Scholarly article on topic 'Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance'

Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance Academic research paper on "Biological sciences"

CC BY
0
0
Share paper
Academic journal
Cell Reports
OECD Field of science
Keywords
{}

Abstract of research paper on Biological sciences, author of scientific article — Zoltán Wiener, Jenny Högström, Ville Hyvönen, Arja M. Band, Pauliina Kallio, et al.

Summary Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Academic research paper on topic "Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance"

Cell Reports

Article

Prox1 Promotes Expansion of the Colorectal Cancer Stem Cell Population to Fuel Tumor Growth and Ischemia Resistance

Graphical Abstract

Authors

Zoltan Wiener, Jenny Hogstrom.....Yinon

Ben-Neriah, Kari Alitalo

Correspondence

kari.alitalo@helsinki.fi

In Brief

Wiener et al. now show that the Proxl transcription factor functions as a stem cell regulator in intestinal adenomas and colorectal cancer (CRC), but not in the normal intestine. Proxl critically contributes to tumor cell survival in hypoxia and to the expansion of the adenoma/ CRC stem cell population via inhibition of the Wnt-target annexin A1 and induction of the actin-binding protein filamin A. The Proxl pathway thus represents an attractive therapeutic target for drug development in CRC.

Highlights Accession Numbers

Proxl expression is dispensable for homeostasis in the normal GSE47568

intestine

A subpopulation of Prox1+ cells has stem cell activity in intestinal adenomas/CRC

Loss of Proxl decreases adenoma/CRC stem cells, tumor cell growth, and survival

Annexin A1 and filamin A mediate Proxl effects on stem cell activity in the tumors

Wiener et al., 2014, Cell Reports 8, 1943-1956 ) ciossMark September 25, 2014 ©2014 The Authors

http://dx.d0i.0rg/l 0.1016/j.celrep.2014.08.034

CelPress

Cell Reports

Article

Prox1 Promotes Expansion

of the Colorectal Cancer Stem Cell Population

to Fuel Tumor Growth and Ischemia Resistance

Zoltan Wiener,16 Jenny Högström,1'6 Ville Hyvonen,1 Arja M. Band,1 Pauliina Kallio,1 Tanja Holopainen,1 Olli Dufva,1 Caj Haglund,3 Olli Kruuna,3 Guillermo Oliver,4 Yinon Ben-Neriah,5 and Kari Alitalo12 *

1Translational Cancer Biology Program, University of Helsinki

2Wihuri Research Institute

Biomedicum Helsinki, 00014 Helsinki, Finland

3Department of Surgery, Helsinki University Central Hospital, 00029 Helsinki, Finland 4Department of Genetics, St Jude Children's Research Hospital, Memphis, TN 38105, USA 5Lautenberg Center for Immunology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel 6Co-first author

'Correspondence: kari.alitalo@helsinki.fi http://dx.doi.org/10.10167j.celrep.2014.08.034

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

SUMMARY

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Proxl-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Proxl deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Proxl also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Proxl is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

INTRODUCTION

Colorectal cancer (CRC) is one of the leading causes of cancer mortality in Western countries. In most CRC patients, an initial mutation occurs in the APC or CTNNB1 gene, leading to activation of the p-catenin/TCF (canonical Wnt) pathway (Fearon, 2011; Fodde and Smits, 2001). In the crypts of the normal gut, the p-catenin/TCF pathway is active in Paneth cells, in transit-amplifying (TA)/progenitor cells that have a limited proliferative capacity, and in Lgr5+ intestinal stem cells (Clevers, 2006). Lgr5+ cells are capable of efficiently initiating adenoma formation upon mutation of the Apc gene (Barker et al., 2009). In addition, progenitors of intestinal epithelial cells can convert to a stem cell-like phenotype and contribute to the initiation of CRC under

inflammatory conditions (Schwitalla et al., 2013). These studies indicate that the acquisition of a stem cell-like phenotype is critical for CRC tumorigenesis.

Intestinal adenomas are highly heterogenous, containing both proliferating and differentiating cells, and they are continuously maintained by a dedicated cell population, the so-called cancer stem cells (Sampieri and Fodde, 2012; Schepers et al., 2012). Although neoplastic cells are characterized by increased cell proliferation and limited cell differentiation capacity, their detailed differentiation pathways in CRC are poorly known. Expression of the Lgr5 gene has been shown to mark a cell population with stem cell properties in mouse intestine and in intestinal adenomas (Barker et al., 2007; Schepers et al., 2012). Furthermore, the intestinal stem cell signature, including LGR5 expression, identifies CRC stem cells and predicts disease relapse also in human CRC patients (Kemper et al., 2012; Mer-los-Suarez et al., 2011).

After the genetic lesion that activates the Wnt signal transduc-tion pathway and abnormal cell proliferation, additional mutations accumulate slowly to promote adenoma progression toward CRC, tumor invasion, and metastasis (Sampieri and Fodde, 2012). Highly elevated Wnt activity after Apc deletion induces expression of the homeobox transcription factor Prox1 in intestinal tumor cells. When Proxl was deleted in mice with an Apc mutation, adenoma growth and development of dysplasia in the tumor epithelium was inhibited (Petrova et al., 2008). Interestingly, Prox1 upregulation after loss of the tp53 tumor suppressor contributes to intestinal tumor progression in some model systems (Elyada et al., 2011).

Here, we analyzed the mechanism of Prox1-induced intestinal adenoma progression in microsatellite-stable tumor models. We found that a subpopulation of Prox1+ cells displays stem cell activity in adenomas/CRC, but not in the normal intestinal epithelium. Furthermore, Proxl deletion reduced the size of the Lgr5+ adenoma and CRC stem cell populations, and stem cell activity and led to reduced growth and decreased tumor cell survival in an unfavorable microenvironment.

RESULTS

Prox1 Expression Is Induced in Lgr5+ Cells upon Apc Gene Deletion

To characterize Proxl-expressing cells in the pathogenesis of intestinal adenomas, we induced an acute deletion of the Apc gene throughout the whole intestinal epithelium in Apcflox/flox; villin-CreER (VApc) mice by a single tamoxifen injection (VApcA/A). As expected, Paneth cells, marking the crypt bottoms in the wild-type (WT) small intestine, were dislocated toward the lumen in most of the Apc-deleted crypts, and expansion of the cell population with an active Wnt signaling pathway was detected by EphB2 immunostaining 6 days after the tamoxifen injection (Figures S1A and S1B) (Batlle et al., 2002). Most crypts contained scattered Prox1+ cell clusters intermingled with lysozyme-positive Paneth cells (Figure S1A). Cyclin D1 marks the proliferating cell population, including the progenitor cells in WT intestine (Gregorieff and Clevers, 2005). In line with previously published results, the cyclin D1+ cell population expanded after the Apc deletion (Sansom et al., 2005) (Figures S1A and S1B). CyclinD1high cells were located frequently close to the Prox1+ cells; however, Prox1+ cells were not cyclin D1high in VApcA/A mice and in tumors from Apcmin/+ mice (Figures S1C and S1D) or from CRC patients (Figure S1E). Similarly to the in vivo experiments, when we isolated intestinal organoids from the VApc mice and induced Apc gene deletion in 3D Matrigel culture by the addition of 4-hy-droxy-tamoxifen (4-OH-Tam), the organoids showed emerging Prox1+ cell clusters 2 days after 4-OH-Tam treatment (Figure S1F). At later time points, Prox1 expression was maintained in isolated cell clusters (Figure S1G).

