Nanog Regulates Other Stem Cell Transciption Factors Nanog controls the expression of Oct4 and SOX2 during early embryogenesis. We have found that inhibition of NanogP8 with allele specific shRNA Np8-1 inhibits expression of Oct4 and SOX2. Thus, targeting one factor may inhibit the others so that single agent targeting may effect multiple downstream pathways. In addition, we have shown that Nanog and NanogP8 affect stemness by reducing spherogenicity, tumorigenicity and metastatic potential in colorectal carcinoma cells (CRC). We have now found that allele-specific inhibition of NanogP8 reduces the size of the side population, while overexpression of Nanog or NanogP8 increases the side population. Thus, Nanog and NanogP8 are instrumental in modulating the stemness of CRC. Nanog and NanogP8 are expressed in clinical liver CRC metastases Ten de-identified samples of colorectal carcinoma resected from patients treated at the NIH Clinical Center were analyzed for expression of Nanog and NanogP8 transcripts and protein. RT-PCR was followed by cutting the cDNA with the restriction endonuclease AlwNI that specifically cuts NanogP8 but not Nanog. Seven liver metastases contained a Nanog-related transcript as did 3 adjacent liver specimens. Six of the 7 specimens contained NanogP8 with and one only Nanog. One of the adjacent liver specimens contained NanogP8 while the 2 others only expressed Nanog transcripts. All results were confirmed by Sanger sequencing. Immunohistochemistry (IHC) was also performed and confirmed that Nanog protein was present in the cytoplasm of the tumor cells that contained detectable Nanog or NanogP8 transcripts but not in CRC that lacked such transcripts. In addition, IHC of sample 9 demonstrated the presence of Nanog protein in a sample where only NanogP8 transcripts were identified. Thus, based on this small sample that needs confirmation and expansion approximately 70% of clinical CRC express a Nanog family member and 60% express NanogP8. NanogP8 Can be Translated into Protein In collaboration with the Veenstra laboratory at NCI-Frederick, we attempted to use tandem MS/MS to identify the NanogP8 protein in CRC cell lines. Extracts of CRC cell lines were immunoprecipitated, isolated by SDS-PAGE, subjected to in gel tryptic digestion and then MS/MS. Four Nanog-related peptides (KTWFQNQRM, KYLSLQQMQELSNILNLSYKQ, KKEDKVPVKK, and KGKQPTSAENSVAKK) were identified in extracts from Clone A overexpressing NanogP8. Interestingly, the last peptide is unique to NanogP8, which includes the shift from Lys (K) in Nanog to Asn (N) in NanogP8 at codon 82. This amino acid change was also reported by Ambady (2) and is consistent with our gene sequencing data. Nanog/NanogP8 proteins were not identified in extracts of CRC cell lines without overexpression by transfection presumably because CRC cell lines express low levels of endogenous proteins. NanogP8 Promoter Activity is increased during 3-D growth consistent with gene expression of NanogP8 We have previously shown that NanogP8 transcripts are increased in spheroids of the CRC cell lines Clone A and CX-1 cultured in serum-free medium (SFM). We tested the postulate that the promoter activity of the NanogP8 gene increases as CRC cells transition from a two-dimensional monolayer culture to a three-dimensional spheroid culture. A lentiviral NanogP8 promoter reporter was obtained from Jeter et al (3). Monolayer cultures of Clone A and CX-1 had low levels of GFP expression compared to expression in spheroids of Clone A and CX-1. Counting GFP positive cells demonstrated a 6- to 14-fold increase in GFP+ cells in spheroids compared to monolayers for Clone A and CX-1, respectively. This is similar to the increase in transcript number assessed by qRT-PCR during transition from 2-D monolayers to 3-D spheroids. Mechanism of Action and Inhibition of 3-D Growth By Transduction with Lentiviral shRNAs to Nanog or NanogP8 To be a successful therapy, inhibition of Nanog by shRNA must inhibit the growth of established tumors in vivo in preclinical models. We obtained a grant from the Department of Defense to support such preclinical work but unfortunately that has not yet started. However, we have been busy performing experiments in vitro with our allele-specific shRNAs and their effects on regression of established 3-D spheroids and cells in suspension to determine the multiplicity of infection (MOI) needed to cause tumor regression. Our previous experiments demonstrated that treatment of CRC in monolayer induced Annexin V expression as an indication of apoptosis. Now we started by taking LV with shRNA to NanogP8 (shNp8-1), Nanog (shNG-1) and a control shRNA (shNEG) and transducing CX-1 and Clone A cells that were then cultured for 24 hr in monolayer and then transitioned to SFM. We first demonstrated that LVshNp8-1 transduction significantly reduced the mass of cells after 72 hr of suspension culture. We then began to transduce CRC growing in suspension in SFM on ULLA plates. Since our LV preparations express GFP, we first determined the fraction of cells transduced in suspension and found that 40% to 75% of CRC cells were transduced stably. We then assessed whether transduction with LV shRNA to Nanog or NanogP8 inhibited growth compared to either untreated CRC cells or cells treated with the control shNEG. The cumulative results of more than 7 different experiments indicated that treatment of CRC in 3-D growth significantly inhibits short term growth by 50% by 72 hr. We then sought to determine how the LV shRNA effects apoptosis in 3-D culture. We previously demonstrated that anoikis apoptosis caused by suspension culture is dependent on activation of Caspase 8 (4). We measured Caspase 3, 8 and 9 activity in Clone A and CX-1 treated with LV shRNA after 4 days of suspension culture, 3 days after LV treatment. Activity of Caspase 3, the executioner caspase, was increased in both CX-1 and Clone A by shRNA to Nanog and NanogP8 beyond what occurs with just suspension culture alone. Activity of Caspase 8 or 9 was increased in Clone A or CX-1, respectively, by shRNA Np8-1. Interestingly, non-transduced cells cells adjacent to transduced cells had similar levels of caspase 9 activity as transduced cells did. Finally, at least in CX-1. Caspase 9 and the intrinsic pathway that uses mitochondrial amplification in apoptosis is important for apoptosis mediated by shNp8-1 and shNG-1. SUMMARY Our work suggests that 1) inhibition of Nanog or NanogP8 inhibits the other core embryonic TFs SOX2 and Oct4, 2) the NanogP8 transcript can be translated so that the retrogene is a functional gene, 3) LV shRNA to Nanog or NanogP8 transduce cells growing in 3-D, 4) an MOI of 8 is sufficient to inhibit CRC short term growth, and 5) inhibition of growth by LV shRNA is mediated by activation of caspases, especially caspase 9 and 3. In vivo experiments will go forward when the DOD IDEA grant is funded. Finally, it is not clear why some tumors produce only Nanog transcripts while others produce either both NanogP8 and Nanog or only NanogP8. However, by producing allele-specific inhibitory shRNA to both genes it is possible to target either or both and offers the possibility of personalizing therapy. While we have not yet determined whether the combination of both allele specific inhibitory shRNAs is better than either alone in the in vitro suspension culture model, we will do this soon. This is important especially since inhibition of either gene works through the intrinsic pathway of apoptosis that is distinct from the extrinsic pathway that is important for anoikis.
