department of experimental surgery, medical …...2013/05/30 · 1 unraveling the role of foxq1 in...

1

Unraveling the role of FOXQ1 in colorectal cancer metastasis

Mohammed Abba1, Nitin Patil

1, Kabeer Rasheed

2, Laura D. Nelson

3, Giridhar Mudduluru

1, Jörg

Hendrik Leupold1 and Heike Allgayer

1*

1 Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, and Department

of Molecular Oncology of Solid Tumors Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany

2 Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore

3 Department of Pediatrics, University of Texas, M.D Anderson Cancer Center, Houston, Texas

*to whom correspondence should be addressed:

Prof. Heike Allgayer

Dept. of Experimental Surgery, Medical Faculty Mannheim,

Ruprecht Karls University Heidelberg and,

Molecular Oncology of Solid Tumors Unit,

German Cancer Research Center (DKFZ), Heidelberg,

Medical Faculty Mannheim, University of Heidelberg,

Theodor Kutzer Ufer 1-3, D-68135

Mannheim, Germany.

e-mail: [email protected]

Abstract word count: 186

Key words: FOXQ1, Twist1, transcriptional regulation, metastasis

Conflict of interest: All authors declare no conflict of interest

on June 18, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 30, 2013; DOI: 10.1158/1541-7786.MCR-13-0024

https://dkfzowa0.dkfz-heidelberg.de/owa/redir.aspx?C=715daa03f8d04139832b978881ebee11&URL=mailto%3aheike.allgayer%40umm.de

http://mcr.aacrjournals.org/

2

Abstract

Malignant cell transformation, invasion and metastasis are dependent on the coordinated

rewiring of gene expression. A major component in the scaffold of these reprogramming

events is one in which epithelial cells lose intercellular connections and polarity and adopt a

motile mesenchymal phenotype, one that is largely supported by a robust transcriptional

machinery consisting mostly of developmental transcription factors.

In this study, we show that the winged helix transcription factor, FOXQ1, contributes to this

process, in part by directly modulating the transcription of Twist1, itself a key mediator of

metastasis, that transcriptionally regulates the expression of important molecules involved in

epithelial to mesenchymal transition (EMT). Forced expression and RNA-mediated silencing

of FOXQ1 led to enhanced and suppressed mRNA and protein levels of Twist1 respectively

with FOXQ1 enhancing the reporter activity of Twist1 in addition directly interacting with its

promoter. Furthermore, enhanced expression of FOXQ1 resulted in increased migration and

invasion in colorectal cancer cell lines, while knock-down studies showed the opposite effect.

Moreover using the in vivo chicken chorioallantoic membrane metastasis assay model, we

showed that FOXQ1 significantly enhanced distant metastasis with insignificant effects on

tumor growth.




3

Introduction

Metastasis is the leading cause of cancer related mortality in almost all cancer types, and

occurs as a result of a coordinated progression of crucial steps encompassing local invasion,

intravasation, survival in the circulation, extravasation, formation of micrometastasis and

colonization (1). Intricately interwoven in this progression cascade is an orchestrated series of

events that disrupt cell to cell and cell-extracellular matrix (ECM) interactions, enhancing

cell motility and liberating epithelial cells from the surrounding tissue. This program involves

in most instances the induction of a new transcriptional program that stimulates and

maintains a mesenchymal phenotype and has come to be recognized as a fundamental

mechanism that induces invasion and metastasis of tumor cells (2-5). Additional molecular

processes are also engaged in the completion of this transition program and include the

expression of specific cell-surface proteins, reorganization and expression of cytoskeletal

proteins, production of ECM-degrading enzymes, and changes in the expression of specific

microRNAs.

FOXQ1 belongs to the large forkhead family of transcription factors that share a conserved

C-terminal forkhead/winged helix DNA-binding domain (6). Most forkhead family members

have confirmed roles in embryonic development and tissue-specific gene expression (6-8).

FOXQ1 itself is a ~ 42kDa protein sharing the same paralogous cluster as FOXF2 and

FOXC1 (9), and has been shown to be involved in hair follicle differentiation, gastrulation

and mucin production in mice (10-12). Recent reports have confirmed it to be a downstream

target of TGF-β, where it enhances mesenchymal transformation that appears to be cell type

dependent (13, 14). Furthermore, it has been found to be over-expressed in colorectal and

breast cancers, where it has been shown to enhance tumorigenicity, anti-apoptotic behavior

and tumor growth (14-16). A direct modulation of p21CIP1/WAF1 expression was shown to

explain the anti-apoptotic effects in colorectal cancer (15), with independent experiments in




4

breast cancer cell lines showing an acquired resistance to chemotherapy-induced apoptosis

upon forced expression, and concurrent correlations to aggressive behavior, especially in

triple negative breast tumors (16). It has also been found to be of prognostic relevance in non-

small cell lung cancer (NSCLC) (17).

Although substantial evidence supports the tumorigenic effect of FOXQ1, and correlation

analyses have linked it to aggressive tumor behavior, the exact mechanisms by which this

occurs have not been properly elucidated. Clearly, it has been shown to stimulate the

expression of the cyclin kinase inhibitor, p21CIP1/WAF1 by binding its core promoter (15), and

to induce a suppression of E-cadherin expression, by supposedly binding to E-box elements

in its promoter, with conflicting reports regarding the validation of the latter (14, 16). As a

transcription factor, it is clear that unraveling its functionality depends to a large extent on

identifying its direct targets, and the dissection of its’ metastatic influence hinges on

ascertaining which one(s) of these targets bear consequences on already known/novel

metastatic molecules or, on metastatic pathways.

