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Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α). Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α. Conclusions Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Abstract Background Smad interacting protein-1 is a transcription factor that is implicated in transforming growth factor-β/bone morphogenetic protein signaling and a repressor of E-cadherin and human telomerase reverse transcriptase. It is also involved in epithelial-mesenchymal transition and tumorigenesis. However, genetic and epigenetic alterations of SIP1 have not been fully elucidated in cancers. In this study, we investigated mutations and promoter hypermethylation of the SIP1 gene in human hepatocellular carcinomas. Methods SIP1 expression was analyzed in HCC cell lines and primary tumors in comparison to normal and non-tumor liver tissues by using semi-quantitative RT-PCR, quantitative real-time RT-PCR and immunohistochemistry. Mutation and deletion screening of the SIP1 gene were performed by direct sequencing in HCC-derived cells. Restoration of SIP1 expression was sought by treating HCC cell lines with the DNA methyl transferase inhibitor, 5-AzaC, and the histone deacetylase inhibitor, TSA. SIP1 promoter methylation was analyzed by the combined bisulfite restriction analysis assay in in silico-predicted putative promoter and CpG island regions. Results We found that the expression of SIP1 was completely lost or reduced in five of 14 (36%) HCC cell lines and 17 of 23 (74%) primary HCC tumors. Immunohistochemical analysis confirmed that SIP1 mRNA downregulation was associated with decreased expression of the SIP1 protein in HCC tissues (82.8%). No somatic mutation was observed in SIP1 exons in any of the 14 HCC cell lines. Combined treatment with DNA methyl transferase and histone deacetylase inhibitors synergistically restored SIP1 expression in SIP1-negative cell lines. Analysis of three putative gene regulatory regions revealed tumor-specific methylation in more than half of the HCC cases. Conclusions Epigenetic mechanisms contribute significantly to the downregulation of SIP1 expression in HCC. This finding adds a new level of complexity to the role of SIP1 in hepatocarcinogenesis.

Abstract Background SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver. Methods Expression of SLIT-ROBO family members was quantified by real-time qRT-PCR in 14 HCC cell lines, 8 normal and 35 tumor tissues from the liver. ANOVA and Pearson's correlation analyses were performed in R environment, and different clinicopathological subgroups were pairwise compared in Minitab. Gene expression matrices of cell lines and tissues were analyzed by Mantel's association test. Results Genewise hierarchical clustering revealed two subgroups with coordinate expression pattern in both the HCC cell lines and tissues: ROBO1, ROBO2, SLIT1 in one cluster, and ROBO4, SLIT2, SLIT3 in the other, respectively. Moreover, SLIT-ROBO expression predicted AFP-dependent subgrouping of HCC cell lines, but not that of liver tissues. ROBO1 and ROBO2 were significantly up-regulated, whereas SLIT3 was significantly down-regulated in cell lines with high-AFP background. When compared to normal liver tissue, ROBO1 was found to be significantly overexpressed, while ROBO4 was down-regulated in HCC. We also observed that ROBO1 and SLIT2 differentiated histopathological subgroups of liver tissues depending on both tumor staging and differentiation status. However, ROBO4 could discriminate poorly differentiated HCC from other subgroups. Conclusion The present study is the first in comprehensive and quantitative evaluation of SLIT-ROBO family gene expression in HCC, and suggests that the expression of SLIT-ROBO genes is regulated in hepatocarcinogenesis. Our results implicate that SLIT-ROBO transcription profile is bi-modular in nature, and that each module shows intrinsic variability. We also provide quantitative evidence for potential use of ROBO1, ROBO4 and SLIT2 for prediction of tumor stage and differentiation status.