Lapatinib is a clinically relevant receptor tyrosine kinase inhibitor that binds to the kinase domains of ERBB1 and ERBB2. ERBB1 and ERBB2 have previously been shown to act upstream of RAS proteins in radiation-induced signal transduction pathways and to play a role in protecting tumor cells from the toxic effects of ionizing radiation. Lapatinib blocked radiation-induced tyrosine phosphorylation of ERBB1, ERBB2 and ERBB3 in parental HCT116 cells and in HCT116 cells expressing H-RAS V12 () (Caron et al, 2005a
; Caron et al, 2005b
). Inhibition of ERBB family receptor function correlated with Lapatinib inhibiting radiation-induced activation of ERK1/2 and AKT (). Lapatinib radiosensitized parental HCT116 cells expressing K-RAS D13 and HCT116 cells expressing H-RAS V12 (). These findings demonstrate that in the presence of expressed mutated active K-RAS and H-RAS proteins, the pan-ERBB receptor inhibitor Lapatinib can act as a radiosensitizer in HCT116 cells.
Lapatinib blocks radiation-induced activation of ERBB1, ERBB2, ERBB3 and activation of ERK1/2 and AKT in HCT116 cells
The development of resistance to ERBB receptor inhibitors has been observed clinically. In many of these studies, resistance to the ERBB tyrosine kinase inhibitor has been due to mutation of the receptor within its catalytic domain so that the inhibitor no-longer can bind and inhibit receptor tyrosine kinase activity. We initially cultured parental HCT116 cells in 10 µM Lapatinib, a concentration which is below the Cmax for this drug in patients although the average plasma profile of a 1500 mg QD dose peaks at ~2.5 µM; within 72h, many cells (> 95%) became detached and died from this drug exposure (data not shown). Cells were cultured in the presence of Lapatinib for a further ~ 3 months until an essentially homogeneous population of cells grew out from the survivors that were adapted to Lapatinib.
In assays to determine cell survival in the absence of serum with a Lapatinib challenge; Lapatinib adapted cells survived to a significantly greater extent than parental cells (; Figure S1
in the presence of serum). Lapatinib adapted cells grew more quickly than parental cells in the presence or absence of Lapatinib (Figure S2
). In general agreement with these findings, Lapatinib resistant cells had a greater level of survival than parental cells in colony formation assays (). When Lapatinib adapted cells were cultured in the absence of Lapatinib for > 10 flask passages (~2 months), no reversion of the resistant phenotype was observed back to the parental phenotype (). Lapatinib adapted cells were cross resistant to multiple chemotherapeutic agents including VP-16, UCN-01, Taxotere, Oxaliplatin and Doxorubicin (Figures S3–S5
). Resistance to Taxotere appeared to be somewhat less than to the other agents. As drug efflux could represent a mechanism of Lapatinib adaptation, particularly as we observed cross-resistance to multiple cytotoxic therapeutic drugs, we performed flow cytometric and immunoblotting analyses to determine the expression of ABC and MDR plasma membrane drug transporters. Little change in the protein levels of any membrane drug transporter was observed, however, comparing wild type and Lapatinib adapted HCT116 cells, arguing that changes in drug efflux was unlikely to be a major component of Lapatinib resistance mechanism under investigation (Fisher and Dent, Unpublished observations).
The generation of Lapatinib resistant HCT116 cells
Based on the above findings, we examined in molecular detail the role of ERBB receptors in Lapatinib resistance. Co-expression of dominant negative ERBB1 (CD533) and dominant negative ERBB2 (CD572) proteins suppressed basal and EGF-stimulated tyrosine phosphorylation of ERBB1 and ERBB2 in immunoprecipitates from parental HCT116 cells (). Co-expression of dominant negative ERBB1 (CD533) and dominant negative ERBB2 (CD572) suppressed basal and EGF-stimulated tyrosine phosphorylation of ERBB1 and ERBB2 in immunoprecipitates from parental HCT116 cells (). To our surprise, however, while co-expression of ERBB1 (CD533) and ERBB2 (CD572) acted in a very similar manner as Lapatinib to inhibit ERBB receptor tyrosine phosphorylation, the dominant negative receptors did not recapitulate the toxic effects of Lapatinib in serum-starved parental or adapted cells (). Further analyses revealed that although parental and Lapatinib adapted cells expressed similar total cellular amounts of ERBB1 as judged by immunoblotting of whole cell lysate, and that stimulated ERBB1 phosphorylation in response to EGF was inhibited equally well by Lapatinib in both parental and adapted cells, the plasma membrane associated levels of ERBB1 in adapted cells were considerably lower in adapted than those in parental cells. These findings were reflected also in a reduced ability of EGF to stimulate ERK1/2 signaling in adapted cells compared to parental cells () Collectively, these findings strongly argue that an ERBB1 receptor mutation has not occurred in Lapatinib adapted HCT116 cells to make these cells resistant to Lapatinib toxicity.