In the search of FOXQ1 targets which could explain its’ metastatic predisposition, we

decided to explore genes whose expressions’ were both down-regulated when FOXQ1 was

suppressed, and conversely upregulated when FOXQ1 was forcibly expressed. The selection

of the investigated genes was based on a selected panel of genes known to be intricately

involved in EMT in addition to a gene pool, whose expression we found to be simultaneously

deregulated in tumor epithelium, stroma and whole-tissue compared to the corresponding

normal equivalents following a microarray analysis of whole and laser-capture

microdissected colorectal cancer tissues (18). This additional gene pool was equally

implicated in epithelial to mesenchymal transition. The investigation was carried out in




5

endogenously high and low FOXQ1-expressing colorectal cancer cell lines and two genes; E-

cadherin and Twist1 showed consistent changes in both experimental set-ups.

In this study, we show that FOXQ1 expression correlates substantially with Twist1 in

colorectal cell lines, and in resected patient tissues, that Twist1 is a direct target of FOXQ1,

that the over-expression and down regulation of FOXQ1 leads to comparable and consistent

changes in Twist expression, and this leads to significant changes in migration, invasion and

in-vivo metastasis, but with no effect on tumor proliferation.

Materials and Methods

Cell Culture

All cell lines, except GEO, a gift from Douglas Boyd (MD Anderson Cancer Center), and

Colo206F from the German Collection of Micro-organisms and Cell Cultures (DSMZ), were

obtained from the ATCC. The Colo206f, SW480 and HeLa cell lines were maintained in

RPMI media supplemented with 10% FCS, GEO was cultivated in DMEM, whereas WiDR

was in maintained in MEM. All cell lines were kept in a humidified incubator at 37oC with

5% CO2.

Plasmids and cloning

The full-length FOXQ1 coding region (IRATp970A1179D) was bought from Imagenes

(Source Bioscience Lifesciences, UK) and was subcloned into the pCDNA 3.1 vector

between the EcoRI and HindIII sites. The EGFP gene from the pEGFP C1 vector was cut out

and cloned into the multiple cloning site of pCDNA 3.1 (NheI/EcoRI) in front of the FOXQ1

gene. This construct was used for all over-expression analysis. The abridged coding sequence

was PCR amplified using the following primer sequences FX08 Fwd: 5’TCG GAT CCA




6

TGA AGT TGG AGG TGT TC 3’; FX08 Rev: 5’ TTC TCG AGA TGA AGG GAA GGA

GGA GC 3’and cloned into a HA tagged pcDNA 3.1 vector in frame with the tag between the

Xho and BamHI sites. This construct was used in the chromatin immunoprecipitation assays.

All transient transfections (with Metafectane) were evaluated at 24hr time points. Stable

Colo206f and GEO cell lines were selected on G418 treatment at a concentration of 400 and

600ug/ml, respectively.

siRNA transfections

Two siRNA’s targeting the 5’AGG GAA CCT TTC CAC ACT ATA 3’ (Hs_FOXQ1_3) and

5’CGC GCG GAC TTT GCA CTT TGA 3’ (Hs-FOXQ1_4) segments of FOXQ1 were

purchased from Qiagen (Hilden, Germany)) were applied at a 50nM end concentration using

the HiperFect transfection reagent and protocol (Qiagen, Germany). A greater knockdown

effect was obtained with Hs-FOXQ1_4, which was then used for all subsequent experiments.

The AllStars negative control siRNA (Qiagen, Hilden, Germany) was utilized, as the name

infers. A validated silencer select siRNA for Twist1 (s14523) was purchased from Life

Technologies. Knock-down effects were evaluated at 48 and 96hr time-points for mRNA and

protein expression, respectively.

Quantitative RT-PCR

RT-PCR was done on the LightCycler480 system (Roche) using the SYBR green detection

system (Thermo Scientific, Waltham, USA). All primers: FOXQ1, Twist1, CDH1, CDH2,

CDH3, Snail1, ETV4, TGFB, CLDN1, LPAR1, CTNNB1, Vimentin, KIAA1199, TBP were

purchased from Qiagen from the Quantitect Primer Assay collection (Qiagen, Hilden,




7

Germany). Relative expression was calculated using the δδCT method with normalization to

the tata-box binding protein (TBP). Absolute quantification was effected using standard

curves generated from a plasmid containing the gene of interest.

Antibodies and Western Blots

FOXQ1 (C-16), E-cadherin (G10), N-cadherin (8C11) and Twist (Twist2C1a) antibodies

were all purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Actin (A2066) was

purchased from Sigma (St Louis, USA) while β-catenin (L87A12) was from Cell Signaling

(Danvers, USA). Anti-HA tag antibody (ab9110) and rabbit polyclonal IgG (ab27478) were

purchased from Abcam (Cambridge, UK).

For Western blotting, cells were harvested and lysed with RIPA-buffer and protease

inhibitors (Complete Mini, Roche Diagnostics, Indianapolis, USA), protein concentrations

determined with the BCA protein assay kit (Thermo Scientific, USA) and samples separated

by SDS-PAGE as previously described (19). Tissue samples were homogenized with the

Mixer Mills tissue lyser (Retsch, Haan, Germany) before processing, as described for cell

lines.

Reporter gene assays

A Twist1 promoter plasmid (product ID S717559) encompassing -742 to +203 nt relative to

the TSS was purchased from Switchgear Genomics (California, USA). Within this construct

were 4 putative ‘GTTT’ binding sequences located at +39 to +43, -184 to -188, -195 to -199

and -604 to -608 relative to the TSS. The mutant forms were generated using the QuikChange

II site directed mutagenesis kit (Agilent Technologies, Santa Clara, USA). Colo206f cells

were plated at a density of 3 x104 cells/well in 96-well plates, and were transfected after a

24hr incubation period using Metafectane (Biontex Laboratories, Martinsried, Germany).




8

Assays were performed in quadruplicates and repeated at least 3 times and reporter signals

were assessed with the LightSwitch luciferase assay kit (SwitchGear Genomics, California,

USA).

Chromatin Immunoprecipitation

The chromatin immunoprecipitation (ChIP) experiments were performed using the ChIP

assay kit (Upstate) from Millipore (Billerica, USA), according to the manufacturer’s

instructions. Colo206f cells were transfected with an empty vector (pcDNA-HA) or HA-

tagged FOXQ1 vector. The putative region of the Twist1 promoter was amplified with the

following primers ChpX Fwd: 5' GTC TCC TCC GAC CGC TTC 3'and ChpX Rev: 5' ATT

CGT CCT CCC AAA CCA TT 3' primers and assayed with both quantitative and semi

quantitative real time PCR.

MTT (3-(4,5-Dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) assay

1x 104 cells were plated in 96 well plates following which 20µl of MTT was added at 0, 24,

48, 72, 96 and 120hr time points. 100 µl of sodium dodecyl sulphate (SDS) diluted in

hydrochloric acid was added 3hrs after the MTT solution (Sigma-Aldrich, St Louis, USA),

followed by measurement of the absorbance at 550 and 640nm. FOXQ1 and vector

transfected stable cell lines (GEO, colo206f), as well as FOXQ1 siRNA and scrambled

transfected cell lines (SW480, WiDr) were evaluated alongside each other.

Migration and invasion assays

Analysis of cell invasion and migration was done using transwell chambers (BD Biosciences,

New Jersey, USA) with and without a (10µg/ml) matrigel coating, respectively. The protocol

is as was previously published (20). Migration was additionally evaluated with the wound




9

healing assay. Briefly cells were seeded in 24 well plates at a density that enabled a

confluency of 80% to be attained 24hrs after plating. A 200ul filter tip was used to gently

scratch the cell monolayer across the center of the well 24hrs after seeding. The cells were

then gently washed with PBS to remove the dislodged cells, and then replenished with fresh

medium, after which the first images were acquired. The cells were incubated for a further

24hrs after which a second set of images were acquired.

In vivo chicken chorio-allantoic membrane (CAM) metastatic assay

This is a well-established model for assessing in-vivo metastasis (21-24). Fertilized special

pathogen free (SPF) eggs were purchased from Charles River (Sulzfeld, Germany) and

incubated for 10 days in a rotary incubator, after which 2x106 FOXQ1 and vector expressing

GEO cells were inoculated onto the upper CAM following a miniaturized dissection of the

shell. After a further 7 days of incubation, the developing chicken embryos were sacrificed,

lungs, liver and the developing tumor on the upper CAM removed. Genomic DNA was

isolated from the liver and lungs and the number of metastasized cells evaluated using human

specific Alu-PCR primers and probes, as previously described (25). The tumors removed

from the upper CAM were weighed to assess in vivo tumor growth.

Statistical analysis

Significance was set at p≤0.05. Two tailed unpaired and paired t –tests were employed when

dealing with independent (cell lines, before/after treatment) and dependent (tumor/normal

patient) variables respectively. Correlation analysis was done with the Pearson’s correlation

coefficient and lambda coefficient of association algorithms. Calculations were made using

Sigmaplot, Microsoft Excel and Graph pad prism 5.0 tools.




10

Results

FOXQ1 is over-expressed in epithelial and stromal tumor compartments alongside other

EMT genes

Following a microarray analysis of whole-tissue and laser-capture microdissected tissues in a

series of 35 colorectal cancer patients in which epithelial, stromal and whole-tissue

compartments were compared, we found FOXQ1 to be simultaneously over-expressed at the

mRNA level in all three tumor compartments as compared to the equivalent normal samples.

In addition to FOXQ1, the genes identified contained many molecules already implicated in

EMT and cellular differentiation, inclusive of TGFB1, TIMP1, ADAMTS1, LPAR1 and

CLDN1 (Supplementary Table 1). Details describing the identification and validation of this

gene pool are elaborated in our recent publication (18). FOXQ1 was chosen for further

evaluation because of its significant expression in all three compartments, its relevance to the

EMT hypothesis as an embryonic transcription factor, and on its relative obscurity in reports

in literature.

Forced expression and knock-down of FOXQ1 modulates Twist1 expression

In the search for possible molecules that were directly affected by FOXQ1 expression, we

performed, using quantitative RT-PCR, an analytical expression screen of genes identified

from the common microarray gene pool (Supplementary Table 1), concurrently incorporating

other well described, EMT related genes. The expression profile of these genes was evaluated

following enhanced expression and knock down of FOXQ1 in endogenously low and high

mRNA expressing cell lines, respectively. Since the mRNA and protein expression of the cell

lines did not always correlate (Figure 1a), over-expression studies were done in Colo206F

and GEO cell lines (Figure 1b), while knock-down experiments were conducted in both high

mRNA expressing (SW480 and HCT15) and high protein expressing (WiDR) cell lines




11

(Figures 1c and d). We observed that of all the genes tested for, CDH1 (E-cadherin) and

Twist1 expression profiles correlated concurrently and significantly with FOXQ1, with

expression levels increasing and decreasing (Twist1 increases, E-cadherin decreases and vice-

versa) with FOXQ1 expression (Figures 1b, c and d). In all situations, there was always a

concomitant cadherin switch, between E-cadherin and either N- (CDH2) or P- (CDH3)

cadherin (Figures 1b, c and d).

Since we observed a systematic correlation of expression between Twist1 and E-cadherin

following FOXQ1 deregulation, and since E-cadherin is a known target of Twist1, we

proceeded to identify if the deregulation of E-cadherin occurred following that of Twist1.

Following the knockdown of FOXQ1 in the SW480 cell line, over a 72hr timeline, we

observed that indeed the expression of Twist1 was affected before that of E-cadherin,

suggesting that the effect of FOXQ1 on E-cadherin was likely mediated through Twist1.

