Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 11;18(1):965.
doi: 10.1186/s12885-018-4852-1.

Development of Acquired Resistance to Lapatinib May Sensitise HER2-positive Breast Cancer Cells to Apoptosis Induction by Obatoclax and TRAIL

Affiliations
Free PMC article

Development of Acquired Resistance to Lapatinib May Sensitise HER2-positive Breast Cancer Cells to Apoptosis Induction by Obatoclax and TRAIL

Alex J Eustace et al. BMC Cancer. .
Free PMC article

Abstract

Background: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.

Methods: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.

Results: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.

Conclusions: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Keywords: AKT; ErbB2; FOXO3a; MCL-1; cFLIP.

Conflict of interest statement

Ethics approval and consent to participate

LPT109096; NCT00524303 is a phase II study which randomized patients with HER2-positive stage II or III invasive breast cancer to treatment with trastuzumab, lapatinib, or both together with chemotherapy. All data were verified by US Oncology Research and GlaxoSmithKline (US Oncology 05–074, GlaxoSmithKline LPT109096, registration NCT00524303). This study was developed by the Breast Committee of US Oncology Research with GlaxoSmithKline and, in accordance with the precepts of the Helsinki Declaration, was approved by the US Oncology Research central institutional review board, Houston, TX, and clinically performed by US Oncology Research. All patients provided written informed consent.

Consent for publication

All authors have read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Percentage apoptosis induction measured by the TUNEL assay and percentage cell growth measured using Viacount on the Guava EasyCyte following treatment with either DMSO or lapatinib at 150 nM and 500 nM for 72 h in a) SKBR3-Par or b) SKBR3-L cells. c) Western blot for total and cleaved PARP following treatment with 150 nM lapatinib for 6, 24 and 48 h. d) fold change in cleaved PARP (relative to total PARP) as measured by densitometry relative to untreated control. Error bars represent the standard deviation of triplicate independent experiments. ‘*’ indicates a p value of < 0.05 calculated by Student’s t-test
Fig. 2
Fig. 2
a A composite of representative images of MCL-1 and BAX expression measured by Western blotting. Densitometry analysis relative to alpha-tubulin in SKBR3-Par, SKBR3-L, HCC1954-Par and HCC1954-L cell lines. ‘*’ indicates a significant increase (p < 0.05) in protein expression relative to the matched parental controls. b Percentage apoptosis induced by obatoclax and/or lapatinib (72 h) in SKBR3-Par and SKBR3-L cells, measured by the TUNEL assay. Error bars represent the standard deviation of triplicate independent experiments. ‘*’ indicates a p value of < 0.05 as calculated by Student’s t-test when comparing obatoclax 300 nM between SKBR3-Par and SKBR3-L cells or when comparing obatoclax (300 nM) and lapatinib (500 nM) between SKBR3-Par and SKBR3-L cells
Fig. 3
Fig. 3
a Percentage apoptosis induction following 72 h treatment with increasing concentrations of TRAIL in SKBR3-Par and SKBR3-L cells. ‘*’ indicates a significant increase (p < 0.05 as calculated by Student’s t-test) in TRAIL induced apoptosis between SKBR3-Par and SKBR3-L cells at the relevant concentration. b Western blot analysis of PARP cleavage relative to total PARP following treatment with 25 ng/mL TRAIL for 6, 24 and 48 h in SKBR3-Par and –L cells. c The effect of 8-h of TRAIL (100 ng/ml) treatment on caspase 3/7 activity normalised to cell viability relative to untreated controls in SKBR3-Par and SKBR3-L cells. Error bars represent the standard deviation of triplicate independent experiments. ‘*’ indicates a significant increase (p < 0.05 as calculated by Student’s t-test) in Caspe3/7 induction by TRAIL in SKBR3-L cells relative to SKBR3-Par cells
Fig. 4
Fig. 4
a Western blot and densitometry for c-FLIP long in SKBR3-Par and SKBR3-L cells. C-FLIP short was not detectable by Western blotting in either SKBR3-Par or SKBR3-L cells. b The effect of 8-h of TRAIL treatment (100 ng/ml) on caspase 8 and caspase 9 activity normalised to cell viability, relative to untreated controls in SKBR3-Par and SKBR3-L cells. c FOXO3a qRT-PCR in SKBR3-Par and SKBR3-L cells. d Western blot and densitometry for p-AKT (s473) relative to total AKT in SKBR3-Par and lapatinib resistant cell lines. e Percentage growth inhibition by LY294002 (LY) and/or TRAIL (TR) (72 h) in SKBR3-Par cells. Error bars represent the standard deviation of triplicate independent experiments. ‘*’ indicates a p value of < 0.05 as calculated by Student’s t-test
Fig. 5
Fig. 5
The effect of TRAIL ligand (5 ng/mL) in combination with obatoclax on proliferation of (a) SKBR3-L and (b) SKBR3-Par cells in a 5-day proliferation assay. Error bars represent the standard deviation of triplicate independent experiments. ‘*’ indicates a p value of < 0.05 as calculated by Student’s t-test

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 2006;66:1630–1639. doi: 10.1158/0008-5472.CAN-05-1182. - DOI - PubMed
    1. Xia W, Gerard CM, Liu L, Baudson NM, Ory TL, Spector NL. Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells. Oncogene [Internet]. 2005;24:6213–6221. doi: 10.1038/sj.onc.1208774. - DOI - PubMed
    1. O’Donovan N, Byrne AT, O’Connor AE, McGee S, Gallagher WM, Crown J. Synergistic interaction between trastuzumab and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells. Invest New Drugs [Internet] 2011;29:752–759. doi: 10.1007/s10637-010-9415-5. - DOI - PubMed
    1. Xia W, Bisi J, Strum J, Liu L, Carrick K, Graham KM, et al. Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers. Cancer Res [Internet] 2006;66:1640–1647. doi: 10.1158/0008-5472.CAN-05-2000. - DOI - PubMed
    1. Foster FM, Owens TW, Tanianis-Hughes J, Clarke RB, Brennan K, Bundred NJ, et al. Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer. Breast Cancer Res [Internet]. 2009;11:R41. Available from: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19563669 - PMC - PubMed

MeSH terms

Substances

Feedback