Identification and Characterization of Nucleolin as a COUP-TFII Coactivator of Retinoic Acid Receptor β Transcription in Breast Cancer Cells

The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 108 to 1 × 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases

PGE2 and Poloxamer Synperonic F108 Enhance Transduction of Human HSPCs with a β-Globin Lentiviral Vector

Lentiviral vector (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. Clinical trials are currently underway to treat sickle cell disease using LVs expressing designed anti-sickling globin genes. However, because of the large size and complexity of the human β-globin gene, LV products often have low titers and transduction efficiency, requiring large amounts to treat a single patient. Furthermore, transduction of patient HSPCs often fails to achieve a sufficiently high vector copy number (VCN) and transgene expression for clinical benefit. We therefore investigated the combination of two compounds (PGE2 and poloxamer synperonic F108) to enhance transduction of HSPCs with a clinical-scale preparation of Lenti/G-AS3-FB. Here, we found that transduction enhancers increased the in vitro VCN of bulk myeloid cultures ∼10-fold while using a 10-fold lower LV dose. This was accompanied by an increased percentage of transduced colony-forming units. Importantly, analysis of immune-deficient NSG xenografts revealed that the combination of PGE2/synperonic F108 increased LV gene transfer in a primitive HSC population, with no effects on lineage distribution or engraftment. The use of transduction enhancers may greatly improve efficacy for LV-based HSPC gene therapy

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs

Use of a TGFβ type I receptor inhibitor in mouse skin carcinogenesis reveals a dual role for TGFβ signaling in tumor promotion and progression

Pharmacological inhibitors of the transforming growth factor β (TGFβ) type I receptor (ALK5) have shown promise in blocking growth of xenotransplanted cancer cell lines but the effect on a multistage cancer model is not known. To test this, we treated mouse skin with SB431542 (SB), a well-characterized ALK5 inhibitor, during a two-stage skin carcinogenesis assay. Topical SB significantly reduced the total number, incidence and size of papillomas compared with 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion alone, and this was linked to increased epidermal apoptosis, decreased proliferation and decreased cutaneous inflammation during promotion. In contrast, the frequency of conversion to squamous cell carcinoma (SCC) was 2-fold higher in papillomas treated with SB. Although there was no difference in tumor cell proliferation in early premalignant lesions, those that formed after SB treatment exhibited reduced squamous differentiation and an altered inflammatory microenvironment similar to SCC. In an inducible epidermal RAS transgenic model, treatment with SB enhanced proliferation and cutaneous inflammation in skin but decreased expression of keratin 1 and increased expression of simple epithelial keratin 18, markers of premalignant progression. In agreement with increased frequency of progression in the multistage model, SB treatment resulted in increased tumor formation with a more malignant phenotype following long-term RAS induction. In contrast to the current paradigm for TGFβ in carcinogenesis, these results demonstrate that cutaneous TGFβ signaling enables promotion of benign tumors but suppresses premalignant progression through context-dependent regulation of epidermal homeostasis and inflammation