(Figure 1e)

The Twist promoter harbors putative FOXQ1 binding motifs

Upon the realization of two potential candidate genes, whose expressions were affected by

FOXQ1, we proceeded to identify the consensus binding sequence of FOXQ1 and find out if

this sequence occurred in the promoter on any of these genes, the presence of which would

signify a potential direct regulatory effect. Overdier and colleagues (26) identified a core

FOXQ1 binding sequence of ‘TGTTTA’, whereas bioinformatic searches within Transfac

suite (www.biobase-international.com) and JASPER CORE database (www.jasper.cgb.ki.se)

had matrix motifs from rat studies only (Figure 2a), with a core ‘GTTT’ motif. Since FOXQ1

is evolutionarily conserved (Figure 2b), and the forkhead DNA binding domain of the human,

mouse and rat FOXQ1 proteins share 100% identity (6), it implied that the core GTTT

sequence may indeed be valid across many species. A detailed analysis of the respective gene



http://www.biobase-international.com/

http://www.jasper.cgb.ki.se/


12

promoters revealed that the Twist1 promoter harbored the cis-elements required for FOXQ1

binding (Figure 2c).

FOXQ1 transcriptionally activates Twist1

Sequel to our findings above, we went further to investigate if FOXQ1 transcriptionally

activated Twist1. Reporter gene assays with the -742 to +203 segment of Twist1 promoter

showed a 1.5- to 2-fold increase in Twist1 expression as compared to the control vector

(Figure 3a). Additionally, titrated increments in the concentration of FOXQ1 led to

increments in reporter activity (Figure 3b). Within this construct were 4 putative ‘GTTT’

binding sequences located at +39 to +43, -184 to -188, -195 to -199 and -604 to -608 relative

to the transcription start site (TSS) (27). Two of these sites were within 7 nt of each other

signifying an enhanced binding probability to this location. To verify if these sites were

important in the observed induction of Twist1 expression, we mutated these positions, with

discernibly decreased effects on the reporter activity at the -184 to -199 position (Figure 3c).

To further corroborate our findings, we performed a chromatin immunoprecipitation assay

(ChIP) specifically looking for enrichment of a short fragment containing the two putative

sites that lay in close proximity to each other, where we observed, after normalizing to the

input, an approximately 3-fold increase in the FOXQ1 consensus-binding sites

immunoprecipitated from over-expressing cell lines as compared to the vector control (Figure

3d and e). No significant induction was observed at the +39 to +43 and -604 to -608 sites

(data not shown).

FOXQ1 expression correlates with Twist1 expression in resected patient tissues

To assess the in situ validity of our hypothesis, we compared, using RT-PCR, the relative

mRNA expression between FOXQ1, Twist1, and other EMT related genes taken from our




13

microarray experiment in an independent cohort of 45 patients. We obtained a coefficient of

association (λ) of 0.66 for FOXQ1 and Twist1, suggesting an in vivo proof of principle. We

tested this association between other genes where a direct regulatory effect of one on the

other has already been established, where we obtained for instance, in the comparison

between Snail and E-cadherin, and between Twist1 and E-cadherin, coefficient of

associations of 0.428 and 0.483, respectively (Figure 4a). We went further to check for a

correlation at the protein level, with similar results (Figure 4b). In a bid to further validate our

data, we searched open databases, and in the R2 database (http://r2.amc.nl), where several

expression profiling data are available, we found that especially in the MAQC studies, the

correlation between FOXQ1 and Twist1 expression had a coefficient of r = 0.9 (Figure 4c).

The MAQC project generated a data set which compared the consistency of microarray data

between laboratories and across platforms (28), and which supports our findings in colorectal

cancer patients.

Forced expression of FOXQ1 did not result in mesenchymal transformation in Colo206F,

GEO and MDCK II cell lines.

In line with published reports that FOXQ1 induced mesenchymal transformations in certain

cell lines and not in others, we analyzed the phenotypic characteristics of 2 colorectal cancer

cell lines (Colo206f; GEO) and the canine MDCK II cell line normally used in EMT studies.

In all of the 3 cell lines, we found no discernible mesenchymal morphological features with

light microscopy that differentiated FOXQ1 and vector expressing cell lines (data not

shown).

FOXQ1 has no significant effect on cell proliferation and in vivo tumor growth.




14

In the bid to unravel the functional significance of FOXQ1 on tumor progression, we

analyzed the effects of FOXQ1 over-expression and knock-down on cell proliferation using

the MTT assay. We found no significant effect on cell proliferation in both sets of

experiments (Figure 5a and 5b). To address the question if this inconsequential effect on cell

proliferation also occurred in vivo, we analyzed the tumor masses that developed on the upper

CAM of eggs inoculated with either FOXQ1 or vector expressing cells where no significant

difference was observed between the groups further supporting the in vitro cell proliferation

assays (Figure 5c).

FOXQ1 enhances tumor migration and invasion.

To dissect the impact that FOXQ1 had on tumor progression, we appraised its influence on

migration and invasion using the Boyden chamber assay, without and with a matrigel coating,

respectively. This was evaluated in four cell lines, two over-expressing FOXQ1 (COLO206F

and GEO) and two in which FOXQ1 expression was suppressed (SW480 and WiDR). We

observed a significant increase in cell migration upon forced expression, and a concurrent

decrease in migration, when FOXQ1 was suppressed (Figure 6a). The same results were

mirrored in the invasion assays, but with a slightly less significance than was obtained for the

migration assay (Figure 6b). Cell migration was additionally evaluated using wound healing

assay (Supplementary Figure 1).

FOXQ1 enhances distant metastasis.

