Transposon Mutagenesis Identifies Genes That Cooperate with Mutant

Transposon mutagenesis identifies genes that PNAS PLUS cooperate with mutant Pten in breast cancer progression

Roberto Rangela, Song-Choon Leeb, Kenneth Hon-Kim Banb,c, Liliana Guzman-Rojasa, Michael B. Manna, Justin Y. Newberga, Takahiro Kodamaa, Leslie A. McNoed, Luxmanan Selvanesand, Jerrold M. Wardb,1, Alistair G. Ruste,2, Kuan-Yew Chinb, Michael A. Blackd, Nancy A. Jenkinsa,b,3, and Neal G. Copelanda,b,3,4

aCancer Research Program, Houston Methodist Research Institute, Houston, TX 77030; bDivision of Genomics and Genetics, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Biopolis, Singapore 138673; cDeparment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 138673; dDepartment of Biochemistry, University of Otago, Dunedin 9016, New Zealand; and eExperimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom

Contributed by Neal G. Copeland, October 4, 2016 (sent for review July 14, 2016; reviewed by Kent W. Hunter and Branden Moriarty) Triple-negative breast cancer (TNBC) has the worst prognosis of identified. Transposon mutagenesis can thus identify genes that any breast cancer subtype. To better understand the genetic forces are functioning at the tips of the cancer evolutionary tree and driving TNBC, we performed a transposon mutagenesis screen in a help deconvolute tumor evolution on a scale that is not yet phosphatase and tensin homolog (Pten) mutant mice and identi- possible through the sequencing of human tumors. fied 12 candidate trunk drivers and a much larger number of To identify genetic drivers of TNBC, we induced Sleeping progression genes. Validation studies identified eight TNBC tumor Beauty (SB) transposition in breast epithelial cells of mice that suppressor genes, including the GATA-like transcriptional repres- were heterozygous for a Pten-null allele. Breast tumors were sor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted subsequently classified using a PAM50 subtyping approach and epithelial-to-mesenchymal transition (EMT) by deregulating multi- found to represent a collection of different breast cancer sub- ple EMT pathway genes, in addition to increasing the expression of GENETICS types including basal-like (45%), luminal A (39%), HER2 (11%), SERPINE1 and SERPINB2 and the subsequent migration, invasion, and normal-like (5%). Cloning and sequencing of the transposon and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC insertion sites in tumors identified 12 candidate trunk drivers and and discovered genes with potential clinical importance in TNBC. a much larger number of progression genes. Subsequent validation studies identified eight TNBC tumor suppressor genes (TSGs), Sleeping Beauty | breast cancer | TRPS1 | metastasis | tumor suppressors including a tumor and metastasis suppressor with clinical relevance in TNBC. reast cancer is the second leading cause of cancer-related Bdeaths in the United States. The Cancer Genome Atlas (TCGA) Significance network has classified breast cancer into four main subtypes: luminal A, luminal B, HER2+, and basal-like (1–5). Basal-like or triple- Triple-negative breast cancer (TNBC) is the most aggressive negative breast cancer (TNBC) constitutes 10–20% of all breast breast cancer subtype. Despite extensive cancer genome- cancers and has a higher rate of distal recurrence and a poorer sequencing efforts, there is still an incomplete understanding prognosis than other breast cancer subtypes. Less than 30% of of the genetic networks driving TNBC. Here, we used Sleeping women with metastatic TNBC survive 5 y and almost all die from Beauty transposon mutagenesis to identify genes that co- their disease despite adjuvant chemotherapy (1, 3–5). Mutations, operate with mutant Pten in the induction of TNBC. We iden- rearrangements, or deletions in highly penetrant genes such as tified 12 candidate TNBC trunk drivers and a larger number of BRCA1, BRCA2, TP53, CDH1, STK11, and PTEN are important progression genes. Subsequent functional validation studies drivers of TNBC (6–8). PTEN is a dual-specificity phosphatase identified eight human TNBC tumor suppressor genes, including that antagonizes the PI3K/AKT pathway through its lipid phos- the GATA-like transcriptional repressor TRPS1,whichwasshown phatase activity and negatively regulates the MAPK pathway to inhibit lung metastasis by deregulating the expression of through its protein phosphatase activity (9, 10). Mutations in multiple serpin and epithelial-to-mesenchymal transition (EMT) PTEN drive epithelial–mesenchymal transition (EMT) and pro- pathway genes. Our study provides a better understanding of mote metastasis in TNBC (11–13). Similarly, in mice, heterozy- the genetic forces driving TNBC and discovered genes with gous deletion of Pten induces mammary tumors with basal-like clinical importance in TNBC. characteristics (14–17). Despite all of the cancer genome-sequencing efforts, there is Author contributions: R.R., S.-C.L., K.H.-K.B., N.A.J., and N.G.C. designed research; R.R., S.-C.L., still an incomplete understanding of the genes and genetic net- L.G.-R., T.K., L.A.M., and L.S. performed research; R.R., S.-C.L., K.H.-K.B., M.B.M., J.Y.N., J.M.W., A.G.R., K.-Y.C., M.A.B., N.A.J., and N.G.C. analyzed data; and R.R., N.A.J., and N.G.C. works driving TNBC. New technologies that would provide a wrote the paper. more complete understanding of the genetics of TNBC are still Reviewers: K.W.H., National Cancer Institute; and B.M., University of Minnesota. needed to deconvolute the complexity of this deadly cancer. Our The authors declare no conflict of interest. laboratory and others have pioneered the use of transposon Freely available online through the PNAS open access option. mutagenesis in mice as a tool for cancer gene discovery (18–26). 1Present address: Global Vet Pathology, Montgomery Village, MD 20886. Transposons induce cancer by randomly inserting into the mouse 2Present address: Tumor Profiling Unit, The Institute of Cancer Research, Chester Beatty genome, mutating, and disrupting potential cancer genes. Trans- Laboratories, London SW3 6JB, United Kingdom. poson insertions in tumors thus serve as molecular tags for the 3N.A.J. and N.G.C. contributed equally to this work. high-throughput cloning and identification of cancer genes. In 4To whom correspondence should be addressed. Email: ncopeland@houstonmethodist. addition, because transposon insertions are PCR-amplified be- org. fore they are sequenced, insertional mutations in cancer genes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that are present in only a small fraction of tumor cells can be 1073/pnas.1613859113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613859113 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 Results from the entire mammary epithelium, which is consistent with SB Mutagenesis Promotes the Development of Multiple Breast Cancer our LacZ reporter assays (Fig. S1 A and B) and those of others Subtypes in Ptenfl/+ Mice. Loss of the TSG PTEN is implicated in (30) who showed that LacZ is expressed in all basal cells and Tg/+ breast cancer progression, clonally selected in TNBC, and favors most luminal cells in the mammary epithelium. K5-Cre the activation of the EMT pathway to promote metastasis (11–13). should therefore induce mammary tumors with both luminal To identify genes that cooperate with PTEN in the progression of and basal cell origins. breast cancer, we crossed Ptenfl/fl mice with K5-Cre transgenic mice SB mutagenesis accelerated mammary tumor formation in + + + to generate K5-CreTg/ ;Ptenfl/ mice. The mice were then crossed Ptenfl/ mice with a median survival of 250 d for SB/Pten–Onc2 to mice carrying one of two conditional SB transposition systems mice and 313 d for SB/Pten–Onc3 mice (Fig. 1A). Tumor latency (SB11fl/fl;T2/Onc2Tg/Tg) (18) or (SB11fl/fl;T2/Onc3Tg/Tg)(27)to for SB/Pten–Onc2 mice was significantly earlier than SB/Pten– + + + + generate K5-CreTg/ ;Ptenfl/ ;SB11fl/ ;T2/Onc2Tg/ (SB/Pten–Onc2) or Onc3 mice (P = 0.003), which may reflect the higher number of + + + + K5-CreTg/ ;Ptenfl/ ;SB11fl/ ;T2/Onc3Tg/ (SB/Pten–Onc3) mice. SB/ transposons carried by SB/Pten–Onc2 mice. Fifty-nine percent of Pten–Onc2 mice carry 350 copies of T2/Onc2, all linked together the tumors were classified as adenocarcinomas, whereas 29% at a single site on chromosome 1, whereas SB/Pten–Onc3 mice were classified as adenosquamous carcinomas and 12% as ade- carry a 30-copy T2/Onc3 transposon concatamer located on nomyoepitheliomas (Dataset S1, Table S1). chromosome 9 (20, 27). By using two different transposon con- Adenocarcinomas were more frequent in SB/Pten–Onc2 mice catamers located on different donor chromosomes, we were able (74%), whereas adenomyoepitheliomas were only identified in to eliminate problems caused by local hopping (28) and achieve SB/Pten–Onc3 mice. Hematoxylin and eosin (H&E) staining of + genome-wide coverage of SB mutagenesis. K5-CreTg/ is active in individual tumors revealed a mixed histology, whereas immu- early mammary progenitors (29). Therefore, K5-driven Cre ex- nohistochemical staining showed that both basal (cytokeratin 14) pression should lead to excision of the conditional floxed allele and luminal (cytokeratin 18) markers were often expressed in the

