RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/− background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357–68. ©2017 AACR.

Ras genes are some of the most frequently mutated in human cancer. According to the catalog of somatic mutations in cancer (COSMIC v77; ref. 1), which represents the most comprehensive database on human cancer mutations currently available, around 20% of the analyzed tumors have activating mutations in any of the 3 Ras genes, with a maximum of 57% incidence for KRAS in pancreatic tumors. Ras signaling may also be activated by other means, notably by inactivation of molecules that limit Ras activity, such as Ras GTPase-activating proteins (RasGAP). Ras proteins are molecular switches that cycle between an inactive GDP-bound form and an active GTP-bound form. They signal through several effector pathways, and RasGAPs stimulate the weak intrinsic GTPase activity of normal (but not mutant) Ras proteins, effectively acting as suppressors of Ras function. Interestingly, less than 1% of the near 10,000 breast cancer samples sequenced in COSMIC have mutations in Ras genes; however, the Ras pathway is significantly activated in a number of breast tumors, in particular of the triple-negative type (2).

Breast cancer is by far the most frequent tumor type in the female population worldwide (25% of all new cases in 2012), and although its mortality rate is not the highest, it is the most frequent cause of cancer death in women (14.7% of all deaths in 2012; ref. 3). Triple-negative breast cancers [TNBC; which are negative for HER2, estrogen receptor (ER)α, and the progesterone receptor] constitute a heterogeneous group of tumors which very often exhibit a basal-like signature (4). Although they represent approximately 15% of all breast cancers, they account for a much higher mortality: they are tumors with a poor prognostic, mainly due to the lack of specific targets for treatment. These triple-negative tumors are enriched for mutations in TP53. Indeed, TP53 is mutated in 36% of all breast cancers, but this proportion increases to 86% in PAM50 basal-like tumors (5). TNBC tumors also bear a highly variable number of genomic alterations, including the presence of a large number of somatic mutations and copy number aberrations (6), suggesting that (i) combinations of mutations interact to drive tumor formation and (ii) most of the mutations found are “passengers,” not related to development of the tumor.

Transposon-induced mutagenesis is an excellent method to identify cancer driver genes. For example, when mobilized by Sleeping Beauty (SB) transposase, the mutagenic T2Onc2 transposon integrates throughout the genome, and cells with insertions in a gene or combination of genes that favor tumorigenesis are positively selected. Using this method, genes involved in multiple tumor types have been identified (reviewed in ref. 7). This analysis has not yet been performed for breast cancer. In this article, we report on generation of transposon-bearing mice that develop mammary tumors, identification of RasGAP genes as the major target of transposon-mediated mutagenesis, and identification of RASA1 hemizygous deletion in human TNBC. Together with the identification of hemizygous deletions in NF1 and RASAL2 in breast cancer (8, 9), our results highlight the importance of RasGAP gene hemizygosity as a driver of elevated Ras signaling and breast cancer in humans.

Transgenic mice generation

Double K5-SB11/T2Onc2 (named as SB/T2) transgenic mice, containing both the SB11 and T2Onc2 transgenes, were generated by interbreeding of heterozygous SB and T2 mice, as described (10). Heterozygous Trp53+/− transgenic mice were obtained by mating conditional mutants Trp53F2-10 mice (11) that carried floxed Trp53 alleles in a FVB genetic background, to K5-Cre transgenic female mice; in this breeding, the maternal K5-Cre transgene causes general cre-mediated deletion in early embryos (12), generating a Trp53-null allele. Triple transgenic K5-SB11/T2Onc2/Trp53+/− mice (SB/T2/p53+/−) were generated by mating heterozygous SB/T2 to heterozygous Trp53+/− mice. In these crosses, different genetic combinations lacking the transgenes T2Onc2 (SB/p53+/−), the transposase SB11 (T2/p53+/−), or both SB and T2Onc2 transgenes (p53 +/−) were also generated and used as control mice for the experiments. Animals were typed by PCR. All procedures involving mice were approved by the Institutional Organism for Animal Welfare (OEBA) and according to the European, Spanish, and local regulations.

Tumor collection

Tumors were excised from the mice and were fractionated. One part was included in formaldehyde for subsequent paraffin embedding for immunohistochemical analysis, and the rest was snap-frozen in liquid N2 for protein/nucleic acid extraction.

Human tumors

Samples and data from patients included in this study were provided by the Biobanco i+12 in the Hospital 12 de Octubre integrated in the Spanish Hospital Biobanks Network (RetBioH; www.redbiobancos.es) following standard operation procedures with appropriate approval of the Ethical and Scientific Commitees. Patient's clinical data are shown in Supplementary Table SII.

Cell lines

MCF10A cells were purchased from ATCC. T47D, HCC1954, and BT474 breast carcinoma cell lines were obtained from Dr. Cristina Sánchez (University Complutense, Madrid, Spain), who purchased them from ATCC and provided vials after first division. All cells lines were authenticated by ATCC by isoenzyme analysis and STR profiles and were kept in culture for less than six months. Cells were periodically analyzed for contamination with a mycoplasma detection kit. All cells were passaged and cultured using the media and recommendations provided by ATCC. MCF10A-p53R175H/shRasa1 cells were grown in plates coated with 0.1% gelatin due to their limited adhesion. For experimentation, all other types of MCF10A cells were also grown in gelatin-coated plates to avoid substrate effect.

Immunohistochemistry

Mouse tissues were fixed in 4% buffered formalin and embedded in paraffin. Five-micrometer-thick sections were used for hematoxylin and eosin (H&E) staining or immunohistochemical preparations. Most of the tumors originated in the mice were fixed and classified by morphology after sectioning and staining with H&E. For immunohistochemistry, slides were deparaffinized and antigen retrieval was performed in microwave with citric acid buffer (pH 6) for mouse tissues and in pressure cooker with Dako Target Retrieval Solution (pH 9; Dako, Agilent Technologies) for human tissues. Endogenous peroxidase was inhibited with hydrogen peroxide (0.3%) in methanol. Unspecific epitopes were blocked with PBS containing 10% of horse serum. The antibodies used in the immunohistochemical analysis were against keratin K5 (PRB-160P; 1:1,000; Covance); RASA1 (sc-63; 1:100), NF1 (sc-67; 1:400), ERα (sc-542; 1:200; Santa Cruz Biotechnology), P53 (M7001; 1:50, Dako), and SB transposase (MAB2798; 1:50; R&D Systems). Signal was amplified with avidin/biotin technology (VECTASTAIN Elite ABC system) and was visualized with DAB peroxidase substrate kit (Vector Laboratories).

Statistical analysis

For Western blotting, bands were quantified by Quantity One software and normalized with respect to β-actin, total ERK, or total AKT expression. P values were determined by using the unpaired, 2-tailed Student t test. For immunohistochemistry, samples were assessed in a blind manner by 2 experts. RASA1 and NF1 staining was quantified from 0 to 3 comparing with normal tissue. When staining was heterogeneous within tumors, 5 different areas were assessed and an average value was calculated. P values were determined by using the χ2 test. In both cases, P < 0.05 was considered significant and data are expressed as mean ± SEM.

