Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma

Jun Hong, Ahmed Katsha, Pengcheng Lu, Yu Shyr, Abbes Belkhiri, Wael El-Rifai

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a poor outcome. Although targeting ERBB2 with trastuzumab has been evaluated in clinical trials, the molecular mechanisms of trastuzumab resistance remain uncharacterized in EAC. The dopamine and cyclic AMP-regulated phosphoprotein of MR32000 (DARPP-32), also known as PPP1R1B, is located together with ERBB2 at the 17q12-q21 amplicon. We evaluated the expression of a transcript variant of DARPP-32 (t-DARPP) and ERBB2 in 141 primary tumors and investigated the role of t-DARPP in trastuzumab resistance using OE19 and OE33 EAC cell models. Overexpression of t-DARPP mRNAwas detected in two-thirds of tumors with a correlation between ERBB2 and t-DARPP overexpression levels (r = 0.58, P < 0.003). Cell viability and clonogenic survival assays showed that t-DARPP increased survival by 40% in response to trastuzumab (P < 0.01). The Annexin-V staining and Western blot analysis indicated that t-DARPP effectively abrogated trastuzumab-induced apoptosis, inhibited cleavage of caspase-3, and blocked trastuzumab-induced dephosphorylation of ERBB2 and AKT proteins. The knockdown of endogenous t-DARPP reversed these effects and sensitized cells to trastuzumab (P < 0.01). The cycloheximide-based protein degradation analysis indicated that t-DARPP extended the half-life of ERBB2, explaining the increase in the basal levels of ERBB2, p-ERBB2(Y1248), and p-AKT(S473). Coimmunoprecipitation and Western blot analysis showed that t-DARPP associated with ERBB2 in a protein complex, and interfered with trastuzumab binding to the ERBB2 receptor. Using EAC-xenografted mouse model, t-DARPP enhanced tumor growth and rendered tumors unresponsive to trastuzumab. This study establishes t-DARPP as a mediator of trastuzumab resistance and underscores its potential importance in clinical trials of EAC.

Original languageEnglish (US)
Pages (from-to)4504-4514
Number of pages11
JournalCancer Research
Volume72
Issue number17
DOIs
StatePublished - Sep 1 2012
Externally publishedYes

Fingerprint

Adenocarcinoma
Phosphoproteins
Neoplasms
Cyclic AMP
Dopamine
Western Blotting
Trastuzumab
Clinical Trials
Survival
Annexin A5
Cycloheximide
Caspase 3
Proteolysis
Half-Life
Cell Survival
Proteins
Apoptosis
Staining and Labeling
Growth

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. / Hong, Jun; Katsha, Ahmed; Lu, Pengcheng; Shyr, Yu; Belkhiri, Abbes; El-Rifai, Wael.

In: Cancer Research, Vol. 72, No. 17, 01.09.2012, p. 4504-4514.

Research output: Contribution to journalArticle

Hong, Jun ; Katsha, Ahmed ; Lu, Pengcheng ; Shyr, Yu ; Belkhiri, Abbes ; El-Rifai, Wael. / Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. In: Cancer Research. 2012 ; Vol. 72, No. 17. pp. 4504-4514.
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abstract = "Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a poor outcome. Although targeting ERBB2 with trastuzumab has been evaluated in clinical trials, the molecular mechanisms of trastuzumab resistance remain uncharacterized in EAC. The dopamine and cyclic AMP-regulated phosphoprotein of MR32000 (DARPP-32), also known as PPP1R1B, is located together with ERBB2 at the 17q12-q21 amplicon. We evaluated the expression of a transcript variant of DARPP-32 (t-DARPP) and ERBB2 in 141 primary tumors and investigated the role of t-DARPP in trastuzumab resistance using OE19 and OE33 EAC cell models. Overexpression of t-DARPP mRNAwas detected in two-thirds of tumors with a correlation between ERBB2 and t-DARPP overexpression levels (r = 0.58, P < 0.003). Cell viability and clonogenic survival assays showed that t-DARPP increased survival by 40{\%} in response to trastuzumab (P < 0.01). The Annexin-V staining and Western blot analysis indicated that t-DARPP effectively abrogated trastuzumab-induced apoptosis, inhibited cleavage of caspase-3, and blocked trastuzumab-induced dephosphorylation of ERBB2 and AKT proteins. The knockdown of endogenous t-DARPP reversed these effects and sensitized cells to trastuzumab (P < 0.01). The cycloheximide-based protein degradation analysis indicated that t-DARPP extended the half-life of ERBB2, explaining the increase in the basal levels of ERBB2, p-ERBB2(Y1248), and p-AKT(S473). Coimmunoprecipitation and Western blot analysis showed that t-DARPP associated with ERBB2 in a protein complex, and interfered with trastuzumab binding to the ERBB2 receptor. Using EAC-xenografted mouse model, t-DARPP enhanced tumor growth and rendered tumors unresponsive to trastuzumab. This study establishes t-DARPP as a mediator of trastuzumab resistance and underscores its potential importance in clinical trials of EAC.",
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AU - Hong, Jun

AU - Katsha, Ahmed

AU - Lu, Pengcheng

AU - Shyr, Yu

AU - Belkhiri, Abbes

AU - El-Rifai, Wael

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