Expression of t-DARPP mediates trastuzumab resistance in breast cancer cells.
OBJECTIVE
We have investigated the role of t-DARPP in trastuzumab resistance in ERBB2-amplified and overexpressed breast cancer cell lines.


METHODS
We have used the HR-5 and HR-6 trastuzumab-resistant cells that were established from tumors that recurred in the presence of trastuzumab therapy following xenografts of BT-474 cells in nude mice. In addition, SKBR-3 cells, engineered for stable expression of t-DARPP, and HCC-1569 cells, which have constitutive expression of t-DARPP and are de novo resistant to trastuzumab, were used.


RESULTS
We reported > or =15-fold up-regulation of mRNA and protein levels of t-DARPP in HR-5 and HR-6 cells compared with their progenitor BT-474 trastuzumab-sensitive cells. The t-DARPP expression was not regulated by changes in its promoter DNA methylation levels. The SKBR-3 cells stably expressing t-DARPP developed resistance to trastuzumab compared with their parental cells and empty vector controls (P < 0.01). The trastuzumab-resistant cell lines showed a significant increase in pAKT (Ser(473)) and BCL2 protein levels. The small interfering RNA knockdown of t-DARPP in all trastuzumab-resistant cells led to a significant reduction in ERBB2, pAKT (Ser(473)), and BCL2 protein levels with a significant decrease in cell viability (P < or = 0.001) and an increase in cleaved caspase-3 levels, indicating the progression of these cells toward apoptosis. The t-DARPP protein was associated with both heat shock protein 90 and ERBB2 forming a potential protein complex. This association may play a role in regulating ERBB2 protein in trastuzumab-resistant cells.


CONCLUSIONS
We conclude that t-DARPP is a novel molecular target that can mediate the therapeutic resistance to trastuzumab in breast cancer cells.
Belkhiri
Abbes
A
Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Dar
Altaf A
AA
Peng
Dun Fa
DF
Razvi
Mohammad H
MH
Rinehart
Cammie
C
Arteaga
Carlos L
CL
El-Rifai
Wael
W
D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000096:biosynthesis*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005260:Female; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D051379:Mice; D020033:Protein Isoforms / Q000096:biosynthesis; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068878:Trastuzumab; D023041:Xenograft Model Antitumor Assays
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C508053:ERBB2 protein, human