Identification of New Tumor Suppressor Genes in Triple-Negative Breast Cancer

Author Manuscript Published OnlineFirst on July 19, 2017; DOI: 10.1158/0008-5472.CAN-17-0785 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Identification of new tumor suppressor genes in triple-negative breast cancer

Roberto Rangel1,2, Liliana Guzman-Rojas1,3, Takahiro Kodama1,4, Michiko Kodama1,5, Justin Y.

Newberg1,6, Neal G. Copeland1,7,8, Nancy A. Jenkins1,7,8

1Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.

2Present address: Department of Immunology, The University of Texas MD Anderson Cancer

Center, Houston Texas, USA.

3Present address: Department of Cardiovascular Sciences, Houston Methodist Research Institute,

Houston, Texas, USA.

4Present address: Department of Gastroenterology and Hepatology, Osaka University Graduate

School of Medicine, Suita Osaka, Japan.

5Present address: Department of Obstetrics and Gynecology, Osaka University Graduate School

of Medicine, Suita Osaka, Japan.

6Present address: Department of Molecular Oncology, Moffit Cancer Center, Tampa, Florida,

USA.

7Present address: Department of Genetics, The University of Texas MD Anderson Cancer

Center, Houston Texas, USA.

8These authors contributed equally to this work

Running title: A two-step tumor suppressor validation in TNBC

Key words: Breast cancer, Sleeping Beauty mutagenesis, tumor suppressors, ZNF326, EMT

Disclosure of Potential Conflicts of Interest: No potential conflicts were disclosed.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 19, 2017; DOI: 10.1158/0008-5472.CAN-17-0785 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Financial support: This work was supported by the Cancer Prevention Research Institute of

Texas (CPRIT) N.G. Copeland (R1112) and N.A. Jenkins (R1113); are also both CPRIT scholars in Cancer Research.

Correspondence should be addressed to:

Nancy A. Jenkins, Ph.D.

Genetics Department

The University of Texas

MD Anderson Cancer Center

1515 Holcombe Blvd., Unit 1010

Houston, TX 77030

Email: [email protected]

Abstract

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step

Sleeping Beauty (SB) forward genetic screen to identify and validate new tumor suppressors

(TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A and ZNF326 showed tumor suppressor (TS) activity in tumor xenograft studies. Subsequent studies showed that

ZNF326 regulated expression of multiple EMT and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal.

ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new tumor suppressor genes in TNBC demonstrate the utility of two-step forward genetic screens in mice, and offer an invaluable tool to identify novel candidate therapeutic pathways and targets.

Introduction

Breast cancer is one of the leading causes of cancer-related deaths in women worldwide. Triple- negative breast cancer (TNBC), which accounts for 10-20% of all breast cancers, does not express estrogen or progesterone receptors and lacks human epidermal growth factor receptor-2

(HER2) amplification. Patients diagnosed with TNBC have a higher risk of disease relapse within 5 years than patients treated for other breast cancer subtypes (1,2). Among breast cancer subtypes, TNBC is the most aggressive, with a high relapse incidence and the most undifferentiated phenotype. Interestingly, residual breast cancer tumors after conventional therapy display mesenchymal as well as cancer stem cell features (3).

EMT is a biological process that allows polarized epithelial cells to acquire a mesenchymal phenotype, which provides advantages for cell migration, invasion and resistance to apoptosis, in addition to increases in the production of extracellular matrix proteins. In cancer, multiple transcription factors regulate the EMT pathway with a coordinated inactivation of tumor suppressor genes (4). Mesenchymal breast tumors contain a small cell population of cancer stem cells (CSCs) that have the capacity to form mammospheres in vitro and maintain tumorigenic activity. It has now been established that a heterogeneous populations of CSCs is responsible for tumor initiation and maintenance (5,6). These CSCs are responsible for metastatic growth in breast cancer, which contributes to the majority of the breast cancer related morbidity and mortality (7,8). To better understand the pathogenesis of TNBC, a comprehensive understating of the genes and pathways that regulate EMT and CSCs in breast cancer is essential.

Recent human cancer genome sequencing studies have provided a genomic mutation landscape of breast cancer (9), thus identification and functional validation of CCGs is a high priority.

Genome-wide insertional mutagenesis is an unbiased and high-throughput method with which to

profile the landscape of driver genes in a mouse model system (10). The Sleeping Beauty (SB)

transposon system has expanded the use of insertional mutagenesis for the study of many types

of cancers (11-20); and discovered candidate cancer genes (CCGs) involved in the EMT and

CSC pathways (21-23). To better understand the genes and pathways involved in the

pathogenesis of TNBC; we performed an siRNA validation study of 40 SB-identified breast

cancer TS genes identified in an SB mutagenesis screen performed in Pten mutant mice (24).

Among the seven CCGs that consistently resulted in increased proliferation in four TNBC cell

lines, four including ADNP, AP2B1, TOMM70A and ZNF326 also showed tumor suppressor

(TS) activity in orthotopic tumor xenograft validation studies. Subsequent studies showed that

ZNF326 is a TS gene that mediates the expression of DACH1, KLF17 and GATA3, and disrupts

the epithelial-mesenchymal transition and cancer stem cell pathways. Our results show that two-

step forward genetic screens in mice are invaluable tools for uncovering CCGs in different signaling pathways involved in TNBC.

Materials and Methods

Cell culture

Human breast cancer cell lines HCC70 (CRL-2315), HCC1569 (CRL-2330), MDA-MB-231

(HTB-26), MDA-MB-468 (HTB-132) and BT-549 (HTB-122) were obtained from the American

Type Culture Collection (Manassas, VA). The cell lines were cultured according to vendor

instructions. All cell lines were purchase between 2012 and 2014 and were frequently tested for

Mycoplasma infection using MycoAlert detection kit (Lonza, Walkersville MD). All Cell lines

were propagated no more than 2 months or 10 passages after resuscitation from stocks and not

further authenticated.

Small interfering RNA (siRNA) screening

siRNA oligonucleotides were purchased from Thermo Fisher Scientific. A list of siRNA

oligonucleotides is provided in Supplementary Table 1. We generated pools of three siRNA

oligos per gene. siRNA transfection was performed using the Lipofectamine RNAiMAX

transfection reagent (Thermo Fisher Scientific) according to the manufacture’s instructions.

Briefly, 4 x 103 cells were seeded into 96-well plates and cultured with medium containing 10%

FBS. After 24 h, cells were transfected with siRNA pooled oligos and incubated at 37°C for 96 hours. Cellular proliferation was then measured using the WST-1 cell proliferation assay system

(Takara). WST-1 regent was added to each well and incubated for 2 hours at 37°C. The absorbance at 450nm and 650nm (reference) was then measured using an Infinite 200 pro

multimode reader (TECAN).

Generation of stable shRNA-expressing cell lines

All lentiviral particles containing GIPZ shRNA or non-targeting control shRNA were purchased from Open Biosystems. A list of shRNAs is provided in Supplementary Table 2. Cells were plated at a density of 1 x 105 cells per 6-well plate one day before infection. The next day, the medium was changed into serum-free medium containing 8ug/ml polybrene (Millipore) and transduced with a three shRNA lentivirus-pool or single shRNA lentivirus clones at an MOI of 6 or 1 respectively. After 48 hours, cells were supplemented with complete medium containing

1.0-1.5 μg/ml puromycin for 7 days (Thermo Fisher Scientific) in order to establish stable cell lines containing lentiviral integrations. For overexpression studies, all cell lines were selected with blasticidin 5 μg/ml, except for MDA-MB-231 (20 μg/ml), for 2 weeks.

Cell migration and invasion assays

Transwell migration assays were performed in a 24-multiwell insert system with a porous polycarbonate membrane (8-μm pore size) according to the manufacturer’s instructions (Cell

Biolabs). Cells were allowed to grow to subconfluency (~75–80%) and were then serum-starved for 24 h. After detachment with trypsin, cells were washed with PBS, resuspended in serum-free medium, and 300 μl of cell suspension (5x105 cell ml-1) was then added to the upper chamber.

500 μl of complete medium was added to the bottom wells of the chamber. The cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated to the lower face of the filters were fixed, stained and quantified according to the manufacturers instructions (Cell Biolabs). Similar inserts coated with Matrigel were used in the invasion assays.

Mammosphere assays

In brief, 500 cells were seeded into ultra low adherent 6-well plates (Corning) and plated in

MammoCult medium (Stem Cell Technologies) supplemented with hydrocortisone, heparin,

amphotericin and gentamicin (SIGMA) for 7-10 days. Mammosphere formation was visible at 7-

10 days and photographs of comparable groups were captured under phase-contrast microscopy

(EVOS XL, ThermoFisher Scientific) at 20X magnification. All experiments were done in

triplicate.

Tumor xenografts

All mouse experiments were approved by the Animal Care and Use Committee (Houston

Methodist Research Institute, Houston TX, USA). Female 6–7-week-old Crl:NU(NCr)-Foxnnu mice were purchase from Charles River Laboratories. Mammary fat pad injections into athymic nude mice were performed using 3 x 106 cells (HCC70, and MDA-MB-231). The human cancer

cells were resuspended in 100 μl of a 1:1 mix of PBS and Matrigel (TREVIGEN). For HCC1569

human breast cancer cells, we injected 4 x 106 cells in 100 μl of a 1:1 mix of PBS and Matrigel.

Injections were done into the fourth mammary gland. Tumors were measured using a digital

caliper and the tumor volume was calculated using the following formula: volume (mm3) = width

x length/2.

qRT–PCR

Total RNA was purified and DNase treated using the RNeasy Mini Kit

(Qiagen). Synthesis of cDNA was performed using SuperScript VILO Master Mix (Life

Technologies). Quantitative PCR analysis was performed on the QuantStudio 12K Flex System

(Life Technologies). All signals were normalized to the levels of GAPDH TaqMan probes.

TaqMan probes were obtained from Life Technologies (Supplementary Table 3). Epithelial

mesenchymal transition (PAHS-090Z) and cancer stem cell (PAHS-176Z) RT-PCR arrays were

purchased from QIAGEN. The assays were performed according to the manufacturer instructions

and the results were evaluated using the QIAGEN data analysis center.

Chromatin immunoprecipitation studies

To determine where ZNF326 binds within the promoter regions, first we retrieve the genomic

sequence (www.ensembl.org) located upstream of the transcriptional start site (TSS) of the gene

including 200 bp downstream. Next, we design PCR primers in which the amplified products

range between 250 – 300 bp and overlap 100 bp (Supplementary Table 4). HCC70 and MDA-

MB-231 cells were fixed in 1% formaldehyde at 37C for 10 min. Cells were washed twice with

ice-cold phosphate-buffered saline containing protease inhibitors, scraped and centrifuged at

4°C. Cell pellets were resuspended in lysis buffer and sonicated (Covaris S220) to shear DNA

with a fragment range of 300 - 400 bp. After sonication, the lysate was centrifuged and the

supernatant was diluted 10-fold with ChIP dilution buffer (EZ-CHIP kit, EMD-MILLIPORE).

Rabbit Anti-V5 tag antibody (Abcam, ab15828), or normal rabbit IgG control (Abcam,

ab171870) were added to the diluted chromatin and incubated overnight at 4°C with rotation.

The antigen-antibody complex was precipitated with protein A/G-agarose and washed

sequentially with low-salt buffer, high-salt buffer and lithium chloride wash buffer, and eluted with elution buffer (1% SDS, 0.1 M NaHCO3 and 200 mM NaCl). Reversal of crosslinking was

performed by heating at 65°C overnight in the presence of NaCl. DNA was purified using DNA-

binding columns provided by the EZ-ChIP kit. The amount of immunoprecipitated DNA was

quantified using high sensitive detection DNA reagent (Q32851, Life Technologies) and

measured using a Qubit 3.0 Fluorometer (Life Technologies). qPCRs were run in triplicate. The

primer list is available in Supplementary Table 4.

Luciferase assays

DACH1, GATA3, KLF17, GAPDH and negative control (RPC) promoter reporter clones were

obtained from Active Motif (LightSwitch Promoter reporter GoClone, Carlsbad, CA). For

luciferase assays, we used the HCC70 and MDA-MB-231 non-targeting control ZNF3260-

shRNA and ectopic expression of recombinant ZNF326. Cells were plated in 96-well plates and

transfection was performed with promoter-reporter plasmids (50 ng/well) using Lipofectamine

2000 (Life Technologies) according to the manufacturers protocol. After 48 hours, we added 100

μl of luciferase assay reagent and incubated for 30 min at room temperature (LightSwitch

luciferase assay system, Active Motif). The cell lysates were transferred to a 96-well plate and each well was read for 2 seconds in a luminometer (Synergy H1 Hybrid Reader, BioTek).

Histological tissue samples

Commercially purchased tissue microarrays (TMAs) included 48 samples of invasive ductal carcinoma (IDC, ER+) and 48 samples of invasive ductal carcinoma (IDC, TNBC), each in duplicates, with corresponding uninvolved tissue from the same patient as control (Pantomics,

Inc., San Francisco, CA, USA; catalog number BRC481-5, BRC482, BRC483 and BRC964).

Briefly, formalin-fixed paraffin embedded tissues were baked at 55°C for 30 min, then

deparaffinized in xylene for 10 minutes, and hydrated in an ethanol gradient (100%, 95%, 80%,

70% distilled water). Tissues were steamed for 30 min in antigen-retrieval solution and then cooled and washed with 0.1% PBS-BSA solution. Tissue sections were then treated with 3% hydrogen peroxide for 20 minutes, followed by blocking in normal horse serum for 30 min at room temperature. Tissues were then incubated with rabbit pre-immune serum, or with rabbit poly- clonal antiserum against ZNF326 (1:500 dilution, 1 h Santa Cruz) at room temperature, followed by incubation with secondary antibody for 30 min, then avidin- biotin complex (ABC) for 30 min, according to the manufacturer’s instructions. The tissue microarray slides were scanned using a Aperio AT2 scanner and analyzed using the tissue image software.

Immunohistological staining of breast tissue microarrays for ZNF326 were scored blindly using the following criteria: 0 = background, 1 = Low, 2 = moderate, 3 = High positive

(Supplementary figure 1).

Statistics

All data are provided as mean ± S.E.M unless otherwise indicated. Statistical analyses were performed using an unpaired Student’s t-test, using GraphPad Prism® 6 software (Version 6.0).

Results

A two-step forward genetic screen identifies multiple triple negative breast cancer tumor

suppressor genes

TNBC has the worst prognosis of any breast cancer subtype. To better understand the genetic

forces driving TNBC, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in

phosphatase and tensin homology (Pten) mutant mice. Pten was used as a sensitizing mutation in

the screen because loss of PTEN has been implicated in breast cancer progression, is clonally

selected in TNBC, and favors the activation of the EMT pathway to promote metastasis (25,26).

The screen identified 12 candidate trunk drivers and a much larger number of progression genes,

many of which were predicted to function as tumor suppressor (TS) genes (24). To provide

additional functional validation for these genes in TNBC, we generated 120 small interfering

RNAs (siRNAs) targeting 40 SB-identified candidate TS genes (3 siRNAs per gene) and then

used them to downregulate the expression of these genes in four human TNBC cell lines (BT549,

HCC70, MAD-MB-231, MDA-MB-468) (Fig. 1A and Supplementary Table 5). We then asked

whether downregulation of these genes increased cell proliferation, since this is one of the most commonly altered processes promoting tumorigenesis observed in tumors with mutations in TS genes (27). Downregulation of seven SB-identified candidate TS genes (ADNP, AP2B1,

ARL6IP5, TOMM70A, ZC3H7A, ZCCHC7 and ZNF326) led to a >15% increase in cell proliferation with all three siRNAs tested, in each of the 4 cell lines assayed, suggesting a high probability of tumor suppressor activity (Fig. 1B).

To further confirm a role for these seven TS genes in TNBC, we used small hairpin RNAs

(shRNAs) delivered by lentivirus to silence the expression of these genes in HCC70 and MDA-

MB-231 cells (Supplementary Fig. S2A and S2B). The cells were then orthotopically injected into the mammary fat pad of athymic nude mice and, after 45 days, the animals were necropsied and tumor volumes measured (Fig. 1C). The silencing of four TS genes (ADNP, AP2B1,

TOMM70A and ZNF326) accelerated tumor growth for both cell lines tested following orthotopic transplant (Fig. 1D and 1E), confirming that they are TS genes.

ZNF326 is a tumor suppressor gene in triple negative breast cancer

Given ZNF326 had the most consistent increase in tumor growth in orthotopic transplants, we repeated the orthotopic transplants using two different shRNAs, in three different TNBC cell lines (Supplementary Fig. 3A-C) to confirm that off-target effects of the shRNA did not cause the increased tumor growth. After injecting the different tumor cell lines with different ZNF326 shRNA constructs, we observed a significantly accelerated tumor growth in nude mice (Fig. 2A-

C), confirming that off-target effects of the shRNA did not cause the increased tumor growth.

Finally, to determine whether ZNF326 overexpression would reduce tumor growth, we overexpressed ZNF326 in MDA-MB-231 cells using a lentivirus construct expressing the

ZNF326 open reading fused to a V5 tag at the 3’ end (Supplementary Fig. 3D). Tumor cells were then injected into the mammary fat pad of nude mice, where we observed a statistically significant reduction in tumor growth in cells overexpressing ZNF326 (Fig. 2D). Collectively, these results show that ZNF326 functions as a TS gene in TNBC.

ZNF326 regulates the expression of multiple genes in the epithelial-mesenchymal transition pathway

ZNF326 encodes a protein containing two C2H2-type zinc finger protein motifs and a glutamic acid-rich domain in the C-terminus. It exhibits DNA-binding activity in a zinc dependent manner and plays a role in regulating cell growth (28,29). Recently, ZNF326 has been biochemically characterized as a core component of the DBIRD complex, a multiprotein complex that acts at the interface between core mRNP particles and RNA polymerase II (RNAPII), and integrates transcript elongation with the regulation of alternative splicing (30). Moreover, siRNA studies have shown that downregulation of ZNF326 in HEK cell lines increases the expression of several epithelial-mesenchymal transition (EMT) pathway genes (30). To determine whether ZNF326 regulates the expression of EMT genes in TNBC cells, we knocked down expression of ZNF326 in HCC70 and HCC1569 cells using three independent ZNF326 shRNAs, and then used an EMT

PCR gene expression array to identify genes regulated by ZNF326. 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. This analysis identified many EMT pathway genes that were deregulated by downregulation of ZNF326 in TNBC cells (Fig. 3A, 3B). As an example, we found an upregulation of Zeb1 and Zeb2 expression, along with a concordant increase in expression of Zeb1 and Zeb2 downstream targets like cadherin-2 and vimentin, supporting a role for ZNF326 in EMT (Fig. 3A, 3B). These results show that ZNF326 regulates multiple genes in the EMT pathway in TNBC cells.

To further explore the biological effects of these expression changes, we performed migration and invasion assays using TNBC cells that have downregulated ZNF326 expression. We

observed increased migration and invasion in HCC70 and MDA-MB-231 cells with reduced

ZNF326 expression compared to control parental cells (Fig. 3C and D). These results show that

ZNF326 negatively regulates cell migration and invasion in addition to controlling the expression

of multiple genes in the EMT pathway.

ZNF326 regulates the expression of multiple genes in the cancer stem cell pathway

Interestingly, ZNF326 was also identified in a transposon screen that identified genes, which can

promote the immortalization of atroglial-like cells (22). Likewise, other studies in embryonic

stem cells have shown that ZNF326 forms complexes with Nanog-Sox2, which plays an important role in stem cell self-renewal (31). Based on these findings, we asked whether ZNF326 might also regulate genes linked to cancer stem cells (CSC) and thereby promote CSC self- renewal. We used two different ZNF326 shRNAs to downregulate ZNF326 expression in MDA-

MB-231 and HCC70 cells, and then used a CSC PCR array that profiles the expression of 84 genes linked to CSCs, to ask whether ZNF326 downregulation affects the expression of any of these genes. We observed a significant upregulation of CXCL8 and THY1 expression, and a significant downregulation of DACH1, DKK1, DLL1, GATA3 and KLF17 expression in ZNF326 shRNA-expressing cells (Fig. 4A and B, upper panels; Supplementary Fig. 4A and B). These results indicate that ZNF326 regulates multiple genes linked to CSCs.

To further explore the biological effects of these expression changes, we performed mammosphere assays using TNBC cells with deregulated ZNF326 expression. We observed an increased number of mammospheres in HCC70 (31 vs 57-60) and MDA-MB-231 (30 vs 50-52) cells with reduced ZNF326 expression, and a decreased number of mammospheres in cells with

increased ZNF326 expression (52 vs 7) (Fig 4C, 4D). We then used orthologous transplants to

determine the relationship between mammosphere formation, tumorigenicity and ZNF326

expression. We found that downregulation of ZNF326 expression in MDA-MB-231

mammospheres greatly increased tumor formation in orthologous transplants, while ZNF326

overexpression in mammospheres decreased tumor formation (Table 1). These results support

the hypothesis that ZNF326 is a TS gene that regulates CSC self-renewal by controlling the

expression of genes important for CSC self-renewal.

ZNF326 is a direct transcriptional activator of genes that regulate CSC self-renewal

To determine whether ZNF326 is a direct transcriptional activator of genes that regulate CSC

self-renewal, we asked whether ZNF326 could bind to the promoter regions of DACH1, KLF17 and GATA3 in chromatin immunoprecipitation (ChIP) assays. ChIP assays using a V5 tag antibody showed binding to multiple sites within the promoter regions of DACH1, KLF17 and

GATA3 in HCC70 and MDA-MB-231 cells expressing recombinant ZNF326-V5 tag protein, but

not in parental cells that lacked the recombinant protein (Fig. 5). To further examine the

transcriptional effects of this binding, we performed luciferase reporter assays. DACH1, KLF17

and GATA3 promoter-luciferase DNA constructs were transfected into ZNF326 knockdown or

ZNF326 overexpressing tumor cells. The promoter constructs contained 1 kb upstream of the transcriptional start site and cloned into the pLightSwitch reporter vector. Subsequent quantification of luciferase activity showed that DACH1, KLF17 and GATA3 luciferase activity

was significantly decreased in ZNF326 knockdown HCC70 or MDA-MB-231 cells compared to

control parental cells. Conversely, DACH1, KLF17 and GATA3 luciferase activity was increased

in tumor cells overexpressing ZNF326 (Fig. 6). These data support the hypothesis that ZNF326 is

a direct transcriptional activator of genes that regulate CSC self-renewal.

ZNF326 expression levels are clinically associated with patient survival

To determine whether ZNF326 expression levels are clinically associated with patient survival, we queried a publically available breast cancer database (32). We found that high ZNF326 expression is associated with increased relapse-free survival in luminal A and luminal B breast cancer patients (Fig. 7A). In TNBC, we also found a similar trend, but the level did not reach

statistical significance. To further explore the expression of ZNF326 protein in breast cancer, we

used commercially available tissue microarrays (TMAs, Pantomics). We compared 48

intraductal carcinomas (IDC) ER+, and 48 IDC TNBC tissue samples, in duplicate, and

compared them with adjacent normal tissue. Interestingly, immunohistochemical analysis

showed that intraductal (IDC) ER+ carcinomas, which are the most predominant luminal A and B

breast cancer subtypes, express high levels of ZNF326 protein compared to normal breast tissue

(Fig. 7B, 7C). In contrast, we observed a marked reduction in ZNF326 protein levels in TNBC

tumors (Fig. 7C), consistent with ZNF326 being an important tumor suppressor in this breast

cancer subtype. Similarly, DACH1 and GATA3 expression is dramatically reduced in basal-like

TNBC (Supplementary Fig. 5A, 5B), in agreement with the reduction in ZNF326 expression we

observed in TNBC in our IHC studies.

Discussion

SB mutagenesis provides a powerful tool for identifying new cancer genes, tumor biomarkers

and therapeutic targets. In previous experiments, we used SB mutagenesis to identify candidate

TS genes that could cooperate with mutant Pten in breast cancer progression (24). Here, we

functionally validated four of these genes (ADNP, AP2B1, TOMM70A and ZNF326) in TNBC

using a combination of in vitro siRNA screening and orthotopic tumor xenografts. Homeobox-

containing, activity-dependent, neuroprotective protein, ADNP, regulates OCT4 and PAX6 expression, and is essential for brain development (33,34). ADNP interacts with components of the BAF complex, the eukaryotic equivalent of the SWI/SNF complex in yeast, which is involved in the regulation of gene expression (35). In cancer cells, ADNP regulates cell growth and differentiation and, under stress conditions, exhibits expression changes leading to cancer progression (36). In contrast, in intestinal cancer cells, down-regulation of ADNP by antisense oligodeoxynucleotides up-regulates the tumor suppressor p53 and reduces the viability of intestinal cancer cells (37). ADNP may therefore also act as an oncogene in certain cellular contexts. AP2B1 is a subunit of the AP2 coat assembly protein complex involved in clathrin- mediated endocytosis. Knockdown studies in breast cancer cells have demonstrated an increase in the number of matrix degrading invadopodia, adhesion structures linked to invasive migration in cancer cells (38), suggesting that AP2B1 may control the balance between the formation of normal cell adhesions and invasive adhesion structures. Translocase of outer mitochondria membrane 70A, TOMM70A is part of a multisubunit complex involved in the recognition, unfolding and translocation of preproteins into the mitochondria. Under normal conditions,

TOMM70A negatively regulates the levels of reactive oxygen species (ROS), but in tumor cells,

ROS drives cancer progression and promotes inflammation (39).

ZNF326 was initially identified as a transcriptional activator involved in neuronal differentiation

(29). Recently, ZNF326 (also called ZNF-protein interacting with nuclear messenger ribonucleoproteins and DBC1 (ZIRD)) was also shown to bind deleted in breast cancer 1

(DBC1) and form part of a protein complex named DBIRD that binds directly to RNA polymerase II (30). DBIRD regulates alternative splicing of a large set of exons embedded in

(A+T)-rich DNA, and is present at the affected exons. Analysis of ZNF326 depleted cells showed a >1.5-fold increase in exon inclusion in >2,800 cases, whereas exon exclusion was observed in only 390 cases. Similar results were observed in DBC1 depleted cells, supporting a close functional relationship between these two proteins. The effect of the depletion was at the level of alternative spicing, as depletion of ZNF326 or DBC1 only affected the expression level of a small number of genes. While the oncogenic effects, if any, of these alternative splicing defects remain to be determined, it would not be surprising if some of the oncogenic effects seen in ZNF326 knockdown TNBC cells were promoted through these splicing defects.

We also showed that knockdown of ZNF326 in TNBC cells resulted in the deregulation of multiple genes involved in EMT, similar to what has previously been observed in ZNF326 HEK knockdown cells (30). In addition, we observed an increase in cell migration and invasion in

ZNF326 TNBC knockdown cells, both biological signatures of EMT activation.

Insertional inactivation of ZNF326 has also been associated with the immortalization of astroglial-like neural stem cells in SB mutagenesis studies (22). Moreover, in embryonic stem cells, ZNF326 has been shown to form complexes with NANOG and SOX2 and play an

important role in stem cell self-renewal (31), suggesting that ZNF326 might also function in CSC

self-renewal. Consistent with this, we found that ZNF326 is a direct transcriptional activator of

genes regulating CSC self-renewal, including DACH1, KLF17 and GATA3. We also observed an increased number and size of mammosphere formation in HCC70 and MDA-MB-231 TNBC cells with reduced ZNF326 expression, and decreased mammosphere formation and size in

TNBC cells with increased ZNF326 expression. Downregulation of ZNF326 expression in

MDA-MB-231 mammospheres also greatly increased tumor formation in orthologous

transplants, while ZNF326 overexpression decreased tumor formation. These results strongly

support the hypothesis that ZNF326 is a TS gene that regulates CSC self-renewal by directly controlling the expression of genes important for CSC self-renewal. Additional support for this hypothesis has been provided by other laboratories who have shown that DACH1 inactivation increases the number of cancer stem cells in glioma and breast cancer, while Gata3 negatively regulates cancer stem cells from luminal progenitors (40-42). Clinical studies have also demonstrated a correlation between poor prognosis in breast cancer patients and reduced DACH1 expression (43). Similar to what we observed for ZNF326 in TNBC cells, forced expression of

DACH1 in glioma cell lines also resulted in decreased proliferation in vitro and tumor growth in vivo (40). Likewise, glioma-DACH1-low cells form spheroids in serum-free medium while parental glioma cells do not. Compared with spheroid-forming glioma-DACH1-low cells, glioma-DACH1-high cells also displayed lower tumorigenicity, indicating that DACH1 decreases the number of tumor-initiating cells. KLF17 is a negative regulator of EMT and metastasis in breast cancer and a tumor suppressor in lung, liver and esophageal cancer (44-47), while GATA3 is a regulator of mammary gland morphogenesis and luminal differentiation in breast tissue

(48,49). In pathological settings, GATA3 is a marker of estrogen receptor (ER) positive breast

cancer in which high levels of GATA3 correlates with high ER expression, while low levels are

strongly associated with ER-negative, higher histological grade, lymph node positivity and a

higher propensity for metastasis (50). Finally, our immunohistochemical tissue array studies

showed high ZNF326 protein levels in luminal A and B ER+ breast cancer subtypes, in contrast to low ZNF326 protein levels in TNBC tumors, consistent with ZNF326 being an important TS gene in this beast cancer subtype. In agreement with the reduced ZNF326 expression we observed in TNBC in tissue array studies, we also showed that DACH1 and GATA3 expression is dramatically reduced in basal-like breast cancers.

In summary, using a combination of siRNA screening and orthologous tumor xenografts, we have identified multiple new TNBC tumors suppressor genes and provided evidence that one of these genes, ZNF326, is a multifunctional TS genes that controls tumor cell growth through effects on RNA splicing, EMT and CSC self-renewal. Our studies demonstrate the power of SB mutagenesis when combined with in vitro and in vivo functional validation for identifying new human TNBC TS genes.

Acknowledgments

We thank E. Freiter and H. Lee for monitoring mice and animal technical assistance and Judith

E. Markovits for the immunohistochemistry studies.

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TABLES

Figure legends

Figure 1. Validation of candidate triple-negative breast cancer tumor suppressor genes identified

by transposon mutagenesis. A, schematic representation of the siRNA proliferation screen used

for candidate TS gene validation. B, seven TS genes were identified that negatively regulate

cellular proliferation in each of the WST-1 proliferation assays. C, stable HCC70 and MDA-

MB-213 cell lines were generated containing three lentiviral shRNAs for each candidate TS gene identified in panel B and orthotopically injected into transplant mice. D, E, orthotopic tumor xenografts validated ADNP, AP2BP1, TOMM70A and ZNF326 as novel TS genes in triple- negative breast cancer. n = 5 mice per group. Error bars represent SEM. (*P < 0.05). (NC shRNA) Negative control shRNA.

