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Further characterization of immune compartments upon DNA vaccination in Î"16HER2 mice

In silico simulation comparing wtHER2 vs delta16HER2. This video simulates the different dynamic behavior of wild type HER2 protein and its splice variant delta16HER2 once inserted into a virtual cellular membrane.

Δ16HER2 is a splice variant of HER2 and defined as the transforming isoform in HER2-positive breast cancer. It has been shown that Δ16HER2 promotes breast cancer aggressiveness and drug resistance. In the present work, we used in silico modeling to identify structural differences between Δ16HER2 and the wild-type HER2 proteins. We then developed DNA vaccines specifically against the Δ16HER2 isoform and showed that these immunotherapies hampered carcinogenesis in a breast cancer transplantable model. However, the vaccines failed to elicit immune protection in Δ16HER2 transgenic mice because of tolerogenic mechanisms toward the human HER2 self-antigen, a scenario commonly seen in HER2+ patients. Thus, we engineered bacteriophages with immunogenic epitopes of Δ16HER2 exposed on their coat for use as anticancer vaccines. These phage-based vaccines were able to break immune tolerance, triggering a protective anti-Δ16HER2 humoral response. These findings provide a rationale for the use of phage-based anti-HER2/Δ16HER2 vaccination as a safe and efficacious immunotherapy against HER2-positive breast cancers.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully unders...

A class of phosphane gold(I) compounds, made of azoles and phosphane ligands, was evaluated for a screening on the regards of Breast Cancer cell panels (BC). The compounds possess N-Au-P or Cl-Au-P bonds around the central metal, and they differ for the presence of aprotic or protic polar groups in the azoles and/or the phosphane moieties to tune their hydrophilicity. Among the six candidates, only the compounds having the P-Au-N environment and not displaying neither the hydroxyl nor carboxyl groups in the ligands were found active. The compounds were screened by MTT tests in SKBR3, A17, and MDA-MB231 cancer cells, and two compounds (namely the 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane, 5, and 4,5-dichloro-imidazolate-1yl-gold(I)-triphenylphosphane, 6) were found very cytotoxic, with the most active with an IC50 value of 3.46 μM in MDA-MB231 cells. By performing enzymatic assays in the treated cells lysates, the residual enzymatic activity of dihydrofolate reductase (DHFR) has been measured after cell treatment for 4 or 12 h in comparison with control cells. Upon 12 h of treatment, the activity of DHFR was significantly reduced in both SKBR3 and A17 cells by compounds 5 and 6, but not in human MDA-MB231 cells; interestingly, it was found remarkably high after 4 h of treatment, revealing a time dependence for the DHFR enzymatic assays. The DHFR inhibition data have been compared to those for the thioredoxin reductase (TrxR), the most recognized molecular target for gold compounds. For this latter, similar residual activities (i.e., 37 and 49% for the match of SKBR3 cells and compound 5 or 6, respectively) were found. Binding studies on the regards of ct-DNA (calf-thymus-DNA) and of plasma transporters proteins, such as BSA (bovine serum albumin) and ATF (apo transferrin), were performed. As expected for gold compounds, the data support strong binding to proteins (Ksv values range: 1.51 ÷ 2.46 × 104 M−1) and a weaker interaction with ct-DNA's minor groove (Ksv values range: 1.55 ÷ 6.12 × 103 M−1).

Lyotropic cubic liquid-crystalline systems have received increasing attention due to their unique microstructural and physicochemical properties as efficient nanocarriers for drug delivery. We report the preparation and characterization of bulk phases and cubosome dispersions of phytantriol loaded with the anticancer drug 5-fluorouracil, in neutral and anionic forms. In both cases, a Pn3m cubic phase was observed. The phytantriol phase behavior can be influenced by the addition of ionic agents, and, to this purpose, a positively charged lipid, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride salt (DOTAP), was included in the studied formulations. It was found to induce a variation of the spontaneous membrane curvature of the phytantriol lipid bilayer, generating a transition from the Pn3m to the Im3m cubic phase. When 5-fluorouracil, in its anionic form (5-FUs), was encapsulated in these latter systems, a further transition to the HII hexagonal phase was observed as a consequence of ...

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNA

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome-protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer.