A recent study reported that PROX1 is part of the Wnthigh CRC stem cell gene signature (de Sousa E Melo et al., 2011). We found that Prox1 was expressed in the earliest histological adenoma-tous lesions, the aberrant crypt foci, of the Apcmin/+ mice, which represent a widely used mouse intestinal adenoma model (Figure 1A). To examine Prox1 expression in the Lgr5+ intestinal stem cells of the adenomas, we produced Apcflox/flox; Lgr5-EGFP-IRES-CreER (LApc) mice and induced Apc deletion in the stem cells (LApcA/A). LApc and Lgr5-EGFP-IRES-CreER mouse intestines show green EGFP signals in the Lgr5+ stem cells at the crypt bottom (Barker et al., 2007). Notably, the cyclin D1high cell clusters were enriched mostly among the Lgr5low and Lgr5~ cell populations in the intestinal adenomas after Apc deletion, confirming that cyclin D1high cells do not accumulate in the stem cell population (Figure 1B). Prox1 was expressed in some of the Paneth cells in the ileum and Prox1 + cells in the normal intestine were often located near the Lgr5+ cells (Figure S1H; Figure 1C), but there was no overlap between the EGFP and Prox1 signals in the untreated intestine or during epithelial repair at 4,6, and 10 days after 6 Gy irradiation (Figure S11 and data not shown). However, 5 days after tamoxifen injection, Prox1 expression was observed also in the Lgr5+ cells of the developing adenomas (Figure 1C). Taken together, these results indicate that Prox1 is induced during the early steps of tumori-genesis in adenoma cells, including Lgr5+ stem cells, both in vivo and ex vivo, but not in the cyclin D1high cells that may represent more differentiated or intestinal progenitor-like cells.

A Subpopulation of the Prox1+ Cells Has Stem Cell Activity in Intestinal Adenomas, but Not in the Normal Intestine

To study if Prox1+ cells have stem cell activity in intestinal adenomas, we produced Prox1-CreER (Srinivasan et al., 2007); Rosa26-tdTomatoflox/Stop/flox; Apcmln/+ mice for lineage-tracing experiments. In this model, a single tamoxifen injection activated the Cre allele, resulting in expression of the tdTomato red fluorescent protein only in the Prox1+ tumor cells 1 day after the tamoxifen injection (Figure 1D), confirming that the activation of the Cre protein is specific to the Prox1-expressing tumor cells. However, 28 days after tamoxifen injection, we observed that the Prox1+ cells had produced adjacent tdTomato+/Prox1 ~ progeny, which occasionally stained for mucin2 of goblet cells or for lysozyme of Paneth cells, indicating that Prox1+ tumor cells can give rise to differentiated cells in the intestinal adenomas (Figures 1D and 1E).

In the Prox1-CreER; Rosa26-tdTomatoflox/Stop/flox; Apcmln/+ mice, Prox1+ epithelial cells were very rarely labeled outside of the tumors after tamoxifen injection. In order to activate the lineage marker more effectively, we used mice harboring a Prox1-CreER bacterial artificial chromosome at an ectopic genomic site (Bazigou et al., 2011). In these mice, only sporadic Prox1 + intestinal epithelial cells were positive for the red lineage marker 7 days after tamoxifen treatment (Figure 1F). Furthermore, Prox1 deletion in the intestinal epithelium of Prox1f'°x/f'°x; villin-CreER mice (VPA/A) did not result in any obvious phenotype (see Supplemental Results). These data indicate that a subpopulation of the Prox1 + cells has stem cell properties in adenomas, but not in normal intestine.

Prox1 Deletion Leads to Loss of Lgr5+ Stem Cells in Intestinal Adenomas

To analyze the significance of Prox1 specifically in adenoma stem cells, we deleted Apc and Prox1 in the Lgr5+ cells of Apcflox/flox; Prox1flox/flox; Lgr5-EGFP-IRES-CreER (LApcP) mice. In order to obtain an efficient deletion, tamoxifen was injected during 2 consecutive days and the size of the Lgr5+ cell population was analyzed 21 days thereafter. Interestingly, the majority of the adenomas contained some Prox1+ cells, suggesting an incomplete deletion of Prox1 in the crypt stem cells (Figure 2A). However, the Prox1+ adenomas contained fewer Lgr5+ cells in tamoxifen-treated LApcP (LApcPA/A) mouse intestines than in the LApcA/A controls (Figure 2B). Furthermore, the number of stem cells was even lower in the Prox1~ crypt-like structures that contained dislocated Lgr5+ cells both in the small and large intestine, indicating that successful Prox1 deletion inhibits the expansion of Lgr5+ cells in the adenomas (Figure 2B).

To further test this hypothesis ex vivo, we used LApcA/A and LApcPA/A organoids. The Wnt-agonist R-Spondin1 is required for the survival and growth of the WT (LApc or LApcP) intestinal organoids (Sato et al., 2009). Without R-Spondin1, only organoids with Apc deletion and resulting active p-catenin/TCF pathway survive beyond 4 days (Wiener et al., 2014). Almost all the Prox1 + cells of LApcA/A organoids were Lgr5-EGFP positive 8 days after the addition of 4-OH-Tam without R-Spondin1 (Figure 2C). However, there was a marked reduction in the number of viable organoids 8 days after the simultaneous deletion of Apc

Apcmin/+

LApcA/A

| Day 0 Day 5 Day 21

\JBcr fc'S ' (H t * «4 k«f * - i

m M* * i$k > & i|r j# l__________1 -

j Day1 Day28

• ;% f ** o Jn§ 4 <<■ At '

«M» 3 X i ' « m : Z a\ •

Muc2 Proxl Lys Proxl

* i ^ 1

p J 'c

" i -4-. ' ^L -J ,

mi t? >

and Proxl in the Lgr5+ cells (Figures 2D-2F). The viable organoids contained some Prox1+ cells at this time point (Figure 2C), suggesting that Proxl deletion was not complete in the organoids. Since Proxl was not expressed in the Lgr5+ stem cells before Apc was deleted, but only after 5 days of Apc deletion in vivo, these data indicate that loss of Proxl expression does

Figure 1. Prox1 Expression and Cell Lineage Tracing in Intestinal Adenomas after Apc Deletion

(A) Proxl expression in an aberrant crypt focus in the Apcminf+ intestine.

(B) Lgr5-EGFP and cyclin D1 staining in LApc mouse intestine before (LApc) and 21 days after the addition of tamoxifen (LApcA/A). Note some overlap of staining in the Lgr5low cell population (dotted areas).

(C) Distribution of the Lgr5-EGFP and Proxl signals in the intestinal epithelium of LApc mice after the injection of a single dose of tamoxifen. Note the lack of overlap between Prox1+ (arrowhead) and Lgr5+ cells at day 0 and the overlap (dotted areas) at days 5 and 21.

(D and E) Immunostaining of adenomas derived from Prox1-CreER; Rosa26-tdTomatoflox/Stop/flox; Apcm'nf+ mice for the indicated proteins after a single tamoxifen injection (cell lineage tracing). The arrows mark tdTomato+/Prox1 ~/Mucin2+ or tdTomato+/Prox1 ~/Lysozyme+ cells. (F) Proxl and tdTomato signals in Prox1-CreER; Rosa26-tdTomatofloxfStopfflox mice 7 days after tamoxifen injection. The arrows indicate the rare labeled Prox1+ cells in the normal intestinal epithelium.

Scale bars represent 20 mm (B and D-F) or 50 mm (A and C). See also Figure S1.

not influence the tumor initiation frequency, but instead, it inhibits the expansion of the Lgr5+ adenoma cell population.