Using the in vivo chicken chorio-allantoic membrane (CAM) metastatic assay, we evaluated

the effect that FOXQ1 expression had on distant tumor metastasis. The liver and lungs of

chicken embryos were dissected following one week of incubation after the inoculation of

FOXQ1- or vector- GEO over expressing cells on the upper CAM. Using real time PCR to




15

detect human specific Alu DNA sequences, we were able to show a significant increase in

both lung and liver metastasis, as compared to the vector control (Figure 7a and 7b).

However, as stated above, we did not observe any significant change in the primary tumor

size itself, indicating that the effect of FOXQ1 on metastasis is proliferation independent.

Discussion

This is the first study to demonstrate that FOXQ1 regulates invasion and metastasis in

colorectal cancer. Metastasis is a highly complex process, consisting of at least 5 different

steps (invasion, intravasation, dissemination in the circulation, extravasation and colonization

of distant sites), each of which depends on the action of diverse molecular events. Over the

years, different key molecules have been identified that contribute significantly to at least one

phase of the metastatic cascade, some playing multiple roles in more than one part of the

process.

The epithelial to mesenchymal transition represents an integral part of metastatic progression

and interestingly, several of the molecules and pathways involved converge on critical and

common end points, one of which is E-cadherin (4, 29). E-cadherin is invariably almost

always down-regulated during EMT with a concomitant switch to a mesenchymal cadherin

that may, or may not be associated with an overt phenotypic mesenchymal transformation

(30, 31). This cadherin switch is modulated by a number of transcriptional regulators, one of

which is Twist1 (32).

In this study, we evaluated the forkhead box q1 transcription factor, a relatively new addition

to embryonic transcription factors that are also involved in metastasis, and show that it

modulates its effects in part by acting on Twist1, whose role in epithelial to mesenchymal




16

transition is well entrenched (33-35). We postulated that FOXQ1 transactivates Twist1,

which in turn suppresses E-cadherin expression, as supported by the concomitant and

consistent deregulation of these genes upon FOXQ1 overexpression/knock-down, by the fact

that Twist1 is an established regulator of E-cadherin expression and finally by the

observation that Twist1 deregulation precedes that of E-cadherin (Figure 1e). It appears that

the expression of FOXQ1 itself is subject to cell specific posttranscriptional and/or

posttranslational regulation that merits further investigation, as supported by the

inconsistencies in FOXQ1 mRNA and protein levels we observed in the colorectal cancer cell

lines we analyzed, and as corroborated by similar observations in breast cancer cell lines,

where for instance, two relatively high FOXQ1 mRNA expressing cells; MDA-MB-468 and

MB-436 had virtually none, and very high protein expression levels, respectively (16). The

cooperation of multiple transcription factors in the modulation of EMT is not new (36-39).

For example, following TGF-beta treatment in NMuMG cells, Snail1 was found to rapidly

enhance Twist1 mRNA and protein expression, as well as induce the nuclear translocation of

Ets1, and both Twist and Ets1 proceeded to induce the expression of ZEB1 by interacting

with different cis-elements in its promoter (36). Snail1, Twist1 and ZEB1 all repress E-

cadherin expression by binding to E-box elements in its promoter. It is likely that the

interaction of FOXQ1 with Twist1 further reinforces the suppression of E-cadherin

transcription, since FOXQ1 itself does not have any binding elements in the E-cadherin

promoter. Additionally, the ensuing cadherin switch could also be likely due to a direct

consequence of Twist1 modulation, as it has been proven to be a direct regulator of both E-

and N-cadherin expression (40, 41). Interestingly, two recent reports have shown that

FOXQ1 was able to directly repress E-cadherin expression by supposedly binding to E-box

elements in its promoter, however, the in vivo validation was inconsistent, where one group

could, and the other could not demonstrate a convincing FOXQ1 enrichment at the proximal




17

E-cadherin promoter containing the E-box (14, 16). Despite the changes in expression of

EMT markers, we observed at both mRNA and protein levels; we failed to observe an overt

phenotypic mesenchymal transformation. This may have been influenced by EMT inhibitors

like Grainyhead-like-2 (GRHL2). GRHL2 gene amplification has been noted in several tumor

types and directly represses the ZEB1 promoter (42). Since ZEB1 is known to be required for

EMT in response to Twist1, it is probable that GRHL2 may have contributed to this outcome,

but further studies are needed to address this postulation.

The forkhead family of transcription factors are known to be involved in tissue specific

transcription (43, 44), and even though we observed an influence of FOXQ1 on Twist1 in 4

different colorectal cancer cell lines, this effect was not present in the canine epithelial

MDCK-II and 293T embryonic kidney cell lines (data not shown), further reiterating this

point.

From our experiments, the effects of FOXQ1 on tumor progression were most marked on cell

migration, but nonetheless significantly impacted invasion and metastasis. As opposed to the

findings of Kaneda and colleagues (15), and in concordance with that of Zhang and

colleagues, and Qiao and colleagues (14, 16), respectively, we did not observe any effect on

cell proliferation, and in vivo tumor growth, even though the findings of the latter two groups

were limited to breast cancer cell lines. Nonetheless, it is important to note that the

experiments on which Kaneda and colleagues made their observations were carried out in the

lung cancer p53-null H1299 cell line, which on the account of this deletion already has an

increased proliferative and tumorigenic propensity (45, 46), and could account for the lack of

concordance to our observations. Given that EMT represents the critical event in the

transition from early to invasive carcinomas (5) and that both over-expression of Twist1 and

the down-regulation of E-cadherin are associated with poor prognosis in colorectal cancer

(47, 48), our finding that FOXQ1 is involved in the regulation of colorectal cancer




18

progression and metastasis is pertinent. However, it is also important to state that Twist1

itself is induced by other strong transcriptional regulators (27, 39, 49, 50), that the effects of

FOXQ1 on Twist1 while significant, were modest, and the observed effects of FOXQ1 on

invasion, migration and metastasis are very likely multifactorial. To ascertain if Twist1 was

entirely responsible for the modulation of these effects, we knocked down Twist1 in FOXQ1

stable over-expressing Colo206f cells and vector control cells. Interestingly, we observed a

significant reduction in both migration and invasion in the Twist1 knockdown cell lines, as

compared to the FOXQ1 stably expressing cells. Still, this knockdown did not completely

eliminate the increase in migration and invasion associated with FOXQ1 overexpression (as

compared to the vector group), suggesting that whereas Twist1 is significant for the increase

in aggressive cell behavior attributable to FOXQ1, other hitherto unidentified molecules also

contribute to these processes (Supplementary Figure 2).

