Lapatinib is approved for the therapy of patients with HER2 positive breast cancer who have progressed on trastuzumab. However, the effectiveness of this compound is limited by both primary and acquired resistance. In order to identify novel mechanisms of resistance to lapatinib we have performed a genome wide loss-of-function shRNA screen. Here we have identified the tumour suppressor PTEN as a mediator of lapatinib sensitivity in vitro
and in vivo
. Previous reports have shown that lapatinib activity is not dependent upon PTEN (19
). However, using an unbiased approach, we clearly demonstrate that loss of PTEN, and the resulting activation of the PI3K pathway, leads to deregulation of lapatinib sensitivity in our model. Consistent with this, we have identified that the two most prevalent breast cancer mutations in PIK3CA (E545K and H1047R) also confer resistance to lapatinib. Therefore, hyperactivation of the PI3K pathway by either loss of PTEN function or by activating mutations of PI3K result in resistance to lapatinib. In addition, our findings are consistent with recently reported observations utilising the anti-HER2 monoclonal antibody trastuzumab (4
). However it must be noted that while overexpression of wt PIK3CA diminished the effectiveness of trastuzumab in BT474 cells it was unable to circumvent the growth inhibitory properties of lapatinib, suggesting that lapatinib may function as a single agent in patients overexpressing wt PIK3CA.
A number of possibilities might explain the differing effect of PTEN loss and lapatinib resistance observed between our group and others, including the efficiency of PTEN knockdown in targeted cell lines, the use of stably infected cell lines to determine the long term effects of PTEN knockdown and lapatinib treatment, and that a 20-fold lower dose of lapatinib was used in the initial screen, reducing the chance of non-specific effects. Be that as it may, a number of studies have identified that PTEN loss does not predict for lapatinib response in patients (19
). Similar results have been observed in trastuzumab resistance whereby no significant correlation has been observed in PTEN loss and time to progression in trastuzumab treated patients (13
). This data indicates that a larger cohort of patients may be needed in order to observe differences in response in PTEN deficient tumours. An additional explanation is the lack of a validated test to determine PTEN loss in human tumours. Until a validated test becomes available it will be difficult to try to establish reliable clinical correlations between PTEN loss and response to lapatinib and other agents. However, subsequent analysis combining both PTEN status and PI3K status has clearly demonstrated the potential of PI3K pathway hyperactivation as a biomarker for trastuzumab efficacy. As such, it will be of critical importance to equally assess PI3K pathway hyperactivation as a predictor to lapatinib response.
Abnormal activation of the PI3K pathway is frequent in breast cancer. Loss-of-function PTEN or PIK3CA mutations have been observed in approximately 20%-25% and 18%-40% of primary breast cancers, respectively (13
). Taking into consideration the near mutual exclusivity between loss-of-function PTEN mutations and PI3K mutations (14
), it is not surprising that deregulation of the PI3K pathway likely occurs in over 50% of breast cancers (29
). In addition, a significant correlation between HER2 overexpression and the presence of PI3K mutations has been described (14
There are several potential implications of these observations. One such implication is that PTEN status and the presence of PI3K activating mutations should be taken into account in clinical studies with anti-HER2 agents since they could predict for resistance. A second consequence of our findings is that hyperactivation of the PI3K pathway may be pharmacologically targeted which could in turn result in reversal of lapatinib resistance. This has been a focus of our study. We have demonstrated a near complete loss of PI3K downstream signalling in BT474 cells harbouring a deregulated PI3K pathway upon treatment with the dual PI3K/mTOR inhibitor NVP-BEZ235 and lapatinib. Interestingly, treatment of NVP-BEZ235 alone in PI3K mutant cell lines was sufficient to inhibit AKT phosphorylation. This is in contrast to cells with PTEN loss where the same NVP-BEZ235 dose fails to completely abrogate AKT activity. Considering PI3K mutant cell lines retain PTEN, this result highlights a collaboration between mechanisms to downregulate signalling through the cascade- NVP-BEZ235 inhibiting PIK3CA and PTEN dephosphorylating its downstream target PIP3. Ultimately, this could impact clinical decision making, where lower doses of NVP-BEZ235 may be selected for patients harbouring activating mutations of PI3K, with higher doses for those individuals with PTEN loss.
Recent data has highlighted the use of the PI3K inhibitors LY294002 and wortmanin in the restoration of trastuzumab sensitivity in PTEN-deficient cells (4
). However, the use of these compounds in the clinic has been limited by their poor pharmacokinetics and excessive toxicity (reviewed in Marone et al. (41
)). Similarly, the use of rapamycin in patients with an activated PI3K pathway has shown promising results in clinical trials(42
). Again, however, patients who rapidly progressed on rapamycin treatment exhibited enhanced PRAS40 phosphorylation, a downstream target of AKT. Although highly promising, this data suggests that rapamycin efficacy in patients is limited due to the inhibition of the negative feedback loop.
Here our data suggests that combination therapy with NVP-BEZ235, which is in early-stage clinical trials, and lapatinib should be considered in patients whose tumours have a defined deregulated PI3K pathway.
Deciphering the molecular basis of response to lapatinib and other HER2 directed therapies is of great importance to maximizing the clinical efficacy of these compounds. In this present study we demonstrate the power of genome wide loss of function screens to identify critical components of lapatinib sensitivity. Furthermore our data justifies the need for future clinical trails to validate the PI3K pathway as a biomarker for lapatinib sensitivity and to explore a combined blockade with anti-PI3K inhibitors and lapatinib in a selected patient population with tumors with HER2 amplification and hyperactivation of the PI3K pathway by PTEN deletion or activating PI3K mutations.