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Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.

ABSTRACT: Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed involving constant and low-frequency background subtraction, segmenting the image to membrane and background pixels followed by evaluation of trastuzumab fluorescence, normalized with the expression level of ErbB2, only in the membrane. The normalized binding of trastuzumab showed a negative correlation with the pericellular density of hyaluronan (r = -0.52) with the effect being the most pronounced in the extreme cases (i.e., low and high hyaluronan densities predicted strong and weak binding of trastuzumab, respectively). Removal of hyaluronan by hyaluronidase digestion unmasked the trastuzumab binding epitope of ErbB2 demonstrated by a significantly increased normalized binding of the antibody. The results show that the accumulation of pericellular hyaluronan plays a crucial role in masking ErbB2.

SUBMITTER: Varadi T

PROVIDER: S-EPMC3460360 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.

Váradi Tímea T Mersich Tamás T Auvinen Päivi P Tammi Raija R Tammi Markku M Salamon Ferenc F Besznyák István I Jakab Ferenc F Baranyai Zsolt Z Szöllősi János J Nagy Peter P

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20120504 8

Although trastuzumab is an efficient drug, primary and acquired resistance is a challenging problem. The authors have previously shown in mouse xenograft experiments that masking ErbB2 by hyaluronan leads to diminished binding of the antibody and consequent resistance. In the current work, they correlated trastuzumab binding with the pericellular density of hyaluronan in ErbB2-overexpressing human breast cancer samples. A method for quantifying the relative binding of trastuzumab was developed i ...[more]

PMID: 22562558

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Project description:BackgroundAlthough trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer.MethodsLncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance.ResultsAFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level.ConclusionExosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.

Project description:Trastuzumab resistance is a severe problem in the treatment of ErbB2-amplified cancer. Although trastuzumab plus pertuzumab is able to partly overcome trastuzumab resistance in ErbB2-overexpressing cancer, its antitumor efficacy remains limited. The present study investigated the antitumor activity of the combination of trastuzumab with H2-18, which is an antibody targetinsg ErbB2 domain I. Cell proliferation and inhibition experiments indicated that H2-18 and trastuzumab synergistically inhibited the proliferation of both the trastuzumab-sensitive gastric cancer cell line, NCI-N87 and the trastuzumab-resistant gastric cancer cell line, NCI-N87-TraRT. Furthermore, H2-18 plus trastuzumab inhibited the growth of NCI-N87-TraRT cells more effectively than trastuzumab plus pertuzumab, both in vitro and in vivo. Compared with trastuzumab plus pertuzumab, H2-18 plus trastuzumab had a potent ability to inhibit NCI-N87-TraRT cells to form colonies. Notably, H2-18 plus trastuzumab was more effective in inducing cell death than trastuzumab plus pertuzumab. The in vivo studies demonstrated that H2-18 plus trastuzumab effectively inhibited the growth of both NCI-N87 and NCI-N87-TraRT xenograft tumors. Further experiments revealed that in NCI-N87-TraRT cells, H2-18 plus trastuzumab was comparable to trastuzumab plus pertuzumab in the inhibition of phosphorylated (p-)HER3, p-AKT and p-ERK. However, compared with trastuzumab plus pertuzumab, H2-18 plus trastuzumab effectively activated ROS production and the phosphorylation of JNK and c-jun in NCI-N87-TraRT cells. Therefore, the superior antitumor efficacy of H2-18 plus trastuzumab over trastuzumab plus pertuzumab may be mainly attributable to the potent cell death-inducing activity. In addition, the in vitro and in vivo antitumor effect of the combination of H2-18, trastuzumab and pertuzumab were further investigated. The results revealed that H2-18 plus trastuzumab plus pertuzumab exhibited a maximal antitumor effect among all the anti-ErbB2 monoclonal antibody combinations tested. In summary, H2-18 plus trastuzumab may have potential as an effective strategy to overcome the resistance to trastuzumab in ErbB2-amplified gastric cancer cell lines.

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Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.

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Binding of trastuzumab to ErbB2 is inhibited by a high pericellular density of hyaluronan.

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