To directly address the connection between Proxl and stem cell number in another ex vivo organoid culture system, we isolated organoids from Apcfloxfflox; villin-CreER (VApc) and Apcf'oxff'ox; Prox1floxfflox; villin-CreER (VApcP) mice, where Apc and/or Proxl are deleted in the whole intestinal epithelium, including the progenitor cells. Deletion of Proxl alone from the whole intestinal epithelium derived from the Proxl f'oxff'ox; villin-CreER mice did not induce morphological changes in the organoids (Figure S2A). However, deletion of both Apc and Proxl (VApcPA/A) from the organoids increased the relative expression level of the progenitor markers Noll and Wdr3 (Van der Flier et al., 2007) and the relative number of cyclin D1high cells in comparison with organoids with only Apc deletion (Figures 3A-3C). As in the tamoxifen-injected VApcA/A mice, the Prox1+ cells were cyclin D1low (Figure 3B). Furthermore, RNAs encoding the Wnt-target intestinal stem cell signature genes Lgr5, Tnfrsf19, and Ascl2 were decreased upon Proxl deletion, whereas c-Myc RNA was not changed (Figure 3C and data not shown). Microarray analysis of VApcA/A and VApcPA/A cultures

Figure 2. Prox1 Deletion from the Lgr5+ Adenoma Cells Inhibits the Expansion of the Stem Cell Pool

(A) Lgr5-EGFP and Proxl signals in untreated LApc mouse intestine and in LApcPD/D intestine 21 days after tamoxifen addition. The arrowhead marks a Proxl" crypt with dislocated Lgr5+ cells (implicating Apc deletion). The asterisks mark Prox1+ crypts.

(B) The number of Lgr5+ cells per the various types of crypts in the small intestine or colon (Kruskal-Wallis test, n = 25 from three mice). The boxplot indicates the minimum, the first quartile, median, third quartile, and maximum.

(C) Lgr5-EGFP and Proxl signals in the LApcD/D and LApcPD/D organoids.

(D and E) Viable LApcD/D and LApcPD/D organoids (D, arrowheads) and their quantification (E). (F) Proxl RNA level.

For(C)-(F), the samples were analyzed 8 days after the addition of 4-OH-Tam and 6 days after the removal of the growth factors. Scale bars represent 20 mm (C) or 100 mm (A and D). Mean + SD are shown (E and F).

7 days after the addition of 4-OH-Tam indicated decreased intestinal stem cell markers and changes in the expression levels of specific Wnt targets in the VApcPA/A organoids (Figure S2B). These data suggested that Proxl deletion decreases the number of adenoma stem cells, resulting in a skewed enrichment of the progenitor population in the organoids 7 days after Apc deletion. Indeed, the Proxl -deleted organoids were less efficient in forming new organoid subcultures when they were dissociated to

small clusters containing approximately four to seven cells (Figure 3D).

PROX1 Silencing Decreases Stem Cells in Human CRC Organoids

To model the effect of PROX1 on stem cell activity in CRC progression, we chose to use the human SW1222 cell line (mutant CRC genes in this cell line are listed in Table S1). This cell line

is enriched for stem cells that can self-renew and differentiate into multiple lineages (Yeung et al., 2010). While the "small colonies'' in SW1222 cell-derived 3D cultures have a limited growth potential and lack lumens, the large glandular "megacol-onies'' produce crypt-like structures consisting of polarized cells surrounding a central lumen (Yeung et al., 2010). Lumens are stem cell-dependent structures present in well-differentiated tumors and likewise in ex vivo and in vitro 3D cultures from human and mouse adenomas and stem cell-containing CRC cell lines (Ashley et al., 2013).

Since the SW1222 cells have heterogenous PROX1 and nuclear p-CATENIN expression levels (Figure S2C), we isolated subclones with a low (PROX1low) or a high (PROX1high) percentage of PROX1+ cells (Figure S2D). We then silenced PROX1 in the PROX1high clone by two different lentiviral short hairpin RNAs (shRNAs) (Figure S2E and data not shown). Interestingly, the PROX1-silenced SW1222-PROX1high cells formed a strikingly reduced number of glandular colonies (Figure 3E), suggesting that PROX1 silencing has a profound effect on CRC stem cell activity. Furthermore, PROX1 silencing decreased the number of lumens in the glandular colonies derived from SW1222-PROX1high cells (Figure 3F). After 12 days of 3D culture, the LGR5 and TNFRSF19 RNAs were decreased, whereas no changes were observed in the expression of other WNT-target genes, such as MYC (Figure 3G). PROX1 silencing in CRC patient-derived organoids also resulted in decreased frequency of new lumen-containing organoids (Figure 3H). Notably, the shPROX1 organoids formed small colonies without lumens (Figure 3H). Similarly to the SW1222 cell line, PROX1 silencing in CRC patient-derived organoids resulted in a marked reduction of LGR5 and TNFRSF19 RNA levels (Figure 3I), even in the presence of a KRAS mutation (see Supplemental Experimental Procedures). LGR5 in situ hybridization further indicated that PROX1 silencing decreases the number of CRC stem cells (Figures 3J and 3K). Notably, there was no difference in the LGR5 RNA in PROX1-silenced SW1222 cells in 2D culture (data not shown), thus ruling out the possibility that LGR5 is a direct PROX1 target. These results suggest that loss of PROX1 decreases the CRC stem cells also in human CRC, and that this is independent of the presence of KRAS mutations.

PROX1+ Tumor Cells Proliferate Less Than Prox1 Tumor Cells, yet PROX1 Deletion Leads to Decreased Cell Proliferation in the Tumors In Vivo

To test the in vivo significance of our findings, we implanted SW1222-PROX1high cells and their PROX1-silenced counterparts subcutaneously into immunocompromised (NOD scid gamma [NSG]) mice and monitored tumor growth. Similarly to the Proxl-deleted intestinal adenomas (Petrova et al., 2008), the PROX1-silenced tumors grew slower than controls transduced with scrambled shRNA (Scr) at this ectopic implantation site (Figure 4A). Notably, we detected 60% fewer tumor nests in the PROX1-silenced samples as compared to Scr controls (200.4 ± 46.8 and 70.9 ± 11.8 tumor nests, respectively; p < 0.01) when they were excised 21 days after the implantation. Unlike the scrambled shRNA-transduced tumors, the PROX1-silenced tumors contained tumor nests with only few glandular structures (Figure 4B). Similarly to the

PROX1-silenced SW1222 cells, subcutaneously implanted Proxl -deleted mouse VApcPA/A organoids grew slower and contained fewer glands than VApcD/D organoids (Figures 4C and 4D). Proxl deletion decreased Lgr5 and Tnfrsf19 RNAs, but not of Myc RNA in the tumors, confirming our ex vivo results (Figure 4E).

Intestinal epithelial progenitor cells proliferate rapidly, but only for a limited number of cell cycles (Gregorieff and Clevers, 2005). Careful analysis of the Prox1 + and Prox1~ cells in control SW1222-PROX1high or in VApcD/D organoid-derived tumors indicated that the Ki67+ proliferating cells were enriched in the Prox1" cell population (Figures S3A and S3B), suggesting that most of the Prox1+ cells are nonproliferative or slowly proliferating. Interestingly, the Prox1+/Lgr5+ cell population had a higher frequency of Ki67+ cells than the Prox1+/Lgr5~ population in the LApcA/A intestine (Figure 4F), indicating that the rapidly proliferating Prox1+ cells are enriched in the Lgr5+ population. Similarly, bromodeoxyuridine labeling of two highly proliferating organoid cultures (VApcA/A and VCKIA/A) showed that only a small proportion of the Prox1 + cells proliferated (Figure S3C). VCKIA/A organoids are deleted of tp53 and Csnklal, encoding casein kinase Ia (CKIa), which phosphorylates p-catenin, targeting it to ubiquitin-mediated destruction; thus, the VCKIA/A organoids display activated Wnt signaling pathway (Elyada et al., 2011). Whereas Prox1+ cells appeared to proliferate slowly, we observed a decreased overall frequency of Ki67+ tumor cells in subcutaneously growing PROX1-silenced SW1222-PROX1high tumors, in Proxl-deleted VApcPA/A organoid tumors in NSG mice, and in the Lgr5+ cells of LApcPA/A intestinal tumors (Figure 4G; Figure S3D). A possible explanation for these apparently contradictory data is that Proxl deletion results in decreased proliferation in the stem cell population, which leads to exhaustion of the CRC stem cell pool and consequently to a decrease of the overall ratio of proliferating cells. Indeed, a slower proliferation rate of the PROX1-silenced SW1222-PROX1high cell-derived organoids was observed only after a 14-day culturing period, when the PROX1+ organoids already had an extensive lumen forming activity, but not at 6 days (Figure S3E). This indicates a delayed effect of PROX1 silencing on the overall cell proliferation in the organoids, in line with the idea that PROX1 influences tumor growth by regulating the size of the proliferating stem cell pool.

