Oncolytic viruses aim to specifically kill tumor cells. A major challenge is the effective targeting of disseminated tumors in vivo. We retargeted herpes simplex virus (HSV) tropism to HER-2 ...oncoprotein p185, overexpressed in ovary and breast cancers. The HER-2-retargeted R-LM249 exclusively infects and kills tumor cells expressing high levels of human HER-2. Here, we assessed the efficacy of systemically i.p. delivered R-LM249 against disseminated tumors in mouse models that recapitulate tumor spread to the peritoneum in women. The human ovarian carcinoma SK-OV-3 cells implanted intraperitoneally (i.p.) in immunodeficient Rag2⁻/⁻;Il2rg⁻/⁻ mice gave rise to a progressive peritoneal carcinomatosis which mimics the fatal condition in advanced human patients. I.p. administration of R-LM249 strongly inhibited carcinomatosis, resulting in 60% of mice free from peritoneal diffusion, and 95% reduction in the total weight of neoplastic nodules. Intraperitoneal metastases are a common outcome in breast cancer: i.p. administration of R-LM249 strongly inhibited the growth of ovarian metastases of HER-2+ MDA-MB-453 breast cells. Brain metastases were also reduced. Cumulatively, upon i.p. administration the HER-2-redirected oncolytic HSV effectively reduced the growth of ovarian and breast carcinoma disseminated to the peritoneal cavity.
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.
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. MamBo89HER2
cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2
cell line gave rise to HER2-negative tumors from which MamBo38HER2
cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2
cells induced the loss of HER2 expression. MamBo38HER2
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 MamBo38HER2
cell tumorigenic ability. Sunitinib inhibited MamBo38HER2
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.
Aerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. ...This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression.
Complex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts.
Complementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue.
Respiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.
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.
Insulin-like Growth Factor Receptor-1 (IGF1R) system sustains the genesis of rhabdomyosarcoma through IGF2 autocrine overexpression. While several IGF1R-targeted strategies have been investigated to ...interphere with rhabdomyosarcoma growth, no attempt to neutralize IGF2 has been reported. We therefore studied the possibility to hamper rhabdomyosarcoma growth with passive and active immune approaches targeting IGF2.
A murine model developing IGF2-overexpressing pelvic rhabdomyosarcoma, along with IGF2-independent salivary carcinoma, was used to investigate the efficacy and specificity of passive anti-IGFs antibody treatment. Active vaccinations with electroporated DNA plasmids encoding murine or human IGF2 were performed to elicit autochthonous anti-IGF2 antibodies. Vaccinated mice received the intravenous injection of rhabdomyosarcoma cells to study the effects of anti-IGF2 antibodies against developing metastases.
Passive administration of antibodies neutralizing IGFs delayed the onset of IGF2-overexpressing rhabdomyosarcoma but not of IGF2-independent salivary carcinoma. A DNA vaccine against murine IGF2 did not elicit antibodies, even when combined with Treg-depletion, while a DNA vaccine encoding the human IGF2 gene elicited antibodies crossreacting with murine IGF2. Mice with anti-IGF2 antibodies were partially protected against the metastatic growth of IGF2-addicted rhabdomyosarcoma cells.
Immune targeting of autocrine IGF2 inhibited rhabdomyosarcoma genesis and metastatic growth.
