<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

CONSTRUCTION PUBLICATIONS Editing Team

<div><p>Renal angiomyolipoma is a kidney tumor in the perivascular epithelioid (PEComa) family that is common in patients with Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) but occurs rarely sporadically. Though histologically benign, renal angiomyolipoma can cause life-threatening hemorrhage and kidney failure. Both angiomyolipoma and LAM have mutations in <i>TSC2</i> or <i>TSC1</i>. However, the frequency and contribution of other somatic events in tumor development is unknown. We performed whole exome sequencing in 32 resected tumor samples (n = 30 angiomyolipoma, n = 2 LAM) from 15 subjects, including three with TSC. Two germline and 22 somatic inactivating mutations in <i>TSC2</i> were identified, and one germline <i>TSC1</i> mutation. Twenty of 32 (62%) samples showed copy neutral LOH (CN-LOH) in <i>TSC2</i> or <i>TSC1</i> with at least 8 different LOH regions, and 30 of 32 (94%) had biallelic loss of either <i>TSC2</i> or <i>TSC1</i>. Whole exome sequencing identified a median of 4 somatic non-synonymous coding region mutations (other than in <i>TSC2/TSC1</i>), a mutation rate lower than nearly all other cancer types. Three genes with mutations were known cancer associated genes (<i>BAP1</i>, <i>ARHGAP35</i> and <i>SPEN</i>), but they were mutated in a single sample each, and were missense variants with uncertain functional effects. Analysis of sixteen angiomyolipomas from a TSC subject showed both second hit point mutations and CN-LOH in <i>TSC2</i>, many of which were distinct, indicating that they were of independent clonal origin. However, three tumors had two shared mutations in addition to private somatic mutations, suggesting a branching evolutionary pattern of tumor development following initiating loss of <i>TSC2</i>. Our results indicate that <i>TSC2</i> and less commonly <i>TSC1</i> alterations are the primary essential driver event in angiomyolipoma/LAM, whereas other somatic mutations are rare and likely do not contribute to tumor development.</p></div

LIst of patient samples available, and mutation findings in <i>TSC2</i> and <i>TSC1</i>.

<p>LIst of patient samples available, and mutation findings in <i>TSC2</i> and <i>TSC1</i>.</p

Copy neutral-LOH on chromosome 16p.

<p>At least 8 different regions of copy neutral LOH were seen in 18 tumor samples with 16p LOH. The blue bars reflect the region of copy neutral LOH for each sample extending from the first to the last SNP with skewed allele frequency (AF <0.4 or >0.6). The gray bars represent the interval between the last SNP with normal AF (0.4 < AF < 0.6) and the first SNP with skewed AF on each side of the region with LOH, and reflect regions with no informative SNP markers to assess LOH.</p

Map of 20 somatic <i>TSC2</i> mutations detected in angiomyolipoma and LAM specimens.

<p>The GAP domain of TSC2 is indicated. Novel variants (n = 13) are in black font; variants previously reported (n = 5) are in blue font. Circle colors reflect different types of mutation, as indicated. Note that in two instances, the mutations differ by a single amino acid position, and hence their circles overlap (p.R749delfs/p.L750delfs, p.Q1588*/p.P1589delfs). Two mutations were seen twice each in two samples.</p

The diagram shows the different kinds of tumor samples analyzed from the different kinds of patients.

<p>S#: Sample number, P#: Patient number.</p

Phylogenetic tree of 16 angiomyolipomas from one TSC patient.

<p>A model of angiomyolipoma development from a progenitor cell in the kidney is shown, including 3 tumors (S20, S23, S24) that had a definite common truncal precursor, and 5 tumors (S14, S15, S17, S18, S19) that may have had a common truncal precursor. Note that the extent of copy neutral LOH in S22 is uncertain due to low tumor purity. CN-LOH denotes copy neutral LOH.</p