Basal like breast cancer (BLBC) is a very aggressive subtype of breast cancer giving few chances of survival, against which cisplatin based therapy is a compromise among the anticancer activity, the resistance development and the severe side effects. With the aim of finding new anticancer agents alternative to cisplatin, seven gold(I) azolate/phosphane compounds were evaluated in vitro by MTT tests in human MDA-MB-231, human mammary epithelial HMLE cells overexpressing FoxQ1, and murine A17 cells as models of BLBC. Two compounds, (4,5-dichloro-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 1 and (4,5-dicyano-1H-imidazolate-1-yl)-(triphenylphosphane)-gold(I) 2 were found very active and chosen for an in vivo study in A17 tumors transplanted in syngeneic mice. The compounds resulted to be more active than cisplatin, less nephrotoxic and generally more tolerated by the mice. This study also provides evidence that both gold(I) complexes inhibited the 19 S proteasome-associated deubiquitinase USP14 and induced apoptosis, while compound 1's mechanism of action depends also on its ability to down-regulate key molecules governing cancer growth and progression, such as STAT3 and Cox-2.

Δ16HER2 is a splice variant of HER2 and defined as the transforming isoform in HER2-positive breast cancer. It has been shown that Δ16HER2 promotes breast cancer aggressiveness and drug resistance. In the present work, we used in silico modeling to identify structural differences between Δ16HER2 and the wild-type HER2 proteins. We then developed DNA vaccines specifically against the Δ16HER2 isoform and showed that these immunotherapies hampered carcinogenesis in a breast cancer transplantable model. However, the vaccines failed to elicit immune protection in Δ16HER2 transgenic mice because of tolerogenic mechanisms toward the human HER2 self-antigen, a scenario commonly seen in HER2+ patients. Thus, we engineered bacteriophages with immunogenic epitopes of Δ16HER2 exposed on their coat for use as anticancer vaccines. These phage-based vaccines were able to break immune tolerance, triggering a protective anti-Δ16HER2 humoral response. These findings provide a rationale for the use of phage-based anti-HER2/Δ16HER2 vaccination as a safe and efficacious immunotherapy against HER2-positive breast cancers.

To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.

In physiological environments (e.g. the blood), nanoparticles (NPs) are surrounded by a layer of biomolecules referred to as a ‘protein corona’ (PC). The most tightly NP-bound proteins form the so-called hard corona (HC), the key bio-entity that determines the NP's biological identity and physiological response. To date, NP-HC has been almost exclusively characterized in vitro, while NP–protein interactions under realistic in vivo conditions remain largely unexplored. In this study, we thoroughly characterized the in vivo HC of a NP formulation that forms around lipid nanoparticles with a lipid composition equal to that of clinically used liposomal amphotericin B (AmBisome®) after the recovery of the NPs from the blood circulation of FVB/N mice 10 minutes post intravenous administration. In vitro HC formed by 10 minutes incubation of NPs in FVB/N mouse plasma was used for comparison. Here we show that the biological identity (i.e. size, zeta-potential and aggregation state) of NPs in vivo is significantly different from that acquired in vitro. Furthermore, the variety of protein species in the in vivo HC was considerably larger. The present work has demonstrated that characterization of the in vivo HC is essential to provide an accurate molecular description of the biological identity of NPs in physiological environments.

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

HER2 tyrosine kinase receptor is a validated target in breast cancer therapy. However, increasing evidence points to a major role of Δ16HER2 splice variant commonly coexpressed with HER2 and identified as a clinically important HER2 molecular alteration promoting aggressive metastatic breast cancer. Consistently, mice transgenic for the human Δ16HER2 isoform (Δ16HER2 mice) develop invasive mammary carcinomas with early onset and 100% penetrance. The present study provides preclinical evidence that Δ16HER2 expression confers de novo resistance to standard anti-HER2-therapies such as Lapatinib and acquired resistance to the selective Src inhibitor Saracatinib in breast cancer. Of note, Dacomitinib, an irreversible small molecule pan-HER inhibitor, was able to completely suppress Δ16HER2-driven breast carcinogenesis. Thus, only Dacomitinib may offer benefit in this molecularly defined patient subset by irreversibly inhibiting Δ16HER2 activation.

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4mgkg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC.