Epigenome engineering: new technologies for precision medicine.

Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1-iPeps and RGD peptides.

Triple negative breast cancers (TNBC) are aggressive malignancies for which chemotherapy is the only treatment option. Many TNBC acquire chemotherapy resistance, notably docetaxel, which has been associated with the overexpression of transcription factors (TFs), such as ENGRAILED1 (EN1). Here, we have developed a tumor delivery system for docetaxel-PGMA-PAA-nanoparticles and interference peptides designed to specifically inhibit EN1 (EN1-iPeps). To promote tumor specific targeting, we functionalized these nanoparticles with EN1-iPeps engineered with RGD sequences. We found that these peptides reduce cell viability and induce apoptosis in TNBC cells with negligible effects on normal cells (EN1-). Moreover, EN1-RGD-iPeps-mediated nanoparticle internalization into breast cancer cells was via integrins and intravenous injection of this nanoformulation increased tumor accumulation. Furthermore, docetaxel nanoparticles functionalized with EN1-RGD-iPeps significantly reduced TNBC growth both in vitro and in vivo without showing toxicity. Our results suggest that this targeted nanoformulation represents a new and safe therapeutic approach for chemoresistant TNBCs.

Aurantoside C Targets and Induces Apoptosis in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a subtype of breast cancers that currently lacks effective targeted therapy. In this study, we found that aurantoside C (C828), isolated from the marine sponge Manihinea lynbeazleyae collected from Western Australia, exhibited higher cytotoxic activities in TNBC cells compared with non-TNBC (luminal and normal-like) cells. The cytotoxic effect of C828 was associated to the accumulation of cell at S-phase, resulting in the decline of cyclin D1, cyclin E1, CDK4, and CDK6, and an increase in p21. We also found that C828 inhibited the phosphorylation of Akt/mTOR and NF-kB pathways and increased the phosphorylation of p38 MAPK and SAPK/JNK pathways, leading to apoptosis in TNBC cells. These effects of C828 were not observed in non-TNBC cells at the concentrations that were cytotoxic to TNBC cells. When compared to the cytotoxic effect with the chemotherapeutic drugs doxorubicin and cisplatin, C828 was found to be 20 times and 35 times more potent than doxorubicin and cisplatin, respectively. These results indicate that C828 could be a promising lead for developing new anticancer agents that target TNBC cells.

Crambescidin 800, Isolated from the Marine Sponge Monanchora viridis, Induces Cell Cycle Arrest and Apoptosis in Triple-Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is currently the only group of breast cancers without an effective targeted therapy. Marine sponges have historically been a source of compounds with anticancer activity. In this study, we screened extracts from twenty marine sponges collected off the coast of Western Australia for cytotoxic activity against TNBC cells. One very active extract derived from the sponge Monanchora viridis was selected for bioactivity-guided fractionation. Through multiple steps of purification, we isolated a potent cytotoxic compound, which was identified as crambescidin 800 (C800). We found that C800 exhibited cytotoxic potency in a panel of breast cancer cells, of which TNBC and luminal cancer cell models were the most sensitive. In addition, C800 induced cell cycle arrest at the G2/M phase, resulting in a decline in the expression of cyclin D1, CDK4, and CDK6 in TNBC cells. This effect was associated with the inhibition of phosphorylation of Akt, NF-κB, and MAPK pathways, resulting in apoptosis in TNBC cells.

Re-expression of Selected Epigenetically Silenced Candidate Tumor Suppressor Genes in Cervical Cancer by TET2-directed Demethylation.

DNA hypermethylation is extensively explored as therapeutic target for gene expression modulation in cancer. Here, we re-activated hypermethylated candidate tumor suppressor genes (TSGs) (C13ORF18, CCNA1, TFPI2, and Maspin) by TET2-induced demethylation in cervical cancer cell lines. To redirect TET2 to hypermethylated TSGs, we engineered zinc finger proteins (ZFPs), which were first fused to the transcriptional activator VP64 to validate effective gene re-expression and confirm TSG function. ChIP-Seq not only revealed enriched binding of ZFPs to their intended sequence, but also considerable off-target binding, especially at promoter regions. Nevertheless, results obtained by targeted re-expression using ZFP-VP64 constructs were in line with cDNA overexpression; both revealed strong growth inhibition for C13ORF18 and TFPI2, but not for CCNA1 and Maspin. To explore effectivity of locus-targeted demethylation, ZFP-TET2 fusions were constructed which efficiently demethylated genes with subsequent gene re-activation. Moreover, targeting TET2 to TFPI2 and C13ORF18, but not CCNA1, significantly decreased cell growth, viability, and colony formation in cervical cancer cells compared to a catalytically inactive mutant of TET2. These data underline that effective re-activation of hypermethylated genes can be achieved through targeted DNA demethylation by TET2, which can assist in realizing sustained re-expression of genes of interest.

