Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers

Abstract

Satellite repeats in heterochromatin are transcribed into noncoding RNAs that have been linked to gene silencing and maintenance of chromosomal integrity. Using digital gene expression analysis, we showed that these transcripts are greatly overexpressed in mouse and human epithelial cancers. In 8 of 10 mouse pancreatic ductal adenocarcinomas (PDACs), pericentromeric satellites accounted for a mean 12% (range 1 to 50%) of all cellular transcripts, a mean 40-fold increase over that in normal tissue. In 15 of 15 human PDACs, alpha satellite transcripts were most abundant and HSATII transcripts were highly specific for cancer. Similar patterns were observed in cancers of the lung, kidney, ovary, colon, and prostate. Derepression of satellite transcripts correlated with overexpression of the long interspersed nuclear element 1 (LINE-1) retrotransposon and with aberrant expression of neuroendocrine-associated genes proximal to LINE-1 insertions. The overexpression of satellite transcripts in cancer may reflect global alterations in heterochromatin silencing and could potentially be useful as a biomarker for cancer detection.

Fig. 1

Massive expression of major satellites in mouse pancreatic tumors. (A) Expression of major satellite in primary tumors, cell lines, and normal tissues, presented as transcripts per million of aligned genomic reads. All tumors and cell lines have Kras^G12D; deleted genes are listed individually (Tp53, Smad4, and Apc). (B) Graphical representation of sequence read contributions from major satellites, averaged among all primary tumors versus normal tissues (pancreas and liver). “Unannotated RNA” indicates reads that aligned to the mouse genome, but not to the mouse reference transcriptome.

Fig. 2

Expression patterns of major satellites in tumors and normal mouse tissues. Northern blot analyses: (A) Three Kras^G12D, Tp53^lox/+ pancreatic primary tumors (Tumors 1 to 3) and a stable cell line (CL3) derived from Tumor 3. (B) CL3 before (0) and after (+) treatment with AZA. (C) CL3 cells cultured in vitro and grown as subcutaneous tumors in vivo. (D and E) RNA-ISH with major satellite probe (purple stain): (D) Normal pancreas, primary PDAC, and liver metastasis. (E) Preneoplastic low-grade PanIN (LP) lesion adjacent to high-grade PanIN (HP) and normal pancreas (N). Higher magnification (400×) of inset low-grade (left) and high-grade (right) PanIN lesions. All images are at 200× magnification (scale bar, 100 μm).

Fig. 3

Overexpression of satellites in human cancers. (A) Breakdown of satellite classes as a percentage of total satellites in human PDAC (black, n = 15) and normal human tissues (white, n = 12). Satellites are ordered from highest in tumors to highest in normal tissue (left to right). Error bars represent SEM. Inset (bar graph, center) shows the differential expression of selected satellite classes enriched in cancer (left, black bars) or normal tissue (right, white bars). (B) HSATII expression in human PDAC, normal pancreas, other cancers (L, lung; K, kidney; O, ovarian; P, prostate), and normal human tissues (1, fetal brain; 2, adult brain; 3, colon; 4, fetal liver; 5, adult liver; 6, lung; 7, kidney; 8, placenta; 9, prostate; and 10, uterus) quantitated by DGE. Satellite expression is shown as transcripts per million (tpm) aligned to human genome. (C and D) RNA-ISH with HSATII probe (red stain): (C) human PanIN (P) and normal adjacent tissue (N). (D) EUS-FNA biopsy of confirmed tumor (T) and normal adjacent tissue (N). All images are at 200× magnification (scale bar, 100 μm).

Fig. 4

Correlation of satellite expression with LINE-1 and cellular transcriptional profile. (A) Linear correlation of mouse major satellite to LINE-1 expression. (B) Fraction of mouse SCGs (blue) versus predicted (red) transcriptional start sites within a given distance of a LINE-1. Enrichment calculations were done at a distance of 10 kbp (black line). (C) Immunohistochemistry of mouse PDAC for the neuroendocrine marker chromogranin A. Tumors are depicted as a function of increasing chromogranin A staining (brown), with the relative level of major satellite expression noted for each tumor (bottom; percentage of all transcripts). Images are at 200× magnification (scale bar, 100 μm). (D) Differentially expressed (Q < 0.05) neuroendocrine genes in human PDACs with high (black) versus low (white) ALR satellite levels. Error bars represent SEM.