Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

A splice isoform of the HER2 receptor that lacks exon 16 (d16HER2) is expressed in many HER2-positive breast tumors, where it has been linked with resistance to the HER2-targeting antibody trastuzumab, but the impact of d16HER2 on tumor pathobiology and therapeutic response remains uncertain. Here, we provide genetic evidence in transgenic mice that expression of d16HER2 is sufficient to accelerate mammary tumorigenesis and improve the response to trastuzumab. A comparative analysis of effector signaling pathways activated by d16HER2 and wild-type HER2 revealed that d16HER2 was optimally functional through a link to SRC activation (pSRC). Clinically, HER2-positive breast cancers from patients who received trastuzumab exhibited a positive correlation in d16HER2 and pSRC abundance, consistent with the mouse genetic results. Moreover, patients expressing high pSRC or an activated "d16HER2 metagene" were found to derive the greatest benefit from trastuzumab treatment. Overall, our results establish the d16HER2 signaling axis as a signature for decreased risk of relapse after trastuzumab treatment. Cancer Res; 74(21); 6248-59. Ó2014 AACR.

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.

ALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors.

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs.

<p class="MsoNormal" style="background: white; margin: 0cm 0cm 0pt; text-align: justify;"><span style="font-size: small;"><span lang="EN-US">The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential.<span style="color: #231f20;"> Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual<span style="mso-spacerun: yes;"> </span></span>tyrosine kinase inhibitor lapatinib. </span><span lang="EN-US">However, patients may develop resistance to</span><span lang="EN-US"> drugs and show</span><span lang="EN-US"> disease progression</span><span lang="EN-US">. Several resistant mechanisms have been explored and are still under investigation. Here, </span><span lang="EN-GB">we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, </span><span lang="EN-US">which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain.<span style="mso-spacerun: yes;"> </span>The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. </span></span></p><span style="font-size: 12pt;" lang="EN-US">Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis.</span>

We reported that the splice variant of human HER2 lacking exon 16 (delta16HER2) represents a highly penetrating HER2 oncogenic alteration identified in human primary breast tumor specimens and is able to influence the response to Trastuzumab. This HER2 variant forms covalent cysteine bonds that generate constitutively active homodimers, thereby activating multiple oncogenic downstream signaling pathways that we recently found to be mediated through activated Src kinase. To examine the ability of delta16HER2 to transform mammary epithelium in vivo and to monitor delta16HER2-driven tumorigenesis in live mice, we generated a FVB transgenic mouse model for the human delta16HER2 isoform. Transgenic female mice developed multifocal mammary tumors with a rapid onset starting at about 12 weeks of age and progressively thereafter, clearly pointing to the candidacy of the delta16HER2 isoform as the transforming form of the human HER2 oncoprotein. Histological and immunohistochemical analysis (IHC) of primary mammary nodules revealed a population of polygonal cells with classical epithelia-like aspects distinctly expressing HER2 and also a population of smaller spindle-shaped cells arranged in fascicles with lower levels of HER2 expression, suggesting the onset of the epithelial-to-mesenchymal transition (EMT). Consistent with these findings, FACS analysis of delta16HER2-positive tumor cells immunomagnetically purified from disaggregated transgenic primary tumors indicated the increased mean fluorescence intensity of HER2 staining with increasing tumor cell size. IHC analysis of the lung metastases that had formed in the majority of female mice revealed monomorphic and classical epithelial tumor cells homogeneously expressing high levels of delta16HER2. FACS and IHC analyses confirmed the lower binding efficacy of Trastuzumab to delta16HER2-overexpressing primary tumor cells cultured both under bidimensional (2D) and tridimensional (3D) conditions as compared to monoclonal reagents directed to different HER2 extracellular domain epitopes. Experiments in both primary and metastatic in vitro and in vivo delta16HER2-positive models are in progress to determine whether delta16HER2-driven tumor aggressiveness and Trastuzumab susceptibility depend not only on genetic changes intrinsic to the tumor cell, i.e., the EMT process, but also on extrinsic tumor surrounding microenvironment-related factors such as an imbalance between extracellular and intracellular pH, redox state and hypoxia. Preliminary FACS and IHC analyses indicate that delta16HER2-positive primary tumor cells are reactive for known epithelial markers as EpCAM, E-cadherin- and ck-18 and, a small subset of these mammary tumor cells, also stain positive for the mesenchymal differentiation markers vimentin, N-cadherin and ck14 significantly indicating an active EMT program. Supported by AIRC and Ministry of Health Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916. doi:1538-7445.AM2012-916