Academic Editor: Bobbie-Jo M. Webb-Robertson
Affilation: Senior Research Scientist , Pacific Northwest National Laboratory, Computational Biology and Bioinformatics, P.0. BOX 999, K9-70 Richland , WA , USA
Determination Of The Proteomic Response To Lapatinib Treatment Using A Comprehensive And Reproducible Ion-current-based Proteomics Strategy
Lapatinib, a small molecule tyrosine kinase inhibitor is currently used in the treatment of HER2-positive breast cancer. The aim of this study was to further understanding of lapatinib response for the development of novel treatment lapatinib-focussed treatment strategies.
HER2-overexpressing SKBR3 breast cancer cells were treated with lapatinib for 12 hours and the resultant proteome analyzed by a comprehensive ion-current-based LC-MS strategy.
Among the 1224 unique protein identified from SKBR3 cell lysates, 67 showed a significant change in protein abundance in response to lapatinib. Of these, CENPE a centromeric protein with increased abundance, was chosen for further validation. Knockdown and inhibition of CENPE demonstrated that CENPE enhances SKBR3 cell survival in the presence of lapatinib.
Based on this study, CENPE inhibitors may warrant further investigation for use in combination with lapatinib.
HER2, a member of the Human Epidermal growth factor Receptor (HER) family, is overexpressed in approximately 25% of breast cancers, resulting in the constitutive activation of tyrosine kinase signalling driving tumour cell growth . This plays a crucial role in cancer pathogenesis and is associated with increased tumour invasiveness and poor prognosis .
Lapatinib (GW572016, GlaxoSmithKline Kline, Research Triangle Park, NC), acts as a dual tyrosine kinase inhibitor of EGFR and HER-2 competing with adenosine triphosphate for its binding site on these receptors. This inhibits phosphorylation of EGFR and HER2, with downstream effects on cell survival and proliferation . In 2007, the US FDA approved lapatinib in combination with capecitabine for second line treatment of HER2-positive breast cancer patients .
Proteomics has been used to identify different breast cancer subtypes , and to identify HER2 signalling proteins . Genomic profiles of lapatinib response in breast cancer have been carried out, however, no proteomic studies have been published to date . Characterisation of cellular responses to lapatinib may have significant importance for the identification of markers of lapatinib response and to identify potential drug targets made available by lapatinib treatment thereby improving efficacy. Identification of drug-responsive proteins via proteomics approaches remains highly challenging, due to the wide dynamic range of a typical cellular proteome and the fact that most regulatory proteins are of lower abundance . In order to achieve high proteomic coverage and accurate quantification, a comprehensive and reproducible ion-current-based proteomic expression profiling strategy developed in our lab , was employed for the quantification of the response of the SKBR3 cell line to lapatinib.
Table I List of proteins identified to have altered protein abundance in response to lapatinib, grouped according to biological function
HER2 and TET2 were chosen for validation of LC-MS/MS data by Western blot. Both proteins were assessed in 7 cell lines (4 sensitive to lapatinib, 3 insensitive), Figure 1B. While TET2 was significantly changed in all 4 lapatinib sensitive cell lines, and not in the insensitive, HER2 only showed a significant increase in the SKBR3 cell line (p<0.05).
Figure 1A) Classifications of the 1224 unique proteins by Biological process, Cellular Compartment and Molecular function B) TET2 and HER2 expression, in the absence (-) or presence (+) of 1µM lapatinib after 12 hours, in lapatinib sensitive cell lines and lapatinib insensitive cell lines (highlighted in bold) C) TET2 and D) HER2 densitometry. Fold change = Control vs. Lapatinib treated. * represents significance at p<0.05 by Students t-test
Figure 2Expression of CENPE, in the absence (-) or presence (+) of 1µM lapatinib after 12 hours, in lapatinib sensitive cell lines and lapatinib insensitive cell lines (highlighted in bold)
A) By western blot including densitometric measurement of protein fold change (control vs. Lapatinib treated).
