Hiwi Mediated Tumorigenesis Is Associated with DNA Hypermethylation

Expression of Piwi proteins is confined to early development and stem cells during which they suppress transposon migration via DNA methylation to ensure genomic stability. Piwi's genomic protective function conflicts with reports that its human ortholog, Hiwi, is expressed in numerous cancers and prognosticates shorter survival. However, the role of Hiwi in tumorigenesis has not been examined. Here we demonstrate that (1) over-expressing Hiwi in sarcoma precursors inhibits their differentiation in vitro and generates sarcomas in vivo; (2) transgenic mice expressing Hiwi (mesodermally restricted) develop sarcomas; and (3) inducible down-regulation of Hiwi in human sarcomas inhibits growth and re-establishes differentiation. Our data indicates that Hiwi is directly tumorigenic and Hiwi-expressing cancers may be addicted to Hiwi expression. We further show that Hiwi associated DNA methylation and cyclin-dependent kinase inhibitor (CDKI) silencing is reversible along with Hiwi-induced tumorigenesis, via DNA-methyltransferase inhibitors. Our studies reveal for the first time not only a novel oncogenic role for Hiwi as a driver of tumorigenesis, but also suggest that the use of epigenetic agents may be clinically beneficial for treatment of tumors that express Hiwi. Additionally, our data showing that Hiwi-associated DNA hyper-methylation with subsequent genetic and epigenetic changes favoring a tumorigenic state reconciles the conundrum of how Hiwi may act appropriately to promote genomic integrity during early development (via transposon silencing) and inappropriately in adult tissues with subsequent tumorigenesis

Assessment of Hiwi target genes.

<p>(A) Top panel: Affymetrix 430 2.0 array of gene expression changes in parental MSCs (x-axis) or Hiwi-MSCs (y-axis). Affymetrix U433 array of gene expression changes in sh-Hiwi MFH cells uninduced (x-axis) or induced for 7 days with doxycycline (y-axis) (middle panel) or untreated (x-axis) and after 7 days of 1 uM 5-azacytidine (y-axis) (bottom panel). Arrow indicates genes used in overlap analysis. (B) Overlap of Tumor Suppressor Genes (TSG) as described. All 19 overlapping TSGs are listed here.</p

CDKIs are decreased in Hiwi–MSCs.

<p>(A) Western blot of indicated proteins reveals that p21 and p27 are decreased in Hiwi-MSCs. (B) Western blot of indicated proteins reveals that p21, p27 and p15 are increased in sh-Hiwi MFH cells that have been induced with doxycycline for 4 or 7 days. (C) IHC analysis of a human sarcoma TMA reveals a tight inverse correlation of p21, p27 and p15 expression (x-axes) to Hiwi expression (y-axis) (R² = 0.764; R² = 0.8679; R² = 0.7539, respectively) but no such correlation for p16 expression (x-axis) to Hiwi expression (y-axis) (R² = 0.5418). Ten cases of each subtype (present in triplicate) were scored from 0 to 2 blindly by sarcoma pathologists. Average scores for each case are plotted here.</p

Down-regulation of Hiwi decreases DNA-methylation and limits tumorigenic growth.

<p>(A) Verification of inducible sh-Hiwi MFH clones by immunohistochemistry. sh-Hiwi MFH clones C and E were induced with doxycycline for 3 days before IHC analysis. (B) Day 21 in osteogenic media. Calcium matrix formation measured by Alizarin Red S stain is restored in sh-Hiwi MFH clones C and E (approximately 50% of cells staining), induced with doxycycline 7 days before addition of differentiation media and continued in doxycycline-spiked media during differentiation. (C) Doxycycline-induced sh-Hiwi MFH cells at 4 weeks in colony forming assay show decreased colony formation, as compared to uninduced sh-Hiwi MFH cells. p<0.001 for clone C and p<0.005 for clone E by Student's T Test (D) Global DNA methylation is decreased in induced sh-Hiwi MFH cells. Cells were induced with doxycycline for 3 days before DNA was collected and assayed. Error bars represent standard error. * = p<0.05 by Student's T Test (E) Untreated sh-Hiwi MFH cells or treated with 1 uM 5-azacytidine at 4 weeks in colony forming assay. 5-azacytidine treatment decreases colony formation capacity. Combined p<0.01 by Student's T Test. All experiments were performed in triplicate. Representative pictures are shown.</p

Hiwi inhibits differentiation and promotes sarcomagenesis.

<p>(A) Immunohistochemical (IHC) analysis of Hiwi on a human sarcoma tissue microarray (TMA). Ten cases of each subtype (present in triplicate) were scored from 0 to 2 blindly by sarcoma pathologists. Representative pictures are shown. (B) Top row: IHC analysis reveals Hiwi-MSC clones (3 and 7) have a distinct clumped morphology and highly express Hiwi. Middle row: Day 21 in osteogenic media. Calcium matrix formation measured by Alizarin Red S stain is decreased in Hiwi-MSCs, approximately 100% versus 5% of cells staining. Bottom row: Day 21 in adipogenic media. Lipid formation measured by Oil Red O stain is decreased in Hiwi-MSCs, approximately 25% versus 1% of cells staining. Experiments were performed in triplicate and representative pictures are shown. (C) Top row: Xenograft tumors derived from Hiwi-MSCs. Middle row: H&E analysis reveals tumors from Hiwi-MSCs are undifferentiated sarcomas. Bottom row: By IHC analysis, tumors from Hiwi-MSCs continue to express Hiwi. All experiments were performed in triplicate. (D) Transgenic mouse model of Hiwi forms sarcomas (left panel) with both well-differentiated and poorly-differentiated sections (H&E panel) and continues to express Hiwi (right panels).</p