Molecular inhibition of ERBB1 and ERBB2 does not recapitulate the effects of Lapatinib in HCT116 cells: Lapatinib resistant cells express less cell surface ERBB1 than parental cells
We then examined the activities of known signaling pathways whose activities could become altered in the adapted HCT116 cell line. However, almost no difference in basal activities of any pathway, or in the basal activity of any pathway in the presence of Lapatinib, could be observed between parental and adapted cells (data not shown). In HCT116 cells expressing H-RAS V12 and effector mutants of H-RAS V12 that had been characterized to specifically activate: the Raf-MEK-ERK pathway (S35); the RAL-GDS pathway (G37); the PI3K-AKT pathway (C40), only H-RAS V12 expression, but not expression of any H-RAS V12 single point mutant that activated a single signaling pathway, suppressed Lapatinib toxicity (). In contrast to our findings with Lapatinib, for example, expression of H-RAS V12, H-RAS V12 S35 and H-RAS V12 C40, but not H-RAS V12 G37, acted to protect HCT116 cells from the toxic effects of radiation in colony formation assays (). After a 1 Gy radiation exposure, approximating to the shoulder of the survival curve, no statistically significant difference between cell survival for cells expressing H-RAS V12 and H-RAS V12 C40 was observed. Cells expressing H-RAS V12 S35 had a greater level of survival than vector control transfected cells however these cells had significantly less survival than cells expressing H-RAS V12 C40 (After a 1 Gy exposure: vector survival 0.70 ± 0.04; H-RAS V12 S35 survival 0.82 ± 0.04; H-RAS V12 C40 survival 0.90 ± 0.03, p < 0.05 difference between each value). The survival of cells expressing H-RAS V12 S35 was not significantly different from wild type HCT116 cells expressing K-RAS D13.
Lapatinib resistance requires multiple pathways downstream of RAS: lack of involvement of “classical” effectors of resistance
In general agreement with our short-term cell killing data using Lapatinib exposure and serum starvation, expression of constitutively active MEK1 EE and constitutively active AKT, to a greater extent than the individual activated kinases, suppressed Lapatinib toxicity in parental cells (). In contrast to the use of activated proteins, expression of dominant negative AKT and/or dominant negative MEK1 did not restore Lapatinib sensitivity in adapted cells (Figure S6
). As inhibition of ERK1/2 and AKT did not restore Lapatinib sensitivity, we explored whether other mechanisms of Lapatinib resistance were present in HCT116 cells.
Lapatinib resistance has been linked to re-activation of the estrogen receptor in breast cancer cells and the estrogen receptor is known to be expressed in colon cancer cells (Xia et al, 2006
; Cho et al, 2007
). However, incubation of adapted cells with the ER inhibitor Tamoxifen did not restore Lapatinib sensitivity (). Similarly, inhibition of NFκB function by over-expression of the IκB super repressor (dominant negative IκB) or inhibition of STAT1 and STAT3 function by expression of a dominant negative STAT3 protein did not restore Lapatinib sensitivity in adapted cells. In some cell types, including colon cancer cells, Src family nonreceptor tyrosine kinases and the insulin like growth factor receptor tyrosine kinase have been linked to the transformed phenotype. However, inhibition of neither Src family kinases using the inhibitor PP2 nor IGF1 receptor function using the inhibitor PPP restored Lapatinib sensitivity (). Of note, inhibition of the IGF1 receptor with PPP caused significant toxicity in parental cells that was abolished in Lapatinib adapted cells arguing that adapted cells were also cross resistant to agents that inhibit the function of other receptor tyrosine kinases that are known to compensate for ERBB1 survival signaling.