Fig. 1. SB mutagenesis promotes the development of multiple mammary tumor subtypes. (A) Kaplan– Meier survival curves of five different genotypic combinations of mice. Pten/SB–Onc2 and Pten/SB– Onc3 mice showed significant tumor acceleration compared with various control mice (Pten/SB–Onc2, P = 0.0003; Pten/SB–Onc3, P = 0.0001). (B and C) H&E or immunohistochemical staining of mammary adenocarcinoma (B) or adenosquamous carcinoma (C). The adenocarcinoma shows areas of less differentiation (Upper H&E). The adenosquamous carcinoma also has areas of no squamous differentiation in- vading muscle (Lower H&E). Both tumors showed a high degree of heterogeneity, expressing both basal (CK14) and luminal (CK18) cytokeratins. Both tumors have low or focal high-proliferation rate tumors, based upon their Ki67 staining (NOTE-B shows a focally high rate), and express high levels of nuclear SBT protein. (Scale bar, 100 μm.) (D) Mammary tumor subtype classification based upon its PAM50 expression signature (31). The heat map displays gene expression data (log scale, right legend) for the PAM50 breast cancer subtype classifier for each mouse tumor (columns). The left side indicates the centroids for each breast cancer subtype. The rows in the heat map represent genes in the PAM50 panel, and columns represent each mammary tumor. Top panels show proliferation scores (blue, low; red, high) and PAM50 subtypes: basal-like (black), Her2 (purple), luminal A (light blue), and normal-like (light green).

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1613859113 Rangel et al. Downloaded by guest on September 26, 2021 Table 1. Most highly mutated CIS genes identified in SB–Pten mammary tumors PNAS PLUS No. of Unique insertion Total no. GKC P value adjusted Gene Chromosome tumors in CIS sites of insertions across genome

Pten 19 25 41 46 <1.00E–16 Axin1 17 10 22 22 <1.00E–16 Fat1 813 12 13<1.00E–16 Nf1 11 18 35 37 <1.00E–16 Rasa1 13 11 19 20 <1.00E–16 Trps1 15 10 20 20 <1.00E–16 Tbl1xr1 310 11 11<1.00E–16 Gnaq 19 10 13 13 <1.00E–16 Erbb2ip 13 4 12 12 <1.00E–16 Pum2 12 7 8 9 <1.00E–16 Cox7b2 57 1 7<1.00E–16 Man1a 10 7 1 7 <1.00E–16 Dcun1d3 7 8 10 10 3.43E–12 Smek1 12 5 7 7 1.66E–05 Fbxw7 3 7 9 9 3.04E–03 Jup 11 7 7 7 6.33E+03 Ipo7 7 6 7 7 0.0127 Mkl1 15 10 11 11 0.0224 Sos2 12 3 7 7 0.0242 Ccny 18 11 11 12 0.0271 Spag9 11 6 8 8 0.0321 Nfib 4 7 8 9 0.0364