SB/T2/p53+/− mice develop spontaneous mammary tumors

To generate tumors, double transgenic mice bearing both a concatemer of T2Onc2 mutagenic transposons and the SB11 transposase under control of the keratin K5 promoter (10) were mated with heterozygous Trp53+/− mice (Fig. 1A). As expected, these Trp53+/− animals are prone to the development of lymphoma. Also, as K5 is expressed in the skin, they also develop skin tumors (10). Interestingly, SB/T2/p53+/− triple transgenic mice preferentially developed mammary tumors (Fig. 1B). This occurs at a higher frequency (41% vs. 19%) and shorter latency (49 vs. 60 weeks) than in control Trp53+/− mice lacking transposition (Fig. 1C). Keratin K5 is expressed in the myoepithelial layer of the mammary gland, and SB11 transposase was detected in this layer of transgenic animals (Fig. 1D). Likewise, mammary tumors showed concurrent expression of K5 and SB11 transposase (Fig. 1D). Thus, transposition of T2Onc2 in K5-expressing cells facilitates development of mammary gland tumors in a Trp53+/− background.

Figure 1.

Generation of mammary tumors in mice by transposon mobilization. A, Schematics of the animal crossings performed to generate the mice. B, Anatomical localization of tumors arisen in female mice for nontransposition (p53+/−; n = 71) and transposition (SB/T2/p53+/−; n = 66) mice. “Others” include thymus, uterus, liver, and ovary. C, Kaplan–Meier analysis of mammary gland tumor-free survival in SB/T2/p53+/− (n = 66) and in control p53+/− mice (n = 71). D, Expression of K5 (top row) and SB11 transposase (bottom row) in normal mammary gland (left column) and SB/T2/p53+/− mammary tumor tissue (middle column) and lack of expression of SB11 in p53-only tumors (right column). All images are at the same magnification. E, Table indicating the histologic adscription of the 32 SB/T2/p53+/− murine mammary tumors. F, Examples illustrating the most common mammary tumor types obtained. Left column, acinar carcinoma. Middle column, solid carcinoma. Right column, papillary carcinoma. Magnifications are indicated. G, Examples of ER-negative (left) and ER-positive (middle and right) tumors. Inset shows an ER-positive, normal mammary epithelia in the same histologic preparation than the ER-negative tumor (bar, 50 μm).

Figure 1.

Generation of mammary tumors in mice by transposon mobilization. A, Schematics of the animal crossings performed to generate the mice. B, Anatomical localization of tumors arisen in female mice for nontransposition (p53+/−; n = 71) and transposition (SB/T2/p53+/−; n = 66) mice. “Others” include thymus, uterus, liver, and ovary. C, Kaplan–Meier analysis of mammary gland tumor-free survival in SB/T2/p53+/− (n = 66) and in control p53+/− mice (n = 71). D, Expression of K5 (top row) and SB11 transposase (bottom row) in normal mammary gland (left column) and SB/T2/p53+/− mammary tumor tissue (middle column) and lack of expression of SB11 in p53-only tumors (right column). All images are at the same magnification. E, Table indicating the histologic adscription of the 32 SB/T2/p53+/− murine mammary tumors. F, Examples illustrating the most common mammary tumor types obtained. Left column, acinar carcinoma. Middle column, solid carcinoma. Right column, papillary carcinoma. Magnifications are indicated. G, Examples of ER-negative (left) and ER-positive (middle and right) tumors. Inset shows an ER-positive, normal mammary epithelia in the same histologic preparation than the ER-negative tumor (bar, 50 μm).

Close modal

We collected 33 mammary gland tumors from 26 SB/T2/p53+/− females. Classification of these tumors based on histology revealed that most lesions were either acinar carcinoma or adenocarcinoma (∼60%). Mixed pattern acinar-solid carcinoma (∼19%) and papillary carcinoma or cystadenocarcinoma (6%) were also frequently seen. Other subtypes, such as solid, tubular, and spindle cell carcinoma occurred in a small number of animals (Fig. 1E and F). Most (84%) tumors generated from SB/T2/p53+/− mice were ER-positive (Fig. 1G). It is interesting to note that mammary tumors generated by KRas activation are mainly ER-positive (13).

RasGAPs are the most frequently mutated genes in transposon-induced mammary tumors

DNA from these 33 mammary tumors was extracted, and sequences flanking the transposon insertion sites were amplified by PCR and then subjected to next-generation Illumina sequencing to identify all transposon integrations. Scrutiny of these integrations using gene-centric Common Insertion Site (gCIS) analysis (14) resulted in identification of 16 CIS, which represent specific sites in the genome that accumulate transposon insertions in independent tumors at a rate significantly higher than expected by chance, and are therefore likely the result of positive selection during tumor development (Table 1). Of the 33 tumors, 26 tumors had transposon insertions in at least one of these CIS. Most of the genes found in these CIS are related to cancer, and the human counterparts of 5 of them (PTEN, NF1, NFIB, SMAD3, and GATA3) are already defined as cancer genes in the Cancer Genes Census (cancer.sanger.ac.uk/census). Moreover, several of the identified genes have already been implicated in breast cancer. For instance, PTEN and GATA3 are two of the most frequently mutated genes in human breast cancer (6, 15–17), and NFIB, which is disrupted by SB in 40% (13 of 33) of tumors undergoes translocations in breast cancers (18). Gene Ontology analysis showed selection for insertion into genes related to cell matrix adhesion, oncogenesis, apoptosis, cell migration, and angiogenesis (Table 2).

Table 1.

Candidate cancer genes present in common insertion sites from murine mammary tumors

Gene symbolGene namePNo. of tumors% Disrupt
Nfib Nuclear factor I/B 13 80 
Nf1 Neurofibromatosis 1 13 100 
Rasa1 RAS p21 protein activator 1 70 
Gata3 GATA-binding protein 3 
Ripk4 Receptor-interacting serine-threonine kinase 4 100 
Ube2f Ubiquitin-conjugating enzyme E2F 8.83E-65 100 
Pten Phosphatase and tensin homolog 6.71E-57 80 
Cul3 Cullin 3 1.49E-46 75 
Smad3 MAD homolog 3 (Drosophila5.37E-37 67 
Zfr Zinc finger RNA-binding protein 1.85E-31 67 
Tead1 TEA domain family member 1 6.53E-27 
Flnb Filamin, beta 1.05E-24 
Pum1 Pumilio 1 (Drosophila6.60E-24 67 
Ankrd11 Ankyrin repeat domain 11 3.15E-20 25 
Ankrd28 Ankyrin repeat domain 28 4.26E-17 100 
Fam135b Family with sequence similarity 135, member B 9.87E-10 
Gene symbolGene namePNo. of tumors% Disrupt
Nfib Nuclear factor I/B 13 80 
Nf1 Neurofibromatosis 1 13 100 
Rasa1 RAS p21 protein activator 1 70 
Gata3 GATA-binding protein 3 
Ripk4 Receptor-interacting serine-threonine kinase 4 100 
Ube2f Ubiquitin-conjugating enzyme E2F 8.83E-65 100 
Pten Phosphatase and tensin homolog 6.71E-57 80 
Cul3 Cullin 3 1.49E-46 75 
Smad3 MAD homolog 3 (Drosophila5.37E-37 67 
Zfr Zinc finger RNA-binding protein 1.85E-31 67 
Tead1 TEA domain family member 1 6.53E-27 
Flnb Filamin, beta 1.05E-24 
Pum1 Pumilio 1 (Drosophila6.60E-24 67 
Ankrd11 Ankyrin repeat domain 11 3.15E-20 25 
Ankrd28 Ankyrin repeat domain 28 4.26E-17 100 
Fam135b Family with sequence similarity 135, member B 9.87E-10 

NOTE: Only genes with transposon insertions in at least three different tumors are shown.