The PROX1- (adherent) cells of the SW480 CRC cell line (SW480A) are unable to initiate subcutaneous tumors in NSG mice, whereas the PROX1+ cells (SW480R) are round, form cell clusters, and are tumorigenic (Petrova et al., 2008). To test if PROX1 silencing in already established CRC xenografts regulates stem cells, we implanted cells from the PROX1+ stem cell-like SW480R subclone, expressing a doxycycline-inducible PROX1 shRNA construct (SW480R-sh) (Petrova et al., 2008), into NSG mice. Doxycycline treatment was started 8 days after subcutaneous injection, when the tumors were already visible. We observed reduced growth of the PROX1-silenced tumors after day 16 (Figures 4H and 4I), at a time point when the LGR5 RNA level had already markedly decreased in the doxycycline-treated tumors (Figure 4J). These data suggest that PROX1 regulates stem cells also in established tumors.

Figure 3. Prox1 Deletion Leads to Reduced Stem Cell Activity in Ex Vivo Organoids

(A) The schematic outline of the mouse organoid experiments. GF, growth factors.

(B) Prox1 and cyclin D1 immunostaining of sections from VApcA/A and VApcPA/A organoids 7 days after the deletion and 5 days after removing R-Spondin1 from the culture medium.

(C) RNA levels of progenitor markers (Nol1, cyclin D1, and Wdr3), stem cell markers (Lgr5 and Tnfrsf19), and Myc in VApcA/A and VApcPA/A organoids analyzed by real-time quantitative PCR (qRT-PCR).

(D) The organoid initiating frequency of VApcA/A and VApcPA/A organoids.

(E) The proportion of glandular colonies derived from 1,000 SW1222-PROX1high cells (transduced with Scr, sh1, or sh2 PROX1 shRNA lentivirus).

(F) The number of lumens in the glandular colonies derived from Scr or shPROX1-transduced (sh1, sh2) SW1222-PROX1high cells at day 14 (n = 10 for sh1 and n = 12forsh2). The lumens (asterisks) were detected by phalloidin staining and counted in optical sections of confocal microscopic images. The boxplot indicates the minimum, the first quartile, median, third quartile, and maximum.

(G) Real-time qPCR of the indicated RNAs from SW1222-PROX1high cell-derived organoids, transduced with shPROX1 or Scr lentivirus and grown in Matrigel for 14 days.

(legend continued on next page)

Figure 4. PROX1 Regulates the Number of Stem Cells via Cell Proliferation

(A) Growth curve of subcutaneous tumors derived from Scr or shPROX1-transduced SW1222-PROX1high cells in NSG mice (n = 10).

(B) PROX1 and E-CADHERIN immunostaining and HE staining of tumor sections 21 days after subcutaneous injection of SW1222-PROX1high cells into NSG mice. The dashed line indicates the tumor border, the arrowheads point to degenerating glandular structures and the asterisk marks necrotic area.

(C-E) Immunostaining, tumor volume (C and D) and RNA quantification (E) of VApcA/A (black columns) and VApcPA/A (red columns) organoid-derived tumors 12 and 28 days after their subcutaneous injection into NSG mice (confocal 3D reconstruction). Note the more complex glandular structure of the VApcA/A tumors (asterisks). (F and G) The percentage of proliferating (Ki67+) cells among the Prox1+ cells in the Lgr5+ and Lgr5~ populations in the intestinal epithelium of LApcA/A mice 21 days after Apc deletion (n = 1012) (F). The percentage of Ki67+ cells in the Lgr5+ cell population in the intestinal epithelium of LApcA/A and LApcPA/A mice (n = 9-11) (G). The boxplots indicate the minimum, the first quartile, median, third quartile, and maximum (F and G). (H-J) Growth of subcutaneous SW480R-sh tumors in NSG mice (H) with or without doxycycline (n = 10). Doxycyline treatment from day 8 (blue arrow) to day 16 (red arrow) after cell implantation was used for the silencing of PROX1. Immunostaining (I) and quantitative RT-PCR (qRT-PCR) (J) analyses at day 16.

Scale bars represent 50 mm (B) and 100 mm (B, D, and I). Mean + SD (C, E, and J) or mean + SEM (A and H) are shown. See also Figure S3.

PROX1 Silencing Increases Annexin A1 Expression in Multiple CRC Models

To determine which genes are responsible for the effect of PROX1 on the adenoma/CRC stem cells, we tested PROX1-

regulated candidate genes based on the microarray data derived from the SW480R subclone (Petrova et al., 2008). Based on our initial results, we focused further on the calcium-dependent phospholipid binding protein Annexin A1 (ANXA1), which has been shown to inhibit breast cancer metastasis (Maschler et al., 2010). PROX1 suppressed ANXA1 RNA (4.21 ± 0.08-fold, mean ± SD) and protein expression in SW480R-sh cells (Figure S4A). Anxa1 was increased after 4-OH-Tam addition to VApc organoids and even further elevated when also Prox1 was deleted (Figure 5A; Figures S4B and S4C), suggesting that Prox1 suppresses Anxa1 expression. Furthermore, the Prox1~

(H) Images and the relative lumen-containing organoid Initiating frequency of Scr and shPROX1 lentlvirus-transduced human organoids derived from CRC patients. The GFP signal shows the lentiviral transduction efficiency.

(I) RNA levels of the indicated genes in human CRC patient-derived organoids, transduced with Scr or shPROX1 lentivirus. Note that while sample 1 contains the G12D clinically relevant mutation in the KRAS gene, samples 2 and 3 are wild-type.

(J and K) LGR5 in situ hybridization and quantification of LGR5+ cells in the Scr and shPROX1-transduced SW1222-PROX1high clone. Scale bars represent 100 mm (B) or 50 mm (E, F, H, and K). Mean + SD (C-E and G-I) or mean + SEM (J) are shown. See also Figure S2.

(legend on next page)

cells showed more intense Anxa1 staining than Prox1 + cells in tumor sections from Apcminf+ mice (Figure S4D).

We observed a mutually exclusive staining for Anxa1 and Prox1 in tumors from VCKIA/A mice and in human CRC samples (Figures 5B and 5C). Furthermore, the Lgr5+ intestinal stem cells in the LApc mice showed very little or no epithelial Anxa1 expression before Apc deletion (Figure S4E). They contained a low level of Anxa1 after tamoxifen injection, whereas the Anxa1high cells were negative for Lgr5 in the resulting adenomas (Figure S4F). Importantly, transfection of a dominant-negative TCF4 (transcription factor 7-like 2 [TCF7L2]) construct, a Wnt pathway suppressor to the PROX1~ subclone of the SW480 CRC cell line (SW480A) (Petrova et al., 2008), led to suppression of ANXA1 expression (Figure 5D). These data indicate that Anxa1 is minimally expressed in the normal intestinal epithelium, where the activation of the Wnt pathway after Apc deletion increases Anxa1 expression, which is then suppressed by the induction of Prox1 in the tumor cells.