Zfp326 5 6 6 6 0.0431 GENETICS Arih1 9 5 9 9 0.0496

same tumor (Fig. 1 B and C), which is suggestive of extensive which likely reflects the strong selective pressure to inactivate the intratumor heterogeneity (Dataset S1, Table S1). Immunohis- wild-type Pten allele present in tumor cells. tochemical analysis also showed that SB transposase (SBT) was T2/Onc2 and T2/Onc3 contain transcriptional stop cassettes in expressed at high levels in tumors, consistent with their SB- both orientations and can inactivate the expression of a TSG if induced origins (Fig. 1 B and C). inserted within one. They also contain promoters and down- To further define the mammary tumor subtypes derived from stream splice donor sites and can deregulate the expression of a SB mutagenesis, gene expression arrays were performed on 21 proto-oncogene if inserted upstream or in the 5′ end, in the same mammary tumors. For each tumor, an “intrinsic subtype” was transcriptional orientation. The pattern of transposon insertions assigned based on the previously described PAM50 subtyping in CCGs can therefore be used to infer whether an insertionally approach (31). Mouse orthologs for the PAM50 genes were mutated gene is an oncogene or TSG. Visual analysis of insertion identified, and the microarray data were used to determine the patterns of the 448 CCGs identified in mammary tumors sug- closest intrinsic subtype centroid for each sample, based on gested that most (97%) are functioning as TSGs. In addition, in Spearman correlation using logged mean-centered expression many cases, only a single transposon insertion was present at a data. A gene proliferation signature was also used to generate a CCG in tumor cells, suggesting that many CCGs are functioning proliferation score for each sample (32). Basal-like (45%) and as haploinsufficient TSGs. This is similar to what has been ob- luminal A (39%) were the most abundant tumor subtypes, al- served in other SB mutagenesis screens performed in solid tu- though HER2 (11%) and normal-like (5%) were detected at mors (18–26). lower frequencies (Fig. 1D). As expected, basal-like tumors were generally more proliferative than the other tumor subtypes. The Comparative Oncogenomic Filtering. To assess the biological rele- SB–Pten mouse model thus provides a resource for functional vance of the 446 SB-identified CCGs with human orthologs to genomic studies across multiple human breast cancer subtypes. human breast cancer, we performed a number of cross-species comparisons. We found that 51 CCGs are listed in the Cancer Identification of Candidate Cancer Genes. To identify genes mu- Gene Census database (35), a catalog of known cancer genes, tated by SB that drive tumor development, we PCR-amplified which is a highly significant overlap (P = 3.61E–18, two-sided and sequenced the transposon insertions from 18 SB/Pten–Onc2 Fisher’s exact test; Fig. 2A and Dataset S1, Table S3). We also and 16 SB/Pten–Onc3 tumors using 454 next-generation se- compared our CCGs to the 127 significantly mutated genes quencing. Using the Wellcome Trust Sanger Institute’s trans- found in 12 major human cancer types (36) and found that 16 are poson common insertion site calling pipeline (33), we identified SB-identified CCGs (P = 1.23E–08, two-sided Fisher’s exact test; 105,540 mapped transposon reads corresponding to 23,137 unique Fig. 2B and Dataset S1, Table S4), indicating that our screen is transposon insertion sites. Using the locus-centric Gaussian kernel selecting for genes that are relevant to many types of cancer. convolution (GKC) (33) and gene-centric common integration site Finally, we used the TCGA breast cancer database (37, 38) to (gCIS) (34) calling methods, we identified 448 statistically signif- identify genes mutated in human breast cancer (i.e., missense, icant candidate cancer genes (CCGs) by combining the two lists of nonsense, sense, and in-frame mutations). Among the 1,375 mu- CIS genes (Dataset S1, Table S2). CISs are genomic regions that tated genes listed in TCGA, 70 are SB-identified CCGs (P = 9.21E– contain more transposon insertions than predicted by chance and 12, two-sided Fisher’s exact test; Fig. 2C and Dataset S1, Table S5). are thus likely to mark the location of CCGs. Pten was the most SB-identified CCGs in mice therefore appear to be highly relevant highly mutated CIS gene (Table 1 and Dataset S1, Table S2), to human breast cancer.

Rangel et al. PNAS Early Edition | 3of10 Downloaded by guest on September 26, 2021 Fig. 2. Comparative oncogenomic and pathway analysis of SB-identified CCGs. Cross-species comparison of SB-identified CCGs against genes listed in (A)the Cancer Gene Census, (B) Pan-cancer 12, and (C) TCGA breast cancer databases. Significant enrichment of SB-identified CCGs was obtained using the Fisher exact test. (D) SB-identified trunk drivers along with mean and median sequence read counts for insertions containing ≥9 sequence reads and mutations in ≥3 tumors. (E) List of the top 12 trunk drivers, along with their mean read counts, predicted effect of transposon insertions on gene expression and gene function. (F) Biological pathway (yellow nodes) interaction network of CCG connections between 14 human orthologs that show a clinical association between RNA abundance and breast cancer patient survival (blue nodes) and reported cancer gene drivers (red nodes), including known breast cancer drivers PTEN, MAP2K4, TBL1XR1, AXIN1, and NF1. Chromatin, chromatin remodeling; EGFR, epidermal growth factor receptor signaling; MAPK, mitogen-activated protein kinase signaling; NTR, neurotrophin signaling; TNF/NF–κB, tumor necrosis factor and nuclear factor kappa B signaling; and WNT, Wnt signaling.

