NANOGP8 Function in Stemness shRNA to NANOG inhibited the expression of parental NANOG and decreased spherogenicity significantly by single CRC Clone A and CX-1 cells. Re-expression of either NANOG or NANOGP8 protein was achieved and re-expression of NANOGP8 increased both spherogenicity and the size of the side population in both Clone A and CX-1 cells. Over-expression of NANOG increased spherogenicity in Clone A cells but not CX-1 cells. In addition, we demonstrated that NANOGP8 is translated. This had not been previously done. The data are included in the manuscript by Zhang et al. Oncogene. 2012 Oct 22. doi: 10.1038/onc.2012.461. [Epub ahead of print] Mechanism of Apoptosis Lentiviral delivered shRNA to NANOG (shNG-1) or NANOGP8 (shNp8-1) inhibits by nearly 50% the three-dimensional (3-D) growth of Clone A, CX-1 and LS 174T human CRC cells within 3 days when added a day after suspension cultures are initiated at a MOI of 5:1 to 16:1. This inhibition of 3-D growth extended to a further 15-30% decrease in regrowth potential when surviving cells were replated in a plating efficiency assay. Transduction efficiency was 45%. This suggested that lentiviral (LV) shNG-1 and/or shNp8-1 may induce apoptosis. We have previously published that suspension culture induces caspase 8-dependent extrinsic pathway apoptosis without activation of caspase 9 and the intrinsic pathway in Clone A and CX-1 cells. When we assessed the effects of LV shNG-1, shNp8-1 or the control LV shNEG on the induction of caspase 3, 8, 9 activity by a cell permeant fluorescent apoptosis assay, shNp8-1 induced activation of caspase 9 in suspension culture to a greater extent than shNG-1. LV delivered shNG-1 or shNp8-1 generally did not cause significant activation of the caspases when CRC were in monolayer culture. Interstingly, occasionally the LV shNEG contro vector did activate caspases in monolayer culture - this may reflect the expression of Toll receptors in monolayer cultures that lead to activation of innate immune mechanisms within CRC cells in monolayer culture. Taken together, the results suggest that shRNA to NANOGs, especially NANOGP8, activate the intrinsic pathway of apoptosis and suggest that LV delivered shNp8-1 and/or shNG-1 may be synergistic with chemo- or targeted therapy. This is currently being written up in a manuscript. We have assessed whether LV shRNA to NANOG and/or NANOGP8 is synergistic with specific aspects of the indirect pathway of apoptosis. Since this pathway involves the participation of BH3 domain proteins and is controlled by the anti-apoptotic Bcl-2 family members as well as Mcl-1 , the effect of LV shRNA on the expression of MCL-1 and Bcl-2 was performed. LV shNp8-1 inhibits the expression of MCL-1 as well as the activation of AKT with LV shNG-1 also inhibiting MCL-1 but to a lesser degree. None of the LV preparations have any effect on Bcl-2 or its family members. The LV shNp8-1 inhibits MCL-1 in the 3 CRC lines Clone A, CX-1, LS 174T. We have tested whether LV shRNA was synergistic with cytotoxic chemotherapy (using Topotecan, the camptothecin as a model) and anti-BCL-2 therapy (cell permeant peptidomimetic ABT-737). Even though both treatments increase NANOG and NANOGP8 gene expression of CRC cells in monolayer cultures the addition of LV shNp8-1 or LV shNG-1 is synergistic with either Topotecan or ABT-737. The LV shNEG does not increase apoptosis in this assay. Also the sequencing of the addition of LV shRNA's either before, simultaneously or after treatment with drug or anti-Bcl-2 agent does not affect the synergism. These results suggest that shRNA to inhibit NANOGP8 or NANOG may inhibit Mcl-1 that is synergistic with an anti-Bcl-2 therapy. A manuscript is under preparation. Preclinical studies will start soon, especially as the anti-BCL-2 therapy is entering the clinic. Regulation of NEDD9 Ingenuity Pathway Analysis of RNA-seq data from CX-1 cells modulated by alterations in NANOGP8 identified NEDD9 as a critical gene. NEDD9 is a scaffolding protein that connects c-src and FAK in adhesion plaques and activates AKT . Thus, NEDD9 may be an anti-apoptotic molecule since it contributes to constitutive activation of FAK. RT-PCR confirmed that NEDD9 expression was inhibited by shRNA to the NANOGs and increased by overexpression of either NANOG in the CRC lines tested. In addition, ChIP assays confirmed that NANOG binds the promoter of NEDD9. Preliminary results with siRNA to NEDD9 are underway to determine whether inhibition of NEDD9 expression affects AKT activation and/or Mcl-1 expression. In addition, we have assessed the role of NANOG and NEDD9 as prognostic factors in a series of nearly 400 primary colon carcinomas in a tissue microarray produced by the Cancer Diagnosis Program, DCTD, NCI. In collaboration with Drs. Scott Lawrence, Stephen Hewitt, Daekwan Seo and Robert Kinders both quantitative immunofluorescence (qIFA) and immunohistochemistry (IHC) were performed to assess NANOG and NEDD9 protein in the primary colon carcinomas. The primary colon carcinomas was divided into a training and test set and analyzed by Kaplan-Meier and then Cox multivariate regression analysis. The data suggest that NANOG and NEDD9 together are a significant prognostic factor in models that contain tumor stage, local invasion, grade, age, and site. These results are bing put into a manuscript. TALEN Evaluation We were chosen to evaluate a TALEN that might remoce NANOG from the genomes of CRCs as a research tool. Interestingly, the company Cellectis who designed the TALEN for our use actually targeted the same sequence in exon 1 as we had found in our most active commercially available shRNA from the TRC consortium. The problem for our TAELN is that the sequence is present not only NANOG but also NANOGP8 as well as rare transcripts of NANOGP4 and NANOGP7. Our strategy for isolating the clones that may have had a loss of NANOG after transfection with the TALEN was to isolate clones that are proliferating more slowly than other clones since inhibition of proliferation is the early sign of NANOG inhibition. Unfortunately, this has really slowed down our evaluation. We have found that we can reduce NANOG protein expression by 75% percent with reduction of parental NANOG by at least 50% and intact NANOGP8. However, one thing that has happened is that the clones have generally increased proliferation after 4 weeks even though NANOG levels have not changed at either the protein or transcript level. Investigation is currently ongoing into adaptive mechanisms. Transduction in Vivo We have had difficulty in transducing CRC growing as xenografts in the sub cutis. Although preliminary intratumoral injection of LV shRNA inhibited tumor growth, it was secondary to innate immunity since the LV shNEG was also active. We have finally demonstrated transduction in vivo in CX-1 cells but it is limited to sporadic islands of 10-15 transduced cells within a small xenograft. As a result, the use of lentiviral delivered shRNA may not be sustainable. As a result, during the coming year we will investigate the use of conditionally replicating adenovirus (CRAd) within tumors. Such CRAds are also known as oncolytic virus because the CRAd may lyse tumor cells but they also may allow the expression of shRNA as an episome. Since a NANOGP8 reporter is quite active within these CRC lines, we will begin to create a NANOGP8-driven CRAd that may also deliver a shRNA.

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Zhang, J; Espinoza, L A; Kinders, R J et al. (2013) NANOG modulates stemness in human colorectal cancer. Oncogene 32:4397-405
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