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The microRNA-371~373 cluster represses colon cancer initiation and metastatic colonization by inhibiting the TGFBR2/ID1 signaling axis

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The vast majority of colorectal cancer (CRC)-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different CRC cell lines and recently established primary cultures enriched in colon cancer stem cells (CSCs) - also known as tumor-initiating cells (TICs) - to identify genes and microRNAs (miRNAs) with regulatory functions in CRC progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, transforming growth factor beta (TGF-β) signaling activity, and reduced expression of the miR-371~373 cluster compared to non-metastatic cultures. TGF-β receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371~373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371~373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor-initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371~373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371~373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371~373 and ID1. Altogether, our results establish the miR-371~373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization.
Metastatic SW620 SC displays prominent TIC properties. Morphologic features (A) and quantification (B) of spheroids formed by SW480 and SW620 parental and SC ells in limiting dilution assays; scale bar, 100 mm. Data presented as mean AE SEM of three independent experiments. C, SOX2 protein expression of SW480 and SW620 parental and SC cells (top) as well as of patient-derived T6, T18, and T20 SCs and adherent counterparts (adh; bottom); representative figure of at least two independent experiments. Sphere formation assays over consecutive passages (P1-P4) for SW480 and SW620 SCs with 1,000 cells per well (D) or different densities (i.e., 1, 2, or 3 cells per well; E) in limiting dilution experiments. Data presented as mean AE SD for D and as mean, with 95% confidence interval for E. F, Colony formation assays were used to determine the clonogenic capacity of SW480 and SW620 SCs. Data presented as mean AE SD; representative figure of three independent experiments. G, In vivo tumor growth in NOD/SCID mice following subcutaneous injection of 100 cells derived from SW480 or SW620 SCs; data presented as mean AE SEM. Representative picture of extracted tumors, 9 weeks after injection; scale bar, 1 cm; tumor incidence is indicated in brackets. H, Relative mRNA and protein expression of SOX2 and LGR5 in SW480 and SW620 SCs; data presented as mean AE SEM (for qPCR) and as representative picture (for Western blot analysis) of three independent experiments. Statistical significance was assessed with an unpaired Student t test for B, D, F, and H, with a x 2 test for E, and with a two-way ANOVA test for G ( ? , P < 0.05; ?? , P < 0.01; and ??? , P < 0.001).
… 
TGFBR2 signaling is enhanced in metastatic SW620 TICs. A, Heatmap depicting median-centered expression intensities of selected TGFb-signaling-related genes, obtained by microarray analysis of SW480 and SW620 SCs (accession number GSE89523). B, Gene set enrichment analysis for the pathway interaction database (PID) TGFBR pathway (c2.cp.v4.0 curated gene sets) using SW480 and SW620 SC microarray data (dataset GSE89523). PID_TGFBRPATHWAY showed a positive normalized enrichment score (NES ? 1.51) in SW620 SCs. C, Relative TGFBR2 mRNA expression (left), TGFBR2 and SMAD2 protein expression as well as SMAD2 phosphorylation (right) of SW480 and SW620 SCs. Data presented as mean AE SEM of three independent qPCR experiments and as representative picture of at least three independent Western blots. D, IHC staining of TGFBR2 (sc-400 antibody, Santa Cruz Biotechnology) on paired colorectal cancer (CRC) tissue microarrays. Matched tumor (CRC; n ? 65) and control mucosa (N; n ? 65) samples were scored for intensity of TGFBR2-positive cells, ranging from 0 (no signal) to 2 (strong signal). When present, the signals were mainly cytoplasmic and located at the luminal side of the cell, both for normal enterocytes and for malignant colorectal cancer cells; scale bar, 200 mm. E, Relapse-free colorectal cancer patient survival, according to TGFBR2 expression in publicly available dataset GSE39582 (28); 95% confidence intervals depicted as colored areas; number of relapse cases (i.e., events) ? 177. F, Self-renewal capacity as well as SMAD2 activation (pSMAD2) and protein expression of SW480 SCs after stimulation with 1 nmol/L TGF-b1. G, Self-renewal capacity of SW620 SCs after lentiviral transduction of either a TGFBR2 shRNA pool or the corresponding control vector. Representative figures of at least three independent Western blots or limiting dilution assays (with 1, 2, or 3 cells per well) for F and G, respectively; data shown as mean with 95% confidence interval. Statistical significance was assessed with an unpaired Student t test for C, with a paired Student t test for D, with the Cox proportional hazard model for E, and with a x 2 test for F and G ( ? , P < 0.05; ?? , P < 0.01; and ??? , P < 0.001).
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Molecular Cell Biology
The miR-371373 Cluster Represses Colon Cancer
Initiation and Metastatic Colonization by
Inhibiting the TGFBR2/ID1 Signaling Axis
Pit Ullmann
1
, Fabien Rodriguez
1
, Martine Schmitz
1
, Steffen K. Meurer
2
,
Komal Qureshi-Baig
1
, Paul Felten
1
, Aur
elien Ginolhac
3
, Laurent Antunes
4
,
Sonia Frasquilho
4
, Nikolaus Z
ugel
5
, Ralf Weiskirchen
2
, Serge Haan
1
, and Elisabeth Letellier
1
Abstract
The vast majority of colorectal cancerrelated
deaths can be attributed to metastatic spreading
of the disease. Therefore, deciphering molecular
mechanisms of metastatic dissemination is a
key prerequisite to improve future treatment
options. With this aim, we took advantage of
different colorectal cancer cell lines and recently
established primary cultures enriched in colon
cancer stem cells, also known as tumor-initiating
cells (TIC), to identify genes and miRNAs with
regulatory functions in colorectal cancer progres-
sion. We show here that metastasis-derived TICs
display increased capacity for self-renewal, TGFb
signaling activity, and reduced expression of the
miR-371373 cluster compared with nonmeta-
static cultures. TGFbreceptor 2 (TGFBR2)and
aldehyde dehydrogenase A1 (ALDH1A1)were
identied as important target genes of the
miR-371373 cluster. In addition, TGFBR2
repression, either by direct knockdown or indi-
rectly via overexpression of the entire miR-
371373 cluster, decreased tumor-initiating
potential of TICs. We observed signicantly
reduced in vitro self-renewal activity as well as
lowered tumor initiation and metastatic out-
growth capacity in vivo following stable over-
expression of the miR-371373 cluster in dif-
ferent colon TIC cultures. Inhibitor of DNA
binding 1 (ID1) was affected by both
TGFBR2 and miR-371373 cluster alterations.
Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371373 and
ID1. Altogether, our results establish the miR-371373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-
renewal and metastatic colonization.
Signicance: These ndings establish the miR-371373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of
tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis.
Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg. Cancer Res; 78(14); 3793808. 2018 AACR.
© 2018 American Association for Cancer Research
Primary tumor Secondary sites
No metastatic outgrowth
miR-371~373
ID1
TGFBR2
miR-371~373
miR-371~373
Loss of miR-371~373 cluster expression and concomitant induction of TGFBR2 and ID1 signaling
promote the self-renewal capacity and metastatic outgrowth potential of disseminated tumor-
initiating cells.
miR-371~373
ID1
TGFBR2
Metastatic outgrowth
Liver
= tumor-initiating cell
Liver
Blood
The miR-371~373 cluster acts as a metastasis suppressor
1
Molecular Disease Mechanisms Group, Life Sciences Research Unit, Univer-
sity of Luxembourg, Belvaux, Luxembourg.
2
Institute of Molecular Patho-
biochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH
University Hospital Aachen, Aachen, Germany.
3
Bioinformatics Core Facility,
Life Sciences Research Unit, University of Luxembourg, Belvaux, Luxem-
bourg.
4
Integrated Biobank of Luxembourg, Luxembourg, Luxembourg.
5
Centre Hospitalier Emile Mayrisch, Rue Emile Mayrisch, Esch-sur-Alzette,
Luxembourg.
Corresponding Author: Elisabeth Letellier, Life Sciences Research Unit, Uni-
versity of Luxembourg, 6 Avenue du Swing, L-4367 Campus Belval, Belvaux,
Luxembourg. Phone: 466-644-6954; Fax: 466-644-6435; E-mail:
elisabeth.letellier@uni.lu
doi: 10.1158/0008-5472.CAN-17-3003
2018 American Association for Cancer Research.
Cancer
Research
www.aacrjournals.org 3793
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Published OnlineFirst May 10, 2018; DOI: 10.1158/0008-5472.CAN-17-3003
Introduction
Colorectal cancer is the third most commonly diagnosed cancer
worldwide, with more than 1.3 million new cases and almost
700,000 deaths per year (1). As more than 90% of cancer-related
deaths can be attributed to metastases (2), it is of utmost clinical
relevance to understand the biology that underlies cancer pro-
gression and metastasis initiation. Clinically detectable macro-
metastases result from a complex series of different steps, includ-
ing cancer cell migration and local invasion, entry into the blood
circulation, arrest at the secondary site, extravasation, and nally
colonization (3). However, most circulating cancer cells die
during metastatic spreading and only a very small fraction of
tumor cells will eventually contribute to the actual colonization of
distant organs. Metastasis formation therefore seems to be restrict-
ed to a rare subpopulation of tumor cells with a distinctive set of
properties, including resistance to anoikis and apoptosis as well as
extensive self-renewal potential (4). In the context of colorectal
cancer, it has been shown that metastasis formation is almost
exclusively driven by a subtype of tumor-initiating cells (TIC)
displaying substantial self-renewal capacity (5).
Although the exact origin and operation of such metastasis-
initiating cells (MIC) remain elusive, MICs were shown to hijack a
variety of different signaling pathways to enhance cellular plas-
ticity and to gain malignant advantages (6). In this context, TGFb
signaling might be of special interest, as it is known to regulate
metastasis formation on multiple levels (7). Importantly, TGFbis
considered both a driving force of epithelialmesenchymal tran-
sition (EMT) and a regulator of stem cell properties (8), making it
an essential inducer of early metastatic events. On the other hand,
the role and regulation of TGFbsignaling in later steps of the
metastatic cascade, such as MIC self-renewal activity or coloniza-
tion of the secondary site, are less well understood.
Several regulators of stemness and self-renewal activity, such as
SRY-box 2 (SOX2), leucine-rich repeat containing G protein-
coupled receptor 5 (LGR5), or aldehyde dehydrogenase 1 family
member A1 (ALDH1A1) have been linked with TIC activity and
are frequently used to isolate TICs, with varying degrees of success
(9). Among these potential TIC regulators, the inhibitor of DNA
binding (ID) family, which is a group of helix-loop-helix proteins
with multiple regulatory functions in normal development,
malignant transformation, and cancer progression (10), has
gained increasing attention. Importantly, simultaneous knock-
down of ID1 and ID3 was shown to abolish in vitro and in vivo self-
renewal activity of colon TICs by blocking the cell-cycle inhibitor
p21 (11). Moreover, ID1 was identied as a TGFbtarget gene in
different cancer types, including breast, prostate, and gastric
cancer (10). While TGFb-induced expression of ID1 was shown
to enhance mammosphere formation and metastatic coloniza-
tion of breast cancer cells (12), little is known about the role of
TGFb/ID1 signaling in regulating colon TIC self-renewal at the
metastatic site.
