Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair

Abstract

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.

Figure 1

IHC analysis of basal-like phenotype markers in Pten^+/− mouse mammary tumors. (a) Representative section showing IHC staining for CK5/6, CK14 and ER in a Pten^+/− mammary tumor (top) and normal breast tissue from a wild-type (Pten^+/+) control mouse (bottom). Arrows indicate tumor cells; filled arrowheads indicate non-neoplastic basal-layer epithelial cells; open arrowhead indicates non-neoplastic luminal-layer epithelial cells. Scale bars, 25 μm. (b) CK5/6/14 IHC staining scores for 13 Pten^+/− mammary tumors. The maximal score for CK5/6 or CK14 staining (Methods) is plotted for each tumor compartment; a score of 1+ (horizontal broken line) is defined as basal-like. 0, tumor compartment present but no staining; np, respective component not present in tumor. See also Supplementary Table 1 online.

Figure 2

Immunohistochemical analysis of PTEN and CK5/14 basal-like phenotype markers in 297 non-hereditary human breast carcinomas. (a) Representative PTEN and CK5/14 IHC in two non-hereditary breast tumors. Top, a PTEN^IHC-positive and CK5/14-negative example; bottom, a PTEN^IHC-loss and CK5/14-positive example. Arrows indicate tumor cells; arrowheads indicate non-neoplastic cells. Asterix denotes the non-neoplastic CK5/14-positive basal epithelial layer enclosing a portion of in situ breast carcinoma, shown here as an example of an internal positive control. Scale bars, 25 μm. (b) Correlation analysis between PTEN loss and the CK5/14-positive basal-like subtype among all tumors (left) and in the ER-negative group (right). *P = 0.0122; **P = 4 × 10⁻⁹.

Figure 3

PTEN status in human BRCA1-mutant hereditary breast cancers. (a–d) PTEN IHC results for three representative PTEN^IHC-null BRCA1-associated hereditary breast tumor specimens (a–c) and one non-hereditary PTEN^IHC-null breast tumor with a PTEN fs108X mutation (d). Scale bars, 25 μm. (e) Fraction of PTEN^IHC-loss (filled) and PTEN^IHC-null (open) tumors among 34 BRCA1-associated hereditary breast tumors. (f) Analysis of PTEN nucleotide sequence mutations in PTEN^IHC-null non-hereditary breast cancers versus PTEN^IHC-null BRCA1-mutant hereditary breast cancers.

Figure 4

Gross PTEN mutations in BRCA1-mutant human cell lines and xenografts. (a,b) HD-aCGH analyses of two model BRCA1-mutant breast cancer cell lines. The location of PTEN exons 1–9 on chromosome 10q23.31 are indicated by blue bars. The log₂ ratios for probes localized in the PTEN gene are plotted in red; those for all other probes are plotted in black (gaps with no probes are present in several regions that contain highly repetitive sequence elements such as intron 2). Arrows indicate regions of focal CNI. Prominent broken horizontal lines are placed at log₂ ratios of 0.3 and −0.3. (c) Two-color FISH assay. The PTEN gene and exons are indicated in blue. Centromeric BAC probes were labeled with spectrum orange–dUTP (red); telomeric BAC probes were labeled with spectrum green–dUTP (green). (d) FISH results for the BRCA1-mutant breast cancer cell lines in a,b. Inset, representative FISH results for one chromosome of a normal lymphocyte metaphase spread. (e) Analysis of BRCA1 mutation (mt) and GPM among 13 breast cancer xenografts and cell lines with any type of PTEN mutation. The basal-like status of these cell lines and xenografts is given in Table 1.

Figure 5

Gross PTEN mutations in five BRCA1-mutant breast tumor biopsies in vivo. (a–e) Analysis of PTEN^IHC-null BRCA1-associated breast tumors by HD-aCGH, revealing PTEN homozygous deletions (a,e), macro-CNI (b) and micro-CNIs (c,d). Probes and the PTEN locus are plotted as in Figure 4a,b. Green bars indicate neighboring genes.