Table 2.

Gene Ontology analysis of the 16 candidate cancer genes using DAVID-NIH revealed enrichment in several canonical signaling pathways and processes

CategoryTermCountPGenes
GOTERM_BP_FAT Regulation of cell–matrix adhesion 1.54E-06 NF1, SMAD3, PTEN, RASA1 
GOTERM_BP_FAT Regulation of apoptosis 0.004 CUL3, NF1, SMAD3, PTEN, RASA1 
PANTHER_BP_ALL Oncogenesis 0.006 CUL3, NF1, SMAD3, PTEN 
GOTERM_BP_FAT Regulation of cell migration 0.009 NF1, SMAD3, PTEN 
GOTERM_BP_FAT Blood vessel morphogenesis 0.014 NF1, PTEN, RASA1 
GOTERM_BP_FAT Posttranscriptional regulation of gene expression 0.014 PUM1, SMAD3, PTEN 
GOTERM_CC_FAT Nuclear lumen 0.028 ANKRD28, TEAD1, SMAD3, ZFR, NFIB 
SP_PIR_KEYWORDS Nucleus 0.032 CUL3, ANKRD28, GATA3, ANKRD11, TEAD1, SMAD3, ZFR, NFIB 
KEGG_PATHWAY MAPK signaling pathway 0.036 NF1, FLNB, RASA1 
GOTERM_BP_FAT Positive regulation of transcription from RNA polymerase II promoter 0.041 TEAD1, SMAD3, NFIB 
CategoryTermCountPGenes
GOTERM_BP_FAT Regulation of cell–matrix adhesion 1.54E-06 NF1, SMAD3, PTEN, RASA1 
GOTERM_BP_FAT Regulation of apoptosis 0.004 CUL3, NF1, SMAD3, PTEN, RASA1 
PANTHER_BP_ALL Oncogenesis 0.006 CUL3, NF1, SMAD3, PTEN 
GOTERM_BP_FAT Regulation of cell migration 0.009 NF1, SMAD3, PTEN 
GOTERM_BP_FAT Blood vessel morphogenesis 0.014 NF1, PTEN, RASA1 
GOTERM_BP_FAT Posttranscriptional regulation of gene expression 0.014 PUM1, SMAD3, PTEN 
GOTERM_CC_FAT Nuclear lumen 0.028 ANKRD28, TEAD1, SMAD3, ZFR, NFIB 
SP_PIR_KEYWORDS Nucleus 0.032 CUL3, ANKRD28, GATA3, ANKRD11, TEAD1, SMAD3, ZFR, NFIB 
KEGG_PATHWAY MAPK signaling pathway 0.036 NF1, FLNB, RASA1 
GOTERM_BP_FAT Positive regulation of transcription from RNA polymerase II promoter 0.041 TEAD1, SMAD3, NFIB 

In our murine tumors, the RasGAPs Nf1 and Rasa1 were among the most frequently mutated genes. Of note, 18 of the 33 analyzed tumors (54%) had transposon insertions in either one or both of the RasGAP genes Nf1 and Rasa1, strongly suggesting activation of the Ras pathway in generation of these tumors. The position and orientation of T2Onc2 insertions in each target gene hints at the type of alteration produced (19); when orientation of the transposon was analyzed, we found that 100% of the transposon insertions in Nf1 and 70% of insertions in Rasa1 were in the opposite orientation, suggesting that gene inactivation has occurred. Both in Nf1 and Rasa1, all insertions were located before or into the functional RASGAP domain (Fig. 2A), effectively disrupting the function of these proteins and resulting in the activation of Ras signaling.

Figure 2.

Inactivation of Rasa1 and Nf1 genes by transposon insertions. A, Gene structure of the murine Nf1 and Rasa1 genes showing the localization of the transposon insertions. Arrows, transcription direction. Arrowheads, the orientation of the transposon for each detected insertion. The functional domains of each protein are represented below, and the exons encompassing the RasGAP domain are indicated. B, Protein expression in mammary tumors induced by transposon insertion. Samples are grouped according to insertions in Rasa1, Nf1, both Nf1 and Rasa1, or none. Insertions in other genes are not considered. C, Relative quantification of the expression of RASA1 protein in tumors without (left) and with (right) transposon insertion in the Rasa1 gene. D, Relative quantification of the expression of NF1 protein in tumors without (left) and with (right) transposon insertion in the Nf1 gene. E and F, Relative quantification of the phosphorylation of ERK in Thr202/Tyr204 (E) and AKT in Ser473 (F) in tumors without (left) and with (right) transposon insertion in the Rasa1 gene. C and D were normalized using β-actin signal, E and F were normalized using total ERK and AKT signals, respectively.

Figure 2.

Inactivation of Rasa1 and Nf1 genes by transposon insertions. A, Gene structure of the murine Nf1 and Rasa1 genes showing the localization of the transposon insertions. Arrows, transcription direction. Arrowheads, the orientation of the transposon for each detected insertion. The functional domains of each protein are represented below, and the exons encompassing the RasGAP domain are indicated. B, Protein expression in mammary tumors induced by transposon insertion. Samples are grouped according to insertions in Rasa1, Nf1, both Nf1 and Rasa1, or none. Insertions in other genes are not considered. C, Relative quantification of the expression of RASA1 protein in tumors without (left) and with (right) transposon insertion in the Rasa1 gene. D, Relative quantification of the expression of NF1 protein in tumors without (left) and with (right) transposon insertion in the Nf1 gene. E and F, Relative quantification of the phosphorylation of ERK in Thr202/Tyr204 (E) and AKT in Ser473 (F) in tumors without (left) and with (right) transposon insertion in the Rasa1 gene. C and D were normalized using β-actin signal, E and F were normalized using total ERK and AKT signals, respectively.

Close modal

Mouse mammary tumors show reduced RasGAP expression

To validate these results, we analyzed a number of tumors by Western blotting (Fig. 2B). Despite the histologic heterogeneity of these lesions, there was an excellent correlation between presence of transposon insertions in Rasa1 and the amount of detected RASA1 protein (Fig. 2C, t test: P < 0.0001). In the case of Nf1, all tumors with insertions showed reduced expression of the protein by Western blotting. Interestingly, some tumors showed reduced NF1 expression, even in the absence of SB-mediated disruption of Nf1 (Fig. 2B and D), suggesting inactivation of Nf1 expression by other mechanisms independent of transposon insertion.

Because disruption of Rasa1 or Nf1 should result in enhanced Ras signaling, we checked for activation of ERK (a known Ras effector) and AKT (an effector of Ras and PI3K) in these tumors. ERK activity (measured as pERK accumulation) was present in 85% (6 of 7) of tumors with Rasa1 and/or Nf1 insertions, as compared with 38% (3 of 8) of tumors without insertions in either gene (Fig. 2B and E). A correlation was also detected between activation of AKT and diminution of RASA1 (Fig. 2B and F), suggesting activation of the Ras pathway in tumors with Rasa1 or Nf1 insertions. These partial correlations may well be associated with ERK activation through oncogenic pathways distinct from elevated Ras signaling associated with disruption of Rasa1 or Nf1. Interestingly, in some tumors with high pERK but lacking insertions in Rasa1 or Nf1, we found insertions (not included in our gCIS list, as they were found in less than three tumors) in other Ras-related genes, such as Rasgrf1 (see Supplementary Table SI). We did not find any correlation between insertions in Rasa1 or Nf1 and expression of EGFR, ERBB2, or ESR1 (data not shown).

