We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.

A novel antibody–drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt–Pt–Ep1). The HFt–Pt–Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4⁺ melanoma cell line, but not to a CSPG4⁻ breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt–Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt–Pt–Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt–Pt–Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt–Pt–Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

The antigenic heterogeneity of primary and metastatic lesions surgically removed from nine patients with nodular melanoma was investigated by using monoclonal antibodies to HLA-A, B antigens, to beta 2-microglobulin, to Ia antigens, and to melanoma-associated antigens (MAA). The latter include three types of membrane-bound MAA and a cytoplasmic MAA. In spite of an homogeneous morphologic appearance, multiple lesions removed from the same patient differed significantly in their reactivity with the panel of monoclonal antibodies in indirect immunofluorescence test. The extent of antigenic heterogeneity did not correlate with melanin synthesis, site of origin of the primary tumor, site of metastatic foci, or treatment, but was less marked in patients carrying the primary tumor. The antigenic heterogeneity of multiple lesions removed from one patient and the independent expression of the various types of MAA investigated suggest that combinations of monoclonal antibodies to MAA may be more effective than single antibodies for radioimaging and immunotherapy.

With a panel of monoclonal antibodies, the effect of recombinant human leukocyte interferons (i.e., IFN-alpha A, IFN-alpha D), of a hybrid leukocyte IFN (i.e., IFN-alpha A/D (Bg1)), and of recombinant fibroblast (beta) IFN on the expression and shedding of four types of melanoma-associated antigens (MAA) and of HLA antigens by the cultured melanoma cell line Colo 38 was investigated. None of the IFN affected the expression of the high m.w. melanoma-associated antigen (HMW-MAA), but all of them increased its shedding. The expression and shedding of the 115,000 MAA and of the 100,000 MAA were increased by IFN; the magnitude of the effect as well as the kinetics were different for the various IFN preparations. The cytoplasmic MAA was the most sensitive to modulation by IFN, because all four types increased its surface expression, its total content, and its shedding. The three types of leukocyte IFN, as well as the fibroblast IFN, were all effective in increasing the expression of HLA-A,B,C antigens, the effect being more marked on the free heavy chain than on the HLA-A,B,C complex. However, only leukocyte IFN enhanced the shedding of the HLA-A,B,C molecular complex. The three types of leukocyte IFN and especially fibroblast IFN enhanced the expression of the gene products of the HLA-D region, the effect being more marked on DC-1 antigens than on HLA-DR antigens. No effect on the shedding of HLA-DR and DC-1 antigens was detected.

Analysis of surgical biopsies with monoclonal antibodies (mAbs) to framework determinants of major histocompatibility complex class I antigens has shown that malignant transformation is frequently associated with a marked loss of these cell surface molecules. The present study sought to determine whether more selective losses of major histocompatibility complex class I expression occur. Multiple specimens from 13 different types of primary and metastatic tumors were tested utilizing mAb BB7.2, which recognizes a polymorphic HLA-A2 epitope. In each case, expression of HLA-A,B,C molecules was determined by testing with mAb W6/32 directed to a framework HLA class I determinant. We have found that in HLA-A2-positive patients (identified by reactivity of their normal tissues with mAb BB7.2), HLA-A2 products are not detectable or are reduced in their expression in 70-80% of endometrial, colorectal, mammary, and renal tumors; in 40-60% of soft-tissue, skin, ovary, urinary bladder, prostate, and stomach tumors; and in 25-30% of melanomas and lung carcinomas tested. All tumors expressed the framework HLA-A,B,C determinant. The HLA-A2 epitope recognized by mAb BB7.2 is located in a portion of the HLA-A2 molecule postulated to react with the T-cell receptor. Immune surveillance to tumors is thought to depend on cytotoxic T cells, which require corecognition of polymorphic HLA class I epitopes, and on natural killer cells, which are, on the contrary, activated by the absence of HLA class I antigens. The selective loss of an HLA class I polymorphic epitope shown in this study may explain the mechanism by which tumor cells escape both T-cell recognition and natural killer cell surveillance.