Mutational inactivation of STAG2 causes aneuploidy in human cancer

Abstract

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.

Fig. 1

STAG2, a gene encoding a subunit of cohesin—a protein complex that regulates sister chromatid separation during cell division—is frequently altered in diverse human cancers. (A) Copy-number plots along the X chromosome for normal human astrocytes (NHAs) and A172, U87MG, and U138MG glioblastoma cells. A genomic deletion between 122.930 and 123.226 Mb encompassing the STAG2 gene is present in U138MG cells. (B to D) Western blots demonstrate complete loss of STAG2 expression in 3 out of 21 glioblastoma, 5 out of 9 Ewing’s sarcoma, and 1 out of 10 melanoma cell lines. (E) Diagram of the STAG2 protein with mutations identified.

Fig. 2

Single-hit genetic inactivation causes loss of STAG2 in diverse human tumor types. (A) STAG2 sequence traces from TC-32 Ewing’s sarcoma cells derived from a female patient. Whereas the genomic DNA is heterozygous for a single nucleotide insertion (T), the mRNA is derived exclusively from the mutant allele on the active X chromosome. (B) Immunohistochemistry identifies frequent loss of STAG2 expression in glioblastoma and Ewing’s sarcoma primary tumors. Scale bar, 100 μm. (C) Number of tumors successfully assessed by immunohistochemistry and the fraction demonstrating complete loss of STAG2 expression.

Fig. 3

Targeted correction of the endogenous mutant allele of STAG2 in human glioblastoma cells restores sister chromatid cohesion. (A) An AAV-targeting vector was used to correct the endogenous nonsense mutation in exon 20 in 42MGBA cells, which left behind a FLOXed splice acceptor–internal ribosome entry site (IRES)–Neo^R gene in the subsequent intron. These “pre-Cre” clones were then infected with adenoviral Cre, which led to excision of the FLOXed splice acceptor–IRES–Neo^R gene in “post-Cre” clones. Black triangles indicate LoxP sites. (B) 42MGBA parental cells and two nonrecombinant clones fail to express STAG2 protein by Western blot. Two pre-Cre KI clones similarly fail to express STAG2 protein because the STAG2 transcript gets spliced to the IRES-Neo^R gene. Three post-Cre KI clones express physiologic levels of corrected STAG2 protein, comparable to the levels in 8MGBA and U87MG glioblastoma cells with unmodified wild-type STAG2 alleles. (C) Examples of mitotic chromosome spreads from STAG2-deficient H4 cells with cohered, parallel, and fully separated sister chromatids. Arrows indicate each sister chromatid in a mitotic chromosome. Arrowhead points to the centromere. Scale bar, 2 μm. (D) Isogenic sets of STAG2-proficient and deficient cells were arrested in mitosis using taxol or nocodazole, Giemsa stained, and assayed for sister chromatid cohesion.

Fig. 4

Correction of mutant STAG2 alleles in human glioblastoma cells does not globally alter gene expression profile but reduces chromosomal instability. (A) Affymetrix GeneChip human gene 1.0 ST arrays were used to generate gene expression profiles in 42MGBA parental cells, two pre-Cre KI clones, and three post-Cre KI clones. The composite expression profile of the STAG2-mutant cells is plotted against the composite expression profile of the STAG2-corrected cells. (B) Imaging of chromosome dynamics using green fluorescent protein–histone H2B in untreated asynchronous cells (left) and quantification of abnormal mitotic figures in 100 anaphase cells (right) demonstrated lagging chromosomes and anaphase bridges (arrowheads) in STAG2-deficient cells. Scale bar, 5 μm. *P < 0.05. (C and D) Isogenic STAG2-proficient and deficient cells were arrested in prometaphase, and karyotypes were prepared using Wright’s stain. Chromosomes were counted in 100 cells for each cell line to determine the diversity of chromosome counts within the cell population. Chromosome counts are shown in fig. S30, and distribution curves from these data are shown here for STAG2-proficient and deficient 42MGBA cells (C) and HCT116 cells (D). (E) Examples of unique chromosomal aberrations present in individual HCT116 STAG2 KO cells.