Identification of Trunk Driver Genes. Most CCGs identified by SB these genes were mostly located throughout the coding regions, mutagenesis are thought to function during late stages of tumor consistent with their TSG function (Fig. 2E and Fig. S2). The one progression (18–26). To identify the CCGs that function early in exception was Jup (also called gamma catenin), which contains a mammary tumor development, we selected transposon insertion sites cluster of insertions on the sense strand near exon 2, suggesting that represented by the highest number of sequencing reads (SB insertions Jup mightfunctionasanoncogene(Fig. S2). Recent reports have in the CCG in ≥3 tumors with ≥9 reads per tumor), arguing that shown that Jup overexpression is crucial for maintaining circulating these insertions would be present in the largest number of tumor tumor cells as clusters and for facilitating homing to the lungs (39). cells. This analysis identified 12 CCGs that we subsequently refer to as trunk drivers (Fig. 2D). Strikingly, 50% are known TSGs, including Altered Signaling Pathways and Cellular Processes. Using Enrichr Pten, Arhgap35, Nf1, Rasa1, Axin1,andApc. Transposon insertions in functional annotation (40), we identified 22 pathways and cellular

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1613859113 Rangel et al. Downloaded by guest on September 26, 2021 processes (FDR P < 0.05) (Dataset S1, Table S6)thatare TRPS1 Is a Tumor Suppressor in TNBC. Down-regulation of Trps1 PNAS PLUS enriched in SB-identified CCGs. Notably, the top six pathways and resulted in the largest acceleration of tumor growth in our cellular processes contained 51% of the SB-identified CCGs with functional validation studies. Trps1 encodes a GATA-like tran- known function. The Wnt pathway contained the most CCGs, with scription factor that regulates gene expression by acting as a loss-of-function mutations detected in Apc, Axin1,andGsk3b, transcriptional repressor (43, 44). Mutations in TRPS1 lead to which are involved in beta-catenin degradation, in addition to Tricho-rhino-phalangeal syndrome, an autosomal-dominant dis- Tcf7l2, Amer1,andSmad4 (41, 42). We also identified numerous order characterized by craniofacial and skeletal malformations insertions in Pten, Rasa1,andNf1, which are important regulators (45). Mutations in TRPS1 have been reported in several human of EGFR, TNF-alpha, and MAPK signaling and are commonly cancers, including leukemia, prostate, colon, endometrial, and altered in patients with breast cancer. breast cancer (46–49). Quantitative immunohistochemistry has also shown that TRPS1 is a prognostic marker in early-stage breast SB-Identified CCGs Have Clinical Relevance. To determine whether cancer and I–II ER+ patients receiving antihormone therapy (50). SB-identified CCGs belonging to the top six signaling pathways Stage II/III breast cancer patients with high expression levels of have clinical relevance, we interrogated a breast cancer expression TRPS1 also have a better survival outcome compared with low microarray dataset that also reports recurrence-free patient sur- TRPS1 expression patients (51), suggesting that tumors arising vival. All CCGs showing a significant association were also ana- from TRPS1-negative/low cells are intrinsically more aggressive. lyzed within each breast cancer subtype. This analysis identified 14 This is in agreement with our TRPS1 expression analysis across CCGs that were significantly associated with patient survival in different human breast cancer subtypes, which showed that TNBC one or more breast cancer subtypes (Fig. 2F and Dataset S1, Table has the lowest expression levels of TRPS1 (Fig. S5). S7). SB-identified CCGs thus appear to have clinical relevance. Down-regulation of TRPS1 in TNBC results from activation of the RAS–RAF–MEK pathway, which is common in basal-like SB-Identified CCGs Act as Tumor Suppressors in Human Breast Cancer. breast cancers (52). This in turn leads to the activation of the Fos To provide additional evidence that SB-identified CCGs are family transcription factor FOSL1 and its downstream target important in human breast cancer, we selected 20 CCGs that are miR-221/miR-222, followed by the down-regulation of TRPS1, mutated in human breast cancer, possess tumor suppressor ac- which is a downstream target of miR-221/miR-222 (52). These tivity in loss-of-function studies, and have been identified as results predict a negative correlation between the expression of CCGs in other SB mutagenesis screens. We then used small TRPS1 and FOSL1/miRNA-221/222 in breast cancer, which we GENETICS hairpin RNAs (shRNAs) delivered by lentivirus to silence the confirmed by quantitative real time (qRT)-PCR (Fig. S6). To de- expression of these CCGs in two TNBC cell lines, HCC70 and termine whether down-regulation of TRPS1 expression accelerates MDA-MB-468, which were selected because they carry mutations tumor growth in vivo, we used two different TRPS1 shRNAs to in PTEN (Figs. S3 and S4). The cells were then orthotopically down-regulate TRPS1 expression in HCC70 and HCC1569 TNBC injected into the mammary fat pad of athymic nude mice, and cells (Fig. S7 A and B). Down-regulation of TRPS1 significantly after 45 d, the animals were necropsied and tumor volumes accelerated tumor growth in nude mice following orthotopic in- measured. The silencing of eight CCGs, including Man1a1, Pkp4, jection (Fig. 3 A and B). Finally, to determine whether TRPS1 Rab10, Rasa1, Trps1, Vps26a, Xpnpep3,andZnf326, accelerated overexpression inhibits tumor growth, we overexpressed TRPS1 in tumor growth, whereas the silencing of R3hcc1l reduced tumor MDA-MB-231 and HCC1954 TNBC cells (Fig. S7 C and D), which growth (Table 2). The silencing of Ppp1r12a and Pum2 induced have reduced TRPS1 expression. We used a lentivirus con- cell death during puromycin selection, suggesting that these genes struct expressing the TRPS1 ORF minus the 3′UTR to avoid are essential for cellular growth. These results show that these miRNA-221/222 targeting degradation. Tumor cells were injected CCGs are enriched for mammary cancer TSGs. into the fat pad of nude mice, and after several weeks, we observed