Increasing evidence shows that miRNAs, which are short non-
coding posttranscriptional repressors of gene expression, play
important mechanistic roles in the regulation of normal and CSC
properties (13). Furthermore, many individual miRNAs have
been linked to TGFbsignaling, colorectal cancer progression, and
metastasis formation (14). The miR-371373 cluster, which is a
short genomic region located on chromosome 19, has originally
been described in the context of human embryonic stem cells (15)
and is known to have diverse functions in cancer. Depending on
the cellular context, miR-371373 cluster members can act
either as tumor suppressors or as oncomiRs by regulating pro-
liferation, migration, and invasion (16). Opposing ndings
concerning miR-371373 expression in colorectal cancer have
been reported: while studies by Ng and colleagues and Yu and
colleagues suggest that miR-372 and miR-373 are frequently
overexpressed in colorectal cancer plasma samples (17, 18),
Tanaka and colleagues have shown that the miR-371373 cluster
is often epigenetically silenced in patients with colorectal cancer
(19). While miR-371373 expression was shown to correlate
with Wnt/b-catenin signaling in different colorectal cancer cell
lines (20), a potential role of this miRNA cluster in the regulation
of TIC self-renewal and metastatic colonization remains to be
determined.
Here, we report a novel molecular mechanism involved
in the control of colon MIC self-renewal at the metastatic
site. By comparing TIC-enriched cultures derived from both
primary and metastatic colorectal cancer cultures, we could
identify the miR-371373 cluster as an efcient inhibitor of
TGFBR2/ID1 signaling and as a potent repressor of metastatic
outgrowth activity.
Materials and Methods
Patients and tissue microarrays
All human tissue samples were collected by the Integrated
Biobank of Luxembourg (IBBL, www.ibbl.lu), as described pre-
viously (21). Patient studies were conducted in accordance with
the declaration of Helsinki and written informed consent as well
as approval from the institutional Ethics Review Panel (ERP-16-
032) and the Comit
e National d'Ethique de Recherche du Lux-
embourg (Reference 201009/09) were obtained. Tissue micro-
array (TMA) blocks were prepared for 65 primary colorectal cancer
samples and paired normal colon counterparts, following stan-
dard procedures (22).
Cell culture
HT-29, HCT 116, LoVo, SW480, and SW620 colorectal cancer
cell lines were purchased from the ATCC and the DSMZ, authen-
ticated via short tandem repeat proling, regularly tested for
mycoplasma, and maintained in recommended conditions. TIC
enrichment was achieved by applying spheroid culture (SC)
conditions, as reported previously (23). Primary cultures T6,
T18, and T20 were established from fresh patient material and
have recently been fully characterized (23). Both patient- and cell
linederived SCs were passaged at least ve times before perform-
ing experiments. Adherent counterparts were cultivated in
DMEM-F12 (Lonza), supplemented with 10% FBS and 100 U/
mL penicillin and streptomycin.
Sphere and colony formation assays
In vitro self-renewal and clonogenic capacity were tested with
sphere and colony formation assays, as described previously (23,
24). Limiting dilution assays were performed in SC conditions
with different cell densities (ranging from 1 to 1,000). Colony and
sphere size was measured under a microscope.
Viral transductions
Ready-to-use lentiviral particles were used (at a multiplicity of
infection of 35) to generate SCs with (i) stable overexpression of
miR-371373 cluster, (ii) stable knockdown of TGFBR2, SOX2,
or ID1 via application of a pool of three different target-specic
Ullmann et al.
Cancer Res; 78(14) July 15, 2018 Cancer Research3794
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short hairpin (sh) RNA constructs, (iii) stable expression of
TGFBR2 30UTR and GFP-containing reporter constructs, or (iv)
respective control vectors (Supplementary Table S1). Transduced
cells were selected with puromycin and transduction efciency
was evaluated via qPCR and uorescence microscopy.
Transient siRNA transfections
Dissociated SCs were cultivated as adherent cells for 24 hours
prior to transfecting a TGFBR2-specic pool of 4 different siRNAs
(Qiagen) or respective negative control siRNA (72 hours, 25
nmol/L); knockdown efciency was assessed via qPCR.
TGFbsignaling activation/inhibition
TGFbsignaling was induced by stimulating adherent cells and
SCs for 1 hour with 400 pmol/L or 1 nmol/L TGFb1 (Abcam),
respectively. Thus, 24 hours after plating, cells were stimulated in
their respective culture condition. Pathway inhibition was
obtained by treating cells with the specic TGFBR1 inhibitor
A-83-01 (Axon Medchem, 4 hours, 10 mmol/L).
In vivo assays
NOD/SCID mice were obtained from Harlan Laboratories (The
Netherlands) and NOD/SCID gamma (NOD.Cg-Prkdc
scid
Il2rg
tm1Wjl
/SzJ; NSG) mice were bred in-house. Approval by the
University's animal care and ethics committee was obtained (14-
MDM-02 and 16-MDM-01-EL) and in vivo experiments were
performed according to applicable laws and regulations. Tumor
formation and in vivo self-renewal assays (serial transplantation)
were performed, as described previously (23). In vivo metastatic
dissemination and colonization potential was assessed by intra-
venously injecting 1 10
6
single cells into the tail vein of NSG
mice and, after 45 weeks, lungs, liver, and kidneys were removed.