Taken together, our results demonstrate that FOXQ1 is a novel modulator of Twist1

expression and a regulator of cancer invasion and metastasis. Certainly, further studies need

to be performed to characterize more of its targets, both direct and indirect, that bear

consequences on metastasis. The fact that it has been found to be significantly over-expressed

in colorectal cancer in two independent studies encourages us to support its further evaluation

as a potential tumor marker, particularly one that is indicative of metastasis




19

Acknowledgement

The authors would like to thank Jürgen Engel and Jacqueline Frohhert for technical

assistance. HA was supported by Alfried Krupp von Bohlen and Halbach Foundation (Award

for Young Full Professors), Essen, Hella-Bühler-Foundation, Heidelberg, Dr. Ingrid zu Solms

Foundation, Frankfurt/Main, the Hector Foundation, Weinheim, Germany, the FRONTIER

Excellence Initiative of the University of Heidelberg, the BMBF, Bonn, Germany, the Walter

Schulz Foundation, Munich, Germany, the Deutsche Krebshilfe, Bonn, Germany, and the

DKFZ-MOST German-Israeli program, Heidelberg, Germany. This manuscript contains part

of the dissertation (Dr. sc.hum) of MA.

Conflict of interest

No conflict of interest declared.




20

References

(1) Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell

2011;144:646-74.

(2) Roussos ET, Keckesova Z, Haley JD, Epstein DM, Weinberg RA, Condeelis JS. AACR special conference on epithelial-mesenchymal transition and cancer

progression and treatment. Cancer Res 2010;70:7360-4.

(3) Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression:

an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7:415-28.

(4) Thiery JP, Sleeman JP. Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 2006;7:131-42.

(5) Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871-90.

(6) Bieller A, Pasche B, Frank S, Glaser B, Kunz J, Witt K, et al. Isolation and characterization of the human forkhead gene FOXQ1. DNA Cell Biol 2001;20:555-61.

(7) Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged

helix/forkhead transcription factors. Genes Dev 2000;14:142-6.

(8) Katoh M, Katoh M. Human FOX gene family (Review). Int J Oncol 2004;25:1495-

500.

(9) Wotton KR, Shimeld SM. Analysis of lamprey clustered Fox genes: insight into Fox gene evolution and expression in vertebrates. Gene 2011;489:30-40.

(10) Goering W, Adham IM, Pasche B, Manner J, Ochs M, Engel W, et al. Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

Cytogenet Genome Res 2008;121:88-95.

(11) Hong HK, Noveroske JK, Headon DJ, Liu T, Sy MS, Justice MJ, et al. The winged helix/forkhead transcription factor Foxq1 regulates differentiation of hair in satin

mice. Genesis 2001;29:163-71.

(12) Potter CS, Peterson RL, Barth JL, Pruett ND, Jacobs DF, Kern MJ, et al. Evidence that

the satin hair mutant gene Foxq1 is among multiple and functionally diverse regulatory targets for Hoxc13 during hair follicle differentiation. J Biol Chem 2006;281:29245-55.

(13) Feuerborn A, Srivastava PK, Kuffer S, Grandy WA, Sijmonsma TP, Gretz N, et al. The Forkhead factor FoxQ1 influences epithelial differentiation. J Cell Physiol

2011;226:710-9.

(14) Zhang H, Meng F, Liu G, Zhang B, Zhu J, Wu F, et al. Forkhead transcription factor foxq1 promotes epithelial-mesenchymal transition and breast cancer metastasis.

Cancer Res 2011;71:1292-301.




21

(15) Kaneda H, Arao T, Tanaka K, Tamura D, Aomatsu K, Kudo K, et al. FOXQ1 is

overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth. Cancer Res 2010;70:2053-63.

(16) Qiao Y, Jiang X, Lee ST, Karuturi RK, Hooi SC, Yu Q. FOXQ1 regulates epithelial-mesenchymal transition in human cancers. Cancer Res 2011;71:3076-86.

(17) Feng J, Zhang X, Zhu H, Wang X, Ni S, Huang J. FoxQ1 overexpression influences

poor prognosis in non-small cell lung cancer, associates with the phenomenon of EMT. PLoS One 2012;7:e39937.

(18) Abba M, Laufs S, Aghajany M, Korn B, Benner A, Allgayer H. Look who's talking: deregulated signaling in colorectal cancer. Cancer Genomics Proteomics 2012;9:15-25.

(19) Leupold JH, Yang HS, Colburn NH, Asangani I, Post S, Allgayer H. Tumor suppressor Pdcd4 inhibits invasion/intravasation and regulates urokinase receptor (u-

PAR) gene expression via Sp-transcription factors. Oncogene 2007;26:4550-62.

(20) Rasheed SA, Efferth T, Asangani IA, Allgayer H. First evidence that the antimalarial drug artesunate inhibits invasion and in vivo metastasis in lung cancer by targeting

essential extracellular proteases. Int J Cancer 2010;127:1475-85.

