XBP1-Independent UPR Pathways Suppress C/EBP-β Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease

Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-β, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-β transcriptional co-factors, GADD45-β and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-β-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-β activity in MCDS chondrocytes may offer therapeutic opportunities

Epigenome erosion and SOX10 drive neural crest phenotypic mimicry in triple-negative breast cancer

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10’s influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10’s normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry—a major contributor to intratumoral heterogeneity

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis

Abstract Background Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. Methods As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated ‘3D on-top cellular assay’ to identify novel growth suppressive mechanisms. Results A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. Conclusion Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells

Ablation of XBP1 does not significantly affect the MCDS phenotype in <i>C/X</i> mice.

<p><i>(A-C)</i> Tibial epiphyseal cryosections from 2 week <i>Wt</i>, <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> and <i>C/X</i> mice stained with <i>(A)</i> haematoxylin and eosin (H&E), or by immunofluorescence using <i>(B)</i> anti-collagen II or <i>(C)</i> anti-collagen X antibodies; B—Bone; HZ—Hypertrophic Zone; PZ—Proliferative Zone; SCO—Secondary Center of Ossification. <i>(D-F)</i> Quantification of growth plate <i>(D)</i> resting zone, <i>(E)</i> proliferative zone, and <i>(F)</i> hypertrophic zone lengths in mutant and <i>Wt</i> mice; N = 3 for each genotype; statistical analysis performed using Student’s <i>t</i> test.</p

Expression of wildtype growth plate zone gene signatures in <i>ColX</i>^<i>N617K</i>, <i>Xbp1</i>^{<i>CartΔEx2</i>}, and <i>C/X</i> hypertrophic zones.

<p>Heatmaps depicting the relative fold difference (log fold change) of microarray probes representing <i>(A)</i> 773 wildtype (<i>Wt</i>) proliferative zone signature genes and <i>(B)</i> 510 <i>Wt</i> hypertrophic zone signature genes following the comparison of <i>C/X</i> versus <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> versus <i>Wt</i>, and <i>Xbp1</i>^{<i>CartΔEx2</i>} versus <i>Wt</i> hypertrophic zones; N = 3. For both heatmaps, each <i>Wt</i> growth plate zone signature gene is represented by a single bar, colour-coded according to relative expression as indicated, with up-regulated probes coloured yellow, and down-regulated probes coloured red.</p

Dysregulated expression of genes involved in ER stress and chondrocyte differentiation.

<p><i>(A)</i> Immunofluorescent analysis for ATF4 in tibial epiphyseal cryosections from 2 week wildtype (<i>Wt</i>), <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> and <i>C/X</i> mice; B—Bone; HZ—Hypertrophic Zone; PZ—Proliferative Zone. <i>(B-H)</i> qPCR with primers specific for <i>(B) Chop</i>, <i>(C) Cebpb</i>, <i>(D) p57</i>^<i>Kip2</i>, <i>(E) Gadd45b</i>, <i>(F) Runx2</i>, <i>(G) Col10a1</i>, and <i>(H) Mmp13</i> on cDNA derived from <i>Wt</i>, <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> and <i>C/X</i> hypertrophic zone aRNA. Plots depict mean fold differences with standard deviation from the mean; N = 3; statistical analysis performed using Student’s <i>t</i> test, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. <i>(I)</i> Schematic diagram of proposed model to explain the molecular pathology of MCDS. Blue boxes depict genes. Red boxes depict biological processes. Green arrows depict activation or up-regulation. Red arrows depict inactivation or down-regulation. Green lines depict increased interaction between proteins. Red lines depict decreased interaction between proteins.</p

Quantitative PCR of mutant and wildtype hypertrophic zones.

<p>qPCR with primers specific for <i>(A) Agc1</i>, <i>(B) Ctgf</i>, <i>(C) Matn1</i>, <i>(D) Creld2</i>, <i>(E) Derl3</i>, <i>(F) Ero1l</i>, <i>(G) Fgf21</i>, <i>(H) Steap1</i>, and <i>(I) p58IPK</i> on cDNA derived from <i>Wt</i>, <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> and <i>C/X</i> hypertrophic zone aRNA. Plots depict mean fold differences with standard deviation from the mean, N = 3, statistical significance was determined using Student’s <i>t</i> test, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001.</p

Microarray analysis of mutant and wildtype hypertrophic zones.

<p><i>(A)</i> Venn diagram depicting the relationship between probes indicating differential gene expression (fold difference ≥ 2.0, adjusted <i>p</i> value ≤ 0.01) following comparisons of <i>C/X</i> versus wildtype (<i>Wt</i>) (blue), <i>Xbp1</i>^{<i>CartΔEx2</i>} versus <i>Wt</i> (yellow), and <i>ColX</i>^<i>N617K</i> versus <i>Wt</i> (red), by whole genome microarray analysis of hypertrophic zone aRNA. <i>(B-D)</i> Ontological analysis of <i>(B)</i> all probes in cohort <i>i</i> in <i>(A)</i>, or those showing <i>(C)</i> up-regulation or <i>(D)</i> down-regulation, by Functional Annotation Clustering, using DAVID v6.7 software, and depicting representative gene ontology terms from each annotation cluster achieving an enrichment score (ES) ≥ 1.3.</p

Apoptosis is elevated in 2 week <i>ColX</i>^<i>N617K</i> and <i>C/X</i> growth plate cartilage.

<p><i>(A)</i> Representative 2 week wildtype (<i>Wt</i>), <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i> and <i>C/X</i> tibial growth plate sagittal cryosections analysed by TUNEL with DAPI counterstaining; HZ—hypertrophic zone. Boxes inset indicate magnified areas of the hypertrophic zones containing TUNEL-positive chondrocytes. <i>(B)</i> TUNEL analysis of at least 6 tibial growth plate sections from each of 3 <i>Wt</i>, <i>Xbp1</i>^{<i>CartΔEx2</i>}, <i>ColX</i>^<i>N617K</i>, and <i>C/X</i> mice, expressed as the number of TUNEL-positive chondrocytes in the hypertrophic zone as a percentage of the total number of chondrocytes per zone (as defined by DAPI-stained nuclei), and showing standard deviation around the mean. <i>(C</i>,<i>D)</i> Representative 2 week <i>(C) ColX</i>^<i>N617K</i> and <i>(D) C/X</i> tibial growth plate cryosections, showing the distribution of TUNEL-positive cells along the antero-posterior axis of <i>ColX</i>^<i>N617K</i> and <i>C/X</i> hypertrophic zones, as demarcated by 10 consecutive columns (1–10) of arbitrary width. Plots depict the number of TUNEL-positive chondrocytes in each column as a percentage of the total number of chondrocytes per column (as defined by DAPI-stained nuclei), from the same <i>ColX</i>^<i>N617K</i> and <i>C/X</i> mice as analysed in <i>(B)</i>, and showing standard deviation around the mean. Statistical analysis performed using Student’s t-test, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001.</p