For the intraperitoneal injection model, NSG mice were grafted
using 57.5 10
5
cells in 0.5-mL PBS. Animals were sacriced 6
7 weeks after injection and the distribution of tumor lesions inside
the peritoneal cavity and at distant organs was assessed by assign-
ing a modied peritoneal cancer index (mPCI) score, as previ-
ously established for mice (25). Briey, the PCI allows assessing
the distribution of cancer in the abdomen and distant organs by
summing lesion size scores (03). The mPCI was adapted to mice
with the following lesion size scores: tumor smaller than 2.0 mm
¼1, 2.1 to 5.0 mm ¼2, and greater than 5.0 mm or conuence ¼
3. Both for tail vein and intraperitoneal injections, tumor inci-
dence was evaluated by counting metastatic nodes on the surface
of affected organs under a microscope.
miRNA mimic transfections and 30UTR reporter assays
The interaction between miR-371373 and TGFBR2 was stud-
ied by using SW620 SCs, stably transduced with a GFP- and
TGFBR2 30UTR-containing reporter construct or the respective
control. Dissociated control and 30UTR reporter cultures were
treated either with negative control or with miR-372-3p and/or
miR-373-3p miRNA mimics (Qiagen, 72 hours, 50 nmol/L).
Spheroids were dissociated and GFP
þ
and total cell numbers
were determined on a Countess II FL Automated Cell Counter
(Life Technologies). GFP
þ
/total cell ratios were normalized to the
negative control mimic group, both for SW620 Ctrl 30UTR and
TGFBR2 30UTR SCs.
RNA extraction and real-time qPCR
RNA extraction, reverse transcription (RT), and real-time
qPCRs were performed, as reported previously (24). Briey,
the miRNeasy Mini Kit (Qiagen) was used to extract total RNA
from patient material (in collaboration with the IBBL), from
SCs and adherent counterparts,aswellasfromextracted
xenografts. RT was done using the miScript II RT Kit (Qiagen),
according to the manufacturer's manual. Specic primer pairs
(Supplementary Table S1) and miScript Primer Assays (Qiagen)
were used to study gene or miRNA expression, respectively.
Quality control and normalization were done in qBase
þ
(Bio-
gazelle), using multiple reference genes (primer sequences
available in refs 23, 24). Samples with threshold cycle (C
t
)
values >30 and/or poor melting curves were considered as not
expressed and were removed from the analysis. For the com-
parison of miR-372-3p and miR-373-3p expression in different
colorectal cancer cell lines, we used the TaqMan miRNA ampli-
cation system (Applied Biosystems), with U6 as a reference
target and the following PCR conditions: 95C10minutes,
followed by 95C 15 seconds, and 60C 1 minute for 50 cycles.
Cell lysis and Western blots
Cells were washed with PBS and lyzed with RIPA buffer
(Thermo Fisher Scientic), supplemented with 1% SDS. After
addition of 1Laemmli buffer, cell lysates were vortexed, cen-
trifuged, heated at 95C for 5 minutes, blotted, and detected,
following standard procedures (24). For all TGFBR2 and SMAD1/
5/9, as well as for corresponding Tubulin, b-Actin, pSMAD2,
SMAD2, and ID1 analyses, Western blots were performed using
Bis-Tris 4%12% gradient gels (Invitrogen) and MES-SDS run-
ning buffer (50 mmol/L 2-(N-morpholino)-ethane sulfonic acid,
50 mmol/L Tris-HCl (pH 7.3), 3.47 mmol/L SDS, and 1.025
mmol/L EDTA), as described previously (26). Primary antibodies
are listed in Supplementary Table S1.
mRNA and miRNA expression arrays and pathway analysis
Microarray gene expression proling experiments were per-
formed and analyzed, as outlined before (23, 24). A false dis-
covery rate (FDR) <0.05 and a |log
2
fold change| >1wereset
as signicance cut-off values for differentially expressed genes
(DEG). Normalized gene expression intensities are available
under accession numbers GSE89523 and GSE102001. Fold
change and Pvalue information of DEGs was used to determine
signicantly deregulated canonical pathways with ingenuity
pathway analysis (IPA, Qiagen). Gene-set enrichment analysis
(27)wasusedtoidentifysignicantly deregulated pathways.
miRNA expression proling was performed using Affymetrix
miRNA chip v4.0 microarrays. Expression intensities were ltered
and normalized, as earlier described for mRNA arrays (23).
Differentially expressed miRNAs (DEM) were identied with the
Bioconductor limma R package, using linear modeling with an
empirical Bayesian approach. FDR <0.05 and |log
2
fold change| >
1 were set as signicance cut-off criteria, resulting in the identi-
cation of 62 DEMs, when comparing SW480 and SW620 SCs.
Normalized expression intensities are available under accession
number GSE89522.
Public datasets and survival analysis
Dataset GSE39582 (28) was used to assess the effect of TGFBR2
expression on relapse-free patient survival. "High" and "low"
groups were separated at the respective median gene expression
value. KaplanMeier curves were generated using the survival,
ggplot2, and ggfortify R packages.
miR-371373/TGFBR2/ID1 Axis Inhibits Metastatic Colonization
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Statistical analysis
Graphs were generated using R 3.2 and GraphPad Prism5
(GraphPad Software). Tumor growth over time was analyzed
with two-way ANOVA tests, limiting dilution assays were evalu-
ated with c
2
tests, and KaplanMeier plots were analyzed with the
Cox proportional hazard model. Student ttests were used to assess
differences between two endpoint conditions. Unless otherwise
stated, experiments were performed in three independent repli-
cates and results are shown as mean SD.