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation.

Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼ 5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.

Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy.

Failure of clinical trials of nonviral vector-mediated gene therapy arises primarily from either an insufficient transgene expression level or immunostimulation concerns caused by the genetic information carrier (e.g., bacteria-generated, double-stranded DNA (dsDNA)). Neither of these issues could be addressed through engineering-sophisticated gene delivery vehicles. Therefore, we propose a systemic delivery of chemically modified messenger RNA (mRNA) as an alternative to plasmid DNA (pDNA) in cancer gene therapy. Modified mRNA evaded recognition by the innate immune system and was less immunostimulating than dsDNA or regular mRNA. Moreover, the cytoplasmic delivery of mRNA circumvented the nuclear envelope, which resulted in a higher gene expression level. When formulated in the nanoparticle formulation liposome-protamine-RNA (LPR), modified mRNA showed increased nuclease tolerance and was more effectively taken up by tumor cells after systemic administration. The use of LPR resulted in a substantial increase of the gene expression level compared with the equivalent pDNA in the human lung cancer NCI-H460 carcinoma. In a therapeutic model, when modified mRNA encoding herpes simplex virus 1-thymidine kinase (HSV1-tk) was systemically delivered to H460 xenograft-bearing nude mice, it was significantly more effective in suppressing tumor growth than pDNA.

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer.

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells.

Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles Encapsulating RNA.

Genome editing tools, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (e.g., CRISPR/Cas9), and their repurposing into epigenetic editing platforms, offer enormous potential as safe and customizable therapies for cancer. Specifically, various transcriptional abnormalities in human malignancies, such as silencing of tumor suppressors and ectopic re-expression of oncogenes, have been successfully targeted with virtually no off-target effects using CRISPR activation and repression systems. In these systems, the nuclease-deactivated Cas9 protein (dCas9) is fused to one or more domains inducing selective activation or repression of the targeted genes. Despite these advances, the efficient in vivo delivery of these molecules into the target cancer cells represents a critical barrier to accomplishing translation into a clinical therapy setting for cancer. Major obstacles include the large size of dCas9 fusion proteins, the necessity of multimodal delivery of protein and gRNAs, and the potential of these formulations to elicit detrimental immune responses.In this context, viral methods for delivering CRISPR face several limitations, such as the packaging capacity of the viral genome, the potential for integration of the nucleic acids into the host cells genome, and immunogenicity of viral proteins, posing serious safety concerns. The rapid development of mRNA vaccines in response to the COVID-19 pandemic has rekindled interest in mRNA-based approaches for CRISPR/dCas9 delivery. Simultaneously, due to their high loading capacity, scalability, customizable surface modification for cell targeting, and low immunogenicity, lipid nanoparticles (LNPs) have been widely explored as nonviral vectors. In this chapter, we first describe the design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing. We outline formulations of LNPs suitable for dCas9 mRNA delivery. Additionally, we provide a protocol for the co-encapsulation of the dCas9-effector mRNAs and gRNA into these LNPs, along with detailed methods for delivering these formulations to both cell lines (in vitro) and mouse models of breast cancer (in vivo).

Writing and rewriting the epigenetic code of cancer cells: from engineered proteins to small molecules.

The epigenomic era has revealed a well-connected network of molecular processes that shape the chromatin landscape. These processes comprise abnormal methylomes, transcriptosomes, genome-wide histone post-transcriptional modifications patterns, histone variants, and noncoding RNAs. The mapping of these processes in large scale by chromatin immunoprecipitation sequencing and other methodologies in both cancer and normal cells reveals novel therapeutic opportunities for anticancer intervention. The goal of this minireview is to summarize pharmacological strategies to modify the epigenetic landscape of cancer cells. These approaches include the use of novel small molecule inhibitors of epigenetic processes specifically deregulated in cancer cells and the design of engineered proteins able to stably reprogram the epigenetic code in cancer cells in a way that is similar to normal cells.

