HER2-Driven Carcinogenesis: New Mouse Models for Novel Immunotherapies

HER2 overexpression is a hallmark of aggressive breast cancer subtypes, and HER2-targeted therapies, such as passive immunotherapy with the humanized monoclonal antibody Trastuzumab, have become standard treatments for these tumor subtypes. However, increasing evidence points to a major role for the Δ16HER2 splice variant, which is commonly coexpressed with the wild-type protein, in cancer progression, metastatic potential and resistance to Trastuzumab treatment. Using our recently derived mouse strain transgenically expressing human Δ16HER2 under the transcriptional control of the MMTV promoter, we showed that this HER2 isoform per se can transform mammary epithelium in vivo. Thus, Δ16HER2 mice provide a new preclinical model in which to study mammary carcinogenesis and the metastatic process, as well as new therapies, including immune-based DNA vaccines. Such vaccines, by virtue of the polyclonal response they induce, might synergize with standard treatments and might ensure targeting of HER2 variants no longer recognized by monoclonal antibodies. In addition, immunological memory might provide long-term anticancer immune protection without side effects associated with many conventional therapies. The efficacy of DNA vaccination against the HER2 oncoantigen has been widely demonstrated in BALB-neuT mice transgenically expressing the activated rat neu oncogene and recapitulating several features of human breast cancers; however, HER2 is a self-tolerated molecule and an effective response to it must circumvent tolerance mechanisms. Here, we retrace the findings that have led to our most promising DNA vaccines encoding human/rat chimeric forms of the HER2 molecule bearing both xenogeneic and syngeneic portions of the protein and able to overcome peripheral tolerance. Preclinical data obtained with our DNA vaccines have provided the rationale for their use in an ongoing phase I clinical trial

HER2 isoforms co-expression differently tunes mammary tumor phenotypes affecting onset, vasculature and therapeutic response

Full-length HER2 oncoprotein and splice variant Delta16 are co-expressed in human breast cancer. We studied their interaction in hybrid transgenic mice bearing human full-length HER2 and Delta16 (F1 HER2/Delta16) in comparison to parental HER2 and Delta16 transgenic mice. Mammary carcinomas onset was faster in F1 HER2/Delta16 and Delta16 than in HER2 mice, however tumor growth was slower, and metastatic spread was comparable in all transgenic mice. Full-length HER2 tumors contained few large vessels or vascular lacunae, whereas Delta16 tumors presented a more regular vascularization with numerous endothelium-lined small vessels. Delta16-expressing tumors showed a higher accumulation of i.v. injected doxorubicin than tumors expressing full-length HER2. F1 HER2/Delta16 tumors with high full-length HER2 expression made few large vessels, whereas tumors with low full-length HER2 and high Delta16 contained numerous small vessels and expressed higher levels of VEGF and VEGFR2. Trastuzumab strongly inhibited tumor onset in F1 HER2/Delta16 and Delta16 mice, but not in full-length HER2 mice. Addiction of F1 tumors to Delta16 was also shown by long-term stability of Delta16 levels during serial transplants, in contrast full-length HER2 levels underwent wide fluctuations. In conclusion, full-length HER2 leads to a faster tumor growth and to an irregular vascularization, whereas Delta16 leads to a faster tumor onset, with more regular vessels, which in turn could better transport cytotoxic drugs within the tumor, and to a higher sensitivity to targeted therapeutic agents. F1 HER2/Delta16 mice are a new immunocompetent mouse model, complementary to patient-derived xenografts, for studies of mammary carcinoma onset, prevention and therapy

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

Promise and failure of targeted therapy in breast cancer

The current molecular targets in breast cancer (BC) clinical trials were identified before the advent of the genomic era and their relevance was confirmed and validated by the introduction of gene profiling. Pioneering molecular analyses and repeated data validations on different gene platforms have thus far served to define 5 subtypes of BC based on their gene signature: luminal A, luminal B, normal-like, HER2-positive, and basal. Luminal A and B tumors are estrogen receptor (ER)-positive, while basal-like are mostly negative for ER, progesterone receptor, and HER2, i.e., triple-negative. Normal-like tumors resemble normal breast tissue and the HER2 subtype is characterized by HER2 overexpression. Here, we summarize current targeted therapeutic options for the luminal, HER2-positive, and basal-like BC subtypes with respect to results observed in clinical trials as a step toward optimizing their appropriate application in the different clinical settings. We give particular consideration to the ER- and HER2-targeted therapies approved for clinical practice with respect to their merits and shortcomings in early and advanced disease, and mention the therapeutic options currently available and potentially promising for the basal-like subtype

Inhibition of the Wnt Signalling Pathway: An Avenue to Control Breast Cancer Aggressiveness

Breast cancer (BC) is the most common tumour in women. Although the introduction of novel therapeutic approaches in clinical practice has dramatically improved the clinical outcome of BC patients, this malignant disease remains the second leading cause of cancer-related death worldwide. The wingless/integrated (Wnt) signalling pathway represents a crucial molecular node relevantly implicated in the regulation of normal somatic stem cells as well as cancer stem cell (CSC) traits and the epithelial–mesenchymal transition cell program. Accordingly, Wnt signalling is heavily dysregulated in BC, and the altered expression of different Wnt genes is significantly associated with cancer-related aggressive behaviours. For all these reasons, Wnt signalling represents a promising therapeutic target currently under clinical investigation to achieve cancer eradication by eliminating CSCs, considered by most to be responsible for tumour initiation, relapse, and drug resistance. In this review, we summarized the current knowledge on the Wnt signalling pathway in BC and have presented evidence implicating the suitability of Wnt targeting in an attempt to improve the outcome of patients without affecting the normal somatic stem cell population

The d16HER2 Splice Variant: A Friend or Foe of HER2-Positive Cancers?

Human epidermal growth factor receptor 2 (ERBB2 or HER2) amplification/overexpression is associated with a particularly aggressive molecular subtype of breast cancer (BC), characterized by a poor prognosis, increased metastatic potential, and disease recurrence. As only approximately 50% of HER2-positive patients respond to HER2-targeted therapies, greater knowledge of the biology of HER2 and the mechanisms that underlie drug susceptibility is needed to improve cure rates. Evidence suggests that the coexistence of full-length, wild-type HER2 (wtHER2) and altered forms of HER2—such as carboxy-terminus-truncated fragments, activating mutations, and splice variants—significantly increases the heterogeneity of HER2-positive disease, affecting its biology, clinical course, and treatment response. In particular, expression of the d16HER2 splice variant in human HER2-positive BC has a crucial pathobiological function, wherein the absence of sixteen amino acids from the extracellular domain induces the formation of stable and constitutively active HER2 homodimers on the tumor cell surface. Notably, the d16HER2 variant significantly influences the initiation and aggressiveness of tumors, cancer stem cell properties, epithelial–mesenchymal transition (EMT), and the susceptibility of HER2-positive BC cells to trastuzumab compared with its wtHER2 counterpart, thus constituting a novel and potentially clinically useful biomarker. The aims of this review are to summarize the existing evidence regarding the pathobiological functions of the d16HER2 variant and discuss its current and future value with regard to risk assessment and treatment choices in HER2-positive disease