Drug repositioning based on gene expression data for human HER2-positive breast cancer.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents approximately 15-30% of all invasive breast cancers. Despite the recent advances in therapeutic practices of HER2 subtype, drug resistance and tumor recurrence still have remained as major problems. Drug discovery is a long and difficult process, so the aim of this study is to find potential new application for existing therapeutic agents. Gene expression data for breast invasive carcinoma were retrieved from The Cancer Genome Atlas (TCGA) database. The normal and tumor samples were analyzed using Linear Models for Microarray Data (LIMMA) R package in order to find the differentially expressed genes (DEGs). These genes were used as entry for the library of integrated network-based cellular signatures (LINCS) L1000CDS2 software and suggested 24 repurposed drugs. According to the obtained results, some of these drugs including vorinostat, mocetinostat, alvocidib, CGP-60474, BMS-387032, AT-7519, and curcumin have significant functional similarity and structural correlation with FDA-approved breast cancer drugs. Based on the drug-target network, which consisted of the repurposed drugs and their target genes, the aforementioned drugs had the highest degrees. Moreover, the experimental approach verified curcumin as an effective therapeutic agent for HER2 positive breast cancer. Hence, our work suggested that some repurposed drugs based on gene expression data can be noticed as potential drugs for the treatment of HER2-positive breast cancer.

PE38-based gene therapy of HER2-positive breast cancer stem cells via VHH-redirected polyamidoamine dendrimers.

Breast cancer stem cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2+ BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin, and 5'UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with anti-HER2 VHHs were used for plasmid delivery. Stem cell proportion of MDA-MB-231, MDA-MB-231/HER2+ and MCF-10A were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression, and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. MDA-MB-231/HER2+ had higher ability to form mammosphere compared to MCF-10A. Our multi-targeted nanosystem efficiently inhibited the mammosphere formation of MDA-MB-231 and MDA-MB-231/HER2+ cells, while it was unable to prevent the mammosphere formation of MCF-10A. In the hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression, and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However, the efficacy of our targeted toxin gene therapy was lower in MDA-MB-231/HER2+ spheroids compared with that in the monolayer culture. the combination of the cell surface, transcriptional, and translational targeting increased the stringency of the treatment.

Targeted Toxin Gene Therapy Of Breast Cancer Stem Cells Using CXCR1 Promoter And bFGF 5'UTR.

BACKGROUND: Breast cancer stem cells (BCSCs) are cells with a higher ability to metastasis and resistance to conventional treatments. They have a phenotype of (CD44high/CD24low) and the unlimited ability for proliferation. Development of strategies to target the BCSC population may lead to the establishment of more effective cancer therapies. Pseudomonas exotoxin A (PE) is a potent cytotoxic protein. CXCR1 promoter provides BCSC and HER2 specificity on transcription level. 5'UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) provides tumor specificity on translation level. Here, we utilized a mutant form of PE encoding DNA "PE38", CXCR1 promoter and bFGF 5'UTR to target BCSCs. METHODS: The stemness of SK-BR-3, MDA-MB-231 and MCF10A cell lines were evaluated based on the expression of the CD44high/CD24low stem cell signature and the ability to form mammospheres. Then, the cell lines were transfected with constructs encoding luciferase/PE38 under the control of the CMV/CXCR1 promoter with or without bFGF 5'UTR. Luciferase protein expression was evaluated using dual-luciferase reporter assay. PE38 transcript expression was measured by real-time PCR, and the cytotoxic effect of PE38 protein expression was determined by MTT assay. RESULTS: The percentage of CD44high/CD24low population did not correlate to mammosphere forming efficiency (MFE). Given that the percentage of CD44 high/CD24 low is not a conclusive BCSC profile, we based our work on the mammosphere assay. However, in comparison with MCF10A, the two tumorigenic cell lines had higher MFE, probably due to their higher BCSC content. Reporter assay and real-time PCR results demonstrated that CXCR1 promoter combined with bFGF 5'UTR increased BCSC-specific gene expression. Meanwhile, tightly regulated expression of PE38 using these two gene regulatory elements resulted in high levels of cell death in the two tumorigenic cell lines while having little toxicity toward normal MCF10A. CONCLUSION: Our data show that PE38, CXCR1 promoter and bFGF 5'UTR in combination can be considered as a promising tool for killer gene therapy of breast cancer.