A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

Abstract

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD) HER1 (4,6,20,36). The need to understand the role of the HER2 receptor, and its growth-regulating ligand(s) (15,27,32), is underscored by the prevalence of HER2 DNA amplification in a variety of human epithelial cancers, including those of ovarian, gastrointestinal, and mammary origin, in which the overexpressed receptor contributes to aggressive tumor growth and reduced patient survival (13, 37). Moreover, newly developed anti-HER2 monoclonal antibodies that bind with high affinity to the receptor's extracellular domain (ECD) result in both in vitro and in vivo growth inhibition of HER2-overexpressing tumors (10,14,17,31,38). One of these murine monoclonal antibodies, muMAb4D5, is now involved in clinical testing for the treatment of patients with HER2-overexpressing breast and ovarian cancers (31).Tumors and cell lines overexpressing transmembrane growth factor receptors often release truncated receptor ECD. The truncated forms of these receptors may arise by proteolytic cleavage of the full-length receptor, as for colony-stimulating factor 1 and interleukin-2 receptors (7,26,34), and/or by alternate splicing of receptor transcript into a form that eliminates the transmembrane and cytoplasmic * Corresponding author. domains, as has been shown for interleukin-4, Fc, and EGF receptors (29,30,33). Soluble HER2 ECD is released from HER2-overexpressing tumor cells in vitro and in vivo (1, 24, 41), and the mechanism for this is thought to involve surface proteolysis of the 185-kDa receptor (41). Largely unexplored are the potential physiological effects of truncated growth factor receptors, which might involve extracellular competition for cognate ligand (5) or intracellular dominant-negative suppression of receptor function by heterodimer formation (11,20).We report here the isolation of clones encoding a truncated ECD form of the HER2 receptor from cDNA libraries prepared from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The truncated HER2 ECD transcript appears to be produced by an alternative RNA processing mechanism in which an exon extends through a splice site utilized by the full-length transcript, providing an in-frame stop codon and an alternate poly(A) addition site. The 2.3-kb truncated HER2 transcript is variably expressed in a panel of human epithelial cancer cell lines and produces intracellular HER2 ECD protein of about 100 kDa. Transfection studies suggest that excess production and intracellular retention of 100-kDa HER2 ECD in tumor cells overexpressing 185-kDa HER2 receptor results in resistance to the growth-inhibiting effects of the anti-HER2 monoclonal antibody ...

Rat Muc4 (sialomucin complex) reduces binding of anti‐ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance

“…One group suggested that intracellular expression of the extracellular domain of ErbB2 interferes with internalized ErbB2/ 4D5 complexes. 58 Others propose that since there is a correlation between ErbB-3 expression and 4D5 sensitivity in some human tumor cells, strong proliferative signals generated from an ErbB2/ ErbB-3 complex may lead to growth dependency during tumor development. 16,17 Lane et al suggest that Herceptin resistant tumor cells utilize alternative signaling pathways to override ErbB2 receptor inhibition.…”

Different mechanisms are implicated in ERBB2 gene overexpression in breast and in other cancers

“…One of the main observations of this study is that breast and ovary cancer cell lines were characterised by a membrane homogeneous staining, whereas in the non-breast cancer cells, the staining was cytoplasmic and heterogeneous ( Figure 1, Table 1). Several truncated intracellular p185 c-erbB-2 fragments have been described (Scott et al, 1993;Esparis-Ongando et al, 1999;Christianson et al, 1998;Molina et al, 2002). These fragments, which signal actively, might be responsible for the cytoplasmic staining observed in most non-breast cancer cells.…”

“…We also have to keep in mind the multiple levels of erbB-2 mRNA and protein expression regulation. Indeed, multiple erbB-2 transcripts have been described which give rise to different proteins (Scott et al, 1993;Doherty et al, 1999a, b;Siegel et al, 1999). Moreover, p185 c-erbB-2 is proteolytically cleaved to generate a soluble extracellular fragment and a constitutively active intracellular fragment (Zabrecky et al, 1991;Pupa et al, 1993;Esparis-Ogando et al, 1999).…”

Dose escalation and pharmacokinetic study of a humanized anti-HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer

“…Circulating shed antigen in this patient had been low. It is possible that tumour clones overexpressing truncated forms of the extracellular domain of the HER2 product appeared (Scott et al, 1993), but confirmation of this would require further analysis.…”