RASA1 and NF1 genes are frequently lost in TNBC

While NF1 is a well-known tumor suppressor gene, and NF1 deletions and mutations have been reported in breast cancer (8, 17), RASA1 has not been previously considered as a breast cancer gene. We used immunohistochemistry to assess expression of RASA1 in 32 human breast tumors. Intensity of RASA1 staining in the tumor area was compared with intensity in the normal mammary tissue and quantified in a blind manner using an arbitrary scale from 0 to 3 (Fig. 3A and Supplementary Table SIII). Expression of RASA1 was reduced in basal or triple-negative tumors versus other types (P < 0.05, Fig. 3C and Supplementary Tables SIII and SIV). This trend was even more evident when ER-negative tumors were compared with ER-positive tumors (P < 0.01, Fig. 3D). For NF1 staining, a trend toward lower expression in ER-negative tumors was also seen (Supplementary Tables SIII and SIV). TP53 is frequently mutated in TNBC, and TP53 staining of these tumors allowed us to identify a significant correlation between low or absent RASA1 staining and mutation in TP53 (P < 0.05, Fig. 3A and B and Supplementary Tables SIII and SIV). Low or absent RASA1 staining also correlated with high Ki67 staining (P < 0.05) and was associated with high-grade (G3) tumors (P < 0.01) and stage III tumors (P < 0.05; Supplementary Table SIV).

Figure 3.

Immunohistochemical staining of human breast tumors using RASA1 and TP53 antibodies. A, Examples of negative, low, moderate, and intense staining (indicated by 0, 1, 2, and 3, respectively) for RASA1 in several human breast tumors. Insets represent different fields in the same slide. N, normal, nontumorigenic area; T, tumor. Scale bar, 100 μm. Tumor numbers are indicated in the bottom right corner. B, TP53 staining in the same human breast tumors as in A. C, Association of RASA1 relative staining with triple-negative subtype. Tumors classified as triple-negative have lower staining of RASA1 than tumors from the other types. D, Association of RASA1 relative staining with ER status. Tumors with ER-negative status present lower staining of RASA1 than tumors with ER-positive status.

Figure 3.

Immunohistochemical staining of human breast tumors using RASA1 and TP53 antibodies. A, Examples of negative, low, moderate, and intense staining (indicated by 0, 1, 2, and 3, respectively) for RASA1 in several human breast tumors. Insets represent different fields in the same slide. N, normal, nontumorigenic area; T, tumor. Scale bar, 100 μm. Tumor numbers are indicated in the bottom right corner. B, TP53 staining in the same human breast tumors as in A. C, Association of RASA1 relative staining with triple-negative subtype. Tumors classified as triple-negative have lower staining of RASA1 than tumors from the other types. D, Association of RASA1 relative staining with ER status. Tumors with ER-negative status present lower staining of RASA1 than tumors with ER-positive status.

Close modal

RASA1 mutations are unusual. According to COSMIC v77 (cancer.sanger.ac.uk), as of May 17, 2016, RASA1 mutations have been found in only 256 of 26,161 tumor samples (1%). RASA1 mutations in breast are even less frequent, with only 8 cases found out of 1,571 samples sequenced. To expand our results, and given that in our mouse tumors T2Onc2 does not cause point mutations but rather disrupts one copy of Rasa1, yielding tumors with low but detectable levels of RASA1 protein, we searched for RASA1 hemizygous loss using the cBio Cancer Genomics Portal (www.cbioportal.org; refs. 17, 20). Analysis of breast cancer samples from The Cancer Genome Atlas (5) confirmed that 27% of breast tumors (289 of 1,080) showed RASA1 gene loss, most of which represented low-level or hemizygous loss (Fig. 4A and Supplementary Table SV). This percentage increased to 84% (90 of 107) when only PAM50 basal tumors were considered. PAM50 HER2-enriched tumors also had allelic losses in 49% (25 of 51) of cases, whereas losses in PAM50-luminal A and B tumors were much less frequent. In general, RASA1 allelic loss was more common in ER-negative (70%) than in ER-positive tumors (14%; P < 0.0001, Fisher exact test, Fig. 4A and Supplementary Table SV). As expected, there was a clear correlation between copy number and expression of RASA1 mRNA (Fig. 4B). Interestingly, we also detected a correlation between copy number of RASA1 and ESR1 expression, both at mRNA and protein levels (Fig. 4C and D), and also between high methylation of ESR1 and hemizygous deletion of RASA1 (Fig. 4E). A similar correlation was found between RASA1 copy loss and PGR1 (but not ERBB2) expression (data not shown). In addition, 75% of PAM50 basal-like tumors showed coincident allelic loss at RASA1 and mutation of TP53 (vs. 21% when all tumors are considered). Enrichment analysis on 974 tumors also revealed a significant correlation between RASA1 copy number loss and mutation of TP53 (P = 3,50E-60, Fisher exact test, Supplementary Table SVI). Similarly, heterozygous deletions in NF1 were more prominent in PAM50 basal (62%, 66 of 107) and HER2-enriched (59%, 30 of 51) tumors as compared with breast cancer when considered as a whole (31%, 334 of 1,080; Supplementary Table SVII). While allelic loss of NF1 in PAM50-basal and ER-negative tumors was statistically significant, no correlation could be found with ESR1 expression (not shown).

Figure 4.

Analysis of RASA1 and NF1 alterations in breast cancer databases. A, Analysis of the GISTIC putative copy number alterations for RASA1 in the The Cancer Genome Atlas breast invasive carcinoma cohort (5). For each tumor subtype, the percentage of each alteration is shown in accumulative bars. IDC, invasive ductal carcinoma. B–E, Analysis of the relation between putative copy number alterations (GISTIC) of the RASA1 gene in cBioportal. B, Levels of RNA expression of RASA1 as measured by RNAseq. C, RNA expression of ESR1. D, Protein expression of ESR1. E, Methylation status of the ESR1 gene. For each column, the number of samples is indicated above. F–I, Analysis of the mRNA expression level of RASA1 (F) and NF1 (G) in PAM50-classified breast tumors from the GOBO database. For each column, the number of samples is indicated above. Analysis of the mRNA expression level of RASA1 (H) and NF1 (I) in breast tumors from the GOBO database, classified according to the ER status.

Figure 4.

Analysis of RASA1 and NF1 alterations in breast cancer databases. A, Analysis of the GISTIC putative copy number alterations for RASA1 in the The Cancer Genome Atlas breast invasive carcinoma cohort (5). For each tumor subtype, the percentage of each alteration is shown in accumulative bars. IDC, invasive ductal carcinoma. B–E, Analysis of the relation between putative copy number alterations (GISTIC) of the RASA1 gene in cBioportal. B, Levels of RNA expression of RASA1 as measured by RNAseq. C, RNA expression of ESR1. D, Protein expression of ESR1. E, Methylation status of the ESR1 gene. For each column, the number of samples is indicated above. F–I, Analysis of the mRNA expression level of RASA1 (F) and NF1 (G) in PAM50-classified breast tumors from the GOBO database. For each column, the number of samples is indicated above. Analysis of the mRNA expression level of RASA1 (H) and NF1 (I) in breast tumors from the GOBO database, classified according to the ER status.

