Additional file 1: Table S1. of Non-coding single nucleotide variants affecting estrogen receptor binding and activity

The list of all ER ChIP-seq datasets in breast cancer. Table S2. List of E2-regulated genes common in vitro, in vivo, and TCGA. Table S3. List of the studies used for the identification of E2-regulated genes. Table S4. Primer sets used for different assays. Table S5. The list of regulatory SNVs in MCF7 Cell line. Table S6. The list of regulatory SNVs in BT474 cell line. Table S7. The list of regulatory SNVs in MDA-MB-134 cell line. Table S8. The list of regulatory SNVs in T47D cell line. Table S9. The list of regulatory SNVs in TAMR cell line. Table S10. The list of regulatory SNVs in ZR75 cell line. Table S11. The list of regulatory SNVs in good prognosis tumors. Table S12. The list of regulatory SNVs in bad prognosis tumors. Table S13. The list of regulatory SNVs in metastatic tumors. Table S14. The allele frequency of top RegSNVs in ER-binding sites with sufficient coverage. (XLSX 3641 kb

Additional file 2: Figure S1. of Non-coding single nucleotide variants affecting estrogen receptor binding and activity

The UCSC gnome browser view of the second intron in IGF1R gene. The index SNP, rs62022087, seems to be located in a region bound by several chromatin-modifying factors based on ENCODE data. Figure S2. The visualization of ChIP-seq reads from multiple cell lines over rs62022087 SNP site in two individual studies: (A) Hurtado et al. {Hurtado, 2011 #23}, (B) Joseph et al. {Joseph, 2010 #15}. Figure S3. The distribution of RegSNVs over the gnome across a panel of breast cancer cell lines, good and bad prognosis tumors. The binding sites from different ER ChIP-seq datasets were extracted and annotated based on their location in the genome. The majority of the binding sites are located in the intergenic and intronic areas. (DOCX 384 kb

Ribosomopathy-like properties of murine and human cancers

<div><p>Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs) and inefficient ribosomal RNA (rRNA) processing. The resulting ribosomal stress induces the canonical p19^<i>ARF</i>/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB) and hepatocellular carcinoma (HCC) unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19^<i>ARF</i>, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19^<i>ARF</i> expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.</p></div

RP transcript deregulation in human cancers.

<p>3D area maps of transcript levels for 77 RPs expressed in HCCs (A), CRCs (B), BCs (C) and PCs (D). To better evaluate differences in the other transcripts which did reach significance for their F-tests, these transcripts–<i>Rps26</i>, <i>Rpl9</i>, <i>Rps27</i>, <i>Rps28</i>, and <i>Rpl21</i>– were excluded from 3D area plots. For each cancer, tumors with matched samples of normal tissue in TCGA were selected for direct comparison (50 for HCC, 41 for CRC, 113 for BC and 52 for PC). Relative expression for each RP transcript was calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.g001" target="_blank">Fig 1</a>. See Figures D-G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.s001" target="_blank">S1 File</a> for 3D area plots of the above matched tumor data together with additional data from unmatched tumor samples. (E, F) Patient survival in HCC and BC inversely correlates with the severity of RP transcript deregulation. Patients were sorted according to their RP transcript deregulation, and survival curves were plotted for the top and bottom 25% of patients with the greatest and least degree of RP transcript deregulation.</p

Incomplete processing of rRNAs in HBs and HCCs.

<p>(A) Normal rRNA processing. Arrows depict regions amplified by qRT-PCR to quantify 18S-ITS1, ITS1-5.8S, 5.8S-ITS2 and ITS2-28S junctional fragments common to all rRNA precursors. (B) Quantification of each of the above four junctions in control livers and HBs. Identically colored dots represent the same tumor RNA sample within the subgroup of tumors that demonstrated abnormal processing of at least one junction. Control livers and tumors with no significant processing differences are depicted in black. (C) Similar quantification of RNA processing in HCCs. Data in (B) and (C) were normalized to levels of total 18S and 28S RNA. Each qRT-PCR reaction was performed in triplicate and the mean is depicted.</p

Relative RP transcript and protein levels differ in murine models of HB and HCC.

