Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here we develop CRISPR interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, and to dissect developmental pathways and model disease.

Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

Constitutive Hedgehog (HH) signaling underlies several human tumors, including basal cell carcinoma (BCC). Recently, Bijlsma and colleagues reported a new biologic function for vitamin D3 in suppressing HH signaling in an in vitro model system. On the basis of that work, we have assessed effects of vitamin D3 on HH signaling and proliferation of murine BCCs in vitro and in vivo. We find that indeed in BCC cells, vitamin D3 blocks both proliferation and HH signaling as assessed by mRNA expression of the HH target gene Gli1. These effects of vitamin D3 on Gli1 expression and on BCC cell proliferation are comparable to the effects of cyclopamine, a known inhibitor of the HH pathway. These results are specific for vitamin D3, because the precursor 7-dehydrocholesterol and the downstream products 25-hydroxy vitamin D3 [25 (OH)D] and 1,25-dihydroxy vitamin D3 [1,25(OH) 2 D] are considerably less effective in reducing either Gli1 mRNA or cellular proliferation. Moreover, these effects seem to be independent of the vitamin D receptor (VDR) because short hairpin RNA knockdown of VDR does not abrogate the anti-HH effects of D3 despite reducing expression of the VDR target gene 24-hydroxylase. Finally, topical vitamin D3 treatment of existing murine BCC tumors significantly decreases Gli1 and Ki67 staining. Thus, topical vitamin D3 acting via its HH inhibiting effect may hold promise as an effective anti-BCC agent.

In vitro and epidemiologic studies favor the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) in preventing skin squamous photocarcinogenesis, but there has been relatively little study of their efficacy in preventing the more common skin basal cell carcinoma (BCC) carcinogenesis. We first compared the relative anti-BCC effects of genetic deletion and NSAID pharmacologic inhibition of cyclooxygenase (COX) enzymes in the skin of Ptch1 +/− mice. We then assessed the effects of celecoxib on the development of BCCs in a 3-year, double-blinded, randomized clinical trial in 60 (PTCH1 +/− ) patients with the basal cell nevus syndrome. In Ptch1 +/− mice, genetic deletion of COX1 or COX2 robustly decreased (75%; P < 0.05) microscopic BCC tumor burden, but pharmacologic inhibition with celecoxib reduced microscopic BCCs less efficaciously (35%; P < 0.05). In the human trial, we detected a trend for oral celecoxib reducing BCC burden in all subjects (P = 0.069). Considering only the 60% of patients with less severe disease (<15 BCCs at study entry), celecoxib significantly reduced BCC number and burden: subjects receiving placebo had a 50% increase in BCC burden per year, whereas subjects in the celecoxib group had a 20% increase (P difference = 0.024). Oral celecoxib treatment inhibited BCC carcinogenesis in PTCH1 +/− mice and had a significant anti-BCC effect in humans with less severe disease. Cancer Prev Res; 3(1); 25-34. ©2010 AACR.

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:  There have been few reports of successful long‐term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long‐term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)+/− mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long‐term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique.

Retinoic acid, acting through the nuclear retinoic acid receptor β2(RARβ2), stimulates neurite outgrowth from peripheral nervous system tissue that has the capacity to regenerate neurites, namely, embryonic and adult dorsal root ganglia. Similarly, in central nervous system tissue that can regenerate, namely, embryonic mouse spinal cord, retinoic acid also stimulates neurite outgrowth and RARβ2 is upregulated. By contrast, in the adult mouse spinal cord, which cannot regenerate, no such upregulation of RARβ2 by retinoic acid is observed and no neurites are extended in vitro. To test our hypothesis that the upregulation of RARβ2 is crucial to neurite regeneration, we have transduced adult mouse or rat spinal cord in vitro with a minimal equine infectious anaemia virus vector expressing RARβ2. After transduction, prolific neurite outgrowth occurs. Outgrowth does not occur when the cord is transduced with a different isoform of RARβ nor does it occur following treatment with nerve growth factor. These data demonstrate that RARβ2 is involved in neurite outgrowth, at least in vitro, and that this gene may in the future be of some therapeutic use.

The Hedgehog signaling pathway plays a key role in directing growth and patterning during embryonic development and is required in vertebrates for the normal development of many structures, including the neural tube, axial skeleton, skin, and hair. Aberrant activation of the Hedgehog (Hh) pathway in adult tissue is associated with the development of basal cell carcinoma (BCC), medulloblastoma, and a subset of pancreatic, gastro-intestinal, and other cancers. This review will provide an overview of what is known about the mechanisms by which activation of Hedgehog signaling leads to the development of BCCs and will review two recent papers suggesting that agents that modulate sterol levels might influence the Hh pathway. Thus, sterols may be a new therapeutic target for the treatment of BCCs, and readily available agents such as statins (HMG-CoA reductase inhibitors) or vitamin D might be helpful in reducing BCC incidence. Epidemiology of Basal cell carcinoma (BCC)Basal cell carcinoma (BCC), the most common of all human cancers, affects close to 1 million Americans a year. Over the past 40 years there has been a dramatic increase in the incidence of BCC, and it is estimated that nearly 30% of Caucasians living in areas of high ambient sun exposure will develop a BCC (Miller and Weinstock, 1994). Furthermore, the incidence of BCC is rising in younger populations, especially among women (Christenson et al., 2005). Although the case fatality rate of BCCs is low, the high incidence and frequent occurrence of multiple primary tumors in affected individuals leads to significant morbidity. BCCs characteristically arise in sun-exposed body areas, most commonly on the head and neck, but also occur on the trunk and extremities. There are three accepted environmental insults for BCC development: ultraviolet radiation (UV), ionizing radiation (IR), and arsenic. Interindividual differences in the susceptibility to BCC development have been recognized for many years. Epidemiologic studies have identified phenotypic features such as fair skin and freckling tendency that are associated with an increased susceptibility to BCCs (Rubin et al., 2005).It is generally accepted that BCC can be caused by UV exposure from the sun. While the risk of squamous cell carcinoma is strongly related to cumulative sun exposure, sunlight's exact role in BCC development remains less clear (Armstrong and Kricker, 2001). Thus, epidemiologic studies have shown that BCC risk correlates better with intermittent sun exposure (i.e. childhood sunburns, weekend sun exposure) than with cumulative lifetime sun exposure (Corona et al., 2001). Hence, the timing, dose, and duration of UV exposure are critical to carcinogenesis, but UV seems to have a different role in SCC versus BCC carcinogenesis development. Consistent with this, a randomized clinical trial of daily sunscreen use showed that sunscreen reduced the incidence of SCC but not BCC. The daily application of an SPF 15 sunscreen to the head, neck, arms, and hands over a 4-5 year period had no e...