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"The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of. human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In. particular, transgenic expression of CDC25A cooperates with HER2 in promoting. mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced. tumorigenesis penetrance. On the basis of this evidence of a synergism between. HER2 and the cell cycle regulator CDC25A in a mouse model of mammary. tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast. cancer and its possible implications in therapeutic response. HER2 status and. CDC25A expression were assessed in 313 breast cancer patients and we found. statistically significant correlation between HER2 and CDC25A (P = .007).. Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and . very aggressive phenotype was identified (P = .005). Importantly, our in vitro. studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on. cell growth and viability relied also on CDC25A expression and that such. inhibition induces CDC25A down-regulation through phosphatidylinositol. 3-kinase\/protein kinase B pathway and DNA damage response activation. In line. with this observation, we found a statistical significant association between. CDC25A overexpression and trastuzumab-combined therapy response rate in two. different HER2-positive cohorts of trastuzumab-treated patients in either. metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P =. .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and . CDC25A that positively modulates HER2-targeted therapy response, suggesting that,. in HER2-positive breast cancer patients, CDC25A overexpression affects. trastuzumab sensitivity."

Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.