<p>(A) RP transcript abundance in hepatocytes (H) versus HB tumors (T). Heat maps are based on averaged RNA-seq data from 4–5 samples in each group with the most abundant transcripts being shown in orange and the least abundant transcripts being shown in blue, with the sum of all transcripts in each group equaling 100%. All transcript levels are expressed as a percent of total, displayed relative to those in WT hepatocytes and do not take into account the fact, as previously shown, that average RP transcript expression was increased 5.2-fold in HBs relative to hepatocytes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref016" target="_blank">16</a>] *: deregulated transcripts in WT tumors vs. WT hepatocytes, ^: deregulated transcripts in KO tumors vs. KO hepatocytes. (B) RP transcript deregulation among WT and KO hepatocytes and HBs. (C) Similar heat maps from livers or HCCs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref013" target="_blank">13</a>]. L: control livers. 3D and 7D: livers obtained 3 and 7 days after removing doxycycline to induce Myc expression. T: initial tumors. 3R and 7R: regressing tumors following doxycycline resumption for 3 or 7 days, respectively. Additional tumor-bearing mice were maintained on doxycycline for 2.5–3 months to allow for complete regression. Doxycycline removal in these mice led to development of recurrent tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref012" target="_blank">12</a>]. *: significant differences in relative expression compared to normal liver; ^: significant differences between recurrent tumor and liver (q-value < 0.05). Relative transcript abundance was expressed as described for panel A and compared with the relative abundance in control livers. (D) RP transcript discordances between HBs and HCCs. “Opposing” directionality occurred when an HB transcript’s direction of change relative to hepatocytes differed between WT and KO HBs. (E) Immunoblots of RPs in WT and KO livers (L) and HB tumors (T). (F) Immunoblots of RPs from livers, collected as described in (C).</p

Reprograming of survival and apoptosis pathways in HBs and HCCs.

<p>(A) Expression of p19^ARF, MDM2 and p53 in total liver (L) and HB (T) lysates from WT and KO mice. (B) Similar immuno-blots from HCCs. (C) Immuno-staining of frozen sections of liver (L) and WT HBs (T) for p53 and MDM2. Using ImageJ software (<a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>), we determined that >80% of Mdm2 and p53 localized to the cytoplasm in both livers and tumors. (D) p53 and MDM2 co-localize to HB cytoplasm. A freshly collected WT HB tumor was fractionated into cytoplasmic, nuclear and nucleolar compartments. Each fraction was tested for the protein markers localizing to these compartments (GAPDH, histone H3 and fibrillarin, respectively) and in parallel for p53, p19^ARF and MDM2. Varying amounts of lysate and exposure times were required to compensate for differential protein expression. (E) Liver and HB cytoplasmic fractions were immuno-precipitated with control IgG or anti-MDM2 IgG. Precipitates were resolved by SDS-PAGE and silver stained. Bracketed regions were excised from lanes 2 and 4 and subjected to trypsin digestion and mass spectrometry. (F) Bcl-2 and Bax expression in mitochondria from WT or KO HBs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref016" target="_blank">16</a>]. The same blot was probed with an antibody for the mitochondrial protein pyruvate dehydrogenase E1α subunit (PDH) as a control for protein loading. The mean up-regulation of Bcl-2 relative to that in livers was 3.8-fold in WT HBs and 2.3-fold in KO HBs. The mean up-regulation of Bax was 11.7-fold in WT HBs and 10.5-fold in KO HBs. (G) Bcl-2 and Bax expression in isolated mitochondria from livers (L), tumors (T) and recurrent HCC tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182705#pone.0182705.ref012" target="_blank">12</a>]. The mean up-regulation of Bcl-2 was 5.5-fold in initial tumors and 6.3-fold in recurrent tumors. Similarly, the mean up-regulation of Bax was 15-fold in initial tumors and 15.6-fold in recurrent tumors.</p