Targeting serous epithelial ovarian cancer with designer zinc finger transcription factors.

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers.

Breastmilk is a novel source of stem cells with multilineage differentiation potential.

The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration.

Unpacking the Complexity of Epithelial Plasticity: From Master Regulator Transcription Factors to Non-Coding RNAs.

Cellular plasticity in cancer enables adaptation to selective pressures and stress imposed by the tumor microenvironment. This plasticity facilitates the remodeling of cancer cell phenotype and function (such as tumor stemness, metastasis, chemo/radio resistance), and the reprogramming of the surrounding tumor microenvironment to enable immune evasion. Epithelial plasticity is one form of cellular plasticity, which is intrinsically linked with epithelial-mesenchymal transition (EMT). Traditionally, EMT has been regarded as a binary state. Yet, increasing evidence suggests that EMT involves a spectrum of quasi-epithelial and quasi-mesenchymal phenotypes governed by complex interactions between cellular metabolism, transcriptome regulation, and epigenetic mechanisms. Herein, we review the complex cross-talk between the different layers of epithelial plasticity in cancer, encompassing the core layer of transcription factors, their interacting epigenetic modifiers and non-coding RNAs, and the manipulation of cancer immunogenicity in transitioning between epithelial and mesenchymal states. In examining these factors, we provide insights into promising therapeutic avenues and potential anti-cancer targets.

Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma.

BACKGROUND: Epigenetic silencing of tumor suppressor genes (TSGs) is a key feature of oncogenesis in hepatocellular carcinoma (HCC). Liver-targeted delivery of CRISPR-activation (CRISPRa) systems makes it possible to exploit chromatin plasticity, by reprogramming transcriptional dysregulation. RESULTS: Using The Cancer Genome Atlas HCC data, we identify 12 putative TSGs with negative associations between promoter DNA methylation and transcript abundance, with limited genetic alterations. All HCC samples harbor at least one silenced TSG, suggesting that combining a specific panel of genomic targets could maximize efficacy, and potentially improve outcomes as a personalized treatment strategy for HCC patients. Unlike epigenetic modifying drugs lacking locus selectivity, CRISPRa systems enable potent and precise reactivation of at least 4 TSGs tailored to representative HCC lines. Concerted reactivation of HHIP, MT1M, PZP, and TTC36 in Hep3B cells inhibits multiple facets of HCC pathogenesis, such as cell viability, proliferation, and migration. CONCLUSIONS: By combining multiple effector domains, we demonstrate the utility of a CRISPRa toolbox of epigenetic effectors and gRNAs for patient-specific treatment of aggressive HCC.

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer.

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability.

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor.

INTRODUCTION: Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations. METHODS: OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments. RESULTS: OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A. In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew. CONCLUSIONS: Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors.

Manipulating the NKG2D Receptor-Ligand Axis Using CRISPR: Novel Technologies for Improved Host Immunity.

The activating immune receptor natural killer group member D (NKG2D) and its cognate ligands represent a fundamental surveillance system of cellular distress, damage or transformation. Signaling through the NKG2D receptor-ligand axis is critical for early detection of viral infection or oncogenic transformation and the presence of functional NKG2D ligands (NKG2D-L) is associated with tumor rejection and viral clearance. Many viruses and tumors have developed mechanisms to evade NKG2D recognition via transcriptional, post-transcriptional or post-translational interference with NKG2D-L, supporting the concept that circumventing immune evasion of the NKG2D receptor-ligand axis may be an attractive therapeutic avenue for antiviral therapy or cancer immunotherapy. To date, the complexity of the NKG2D receptor-ligand axis and the lack of specificity of current NKG2D-targeting therapies has not allowed for the precise manipulation required to optimally harness NKG2D-mediated immunity. However, with the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, novel opportunities have arisen in the realm of locus-specific gene editing and regulation. Here, we give a brief overview of the NKG2D receptor-ligand axis in humans and discuss the levels at which NKG2D-L are regulated and dysregulated during viral infection and oncogenesis. Moreover, we explore the potential for CRISPR-based technologies to provide novel therapeutic avenues to improve and maximize NKG2D-mediated immunity.