Based on our relative lack of success at precisely defining the signaling pathways downstream of ERBB1 and ERBB2 that could be mediating Lapatinib adaptation, we next determined the proximal downstream molecular mechanisms by which serum starved and Lapatinib treated cells die, and the mechanisms by which adaptation was gained. Adapted HCT116 cells expressed higher levels of MCL-1, BCL-XL and p53 than parental cells; these cells expressed lower levels of BAX and BAK than parental cells (, upper blotting section). No obvious changes in the protein expression of CD95, FAS ligand, pro-caspase 8, pro-caspase 9, pro-caspase 3, Apaf-1, A10, Smac/DIABLO, c-FLIP-s, XIAP, BCL-2, BID, BIM, NOXA or PUMA were noted based on immunoblotting analyses (data not shown). Based on the established concept of the so called “apoptotic rheostat,” in which BCL-2 family proteins act in a dynamic balance to suppress the pro-apoptotic signals generated by BH3 domain proteins such as BAX and BAK, our data suggest that adapted cells could be more resistant to Lapatinib than parental cells because they express more of the mitochondrial protective proteins BCL-XL and MCL-1 and that they express less of the mitochondrial toxic proteins BAX and BAK.
Lapatinib resistance is mediated by increased expression of MCL-1, decreased activation of BAK and mutation of p53
As we observed changes in the expression of proteins who act at the mitochondrion to modulate mitochondrial stability, we next determined whether activation of caspase proteases, and specifically pro-caspase 9, played a role in Lapatinib toxicity. To our surprise, inhibition of caspase function only modestly suppressed Lapatinib toxicity in parental cells treated with Lapatinib (, lower graphical section). In contrast, inhibition of caspases significantly reduced serum-withdrawal –induced cell killing (Figure S7
). Inhibition of cathepsin, calpain and serine protease function also caused similar very modest effects on promoting cell survival in Lapatinib treated cells (data not shown). Over-expression of BCL-XL abolished Lapatinib toxicity in parental cells (). Finally, we tested whether apoptosis inducing factor (AIF) played a role in Lapatinib toxicity. Knock down of AIF expression reduced Lapatinib toxicity in parental HCT116 cells, and knock down of AIF expression combined with pan-caspase inhibition almost eliminated Lapatinib toxicity ().
Knock down of MCL-1 expression, to a greater extent than that of BCL-XL, reverted Lapatinib sensitivity in adapted cells (). In we noted that the expression levels of pro- and anti-apoptotic proteins were altered comparing parental and adapted HCT116 cells. In parental cells, Lapatinib treatment caused release of AIF into the cytosol whereas in adapted cells, no AIF release was observed (). Thus the induction of cell killing by Lapatinib in parental cells correlated with activation of BAK and BAX. Knock down of BAK activation in adapted cells significantly reduced the reversion of their resistant phenotype by reduced MCL-1 expression ().
Knock down of BAK re-reverts Lapatinib adapted cells after siMCL-1 exposure
In , we noted that the expression of p53 was elevated, even though the protein levels of a p53 target protein, BAX, were reduced. In cells that express a mutated p53 protein, the expression of total p53 within a cell is often noted to be elevated. Thus, parental HCT116 cells which express a wild type p53 protein may have in part survived and adapted to Lapatinib exposure by mutating one of their p53 alleles.
Native p53 proteins were immunoprecipitated from parental and Lapatinib resistant HCT116 cells using an antibody that specifically recognizes mutated forms of p53, as judged by recognition of mutant p53 tertiary structure within the DNA binding domain of p53. The p53 proteins were then separated on denaturing SDS PAGE and immunoblotted; Lapatinib resistant cells, but not parental cells, immunoprecipitated a greater amount of “mutant” p53 (). Total poly A mRNA was isolated from adapted HCT116 cells and amplified and sequenced using primers specific for the DNA binding domain of p53. We noted, however, that adapted HCT116 cells did not contain a mutation in p53, suggesting that either our antibody was recognizing an alteration in p53 tertiary conformation in adapted cells unrelated to p53 mutation or that p53 mutation had occurred in a domain unrelated to the DNA binding domain of p53 but that was affecting the tertiary conformation of the DNA binding domain. These findings argue that Lapatinib adaptation in HCT116 cells is mediated by changes in the expression of multiple mitochondrial protective proteins, rather than mutation of ERBB receptors.