Table 2. Functional validation screening of CCGs Predicted effect Tumor growth, gene knockdown CIS ortholog on gene function Function or pathway vs. control

AFTPH Disruption Membrane trafficking No significant difference ASH1L Disruption Histone methyltransferase No significant difference EP400 Disruption Transcriptional activation No significant difference ERBB2IP Disruption EGFR signaling No significant difference LPP Disruption Cell adhesion No significant difference MAN1A1 Disruption Metabolism Acceleration NIPBL Disruption Cell cycle No significant difference PKP4 Disruption Cell adhesion Acceleration PPP1R12A Disruption Regulation of actin cytoskeleton Cell death PUM2 Disruption Translational control Cell death R3HCC1L Activation Unknown Decrease RAB10 Disruption Cytoskeletal signaling Acceleration RASA1 Disruption EGFR and FGFR signaling Acceleration SOS2 Disruption PI3K-Akt signaling No significant difference TRPS1 Disruption Transcriptional repressor Acceleration VPS26A Disruption WNT signaling Acceleration XPNPEP3 Disruption Aminopeptidase Acceleration YTHDF3 Disruption mRNA processing No significant difference ZNF143 Activation Transcriptional activation No significant difference ZNF326 Disruption Transcriptional activation Acceleration

Genes in bold indicate a significant tumor growth difference.

Rangel et al. PNAS Early Edition | 5of10 Downloaded by guest on September 26, 2021 knocked down TRPS1 expression in HCC70 TNBC cells using two independent TRPS1 shRNAs and then used an EMT PCR gene expression array to identify genes that might be regulated by TRPS1. The EMT PCR array profiles the expression of 84 key genes that either change their expression during the process of EMT or regulate the expression of these genes. We observed a significant up-regulation of BMP2, MMP2, MMP9, SERPINE1, SNAI2, TFPI2, TGFB2, and ZEB1 and a significant down-regulation of COL5A, FN1, KRT14, SNAI1, SNAI3, and SOX10 using this array (Fig. 4A). These results indicate that TRPS1 regulates multiple genes in the EMT pathway. To further explore the biological effects of these expression changes, we performed migration and invasion assays using TNBC cells that over- or underexpress TRPS1. We observed increased migration and invasion of HCC70 cells that had reduced TRPS1 expression (Fig. 4 B and C) and decreased migration and invasion of MDA-MB-231 and HCC1954 cells that had increased TRPS1 expression (Fig. 4 B and C). Collectively, these results show that TRPS1 regulates multiple genes in the EMT pathway that have effects on cell migration and invasion and are consistent with previous studies that measured the effects of miR-221/222 expression on the migration and invasion of tumor cells (52).

SERPINE1 and SERPINB2 Expression Is Negatively Regulated by TRPS1. SERPINE1 was the gene whose expression was most increased in TRPS1 knockdown cells (Fig. 4A). SERPINE1 encodes endo- thelial plasminogen activator inhibitor-1 (PAI1), a member of theserineproteaseinhibitorfamily that inhibits tissue-type plasminogen activator (PLAT) and urokinase-type plasminogen activator (PLAU), which breaks down fibrin clots. Interestingly, Fig. 3. TRPS1 reduces tumor growth and suppresses metastasis in breast high SERPINE1 expression has been shown to enhance cell cancer. (A and B) Inactivation of TRPS1 in HCC70 and HCC1569 TNBC cell lines migration and apoptosis resistance in head and neck carcinoma accelerates tumor growth in orthotopic xenografts. (C and D) Ectopic over- patients (53), whereas its down-regulation in nasopharyngeal expression of TRPS1 reduces tumor growth in HCC1954 and MDA-MB-231 carcinoma has been associated with reduced metastasis (54). orthotopic xenografts. n =10 mice per group. One-sided unpaired t test was This led us to explore the effects of TRPS1 knockdown on the run for all experiments. (E) TRPS1 expression in MDA-MB-231 cells reduces expression of other serpin family members, which showed that metastasis. Tumor lung colonization was measured by bioluminescent im- aging. The panels show mouse images at different time points. (F) Line graphs represent three time-point measurements of the radiance average from five injected animals. All data were evaluated using two-sided t test. Error bars represent SEM.

a statistically significant reduction in tumor growth (Fig. 3 C and D). These results show that TRPS1 functions as a TSG in TNBC.

TRPS1 Is a Breast Cancer Metastasis TSG. miR-221/222 promotes EMT in breast cancer cells in part by down-regulating TRPS1 expression, which is a direct transcriptional repressor of ZEB2 and a driver of EMT (52). miR-221/222 expression also has been shown to enhance the migration and invasion of nontransformed human mammary epithelial MCF10A cells, whereas synthetic oligo inhibitors of miR-221/222 attenuate the migration and invasion of MDA-MD-231 cells through the basement membrane matrix (52). To determine whether TRPS1 is a breast cancer metastasis TSG, we examined the ability of MDA-MB-231 TNBC cells overexpressing the TRPS1 ORF, minus the 3′UTR, to colo- nize the lung. TRPS1-overexpressing cells were injected into the mammary fat pad of athymic nude mice, and lung metastatic progression was monitored by bioluminescence. Five weeks after injection, we observed a significant decrease in luciferase activity in the lungs of injected mice compared with control mice (Fig. 3 E and F). These results indicate that TRPS1 is a TSG that in- Fig. 4. TRPS1 regulates the expression of genes in the EMT pathway. hibits lung metastasis. (A) EMT gene expression in HCC70 TRPS1 knockdown cells. Two independent TRPS1 shRNA knockdown clones showed consistent mRNA expression levels TRPS1 Regulates the Expression of Multiple Genes in the EMT over control. (B) Migration and (C) invasion assays of TRPS1 shRNA knockdown Pathway. To determine whether other EMT pathway genes in in HCC70 cells and TRPS1 overexpression in MDA-MB-231 and HCC1954 cells. addition to ZEB2 are transcriptionally regulated by TRPS1,we Data represent means ± SEM of three independent experiments.