Oncolytic virotherapy exploits the ability of viruses to infect, replicate into, and kill tumor cells. Among the viruses that entered clinical trials are HSVs. HSVs can be engineered to become ...tumor-specific by deletion of selected genes or retargeting to tumor-specific receptors. A clinically relevant surface molecule is HER-2, hyperexpressed in one fourth of mammary and ovary carcinomas, and associated with high metastatic ability. As a previously undescribed strategy to generate HSV recombinants retargeted to HER-2 and detargeted from natural receptors, we replaced the Ig-folded core in the receptor-binding virion glycoprotein gD with anti-HER-2 single-chain antibody. The recombinant entered cells solely via HER-2 and lysed HER-2-positive cancer cells. Because of the high specificity, its safety profile in i.p. injected mice was very high, with a LD₅₀ >5 x 10⁸ pfu, a figure at least 10,000-fold higher than that of corresponding WT-gD carrying virus (LD₅₀ almost equal to 5 x 10⁴ pfu). When administered intratumorally to nude mice bearing HER-2-hyperexpressing human tumors, it strongly inhibited progressive tumor growth. The results provide a generally applicable strategy to engineer HSV recombinants retargeted to a wide range of receptors for which a single-chain antibody is available, and show the potential for retargeted HSV to exert target-specific inhibition of human tumor growth. Therapy with HER-2-retargeted oncolytic HSV could be effective in combined or sequential protocols with monoclonal antibodies and small inhibitors, particularly in patients resistant to HER-2-targeted therapy because of alterations in HER-2 signaling pathway, or against brain metastases inaccessible to anti-HER-2 antibodies.
Human sarcomas arise suddenly, thus preempting the study of preneoplastic and early neoplastic lesions. To explore the natural history of these tumors we studied male mice carrying a heterozygous ...deletion of p53 and an activated HER-2/neu transgene (BALB-p53Neu mice), that develop urethral rhabdomyosarcomas with nearly full penetrance and early onset (4 months of age). Among genes prominently upregulated in preneoplastic tissue, and more highly expressed in tumors, we found the insulin-like growth factor 2 (Igf2) and tumor suppressors, p19Arf and p21Cip1. In urethral tissues of male mice p53 was less expressed than in female mice, whereas HER-2/neu was more expressed, a combination not found in other skeletal muscles of the same mice that could contribute to the anatomic and sexual specificity of BALB-p53Neu rhabdomyosarcoma. Upregulation of p19Arf and p21Cip1 was additively determined by HER-2/neu activation and by p53 inactivation. Silencing of p19Arf or p21Cip1 in rhabdomyosarcoma cell lines can inhibit cell growth and motility, thus suggesting that these genes can contribute to growth autonomy and malignancy of tumor cells. In vivo injection of gene-silenced cells highlighted selective variations in organ-specific metastatic ability, indicating that overexpression of p19Arf and p21Cip1 controlled both tumor cell-intrinsic properties and microenvironmental interactions. The onset of pelvic rhabdomyosarcoma in BALB-p53Neu male mice is triggered by the coincidental overexpression of HER-2/neu and hypoexpression of the residual p53 allele, that foster p53 loss, Igf2 autocriny and overexpression of p19Arf and p21Cip1, a phenotype that could provide novel potential targets for cancer prevention and therapy.
Rhabdomyosarcomas (RMS) are tumors of the skeletal muscle lineage. Two main features allow for distinction between subtypes: morphology and presence/absence of a translocation between the PAX3 (or ...PAX7) and FOXO1 genes. The two main subtypes are fusion-positive alveolar RMS (ARMS) and fusion-negative embryonal RMS (ERMS). This review will focus on the role of receptor tyrosine kinases of the human epidermal growth factor receptor (EGFR) family that is comprised EGFR itself, HER2, HER3 and HER4 in RMS onset and the potential therapeutic targeting of receptor tyrosine kinases. EGFR is highly expressed by ERMS tumors and cell lines, in some cases contributing to tumor growth. If not mutated, HER2 is not directly involved in control of RMS cell growth but can be expressed at significant levels. A minority of ERMS carries a HER2 mutation with driving activity on tumor growth. HER3 is frequently overexpressed by RMS and can play a role in the residual myogenic differentiation ability and in resistance to signaling-directed therapy. HER family members could be exploited for therapeutic approaches in two ways: blocking the HER member (playing a driving role for tumor growth with antibodies or inhibitors) and targeting expressed HER members to vehiculate toxins or immune effectors.
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