The Circadian Rhythm Gene <i>Arntl2</i> Is a Metastasis Susceptibility Gene for Estrogen Receptor-Negative Breast Cancer

<div><p>Breast cancer mortality is primarily due to metastasis rather than primary tumors, yet relatively little is understood regarding the etiology of metastatic breast cancer. Previously, using a mouse genetics approach, we demonstrated that inherited germline polymorphisms contribute to metastatic disease, and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. In this study, a backcross between a highly metastatic (FVB/NJ) and low metastatic (MOLF/EiJ) mouse strain identified <i>Arntl2</i>, a gene encoding a circadian rhythm transcription factor, as a metastasis susceptibility gene associated with progression, specifically in estrogen receptor-negative breast cancer patients. Integrated whole genome sequence analysis with DNase hypersensitivity sites reveals SNPs in the predicted promoter of <i>Arntl2</i>. Using CRISPR/Cas9-mediated substitution of the MOLF promoter, we demonstrate that the SNPs regulate <i>Arntl2</i> transcription and affect metastatic burden. Finally, analysis of SNPs associated with <i>ARNTL2</i> expression in human breast cancer patients revealed reproducible associations of <i>ARNTL2</i> expression quantitative trait loci (eQTL) SNPs with disease-free survival, consistent with the mouse studies.</p></div

Distal chromosome 6 is a metastasis modifier.

<p>(A) Survival plot of FVB/NJ x (MOLF/EiJ x MMTV-PyMT) mice. p-value calculated by Log-Rank test. FVB = FVB/NJ x MMTV-PyMT; MOLF = MOLF/EiJ x MMTV-PyMT. (B) Surface metastasis count of FVB/NJ x (MOLF/EiJ x MMTV-PyMT) mice. p-value calculated by Mann-Whitney test. FVB = FVB/NJ x MMTV-PyMT; MOLF = MOLF/EiJ x MMTV-PyMT. (C) Depiction of breeding scheme. (D) LOD score plot for metastasis susceptibility, latency and tumor burden loci in the backcross. The y-axis represents the LOD score observed across the genome. The mouse chromosomes are presented head-to-tail on the x-axis. The lower horizontal dashed line represents the suggestive LOD score for a quantitative trait locus after correction for multiple testing by permutation testing. The upper dashed line is the significance threshold after permutation testing. (E) Approximate location of metastasis-associated eQTL candidate genes on distal chromosome 6. (F) The Gene expression-based Outcome for Breast Cancer Online (GOBO) database was queried for the eQTL genes on distal chromosome 6. Distant metastasis-free survival (DMFS) plotted as Kaplan-Meier curves for patients with ER+ and ER- tumors expressing high (blue), intermediate (red), or low (gray) levels of the eQTL genes.</p

Human eQTL SNPs associated with ARNTL2 expression that are prognostic for ER- breast cancer.

<p>Human eQTL SNPs associated with ARNTL2 expression that are prognostic for ER- breast cancer.</p

Tumor-autonomous effect of Arntl2.

<p>(A) <i>Arntl2</i>^+/+ and <i>Arntl2</i>^-/- mice were bred to MMTV-PyMT+ mice to generate spontaneous primary tumors and lung metastases. At the time of euthanasia, autochthonous PyMT-induced tumors were weighed. N.S. = not significant. (B) Surface lung metastasis count of spontaneous metastases from (A). P-value calculated by two-tailed Mann-Whitney test (C) Primary tumor weight of orthotopically injected 4T1 cells into F1 hybrids from <i>Arntl2</i>^+/+ and Arntl2^+/- mice crossed with BALB/cJ mice. N.S. = not significant. (D) Surface lung metastasis count of the injected mice in (C). N.S. = not significant.</p

SNPs within the <i>Arntl2</i> promoter modulate chromatin binding.

