The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

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

Multiorgan Metastasis of Human HER-2+ Breast Cancer in Rag2−/−;Il2rg−/− Mice and Treatment with PI3K Inhibitor

In vivo studies of the metastatic process are severely hampered by the fact that most human tumor cell lines derived from highly metastatic tumors fail to consistently metastasize in immunodeficient mice like nude mice. We describe a model system based on a highly immunodeficient double knockout mouse, Rag2−/−;Il2rg−/−, which lacks T, B and NK cell activity. In this model human metastatic HER-2+ breast cancer cells displayed their full multiorgan metastatic potential, without the need for selections or additional manipulations of the system. Human HER-2+ breast cancer cell lines MDA-MB-453 and BT-474 injected into Rag2−/−;Il2rg−/− mice faithfully reproduced human cancer dissemination, with multiple metastatic sites that included lungs, bones, brain, liver, ovaries, and others. Multiorgan metastatic spread was obtained both from local tumors, growing orthotopically or subcutaneously, and from cells injected intravenously. The problem of brain recurrencies is acutely felt in HER-2+ breast cancer, because monoclonal antibodies against HER-2 penetrate poorly the blood-brain barrier. We studied whether a novel oral small molecule inhibitor of downstream PI3K, selected for its penetration of the blood-brain barrier, could affect multiorgan metastatic spread in Rag2−/−; Il2rg−/− mice. NVP-BKM120 effectively controlled metastatic growth in multiple organs, and resulted in a significant proportion of mice free from brain and bone metastases. Human HER-2+ human breast cancer cells in Rag2−/−;Il2rg−/− mice faithfully reproduced the multiorgan metastatic pattern observed in patients, thus allowing the investigation of metastatic mechanisms and the preclinical study of novel antimetastatic agents

HER2 splice variants and their relevance in breast cancer

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. 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

Laparoscopic management of a large urethral leiomyoma

Introduction and hypothesis: A 42-year-old female presented with a 12-cm mass bulging the anterior vaginal wall and causing urgency urinary incontinence and bulk symptoms. Methods: Imaging showed a tumor originating from the dorsal and cranial part of the urethra and developing in the vesicouterine space and vesicovaginal septum, dislocating the bladder ventrally and the uterus cranial-dorsally. Results: Tranvaginal biopsy showed a benign leiomyoma. A laparoscopic approach with development of the vesicouterine space permitted a safe partial morcellation of the myoma. After the bladder and vaginal wall had been completely freed, further caudal dissection was conducted with isolation of the distal cranio-dorsal portion of the urethra. The dissection plane with the vaginal wall was developed up to the caudal margin of the urethral myoma almost corresponding to the vulvar plane, and total excision of the lesion was performed. Conclusion: Laparoscopic management of urethral leiomyomas that develop into the vesicouterine space and vesicovaginal septum is feasible and safe also for very large lesions. © 2019, The International Urogynecological Association

Role of d16HER2 splice variant in HER2-positive breast cancer

Almost 90% of human primary breast cancers (BCs) express 4-9% of total wild-type (WT) HER2 as a splice variant lacking exon 16 (d16HER2). Consequent in-frame activating deletion of 2 cysteine residues causes an imbalanced conformation resulting in receptor constitutive homodimerization, enhanced signaling activity, transformation and altered trastuzumab (T) binding. We recently produced a human d16HER2 transgenic (tg) mouse characterized by a rapid multifocal mammary tumors onset and the expression of active d16HER2 dimers on tumor cells, whose downstream signaling was found coupled to multiple activated nodes that include Src kinase. In order to dissect d16HER2 role in the aggressiveness and in the susceptibility to anti-HER2 therapy, we focused on the generation of stable d16HER2-expressing mammary tumor cell lines to be used in different bioassays. An immunomagnetic purification procedure was applied to generate primary homogeneously d16HER2-expressing cell cultures. These purified tumor cell lines were analyzed by flow cytometry and immunofluorescence and their migration/invasion ability was assessed through Boyden chamber test. d16HER2 downstream signaling was evaluated by western blot and T, Lapatinib (L) and Dasatinib (D) drugs sensitivity was measured with WST-1 and soft-agar bioassays. As controls, we compared in vitro d16HER2-models oncogenic features to those of the human BC BT474, which also co-expresses a low amount of d16HER2 transcript (4%), and to a murine mammary carcinoma cell line (wtHER2), derived from a primary mammary tumor developed in human WT HER2 tg mice. We found that d16HER2 in vitro models expressed high levels of stable homodimers combined with the recruitment of activated Src, STAT3, MAPK and Akt, as in vivo primary mammary tumors. Both in bidimensional (2D) and matrigel-cultured tumor cells, we confirmed the T lower binding capability for d16HER2 than other anti-HER2 MAbs directed to different extracellular domain epitopes. d16HER2 tumor cells had an enhanced migratory and invasive capacity compared to wtHER2 and BT474 cells and, notably, were completely resistant to T treatment and less responsive to L. In virtue of a highly activated Src kinase expression in d16HER2-positive models, we tested in 2D the therapeutic effects of D and observed that d16HER2-cells were significantly more sensitive than wtHER2 and BT474 cells. Preliminary 3D-drug susceptibility assays showed that the sensitivity of d16HER2 cells increased for all the tested drugs, if assessed in a suitable environment such as soft-agar. Our findings further indicate that the constitutive expression of d16HER2 variant identifies an aggressive tumor phenotype and confirm, at least in vitro in 2D conditions, that this isoform is resistant to T and L, whereas is sensitive to D. Further analyses are ongoing to analyze in vivo drug sensitivity of d16 in comparison to WT HER2 model