation. One explanation for this could be that these cells have activation of parallel pathways, which are able to compensate for the loss of PI3K signals. For example, Hs578t cells are known to express activated forms of the H-Ras oncogene [36
]. Another resistant cell line (both biochemically and biologically) is MDA-MB-231 which is known to harbor K-Ras mutations [35
]. The clinical importance of these observations is apparent by the finding that Ras mutations confer resistance to additional signal transduction inhibitors such as anti-EGFR therapies [39
]. Importantly, in these two breast cancer cell lines examined, the resistance to PI3K inhibitors could be overcome by the simultaneous addition of a MEK inhibitor. Part of the reason for this could be the additional input to S6 S235,236 phosphorylation through the MEK-ERK-p90rsk pathway seen in the context of activated KRas. This observation has previously been seen in HEK 293E cells expressing activated NRas [32
]. However, this residual phosphorylation could also be abolished using the p90rsk inhibitor fmk, without having any large effect on cell cycle profile, suggesting that additional MEK-dependent pathways downstream of Ras are important for this. Therefore the rational combination of targeted therapies in the appropriate genetic contexts is likely to extend the patient populations that will respond to these agents. We have previously shown that high PKB/Akt phosphorylation predicts poor response to EGFR inhibitors in patients with glioblastoma [49
]. These patients would therefore be candidates for combination therapy with a PI3K or PKB/Akt inhibitor. In support of this a strong synergistic response between LY294002 and gefitinib was previously demonstrated in glioblastoma xenograft tumors [50
Although PIK-75 was very effective at killing all cell types examined, several lines of evidence suggest that this compound is unlikely to mediate its cell death responses through inhibition of PI3K. First, PI-103 is as potent at inhibiting p110α, but does not cause such rapid and potent inhibition of cell number. Second, PIK-75 results in inhibition of cell number in MDA-MB-231 cells, which are not biochemically affected by this compound. Third, PIK-75 causes apoptosis, in contrast to PI-103 (as shown for SKBr3 cells ). Fourth, in some cell lines PIK-75 appears to be causing arrest of the cell cycle at G2/M. This is in contrast with the G1 arrest as seen here with PI-103, and also more typically seen with previous studies showing inhibition of PI3K [51
]. Given the potent and somewhat selective ability of PIK-75 to induce cell death, the task of finding its additional cellular target(s) remains an interesting question.
The cell lines described here may well reflect the behavior of tumor sub-types with respect to p110 isoform inhibition. Our findings that some small molecules described are able to inhibit PI3K and induce cell cycle arrest differentially among the breast cancer cell lines studied, indicates that these compounds may be able to operate within a therapeutic window. The behavior of sensitive and resistant cell lines described here in xenograft models will further support the validity of these compounds as therapeutics. Taken together, our data reinforces the importance of PI3K in maintaining normal cell growth and suggests that p110 selective drugs may be of value as targeted therapies.