(21) Chambers AF, Ling V. Selection for experimental metastatic ability of heterologous

tumor cells in the chick embryo after DNA-mediated transfer. Cancer Res 1984;44:3970-5.

(22) Dexter DL, Lee ES, DeFusco DJ, Libbey NP, Spremulli EN, Calabresi P. Selection of

metastatic variants from heterogeneous tumor cell lines using the chicken chorioallantoic membrane and nude mouse. Cancer Res 1983;43:1733-40.

(23) Wilson SM, Chambers AF. Experimental metastasis assays in the chick embryo. Curr Protoc Cell Biol 2004;Chapter 19:Unit.

(24) Brenner JC, Ateeq B, Li Y, Yocum AK, Cao Q, Asangani IA, et al. Mechanistic

rationale for inhibition of poly(ADP-ribose) polymerase in ETS gene fusion-positive prostate cancer. Cancer Cell 2011;19:664-78.

(25) van der Horst EH, Leupold JH, Schubbert R, Ullrich A, Allgayer H. TaqMan-based quantification of invasive cells in the chick embryo metastasis assay. Biotechniques 2004;37:940-2, 944, 946.

(26) Overdier DG, Porcella A, Costa RH. The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the

recognition helix. Mol Cell Biol 1994;14:2755-66.

(27) Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, et al. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol 2008;10:295-305.

(28) Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC, et al. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform

reproducibility of gene expression measurements. Nat Biotechnol 2006;24:1151-61.




22

(29) Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin

promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008;68:3645-54.

(30) Shah GV, Muralidharan A, Gokulgandhi M, Soan K, Thomas S. Cadherin switching and activation of beta-catenin signaling underlie proinvasive actions of calcitonin-calcitonin receptor axis in prostate cancer. J Biol Chem 2009;284:1018-30.

(31) Wheelock MJ, Shintani Y, Maeda M, Fukumoto Y, Johnson KR. Cadherin switching. J Cell Sci 2008;121:727-35.

(32) Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004;117:927-39.

(33) Ansieau S, Morel AP, Hinkal G, Bastid J, Puisieux A. TWISTing an embryonic transcription factor into an oncoprotein. Oncogene 2010;29:3173-84.

(34) Eckert MA, Lwin TM, Chang AT, Kim J, Danis E, Ohno-Machado L, et al. Twist1-induced invadopodia formation promotes tumor metastasis. Cancer Cell 2011;19:372-86.

(35) Yang F, Sun L, Li Q, Han X, Lei L, Zhang H, et al. SET8 promotes epithelial-mesenchymal transition and confers TWIST dual transcriptional activities. EMBO J

2012;31:110-23.

(36) Dave N, Guaita-Esteruelas S, Gutarra S, Frias A, Beltran M, Peiro S, et al. Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during

epithelial to mesenchymal transition. J Biol Chem 2011;286:12024-32.

(37) Byles V, Zhu L, Lovaas JD, Chmilewski LK, Wang J, Faller DV, et al. SIRT1 induces

EMT by cooperating with EMT transcription factors and enhances prostate cancer cell migration and metastasis. Oncogene 2012;31:4619-29.

(38) Alves CC, Rosivatz E, Schott C, Hollweck R, Becker I, Sarbia M, et al. Slug is

overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin. J Pathol 2007;211:507-15.

(39) Lander R, Nordin K, LaBonne C. The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1. J Cell Biol 2011;194:17-25.

(40) Moreno-Bueno G, Portillo F, Cano A. Transcriptional regulation of cell polarity in

EMT and cancer. Oncogene 2008;27:6958-69.

(41) Peinado H, Portillo F, Cano A. Transcriptional regulation of cadherins during

development and carcinogenesis. Int J Dev Biol 2004;48:365-75.

(42) Cieply B, Riley P, Pifer PM, Widmeyer J, Addison JB, Ivanov AV, et al. Suppression of the epithelial-mesenchymal transition by Grainyhead- like-2. Cancer Res

2012;72:2440-53.

(43) Choi VM, Harland RM, Khokha MK. Developmental expression of FoxJ1.2, FoxJ2,

and FoxQ1 in Xenopus tropicalis. Gene Expr Patterns 2006;6:443-7.




23

(44) Hoggatt AM, Kriegel AM, Smith AF, Herring BP. Hepatocyte nuclear factor-3

homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes. J Biol Chem 2000;275:31162-70.

(45) Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Jr., Butel JS, et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992;356:215-21.

(46) Harvey M, McArthur MJ, Montgomery CA, Jr., Butel JS, Bradley A, Donehower LA. Spontaneous and carcinogen- induced tumorigenesis in p53-deficient mice. Nat Genet

1993;5:225-9.

(47) Gomez I, Pena C, Herrera M, Munoz C, Larriba MJ, Garcia V, et al. TWIST1 is expressed in colorectal carcinomas and predicts patient survival. PLoS One

2011;6:e18023.

(48) Karamitopoulou E, Zlobec I, Patsouris E, Peros G, Lugli A. Loss of E-cadherin

independently predicts the lymph node status in colorectal cancer. Pathology 2011;43:133-7.

(49) Dupont J, Fernandez AM, Glackin CA, Helman L, LeRoith D. Insulin-like growth

factor 1 (IGF-1)-induced twist expression is involved in the anti-apoptotic effects of the IGF-1 receptor. J Biol Chem 2001;276:26699-707.

(50) Tan EJ, Thuault S, Caja L, Carletti T, Heldin CH, Moustakas A. Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition. J Biol Chem 2012;287:7134-

45.




