br Fig Mutation of the newly
Fig. 4. Mutation of the newly discovered AT-hook-like ‘core’ motif of PTOV1 reduces the protein transcription capacity. The plasmid containing GFP/full-lenght PTOV1 mutated at the AT-hook (EGGP), or wild-type plasmid, and the control GFP plasmid were transfected in HEK293T cells. (A) Cells were analyzed for gene expression by real-time PCR 72 h after transfection. The graph shows the relative expression of PTOV1, ALDH1A1 and CCNG2 as compared to their endogenous levels (cells transfected with the GFP plasmid, CTL). (B) The levels of expression of GFP-PTOV1 (wild-type) was taken as control to compare to mutated GFP-PTOV1 from the same experiment as in A to show the eﬀect that mutant exogenous PTOV1 has on the expres-sion of downstream ALDH1A1 and CCNG2 genes in comparison to wild-type PTOV1.
(C) Western blot analysis of Ifenprodil hemitartrate transfected with wild-type or mutant GFP-PTOV1 plasmids, shows similar levels of expression of the exogenous PTOV1, as indicated by the GFP antibody signals. Vinculin is shown as protein loading control.
expression of ALDH1A1 (Spearman 0.46, p < 0.0001) and CCNG2 (Spearman 0.68, p < 0.0001) (GSE46691). Moreover, in a cohort of patients with prostate carcinoma including data derived from 10 studies [35–44], PTOV1, ALDH1A1 and CCNG2 genes also showed a highly significant co-occurrence of alterations at their DNA (Table 1). These observations suggest that the expression of PTOV1, ALDH1A1 and CCNG2 genes is associated for a coordinated activity in aggressive prostate tumors.
Because PTOV1 can strongly and specifically bind to DNA in vitro and to chromatin in vivo, we searched for PTOV1 association with DNA in mitotic tumors cells using immunohistochemical analysis in diﬀerent types of tumors. In xenografted mice tumors derived from LNCaP cells, a strong signal for PTOV1 was detected in mitotic cells where the staining appears associated to condensed chromosomes (Fig. 5B). Si-milarly, in colon carcinoma tissues immunohistochemical analysis re-veals a clear accumulation of PTOV1 in the nuclei of mitotic cells, confirming its strong association with chromatin in aggressive tumor cells (Fig. 6A). Interrogating publicly available datasets of patients with colon carcinomas (GSE24551, GSE14333), high levels of PTOV1, ALDH1A1 and CCNG2 expression correlated with poor relapse-free survival and event-free survival, although for ALDH1A1 the association did not reach significance (Fig. 6B). Furthermore, im-munohistochemical analyses of high grade urothelial bladder carci-noma and ductal breast carcinoma, previously shown to express high levels of PTOV17, also revealed a clear association of the protein with condensed mitotic DNA (Fig. 7).
Here, we provide new insights into the mechanisms used by the
protein PTOV1 to regulate the expression of ALDH1A1 and CCNG2, relevant factors involved in tumor progression. Specifically, we show that PTOV1 is able to directly bind to the promoter sequences of these genes through a newly unveiled protein motif localized within the A domain of the protein . This finding impacts on several aspects of the action of PTOV1 in cancer progression.
First, our findings define PTOV1 as a new nucleic acid binding protein containing two distinct motifs: one extended (eAT-hook) at the N-terminal  and a second AT-hook-like motif, identified and char-acterized in this work, within the A domain of PTOV1. The previously described eAT-hook does not bind to any of the DNA sequences tested here, in line with previous results that this motif has a higher aﬃnity for RNA sequences . In contrast, the newly identified AT-hook-like strongly binds to DNA sequences from the ALDH1A1 and CCNG2 pro-moters but not to ABCB1 promoter or internal HES1 gene sequences, indicating its specificity. Of note, mutations of the ‘core’ sequence in the AT-hook-like motif, decreases the expression of ALDH1A1 and CCNG2 indicating that this motif is functionally relevant for the transcriptional activity of PTOV1. Since PTOV1 was shown to function both at specific promoter sites to regulate transcription [17,18] and at ribosomes  to regulate mRNA translation, our present findings reveal potential new ways to selectively mitigate the nuclear or cytoplasmic functions of PTOV1 in cancer cells: the identification of inhibitors of these ‘micro-handles’ in the protein could prevent its specific binding to either DNA or RNA, causing functionally diverse consequences on cell fate.