ANXA1 Silencing Mimics the Effects of PROX1 in CRC

We next tested the possibility that the strong ANXA1 suppression mediates the effects of PROX1, such as the associated rearrangement of the actin cytoskeleton, changes in cell shape, and adherence to the culture plates (Petrova etal., 2008). Indeed, ANXA1 overexpression in the SW480R subclone induced an elongated cell shape and increased the number of tightly adherent cells, whereas ANXA1 suppression resulted in rearrangement of the actin cytoskeleton, rounded cell shape, and decreased adherence (Figures S5A-S5C). Interestingly, Anxa1 silencing in VApcA/A organoids increased the Lgr5 and Tnfrsf19 stem cell marker RNAs, without affecting Myc (Figure 5E). Although ANXA1 silencing had no effect on PROX1 expression in SW1222-PROX1low or SW480 cells in 2D culture (data not shown), it resulted in increased lumen formation (Figure 5F), upregulation of TNFRSF19 and LGR5 RNAs, and increase of LGR5+ cells in 3D organoids derived from shANXA1-transduced cells (Figures 5G and 5H). In contrast, ANXA1 overexpression in SW1222-PROX1high cells decreased the proportion of glandular colonies, the number of organoid lumens, and LGR5 and TNFRSF19 RNA levels when compared to the controls (Figures S5D-S5F). Furthermore, the ANXA1-silenced SW1222-PROX1low cell-derived tumors grew faster than control tumors, contained more Ki67+ cells, and were composed of larger tumor nests with multiple lumens organized into labyrinth-like

structures (Figure 5I; Figure S5G). Similar to the effect of PROX1, ANXA1 silencing in SW1222 cell-derived organoids was associated with enhanced cell proliferation only after 16 days, when the organoids showed extensive outpocketing and lumen formation, but not at day 6 (Figure S5H). These data indicate that ANXA1 silencing mimics the effects of PROX1 expression, leading to an expansion of the stem cell pool via increased proliferation.

Silencing the Actin-Binding Protein FILAMIN A Decreases Stem Cells in CRC

Reanalysis of the microarray data derived from the SW480R subclone showed that focal adhesion and regulation of actin cytoskeleton were the top Kyoto Encyclopedia of Genes and Genomes gene pathway categories affected when PROX1 was silenced (data not shown). Thus, we searched for PROX1-regu-lated genes directly affecting the cytoskeleton among published CRCstem cell gene sets(de Sousa E Meloet al., 2011) and prognosis markers in CRC subgroups (Sadanandam etal., 2013). We found the gene encoding the actin-binding protein filamin A (FLNA) in both gene sets. FLNA connects actin filaments to transmembrane receptors, including p1 integrin, modulates cell migration, and functions as a central mechanotransduction element of the cytoskeleton (Ehrlicher et al., 2011; Zhou et al., 2010). Interestingly, we found highly increased Flna expression in the Prox1+ and Lgr5+ tumor cells (Figure 6A and data not shown) and a correlation between PROX1 and FLNA levels in the SW1222-PROX1high cell-derived organoids (Figure S5I). FLNA was also upregulated in the ANXA1-silenced organoids (Figures 6B and 6C). Furthermore, PROX1 silencing in the SW1222-PROX1high and CRC patient-derived organoids resulted in markedly decreased FLNA RNA (Figure 6D) and protein (Figure 6E).

Because both PROX1 and ANXA1 regulated FLNA expression in intestinal adenomas and in CRC 3D organoids, we studied FLNA function by silencing its expression in VApcA/A organoids and in SW1222-PROX1high cells. This led to reduced Lgr5 and Tnfrsf19 RNAs (Figure 6F), to a decrease of SW1222 cell-derived glandular colonies and lumens (Figure 6G), and to a decreased number of Ki67+ cells in the organoids (Figure 6H). Surprisingly, FLNA silencing did not affect the growth of the cells in 2D culture conditions (data not shown). Overall, these results suggest a model where the loss of Prox1 leads to an elevated Anxa1 level and a reduction in Flna, which limit the expansion

Figure 5. ANXA1 Silencing in CRC Leads to Elevated Stem Cell Activity and Tumor Growth

(A) Immunostaining of VApcA/A and VApcPA/A organoids for the indicated proteins.

(B and C) Immunoperoxidase staining of sections from (B) VCKIA/A tumors and (C) from a tumor of a CRC patient (n = 5).

(D) ANXA1 expression in SW480 cells transfected with a dominant-negative dnTCF4-FLAG or an unrelated control construct (TIE1-FLAG). The arrowheads indicate transfected cells.

(E) RNA analysis of Scr and shAnxa1-transduced VApcA/A organoids (black and green columns, respectively).

(F) Number of lumens in SW1222-PROX1low subclone-derived organoids, transduced with either Scr or shANXA1-expressing lentivirus, stained with fluorescent phalloidin and counted from optical sections of confocal images. The white asterisks mark the lumens (n = 11 for sh(1) and n = 25 forsh(2)). The boxplot indicates the minimum, the first quartile, median, third quartile and maximum.

(G and H) RNA levels of the indicated transcripts (G) and LGR5 in situ hybridization (H) (red signal) in SW1222-PROX1 low-derived organoids, grown in Matrigel for 16 days.

(I) Growth curves (n = 6), histological analysis and immunostaining of SW1222-PROX1low tumors growing subcutaneously in NSG mice.

For the staining, the tumors were isolated on day 11 after the implantation. Scale bars represent 100 mm (A-D) or 50 mm (F, H, and I). Mean + SD (E and G) or

mean + SEM (H and I) are shown. See also Figures S4 and S5.

Figure 6. Silencing the Actin-Binding Protein Filamin A Inhibits Stem Cell Activity

(A) Immunostaining forfilamin A (Flna) and Prox1 in an intestinal adenoma of Apcmlnf+ mice.

(B and C) FLNA staining (B) and qRT-PCR (C) from scrambled and shANXA1-transduced SW1222-PROX1low cell-derived organoids 14 days after 3D seeding.

(D) FLNA RNA in CRC patient-derived and SW1222-PROX1high cell-derived organoids, transduced with Scr or shPROX1 lentivirus (qRT-PCR).

(E) Immunostaining for PROX1 and FLNA in Scr or shPROX1-transduced, SW1222-PROX1high cell-derived organoids, cultured for 7 days in Matrigel (confocal 3D reconstructions).

(F) The levels of the indicated RNAs in VApcA/A organoids 10 days after transduction with Scr or shFlna lentivirus (qRT-PCR).

(G) The proportion of glandular colonies and the number of lumens (n = 8 for sh(1) and n = 14 for sh(2)) in SW1222 cell-derived organoids, transduced with the indicated lentivirus.

(H) The percentage of Ki67+ cells in Scr (black box) or shFLNA-transduced (gray box) SW1222 cell-derived organoids after 9 days of 3D culturing, counted from confocal optical sections (n = 12).

The boxplots in (G) and (H) indicate the minimum, the first quartile, median, third quartile, and maximum. Mean + SD are shown (C, D, F, and G). Scale bars represent 50 mm (A and B) or 25 mm (E). See also Figure S5.

of the adenoma/CRC stem cell population via decreasing cell proliferation.

PROX1 Silencing Reduces Cell Survival in Hypoxic Tumor Xenografts

Interestingly, when grown to a large size in Matrigel culture (>150 mm diameter, >5 days after subculture) or injected subcu-taneously into NSG mice, Prox1 -deleted VApcPA/A organoids contained more apoptotic cells than VApcA/A organoids (Figure 7A). Also, we detected an intense staining for the apoptosis marker active CASPASE-3 inside the PROX1-silenced SW1222-

PROX1high cell-derived subcutaneously growing tumors that were positive for the hypoxia marker carbonic anhydrase IX, but not at the tumor margin (Figure 7B and data not shown), suggesting reduced tumor cell survival in the ischemic/hypoxic tumor interior. Interestingly, we found no difference in the endo-mucin+ bloodvessel density between Scr and shPROX1 tumors, even when vascular endothelial growth factor (VEGF) was over-expressed in the tumor cells (Figures S6A and S6B). However, the shPROX1 tumors were smaller than the Scr tumors, even when VEGF was overexpressed in the tumor cells and VEGF failed to rescue the large necrotic areas inside the

PROXI-silenced tumors (Figures S6A and S6B). Thus, the presence of the large necrotic areas in the shPROXI tumors could not be explained by the lack of angiogenic factors.

