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.


Introduction
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 [1]. This plays a crucial role in cancer pathogenesis and is associated with increased tumour invasiveness and poor prognosis [2][3] [4].

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 [5]. In 2007, the US FDA approved lapatinib in combination with capecitabine for second line treatment of HER2-positive breast cancer patients [6].
Proteomics has been used to identify different breast cancer subtypes [7] [8], and to identify HER2 signalling proteins [9]. Genomic profiles of lapatinib response in breast cancer have been carried out, however, no proteomic studies have been published to date [10] [11]. 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 drugresponsive 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 [12] [13]. In order to achieve high proteomic coverage and accurate quantification, a comprehensive and reproducible ioncurrent-based proteomic expression profiling strategy developed in our lab [14][15] [16], was employed for the quantification of the response of the SKBR3 cell line to lapatinib.

Materials and Methods
Cell Culture

LC-MS/MS
Cell lysates from the SKBR3 cell line (+/-12 hours 1µM lapatinib, n=6 biological replicates) were tryptically digested using an on-pellet-digestion procedure described previously [15]. A customised nano-LC system [15], was used to separate peptides during a 5-      Table 1 end of the treatment range the combination of lapatinib and GSK23295A displayed synergy ( Figure 4C).

Discussion
( 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 [26] [27]. As kinesins and kinesin-like proteins represent  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 promising molecular targets in cancer it was decided to investigate the effect of CENPE inhibition on lapatinibtreated SKBR3 cells [28].
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 [29]. siRNA knockdown of CENPE results in arrest at the G2/M phase of the cell cycle [30]. CENPE inhibition by siRNA had a greater effect on lapatinibtreated 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 [20] and GSK923295A [31] 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 [32]; 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.