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

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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

<i>Arid4b</i> germline amino acid sequence variants.

<p>Substitutions in the AKR allele are shown relative to a consensus sequence that is identical between DBA, FVB, and C57Bl/6. The+symbol indicates conserved substitutions.</p

Cellular processes regulated by <i>Arid4b</i> knockdown.

<p>Biological processes are listed in order of statistical significance after applying a cutoff based on z-scores with an absolute value greater than 2. Negative z-scores indicate downregulation; positive z-scores indicate upregulation.</p

High expression of <i>ARID4B</i> is associated with poor clinical outcome.

<p>In patients with ER-positive tumors who were node negative at diagnosis (A) distant metastasis-free survival (DMFS) was significantly lower (p = .009) in patients with high expression (blue) compared to middle (red) or low (gray) expression of <i>ARID4B</i>, and multivariate analysis of 440 cases (B) was performed to determine metastatic progression hazard ratios of 0.54 and 0.42 for median and low <i>ARID4B</i> terciles, respectively, compared to the high <i>ARID4B</i> tercile. The association of high <i>ARID4B</i> with poor DMFS was also highly significant (p = 3.05×10∧−4) among ER-positive patients in the absence of adjuvant therapy (C) with similar hazard ratios (D) of 0.53 and 0.49 for middle and low <i>ARID4B</i> groups compared to high <i>ARID4B</i>.</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

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

<i>Arid4b</i> expression is correlated with tumor growth and metastasis.

<p>Gene expression microarray data from 18 of the AKXD recombinant inbred strains shows expression of <i>Arid4b</i> is positively correlated with tumor burden (A) and metastatic density (B). The formula used to calculate metastatic density was ln[(metastases/um²)×10⁸].</p

image_6_Sialic Acid Metabolism: A Key Player in Breast Cancer Metastasis Revealed by Metabolomics.jpeg

<p>Metastatic breast cancer is currently incurable. It has recently emerged that different metabolic pathways support metastatic breast cancer. To further uncover metabolic pathways enabling breast cancer metastasis, we investigated metabolic differences in mouse tumors of differing metastatic propensities using mass spectrometry-based metabolomics. We found that sialic acid metabolism is upregulated in highly metastatic breast tumors. Knocking out a key gene in sialic acid metabolism, Cmas, inhibits synthesis of the activated form of sialic acid, cytidine monophosphate-sialic acid and decreases the formation of lung metastases in vivo. Thus, the sialic acid pathway may be a new target against metastatic breast cancer.</p