Interleukin-15 is required for immunosurveillance and immunoprevention of HER2/neu-driven mammary carcinogenesis

We previously demonstrated that HER2/neu-driven mammary carcinogenesis can be prevented by an interleukin-12 (IL-12)-adjuvanted allogeneic HER2/neu-expressing cell vaccine. Since IL-12 can induce the release of interleukin-15 (IL-15), in the present study we investigated the role played by IL-15 in HER2/neu driven mammary carcinogenesis and in its immunoprevention

Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation

20siBruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.openopenGrassilli, Emanuela; Pisano, Fabio; Cialdella, Annamaria; Bonomo, Sara; Missaglia, Carola; Cerrito, Maria Grazia; Masiero, Laura; Ianzano, Leonarda; Giordano, Federica; Cicirelli, Vittoria; Narloch, Robert; D'Amato, Filomena; Noli, Barbara; Ferri, Gian Luca; Leone, Biagio; Stanta, Giorgio; Bonin, Serena; Helin, Kristian; Giovannoni, Roberto; Lavitrano, MarialuisaGrassilli, Emanuela; Pisano, Fabio; Cialdella, Annamaria; Bonomo, Sara; Missaglia, Carola; Cerrito, Maria Grazia; Masiero, Laura; Ianzano, Leonarda; Giordano, Federica; Cicirelli, Vittoria; Narloch, Robert; D'Amato, Filomena; Noli, Barbara; Ferri, Gian Luca; Leone, Biagio; Stanta, Giorgio; Bonin, Serena; Helin, Kristian; Giovannoni, Roberto; Lavitrano, Marialuis

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation

Bruton’s tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumor tissue samples. p65BTK protein is expressed, through hnRNPK-dependent and IRES-driven translation, from a transcript containing an alternative first exon in the 5’UTR, and is post-transcriptionally regulated, via hnRNPK, by the MAPK pathway. p65BTK is endowed with strong transforming activity that depends on active ERK1/2 and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approac

Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2

INTRODUCTION: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. METHODS: FVB-huHER2 were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. RESULTS: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-gamma production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. CONCLUSIONS: Anti-huHER2 antibodies elicited in the tolerant host exert antitumoral activity

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

Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches

HER2 cancer vaccine optimization by combining Drosophila S2 insect cell manufacturing with a novel VLP-display technology

Breast cancer is a widespread oncology indication affecting more than 1.3 million people worldwide annually, 20%-30% of which are HER2 positive. HER2 is a tyrosine kinase receptor that is frequently overexpressed in several solid-tumor cancers (incl. breast, prostate, gastric, esophageal and osteosarcoma) where it denotes an aggressive phenotype, high metastatic rate, and poor prognosis. In a human context, passive HER2-targeted immunotherapy using monoclonal antibodies (mAb, e.g. Trastuzumab and Pertuzumab) has proven to be an effective treatment modality, which has dramatically improved clinical outcomes. Unfortunately, mAb therapy is very expensive and the repeated injections of high doses can be associated with severe side-effects that reduce efficacy. Vaccines are highly cost-effective, but overall progress in development of anti-cancer vaccines based on cancer-associated antigens (e.g. HER2) has been hampered by inherent immune-tolerogenic mechanisms rendering the immune system incapable of reacting against the body’s own cells/proteins (i.e. self-antigens). Consequently, many attempts to develop anti-cancer vaccines have failed in clinical trials due to insufficient immunogenicity. To circumvent this central issue, we have developed a proprietary virus-like particle (VLP)-based vaccine delivery platform. Notably, the VLP-platform is currently the only available technology to effectively facilitate multivalent “virus-like” display of large/complex vaccine antigens. This is key to overcome immune-tolerance and enable induction of therapeutically potent antibody responses directed against cancer-associated self-antigens. In this talk I will discuss the non-viral Drosophila S2 insect cell production system and how it was applied to the production of hHer2/neu antigen, including using advanced production methods such as perfusion for clinical material manufacture. Furthermore, I will present our data from a transgenic mouse model for spontaneous breast cancer development, where high-density display of the HER2 extracellular domain on the surface of virus-like particles (VLPs) enables induction of therapeutically potent anti-HER2 responses. Split-protein tag/catcher conjugation was used to facilitate directional covalent attachment of HER2 to the surface of icosahedral bacteriophage-derived VLPs, thereby harnessing the VLP platform to effectively overcome B-cell tolerance. Vaccine efficacy was demonstrated both in prevention and therapy of mammary carcinomas in HER2 transgenic mice. Thus, the HER2-VLP vaccine shows promise as a new strategy for treatment of HER2-positive cancer. The modular VLP system may also represent an effective tool for development of self-antigen based vaccines against other non-communicable diseases

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches

Mutation analysis of the Fanconi anaemia A gene in breast tumours with loss of heterozygosity at 16q24.3

The recently identified Fanconi anaemia A (FAA) gene is located on chromosomal band 16q24.3 within a region that has been frequently reported to show loss of heterozygosity (LOH) in breast cancer. FAA mutation analysis of 19 breast tumours with specific LOH at 16q24.3 was performed. Single-stranded conformational polymorphism (SSCP) analysis on cDNA and genomic DNA, and Southern blotting failed to identify any tumour-specific mutations. Five polymorphisms were identified, but frequencies of occurrence did not deviate from those in a normal control population. Therefore, the FAA gene is not the gene targeted by LOH at 16q24.3 in breast cancer. Another tumour suppressor gene in this chromosomal region remains to be identified. © 1999 Cancer Research Campaig