Results
Metastatic SW620 spheroid cultures are highly enriched in
colon TICs
In this study, we investigated the molecular mechanisms
underlying the tumorigenic properties of colon MICs. We took
advantage of the SW480/SW620 cell line pair, which represents
an accepted model to study metastatic progression (29) and
which has proven to be a viable approach to discover colorectal
cancer- and metastasis-relevant miRNAs (30). Both cell lines are
derived from the same patient, but at different stages of the
disease, that is, from the primary tumor (SW480) and from a
lymph node metastasis (SW620), respectively. With the aim of
extending the study of Hewitt and colleagues to the context of
colon TICs, both cell lines were functionally enriched by apply-
ing SC methodology, as described previously (23). Indeed,
sustained cultivation under SC conditions resulted in the emer-
gence of morphologic changes; whereas parental cells were only
able to form loosely packed aggregates, long-term SCs reliably
gave rise to compact tumor spheres (Fig. 1A). Limiting dilution
analysis unveiled that SW480 and SW620 SCs display enhanced
in vitro self-renewal potential, compared with the parental cell
lines (Fig. 1B). Of note, in vivo tumor formation assays did not
only conrm the ndings of Hewitt and colleagues (i.e., elevated
tumor size for parental SW620 cells; data not shown), but also
revealed increased tumor incidence after injection of SW620
compared with SW480 cells (Supplementary Table S2). Inter-
estingly, SOX2, which is one of the most noted pluripotency
markers and which has also been associated with cancer stem cell
properties (31), was highly expressed in SW620 SCs as well as in
our recently established TIC cultures T6, T18, and T20, which are
derived from colorectal cancer patient tissue (Fig. 1C; ref. 23).
Taken together, it can thus be assumed that sustained cultivation
under SC conditions allows for the enrichment of colon TICs. To
further focus on the differences between primary and metastatic
TICs, we thoroughly compared SW480 and SW620 SCs regarding
in vitro and in vivo self-renewal ability. Different sphere and
colony formation assays demonstrated that SW620 SCs consis-
tently display enhanced clonogenicity and self-renewal capacity,
while maintaining similar proliferative potential, compared with
their nonmetastatic SW480 SC counterparts (Fig. 1DF; Supple-
mentary Fig. S1AS1C). Furthermore, tumor size and, most
importantly, tumor incidence were increased for SW620 SCs,
when a limited number of 100 cells were subcutaneously
injected into immunodecient mice (Fig. 1G; Supplementary
Table S2). Besides SOX2, gene and protein expression of LGR5,
an additional marker commonly used for the identication of
intestinal stem cells and TICs (32), was signicantly increased in
SW620 SCs (Fig. 1H). Altogether, it is noticeable that metastatic
SW620 SCs clearly display higher TIC potential compared with
their primary SW480 counterparts, underlining the applicability
of our SW480/SW620 SC model to study secondary tumor
initiation.
Enhanced TGFbsignaling and TGFbR2 expression in
metastatic SW620 TICs
With the aim to decipher molecular differences between pri-
mary and metastatic TICs, we performed gene expression proling
experiments, noting that several genes associated with TGFb
signaling, including TGFB2, TGFbreceptor 2 (TGFBR2), and
TGFBR3, were strongly upregulated in SW620, compared with
SW480 SCs (Fig. 2A). In addition, two important TGFbsignaling
repressors, namely SMAD family member 7 (SMAD7) and Spe-
cic E3 Ubiquitin Protein Ligase 2 (SMURF2), were downregu-
lated in the metastatic TIC condition (Fig. 2A). In line with these
ndings, our microarray data pointed out a general upregulation
of TGFb-relevant genes in SW620, compared with SW480 SCs
(Fig. 2B), which was further conrmed using the IPA search tool
(data not shown). Enhanced gene and protein expression of
TGFBR2 as well as phosphorylation of its downstream effector
SMAD2 further emphasized increased TGFbactivity in SW620,
compared with SW480 SCs (Fig. 2C). Interestingly, TGFbsignal-
ing is known to have a dual role in cancer and can switch between
tumor-suppressive and oncogenic activity, depending on the
cellular framework (33). In the context of colorectal cancer,
alterations of different TGFbsignaling-related genes, including
SMAD4 and TGFBR2, have been linked to cancer initiation and
progression (34). Accordingly, we could observe signicantly
higher TGFBR2 protein expression in tissue sections of 65 pa-
tients with colorectal cancer, compared with matching nonneo-
plastic colon tissue samples (Fig. 2D). Furthermore, we detected a
signicant correlation between high TGFBR2 gene expression and
shorter disease-free survival (Fig. 2E). To test whether TGFb
signaling is also regulating colon TIC self-renewal, we both
stimulated SW480 SCs with TGFb1 and performed a stable
knockdown of TGFBR2 in SW620 SCs by lentiviral transduction
of an shRNA pool (Fig. 2FG; Supplementary Fig. S2 for TGFBR2
knockdown efciency). While pathway activation via TGFb1
stimulation resulted in increased self-renewal activity (Fig. 2F),
stable repression of TGFBR2 led to signicantly reduced sphere
formation capacity (Fig. 2G), highlighting the key regulatory role
of TGFbsignaling in maintaining TIC properties. Overall, our data
provide strong evidence that activated TGFbsignaling and, in
particular, high levels of TGFBR2 are clinically relevant for
patients with colorectal cancer and correlate with colon tumor-
initiating potential.
miR-371373 cluster members inhibit TGFBR2 expression
Over the past decade, numerous studies have shown that
miRNAs are important regulators of colorectal cancer develop-
ment and progression (14). Nevertheless, their exact roles in
regulating colon TIC potential and colorectal cancer metastasis
initiation remain poorly understood. Accordingly, we performed
miRNA expression proling of SW480 and SW620 SCs to identify
potential regulators of metastatic TIC properties. Strikingly, a
drastic downregulation of all 4 mature miR-371373 cluster
members was detected in SW620 SCs (Fig. 3A; Supplementary
Fig. S3A; top 20 DEMs are listed in Supplementary Table S3). As
the functional role of the miR-371373 cluster in colorectal
cancer initiation and progression has not been fully elucidated
yet, we decided to focus on its potential link to the observed
increase in TGFbactivity and metastatic TIC potential.