<p>(A) Differential binding of MOLF and FVB promoter DNA probes to chromatin proteins. Relative densitometry compared to beads only is reported above each sample. Densitometry was calculated using ImageJ. (B) Relative mRNA expression of <i>Stkl3</i> in wildtype and heterozygous CRISPR 6DT1 cells. (C) Working model depicting differential chromatin binding of MOLF and FVB to regions of the nucleus. LADs = laminar-associated domains (repressive state).</p

Genome editing of the <i>Arntl2</i> promoter.

<p>(A) Haplotypes for the <i>Arntl2</i> putative promoter from the FVB, MOLF, PWK and WSB inbred strains. Brain RNAseq data shows that wild-derived PWK/PhJ carries the same haplotype as MOLF/EiJ, and WSB/EiJ shares 9 of the 10 FVB/NJ SNPs at this location. (B) Relative expression of <i>Arntl2</i> in brain RNA from the PWK and WSB strains. (C) Confirmation of genomically-edited 6DT1 cells. (D) Relative mRNA expression of <i>Arntl2</i> in CRISPR-engineered 6DT1 cells as measured by qPCR. (E) Primary tumor weight of injected CRISPR-engineered WT and heterozygous substituted cells in FVB mice. N.S. = not significant. (F) Surface lung metastasis count on injected cells in (E). P-value calculated by two-tailed Mann-Whitney test.</p

Effect of Arntl2 expression <i>in vivo</i>.

<p>(A) Relative <i>Arntl2</i> mRNA expression in 4T1 cells with control (scramble) and <i>Arntl2</i> shRNA cell lines. (B) Protein expression of ARNTL2 in transiently transfected HEK293 cells with shRNA and overexpression vector expressing myc-tag. Actin is the loading control. (C) Weight of primary tumor from orthotopically injected BALB/cJ mice with 4T1 control and C7 and C11 <i>Arntl2</i> shRNA constructs. N.S. = not significant. (D) Count of surface pulmonary nodules from (C). P-value calculated by two-tailed Mann-Whitney test. (E) Primary tumor weight of <i>Arntl2</i> overexpressing 4T1 cells. N.S. = not significant. (F) Lung metastases from (E), p-value calculated by one-tailed Mann-Whitney test.</p

Knockdown of Tpx2 does not induce prototypical MET in 6DT1 cells.

<p>A) E-cadherin mRNA levels in 6DT1 shCtrl, 6DT1-shTpx2#1, and 6DT1-shTpx2#2 cells were measured by qRT-PCR and displayed relative to levels in shRNA control cells. Error bars represent standard deviations. B) Western blot analysis for E-cadherin, beta-catenin, N-cadherin, and vimentin of cell lines described in a). Beta-actin serves as loading control. 67NR and 4T1 cells serve as positive and negative controls for the different EMT markers. C) 6DT1-shCtrl, 6DT1-shTpx2#1 and 6DT1-shTpx2#2 cells (and 67NR and 4T1 cells as controls) were immunofluorescently stained for E-cadherin and actin cytoskeleton was labeled with phalloidin (green). DAPI was used for nuclear staining. Confocal images are shown at 63x magnification.</p

Network modules from the cross species analysis.

<p>A) Proliferation associated network centered around the <i>Tpx2</i> gene. B) Immune cell network containing the <i>Il10ra</i> gene. Both Figures are adapted from Hu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111813#pone.0111813-Hu1" target="_blank">[3]</a>.</p

Partial Tpx2 ablation in 6DT1 cells does not change expression levels of Tpx2 network hub genes.

<p>6DT1 mouse mammary carcinoma cells were lentivirally transduced with pLKO-Scrambled (shCtrl), pLKO-Tpx2#1 (shTpx2#1), or pLKO-Tpx2#2 (shTpx2#2) and stable, polyclonal pools generated. qRT PCR was carried out to measure mRNA levels of <i>Tpx2</i> and eight genes previously identified as ‘hubs’ of the Tpx2 gene expression network <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111813#pone.0111813-Hu1" target="_blank">[3]</a>. Gene expression levels are displayed relative to levels in shCtrl control cells, error bars represent standard deviations, asterisks indicate p-values <0.05. Only <i>Tpx2</i> was significantly down regulated in both shTpx2 cell lines (red asterisks).</p