Determination of the Proteomic Response to Lapatinib Treatment using a comprehensive and reproducible ion-current-based proteomics strategy

Abstract

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.

Keywords: Breast; CENPE; Cancer; HER2; LC-MS; lapatinib.

Figure 1

A) 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 1

A) 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 2

Expression 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 3

A) 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

Figure 4

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