B) qRT-PCR measurement of expression changes of mRNA (control vs. Lapatinib treated)
* represents significance at p<0.05 ** at p<0.01 by Students t-test
Figure 3A) CENPE protein expression in response to a 12 hour treatment with 150nM Afatinib, 150nM Trastuzumab (Her), 150nM Trastuzumab + 1µM Lapatinib (Lap) and 1µM Lapatinib + 20µM Capecitabine (Cap) with densitometric measurement of fold change (control vs. drug treated) in B) the SKBR3 cell line and C) the BT474 cell line. * represents significance at p<0.05 ** at p<0.01 by Students t-test
A) The effect of CENPE knockdown by siRNA, with and without 100nM Lapatinib, on cell viability. Knockdown of CENPE expression confirmed by western blot.
B) Effect on cell viability (after 5 days) by the CENPE inhibitor UA62784, alone and in combination with 50nM Lapatinib.
C) Effect on cell viability (after 5 days) by the CENPE inhibitor GSK923295A, alone and in combination with 50nM Lapatinib. * represents significance at p<0.05 ** at p<0.01 by Students t-test
In order to further understand how lapatinib treatment affects HER2 positive cells we studied lapatinib-sensitive SKBR3 cells, in the absence and presence of lapatinib, by LC-MS using a highly optimised and reproducible ion-current strategy .
This approach resulted in the identification of 67 proteins that had altered abundance in response to lapatinib treatment. Western blotting analysis of two proteins, HER2 and TET2, confirmed the accuracy of MS results for the SKBR3 and highlighted the importance of validating results in multiple cells lines. CENPE, a mitotic checkpoint protein, acts as a kinesin-like motor protein aiding in the segregation of chromosomes and plays a role in the mitotic checkpoint by binding to and regulating activation of BUBR1 [21, 22]. CENPE is present in normal cells at low levels during G1 and accumulates during late G2 and M-phases . It is over expressed in invasive breast tumours compared to normal breast tissue . There is no known association of CENPE with HER2 nor with lapatinib response. In this study, CENPE demonstrated increased protein and mRNA abundance in lapatinib sensitive breast cancer cells after treatment with Lapatinib. This CENPE response seems to be specific to lapatinib as other HER2 targeting drugs, namely afatinib and trastuzumab, did not result in increased CENPE expression. Lapatinib is currently administered with capecitabine  and is undergoing testing in combination with trastuzumab , the combination of either drug with lapatinib resulted in increased CENPE protein. This suggests that lapatinib, even when administered with additional anti-cancer agents, will result in increased CENPE expression.
Previous studies have shown that it is possible to target alterations that occur in a cell in response to a drug, further sensitising the treated cells to that drug . As kinesins and kinesin-like proteins represent promising molecular targets in cancer it was decided to investigate the effect of CENPE inhibition on lapatinib-treated SKBR3 cells .
Reduction of CENPE expression has been implicated in tumour formation, however, it seems to have contradictory roles, both promoting tumourogensis at low levels of genomic instability (specifically ploidy) and inhibiting tumourogensis when a higher threshold is reached . siRNA knockdown of CENPE results in arrest at the G2/M phase of the cell cycle . CENPE inhibition by siRNA had a greater effect on lapatinib-treated cells than lapatinib alone. As small molecule inhibitors and monoclonal antibodies remain the current platform for targeted therapies, and may represent more efficient inhibition of target activity than siRNA, two small molecule drugs, UA62784  and GSK923295A  were applied to SKBR3 cells alone and in combination with lapatinib. Initial publication of UA62784 data suggest that it is a specific inhibitor of CENPE but this has subsequently been challenged ; no such controversy exists with regards to GSK923295A. Both UA62784 and GSK923295A demonstrated synergy in combination with lapatinib. The data suggests CENPE inhibition in combination with lapatinib may, with further investigation, be a novel treatment strategy. Should UA62784 ultimately prove to be a microtubule inhibitor, as suggested, lapatinib may sensitise HER2 positive breast cancer cells to a wider range of microtubule and mitotic checkpoint protein inhibitors.
The authors wish to thank the Science Foundation Ireland, Strategic Research Cluster award to Molecular Therapeutics for Cancer Ireland (award 08/SRC/B1410) for funding this work .