Although ANXA1 silencing mimicked several of the effects of PROX1 in the intestinal adenoma and CRC models, the apoptosis rate in the ANXAI-silenced subcutaneously growing tumors was not significantly affected when analyzed 9 or 24 days after implantation (data not shown). This raised the possibility that in unfavorable conditions also alternative pathways contribute to the apoptosis in the shPROX1 tumors. Autophagy is an essential cellular process for the survival of cells under hypoxia or nutrient deprivation (Sato et al., 2007). Interestingly, PROX1 silencing in the SW480R cells resulted in a decrease of the autophagy-associated LC3-II protein both in normal medium and in starvation conditions (Figures 7C and 7D). Furthermore, lack of PROX1 prevented the accumulation of the LC3-contain-ing early autophagosomes in the presence of the lysosomal inhibitor bafilomycin A or chloroquine, which inhibit the fusion of autophagosomes with the lysosomes (Figure 7D). Importantly, we observed a decreased number of LC3-containing early auto-phagosomes under hypoxia in lentivirally PROX1-silenced SW480R or SW1222-PROX1high cells when their fusion with lysosomes was inhibited (Figure 7E), suggesting that PROX1 enhances the autophagic flux. The addition of a low concentration of chloroquine or bafilomycin A resulted in loss of the Lgr5+ cells after Apc deletion (Figure 7F). Of note, this chloroquine concentration inhibited the formation of VApcA/A organoid colonies upon subculture, but not their survival when added 2 days after organoid plating (Figure 7G). Furthermore, the addition of chloroquine or bafilomycin at a low dose to the LApcA/A organoid cultures reduced the proportion of viable organoids to the same level as Prox1 deletion in the LApcPA/A cultures, thus highlighting the important role of autophagy in promoting tumor cell survival specifically in the Prox1+ cells (Figure 7H).

DISCUSSION

Prox1 is critical for the fate of several types of stem and progenitor cells (Elsir et al., 2012). Here, we show that Prox1 is not expressed in the Lgr5+ stem cells of normal intestinal crypts, but is induced soon after the initiating mutation in intestinal tumori-genesis. We show that the Prox1+ cells give rise to more differentiated Prox1" cells in the adenomas, but not in the normal intestine, indicating that a subpopulation of the Prox1+ cells has cancer stem cell activity. Upon Prox1 deletion, the number of stem cells declined and this was reflected later on as decreased overall tumor cell proliferation and tumor growth.

Stem-like cells provide an important drug target in cancer, as they are able to persist in tumors as a distinct population, self-renew, and differentiate, and they are associated with tumor relapse (Sampieri and Fodde, 2012). Therefore, development of specific therapies targeted at cancer stem cells may improve survival. In elegant studies, Schepers et al. demonstrated that the Lgr5+ cells in intestinal adenomas have stem cell properties (Schepers et al., 2012). Interestingly, recent studies indicated that intestinal adenomas contain fewer stem cells than Lgr5+ cells, suggesting that only a subpopulation of the Lgr5+ cells function as stem cells (Kozar et al., 2013). Furthermore, Myant

et al. have shown that the Rac1 GTPase is an important regulator of the proliferation of the Lgr5+ cell population in intestinal adenomas (Myant et al., 2013). Although the Lgr5+ cells may be dispensable for homeostasis of the normal intestine and for increased proliferation of intestinal epithelium after Apc deletion (Metcalfe et al., 2014), the expansion of cells derived from the Lgr5+ cells in adenomas after Apc deletion clearly indicates that they contribute to tumor growth (Schepers et al., 2012). Importantly, Prox1 deletion not only decreased the number of Lgr5+ cells in intestinal adenomas in vivo, but it also reduced their proliferation, the organoid-initiating frequency and the number of megacolonies and lumens in the glandular CRC organoids, which all represent indicators of cancer stem cells (Ashley et al., 2013). Furthermore, PROX1 silencing after the tumor establishment resulted in a decrease of the LGR5+ tumor cell marker before a decline in the overall tumor growth rate.

Mechanistically, we show that the phospholipid-binding protein Anxa1 is increased by the Wnt pathway activation after Apc deletion, while Prox1 suppresses its expression. ANXA1 and PROX1 showed also mutually exclusive expression patterns in human CRC samples. Strikingly, the silencing of ANXA1 in the PROX1~ cells mimicked the effects of PROX1 in the CRC cells. Among its other effects, ANXA1 inhibits the proinflammatory phospholipase A2, which has been shown to stimulate the proliferation of CRC cells by producing various lipid mediators and which regulates intercellular junctions and the actin cytoskeleton (Cristante et al., 2013; Parente and Solito, 2004; Surrel et al.,

2009). Both ANXA1 silencing and PROX1 expression increased stem cell markers and the number of LGR5+ cells in tumor orga-noidsand enhanced lumen formation, proliferation of tumor cells, and tumor growth in vivo. Furthermore, ANXA1 silencing and PROX1 expression increased FLNA, which stabilizes the cortical 3D actin networks, links them to the transmembrane receptor p1 integrin, and functions as a central mechanotransduction element of the cytoskeleton (Ehrlicher et al., 2011; Zhou et al.,

2010). The role of the actin cytoskeleton in the expansion of the adenoma stem cell population was supported by our FLNA results, showing that silencing this actin-binding protein, which was expressed in the Lgr5+ stem cells of intestinal adenomas, dramatically inhibited the activity and proliferation of the adenoma/CRC stem cells in 3D, but not in 2D, culture conditions.

FLNA is one of the markers of a CRC subtype characterized by poor disease-free survival (Sadanandam et al., 2013). FLNA expression is increased in a number of cancers, and it has been recently shown to boost the hypoxia response and tumor progression (Zheng et al., 2014). Consistent with this, the FLNA-expressing PROX1+ tumor cells were more resistant to apoptosis than the FLNAlow PROX1_ tumor cells both in vivo and in the organoids. There was a striking increase in apoptosis, particularly in the central parts of the PROX1-silenced tumors growing subcutaneously in mice. However, the sensitivity of the PROX1~ tumor cells to apoptosis was not due to insufficient expression of angiogenic growth factors, as shown by the inability of VEGF overexpression to rescue the difference. Instead, the analysis of autophagy markers and use of lysosomal inhibitors of autophagy indicated that PROX1 expression sustains autophagy, which is known to be essential for the survival of CRC cells (Sato et al., 2007). This finding is particularly

Figure 7. Loss of PROX1 Reduces CRC Stem Cell Survival under Unfavorable Conditions

(A) Percentage of caspase-3+ apoptotic cells among the E-cadherin+ epithelial cells in VApcA/A (black columns) and Prox1-deleted (VApcPA/A; red columns) organoids 8 days after the addition of 4-OH-Tam and 6 days after the removal of R-Spondin1 (right panel, n = 14) or growing subcutaneously in NSG mice (left panel, n = 22).

(B) Active caspase-3 staining of subcutaneously growing Scr and shPROX1-transduced SW1222-PROX1high tumors in NSG mice. Note that the shPROX1-transduced tumor interior is highly apoptotic (asterisk).

(Cand D) Immunoblotting of SW480R-sh cells forthe indicated proteins. Cells were either cultured in complete medium (C) or in starvation medium lacking amino acids 8 hr before protein isolation (D) in the absence or presence of 100 nM bafilomycin A or 30 mM chloroquine.

