The developmental gene Chordin is amplified and expressed in human cancers

Abstract

Chordin (CHRD) is a secreted protein important in early development, yet a role for CHRD in human disease has not been identified. In this study we investigated CHRD in cancer and normal adult tissues using the wealth of genome-wide data available in public databases. We found that Chordin is amplified in the DNA of specific cancers such as lung squamous cell and others, although copy number variation did not strictly correlate with higher mRNA expression. In some cancers, such as renal and stomach carcinomas, increased CHRD expression significantly correlated with poor survival. In normal adult human tissues, CHRD mRNA was highest in hepatocytes. Crossveinless-2/BMPER, a component of the Chordin morphogenetic pathway expressed at the opposite side in embryos, was expressed in liver stellate cells. This raises the intriguing possibility that a BMP gradient might be established in the extracellular matrix of the space of Disse that surrounds portal sinusoid capillaries.

Keywords: Bone morphogenetic proteins; Chordin; Crossveinless 2; TCGA; hepatocytes; lung squamous cell carcinoma; renal carcinoma; scRNA-seq; stellate cells; stomach carcinoma.

Figure 1.

CHRD amplification in cancer does not correlate with higher mRNA expression. (a) Alteration frequencies indicate that CHRD DNA amplification is most common across pulmonary cancer subtypes. CHRD amplification was observed across multiple cancers except skin cutaneous melanoma and small cell lung cancer. This data was extracted from the cBioportal for Cancer Genomics and encompasses 4,417 patients across 11 TCGA studies. (b) A diversity of cancers harbor gain-of-function CHRD CNVs in 3,968 patients from 33 TCGA studies. CHRD CNVs are primarily gain-of-function with uveal melanoma serving as the only exception. (c) Chordin amplification is not significantly associated with increased mRNA expression in a subset of lung squamous cell carcinoma. Lung squamous cell carcinoma, which possesses high CHRD DNA amplification (red circles), had comparable CHRD mRNA expression compared to tumors with infrequent amplification (thyroid carcinoma). This pattern differs from what is observed in a well-established amplified oncogene, MYC, in which DNA amplification correlates with higher mRNA expression. In these data from TCGA, red and blue circles in the boxplot indicate the presence or absence of amplification, respectively. The black center line denotes the median value (50^th percentile), while the red and blue boxes contain the 25^th to 75^th percentiles of the dataset. The black whiskers denote the 5^th and 95^th percentiles, and values beyond these upper and lower bounds are considered outliers. The single red asterisk indicates a p-value <.05 obtained via two-tailed Student’s t-test.

Figure 2.

High CHRD expression correlates with decreased survival in renal and stomach carcinoma. (a) Box and whisker plot of CHRD RNA expression in renal carcinomas from 877 patients. Values above the black whisker (95^th percentile) denote outliers and are colored in red. High CHRD RNA expression (≥6 RPKM) is significantly correlated with decreased survival probability. Kaplan-Meier plots of patients with high and low CHRD expression possessed a 5-year survival rate of 55% and 74%, respectively (p = 6.1 × 10⁻⁷). (b) Box and whisker plot of CHRD RNA expression in stomach carcinomas from 354 patients. High CHRD RNA expression (≥5 RPKM) is significantly correlated with decreased survival probability. Kaplan-Meier plots of patients with high and low CHRD expression possessed a 5-year survival rate of 23% and 52%, respectively (p = .0055). The double red asterisks indicate an obtained p-value <.001 via two-tailed Student’s t-test.

Figure 3.

Single-cell CHRD expression in hepatocytes and BMPER/CV2 in hepatic stellate (Ito) cells suggests a BMP-signaling gradient in adult liver tissue. (a) Tissues with the highest reproducible CHRD mRNA expression include female reproductive structures (red bars), brain regions (green bars), and gastrointestinal organs (blue bars). Hepatic tissue contained the highest CHRD RNA expression with 130 nTPM. This consensus dataset was created by combining the HPA, GTEx, and FANTOM transcriptomic databases available through the Human Protein Atlas. (b) Single-cell hepatic CHRD expression data is limited exclusively to hepatocytes (blue coordinates) according to UMAP analysis. Z-scores were generated from single-cell RNA expression values and represented through a logarithmic heatmap. Cell types with a positive z-score possess significantly higher CHRD expression. Z-scores were converted to p-values to determine cell type CHRD expression significance * p < .05, ** p < .01, indicated by red asterisks in the figure. (c) Conversely, CV2/BMPER single-cell expression is absent in hepatocytes and limited to mesenchymal stellate cells (green coordinates). Only stellate cells possessed a positive z-score reflecting a significantly higher CV2/BMPER expression. Single-cell RNA data was acquired from thousands of single-cell expression collections deposited in the Human Protein Atlas.

Figure 4.

Diagram indicating the known biochemical functions of CHRD and CV2 in BMP morphogen signaling. Chordin is a protein that contains 4 VWF-C modules that bind BMPs. Twisted gastrulation is a co-factor that bridges the binding of BMP to Chordin modules and is shown as a dimer on top of BMP. At the high-BMP side of a BMP gradient the protein CV2, which has five VWF-C domains, binds to and concentrates BMP-Tsg-CHRD complexes, facilitating their cleavage by Tolloid metalloproteinases (shown by scissors). Once CHRD is cleaved, BMPs are liberated and signal through BMPR. While CHRD complexes readily diffuse between cells, CV2 contains a Vwf-D domain (shown here as an oval) that anchors it to Glypicans (called Dally in Drosophila) in the plasma membrane of the cells that secrete CV2. Because CHRD is expressed in hepatocytes (red) an CV2 in stellate cells (blue), we propose that these proteins may generate a BMP gradient within the perisinusoidal space of Disse in normal liver tissue.