Close modal

We also performed expression meta-analysis using the GOBO online database (http://co.bmc.lu.se/gobo; ref. 21), which includes more than 1,800 patients from 10 breast cancer studies. Again, in agreement with our results, expression of NF1 and RASA1 was significantly reduced in ER-negative tumors, and in particular in basal tumors (P < 0.00001; Fig. 4F–I). Moreover, low expression of NF1 and, in particular of RASA1, correlated with high-grade (G3) tumors (P < 0.0001, data not shown).

Concomitant TP53 mutation and inactivation of RASA1 malignize human breast cells

To validate in vitro these observations, we permanently inactivated RASA1 by lentiviral-mediated shRNA interference in several breast cell lines. In T-47D, BT-474, and HCC-1954 cells, inactivation of RASA1 with 2 different shRNAs (sh1 and sh2) resulted in activation of ERK and/or AKT, confirming that partial deletion of RASA1 is enough to activate the Ras pathway (Fig. 5A). Interestingly, in SUM159 or MDA-MB-231 cells, which already have constitutive activation of the Ras pathway, this effect was not seen (data not shown). As all the breast cancer cell lines tested had mutations in TP53, and most triple-negative tumors present concomitant deletion of RASA1 and mutations in the TP53 gene, we next investigated the relation of TP53 and RASA1 using MCF10A cells, a TP53-wild type immortalized nonmalignant mammary epithelial cell line that is often used as a model of normal human mammary gland. In these cells, interference using sh1 almost completely suppressed the expression of RASA1, whereas sh2 reduced its expression to around 50% (Fig. 5B). For both shRNAs, phosphorylation of ERK was weak, as also was upon introduction of a R175H TP53 mutation (the most frequent mutation in breast tumors). However, combination of p53R175H expression and RASA1 knockdown caused a strong increase in the activation of the Ras pathway, as seen by strong phosphorylations of ERK and AKT (Fig. 5B). Cells in which only RASA1 had been inactivated did not show signs of increased malignancy, but co-occurrence of TP53 mutation and RASA1 inactivation resulted in increased malignancy, as indicated by several endpoints reminiscent of epithelial–mesenchymal transition (EMT): cells abandoned their cobblestone-like appearance, lost adherence, and acquired an elongated, fibroblastoid aspect (Fig. 5C). Cytometric analysis confirmed that both types of p53R175H/shRASA1 cells strongly reduced EpCAM and CD49f expression (Fig. 5D), which is indicative of a transition from an epithelial to a mesenchymal phenotype (22). Moreover, p53R175H/shRASA1 cells lost E-cadherin and upregulated N-cadherin expression (Fig. 5B and E). Finally, double p53R175H/shRASA1 cells also acquired invasive properties, as seen by invasion chamber assays using Matrigel (Fig. 5F). Interestingly, the two interfering shRNAs exerted similar but not identical effects on MCF10A cells in the context of the p53 mutation: almost total inactivation of RASA1 by sh1 resulted in almost total disappearance of EpCAM and E-cadherin, whereas partial inactivation of RASA1 by sh2 resulted in partial EpCAM loss and partial inactivation of E-cadherin, with the remaining E-cadherin–expressing cells exhibiting a spiky, discontinuous pattern of E-cadherin, instead of the continuous staining seen in the control cells (Fig. 5E). On the contrary, both total and partial inactivation of RASA1 coupled with p53R175H resulted in activation of the Ras pathway, induction of N-cadherin and invasivity to similar extents, indicating that loss of RASA1, even if it is not complete, results in increased malignancy of human mammary cells when in coexistence with mutated TP53.

Figure 5.

Effects of the inactivation of RASA1 RNA in human mammary cell lines. Two different shRNAs for RASA1 (sh1 and sh2) and a nonspecific shRNA (n-s) were used. A, Western blot analysis showing downregulation of RASA1 protein and phosphorylation of ERK1/2 (Thr202/Tyr204) and AKT (Ser473) by RASA1 shRNAs in HCC-1954, BT-474, and T-47D cell lines. B, Western blot analysis showing the effect of RASA1 inactivation in MCF10A cells. Both sh1 and sh2 shRNAs reduced RASA1 expression to different extents. Cells infected with n-s, sh1, or sh2 constructs had no significant changes, whereas co-introduction of these shRNAs with p53R175H caused activation of ERK and AKT, downregulation of E-cadherin, and upregulation of N-cadherin. C, Photographs of exponentially growing MCF10A cells. Cells expressing nonspecific (not shown), sh1, or sh2 shRNAs did not show any difference with controls. Cells expressing p53R175H had a more rounded appearance; cells co-expressing p53R175H and either sh1 or sh2 RASA1 shRNAs adopted a fibroblastoid appearance and lost adhesion to the substrate. Bar, 100 μm. D, Flow cytometric analysis for EpCAM and CD49f (integrin α6) for each cell type. MCF10A control cells, as well as sh1, sh2, and nonspecific sh (not shown) present almost exclusively an EpCAM+/CD49fhi phenotype. Introduction of p53R175H partially shifts cells toward an EpCAM/CD49fmed/low phenotype, whereas co-introduction of p53R175H and sh1 or sh2 resulted in a majority of EpCAM/CD49fmed/low cells. Data are shown as mean ± SEM. E, Expression of E-cadherin or N-cadherin in the different MCF10A cells was detected by immunofluorescence. Cells infected with nonspecific, sh1, or sh2 RASA1 shRNAs (not shown) behaved as control, expressing only E-cadherin. Cells expressing p53R175H slightly diminished E-cadherin expression and showed small patches of N-cadherin expression; cells with both p53R175H and sh1 or sh2 greatly increased N-cadherin expression and reduced E-cadherin expression, in particular p53R175H/sh2 cells, which totally reversed the control phenotype. Bar, 100 μm. F, Invasion assays on Matrigel. Graphic shows invasion indexes normalized to control cells.

Figure 5.

Effects of the inactivation of RASA1 RNA in human mammary cell lines. Two different shRNAs for RASA1 (sh1 and sh2) and a nonspecific shRNA (n-s) were used. A, Western blot analysis showing downregulation of RASA1 protein and phosphorylation of ERK1/2 (Thr202/Tyr204) and AKT (Ser473) by RASA1 shRNAs in HCC-1954, BT-474, and T-47D cell lines. B, Western blot analysis showing the effect of RASA1 inactivation in MCF10A cells. Both sh1 and sh2 shRNAs reduced RASA1 expression to different extents. Cells infected with n-s, sh1, or sh2 constructs had no significant changes, whereas co-introduction of these shRNAs with p53R175H caused activation of ERK and AKT, downregulation of E-cadherin, and upregulation of N-cadherin. C, Photographs of exponentially growing MCF10A cells. Cells expressing nonspecific (not shown), sh1, or sh2 shRNAs did not show any difference with controls. Cells expressing p53R175H had a more rounded appearance; cells co-expressing p53R175H and either sh1 or sh2 RASA1 shRNAs adopted a fibroblastoid appearance and lost adhesion to the substrate. Bar, 100 μm. D, Flow cytometric analysis for EpCAM and CD49f (integrin α6) for each cell type. MCF10A control cells, as well as sh1, sh2, and nonspecific sh (not shown) present almost exclusively an EpCAM+/CD49fhi phenotype. Introduction of p53R175H partially shifts cells toward an EpCAM/CD49fmed/low phenotype, whereas co-introduction of p53R175H and sh1 or sh2 resulted in a majority of EpCAM/CD49fmed/low cells. Data are shown as mean ± SEM. E, Expression of E-cadherin or N-cadherin in the different MCF10A cells was detected by immunofluorescence. Cells infected with nonspecific, sh1, or sh2 RASA1 shRNAs (not shown) behaved as control, expressing only E-cadherin. Cells expressing p53R175H slightly diminished E-cadherin expression and showed small patches of N-cadherin expression; cells with both p53R175H and sh1 or sh2 greatly increased N-cadherin expression and reduced E-cadherin expression, in particular p53R175H/sh2 cells, which totally reversed the control phenotype. Bar, 100 μm. F, Invasion assays on Matrigel. Graphic shows invasion indexes normalized to control cells.

