<i>MYC</i> and <i>PVT1</i> synergize to regulate RSPO1 levels in breast cancer

<p>Copy number gain of the 8q24 region including the v-myc avian myelocytomatosis viral oncogene homolog (<i>MYC)</i> oncogene has been observed in many different cancers and is associated with poor outcomes. While the role of <i>MYC</i> in tumor formation has been clearly delineated, we have recently shown that co-operation between adjacent long non-coding RNA plasmacytoma variant transcription 1 (<i>PVT1)</i> and <i>MYC</i> is necessary for tumor promotion. Chromosome engineered mice containing an increased copy of <i>Myc-Pvt1</i> (Gain <i>Myc-Pvt1</i>) accelerates mammary tumors in <i>MMTV-Neu</i> mice, while single copy increase of each is not sufficient. In addition, mammary epithelium from the Gain <i>Myc-Pvt1</i> mouse show precancerous phenotypes, notably increased DNA replication, elevated -<i>H2AX</i> phosphorylation and increased ductal branching. In an attempt to capture the molecular signatures in pre-cancerous cells we utilized RNA sequencing to identify potential targets of supernumerary <i>Myc-Pvt1</i> cooperation in mammary epithelial cells. In this extra view we show that an extra copy of both <i>Myc</i> and <i>Pvt1</i> leads to increased levels of <i>Rspo1</i>, a crucial regulator of canonical β-catenin signaling required for female development. Human breast cancer tumors with high levels of <i>MYC</i> transcript have significantly more <i>PVT1</i> transcript and <i>RSPO1</i> transcript than tumors with low levels of MYC showing that the murine results are relevant to a subset of human tumors. Thus, this work identifies a key mechanism in precancerous and cancerous tissue by which a main player in female differentiation is transcriptionally activated by supernumerary <i>MYC</i> and <i>PVT1</i>, leading to increased premalignant features, and ultimately to tumor formation.</p

Relative free energy profiles across the ε−ζ reaction coordinates in B- and 5mCB-DNA.

<p>The plots show the changes in free energy (y-axis) across the ε−ζ coordinate range (x-axis) that define the BI and BII sub-states. (A) Overall relative free energy profiles for unmethylated B-DNA (<i>black</i>) and methylated 5mCB-DNA (<i>red</i>). (B) Relative free energy profiles for unmethylated and methylated CpG and GpC steps. <i>Black</i>, CpG steps of B-DNA; <i>red</i>, 5mCpG steps of 5mCB-DNA; <i>blue</i>, GpC steps of B-DNA; <i>green</i>, Gp5mC steps of 5mCB-DNA.</p

Schematics of torsional angles in a nucleotide phosphate backbone.

<p>α and γ angles determine canonical/non-canonical backbone conformations; ε and ζ define BI and BII sub-states of B-DNA.</p

MM/PBSA analysis of B, methylated B (BM), Z and methylated Z (ZM) DNA.

1<p>SE, standard error.</p>2<p>All energy values are in kcal/mol.</p

Scatter plots of α vs. γ for CpG and GpC steps.

<p>(A) GpC steps in B-DNA; (B) CpG steps in B-DNA; (C) Gp5mC steps in 5mCB-DNA; (D) 5mCpG steps in 5mCB-DNA. The plots are color-coded based on the density of points.</p

The α/γ distributions in B- and Z-DNA.

<p><b>Landscape of the combined distributions of phosphate torsion angles along the α/γ space.</b> (A) B-DNA; (B) 5mCB-DNA; (C) Z-DNA; (D) 5mCZ-DNA. The plots are color-coded based on the density of points. The results from the two independent simulations for each state are combined, giving 1.6 million points. The color bars on panels B and D show the density values for B- (panels A and B) and Z-DNA (panels C and D) simulations.</p

Summary of somatic mutations detected by WGS.