Ullmann et al.
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Figure 1.
Metastatic SW620 SC displays prominent TIC properties. Morphologic features (A) and quantication (B) of spheroids formed by SW480 and SW620 parental
and SC ells in limiting dilution assays; scale bar, 100 mm. Data presented as mean SEM of three independent experiments. C, SOX2 protein expression of
SW480 and SW620 parental and SC cells (top) as well as of patient-derived T6, T18, and T20 SCs and adherent counterparts (adh; bottom); representative gure of at
least two independent experiments. Sphere formation assays over consecutive passages (P1P4) for SW480 and SW620 SCs with 1,000 cells per well (D) or different
densities (i.e., 1, 2, or 3 cells per well; E) in limiting dilution experiments. Data presented as mean SD for Dand as mean, with 95% condence inter val for E.F, Colony
formation assays were used to determine the clonogenic capacity of SW480 and SW620 SCs. Data presented as mean SD; representative gure of three
independent experiments. G, In vivo tumor growth in NOD/SCID mice following subcutaneous injection of 100 cells derived from SW480 or SW620 SCs; data
presented as mean SEM. Representative picture of extracted tumors, 9 weeks after injection; scale bar, 1 cm; tumor incidence is indicated in brackets. H,
Relative mRNA and protein expression of SOX2 and LGR5 in SW480 and SW620 SCs; data presented as mean SEM (for qPCR) and as representative
picture (for Western blot analysis) of three independent experiments. Statistical signicance was assessed with an unpaired Student ttest for B,D,F,andH, with
ax
2
test for E, and with a two-way ANOVA test for G(,P<0.05; ,P<0.01; and ,P<0.001).
miR-371373/TGFBR2/ID1 Axis Inhibits Metastatic Colonization
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Figure 2.
TGFBR2 signaling is enhanced in metastatic SW620 TICs. A, Heatmap depicting median-centered expression intensities of selected TGFb-signalingrelated genes,
obtained bymicroarray analysis of SW480and SW620 SCs (accessionnumber GSE89523). B, Gene set enrichment analysisfor the pathway interactiondatabase (PID)
TGFBR pathway (c2.cp.v4.0 curated gene sets) using SW480 and SW620 SC microarray data (dataset GSE89523). PID_TGFBRPATHWAY showed a positive
normalized enrichment score (NES ¼1.51) in SW620 SCs. C, Relative TGFBR2 mRNA expression (left), TGFBR2 and SMAD2 protein expression as well as SMAD2
phosphorylation (right) of SW480 and SW620 SCs. Data presented as mean SEM of three independent qPCR experiments and as representative picture of at
least three independent Western blots. D, IHC staining of TGFBR2 (sc-400 antibody, Santa Cruz Biotechnology) on paired colorectal cancer (CRC) tissue microarrays.
Matched tumor (CRC; n¼65) and control mucosa (N; n¼65) samples were scored for intensity of TGFBR2-positive cells, ranging from 0 (no signal) to 2
(strong signal).When present, the signals were mainly cytoplasmic and located at the luminalside of the cell, both for normal enterocytes and for malignant colorectal
cancer cells; scale bar, 200 mm. E, Relapse-free colorectal cancer patient survival, according to TGFBR2 expression in publicly available dataset GSE39582 (28);
95% condence intervals depicted as colored areas; number of relapse cases (i.e., events) ¼177. F, Self-renewal capacity as well as SMAD2 activation (pSMAD2)
and protein expression of SW480 SCs after stimulation with 1 nmol/L TGF-b1. G, Self-renewal capacity of SW620 SCs after lentiviral transduction of either a TGFBR2
shRNA pool or the corresponding control vector. Representative gures of at least three independent Western blots or limiting dilution assays (with 1, 2, or 3
cells per well) for Fand G, respectively; data shown as mean with 95% condence interval. Statistical signicance was assessed with an unpaired Student ttest
for C, with a paired Student ttest for D, with the Cox proportional hazard model for E, and with a x
2
test for Fand G(,P<0.05; ,P<0.01; and ,P<0.001).
Ullmann et al.
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Figure 3.
miR-371373 cluster inhibits TGFBR2 expression in colon TICs. A, Heatmap depicting median-centered expression intensities of all mature members of the
miR-371373 cluster, obtained by miRNA microarray analysis of SW480 and SW620 SCs (dataset GSE89522). B, Schematic representation of miR-372-3p and
miR-373-3p binding sites in the 30UTR of their potential target gene TGFBR2. Binding site inf ormation and 30UTR length were retrieved from TargetScan v6.2 (www.
targetscan.org). C, Reporter assay showing the relative fraction of GFP
þ
cells following miRNA mimic treatment of SW620 SCs that were transduced with a control
vector (left) or with a GFP-containing TGFBR2 30UTR vector (right), respectively. Data presented as mean SD; representative gure of three independent
experiments. Relative TGFBR2 mRNA (D) and TGFBR2 protein expression (E) following stable overexpression of the miR-371373 cluster in different SCs.