Close modal

Collectively, our results indicate that decreased RASA1 expression associated with transposon insertion leads to mammary tumor formation in Trp53+/− mice and strongly suggest that hemizygous loss of RASA1 (frequently associated to TP53 mutation) is a common oncogenic driver in basal and other TNBCs.

Transposon-mediated generation of tumors in a Trp53-heterozygous background has allowed us to identify inactivation of RasGAP genes as a frequent event in murine mammary tumors. Since its initial development as a tool for identification of cancer-promoting genes in transgenic mice (23, 24), transposon technology has been successfully used by many laboratories to identify genes causing cancer in a variety of tissues, and many of these have proved to be of clinical significance (reviewed in ref. 7). Our screen has identified a number of gCIS that are known to function as tumor suppressor genes and are subject to loss-of-function mutations in human breast cancer. Thirty percent of the genes identified in our screen are included in the cancer gene census, among them PTEN, which is mutated in a high percentage of human breast tumors, in particular in TNBC (25). Combined deletion of Pten and Trp53 in mouse mammary epithelium results in the development of claudin-low type tumors (26).

Ras genes are some of the most frequently mutated genes in human cancer, but mutations in breast cancer are rare. Despite this, several lines of evidence suggest involvement of elevated Ras signaling in a subset of breast tumors. (i) The Ras transcriptional signature is highly prevalent in triple negative or basal-like breast cancer. Indeed, integrative analysis suggests widespread activation of the Ras pathway in TNBC (2, 25). (ii) Breast cancer cell lines of the basal type have an activated Ras-like transcriptional program and are particularly sensitive to MAPK/ERK inhibitors (27, 28). (iii) Active, mutant Ras can transform mammary cells: human mammary cells transduced with a mutant KRAS gene generate invasive ductal carcinomas (29) and MMTV-RAS or WAP-RAS transgenic mice develop mammary tumors, which appear earlier and are more malign in a Trp53+/− background (reviewed in ref. 30). Furthermore, there are several reports that hint in particular to RASA1 as a breast cancer gene: for instance, downregulation of RASA1 is associated with poor survival of patients with breast invasive ductal carcinoma (31), and loss of Chr 5q14 (where the RASA1 gene is located) has been noted before in breast tumors (32–34). Moreover, chromosome 5q loss is a characteristic marker of the integrative cluster IntClust 10. This IntClust 10 is 1 of 10 groups that result from the characterization of human breast tumors according to genomic and transcriptomic landscapes (35, 36) and includes mostly triple-negative tumors from the core basal-like intrinsic subtype. Interestingly, these tumors have the highest rate of TP53 mutations (36).

More than 50% of the murine tumors that we have analyzed showed transposon insertions in either one or both of the RasGAP genes Nf1 and Rasa1, suggesting a strong selective pressure toward RasGAPs inactivation for tumorigenesis. There are some classical studies that have already established a relationship between NF1 and human breast cancer (37–40), and recently, high-throughput studies have confirmed involvement of NF1 mutations in this disease (8, 17), so it is conceivable that RASA1 (which is a protein that shares its main function with NF1) is also involved in the development of breast tumors. Our results also synergize with recent reports on RasGAPs alterations in other tumor types, as for instance RASA2 in melanoma (41), RASALl1 in colorectal cancer (42), and RASAL2 also in breast cancer (9, 43). RASA1 itself has been linked to colorectal and prostate tumorigenesis [refs. 44, 45; see also the study by Maertens and Cichowski (46) for a recent review]. In addition, other molecules downstream of Ras could also facilitate breast oncogenesis: for example, DUSP4, a negative regulator of ERK activity, acts as a tumor suppressor in basal-like breast cancer (47). Interestingly, a recently reported breast cancer transposon screen has also identified Rasa1 as a potential breast tumor suppressor gene in a Pten-mutant background (48).

Our results also confirm that downregulation of RASA1, when accompanied by the presence of a mutated TP53, is sufficient to induce an EMT response in MCF10A cells. This response probably requires a mutation in TP53, as cells bearing wild-type TP53 did not show any sign of malignancy upon RASA1 inactivation, nor did cells with p53R175H and wild-type RASA1 (Fig. 5 and data not shown). These results agree with the strong correlation between RASA1 loss and mutation of TP53 that we have detected (Supplementary Table SVI). Interestingly, partial suppression of RASA1 expression also induces significant traits of malignancy in MCF10A p53R175H cells, suggesting that haploinsufficiency of RASA1 can malignize breast cells in the context of a TP53 mutation.

The murine tumors generated in this study have arisen from cells that express or previously expressed keratin K5. While keratin K5 is associated with basal myoepithelial cells of the differentiated gland, it is also expressed by primitive cells within the epithelial hierarchy, including bipotential cells that can generate luminal and basal cell types (49–51). Indeed, our transposon-induced tumors are not myoepithelial tumors and therefore they are likely to arise from some form of bipotent or multipotent progenitor cell. K5-positive mammary stem cells, which give rise both to luminal and basal populations are long-lived, and capable of considerable expansion (49). The longevity of these cells makes them likely targets for acquisition of mutations associated with continuous mobilization of mutagenic transposon by K5 promoter–driven transposase. Moreover, the exacerbated expansion of clonogenic stem/progenitor cells in both luminal and basal mammary epithelial cell layers that results as a consequence of p53 inactivation (52) seems to facilitate transposon-induced tumorigenesis, as mammary tumors were not common in SB/T2 Trp53WT transgenic mice (10).

In conclusion, deletion of RASA1 (probably in association with TP53 mutation) is likely to promote development and/or progression of basal subtype breast cancer in humans. Together with the recently reported losses of other RasGAPs such as NF1 and RASAL2 (8, 9), our results support the possible activation of the Ras pathway, independently of mutations in Ras, as a driver of tumorigenesis in human breast tumors.

A.J. Dupuy has ownership interest (including patents) in Intrexon and Ziopharm. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C. Suárez-Cabrera, R.M. Quintana, A. Ramírez, M. Navarro

Development of methodology: C. Suárez-Cabrera, R.M. Quintana, A.J. Dupuy

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Suárez-Cabrera, R.M. Quintana, J.P. Alameda, A. Maroto, J. Salamanca, A.J. Dupuy, A. Ramírez, M. Navarro

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Suárez-Cabrera, R.M. Quintana, A. Bravo, M.L. Casanova, A. Page, J.M. Paramio, A.J. Dupuy, M. Navarro

Writing, review, and/or revision of the manuscript: C. Suárez-Cabrera, R.M. Quintana, A. Bravo, A.J. Dupuy, A. Ramírez, M. Navarro

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.M. Paramio, A. Maroto, M. Navarro

Study supervision: A. Ramírez, M. Navarro

We would like to thank Berta Hernanz and Rebeca Sanz for her excellent technical help; Federico Sanchez-Sierra and Pilar Hernández Lorenzo for their assistance with the histologic processing of the samples; and Jesús Martínez, Edilia de Almeida, and the personnel of the CIEMAT Animal Unit for their care of the mice used in this work. Thanks to Dr. Cristina Sánchez (Universidad Complutense) for providing cells. We want to particularly acknowledge the patients enrolled in this study for their participation and the Biobanco i+12. Special thanks to Dr. Sean E. Egan and Jes R. Adams (Toronto, Canada) for discussions and help with manuscript preparation.

