Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer - PubMed (original) (raw)

Convergent structural alterations define SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeler as a central tumor suppressive complex in pancreatic cancer

A Hunter Shain et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Defining the molecular genetic alterations underlying pancreatic cancer may provide unique therapeutic insight for this deadly disease. Toward this goal, we report here an integrative DNA microarray and sequencing-based analysis of pancreatic cancer genomes. Notable among the alterations newly identified, genomic deletions, mutations, and rearrangements recurrently targeted genes encoding components of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, including all three putative DNA binding subunits (ARID1A, ARID1B, and PBRM1) and both enzymatic subunits (SMARCA2 and SMARCA4). Whereas alterations of each individual SWI/SNF subunit occurred at modest-frequency, as mutational "hills" in the genomic landscape, together they affected at least one-third of all pancreatic cancers, defining SWI/SNF as a major mutational "mountain." Consistent with a tumor-suppressive role, re-expression of SMARCA4 in SMARCA4-deficient pancreatic cancer cell lines reduced cell growth and promoted senescence, whereas its overexpression in a SWI/SNF-intact line had no such effect. In addition, expression profiling analyses revealed that SWI/SNF likely antagonizes Polycomb repressive complex 2, implicating this as one possible mechanism of tumor suppression. Our findings reveal SWI/SNF to be a central tumor suppressive complex in pancreatic cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Genomic DNA copy number alterations in pancreatic cancer. (A) GISTIC plot of the 70 pancreatic cancer specimens, integrating frequency and amplitude to identify significant amplifications (red) and deletions (blue) across the genome (ordered by chromosome). The threshold for significance is determined by the false discovery rate (_q_-value < 0.25). Known/candidate driver genes within selected peaks are indicated. Question marks identify newly identified loci that are depicted in greater detail in

Fig. S1

. (B) ARID1B is focally deleted in pancreatic cancer. The heat map shows gains and losses (red and blue, respectively; log2 ratio scale shown) for the 70 pancreatic cancer specimens across a subregion of 6q25.2-q25.3. CGH array probes are ordered by chromosome position, and samples are ordered by focality and amplitude of loss. Three focal deletions, including two homozygous deletions, of ARID1B are noted (far right). (C) Semifocal deletions span an ∼870-kb region within 1p36.11, harboring ARID1A (along with 24 other genes).

Fig. 2.

Fig. 2.

Deletions target SWI/SNF genes PBRM1, SMARCA4, and SMARCA2 in pancreatic cancer. (A) Array CGH profile of xenograft 287 across a subregion of 3p21.1 identifies a homozygous deletion ∼10 kb upstream of PBRM1 transcriptional start. Tumor/normal log2 ratios of CGH array probes are plotted by chromosome Mb position. (B) Reduced PBRM1 expression in xenograft 287 and in the PL45 cell line (harboring a homozygous nonsense mutation); samples are identified by a star. Transcript levels are depicted by a log2 ratio scale, separately median-centered for xenografts (Upper) and cell lines (Lower), and shown for two array probes and ordered by their average. (C) PBRM1 protein is undetected by Western blot in PL45 (star), among a panel of pancreatic cell lines assayed. GAPDH serves as a loading control. (D) Homozygous deletion overlapping the 5′ end of SMARCA4 is identified in Capan2 by high-resolution CGH array (probes represented by blue dots) and by SNP array (green triangles) (16). (E) Reduced SMARCA4 transcript in Capan2 and PANC1 cells. (F) SMARCA4 protein is undetected by Western blot in Capan2, Hs700T, and PANC1 cells. (G) Schematic depiction of RNAseq-identified internal duplication of SMARCA4 exons 25–28 in PANC1 cells (detailed further in

Fig. S2_C_

). (H) Heat map of copy number alterations across a subregion of 9p24.3-p24.1 reveals focal deletions targeting SMARCA2 (far right). Samples labeled in red text are included from publicly available SNP array data (16) (Experimental Procedures).

Fig. 3.

Fig. 3.

SWI/SNF subunit expression in pancreatic cancer lines. Western blots of ARID1A, ARID1B, PBRM1, SMARCA2, and SMARCA4, done as two separate blots (bracketed). HPDE lysates appear on both blots to facilitate comparison, and GAPDH serves as a loading control. Cell lines with SWI/SNF subunit complete deficiency are identified by a star. Underlying genomic alterations/mutations are annotated as follows: D, homozygous deletion; M, deleterious point mutation (Table 1); R, internal gene rearrangement (detailed in main text); U, undetermined.

Fig. 4.

Fig. 4.

Re-expression of SMARCA4 in deficient lines reduces cell growth. (A) Western blot confirming re-expression of transduced SMARCA4 or SMARCA4 (K798R) mutant in PANC1 and Hs700T cells, and overexpression in HPDE cells (protein levels quantified relative to pBABE empty vector). Lamin A/C and GAPDH serve as loading controls. (B) Cell growth of PANC1, Hs700T, and HPDE cells 5 d after transduction of SMARCA4 or SMARCA4 (K798R), quantified relative to pBABE empty vector. Error bars represent SDs. P values (two-sided Student t test) are indicated. (C) Western blot of PANC1 cells 15 and 30 d after transduction of empty vector (pBABE), SMARCA4, or SMARCA4 (K798R) shows selective loss of SMARCA4 expression with prolonged cell culture. Blots shown are representative of independently replicated experiments. (D) Senescence-associated β-galactosidase staining of transduced PANC1 cells, confirming increased senescence with SMARCA4 re-expression.

Fig. 5.

Fig. 5.

Schematic summary of SWI/SNF subunit alterations in pancreatic cancer. Enzymatic and putative DNA-binding subunits are shown, with the arrows denoting possible pairings within SWI/SNF complexes. Subunit-specific (and total) frequencies of deletion/mutation are indicated. Details of the analysis are provided in

SI Text

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Fig. P1.

Fig. P1.

Genomic aberrations target SWI/SNF chromatin remodeling complex in pancreatic cancer. In aggregate, SWI/SNF subunits are altered in one-third of pancreatic cancers, compromising possible tumor-suppressive functions that include growth suppression, senescence (or cellular aging processes), and antagonism of polycomb repressive complexes that are responsible for inhibiting other genes.

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