(E) The number of LC3+ granules in the indicated cell lines transduced with Scr or shPROX1 lentivirus and cultured in starvation medium containing 100 nM bafilomycin in hypoxia for 8 hr (n = 50).

(F) Flow cytometric analysis of the Lgr5-EGFP+ cells in LApcA/A organoids, 9 days after the Apc deletion and 3 days after the addition of 15 mM chloroquine or 0.2 mM bafilomycin A.

(G) The plating efficiency and survival percentage of VApcA/A organoids in the presence or absence of chloroquine or bafilomycin A. Note that for organoid survival chloroquine or bafilomycin were added 2 days after organoid splitting for 3 days.

(legend continued on next page)

interesting considering that hypoxia-induced autophagy can promote tumor cell survival and adaptation to antiangiogenic treatment, which is used in CRC therapy (Hu et al., 2012). Our results are in agreement with the findings of Ragusa et al., who show that PROX1 promotes the metabolic adaptation of CRC cells in unfavorable microenvironments, and thus critically contributes to the metastatic outgrowth of CRCs (Ragusa et al., 2014).

In summary, we show here that PROX1 is induced in intestinal stem cells in adenoma/CRC soon after the activation of the Wnt pathway. While the APC mutation induces both PROX1 and ANXA1 expression in the epithelium, PROX1 restricts ANXA1 levels and induces FLNA, which stimulates cell proliferation and promotes stem cell activity in the adenomas, and may counteract stem cell exhaustion during tumor growth. The net effect is the expansion of the adenoma/CRC stem cell population and increased tumor growth. In the hypoxic parts of tumor transplants, PROX1 promotes tumor cell survival by increasing auto-phagy. Based on this study, PROX1 regulates the number of adenoma/CRC stem cells without affecting the homeostasis of the normal intestine, thus providing an attractive therapeutic target pathway for drug development in CRC.

EXPERIMENTAL PROCEDURES

A detailed description of the experimental procedures is provided in Supplemental Experimental Procedures.

Intestinal Crypt/Organoid Cultures

The National Board for Animal Experiments at the Provincial State Office of Southern Finland approved all experiments performed with mice. Intestinal crypts from Apcflox/flox; villin-CreER, Apcflox/flox; Prox1flox/flox (Harvey et al., 2005); villin-CreER, Csnk1a1flox/flox; tp53flox/flox; villin-CreER (Elyada et al., 2011), Apcflox/flox; Lgr5-EGFP-IRES-CreER (Barker et al., 2007) and Apcflox/flox; Prox1flox/flox; Lgr5-EGFP-IRES-CreER mice were isolated and cultured as described previously (Sato et al., 2009, 2011). To activate the endogenous p-catenin/TCF pathway in mouse organoids, cultures were treated with 300 nM 4-hydroxy-tamoxifen (4-OH-Tam) for 48 hr. Organoids with the endog-enously active p-catenin/TCF pathway were then selected and cultured in growth factor-deficient medium.

Human Organoid Cultures

For 3D culture, SW1222 or patient-derived CRC cells were extensively trypsi-nized, embedded into Matrigel (500-2,000 cells/50 ml Matrigel/well), and grown for 3-16 days. The ethics committee of the Department of Surgery at Helsinki University Hospital approved all experiments involving patient samples, and informed consent was obtained from the patients. Tissue samples isolated from CRC patients were processed according to a previously published method (Sato et al., 2011).

In Vivo Experiments

Mice were injected with 2 mg tamoxifen (Sigma) dissolved in 200 ml sunflower oil (Sigma) at the age of 8-9 weeks. The mice were euthanized at the indicated time points. The mice were on the C57Bl/6 background. In all experiments littermate controls were used.

Statistical Analysis

Statistical comparison of two groups was done by two-tailed unpaired or paired t test using the SPSS software unless otherwise indicated. For nonpara-

metric tests, the Mann-Whitney U test was used, and the data are presented as boxplots, showing the five statistics (minimum, first quartile, median, third quartile, maximum). The statistical significance is marked by *p < 0.05, **p < 0.01, and ***p< 0.005.

ACCESSION NUMBERS

The Gene Expression Omnibus accession number for the microarray data reported in this paper is GSE47568.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Results, Supplemental Experimental Procedures, six figures, and one table and can be found with this article online at http://dx.doi.org/10.1016Zj.celrep.2014.08.034.

AUTHOR CONTRIBUTIONS

Z.W., J.H., V.H., A.M.B. and K.A. designed research; Z.W., J.H., V.H., A.M.B., P.K.,T.H., and O.D. performed research; C.H., O.K., G.O., and Y.B.-N. contributed new reagents/analytic tools; Z.W., J.H., V.H., and K.A. analyzed data; and Z.W., J.H., and K.A. wrote the paper.

ACKNOWLEDGMENTS

We thank Dr. Leif Andersson for the consultations on histopathology, Dr. Taija Makinen for the Prox1-CreER mice, Dr. Darren Tyson for the ANXA1-modu-lating constructs, Dr. David Calderwood for the FLNA antibody, Dr. Meenhard Herlyn (Wistar Institute) for the SW1222 cell line, Dr. Tatiana Petrova for discussions of PROX1 functions in CRC, Dr. Pekka Katajisto, Dr. Tuomas Tammela, and Dr. Timo Otonkoski for comments on the manuscript, and Lari Pyoria, Kirsi Lintula, KatjaSalo, Laura Raitanen, and TapioTainola for their help with the experiments. The Biomedicum Imaging Unit is acknowledged for microscopy services. This work was funded by the Sigrid Juselius Foundation, the Finnish Cancer Organizations, and the Academy of Finland (262976). Z.W. was supported by the Sigrid Juselius Foundation and by the Marie-Curie Intra-Euro-pean Fellowship (PIEF-GA-2009-236695).

Received: October 23, 2013 Revised: January 14, 2014 Accepted: August 17, 2014 Published: September 18, 2014

REFERENCES

Ashley, N., Yeung, T.M., and Bodmer, W.F. (2013). Stem cell differentiation and lumen formation in colorectal cancer cell lines and primary tumors. Cancer Res. 73, 5798-5809.

Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. (2007). Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-1007.

Barker, N., Ridgway, R.A., van Es, J.H., van de Wetering, M., Begthel, H., van den Born, M., Danenberg, E., Clarke, A.R., Sansom, O.J., and Clevers, H. (2009). Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608-611.

Batlle, E., Henderson, J.T., Beghtel, H., van den Born, M.M., Sancho, E., Huls,

G., Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T., and Clevers,

H. (2002). Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251-263.

(H) Relative survival rate of LApcD/D or LApcPD/D organoids 8 days after 4-OH-Tam and 6 days in R-Spondinl-free medium. Chloroquine (15 mM) or bafilomycin A (0.2 mM) were added 4 days after 4-OH-Tam for 4 days.

The boxplots in (A) and (E) indicate the minimum, the first quartile, median, third quartile, and maximum. Mean + SD are shown for (F)-(H). Scale bars represent 100 mm. See also Figure S6.

Bazigou, E., Lyons, O.T., Smith, A., Venn, G.E., Cope, C., Brown, N.A., and Makinen, T. (2011). Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice. J. Clin. Invest. 121, 2984-2992. Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell 127, 469-480.

Cristante, E., McArthur, S., Mauro, C., Maggioli, E., Romero, I.A., Wylezinska-Arridge, M., Couraud, P.O., Lopez-Tremoleda, J., Christian, H.C., Weksler, B.B., et al. (2013). Identification of an essential endogenous regulator of blood-brain barrier integrity, and its pathological and therapeutic implications. Proc. Natl. Acad. Sci. USA 110, 832-841.

de Sousa E Melo, F., Colak, S., Buikhuisen, J., Koster, J., Cameron, K., de Jong, J.H., Tuynman, J.B., Prasetyanti, P.R., Fessler, E., van den Bergh, S.P., et al. (2011). Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients. Cell Stem Cell 9, 476-485.