<p>(A) Stacked bar graphs representing total number of C/G and T/A context somatic mutations in the indicated granddaughter subclones (black and white bars, respectively). Sequences from granddaughter clone AG3 were used as a baseline to call mutations in AA3 (i.e., mutations for AG3 are not shown in bar format because WGS data from another control granddaughter clone were not available for comparison). (B) Pie charts representing the proportion of each type of cytosine mutation across the genome in the indicated granddaughter clones. Red, blue, and black wedges represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Stacked bar graphs representing the observed percentage of C-context somatic trinucleotide mutations detected in each granddaughter clone from the B panel. (D) Stacked bar graphs representing the extracted mutation signatures from WGS data. (E) The relative proportion that each extracted mutation signature contributes to the overall base substitution spectrum in the indicated granddaughter clones.</p

SNP analyses to estimate new mutation accumulation.

<p>(A) A dynastic tree illustrating the relationship between mother, daughter, and granddaughter clones used for SNP and WGS experiments. The red, dashed box around the daughter clones denotes 10 cycles of Dox-treatment. (B) A histogram summarizing the SNP alterations observed in granddaughter clones by microarray hybridization. Red, blue, and black colors represent C-to-T, C-to-A, and C-to-G mutations, respectively. (C) Sanger sequencing chromatograms confirming representative cytosine mutations predicted by SNP analysis. The left chromatogram shows a G-to-A transition (C-to-T on the opposite strand) and the right chromatogram a C-to-G transversion. (D) A histogram plot of the total number of copy number (CN) alterations in the indicated categories in A3B-eGFP exposed granddaughter clones in comparison to eGFP exposed controls, which were normalized to zero in order to make this comparison. (E) A dot plot and best-fit line of data in panel B versus data in panel D.</p

A3B induction optimization and targeted sequencing results.

<p>(A, B) Dose response curves indicating the relative colony forming efficiency (viability index) of T-REx-293 A3B-eGFP daughter clones treated with the indicated Dox concentrations (n = 3; mean viability +/- SD of biological replicates). The dotted lines show the Dox concentration required to induce 80% cell death (2 or 1 ng/mL for C- and A-series daughter clones, respectively). (C) A schematic representation of the experimental workflow depicting the viability index of a population of cells induced to express A3B-eGFP and recover over time. Dox treatment occurs on day 1, maximal death is observed on days 3 or 4, and each population typically rebounds to normal viability levels by days 6 or 7. (D-G) A summary of the base substitution mutations observed in <i>MYC</i> (241 bp) and <i>TP53</i> (176 bp) by 3D-PCR analysis of genomic DNA after 10 rounds of A3B-eGFP or eGFP exposure. Red, blue, and black columns represent the absolute numbers of C-to-T, C-to-A, and other base substitution types in sequenced 3D-PCR products, respectively. Asterisks indicate cytosine mutations occurring in 5’-TC dinucleotide motifs. The adjacent pie graphs summarize the base substitution mutation load for each 3D-PCR amplicon. The number of sequences analyzed is indicated in the center of each pie graph.</p

Germline mutations are affected by transcription.

<p><i>Panel A</i>, HGMD dataset; <i>y-axis</i>, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003816#pgen-1003816-g001" target="_blank">Figure 1C</a>; <i>x-axis</i>, ratio of mutated NGNN sequences in protein coding genes containing the P2-guanine base on the non-transcribed (<i>NT</i>) <i>vs</i>. transcribed (<i>T</i>) strand; <i>solid circles</i>, HGMD dataset (<i>r</i>² 0.32, P(α)_0.05 0.991, P<0.001); <i>open circles</i>, 1000 Genomes Project dataset. <i>Panel B</i>, inherited splicing mutations dataset; <i>top</i>, cartoon of exon-intron boundaries showing the conserved GT and AG bases at the donor (<i>ds</i>) and acceptor (<i>as</i>) splice sites; <i>bottom</i>, graph of splicing mutations; <i>y-axis</i>, number of SBSs; <i>x-axis</i>, position of SBSs relative to +/−20 nt of splice junctions; <i>Panel C</i>, model for sequence-dependent SBSs in cancer and human inherited disease. In the first step, an electron is lost from within a tetranucleotide sequence, leaving a hole. In the second step, the hole migrates to and from various competing sites, including nearby bases and chromatin-associated amino acids (not shown), eventually being trapped by a guanine base. The resulting guanine radical cation either causes DNA-protein crosslinking or undergoes subsequent chemical modifications. If the modified base is not corrected by DNA repair, it may give rise to a mutation (X-Y base-pair) as a result of error-prone DNA polymerase synthesis during DNA replication (dashed arrow).</p