This research was supported by grants from the Spanish Government and co-funded by Fondo Europeo de Desarrollo Regional (FEDER): PI14/01403 from the Instituto de Salud Carlos III to A. Ramírez; PI13/02580 from the Instituto de Salud Carlos III to M.L. Casanova; RD12/0036/0009 from the Instituto de Salud Carlos III, S2011/BMD2470 from the Comunidad Autónoma de Madrid, SAF-2015-66015R from the Ministerio de Economía y Competitividad, and PIE15/00076 from the Instituto de Salud Carlos III to J.M. Paramio. Biobanco 1+12 is supported by Instituto de Salud Carlos III.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Forbes
SA
,
Beare
D
,
Gunasekaran
P
,
Leung
K
,
Bindal
N
,
Boutselakis
H
, et al
COSMIC: exploring the world's knowledge of somatic mutations in human cancer
.
Nucleic Acids Res
2015
;
43
(
D1
):
D805
11
.
2.
Giltnane
JM
,
Balko
JM
. 
Rationale for targeting the Ras/MAPK pathway in triple-negative breast cancer
.
Discov Med
2014
;
17
:
275
83
.
3.
Ferlay J
SI
,
Ervik
M
,
Dikshit
R
,
Eser
S
,
Mathers
C
,
Rebelo
M
, et al
GLOBOCAN 2012 v1.0, Cancer incidence and mortality worldwide: IARC CancerBase No. 11 [Internet]
.
Lyon, France
:
International Agency for Research on Cancer
; 
2013
.
4.
Bertucci
F
,
Finetti
P
,
Cervera
N
,
Esterni
B
,
Hermitte
F
,
Viens
P
, et al
How basal are triple-negative breast cancers?
Int J Cancer
2008
;
123
:
236
40
.
5.
Ciriello
G
,
Gatza Michael
L
,
Beck Andrew
H
,
Wilkerson Matthew
D
,
Rhie Suhn
K
,
Pastore
A
, et al
Comprehensive molecular portraits of invasive lobular breast cancer
.
Cell
2015
;
163
:
506
19
.
6.
Shah
SP
,
Roth
A
,
Goya
R
,
Oloumi
A
,
Ha
G
,
Zhao
Y
, et al
The clonal and mutational evolution spectrum of primary triple-negative breast cancers
.
Nature
2012
;
486
:
395
9
.
7.
DeNicola
G
,
Karreth
F
,
Adams
D
,
Wong
C
. 
The utility of transposon mutagenesis for cancer studies in the era of genome editing
.
Genome Biol
2015
;
16
:
229
.
8.
Wallace
MD
,
Pfefferle
AD
,
Shen
L
,
McNairn
AJ
,
Cerami
EG
,
Fallon
BL
, et al
Comparative oncogenomics implicates the neurofibromin 1 gene (NF1) as a breast cancer driver
.
Genetics
2012
;
192
:
385
96
.
9.
McLaughlin
SK
,
Olsen
SN
,
Dake
B
,
De Raedt
T
,
Lim
E
,
Bronson
RT
, et al
The RasGAP gene, RASAL2, is a tumor and metastasis suppressor
.
Cancer Cell
2013
;
24
:
365
78
.
10.
Quintana
RM
,
Dupuy
AJ
,
Bravo
A
,
Casanova
ML
,
Alameda
JP
,
Page
A
, et al
A transposon-based analysis of gene mutations related to skin cancer development
.
J Invest Dermatol
2013
;
133
:
239
48
.
11.
Jonkers
J
,
Meuwissen
R
,
van der Gulden
H
,
Peterse
H
,
van der Valk
M
,
Berns
A
. 
Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer
.
Nat Genet
2001
;
29
:
418
25
.
12.
Ramirez
A
,
Page
A
,
Gandarillas
A
,
Zanet
J
,
Pibre
S
,
Vidal
M
, et al
A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination
.
Genesis
2004
;
39
:
52
7
.
13.
Wright
KL
,
Adams
JR
,
Liu
JC
,
Loch
AJ
,
Wong
RG
,
Jo
CE
, et al
Ras signaling is a key determinant for metastatic dissemination and poor survival of luminal breast cancer patients
.
Cancer Res
2015
;
75
:
4960
72
.
14.
Brett
BT
,
Berquam-Vrieze
KE
,
Nannapaneni
K
,
Huang
J
,
Scheetz
TE
,
Dupuy
AJ
. 
Novel molecular and computational methods improve the accuracy of insertion site analysis in sleeping beauty-induced tumors
.
PLoS One
2011
;
6
:
e24668
.
15.
Banerji
S
,
Cibulskis
K
,
Rangel-Escareno
C
,
Brown
K
,
Carter
S
,
Frederick
A
, et al
Sequence analysis of mutations and translocations across breast cancer subtypes
.
Nature
2012
;
486
:
405
9
.
16.
Usary
J
,
Llaca
V
,
Karaca
G
,
Presswala
S
,
Karaca
M
,
He
X
, et al
Mutation of GATA3 in human breast tumors
.
Oncogene
2004
;
23
:
7669
78
.
17.
CancerGenomeAtlasNetwork
. 
Comprehensive molecular portraits of human breast tumours
.
Nature
2012
;
490
:
61
70
.
18.
Persson
M
,
Andrén
Y
,
Mark
J
,
Horlings
HM
,
Persson
F
,
Stenman
G
. 
Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck
.
Proc Natl Acad Sci
2009
;
106
:
18740
4
.
19.
Collier
LS
,
Largaespada
DA
. 
Hopping around the tumor genome: transposons for cancer gene discovery
.
Cancer Res
2005
;
65
:
9607
10
.
20.
Cerami
E
,
Gao
J
,
Dogrusoz
U
,
Gross
BE
,
Sumer
SO
,
Aksoy
BA
, et al
The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data
.
Cancer Discov
2012
;
2
:
401
4
.
21.
Ringnér
M
,
Fredlund
E
,
Häkkinen
J
,
Borg
Å
,
Staaf
J
. 
GOBO: gene expression-based outcome for breast cancer online
.
PLoS One
2011
;
6
:
e17911
.
22.
Sarrio
D
,
Franklin
CK
,
Mackay
A
,
Reis-Filho
JS
,
Isacke
CM
. 
Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties
.
Stem Cells (Dayton, Ohio)
2012
;
30
:
292
303
.
23.
Collier
LS
,
Carlson
CM
,
Ravimohan
S
,
Dupuy
AJ
,
Largaespada
DA
. 
Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse
.
Nature
2005
;
436
:
272
6
.
24.
Dupuy
AJ
,
Akagi
K
,
Largaespada
DA
,
Copeland
NG
,
Jenkins
NA
. 
Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system
.
Nature
2005
;
436
:
221
6
.
25.
Craig
DW
,
O'Shaughnessy
JA
,
Kiefer
JA
,
Aldrich
J
,
Sinari
S
,
Moses
TM
, et al
Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities
.
Mol Cancer Ther
2013
;