24

Figure Legends:

Figure 1: Analytical screen of genes affected by FOXQ1 expression: a) Endogenous mRNA

and protein expression in cell lines; b) Over-expression of FOXQ1 in Colo206f and Geo cell

lines with evaluation of relative mRNA expression changes of the candidate genes by

quantitative real time PCR; c) si-RNA mediated suppression of FOXQ1 expression with

evaluation of the same genes as in b); d) Evaluation of protein expression changes upon

FOXQ1 over-expression in GEO and knock down in WiDr cell lines respectively; e)

Evaluation of the knock-down of FOXQ1 over a 72hr time-line in SW480 cells showing that

the effects of suppression are first observed with Twist1 before CDH1, indicating that the

effects of FOXQ1 on CDH1 are likely secondary to that of Twist1. Relative mRNA

expression is represented on the y axis. (* = p< 0.05; ** = p< 0.01; ***= p< 0.001).

Figure 2: Identification of FOXQ1 binding motifs: a) Consensus binding motifs of FOXQ1 as

predicted by Transfac and Jasper databases for rat; no human matrices were identified; b) The

FOXQ1 coding sequence is evolutionary conserved; c) Putative binding sites in the core

Twist1 promoter.

Figure 3: Transcriptional regulation of Twist1 by FOXQ1 a) Reporter gene assays showing

increased luciferase activity with the Twist1 promoter upon expression of Foxq1; b) Twist 1

induction in a concentration-dependent manner; c) Reporter assays with-wild type and mutant

constructs, with schematic representation of the mutants; d) Quantitative real time PCR

evaluation for enrichment of a fragment of the Twist promoter following ChIP with anti-HA

and IgG antibodies and normalization to the respective input; e) Evaluation of the enrichment

using semi-quantitative PCR. (* = p< 0.05; ** = p< 0.01).




25

Figure 4: In-vivo mRNA expression heatmap of EMT genes in a cohort of 45 resected patient

tissues. The relative tumor/normal expression values of each gene after normalisation to 3

house-keeping genes (SDHA, TBP and B2M) were imported into the MeV tool of the TM4

software suite (www.tm4.org). The colour bar at the top represents the range of expression

values and, each row represents the profile of an individual patient, and each column the

profile of a gene across patients. Grey boxes signify samples where the values were not

determined.; b) In-vivo correlation of protein expression in 12 patient samples with Western

blot showing predominant expression in tumor tissues; c) Y-Y correlation plot of FOXQ1

and Twist1 gene expression as was obtained from the MAQC control project (GEO ID:

GSE5350) encompassing 120 reference RNA samples. The red dots represent the expression

log2 transformed expression values of FOXQ1 and the blue dots, the corresponding values of

Twist1. The coloured boxes at the bottom of the panel indicate the reference samples that

were used; correlation coefficient r = 0.905.

Figure 5: Effects of FOXQ1 on cell proliferation: a) Cellular growth curve of Colo-206F and

GEO cell lines stably expressing EGFP or FOXQ1. A total of 1 × 104 cells of each cell line

were seeded in 96-well plates and evaluated at 0, 24, 48, 72, 96 and 120hr time points using

MTT assay; b) Cellular growth of SW480 and WiDR cell lines transfected with a scrambled

or FOXQ1 specific si-RNA. These cells were initially seeded in 6 well plates, transfected, and

24 hrs after transfection trypsinized and reseeded in 96 well plates. MTT was performed in

the same manner as for the over-expressing cell lines; c) Box plots representing the weight of

tumor masses excised from the upper chorioallantoic membrane of 17 day incubated eggs

following inoculation of FOXQ1 or vector expressing GEO and Colo206F cells, respectively.

For GEO cells; vector n=4, FOXQ1, n=5; for Colo206F cells, n=7 for both vector and



http://www.tm4.org/


26

FOXQ1 expressing cells. The observed difference in tumor weight between cell lines was

accounted for by the different sizes of the tumors that formed for the two cell lines (inset).

Figure 6: FOXQ1 promotes cell migration and invasion: a) Boyden chamber migration assays

in two over-expressing and two knock-down cells. 5, 000 cells were seeded in transwell

chambers in serum deprived medium and migration towards serum containing media was

measured by the Cell Titer-Glo assay. Values represent the percentage of migrated cells in

relation to the total number of cells; b) Invasion assays with 10ug/ml matrigel, and also

evaluated with Cell Titer Glo. Migration and invasion were assessed at 24hr time points. (* =

p< 0.05; ** = p< 0.01)

Figure 7: FOXQ1 promotes long distance metastasis in the in vivo CAM assay model; a) lung

and b) liver metastasis were evaluated quantitatively by RT PCR using primers and a probe

specific for the human YB8 Alu family. Genomic DNA isolated from the harvested organs 7

days after the inoculation of cells onto the upper CAM was used for the assay (n=7 and n=8

for vector and FOXQ1 groups, respectively). (* = p< 0.05; ** = p< 0.01)




27




28




29




30




31




32




33




Published OnlineFirst May 30, 2013.Mol Cancer Res Mohammed Abba, Nitin Patil, Kabeer Rasheed, et al. Unraveling the role of FOXQ1 in colorectal cancer metastasis

Updated version

10.1158/1541-7786.MCR-13-0024doi:

Access the most recent version of this article at:

Material

Supplementary

http://mcr.aacrjournals.org/content/suppl/2013/05/30/1541-7786.MCR-13-0024.DC1

Access the most recent supplemental material at:

Manuscript

Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mcr.aacrjournals.org/content/early/2013/05/30/1541-7786.MCR-13-0024To request permission to re-use all or part of this article, use this link



http://mcr.aacrjournals.org/lookup/doi/10.1158/1541-7786.MCR-13-0024

http://mcr.aacrjournals.org/content/suppl/2013/05/30/1541-7786.MCR-13-0024.DC1

http://mcr.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mcr.aacrjournals.org/content/early/2013/05/30/1541-7786.MCR-13-0024


department of experimental surgery, medical …...2013/05/30 · 1 unraveling the role of foxq1 in...

Documents