Ehrlicher, A.J., Nakamura, F., Hartwig, J.H., Weitz, D.A., and Stossel, T.P. (2011). Mechanical strain in actin networks regulates FilGAP and integrin binding to filamin A. Nature 478, 260-263.

Elsir, T., Smits, A., Lindstrom, M.S., and Nister, M. (2012). Transcription factor PROX1: its role in development and cancer. Cancer Metastasis Rev. 31, 793-805.

Elyada, E., Pribluda, A., Goldstein, R.E., Morgenstern, Y., Brachya, G., Cojo-caru, G., Snir-Alkalay, I., Burstain, I., Haffner-Krausz, R., Jung, S., et al. (2011). CKIa ablation highlights a critical role for p53 in invasiveness control. Nature 470, 409-413.

Fearon, E.R. (2011). Molecular genetics of colorectal cancer. Annu. Rev. Pathol. 6, 479-507.

Fodde, R., and Smits, R. (2001). Disease model: familial adenomatous polyposis. Trends Mol. Med. 7, 369-373.

Gregorieff, A., and Clevers, H. (2005). Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev. 19, 877-890.

Harvey, N.L., Srinivasan, R.S., Dillard, M.E., Johnson, N.C., Witte, M.H., Boyd, K., Sleeman, M.W., and Oliver, G. (2005). Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity. Nat. Genet. 37, 1072-1081.

Hu, Y.L., DeLay, M., Jahangiri, A., Molinaro, A.M., Rose, S.D., Carbonell, W.S., and Aghi, M.K. (2012). Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res. 72, 1773-1783.

Kemper, K., Prasetyanti, P.R., De Lau, W., Rodermond, H., Clevers, H., and Medema, J.P. (2012). Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells. Stem Cells 30, 2378-2386. Kozar, S., Morrissey, E., Nicholson, A.M., van der Heijden, M., Zecchini, H.I., Kemp, R., Tavare, S., Vermeulen, L., and Winton, D.J. (2013). Continuous clonal labeling reveals small numbers of functional stem cells in intestinal crypts and adenomas. Cell Stem Cell 13, 626-633.

Maschler, S., Gebeshuber, C.A., Wiedemann, E.M., Alacakaptan, M., Schreiber, M., Custic, I., and Beug, H. (2010). Annexin A1 attenuates EMT and metastatic potential in breast cancer. EMBO Mol. Med. 2, 401-414. Merlos-Suarez, A., Barriga, F.M., Jung, P., Iglesias, M., Cespedes, M.V., Ros-sell, D., Sevillano, M., Hernando-Momblona, X., da Silva-Diz, V., Muhoz, P., et al. (2011). The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8, 511-524. Metcalfe, C., Kljavin, N.M., Ybarra, R., and deSauvage, F.J. (2014). Lgr5 stem cells are indispensable for radiation-induced intestinal regeneration. Cell Stem Cell 14, 149-159.

Myant, K.B., Cammareri, P., McGhee, E.J., Ridgway, R.A., Huels, D.J., Cordero, J.B., Schwitalla, S., Kalna, G., Ogg, E.L., Athineos, D., et al. (2013). ROS production and NF-kB activation triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation. Cell Stem Cell 12, 761-773.

Parente, L., and Solito, E. (2004). Annexin 1: more than an anti-phospholipase protein. Inflamm. Res. 53, 125-132.

Petrova,T.V., Nykanen, A., Norrmen,C., Ivanov, K.I., Andersson, L.C., Haglund,

C., Puolakkainen, P., Wempe, F., von Melchner, H., Gradwohl, G., et al. (2008). Transcription factor PROX1 induces colon cancer progression by promoting the transition from benign to highly dysplastic phenotype. Cancer Cell 13,407-419. Ragusa, S., Cheng, J., Ivanov, K.I., Zangger, N., Ceteci, F., Bernier-Latmani, J., Milatos, S., Joseph, J.-M., Tercier, S., Bouzourene, H., et al. (2014). PROX1 Promotes Metabolic Adaptation and Fuels Outgrowth of Wnthigh Metastatic Colon Cancer Cells. Cell Rep. Published online September 18, 2014. http://dx.doi.org/10.1016Zj.celrep.2014.08.041.

Sadanandam, A., Lyssiotis, C.A., Homicsko, K., Collisson, E.A., Gibb, W.J., Wullschleger, S., Ostos, L.C., Lannon, W.A., Grotzinger, C., Del Rio, M., et al. (2013). A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat. Med. 19, 619-625. Sampieri, K., and Fodde, R. (2012). Cancer stem cells and metastasis. Semin. Cancer Biol. 22, 187-193.

Sansom, O.J., Reed, K.R., van de Wetering, M., Muncan, V., Winton, D.J., Clevers, H., and Clarke, A.R. (2005). Cyclin D1 is not an immediate target of beta-catenin following Apc loss in the intestine. J. Biol. Chem. 280, 2846328467.

Sato, K., Tsuchihara, K., Fujii, S., Sugiyama, M., Goya, T., Atomi, Y., Ueno, T., Ochiai, A., and Esumi, H. (2007). Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Res. 67, 9677-9684.

Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange,

D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265.

Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., and Clevers, H. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141,1762-1772. Schepers, A.G., Snippert, H.J., Stange, D.E., van den Born, M., van Es, J.H., van de Wetering, M., and Clevers, H. (2012). Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730-735. Schwitalla, S., Fingerle, A.A., Cammareri, P., Nebelsiek, T., Goktuna, S.I., Ziegler, P.K., Canli, O., Heijmans, J., Huels, D.J., Moreaux, G., et al. (2013). Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. Cell 152, 25-38.

Srinivasan, R.S., Dillard, M.E., Lagutin, O.V., Lin, F.J., Tsai, S., Tsai, M.J., Samokhvalov, I.M., and Oliver, G. (2007). Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev. 21, 2422-2432.

Surrel, F., Jemel, I., Boilard, E., Bollinger, J.G., Payre, C., Mounier, C.M., Tal-vinen, K.A., Laine, V.J., Nevalainen, T.J., Gelb, M.H., and Lambeau, G. (2009). Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators. Mol. Pharmacol. 76, 778-790. Van der Flier, L.G., Sabates-Bellver, J., Oving, I., Haegebarth, A., De Palo, M., Anti, M., Van Gijn, M.E., Suijkerbuijk, S., Van de Wetering, M., Marra, G., and Clevers, H. (2007). The Intestinal Wnt/TCF Signature. Gastroenterology 132, 628-632.

Wiener, Z., Band, A.M., Kallio, P., Hogstrom, J., Hyvonen, V., Kaijalainen, S., Ritvos, O., Haglund, C., Kruuna, O., Robine, S., et al. (2014). Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-p. Proc. Natl. Acad. Sci. USA 111, E2229-E2236. Yeung, T.M., Gandhi, S.C., Wilding, J.L., Muschel, R., and Bodmer, W.F. (2010). Cancer stem cells from colorectal cancer-derived cell lines. Proc. Natl. Acad. Sci. USA 107, 3722-3727.

Zheng, X., Zhou, A.X., Rouhi, P., Uramoto, H., Boren, J., Cao, Y., Pereira, T., Akyurek, L.M., and Poellinger, L. (2014). Hypoxia-induced and calpain-depen-dent cleavage of filamin A regulates the hypoxic response. Proc. Natl. Acad. Sci. USA 111, 2560-2565.

Zhou, A.X., Hartwig, J.H., and Akyurek, L.M. (2010). Filamins in cell signaling, transcription and organ development. Trends Cell Biol. 20, 113-123.