Dataset Information

Protein kinase A activation confers resistance to trastuzumab in human breast cancer cell lines.

ABSTRACT: Trastuzumab is a monoclonal antibody targeted to the Her2 receptor and approved for treatment of Her2-positive breast cancer. Among patients who initially respond to trastuzumab therapy, resistance typically arises within 1 year. BT/Her(R) cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/Her(R) cells is failure to downregulate phosphoinositide 3-kinase/Akt signaling on trastuzumab binding. The current work addresses the mechanism of sustained signaling in BT/Her(R) cells, focusing on the protein kinase A (PKA) pathway.We performed microarray analysis on BT/Her(R) and BT474 cell lines to identify genes that were upregulated or downregulated in trastuzumab-resistant cells. Specific genes in the PKA pathway were quantified using reverse transcription-PCR and Western hybridization. Small interfering RNA transfection was used to determine the effects of gene knockdown on cellular response to trastuzumab. Electrophoretic mobility shift assays were used to measure cyclic AMP-responsive element binding activity under defined conditions. Immunohistochemistry was used to analyze protein expression in clinical samples.BT/Her(R) cells had elevated PKA signaling activity and several genes in the PKA regulatory network had altered expression in these cells. Downregulation of one such gene, the PKA-RIIalpha regulatory subunit, conferred partial trastuzumab resistance in Her2-positive BT474 and SK-Br-3 cell lines. Forskolin activation of PKA also produced significant protection against trastuzumab-mediated Akt dephosphorylation. In patient samples, PKA signaling appeared to be enhanced in residual disease remaining after trastuzumab-containing neoadjuvant therapy.Activation of PKA signaling may be one mechanism contributing to trastuzumab resistance in Her2-positive breast cancer. We propose a molecular model by which PKA confers its effects.

SUBMITTER: Gu L

PROVIDER: S-EPMC2787631 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Protein kinase A activation confers resistance to trastuzumab in human breast cancer cell lines.

Gu Long L Lau Sean K SK Loera Sofia S Somlo George G Kane Susan E SE

Clinical cancer research : an official journal of the American Association for Cancer Research 20091117 23

<h4>Purpose</h4>Trastuzumab is a monoclonal antibody targeted to the Her2 receptor and approved for treatment of Her2-positive breast cancer. Among patients who initially respond to trastuzumab therapy, resistance typically arises within 1 year. BT/Her(R) cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/Her(R) cells is failure to downregulate phosphoinositide 3-kinase/Akt signaling on trastuzumab binding. The current work addresses th ...[more]

PMID: 19920112

Similar Datasets

Project description:BACKGROUND:Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. METHODS:We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. RESULTS:Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. CONCLUSIONS:Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Project description:BACKGROUND/PURPOSE:TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS:To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS:TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS:Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.

Dataset Information

Protein kinase A activation confers resistance to trastuzumab in human breast cancer cell lines.

Publications

Protein kinase A activation confers resistance to trastuzumab in human breast cancer cell lines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets