FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression

Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

FOXO transcription factors both suppress and support

breast cancer progression.

Marten Hornsveld1,2,$, Lydia M.M. Smits1,2, Maaike Meerlo1,2, Miranda van

Amersfoort3, Marian J.A. Groot Koerkamp4, Dik van Leenen2, David E.A. Kloet2,

Frank C.P. Holstege4, Patrick W.B. Derksen3, Boudewijn M.T. Burgering1,2#, Tobias

B. Dansen2#

1Oncode Institute and 2Center for Molecular Medicine, Molecular Cancer

Research, University Medical Center Utrecht, Utrecht University, The

Netherlands.

3Department of Pathology, University Medical Center Utrecht, Utrecht University,

The Netherlands.

4Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

#Equal contribution.

$present address: Leiden University Medical Center, Cell and Chemical Biology

department, Leiden, The Netherlands

The authors declare that there is no conflict of interest

Correspondence: University Medical Center Utrecht Center for molecular medicine, Molecular Cancer Research Universiteitsweg 100 3584CG Utrecht, The Netherlands Tel: +31 887568918 E-mail: TBD: [email protected] & BMTB: [email protected]

Running title: FOXOs both suppress and support breast cancer progression.

Abstract

FOXO transcription factors are regulators of cellular homeostasis and putative

tumor suppressors, yet the role of FOXO in cancer progression remains to be

determined. The data on FOXO function, particularly for epithelial cancers, are

fragmentary and come from studies that focused on isolated aspects of cancer. To

clarify the role of FOXO in epithelial cancer progression, we characterized the

effects of inducible FOXO activation and loss in a mouse model of metastatic

invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function

suppressed tumor growth and metastasis. We show that the multitude of cellular

processes critically affected by FOXO function include proliferation, survival,

redox homeostasis, and PI3K-signaling, all of which must be carefully balanced for

tumor cells to thrive.

Significance

FOXO proteins are not solely tumor suppressors but also support tumor growth

and metastasis by regulating a multitude of cellular processes essential for

tumorigenesis.

Introduction

The PI3K-AKT/PKB pathway is commonly hyper-activated in cancer and

negatively regulates nuclear localization and thereby the activity of Forkhead Box

O family of transcription factors (FOXO1, FOXO3, FOXO4). When active, FOXOs

control a plethora of target genes involved in redox homeostasis, cell proliferation,

differentiation, metabolism and apoptosis (1).

FOXOs are putative tumor suppressors as combined loss of Foxo1, Foxo3

and Foxo4 in adult mice results in increased hematopoietic lineage specific tumors

and FOXO activation arrests the cell cycle and induces apoptosis or anoikis in

multiple types of tumor cells (2-5). Despite their tumor suppressive roles,

mutational loss of all FOXO alleles is statistically unlikely and has not been

reported in cancer. Apparently, AKT/PKB mediated inhibition of FOXO is either

sufficient to avoid tumor suppression, or the maintenance of a certain level of

FOXO activity is beneficial to tumor cells. It remains however to be established

what level of activity and under what conditions FOXO activity then would be

required.

It has been suggested that FOXOs may also contribute to tumorigenesis,

and a more active mutant form of FOXO1 has been reported in cancer (3,5-10)

Additionally, FOXOs have been implicated in a drug resistance signaling feedback

loop that re-establishes RTK-PI3K-AKT signaling after pharmacological inhibition

of AKT/PKB (11-13). In line with these contrasting roles for FOXOs in cancer,

studies aimed to use FOXO expression levels and localization as prognostic

markers for cancer patient survival yielded diametrically opposing results (14).

The current understanding of the role of FOXOs in cancer is clearly incomplete,

scattered over many different studies, in various types of cancer and limited to

only the effects of FOXO loss or gain of function in isolated aspects of cancer (3).

Studies designed to characterize the effects of both acute FOXO gain and loss of

function within one cancer model covering the complete disease spectrum from

tumor initiation to metastasis are lacking.

To clarify the role of FOXOs in cancer we characterized inducible FOXO

activation in a mouse model for metastatic invasive lobular carcinoma (mILC) that

allows live monitoring of both primary tumor growth and metastasis (15,16). Our

study reveals that FOXOs both support and suppress metastatic breast cancer

progression and hence are not classical tumor suppressors. FOXO activity is

actually required for efficient proliferation and dissemination, which suggests that

FOXO inhibitors might be of use as cancer therapeutics.

Materials and Methods

Cell culture

The Luciferase expressing Mouse invasive lobular carcinoma cell line (mILC1) has

been described before and was originally derived from tumors that developed in

female K14cre;Cdh1F/F;Trp53F/F mice and cultured as described before (15). The

identity of these cells was regularly checked by Luciferase expression and PCR for

CdhD/F and p53 and these cells behaved as expected in the transplantation studies.

mILC1 cells used for the transplantation assays were in culture for two weeks

prior to surgery. Cells virally transduced to express iFOXO3, iFOXO3.A3 or

shFOXOs were cultured and selected for three weeks prior to freezing. MCF7,

MDA-MB231 and SKBR3 cell lines were acquired from the ATCC and verified by

STR analysis before freezing (2013-2014). Cells were passaged two times a week

for about two months after which fresh vials were thawed. All cells used tested

negative for mycoplasma (tests were performed routinely every ~3 months).

Cells were treated with 100nM Doxycline (Dox). 200µM Trolox, 20µM EUK-134 or

1mM N-acetyl cysteine (NAC) was used for daily antioxidant treatment.

3D spheroid cultures were established in 20 µL drops containing 40% matrigel,

and 1,5mg/ml rat tail collagen I (Ibidi). For colony formation assays 2000

cells/well were plated, subsequently fixed with methanol and stained with 0.5%

crystal violet. Fresh tumor tissue digestion into single cells was performed with

collagenase and dispase. 50µg/mL neomycin and 100µg/mL primocin

(Invivogen) were used to dispense of stromal cells and avoid infection.

NADP+/NADPH ratio was determined using Amplite™ Fluorimetric

NADP/NADPH Ratio Assay Kit (AAT Bioquest).

Constructs, lentiviral transduction and transfections

Third generation packaging vectors and HEK293T cells were used to generate

lentiviral particles (17). Lentiviral cDNA expression vectors expressing FOXO3

and FOXO3.A3 were generated using Gateway cloning in the pINDUCER20 and

selected with Neomycin (Addgene #44012)(18). Foxo1 and Foxo3 knockdown

done using the lentiviral FH1tUTG Dox inducible vector (19,20). The Lentiviral

constructs pLV-CMV-PIK3CA-IRES-Puromycin, pLV-CMV-PIK3CAH1047R-IRES-

Puromycin, pCDH-myr-HA-PKB-puromycin (Addgene #46969) pLV-CMV-

RICTOR-IRES-Hygromicin and pINDUCER20-GFP-FOXO3mt were used to express

PI3K, PI3KH1047R, myrPKB, RICTOR and FOXO3mt respectively.

Immunoblotting, immunofluorescence & antibodies

For Western blot cells were lysed in sample buffer containing 0.2% m/v SDS, 10%

v/v glycerol, 0.2% v/v b-mercaptoethanol, 60mM Tris pH6.8. Protein

concentration was determined using Bradford (Biorad) and equal concentrations

of protein were used for Western blot. Proteins were detected using 6-15% SDS-

PAGE gels and subsequent Western-blot analysis with primary antibodies

(Supplemental Table 1) detected by secondary HRP conjugated. Unless stated

otherwise, non-specific bands were used as a loading control. For

immunofluorescence, cells were fixed with 3,7% Paraformaldehyde in PBS,

permeabilized with 0.1% v/v Triton in PBS and blocked with 2% m/v BSA (Sigma)

and 2% v/v NGS (Sigma). Cells were subsequently incubated with primary

antibody and visualized with Alexa-488 secondary antibody and Hoechst33342

(sigma).

Orthotopic Transplantation assays

10000 cells were orthotopically transplanted in the inguinal (4th) mouse

mammary gland of Hsd:Athymic Nude-Foxn1nu (Harlan) recipient mice as

described previously (21). Primary tumors were allowed to develop to a volume

of 50-100mm3 (~100mm3) at which point expression or knockdown of FOXOs

was induced by feeding Dox-containing chow (230mg/kg) (Ssniff). Alternatively,

FOXO expression or loss was induced 2 weeks after transplantation. Tumor

volumes and lung metastases were followed in time as described using a Biospace

φ imager (Biospace) and digital caliper (Mitotyo) (21). All animal experiments

have been conducted in accordance with the approval of the Utrecht University

Institutional Animal Care and Use Committee (DEC-ABC no. 2012.III.12.135).

Immunohistochemistry

Tissues were fixed in 4 % formaldehyde for 24 hours followed by dehydration in

ethanol and paraffin embedding. Rehydrated slides were blocked for endogenous

peroxidase activity in phosphate buffer (pH5.8) containing 1.5% hydrogen

peroxide. Antigen retrieval was performed by 20 minutes boiling in 10mM citrate

buffer (pH6). Primary and secondary HRP-conjugated antibodies were incubated

overnight or 1 hour at 4°C respectively. Staining of slides was performed using

diaminobenzidine (DAB) and hematoxylin. Relative DAB intensity was

determined using color deconvolution in ImageJ and optical density calculation.

RT-qPCR & Microarray mRNA profiling

mRNA was isolated with the Qiagen RNeasy kit (Qiagen). cDNA synthesis was

performed using the iScript cDNA synthesis kit (BioRad). Real-time PCR was

performed using SYBR green FastStart master mix (Roche) in the CFX Connect

Real-time PCR detection system (BioRad). Target genes were amplified using

specific primer pairs (Supplemental table 2) and specificity was confirmed by

analysis of the melting curves. Target gene expression levels were normalized to

Gapdh, Pbdg and Hrnpn1a levels. Genomic DNA from tumors and cell lines was

isolated using the DNeasy kit (Qiagen).

Quality control of total mRNA samples for microarray analysis was performed

using a 2100 Bioanalyzer (Agilent). The mRNA of the parental mILC1 cell line was

used as reference pool. Microarrays used were Mouse Whole Genome Gene

Expression Microarrays V1 (Agilent Technologies) and analyzed using MAANOVA.

Raw microarray data will be made publicly available (GSE#). p-value is calculated

after family-wise error correction (FWER) and 10000 permutations, p-values

<0.05 were considered significant. Pathway activity prediction was performed

with all significantly changed genes with a 2log change of >0.5 using the Ingenuity

Pathway Analysis platform. z-scores indicate the predicted pathway activity based

on gene expression levels of genes annotated to be functionally involved in the

pathway (qiagenbio-informatics.com/products/ingenuity-pathway-

analysis)(22). The microarray data have been deposited at the GEO repository

under accession number GSE108842. To review GEO accession GSE108842:

Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE108842

Enter token urmlkyauxxqjjgz into the box

Flow cytometry

Cells were stained for apoptosis with Annexin-V-FITC (IQ Products) and

propidium iodide (Sigma-Aldrich) and quantified using either a FACSCalibur or a

FACSCelesta flow cytometer (BD Biosciences). Anoikis was assessed by culturing

50000 cells/mL for 24 hours in 6-well ultra-low cluster polystyrene culture dishes

(Corning) prior to apoptotic analysis as above. Oxidative stress was assessed by

treatment for 30 minutes with 5µM CellRox deep red (Thermofisher) or CM-

H2DCFA (Thermofisher) and cytometric analysis. Cell cycle profiles were

generated based on DNA content as measured by Propidium Iodide staining after

fixation with ethanol and RNAse treatment.

Live cell imaging and tracking

Cells were cultured in Lab-Tek II 8-well imaging chambers and treated with Dox

72 hours prior to imaging. mILC1-iFOXO3.A3 cells were treated with Dox 8 hours

prior to imaging. DIC imaging was performed on an Olympus Real-Time imager

microscope for 48 hours at 37°C and 5% CO2. Cell tracking and quantification of

cell migration was performed using ImageJ.

Results

FOXO3 activation represses tumor growth and metastasis

To determine how FOXO levels and activity affect invasive breast cancer we

established mILC1 cell lines constitutively expressing luciferase, combined with

either Dox-inducible wild-type FOXO3 (iFOXO3) or the AKT/PKB resistant,

constitutively active mutant FOXO3.A3 (iFOXO3.A3). Since individual FOXOs

function is largely redundant and FOXO3 is most ubiquitously expressed we opted

for this paralog. High FOXO3 and FOXO3.A3 expression was observed in mILC1-

iFOXO3 and mILC1-iFOXO3.A3 cells after Dox treatment. As expected,

phosphorylation of the threonine-32 PKB/AKT site in FOXO3 was only detected

on FOXO3 and not on FOXO3.A3 (Figure 1A). FOXO3 was largely sequestered in

the cytoplasm and FOXO3.A3 was localized in the nucleus, confirming that

Akt/Pkb negatively regulates FOXO3 under basal conditions in mILC1 cells

(Figure 1B). Colony outgrowth was impaired in both mILC1-iFOXO3 and mILC1-

iFOXO3.A3 cells (Figure 1C). No significant increase in apoptosis was measured in

both mILC1-iFOXO3 and mILC-iFOXO3.A3 cells after 24 hours (Figure 1D).

Induction of FOXO3.A3 for 48 hrs did however induce a G1 cell cycle arrest (Figure

1E). A small increase in G1 cells was observed in mILC1-iFOXO3 cells. Expression

of the FOXO target Cdkn1b/p27 was detected after both FOXO3 and FOXO3.A3

induction (Figure 1F). Combined, these results indicate that colony formation is

restricted upon FOXO3 expression through cell cycle inhibition. In case of iFOXO3

the cell cycle effect appears temporary despite p27 induction after 16 hours.

Colony outgrowth is more efficient compared to mILC1-iFOXO3.A3 cells and only

a mild G1 cell cycle arrest is observed after 48 hrs. This could be attributed to the

observed high Akt/Pkb mediated phosphorylation and inactivation of FOXO3.

These results confirm that the well-described roles of FOXOs in regulation of the

cell cycle are conserved in mILC1 cells.

Orthotopic transplantation of mILC1 cells is a robust model for mILC and

the aggressive nature of this tumor type allows studying both primary tumor

growth and formation of metastasis. In order to verify the observed tumor

suppressive effects of FOXO3 and FOXO3.A3 expression in vivo, we orthotopically

transplanted 10.000 cells into the inguinal mammary glands of recipient mice.

Expression of FOXO3 and FOXO3.A3 was subsequently induced by feeding mice

Dox-containing chow either early in tumor growth (2 weeks after surgery) or at a

later stage at which tumors reached a size of ~100mm3 (measured weekly). Dox

alone has been shown not to influence mILC1 tumor growth in this model (4).

No effect on primary tumor growth was observed upon induction of FOXO3

(Figure 1G), whereas early expression of FOXO3.A3 did hamper initial primary

tumor outgrowth. Late expression of FOXO3.A3 resulted in collapse of the tumor

(Figure 1H). However, primary tumor growth was re-established upon late

FOXO3.A3 expression, despite continuous Dox administration.

Immunohistochemical analysis revealed that reduction of tumor volume

correlated with inhibition of cell proliferation (Ki67) and induction of apoptosis

(cleaved caspase 3) (Supplemental Figure 1A).

Detection of luciferase activity in the mouse outside the primary tumor

location, primarily the lungs, was used to determine metastasis of mILC1 tumors.

No significant difference in metastasis free survival was detected for mice bearing

tumors in which FOXO3 or FOXO3.A3 was induced during early tumor growth. In

contrast, late FOXO3 or FOXO3.A3 induction significantly delayed metastasis

detection (Figure 1I&J). The average primary tumor volumes were significantly

larger when metastases were first detected in Dox treated mice (Supplemental

Figure 1B&C). This suggests that FOXO3 activation not only restrained primary

tumor growth and thereby delayed the onset of metastasis, but that tumors also

needed larger volume and cell numbers for metastasis to arise. Together these

results show that AKT/PKB mediated FOXO inhibition facilitates both cancer

growth and metastasis.

mILC1-iFOXO3.A3 tumors adapt to FOXO3.A3 expression.

Although induction of FOXO3.A3 in mILC1 initially led to reduced tumor growth

and delayed metastasis, these effects were temporary, which suggests that mILC1-

iFOXO3.A3 cells either adapt to high FOXO3.A3 levels or that there is negative

selection for cells expressing high FOXO3.A3 levels. To test this, we established

cell lines from mILC1-iFOXO3.A3 cells isolated from untreated, early and late

treated tumors. mILC1-iFOXO3.A3 cell lines established from Dox treated mice

overcome the FOXO3.A3 mediated inhibition of colony formation. In contrast,

colony formation was still abolished upon FOXO3.A3 induction in cells isolated

from mILC1-FOXO3.A3 tumors from untreated mice. All these cell lines had

retained neomycin resistance, indicating that the pINDUCER20-FOXO3.A3

construct was still present (Figure 2A). This was further confirmed by

quantification of the relative amount of viral integrations of the construct in the

genome of cell lines established from the tumors from all groups. Strong selection

for cells carrying low amounts of viral integrations of the FOXO3.A3 construct was

observed in Dox treated tumors (Figure 2B), but the construct was indeed not

completely lost and super-endogenous levels of FOXO3.A3 protein were still

observed after Dox treatment (Figure 2C). Similar selection for low, but super-

endogenous levels of FOXO3.A3 expression was observed in mILC1-iFOXO3.A3

cells cultured in the presence of Dox for 3 weeks (Figure 2D). Although expression

levels of FOXO3.A3 are low in cells isolated from mILC1 tumors continuously

treated with Dox, FOXO3.A3 localization was still capable of nuclear accumulation

(Figure 2E).

Taken together, we observed a strong selection for cells expressing low

(but higher than endogenous Foxo3) levels of FOXO3.A3, we did however not find

tumors that lost inducible FOXO3.A3 expression altogether.

FOXOs support primary tumor growth and metastasis formation

As outlined above, our data confirmed that hyper-activation of FOXOs suppresses

tumor growth and metastasis in vivo, but also suggests that complete loss of FOXO

activity is not selected for during tumor progression. We therefore set out to

determine the effects of induced FOXO loss in mILC. As FOXO transcription factors

are highly redundant in their function we first determined the endogenous

expression levels of Foxo1, Foxo3 and Foxo4 in mILC1. Both Foxo1 and Foxo3 were

expressed but Foxo4 mRNA was hardly detected (Supplemental figure 2A).

Conveniently, Foxo1 and Foxo3 share sufficient sequence overlap that they can be

targeted by the same shRNA (Supplemental figure 2B (20,23,24). As shRNA#3 is

predicted to be the most preferred shRNA based on targeting of both mouse and

human FOXOs with the least off-targets, we expressed shRNA#3 in a Dox-

inducible fashion in mILC1 cells (shFOXOs)

(portals.broadinstitute.org/gpp/public). Efficient knockdown of both Foxo1 and

Foxo3 was observed upon activation of shFOXOs expression (Figure 3A &

Supplemental figure 2B&C). Several phenotypes described further in this study

using shRNA#3 below were corroborated using shRNA#1 and shRNA#2

(Supplemental figure 2D-G). Although Foxo knockdown reduced colony

outgrowth (Figure 3B & Supplemental figure 2D), it did not induce apoptosis or

lead to a cell cycle arrest (Figure 3C&D Supplemental figure 2E). However, we

observed an overall increase in cell cycle time as measured by video time-lapse

microscopy (Figure 3E). Whereas Cdkn1b/p27 was induced upon FOXO3

activation, inactivation of FOXOs in mILC1 cells resulted in the expression of

Cdkn1a/p21 indicating that FOXO loss and FOXO activation block cell cycle

progression through different mechanisms (Figure 3F).

To determine the effect of FOXO loss in vivo, we orthotopically transplanted

mILC1-shFOXOs cells into the inguinal mammary gland of recipient mice. As it was

previously reported that shLuc expression does not affect tumor growth in our

mILC1 model, we did not include this in the current study (21). Early FOXO loss

hampered tumor initiation strongly, delaying primary tumor growth by

approximately 4 weeks. Late expression of shFOXOs in primary tumors of

~100mm3 resulted in efficient but temporary growth inhibition (Figure 3G).

Immunohistochemical analysis of Foxo1, Ki67, phospho-Histone H3 and cleaved

caspase3 levels in mILC1-shFOXOs tumors treated with Dox for 7 days after the

tumors had reached ~100mm3, revealed that the inhibition of primary tumor

growth correlates with reduced proliferation (Supplemental figure 3A).

Strikingly, the detection of metastasis was significantly delayed when

FOXO was knocked down early in tumor growth compared to control tumors. Late

FOXO knockdown in established tumors delayed detectable metastasis in mice

that had not presented with metastasis up to that point (Figure 3H). Average

primary tumor volumes were significantly larger when metastases were first

detected in Dox treated mice, indicating that loss of Foxo1 and Foxo3 expression

not only restrained primary tumor growth but also metastasis formation

(Supplemental Figure 3B). Together these results show mILC1 tumors initially

depend on the presence of Foxos for both efficient growth and metastasis.

Tuning FOXO activity is required for efficient cancer cell growth

Similar to mILC1-FOXO3.A3 tumors, the suppressive effects on tumor growth

observed in mILC1-shFOXOs tumors were temporary. We quantified the relative

amount of viral integrations of the FH1tUTG-shFOXOs construct in the genome of

mILC1-shFOXOs cells isolated from untreated, early and late treated tumors by

real-time qPCR. In contrast to mILC1-iFOXO3.A3 tumors, quantification of viral

integrations in the genome of mILC1-shFOXOs tumor cells showed that only in

early treated tumors selection for cells with significantly lower amounts of viral

integrations occurred (Figure 4A). GFP expression levels corresponded to the

relative amount of viral integrations (Figure 4B). Efficient Foxo1 and Foxo3

knockdown was still induced in Dox treated mILC1-shFOXOs cells derived from

the Dox treated tumor groups, however, slightly higher residual FOXO levels were

detected in cell lines derived from early treated tumors (Figure 4C). Similar to the

cell lines isolated from tumors, mILC1-shFOXOs cells cultured in the constant

presence of Dox for 3 weeks still showed efficient FOXO knockdown (Figure 4D)

Interestingly, colony forming capacity of tumor cell lines carrying lower amounts

of viral integrations was no longer impaired, suggesting that a slight reduction in

knockdown efficiency, and therefore residual Foxo1 & Foxo3 expression, is already

sufficient to overcome the tumor suppressive effect of FOXO loss (Figure 4E). In

order to test whether our findings apply also to human breast cancer we

established MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines carrying the

shFOXOs and iFOXO3.A3 inducible constructs and found that similar to mILC1

cells, human breast cancer cells are also sensitive to both loss and over-activation

of FOXO (Figure 4F-I)

As our results suggest that mILC1 cells could rely on balancing FOXO

activity to thrive, we determined whether there is indeed a window of FOXO

expression that supports the growth of mILC1, MCF7, SKBR3 and MDA-MB-231

cells. To this end we titrated Dox in shFOXOs and iFOXO3.A3 cells, to gradually

change FOXO levels. Indeed, we find the highest colony formation capacity when

FOXO levels are near or slightly above the endogenous level (Figure 4J-L). In

contrast to the common idea that FOXOs are tumor suppressors, our results now

establish that breast cancer cells rely on the presence of FOXOs for efficient tumor

growth and metastasis.

FOXOs provide tumor cells with motility, invasiveness and anoikis resistance

Since our data indicate that cancer cells preferably retain FOXOs, we sought to

determine in which processes and steps of tumor progression FOXOs are most

important. For cells to become metastatic they need to be motile and able to

survive dissemination from the primary tumor. FOXOs have been suggested to be

involved in regulating cell motility and migration in breast and colon cancer cells

(6,8). In contrast, others and we have previously shown that FOXO activation

induces anoikis in cancer cells (4,25). We therefore determined whether the

observed decrease in metastatic potential upon both loss and gain of FOXO activity

correlates with changes in cell migration or prevention of anchorage independent

survival.

Live tracking microscopy of mILC-shFOXOs and mILC1-iFOXO3.A3 in 2D

culture showed a significant inhibition of cell motility upon loss but not upon

activation of FOXO (Figure 5A). Large multicellular structures that invade into the

matrix are formed when mILC1, MDA-MB-231 and SKBR3 cells grow as spheroids

in a mixture of matrigel and collagen. Loss of FOXO results in a marked reduction

of this invasive growth whereas forced expression of FOXO3.A3 abolishes

spheroid formation or leads to the collapse of the 3D structure when induced after

the formation of invasive protrusions (Figure 5B & Supplemental figure 4).

It was previously shown that FOXO hyper activation can induce anoikis (4),

but the effect of FOXO loss on anoikis has not been studied thus far. Anoikis was

increased in mILC1-shFOXOs cells when transferred to suspension after 72 hours

of FOXO knock down (Figure 5C & Supplemental figure 2F). Similar to mILC1,

anoikis resistance of MDA-MB-231 and SKBR3 was abolished upon loss of FOXOs

(Figure 5D). Anoikis induction in response to FOXO3 hyper activation involves the

upregulation of Bim and Bmf expression. These FOXO targets are indeed down-

regulated upon FOXO knockdown in mILC1, MCF7, MDA-MB-231 and SKBR3 cells

and hence does not explain the concurring increase in anoikis (Figure 5E-G). The

expression levels of the anti-apoptotic Bcl2 and Bcl-xl were however also reduced

after FOXO knockdown, which could underlie the observed sensitivity to anoikis

(Figure 5E-G). Indeed, exogenous expression of BCL2 or BCL-XL in mILC1-

shFOXOs and mILC1-iFOXO3.A3 re-established anoikis resistance in these cells

(Figure 5H-J). Collectively, these results show that mILC1 cells rely on the

presence of FOXOs in order to invade and circumvent anoikis, providing a

rationale for the observed reduction in metastasis upon loss of FOXO in vivo. How

FOXOs influence invasion and migration at the molecular level remains to be

elucidated. Changes in canonical EMT and migration markers (5) in a FOXO-

dependent manner were not observed as discussed below.

FOXOs maintain cancer cell redox homeostasis

Anchorage independent survival in cancer is dependent on active growth factor

signaling and the ability to maintain redox homeostasis (26,27). FOXOs play key

roles in redox signaling and redox homeostasis (9,28-30). FOXO knockdown led

to induction of stress activated protein kinase (JNK and p38) activity, suggesting

that FOXO loss leads to elevated ROS levels (Figure 6A). Next, we determined

whether stress kinase activation correlates with increased levels of ROS. CellRox

fluorescence intensity was increased after FOXO knockdown indicating that ROS

levels increase upon FOXO loss (Figure 6B). Similar to mILC1, loss of FOXOs

resulted in increased ROS levels in MCF7, MDA-MB-321 and SKBR3 cells

(Supplemental Figure 5A). The NADPH/NADP+ ratio decreased after FOXO

knockdown supporting the notion that an overall more oxidative environment

prevails in cells after loss of FOXO (Figure 6C). Interestingly, no changes in the

expression of FOXO target genes Catalase and Sod2 were detected, suggesting that

the redox imbalance is not caused by reduced expression of proteins involved in

ROS detoxification (Supplemental figure 5B-D).

Although FOXOs clearly affect redox homeostasis in mILC1 cells,

compounds that act to boost antioxidant function in various ways, e.g. Trolox (free

radical species scavenger), N-acetyl cysteine (GSH precursor) or EUK134

(Superoxide and hydrogen peroxide scavenger), did not rescue cell proliferation

or anoikis induction by FOXO loss (Figure 6D&E). Taken together, these data

indicate that although FOXOs are essential for cellular homeostasis in mILC1, the

role of FOXOs in cancer is evidently more complex than just providing tumor cells

with sufficient reductive capacity.

FOXOs tune growth factor signaling activity

Growth factor signaling is fundamental to tumor progression and anchorage

independent survival is strongly dependent on RAS and PI3K pathway activity

(4,25,26,31). Although the precise regulation of growth factor feedback signaling

by FOXO remains to be fully characterized, it is clear that FOXOs are key regulators

of PI3K-pathway activity in response to pharmacological inhibition of PI3K (11-

13,32). We therefore set out to determine the effects of FOXO activation and loss

on PI3K-pathway activity.

Expression of FOXO3.A3 increases PI3K-PKB activity, whereas loss of FOXO

correlated with a marked reduction in Pkb-S308 and Pkb-S473 phosphorylation

(Figure 7A and Supplemental Figure 2G). To validate these observations in vivo we

stained tissue sections from mILC1-iFOXO3.A3 and mILC1-shFOXOs tumors for

Pkb and Pkb-S473 phosphorylation. Induction of FOXO3.A3 or shFOXOs

correlated with increased and reduced levels of Pkb-S473 respectively (Figure

7B). FOXO activation induced mRNA expression of a panel of growth factor

signaling feedback components including Met, Erbb2, Erbb3, Insr, Igf-1r, Pdgfra,

Fgfr1, Fgfr3, Irs1, Irs2, Rictor, Sesn1 and Sesn3 (Figure 7C). FOXO knockdown

indeed resulted in decreased mRNA expression of most of these gene transcripts.

(Figure 7C). Western blot analysis confirmed that FOXOs control the protein

expression levels of Met, Igf-1r, Insr, Erbb2, Erbb3, Irs1, Irs2, Rictor and Pdgfra

(Figure 7D). The role of FOXOs as important mediators of PI3K-PKB feedback

signaling was confirmed in MCF7, MDA-MB-231 and SKBR3 human breast cancer

cells (Supplemental figure 6A&B).

Co-expression of an shRNA-insensitive mutant of wild-type FOXO3

(FOXO3mt) in mILC1-shFOXOs both restored PKB activity and prevented stress

kinase activation (Supplemental figure 7A). Colony formation was however not

rescued, which is in line with our observations in Figure 1C that FOXO

overexpression blocks proliferation. This suggests that in the context of high FOXO

expression the ensuing high PI3K/PKB activity is not sufficient to bypass the FOXO

mediated arrest.

To test whether loss of active PI3K/PKB underlies the inhibition of colony

formation upon shFOXO expression, constitutively active variants of PI3K

(PI3KH1047R), PKB, (myrPKB) or RICTOR were co-expressed. This could however not

rescue shFOXO mediated anoikis and proliferation, even in the presence of

antioxidants (Supplemental figure 7B-G). These results suggest that FOXO

controls other crucial survival pathways PI3K/PKB signaling and the cellular

redox state.

Indeed, microarray analysis of mRNA expression changes in mILC1-

shFOXOs and mILC1-iFOXO3.A3 revealed that the expression levels of thousands

of genes are influenced by FOXOs, including the most well established FOXO target

genes (Supplemental figure 8A)(33,34). Subsequent pathway prediction analysis

using all significantly changed genes including non-FOXO target genes,

underscores that many pathways and processes additional to the above studied

cell proliferation, apoptosis, stress and RTK pathways in cancer cells are

attenuated upon aberrant FOXO expression (Supplemental Figure 8A-E &

Supplemental Table 3). Of note, and in line with the observed loss of viability in

the context of both shFOXOs and iFOXO3.A3, several pathways are regulated in

the same direction upon loss and overexpression of FOXO. Despite the earlier

described roles for FOXO in cell migration and EMT (5) we found no evidence of

transcriptional control of these programs by FOXO (Supplemental figure 8F)

Our findings suggest that carefully tuning FOXO activity is essential for

tumor cells to thrive, as ectopic hyper activation, complete loss of FOXO or hyper

activation of GFR signaling all hamper tumorigenesis due to imbalances in

proliferation, survival, migration, redox homeostasis and PI3K-pathway activity.

(Figure 7E).

Discussion

FOXOs both support and suppress cancer progression

This study for the first time characterizes the effects of both induced FOXO

activation and loss during tumor progression in a model for epithelial cancer (i.e.

metastatic breast cancer). We show that FOXO3 has tumor suppressive potential

at various stages of cancer in this model. Elevated FOXO3 levels had no effect on

primary tumor growth but did delay metastasis. The expression of constitutively

nuclear and hyperactive FOXO3.A3 strongly affected both primary tumor growth

and metastasis, underlining that attenuation of FOXO activity by AKT/PKB is

essential for tumor cells to efficiently proliferate and survive. Cells expressing high

levels of FOXO3.A3 were negatively selected for, but cells with levels higher than

endogenous FOXO3 were retained in the tumor meaning that FOXO activity is not

tumor suppressive per se and could contribute to tumor growth or survival.

Indeed, induced loss of FOXOs revealed that mILC1 tumors are also highly

dependent on endogenous FOXOs for tumor growth, metastasis, and cellular

homeostasis. These observations strongly argue against a general role for FOXOs

as classical tumor suppressors. Interestingly, it takes longer for early treated

mILC1 tumors to grow out upon FOXO loss than upon FOXO3.A3 overexpression,

suggesting that loss of FOXO is a more complex problem for mILC cells to

overcome. Based on these observations we conclude that FOXOs can both

suppress and support tumor progression and that tuning FOXO activity is

essential for tumor growth and metastasis. The key observations in this

manuscript were corroborated in human breast cancer cell lines, suggesting that

the conclusions can be extended to human cancer.

FOXOs in metastasis formation.

An unexpected finding in this study is that both loss and gain of FOXO activity

hampers metastasis formation. A potential explanation might be that metastasis

is delayed in both situations because primary tumor growth is also delayed and

that a tumor needs to reach a certain size or stage before it can metastasize.

However, primary tumor volume at the time of the first detected metastasis was

larger both upon FOXO loss and gain of activity (Supplemental Figures 1 and 3),

arguing against this explanation. Another rationale could be that these

observations may stem from the different roles of FOXO at different stages of

metastasis. It could be that that both the anoikis promoting as well as the anti-

proliferative effects of FOXO3.A3 hyper activation may hamper both

dissemination of tumor cells and efficient outgrowth of distant metastasis to form

detectable tumors, whereas loss of FOXO prevents efficient survival during EMT

due to its roles in motility, invasion, anchorage independent growth, PI3K

feedback signaling and cellular redox homeostasis. Adverse conditions like high

levels of reactive oxygen species or nutrient depletion (as has been reported to

occur during EMT) have been reported to activate FOXOs even in the presence of

growth factor signaling (35-37) to maintain homeostasis. It is therefore not

unlikely that loss of these FOXO functions delay the metastasis in the mILC1-

shFOXOs tumors. An observation that somewhat supports this idea is that late

induction of loss of FOXO in mILC1-shFOXOs tumors seems to delay metastasis

efficiently, but in only about half of the mice (Figure 2H). It could be that in the

other mice EMT and establishment of (not yet detectable) micro-metastasis had

already occurred and that these are less dependent on FOXO activity for survival.

Interestingly, expression of genes involved in canonical EMT and invasion in

response to FOXO activity did not change in a way that could explain the observed

effects on invasion and migration (5,8,38). This further strengthens the idea that

the role of FOXO in metastasis lies more in regulation of survival during anchorage

independent growth, PI3K-PKB feedback signaling and proliferation and

outgrowth of distant metastasis. It could however be that FOXOs regulate EMT at

earlier stages of tumorigenesis. After all, the mILC1 breast cancer model is already

prone to metastasis before FOXO3.A3 or shFOXO expression is induced.

Due to the role of FOXO in a wide variety of cellular processes, it can be

expected that our attempts to re-establish proliferation and anoikis resistance

after FOXO knock down with antioxidants and/or growth factor signaling

components were unsuccessful in tissue culture experiments. Likely, the right

balance of induction of processes otherwise regulated by FOXO must be found, a

complex process that is difficult, if not impossible, to faithfully mimic using

experimental conditions. It could for instance be that tumor cells benefit most

from oscillating or regulatable levels of FOXO activity to maintain (redox)

homeostasis and PI3K-PKB feedback signaling without the induction of growth

arrest.

Targeting FOXOs in cancer?

FOXOs regulate a variety of vital cellular processes and it therefore may

seem not surprising that tumor cells retain FOXO activity. However, most, if not

all, tumor cells have loss of function of the p53, also a transcription factor that

besides its role as a tumor suppressor also has a multitude of functions in cellular

homeostasis, many of which overlap with FOXO (39). It would therefore be

interesting to study to what extent tumor cells rely on FOXO activity when, unlike

in mILC1 cells, p53 function is intact. It is tempting to speculate that in the absence

of p53, tumor cells could rely more on FOXO activity. Since, unlike p53, mutational

loss of all FOXOs has not been reported in cancer (40), this would make FOXO an

interesting therapeutic target. Our observations that loss of FOXO is detrimental

to tumor growth and metastasis were unexpected and might go against the

rationale for the development of several small molecules that aim to promote

FOXO activity either directly (41) or through inhibition of PI3K/AKT for cancer

therapy (42,43). Although we do not currently know how our observations on the

requirement for FOXO extend to other cancer types it is tempting to speculate that

compounds that can inhibit FOXOs could be effective in cancer treatment. Of note,

recently efforts have been made to make such FOXO directed compounds for the

treatment of type II diabetes, illustrating that the development of FOXO inhibitors

is certainly possible(44,45).

Acknowledgements

This work was supported by Dutch Cancer Society (KWF Kankerbestrijding)

grants UU2014-6902 and UU2009-4490 to TBD and UU2014-7201 and UU2017-

10456 to PWBD. BMTB, MH, AMMS and MM are part of the Oncode Institute which

is partly financed by the Dutch Cancer Society (KWF Kankerbestrijding) and was

funded by the gravitation program CancerGenomiCs.nl from the Netherlands

Organisation for Scientific Research (NWO). We are grateful for input from our

colleagues from the Dansen and Burgering labs. The authors declare no conflict of

interest.

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Figure Legends

Figure 1: FOXO3 activation inhibits tumor growth and metastasis

A FOXO3 expression and FOXO3-T32 phosphorylation in mILC1-iFOXO3 and

mILC1-iFOXO3.A3 cells (16 hours Dox). B Immunofluorescence staining for

FOXO3 (white) and DNA/Hoechst (Blue) in mILC1-iFOXO3 and mILC1-FOXO3.A3

cells (8 hours Dox). C Colony formation capacity of mILC1-iFOXO3 and mILC1-

iFOXO3.A3 cells (7 days Dox (typical result). D Apoptosis in mILC1-iFOXO3 and

mILC1-iFOXO3.A3 (24 hours Dox). Data represent the average of 3 experiments

±SD. E Cell cycle profile of mILC1-iFOXO3 and mILC1-iFOXO3.A3 cells before and

after 48 hours Dox. Shown is a representative histogram and the average

percentage of cells with 2n DNA (G1) (n=3) ±SD F Cdkn1a and Cdkn1b protein

levels in mILC1-iFOXO3 and mILC1-iFOXO3.A3 cells (8 or 16 hours Dox). G & H

Primary tumor growth of orthotopically transplanted mILC1-iFOXO3 and mILC1-

iFOXO3.A3 cells. Black lines indicate untreated mice (n=10), green lines indicate

early (n=10, green arrow indicates start of treatment) and blue lines indicate late

Dox treated mice (n=10, blue arrow indicates start of treatment). Data represent

the mean tumor volume ±SEM, Holm-Sidak corrected t-test p<0.005 =**, p<0.0005

=***. I & J Kaplan-Meier curve representing metastasis free survival, measured in

days until metastasis was detected by bioluminescence imaging in control mice

(n=10), early (n=10, green arrow indicates start of treatment) and late Dox treated

mice (n=10, blue arrow indicates start of treatment), Mantel-Cox test mILC1-

iFOXO3 p=0.0015 (**), mILC1-iFOXO3.A3 p<0.0001=***.

Figure 2: mILC1-iFOXO3.A3 tumors adapt to FOXO3.A3 expression

A Colony formation capacity of mILC1-iFOXO3.A3 tumor cells isolated from

control (tumor#2 & 3), early (tumor#5 & 6) and late Dox treated tumors (tumor#8

& 9) cultured in the presence of Dox (Dox) or Neomycin (Neo) for 7 days. B

Quantitative PCR analysis of genomic DNA for viral integrations of pINDUCER-20-

iFOXO3.A3 in tumor cells isolated from control (tumor# 1, 2 & 3), early (tumor#4,

5 & 6) and late Dox treated tumors (tumor#7, 8 & 9). Light grey bars represent

relative integration levels of the rtTA3G-IRES-Neo cDNA contained in the

pINDUCER20 construct. Dark grey bars represent relative integration levels

FOXO3.A3 cDNA contained in the pINDUCER20-iFOXO3.A3 construct (*= t-test

p<0.05). C Foxo3 protein levels in corresponding tumor cells (24 hours Dox). D

Western blot analysis of Foxo3 levels in mILC1-iFOXO3.A3 cells (24 hours or 3

weeks Dox) E Immunofluorescence staining for FOXO3 (white) and DNA/Hoechst

(Blue) of the mILC1-iFOXO3.A3 parental cell line either treated with Dox for 16

hours or continuously for 3 weeks and mILC1-FOXO3.A3 tumor derived cells

treated with Dox for 16h. Both equal and optimal exposure settings are shown to

compare both levels and localization. Yellow arrows indicate cells with nuclear

localization of FOXO3.

Figure 3: FOXOs are essential for tumor growth and metastasis

A Foxo1 and Foxo3 protein levels in mILC1-shLuc and mILC1-shFOXOs cells after

24, 48 and 72 hours of Dox. B Colony formation capacity of mILC1-shLuc and

mILC1-shFOXOs cells (7 days Dox, typical result) C Apoptosis in mILC1-shLuc and

mILC1-shFOXOs cells (72 hours Dox). Data represent the average of 3 experiments

±SD. D Cell cycle profiles of mILC1-shFOXOs cells treated with Dox for 72 and 96

hours. Shown is a representative histogram and the average percentage of cells

with 2n DNA (G1) (n=3) ±SD E Quantification of time-lapse microscopy imaging of

the cell cycle time in untreated or 72 hours Dox treated mILC1-shFOXOs cells. F

Cdkn1a/p21 and Cdkn1b/p27 protein levels in mILC1-shLuc and mILC1-shFOXOs

cells after 24, 48 or 72 hours of Dox. G Primary tumor growth of mILC1-shFOXOs

cells in mice. Black lines indicate untreated mice (n=10), green lines indicate early

(n=10, green arrow indicates start of treatment) and blue lines late Dox treated

mice (n=10, blue arrow indicates start of treatment). Data represent the mean

tumor volume ±SEM, Holm-Sidak corrected t-test p<0.005 =**, p<0.0005 =***. H

Kaplan-Meier curve representing metastasis free survival, measured in days until

metastasis were detected by bioluminescence imaging in control mice (n=10),

early treated (n=10, green arrow indicates start of treatment) and late Dox treated

mice (n=10, blue arrow indicates start of treatment), Mantel-Cox p<0.0003 (***).

Figure 4: Tuning FOXO activity is critical for tumor maintenance

A Quantitative PCR analysis of genomic DNA for viral integrations of FH1tUTG-

shFOXOs in tumor cells isolated from tumors of mILC1-shFOXOs control (Tumor#

10, 11 & 12), early (tumor#13, 14 & 15) and late Dox treated tumors (tumor#16,

17 & 18). Relative integration levels of the GFP-T2A-tTA cassette (light grey bars)

and the region around the shFOXO sequence (dark grey bars) in the FH1tUTG-

shFOXOs construct (*= t-test p<0.05). B GFP protein levels in corresponding

tumor cell lines. C Foxo1 and Foxo3 protein levels in corresponding tumor cells

(72 hours Dox). D Foxo3 protein levels in mILC1-shFOXOs cells (72 hours or 3

weeks Dox). E Colony formation capacity of mILC1-shFOXOs tumor cells isolated

from control, early and late Dox treated tumors, cultured in the presence of Dox

for 7 days. F-H FOXO1 and FOXO3 protein expression in MCF7-

shluc/shFOXOs/iFOXO3.A3, MDA-MB-231-shluc/shFOXOs/iFOXO3.A3, SKBR3-

shluc/shFOXOs/iFOXO3.A3 human breast cancer cells (48 and 72 hours/16 hours

Dox). Below: Colony formation capacity of MCF7-shluc/shFOXOs/iFOXO3.A3,

MDA-MB-231-shluc/shFOXOs/iFOXO3.A3, SKBR3-shluc/shFOXOs/iFOXO3.A3

cells after 7 days of Dox (typical result). I-L FOXO3 protein levels and colony

formation capacity of mILC1/MCF7/SKBR3/MDA-MB-231-shFOXOs and -

iFOXO3.A3 cells treated with a range of different Dox concentrations (24 hours for

protein samples, 7 days for colony assay).

Figure 5: FOXOs support cell motility, invasion and anchorage independent

survival.

A Cell migration speed in µm/frame (5 min) determined by live tracking

microscopy of mILC1-shFOXOs (72 hours Dox) and mILC1-FOXO3.A3 cells (8

hours Dox). (****= t-test p<0.0005) B Spheroid cultures of mILC1-shFOXOs and

mILC1-FOXO3.A3 in Matrigel containing collagen I cultured in the presence of 1 or

7 days Dox. C Anoikis in mILC1-shLuc, mILC1-shFOXOs (72 hours Dox prior to

transfer to suspension) and mILC1-iFOXO3 and mILC1-iFOXO3.A3 cells (24 hours

Dox in suspension). Data represent the average of 3 experiments ±SD (*= t-test

p<0.05). D Apoptosis analysis of MCF7-shFOXOs/iFOXO3.A3, MDA-MB-231-

shFOXOs/iFOXO3.A3, SKBR3-shFOXOs/iFOXO3.A3 cells after 72 hours hours

cultured under adherent (adh) and 24h suspension (sus) conditions. A

representative experiment is shown with the average of 3 technical replicates

±SD. E RT-qPCR analysis of Bcl2-family gene expression levels in mILC1-shFOXOs

cells. mILC1-shFOXOs cells (adh) were cultured in the presence of Dox for 96

hours (adh+Dox) or transferred to suspension for 24 hours (sus) after 72 hours of

Dox (sus+Dox). Data represent the average of 3 experiments ±SD, data is

normalized to Gapdh mRNA expression. F-G Bmf, Bim, Bcl-xl, Bcl2 and Foxo1

protein levels. mILC1-shFOXOs cells (adh) were cultured in the presence of Dox

for 96 hours (adh+Dox) or transfered to suspension for 24 hours (sus) after 72

hours of Dox (sus+Dox). Lanes were cropped out at the indicated dashed line. H

Foxo1, Foxo3, BCL2 & BCL-XL protein levels in mILC1-shFOXOs and mILC1-

FOXO3.A3 cells with exogenous BCL2 or BCL-XL expression. I & J Anoikis

induction in mILC1-shFOXOs and mILC1-iFOXO3 and mILC1-iFOXO3.A3 cells with

exogenous BCL2 or BCL-XL expression cultured in suspension for 24 hours.

mILC1-shFOXOs were pretreated with Dox for 72 hours. mILC1-iFOXO3 and

mILC1-FOXO3.A3 were treated with Dox upon transfer to suspension. Data

represent the average of 3 experiments ±SD (*= t-test p<0.05).

Figure 6: FOXOs are required for maintaining cellular redox homeostasis

A Stress-kinase activation in mILC1-shFOXOs cells upon Dox treatment. B ROS

levels in mILC1-shFOXOs cells (72 hours Dox). Representative cytometry

histogram and the average of three independent experiments ±SD (*= t-test

p<0.05). C NADP+/ NADPH ratio. Data represents the average of 3 experiments

±SD (*= t-test p<0.05). D Colony forming capacity of mILC1-shFOXOs cells (7 days

Dox) in combination with 200µM Trolox, 20µM Euk-134 or 1 mM N-acetyl

cysteine (NAC). E Anoikis in mILC1-shFOXOs cells cultured in suspension for 16

hours, after 72 hour pretreatment with Dox alone or in combination with 200µM

Trolox, 20µM Euk-134 or 1 mM NAC.

Figure 7: FOXOs maintain PI3K pathway activity

A Foxo1, Foxo3, Pkb, Phospho-Pkb-S308, phospo-Pkb-S473 protein levels in

mILC1-shLuc, mILC1-shFOXOs (both 72 hrs Dox), mILC1-iFOXO3 and mILC1-

iFOXO3.A3 (both 16 hrs Dox) cells. Total Pkb levels were used to control for

loading. B Total Pkb and phospo-Pkb-S473 protein levels in mILC1-iFOXO3.A3 and

mILC1-shFOXOs tumors (7 days Dox). Representative pictures with the

corresponding relative DAB intensity (RDI). C RT-qPCR for Egfr, Met, Igf1r, Insr,

Irs1, Irs2, Rictor, Fgfr1, Fgfr3, Pdgfra, Erbb2, Erbb3, Sesn1, Sesn2 and Sesn3 mRNA

expression levels in mILC1-shLuc, mILC1-shFOXOs (both 72hrs Dox) and mILC1-

iFOXO3.A3 cells (16hrs Dox). Graph represents the average of 3 independent

experiments ±SD, ND = not detected. D Irs1, Irs2, Met, Rictor, Igf-1r, Insr, Pdgfra,

Erbb2 and Erbb3 protein levels in mILC1-shLuc, mILC1-shFOXOs (both 72 hrs

Dox) and mILC1-iFOXO3.A3 cells (16 hrs Dox). E Graphic model illustrating the

consequences of differential FOXO activity in tumor progression and in breast

cancer cells based on literature combined with the findings presented in this

manuscript (White = low, Dark blue = high).

Supplemental Figure 1: Immunohistochemistry analysis of mILC1-

iFOXO3.A3 tumors & primary tumor size upon detection of metastasis.

A Expression of FOXO3, Ki67 (cell proliferation marker) and cleaved caspase 3

(apoptosis marker) was determined in tumors either untreated or treated with

Dox for 7 days. Treatment of mILC1-iFOXO3.A3 with Dox for 7 days resulted in

high levels of FOXO3 expression, low levels of Ki67 expression and increased

levels of cleaved caspase 3 (Scalebar FOXO3 & Ki67 = 30µm, Cleaved caspase =

100µm). Indicating that the observed decrease in tumor size correlates to reduced

cell proliferation and increased apoptosis. B&C Primary tumor volumes of mILC1-

iFOXO3 & iFOXO3.A3 tumors upon metastasis detection by bioluminescent

imaging. Black dots represent control tumors (n=8 & n=8), green dots represent

early (n=6 & n=6) and blue dots represent late treated tumors (n=6 & n=8). Graph

represents the average ±SD, t-test p<0.005=** .

Supplemental figure 2: Foxo1, Foxo3 and Foxo4 expression levels and

depletion in mILC1

A RT-qPCR analysis of Foxo1, Foxo3 and Foxo4 expression in mILC1 cells. Graph

represents the average of 3 experiments ±SD, mRNA levels are relative to Gapdh

mRNA levels. B Western blot analysis of Foxo1 and Foxo3 expression in mILC1 72

hours after transduction of shLuc, shFOXOs#1, shFOXOs#2, shFOXOs#3.

Nonspecific background signal was used to control for loading. C RT-qPCR

analysis of Foxo1, Foxo3 and Foxo4 expression after shFOXOs#3 induction in

mILC1-shFOXOs cells. Graph is a representative experiment, average of 3

replicates ±SD, mRNA levels are normalized to Gapdh mRNA levels. D Colony

formation capacity of mILC1-shLuc and mILC1-shFOXOs cells after 7 days of Dox.

Shown is a representative experiment. E Flow cytometric analysis of Hoechst

staining for DNA content in shRNA transduced mILC1 after 72 hours. Shown is a

representative histogram and the average percentage of cells with S/G2/M ±SD F

Flow cytometric analysis of shRNA transduced mILC1 with Propidium Iodide (PI)

transferred to suspension culture for 24 hours after 72 hours. A representative

experiment is shown with the average of 3 technical replicates ±SD. G Western

blot analysis of Pkb, phosphor-Pkb-S308 and phosphor-Pkb-S473 expression in

mILC1 72 hours after transduction of shLuc, shFOXOs#1, shFOXOs#2, shFOXOs#3.

Pkb was used to control for loading

Supplemental figure 3: Immunohistochemistry analysis of mILC1-shFOXOs

tumors & primary tumor size upon detection of metastasis.

A Expression of FOXO1, Ki67 phosphorylated Histone H3 and cleaved caspase 3

was determined in tumors either untreated or treated with Dox for 7 days.

Treatment of mILC1-shFOXOs with Dox for 7 days resulted in reduced levels of

FOXO1 expression, low levels of Ki67 & pH3 expression and no changes in cleaved

caspase 3 levels. Indicating that the observed delay in tumor growth correlates to

reduced cell proliferation. B Primary tumor volume of mILC1-shFOXOs cells upon

metastasis detection by bioluminescent imaging. Black dots represent control

tumors (n=6), green dots represent early treated (n=6) and blue dots represent

late treated tumors (n=7). Graph represents the average ±SD, t-test p<0.05=*

Supplemental figure 4: 3D Spheroid formation of human breast cancer cells

A 10 day 3D Spheroid cultures of MCF7/MDA-MB-231/SKBR3-shFOXOs and -

iFOXO3.A3 in Matrigel containing collagen I cultured in the presence of Dox after

1 or after 7 days for shFOXOs and iFOXO3.A3 respectively.

Supplemental figure 5: Expression of catalase and Sod2 in mILC1

A Representative Flow cytometric analyses of CM-H2DCFA oxidation in

MCF7/MDA-MB-231/SKBR3-shFOXOs cells (Grey) treated with Dox for 72 hours

(green) or 100µM H2O2 as a control for oxidative stress (Blue). B&C RT-qPCR

analysis of Catalase and Sod2 expression after shLuc, shFOXOs and iFOXO3.A3

induction in mILC1 cells. The graph presents the average of 3 independent

experiments ±SD, mRNA levels are normalized to Gapdh mRNA levels. D

Westernblot analysis of Catalase, Sod2 and FOXO3 levels in shLuc, shFOXOs and

iFOXO3.A3 before and after Dox treatment for the indicated times. Nonspecific

background staining was used to visualize protein loading.

Supplemental Figure 6: FOXOs control PI3K signaling feedback in human

cancer cells.

A RT-qPCR of RICTOR, IRS1, IRS2, ERBB2, ERBB3, INSR, IGF-1R, EGFR mRNA

expression levels in MCF7/MDA-MB-231/SKBR3-shFOXOs and -iFOXO3.A3 cells.

shFOXOs cells were treated with Dox for 72 hours, iFOXO3.A3 were treated with

Dox for 16 hours. Shown is a representative experiment with the average of 3

technical replicates ±SD. B Western blot analysis of FOXO1, FOXO3, PKB, pPKB-

S308, pPKB-S473, IGF-1R, RICTOR, INSR and ERBB3 levels MCF7/MDA-MB-

231/SKBR3-shFOXOs and -iFOXO3.A3 cells. shFOXOs cells were treated with Dox

for 72 hours, iFOXO3.A3 were treated with Dox for 16 hours. PKB signal was used

to control for loading.

Supplemental figure 7: Restoring GFR-signaling in mILC1-shFOXOs cells is

insufficient to rescue cell proliferation and stress kinase induction.

A Western blot analysis of Foxo1, Foxo3, cJun, phospo-cJun, Pkb, pPkb-S308 and

pPkb-S473 levels in mILC1-shFOXOs co-expressing a hairpin insensitive GFP-

FOXO3 (FOXO3mt). Total Pkb levels were used to control for loading. B Western

blot analysis of Pkb, pPkb-S308, pPkb-S473 and Foxo1 levels in mILC1-shFOXOs

cell constitutively expressing PIK3CA or oncogenic PIK3CAH1047R. Total Pkb levels

were used to control for loading. C Western blot analysis of Rictor, PIK3CA, cJun,

phospho-cJun and Foxo1 levels in mILC1-shFOXOs cell constitutively expressing

oncogenic PIK3CAH1047R. Nonspecific background signal was used to control for

loading. D Western blot analysis of Rictor, Akt, Akt-S473 and Foxo1 levels in cells

constitutively expressing RICTOR, myristoylated PKB (myrPKB) or both RICTOR

and myrPKB. Total Pkb levels were used to control for loading. E Flow cytometric

analyses of Annexin-V-FITC and PI stained mILC1-shFOXOs constitutively

expressing PIK3CAH1047R, myrPKB, RICTOR, myrPKB & RICTOR or FOXO3mt cultured

in suspension in the presence of Dox for 16 hours. F Colony forming capacity of

mILC1-shFOXOs constitutively expressing PIK3CAH1047R, myrPKB, RICTOR, myrPKB

& RICTOR or FOXO3mt cultured in suspension in the presence of Dox for 7 days. G

Colony forming capacity of mILC1-shFOXOs constitutively expressing myrPKB,

RICTOR or myrPKB & RICTOR cultured in the presence of Dox for 7 days in

combination with 1mM NAC or 200µM Trolox.

Supplemental figure 8: FOXOs mediated gene expression influences a

plethora of cellular processes.

A Heatmap representing the 2Log canonical FOXO target gene mRNA expression

in mILC1-iFOXO3.A3 and mILC1-shFOXOs treated with Dox for 8, 16, 48 and 72

hours. B Venn diagram representing all differentially expressed genes in mILC1-

iFOXO3.A3 cells after 8 hours (blue) or 16 hours (yellow) of Dox treatment. C Venn

diagram representing all differentially expressed genes in mILC1-shFOXOs cells

after 48 hours (green) or 72 hours (red) of Dox treatment. D Venn diagram

combining all differentially expressed genes in mILC1-iFOXO3.A3 cells after 8

hours (blue), 16 hours (yellow) and mILC1-shFOXOs cells after 48 hours (green)

and 72 hours (red) of Dox treatment. Bold numbers represent the overlapping

gene-sets used for pathway activity prediction. E Heatmap representing pathway

activity z-scores (blue: <-0.4, pathway inactive, black: activity status not inferred

but altered pathway, yellow: >0.4, pathway active) generated by Ingenuity

Pathway Analysis based on differential gene expression in at least three

overlapping data-sets of the mILC1-iFOXO3.A3 and mILC1-shFOXOs treated with

Dox for 8, 16, 48 and 72 hours. F Heatmap representing the 2Log gene mRNA

expression mILC1-iFOXO3.A3 and mILC1-shFOXOs treated with Dox for 8, 16, 48

and 72 hours of canonical EMT/metastasis genes combined with genes previously

linked to FOXO and metastasis.

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 1 A B C mILC1-iFOXO3 mILC1-iFOXO3.A3 FOXO3 FOXO3+Hoechst FOXO3 FOXO3+Hoechst mILC1 mILC1 20μm iFOXO3 iFOXO3.A3 iFOXO3 iFOXO3.A3 + + FOXO “ON” 100kDa

FOXO3 Control Untreated 100kDa pFOXO3-T32

Loading FOXO “ON” FOXO 8h “ON” FOXO

D 10 E F Control FOXO “ON” Control 48h FOXO “ON” 8 iFOXO3 iFOXO3.A3 - 8h - 16h - 8h - 16h FOXO “ON” 25kDa cell # cell

nn exin-V) 6 p21 iFOXO3 G1: 54±2% G1: 61±6% 4 2n 4n 2n 4n 25kDa p27

2 Loading % apoptosis (a apoptosis % cell # cell

0 iFOXO3.A3 G1: 52±3% G1: 81±9% iFOXO3 iFOXO3.A3 2n 4n 2n 4n

G mILC1-iFOXO3 H mILC1-iFOXO3.A3 600 control 600 control FOXO3 “ON” at 2wks FOXO3.A3 “ON” at 2wks 3 3 ) 3 FOXO3 “ON” at ~100mm 3 FOXO3.A3 “ON” at ~100mm

400 400

200 200 *** Tumor volume (mm volume ) Tumor Tumor volume (mm volume Tumor ** *** ** ** *** 0 0 *** 0 20 40 60 0 20 40 60 Days post transplantation Days post transplantation I J Metastasis free survival mILC1-iFOXO3 Metastasis free survival mILC1-iFOXO3.A3 100 100 re e re e *** *** is f as is is f as is as t as t 50 50 me t me t % % control control FOXO3 “ON” at 2wks FOXO3.A3 “ON” at 2wks FOXO3 “ON” at ~100mm3 FOXO3.A3 “ON” at ~100mm3 0 0 0 20 40 60 80 0 20 40 60 80 Days post transplantation Days post transplantation

A mILC1-iFOXO3.A3 tumor lines B 2.0 control dox at 2wks dox at ~100mm3 rtTA3G-IRES-Neo 2 3 5 6 8 9 FOXO3.A3 cDNA 1.5 ti ons - gr a nter

i 1.0

Neo e v iral

ti v 0.5 * * rela ** Dox * * 0.0 iF3A3 1 2 3 4 5 6 7 8 9 mILC1 untreated dox at 2wks dox at ~100mm3 parental mILC1-iFOXO3.A3 tumor lines C mILC1-iFOXO3.A3 tumor lines D untreated dox at 2wks dox ~100mm3 mILC1-FOXO3.A3 2 3 5 6 8 9 - -24h3wk dox + + + + ++ dox 100kDa 100kDa FOXO3 FOXO3 100kDa FOXO3 FOXO3 long exp.

Loading Loading E mILC1-iFOXO3.A3 Parental cell line Untreated tumor lines Dox at 2wks tumor lines Dox at ~100mm3 tumor lines untreated 16h dox 3wks cont. dox #2 + 16h dox #3 + 16h dox #5 + 16h dox #6 + 16h dox #8 + 16h dox #9 + 16h dox FOXO3 Equal exp. Equal FOXO3 Optimal exp. Hoechst FOXO3+

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Figure 3Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A B C 10 mILC1 shLuc shFOXOs ctrl shLuc shFOXOs Dox 8 0 24h 48h 72h 0 24h 48h 72h Dox ctrl 75kDa Foxo1 6 nn exin-V) 100kDa Foxo3 4 Loading Dox 2 % apoptosis (a apoptosis %

0 shLuc shFOXOs D mILC1-shFOXOs E F Control: mILC1-shFOXOs mILC1 G1: 57±4% 50 **** shLuc shFOXOs 40 0 24h 48h 72h 0 24h 48h 72h Dox

cell number cell 25kDa p21 72h FOXO “OFF” 1 1.01 1.06 1.01 0.93 1.91 3.19 2.84 G1: 55±2% 30

e (hours) im e p27 25kDa 20 1 1.02 1.17 1.381.201.15 1.29 1.25 cell number cell 96h FOXO “OFF” Loading Cell cycle t cycle Cell 10 G1: 54±2%

0 Control FOXO “OFF” cell number cell 2n 4n G H mILC1-shFOXOs Metastasis free survival mILC1-shFOXOs 400 100 control FOXO “OFF” at 2 wks *** 3 )

3 FOXO “OFF” at ~100mm

300 re e is f as is

200 as t 50 **

me t control 100 ** % FOXO “OFF” **** at 2 wks Tumor volume (mm volume Tumor **** FOXO “OFF” ** **** **** at ~100mm3 0 0 0 20 40 60 0 20 40 60 80 100 Days post transplantation Days post transplantation

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Figure 4Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A B mILC1-shFOXOs tumor lines 2.0 untreated dox at 2wks dox at ~100mm3 GFP-T2A-tTA 10 11 12 13 14 15 16 17 18

ti ons shFOXOs 1.5 GFP

gr a 25kDa Loading nter

i 1.0 * * e v iral 0.5 * C ti v mILC1-shFOXOs tumor lines rela 0.0 untreated dox at 2wks dox ~100mm3 10 11 12 13 14 15 16 17 18 10 11 12 13 14 15 16 17 18 untreated dox at 2wks dox at ~100mm3 + +++++ + + + dox parental mILC1-shFOXOs tumor lines 75kDa Foxo1 mILC1-shFOXO 100kDa Foxo3 D Loading mILC1-shFOXOs - 72h 3wk dox E mILC1-shFOXOs tumor lines 75kDa FOXO1 untreated dox at 2wks dox ~100mm3 10 11 12 13 14 15 16 17 18 100kDa FOXO3 Loading MCF7 F dox shLuc shFOXOs iF3.A3 ---48 72 48 72 16 hours dox 75kDa FOXO1

100kDa FOXO3 mILC1-shFOXOs mILC1-iFOXO3.A3 I 10 5 2.5 1.25 0.63 - 1052.51.250.63 ng/mL dox Loading 100kDa FOXO3 shLuc shFOXOs iF3.A3 0.03 0.02 0.02 0.26 0.95 1 1.75 1.58 1.73 2.38 3.38 Loading

dox

G SKBR3 J MCF7-shFOXOs MCF7-iFOXO3.A3 shLuc shFOXOs iF3.A3 10 5 2.5 1.25 0.63 - 1052.51.25 100 ng/mL dox ---48 72 48 72 16 hours dox 100kDa FOXO3 75kDa FOXO1 0.67 0.85 0.82 0.81 0.781 1.42 1.75 1.90 2.10 3.04 Loading 100kDa FOXO3 Loading shLuc shFOXOs iF3.A3

K MDA-MB-231-shFOXOs MDA-MB-231-iFOXO3.A3 10 5 2.5 1.25 0.63 - 1052.51.25 100 ng/mL dox

dox 100kDa FOXO3 0.51 0.61 0.88 0.98 1.061 0.93 1.11 1.44 1.96 2.83 H MDA-MB-231 Loading shLuc shFOXOs iF3.A3 ---72 72 16 hours dox 75kDa FOXO1

FOXO3 100kDa L SKBR3-shFOXOs SKBR3-iFOXO3.A3 Loading 10 5 2.5 1.25 0.63 - 1052.51.25 100 ng/mL dox FOXO3 shLuc shFOXOs iF3.A3 100kDa 0.46 0.68 0.73 0.89 0.851 1.20 1.15 3.23 7.38 9.56 Loading

dox

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2018; DOI: 10.1158/0008-5472.CAN-17-2511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 5 mILC1-shFOXOs mILC1-iFOXO3.A3 A B FOXO “OFF” FOXO”ON” 2.0 from day 1 from day 1 from day 7

min) 1.5

mm/ **** 1.0 e speed ( 0.5 ve rag A 0.0 untreated FOXO “OFF” FOXO “ON” 50μm C D E 60 Sus 60 adh 10 mILC1-shFOXOs Sus + dox * adh FOXO “OFF” adh sus sus FOXO “OFF” 8 adh FOXO “OFF” ss ion sus 40 * 40 sus FOXO “OFF”

expre 6 * A anoikis 4 % % 20 % apoptosis 20 2 Relative mRN Relative

0 0 0 shLuc shFOXOs iFOXO3 iF3.A3 MCF7 MM231 SKBR3 Mcl1 Bcl2 Bcl-xl Bim Bmf Bid Puma Noxa

F G MCF7 MDA-MB-231 SKBR3 adh sus adh sus adh sus mILC1-shFOXOs + + + + + + FOXO “OFF” adh sus + + FOXO “OFF” 25kDa BCL2 Bmf 37kDa 25kDa BCL-XL 25kDa BimEL 25kDa BIM-EL 37kDa Bcl-xl 15kDa BIM-L BIM-S 25kDa Bcl2 25kDa BMF 75kDa Foxo1 75kDa FOXO1 Loading 100kDa FOXO3

H I 40 J 40 sus sus mILC-shFOXOs mILC-iFOXO3.A3 sus FOXO “OFF” sus FOXO “ON” BCL2 BCL-XL BCL2 BCL-XL + + + + + + Dox 30 30 75kDa Foxo1 100kDa Foxo3 20 20

25kDa BCL2 * * * * 37kDa 10 10 % anoikis (a nnexin-V) BCL-XL % anoikis (a nnexin-V)

Loading 0 0 Ctrl BCL2 BCL-XL Ctrl BCL2 BCL-XL mILC1-shFOXOs mILC1-iFOXO3.A3

mILC1-shFOXOs A 24h 48h 78h FOXO “OFF” B C 37kDa cJun 2 1.0 200 Contol FOXO “OFF” * 1 1.54 4.58 4.56 ce 37kDa p-cJun ratio 1 1.72 5.87 5.93 + DP Jnk 1 0.5 * 37kDa 1 0.77 2.74 3.43 Cellrox fuorescen p-Jnk number Cell 37kDa 1 1.51 4.43 5.97 NADPH/N A p38 Relative 37kDa 10 101 102 103 - FOXO - FOXO 1 1.78 2.56 6.12 CellRox uorescence intensity “OFF” “OFF”

37kDa p-p38 0.961 2.24 3.12 Loading D E 40 mILC1-shFOXOs Sus Trolox EUK-134 NAC sus + FOXO “OFF” 30 Control 20

FOXO 10 “OFF” % anoikis (a nnexin-V) 0 Ctrl Trolox EUK-134 NAC

A B pPkb-S473 C untreated dox FOXO RDI: 1 RDI:2,41 “OFF” “ON” Sesn3 shLuc shFOXOs iFOXO3 iFOXO3.A3 Sesn2 - 72h - 72h - 16h - 16h dox Sesn1 pPkb-S308 50kDa Erbb3 RDI: 1,11 RDI: 0,66 Erbb2 pPkb-S473 50kDa Pdgfra

75kDa Foxo1 Fgfr3

100kDa shFOXOs iFOXO3.A3 Fgfr1 Foxo3 30mm Rictor Pkb Irs2 50kDa Pkb untreated dox RDI: 1 RDI: 0,97 Irs1 Insr D Igf1 c-Met shLuc shFOXOs iFOXO3 iFOXO3.A3 Egfr ---48h72h 48h72h 16h- 16h dox RDI: 0,97 RDI: 1.02 150kDa Irs1 -2 4 1 1.15 1.12 1 0.38 0.45 1 0.96 1 0.67 2Log mRNA change 150kDa Irs2 shFOXOs iFOXO3.A3 1 0.91 0.89 1 0.29 0.02 1 1.62 1 3.13 100mm

150kDa c-Met E 1 0.92 1.12 1 0.27 0.22 1 0.89 1 0.73 shFOXOs Endogenous iFOXO3 iFOXO3.A3 200kDa Rictor 1 0.96 0.92 1 0.36 0.36 1 1.09 1 1.34 low FOXO activity high 150kDa Igf-1r 1 1.03 1.03 1 1.00 1.03 1 0.80 1 2.94 Tumor growth

150kDa Insr Metastasis 1 1.04 1.03 1 1.32 1.14 1 2.45 1 4.05 200kDa Pdgfra Proliferation 1 0.85 0.97 1 0.79 0.77 1 1.13 1 1.48 150kDa Erbb2 Anoikis resistance 1 1.04 1.09 1 0.91 0.37 1 1.41 1 3.69 150kDa Erbb3 ROS 1 0.96 0.97 1 1.14 0.80 1 2.39 1 3.56 Loading PI3K signaling

FOXO transcription factors both suppress and support breast cancer progression.

Marten Hornsveld, Lydia M Smits, Maaike Meerlo, et al.

Cancer Res Published OnlineFirst February 12, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-2511

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