12
:
104
16
.
26.
Liu
JC
,
Voisin
V
,
Wang
S
,
Wang
DY
,
Jones
RA
,
Datti
A
, et al
Combined deletion of Pten and p53 in mammary epithelium accelerates triple‐negative breast cancer with dependency on eEF2K
.
EMBO Mol Med
2014
;
6
:
1542
60
.
27.
Mirzoeva
OK
,
Das
D
,
Heiser
LM
,
Bhattacharya
S
,
Siwak
D
,
Gendelman
R
, et al
Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition
.
Cancer Res
2009
;
69
:
565
72
.
28.
Hoeflich
KP
,
O'Brien
C
,
Boyd
Z
,
Cavet
G
,
Guerrero
S
,
Jung
K
, et al
In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models
.
Clin Cancer Res
2009
;
15
:
4649
64
.
29.
Nguyen
LV
,
Pellacani
D
,
Lefort
S
,
Kannan
N
,
Osako
T
,
Makarem
M
, et al
Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells
.
Nature
2015
;
528
:
267
71
.
30.
Menezes
ME
,
Das
SK
,
Emdad
L
,
Windle
JJ
,
Wang
XY
,
Sarkar
D
, et al
Genetically engineered mice as experimental tools to dissect the critical events in breast cancer
.
Adv Cancer Res
2014
;
121
:
331
82
.
31.
Liu
Y
,
Liu
T
,
Sun
Q
,
Niu
M
,
Jiang
Y
,
Pang
D
. 
Downregulation of Ras GTPaseactivating protein 1 is associated with poor survival of breast invasive ductal carcinoma patients
.
Oncol Rep
2015
;
33
:
119
24
.
32.
Johannsdottir
HK
,
Jonsson
G
,
Johannesdottir
G
,
Agnarsson
BA
,
Eerola
H
,
Arason
A
, et al
Chromosome 5 imbalance mapping in breast tumors from BRCA1 and BRCA2 mutation carriers and sporadic breast tumors
.
Int J Cancer
2006
;
119
:
1052
60
.
33.
Adélaïde
J
,
Finetti
P
,
Bekhouche
I
,
Repellini
L
,
Geneix
J
,
Sircoulomb
F
, et al
Integrated profiling of basal and luminal breast cancers
.
Cancer Res
2007
;
67
:
11565
75
.
34.
Hu
X
,
Stern
HM
,
Ge
L
,
O'Brien
C
,
Haydu
L
,
Honchell
CD
, et al
Genetic alterations and oncogenic pathways associated with breast cancer subtypes
.
Mol Cancer Res
2009
;
7
:
511
22
.
35.
Curtis
C
,
Shah
SP
,
Chin
SF
,
Turashvili
G
,
Rueda
OM
,
Dunning
MJ
, et al
The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups
.
Nature
2012
;
486
:
346
52
.
36.
Dawson
S-J
,
Rueda
OM
,
Aparicio
S
,
Caldas
C
. 
A new genome-driven integrated classification of breast cancer and its implications
.
EMBO J
2013
;
32
:
617
28
.
37.
Madanikia
SA
,
Bergner
A
,
Ye
X
,
Blakeley
JO
. 
Increased risk of breast cancer in women with NF1
.
Am J Med Genet
2012
;
158A
:
3056
60
.
38.
Wang
X
,
Levin
AM
,
Smolinski
SE
,
Vigneau
FD
,
Levin
NK
,
Tainsky
MA
. 
Breast cancer and other neoplasms in women with neurofibromatosis type 1: a retrospective review of cases in the Detroit metropolitan area
.
Am J Med Genet
2012
;
158A
:
3061
4
.
39.
Salemis
NS
,
Nakos
G
,
Sambaziotis
D
,
Gourgiotis
S
. 
Breast cancer associated with type 1 neurofibromatosis
.
Breast Cancer (Tokyo, Japan)
2010
;
17
:
306
9
.
40.
Guran
S
,
Safali
M
. 
A case of neurofibromatosis and breast cancer: loss of heterozygosity of NF1 in breast cancer
.
Cancer Genet Cytogenet
2005
;
156
:
86
8
.
41.
Arafeh
R
,
Qutob
N
,
Emmanuel
R
,
Keren-Paz
A
,
Madore
J
,
Elkahloun
A
, et al
Recurrent inactivating RASA2 mutations in melanoma
.
Nat Genet
2015
;
47
:
1408
10
.
42.
Ohta
M
,
Seto
M
,
Ijichi
H
,
Miyabayashi
K
,
Kudo
Y
,
Mohri
D
, et al
Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression
.
Gastroenterology
2009
;
136
:
206
16
.
43.
Feng
M
,
Bao
Y
,
Li
Z
,
Li
J
,
Gong
M
,
Lam
S
, et al
RASAL2 activates RAC1 to promote triple-negative breast cancer progression
.
J Clin Invest
2014
;
124
:
5291
304
.
44.
Sun
D
,
Yu
F
,
Ma
Y
,
Zhao
R
,
Chen
X
,
Zhu
J
, et al
MicroRNA-31 activates the RAS pathway and functions as an oncogenic MicroRNA in human colorectal cancer by repressing RAS p21 GTPase activating protein 1 (RASA1)
.
J Biol Chem
2013
;
288
:
9508
18
.
45.
Berndt
SI
,
Wang
Z
,
Yeager
M
,
Alavanja
MC
,
Albanes
D
,
Amundadottir
L
, et al
Two susceptibility loci identified for prostate cancer aggressiveness
.
Nat Commun
2015
;
6
:
6889
.
46.
Maertens
O
,
Cichowski
K
. 
An expanding role for RAS GTPase activating proteins (RAS GAPs) in cancer
.
Adv Biol Regul
2014
;
55
:
1
14
.
47.
Balko
JM
,
Cook
RS
,
Vaught
DB
,
Kuba
MG
,
Miller
TW
,
Bhola
NE
, et al
Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance
.
Nat Med
2012
;
18
:
1052
9
.
48.
Rangel
R
,
Lee
S-C
,
Hon-Kim Ban
K
,
Guzman-Rojas
L
,
Mann
MB
,
Newberg
JY
, et al
Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression
.
Proc Natl Acad Sci
2016
;
113
:
E7749
58
.
49.
Rios
AC
,
Fu
NY
,
Lindeman
GJ
,
Visvader
JE
. 
In situ identification of bipotent stem cells in the mammary gland
.
Nature
2014
;
506
:
322
7
.
50.
Moumen
M
,
Chiche
A
,
Deugnier
M-A
,
Petit
V
,
Gandarillas
A
,
Glukhova
MA
, et al
The proto-oncogene Myc is essential for mammary stem cell function
.
Stem Cells
2012
;
30
:
1246
54
.
51.
Wang
D
,
Cai
C
,
Dong
X
,
Yu
QC
,
Zhang
X-O
,
Yang
L
, et al
Identification of multipotent mammary stem cells by protein C receptor expression
.
Nature
2015
;
517
:
81
4
.
52.
Chiche
A
,
Moumen
M
,
Petit
V
,
Jonkers
J
,
Medina
D
,
Deugnier
M-A
, et al
Somatic loss of p53 leads to stem/progenitor cell amplification in both mammary epithelial compartments, basal and luminal
.
Stem Cells
2013
;
31
:
1857
67
.