Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Oct 20;6(32):33134-45.
doi: 10.18632/oncotarget.5311.

t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib

Jessica L Christenson  1   2 , Erin C Denny  1   3 , Susan E Kane  1
Affiliations

t-Darpp overexpression in HER2-positive breast cancer confers a survival advantage in lapatinib

Jessica L Christenson et al. Oncotarget. .

Abstract

Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3-4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis.

Keywords: BIM; HER2; lapatinib; resistance; t-Darpp.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare having no competing interests.

Figures

Figure 1
Figure 1. SK/LapR cells
A. Cell growth in the indicated cell lines was measured by CellTiter-Glo luminescence assay after a 5-day exposure to 0.1% DMSO or 2 μM lapatinib. Data was normalized to the mean luminescence of DMSO-treated cells; mean ± standard deviation. B. Proliferation in lapatinib was quantified by SRB assay after 5-day exposure to either 0.1% DMSO or increasing concentrations of lapatinib. Data was normalized to the mean absorbance of DMSO-treated cells; mean ± standard deviation. C. Darpp-32 and t-Darpp protein levels were measured by Western analysis. α-Tubulin was used as a loading control. D. Western data was quantified using ImageJ software. Relative Darpp-32 and t-Darpp levels were calculated and each individual ratio from replicate experiments was plotted. The bar indicates the mean. E. Darpp-32 and t-Darpp mRNA levels were measured by SYBR Green quantitative RT-PCR. Data was normalized to GAPDH and SKBR3 expression levels; mean ± standard deviation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ╪ ╪p ≤ 0.0001 for each cell line compared to SKBR3 cells.
Figure 2
Figure 2. Lapatinib sensitivity in cell lines overexpressing t-Darpp
SK/HerR trastuzumab-resistant cells overexpress endogenous t-Darpp and SK.tDp cells overexpress stably transfected exogenous t-Darpp. SK.empty (SK.e) cells carry a stably transfected empty vector control. A. Darpp-32 and t-Darpp protein levels were measured by Western analysis in 0.1% DMSO (−) or 2 μM lapatinib (+) for 24 hours. β-Actin was used as a loading control. B. Proliferation in lapatinib was determined by SRB assay after 7-day exposure to either 0.1% DMSO or increasing concentrations of lapatinib. Data was normalized to the mean absorbance of DMSO-treated cells; mean ± standard deviation.
Figure 3
Figure 3. Survivin and BIM expression in response to lapatinib
Western analysis of Survivin and BIM protein levels after 24 hour exposure to 0.1% DMSO (−) or 2 μM lapatinib (+) in lapatinib-resistant cells (A), cells overexpressing t-Darpp (B), or SK/HerR cells transiently transfected with siRNA targeted to GFP (siCtrl) or Darpp-32/t-Darpp (siDp) for 72 hours (C). α-Tubulin was used as a loading control. Protein expression was quantified using ImageJ software. Data was normalized to α-Tubulin levels and expressed as the fold change in protein level after lapatinib treatment, relative to the DMSO control, for each cell line.
Figure 4
Figure 4. Colony formation by SK/HerR cells exposed to lapatinib
SKBR3 cells stably expressing EGFP (SKBR3.EGFP) and SK/HerR cells stably expressing mCherry (SK/HerR.mCherry) were co-cultured at a ratio of 1:1 and continuously exposed to 0.1% DMSO, 0.6 μM lapatinib or 1.0 μM lapatinib for 5 weeks. DMSO-treated cells were split twice-weekly (1:4 dilution). Lapatinib-treated cells were grown without passaging. Each experiment was run in triplicate. A. Co-cultured cells were imaged weekly for fluorescence (10x magnification, scale bar = 200 μm). Shown are representative fields for each condition and time point. B. The percentage of EGFP-positive and mCherry-positive cells in each co-culture was quantitatively measured weekly via flow cytometry. Data was normalized to the mean percentage of EGFP-positive and mCherry-positive cells, respectively, in DMSO-treated co-cultures; mean ± standard deviation. C. For each co-culture the number of fluorescent colonies was counted after 5 weeks in lapatinib; mean ± standard deviation, *p ≤ 0.05, ***p ≤ 0.0001.
Figure 5
Figure 5. Colony formation by SK.tDp cells exposed to lapatinib
SKBR3 cells stably expressing EGFP (SKBR3.EGFP) and SK.tDp cells stably expressing mCherry (SK.tDp2A.mCherry) were co-cultured at a ratio of 1:1 and continuously exposed to 0.1% DMSO, 0.6 μM lapatinib or 1.0 μM lapatinib for 5 weeks. DMSO-treated cells were split twice-weekly (1:4 dilution). Lapatinib-treated cells were grown without passaging. Each experiment was run in triplicate. A. Co-cultured cells were imaged weekly for fluorescence (10x magnification, scale bar = 200 μm). Shown are representative fields for each condition and time point. B. The percentage of EGFP-positive and mCherry-positive cells in each co-culture was quantitatively measured weekly via flow cytometry. Data was normalized to the mean percentage of EGFP-positive and mCherry-positive cells, respectively, in DMSO-treated co-cultures; mean ± standard deviation. C. For each co-culture the number of fluorescent colonies was counted after 5 weeks in lapatinib; mean ± standard deviation, **p ≤ 0.01.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nahta R. Molecular mechanisms of trastuzumab-based treatment in HER2-overexpressing breast cancer. ISRN Oncol. 2012;2012:428062. - PMC - PubMed
    1. Fiszman GL, Jasnis MA. Molecular mechanisms of trastuzumab resistance in HER2 overexpressing breast cancer. Int J Breast Cancer. 2011;2011:352182. - PMC - PubMed
    1. Frampton JE. Lapatinib: a review of its use in the treatment of HER2-overexpressing, trastuzumab-refractory, advanced or metastatic breast cancer. Drugs. 2009;69:2125–2148. - PubMed
    1. Moy B, Kirkpatrick P, Kar S, Goss P. Lapatinib. Nat Rev Drug Discov. 2007;6:431–432. - PubMed
    1. Verma S, Miles D, Gianni L. Updated overall survival results for EMILIA, a phase 3 study of trastuzumab emtansine (T-DM1) vs capecitabine and lapatinib in HER2-positive locally advanced or metastatic breast cancer. 2012 ESMO Congress. 2012 Oct 1;

Publication types

MeSH terms