Data presented as mean SD for D; representative gures of three (D)andtwo(E) independent experiments, respectively. Effects were further validated
in two additional independent clones for T18 SCs. Statistical signicance was assessed with an unpaired Student ttest for Cand D; ns, not signicant; ,P<0.05;
,P<0.01; and ,P<0.001.
miR-371373/TGFBR2/ID1 Axis Inhibits Metastatic Colonization
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Interestingly, TGFBR2 has been described as a target gene of
individual miR-371373 cluster members (20, 35) and displays
4 potential binding sites for both miR-372-3p and miR-373-3p in
its 30untranslated region (30UTR; N.B. these two miRNAs belong
to the same family and share the same seed sequence; Fig. 3B). In
line with previous ndings (19), we could detect varying degrees
of miR-372 and miR-373 expression in different colorectal cancer
cell lines and we were able to link reduced miR-371373 expres-
sion levels to epigenetic silencing (Supplementary Fig. S3B and
S3C). Using GFP reporter assays, we were able to validate the
interaction of miR-371373 with the 30UTR of TGFBR2 in SW620
SCs (Fig. 3C). While further studies are needed to fully understand
which miR-371373 cluster member has the biggest effect, we
could observe a stronger TGFBR2 downregulation after miR-372-
3p than after miR-373-3p transfection (Fig. 3C), which is in
agreement with the results of Zhou and colleagues (20). Most
importantly, stable overexpression of the entire miR-371373
cluster in different SCs consistently resulted in strongly repressed
TGFBR2 expression, both on gene and protein levels (Fig. 3DE;
Supplementary Fig. S3DS3G for overexpression efciency). Con-
sequently, the here reported loss of miR-371373 cluster expres-
sion in metastasis-derived SCs might be responsible for their
increased TGFBR2 expression and enhanced TIC properties.
The miR-371373 cluster represses TIC properties
Consistent with its inhibitory impact on TGFBR2 expression,
stable overexpression of the miR-371373 cluster signicantly
suppressed in vitro self-renewal and colony formation capacity,
without affecting cellular proliferation (Fig. 4A; Supplementary
Fig. S4AS4C). Moreover, we could show that the miR-371373
cluster efciently blocks the TIC-promoting effect of TGFb1
stimulations (Fig. 4B), emphasizing that this miRNA cluster most
probably acts via modulation of TGFbsignaling activity. Next, the
tumor-suppressive role of this miRNA cluster was conrmed in a
series of in vivo tumor formation experiments. Lentiviral trans-
duction of miR-371373, both in T6 and T18 patient-derived
(Fig. 4C) as well as in SW620 (Fig. 4D) SCs, resulted in signif-
icantly reduced tumor growth. Of note, overexpression of miR-
372-3p and miR-373-3p was maintained in extracted xenografts
(Supplementary Fig. S5A). The inhibitory effect of the miR-
371373 cluster on the metastatic colonization potential of
colon TICs was assessed by performing intravenous injections of
miR-371373overexpressing cells, consistently resulting in
reduced in vivo metastatic outgrowth activity, compared with
injections of respective control cells (Fig. 4E; Supplementary Fig.
S5B). SW620 SCs merely gave rise to kidney metastases and hence
only poorly reected the actual situation of patients with stage IV
colorectal cancer (Supplementary Fig. S5B). On the other hand,
injection of primary T18 cells showed a pathophysiologically
more relevant response, leading to an extensive number of met-
astatic nodes in the lungs and liver. In line with our in vitro data,
this process could be largely blocked by overexpressing miR-
371373 prior to injection (Fig. 4E). In parallel, we used a
peritoneal carcinomatosis model (i.e., intraperitoneal injections)
to further study the effect of miR-371373 on the metastatic
outgrowth potential of colon TICs. In contrast to tail vein assays,
in which colonization of the lungs is often due to hematogenous
embolization, the intraperitoneal injection model has the advan-
tage of studying the dissemination of free cancer cells as well as
their metastatic outgrowth activity at distant organs. In addition,
carcinomatosis is clinically relevant as it can be observed in 20%
30 % of patients with late-stage colorectal cancer and is associated
with poor prognosis (36). Clinical studies have shown that
patients with free cancer cells do not always experience peritoneal
metastasis, suggesting that their occurrence is governed by specic
signaling pathways. Accordingly, we decided to investigate wheth-
er the miR-371373 cluster could potentially inuence the dis-
semination of free cancer cells as well as their metastatic coloni-
zation capacity at distant sites. Strikingly, stable overexpression of
miR-371373 led to signicantly decreased carcinomatosis as
indicated by both a reduced mPCI score and decreased tumor
incidence at distant organs (Fig. 4F; Supplementary Table S4A).
While the miR-371373 cluster has clearly been associated with
the regulation of early metastasis (16), its role in late metastatic
events remains to be revealed. Accordingly, we wanted to assess
whether the miR-371373 cluster specically represses tumor
initiation potential, which is a key requirement for metastatic
outgrowth at the secondary site. To this end, serial transplantation
experiments, which represent the gold standard for the evaluation
of TIC potential, were performed to study the impact of miR-
371373 on in vivo self-renewal potential. Strikingly, injection of
low cell doses resulted in reduced in vivo self-renewal capacity of
T6 and SW620 SCs after stable overexpression of the miR-
371373 cluster, further highlighting its antitumorigenic and
TIC-repressing properties (Fig. 4G). Taken together, our results
clearly demonstrate a novel role for the miR-371373 cluster as a
potent inhibitor of colon TIC activity and hence secondary tumor
initiation capacity.
The miR-371373 cluster represses ALDH1A1 in a TGFb
signaling-independent manner
In search of further downstream effectors that might explain the
described functional effects of miR-371373 on the phenotype of
colon MICs, we performed microarray gene expression analysis
(accession number GSE102001) of SW620 and T18 SCs, follow-
ing stable overexpression of the miR-371373 cluster. By com-
paring the list of miR-371373responsive genes with the pre-
viously identied genes that were upregulated in metastatic SCs
(cf. Fig. 2, comparison between SW480 and SW620 SCs; accession
number GSE89523), we could single out a small set of genes that
are both putative miR-371373 target genes and potential reg-
ulators of metastasis initiation (Fig. 5A). Among the latter was not
only TGFBR2 but also the accepted TIC marker ALDH1A1, which
has been described as a regulator of self-renewal activity and
tumor initiation in various cancer types, including colorectal
cancer (37). Strikingly, we could not only detect increased
ALDH1A1 expression in SW620 compared with SW480 SCs (Fig.