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Fig. 5. TRPS1 directly regulates the expression of SERPINE1 and SERPINB2.(A) Gene expression analysis of different serpin family members. SERPINE1 and SERPINB2 were up-regulated after TRPS1 depletion in HCC70 cells. qRT-PCR validation was performed in triplicates. *P < 0.001. (B) Increased serpin protein levels in the supernatants of TRPS1 knockdown cells were detected by ELISA, *P < 0.001. (C and D) Serpin expression levels are regulated by TRPS1 in tumor xerographs. TRPS1 and SERPINE1 and SERPINB2 expression levels are negatively correlated in (C) HCC70 (n = 10 per group) and (D) MDA-MB-231 (n = 7) tumor xenografts. mRNA expression levels were detected by qRT-PCR. (E) ChIP assays confirmed that TRPS1 binds to SERPINB2 and SERPINE1 promoters. (Top) Possible TRPS1 binding sites in SERPINB2 and SERPINE1 promoters and control regions. (Bottom) Fold enrichment of TRPS1 binding to SERPINE1 and SERPINB2 promoters. To validate anti-TRPS1 specificity, we used TRPS1 knockdown (shTRPS1) and nontargeted control (NTC) cells. The ZEB2 promoter was included as a positive control as previously described (52). (F and G) Luciferase reporter expression assays were performed using SERPINE1 and SERPINB2 promoters in (F) HCC70 control and TRSP1 shRNA cells and (G) MDA-MB-231 control and TRPS1-overexpressing cells. ZEB2 and GAPDH promoters were used as positive controls. Random control promoter (RCP) was used as a negative control. All promoters contained 1,000 base pairs of DNA. In all experiments, the results are reported as means ± SEM of three independent experiments. P values were calculated using two-sided t test.

SERPINB2 is also highly overexpressed in TRPS1 knockdown 24 h and then collected the supernatants. Sandwich ELISA cells (Fig. 5A). Plasmin from reactive brain stroma provides a showed that SERPINE1 and SERPINB2 proteins are highly defense against metastatic invasion of lung and breast cancer secreted from TRPS1 knockdown TNBC cells (Fig. 5B). Finally, cells (55). Plasmin does this by converting membrane-bound we used qRT-PCR to confirm that SERPINE1 and SERPINB2 astrocytic FasL into a paracrine death signal for cancer cells and expression is negatively regulated by TRPS1 in orthotopic tumor by inactivating the axon guidance molecule L1CAM, which xenografts (Fig. 5 C and D). These results indicate that the sup- metastatic cells express for spreading along brain capillaries and pressive effects of TRPS1 on tumor metastases are mediated in for metastatic outgrowth. Importantly, brain metastatic lung part through the suppressive effects of TRPS1 on SERPINE1 and and breast cancer cells that express high levels of neuroserpin SERPINB2 expression. and serpin B2 inhibit plasmin generation and its metastasis- suppressive effects (55). TRPS1 Is a Direct Transcriptional Repressor of SERPINE1 and To confirm that SERPINE1 and SERPINB2 are secreted from SERPINB2. To determine whether TRPS1 is a direct transcriptional TRPS1 knockdown cells, we grew the cells in serum free-media for repressor of SERPINE1 and SERPINB2, we asked whether TRPS1