Figure 2. ZNF326 is a tumor suppressor gene in triple-negative breast cancer. A-C, shRNA- induced inactivation of ZNF326 in HCC70, HCC1569 and MDA-MB-231 TNBC cells accelerated tumor growth in orthotopic xenograft assays. D, ectopic overexpression of ZNF326 inhibited tumor growth in MDA-MB-231 orthotopic xenograft assays. n =10 mice per group.

Error bars represent SEM (*P < 0.05). (NC shRNA) Negative control shRNA.

Figure 3. ZNF326 regulates the expression of multiple genes in the EMT pathway. A-B, shRNA knockdown of ZNF326 in HCC70 and HCC1569 TNBC cells resulted in the deregulation of multiple genes in the EMT pathway. Three independent ZNF326 shRNAs showed consistent mRNA expression levels over control. (*) 1.5-fold gene expression difference over control was

considered as significant. C, migration and (D) invasion assays of stable ZNF326-shRNA

knockdown HCC70 and MDA-MB-231 tumor cells. Data represents means ± SEM of three

independent experiments (*P < 0.05). (NC shRNA) Negative control shRNA.

Figure 4. ZNF326 controls the expression of genes important for cancer stem cell self-renewal.

Cancer stem cell gene expression studies in ZNF326 knockdown (A) MDA-MB-231 and (B)

HCC70 cells. Three independent shRNA ZNF326 clones showed consistent mRNA expression

level changes relative to control cells. (*) 1.5-fold gene expression difference over control was

considered as significant. C-D, number of mammosphere-forming cells in control and ZNF326

shRNA knockdown and ZNF326 overexpression MDA-MB-231 and HCC70 tumor cells,

respectively. The values are the means ±SEM from three independent experiments (* P < 0.05).

Representative microphotographs of the mammospheres formed by 500 control and 500 ZNF326

shRNA knockdown and ZNF326 overexpression tumor cells. Scale bar 100 μm. (NC shRNA)

Negative control shRNA.

Figure 5. ZNF326 binds to the promoter regions of DACH1, KLF17 and GATA3. ChIP assays confirm that ZNF326 binds to the promoter regions of (A) DACH1, (B) KLF17 and (C) GATA3.

Top panels show the promoter regions (BS1-BS4) that were assayed form ZNF326 binding, while the bottom panels show the extent of ZNF326 binding to each of the promoter regions. r, cells expressing recombinant ZNF326-V5 protein; p, parental cells lacking the recombinant

ZNF326-V5 protein. In all experiments, data represents means ± SEM of three independent experiments.

Figure 6. ZNF326 regulates DACH1, KLF17 and GATA3 expression in TNBC cells. Luciferase reporter expression assays were performed using DACH1, KLF17 and GATA3 promoters. A,

MDA-MB-231 control and ZNF326 shRNA-expressing cells; (B) MDA-MB-231 control and

ZNF326 overexpressing cells; (C) HCC70 control and ZNF326 shRNA-expressing cells; (D)

HCC70 control and ZNF326 overexpressing cells, were transfected with luciferase reporter constructs under the control of the DACH1, KLF17 or GATA3 promoters. A GAPDH promoter was used as positive control and a random control promoter (RCP) was used as a negative control. All promoters contained 1000 base pairs. In all experiments, data represents means ±

SEM of three independent experiments (*P < 0.05).

Figure 7. ZNF326 expression levels are clinically associated with patient survival. A, Kaplan-

Meier plots depicting the recurrence free survival probability of patients with different breast cancer subtypes based on ZNF326 expression levels. Luminal A (n=1764), luminal B (n=1002),

HER2 (n=208) and TNBC (n = 580). HR, hazard ratio. Log-rank test was used in the analysis. B,

ZNF326 expression levels in ER+ and TNBC. Percentage distribution of ZNF326 protein expression determine by IHC score. C, representative ZNF326 expression in normal breast epithelium, in situ ductal carcinoma (IDC), in situ lobular carcinoma and TNBC using ZNF326- specific antibodies. Scale bar 200 μm.

Identification of new tumor suppressor genes in triple-negative breast cancer

Roberto Rangel, Liliana Guzman-Rojas, Takahiro Kodama, et al.

Cancer Res Published OnlineFirst July 19, 2017.

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