Laboratory of Molecular Design
Breast Cancer miRNA Modulation

      Aggressive breast cancer (BC) will attack 200,000 women in the US in 2010, and will take the lives of 40,000. Breast cancer cells typically display reduced levels of tumor suppressor proteins such as PDCD4, TPM1, and PTEN, and elevated levels of proliferative proteins such as H-Ras and AIB1. Loss of suppressor activity allows increased breast cancer cell proliferation, survival, microfilament destabilization, metastatic transformation, and invasion of surrounding tissues and blood vessels. We hypothesize that reduction of miR-21 activity or restoration of let-7 and miR-17-5p activity will restore homeostasis in breast cancer cells. miR-21 is a microRNA (miRNA) that inhibits the translation of PDCD4, TPM1 [1], and PTEN mRNAs. We hypothesize that knockdown of elevated miR-21 by a systemic drug that targets breast cancer cells specifically would offer a novel therapy for disseminated drug-resistant disease. In contrast, let-7 and miR-17-5p function as tumor suppressors and are underexpressed in breast cancer. let-7 family miRNAs inhibit HRAS proto-oncogene, both in vitro and in vivo. The differential expression of some let-7 isoforms correlates with progesterone receptor status, lymph node metastasis, or high proliferation index in breast cancer. miR-17-5p downregulates AIB1 protein, which acts as a rate-limiting factor for estrogen-mediated and E2F1-mediated growth in breast cancer cells. We hypothesize that restoration of let-7 and miR-17-5p activity by systemic miRNA mimics that target breast cancer cells specifically could exhibit therapeutic efficacy. Peptide nucleic acid (PNA) is a neutral polyamide derivative that resists nucleases and proteases, yet binds tightly to RNA targets with single mismatch specificity. Thus, PNA would be a powerful anti-miR agent or miRNA mimic if it could enter the cytoplasm of breast cancer cells. Most breast cancer cells overexpress insulin-like growth factor 1 (IGF1) receptor and epidermal growth factor (EGF) receptor. We have shown that PNA-IGF1 analogs are endocytosed specifically into breast cancer cells. Hence, a small EGF analog might target PNA oligomers for EGFR internalization by diseased cells, bypassing normal cells. We have designed a let-7 PNA oligomer with a cyclized EGF peptide that enters breast cancer cells. This design provides a dramatic innovation for breast cancer therapy, which will be extended to miR-21 and miR-17-5p. Specific Aim 1: Determine by confocal fluorescence microscopy whether fluorophore-PNA-EGF analogs endocytose into the cytoplasm of breast cancer cells that overexpress EGFR protein, and hybridize specifically to their miRNA targets. Specific Aim 2: Determine by Western blot whether anti-miR-21 PNA-EGF analogs will restore translation of PDCD4, TPM1, and PTEN mRNAs. Do let-7 PNA-EGF analogs knock down HRAS mRNA translation? Do miR-17-5p PNA-EGF analogs knock down AIB1 mRNA translation? Specific Aim 3: Determine whether treatment with anti-miR-21, let-7 mimic, or miR-17-5p mimic will decrease breast cancer cell proliferation, survival, microfilament disruption, metastatic transformation, and invasion of adjacent cells.

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