Rangel et al. PNAS Early Edition | 7of10 Downloaded by guest on September 26, 2021 couldbindtotheSERPINE1 and SERPINB2 promoters using TRPS1 Expression Levels Are Clinically Associated with Patient chromatin immunoprecipitation (ChIP) assays. Because TRPS1 Survival. Finally, to determine whether TRPS1 expression levels represses transcription from GATA-containing binding sites (43, are clinically associated with patient survival, we queried a 56–59), we searched for conserved GATA sites in the promoter publically available breast cancer database (60). We found that regions of SERPINE1 and SERPINB2. We then designed oligo- high TRPS1 expression is associated with increased relapse-free nucleotides specific for these binding sites. As a negative control, survival but only in luminal A (ER+) breast cancer patients (Fig. we targeted a serpin promoter region that lacked a GATA binding S8A). This is in agreement with previous reports showing that site. ChIP assays showed that TRPS1 could bind to the SERPINE1 TRPS1 is a positive prognostic marker in ER+ breast cancer and SERPINB2 promoters but only in parental HCC70 cells and (51). Luminal B and basal-like breast cancer patients with re- not in cells expressing TRPS1 shRNA (Fig. 5E). To further examine duced expression of TRPS1 showed a negative trend in survival, TRPS1 repressor activity, we performed luciferase reporter assays. but the results do not reach statistical significance (Fig. S8A). SERPINE1 and SERPINB2 promoter-luciferase DNA constructs Interestingly, SERPINE1, SERPINB2, and FOSL1 expression were transfected into TRPS1 knockdown HCC70 or TRPS1-over- was inversely correlated with TRPS1 expression in ER– breast expressing MDA-MB-231 cells. Subsequent quantification of lu- cancer (Fig. S8B) (61), in agreement with our findings. Collec- ciferase activity showed that SERPINE1 and SERPINB2 luciferase tively, these results suggest that high TRPS1 expression in ER+ activity was significantly increased in TRPS1-deficient HCC70 cells breast cancer represses EMT and serpin gene expression, leading compared with control cells. Conversely, SERPINE1 and SERPINB2 to reduced tumor growth and metastases. However, in patients – luciferase activity was significantly decreased in MDA-MB-231 with ER breast cancer, where FOSL1 levels are high and cells overexpressing TRPS1. These results are in agreement mir221/222 expression is increased, reduced TRPS1 expression with our in vitro and in vivo studies showing that TRPS1 is leads to an increase in EMT and serpin gene expression, with a transcriptional repressor of SERPINE1 and SERPINB2 concomitant increased tumor growth and tumor metastasis (Fig. 5 F and G). (Fig. S8C). To determine whether SERPINE1 and SERPINB2 are im- Discussion portant for the tumor acceleration observed in TRPS1 knockdown cells, we performed a rescue experiment by disrupting Here we have used SB mutagenesis to identify genes that co- SERPINE1 and SERPINB2 expression in TRPS1 knockdown operate with mutant Pten in the induction of breast cancer. HCC70 cells and then measuring the effect of this disruption on Previous studies have shown that Pten mutant mice develop tumor growth in transplanted mice (Fig. 6A). As shown in Fig. well-differentiated adenocarcinomas with prominent stromal 6B, knockdown of SERPINE1 or SERPINB2 in TRPS1 knock- proliferation (16) in addition to well-differentiated fibroade- down cells inhibited tumor growth, confirming that SERPINE1 nomas and pleomorphic adenocarcinomas (15). By using gene or SERPINB2 is important for the tumor acceleration seen in expression microarrays and molecular signatures that are as- TRPS1 knockdown cells. sociated with different breast cancer subtypes, we were able to identify multiple mammary tumor subtypes in our SB–Pten model. The two major subtypes we identified were luminal A and basal-like tumors, suggesting that transposon mutagenesis is occurring in all mammary epithelium cell populations. Con- sistent with this, studies in K5Cre transgenic mice have identified Cre activity in early mammary cell progenitors (30). K5Cre should therefore inactivate Pten and activate SB transposition in both luminal and basal cell progenitors and might explain the development of different breast cancer subtypes in our SB mouse model. Cloning and sequencing of SB insertion sites in tumors led to the identification of 12 candidate trunk drivers and a much larger number of tumor progression genes. Comparative oncogenomic filtering showed that these genes are enriched for genes causally associated with human cancer, including breast cancer and cancers of many other cell types. Strikingly, 6 of the 12 trunk drivers identified by SB are known TSGs, including Pten, Arhgap35, Nf1, Rasa1, Axin1,andApc. Pathway analysis showed that these genes function in multiple signaling pathways and cellular processes important in cancer. Notably, the top six pathways and cellular processes contained 51% of the SB- identified cancer genes, with the Wnt pathway showing a very high enrichment for mutations in negative regulators of β-catenin. We also identified a high frequency of mutation in Map2k4 and Mapk8. These genes are important components in the activation of apoptosis in response to stress (62), which might explain why these genes are inactivated in SB–Pten tumors. Finally, we also identified a high frequency of transposon insertions in chromatin-remodeling and -modifying enzymes. Collectively, these data suggest that SB is targeting cooperative signaling networks that promote tumor growth without induc- Fig. 6. Serpins mediate tumor growth in TRPS1-deficient tumor cells. ing apoptosis. (A) qRT-PCR analysis of TRPS1, SERPINB2, and SERPINE1 expression in stable An interesting finding in our studies was the identification of lentivirus-transfected HCC70 cells. (B) Tumor growth is significantly reduced tumor suppressors that have already been described in human in serpin-deficient TRPS1 shRNA tumor cells. n = 8 mice per group. Two-sided breast cancer. These results indicate that humans and mice have t test was run for all experiments. Error bars represent SEM. similar selection pressures and provide evidence that these

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1613859113 Rangel et al. Downloaded by guest on September 26, 2021 mutations are important driving events in tumorigenesis. Al- In summary, our studies have provided insights into the ge- PNAS PLUS though SB mutagenesis can uncover hundreds of genes driving netic and evolutionary forces driving breast cancer and identified tumor progression and enable system-level studies, this tech- eight TNBC TSGs. Our SB–Pten mouse model can be used to nology also possesses a challenge for functional validation. Here, study many subtypes of breast cancer, including basal-like, lu- we show that we can use high-throughput shRNA screens in minal A, HER2, and normal-like. Finally, our studies have human breast cancer cells in conjunction with SB mutagenesis to helped to elucidate an important signaling pathway in TNBC validate genes important for human breast cancer progression. with potential clinical importance to a disease that currently Among 20 candidate TSGs we assayed using this high-through- has limited treatment options. put approach, the silencing of eight genes was found to accel- erate tumor growth, whereas the silencing of one gene reduced Materials and Methods tumor growth. The silencing of two genes induced cell death Mice. SB–Pten mice were generated by crossing the following mice: K5-Cre + during puromycin selection, suggesting that they are essential transgenic mice (K5-CreTg/ ) (69), Pten conditional knockout mice (C;129S4- Ptentm1Hwu/J) (15), Rosa26-loxP-STOP-LoxP-SB11 transposase conditional genes. Gene inactivation in orthotopic tumor xenografts there- tm2(sb11)Njen fore appears to be a highly efficient platform for functional knock-in mice [Gt(ROSA)26Sor ] (70), T2/Onc2 (SB-6113) trans- validation studies of candidate TSGs. poson transgenic mice [TgTn(sb-T2/Onc2)6113Njen] (20), and T2/Onc3 (SB- 12740) transposon transgenic mice [TgTn(sb-T2/Onc3)12740Njen] (27). To A major finding of our screen was the discovery and func- examine the expression of Cre within the mammary epithelium, we used the + tional validation of TRPS1 as a metastasis tumor suppressor in following mice: K5-Cre transgenic mice (K5-CreTg/ ) (69) and LacZ transgenic human TNBC. Consistent with these results, in SB–Pten tu- reporter mice [B6;129S4-Gt(ROSA)26Sortm1Sor/J; Jackson Laboratory, Stock mors, Trsp1 was insertionally mutated only in basal-like tumors. No. 003309]. Mice were housed in a specific pathogen-free facility with a This is in contrast to what has been reported in ER+ breast 12-h light/12-h dark cycle. Mice with the selected genotypes were aged and cancer, where increased TRPS1 expression correlates with monitored twice a week for mammary tumor development. Kaplan–Meier improved survival and a favorable response to antihormone analysis was performed, and survival curves were generated using Prism6 therapy. Remarkably, tumor cells from ER+ breast cancer pa- software (GraphPad). Tumors >5 mm in diameter were dissected at nec- tients after antihormone therapy have decreased TRPS1 expres- ropsy; half of the tumor was frozen for DNA sequence analysis, and the other half was fixed with 4% (vol/vol) paraformaldehyde. Fixed tissues were sion and increased expression of mesenchymal markers (63), then processed to paraffin blocks. H&E staining was used to characterize suggesting that breast tumors with low TRPS1 expression might tumor histopathology. All mouse procedures were approved by the In-

be more resistant to chemotherapy and have a higher probability stitutional Animal Care and Use Committee, Institute of Molecular and Cell GENETICS to metastasize. Biology, Singapore, and the Animal Care and Use Committee, Houston TRPS1 is a GATA-like transcription factor, which functions as Methodist Research Institute, Houston. a transcriptional repressor or activator, depending on cell type, stage of development, or pathological conditions. In early hair Cell Culture, Functional Validation Screening, and Generation of Cell Clones. follicle progenitors, TRPS1 is a transcriptional activator of genes Human breast cancer cell lines HCC70 (CRL-2315), HCC1569 (CRL-2330), that inhibit Wnt signaling (56), whereas it is a repressor of HCC1954 (CRL-2338), MDA-MB-231 (HTB-26), MDA-MB-468 (HTB-132), and RUNX2, a gene important for osteoclast differentiation and BT-549 (HTB-122) were obtained from the American Type Culture Collection. Trsp1 MDA-MB-231 Luciferase (AKR-231) was obtained from Cell Biolabs. All cell maturation (57). Moreover, in odontoblast development, is lines were cultured according to the vendor’s instructions. All cell lines usage an inhibitor of Dspp expression (58), whereas in chondrocytes, was approved by the IRB at the Houston Methodist Research Institute. Trps1 controls proliferation and survival by repressing Stat3 ex- Lentiviral shRNA particles were originally obtained from Open Biosystems. pression (59). In pathological conditions, TRPS1 represses the For the functional validation screening, we plated MDA-MB-468 and HCC70 expression of ZEB2, a component of the EMT pathway (52). cells at a density of 5 × 105 cells per well in a six-well plate the night before Studies by Stinson et al. (52) have demonstrated that mir221/222 lentiviral transduction. The next day, the cells were infected overnight in targets the 3′UTR of the TRPS1 mRNA transcript. This degra- serum-free medium containing polybrene (8 μg/mL) mixed with lentiviral dation leads to increased ZEB2 expression as well as increased particles at a multiplicity of infection (MOI) of 6, consisting of individual migration and invasion of tumor cells. In our studies, we show shRNA NTC or pools of three CCG shRNAs. After overnight incubation, com- that TRPS1 regulates many other genes in the EMT pathway in plete medium was added. At 48 h postinfection, puromycin selection medium was added (1.5 μg/mL), and the medium was change every 2 d until >95% of addition to ZEB2. We also show that TRPS1 directly represses the cells expressed GFP fluorescence. All shRNA lentiviral clones were obtained the expression of SERPINE1 and SERPINB2 in TNBC. Recent from Open Biosystems (Dataset S1, Table S8). For the generation of tumor cells studies using a breast metastasis model have shown that SERPINB2 expressing single shRNA clones, we used shRNA-NTC (RHS4348), shRNA-TRPS1 protects metastatic cells from plasminogen activator plasmin in (V3LHS_366300, V3LHS_366303), and TRPS1-ORF (OHS5900-224626817). the brain, providing a metastatic advantage (55). Another serpin Moreover, we used lentivirus expressing shRNA-SERPINE1, shRNA-SERPINB2, family member, Serpine2, was initially discovered in a model of and shRNA-TFPI2 with a blasticidin resistant marker. The virus particles were breast cancer metastasis to bone and later reported to facilitate obtained from GenTarget Inc. For stable shRNA knockdown studies, HCC70 tumor cells to form vascular-like networks enabling perfusion and HCC1569 cells were selected with 1.5 μg/mL of puromycin for 1 wk. For and lung metastasis (64, 65). Likewise, SERPINE1 has been used knockdown or overexpression studies, all cell lines were selected with blasticidin at 5 μg/mL, except for MDA-MB-231 (20 μg/mL), for 2 wk. as a prognostic marker of breast cancer and evaluated during chemotherapy administration (66, 67). In rescue experiments, we ACKNOWLEDGMENTS. We thank K. Rogers, S. Rogers and the Institute of demonstrated that tumor growth in TRPS1 knockdown cells is Molecular and Cell Biology histopathology core for performing histological mediated by SERPINE1 and SERPINB2. This is in agreement analysis, and P. Cheok, N. Lim, D. Chen, C. Wee, E. Freiter, and H. Lee for with mouse serpine1 knockout studies, which have also revealed a monitoring mice and animal technical assistance. The Biomedical Research Council, Agency for Science, Technology and Research (A-STAR), Singapore, defect in tumor growth (68). Collectively, our studies suggest that and The Cancer Research Institute of Texas (CIPRIT), supported this research. TRPS1 is a master regulator of several gene networks that reduce A.G.R. was supported by the Cancer Research UK and the Wellcome Trust. tumor growth and metastasis. Both N.G.C. and N.A.J. are CPRIT Scholars in Cancer Research.

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