5B), but we also observed consistently higher ALDH1A1 levels in
different primary SCs, compared with their respective adherent
counterparts (Fig. 5C). Moreover, lentiviral transduction of the
miR-371373 cluster resulted in reduced ALDH1A1 expression,
both on gene (Fig. 5D) and protein level (Fig. 5E), further
highlighting the potential regulatory link between miR-
371373 and ALDH1A1. However, no miR-371373binding
sites were predicted in the 30UTR of ALDH1A1 (TargetScan v6.2),
suggesting either an indirect regulatory connection between this
miRNA cluster and ALDH1A1 or the presence of noncanonical
binding sites. As we have shown TGFBR2 to be a direct miR-
371373 target gene in our SCs (Figs. 3 and 5A), we hypothesized
that ALDH1A1 might be regulated via miR-371373induced
modulation of TGFbsignaling activity. However, neither pathway
stimulations with TGFb1 nor siRNA-mediated knockdown of
Ullmann et al.
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Figure 4.
miR-371373 cluster represses TIC properties. A, Self-renewal capacity of different SCs after stable overexpression of the miR-371373 cluster, assessed by
limiting dilution analysis (with 1, 2, or 3 cells per well). Data shown as mean with 95% condence interval; representative gure of at least three independent
experiments. B, Effect of stable miR-371373 cluster overexpression and/or 1 nmol/L TGFb1 stimulation on the self-renewal capacity of T18 SCs. Representative
gure of two independent experiments, shown as mean with 95% condence interval. C, Weight of extracted tumors, 9 weeks after subcutaneous injection of
100 cells derived from primary T6 and T18 SCs, following lentiviral transduction of miR-371373 cluster or corresponding control vector, respectively; n¼5
for T6 and n¼6 for T18; data shown as mean SD. D, In vivo tumor growth in NSG mice, follow ing subcutaneous injection of 100 SW620 SC-derived cells, following
lentiviral transduction of miR-371373 cluster or respective control vector. Data presented as mean SEM; n¼5. Representative picture of extracted
tumors, 9 weeks after injection. E, Quantication and representative picture of in vivo liver and lung metastasis formation in NSG mice. Hematoxylin and eosinstained
tissue sections were performed following intravenous injection of 1 10
6
T18 SC cells that were transduced with miR-371373 cluster or control vector,
respectively. Metastatic nodes (arrows in lung sections) per organ (liver) or per lobe (lung) were counted under a microscope. Incidence of liver metastasis is
indicated in brackets. Data presented as mean SD; n¼3 mice per group; scale bar, 200 mm. F, Tumor distribution in mice measured by the modied PCI score after
intraperitoneal engraftment with 7.5 10
5
T6 SC cells overexpressing the miR-371373 cluster, or respective control cells. Data presented as mean SD;
representative pictures of peritoneal cavities; n¼8 mice. G, Serial in vivo limiting dilution experiment with T6 and SW620 SCs, following stable overexpression of
miR-371373 cluster or corresponding control vector. After a rst round of xenotransplantation, extracted tumors were dissociated and different cell densities
were subcutaneously injected into secondary recipient NSG mice. Secondary tumor incidence was evaluated after 12 weeks. Statistical signicance was assessed with
ax
2
test for A,B,andG, a paired Student ttest for C, a two-way ANOVA test for D, and an unpaired Student ttest for Eand F.,P<0.05; ,P<0.001.
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Figure 5.
The miR-371373 cluster represses ALDH1A1 in a TGFbsignaling-independent manner. A, Identication of miR- 371373responsive genes of interest was done by
performing gene expression microarray analysis (dataset GSE102001) after stable overexpression of miR-371373 in SW620 and T18 SCs. A list of 28 genes,
considered as both responsive to miR-371373 and relevant in the context of metastatic SCs, was identied by comparing the 172 commonly downregulated
genes (i.e., potential miR-371373 target genes) to the list of 429 upregulated genes in SW620, compared with SW480 SCs (from dataset GSE89523). B, Relative
ALDH1A1 mRNA expression in SW480 and SW620 SCs; data presented as mean SEM of three independent experiments. C, Relative ALDH1A1 expression (fold
change) for T6, T18, and T20 adherent and spheroid cultures. Previously published microarray data (23) available in the ArrayExpress database (accession
number E-MTAB-3575). Relative ALDH1A1 mRNA (D) and ALDH1A1 protein expression (E) after stable overexpression of miR-3 71373 cluster or respective control
vector in different SCs; data presented as mean SEM for Dand as a representative gure of three independent experiments for E. Effect of TGFBR2 siRNA
(25 nmol/L, 72 hours) and/or TGFb1 stimulation (400 pmol/L, 1 hour) on the relative expression of ALDH1A1 mRNA (F) and on SMAD2 activation (pSMAD2) as
well as on SMAD2 and ALDH1A1 protein expression of different adherent cultures (G). Representative gure of two (F) or three (G) independent experiments,
respectively; qPCR data shown as mean SD. Statistical signicance was assessed with an unpaired Student ttest for B,C,D,andF; ns, not signicant; ,P<0.05;
,P<0.001.
Cancer Res; 78(14) July 15, 2018 Cancer Research3802
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TGFBR2 (Supplementary Fig. S6A and S6B for knockdown ef-
ciency) had any signicant effect on ALDH1A1 expression (Fig.
5FG). Of note, we also investigated the relationship between
miR-371373/TGFBR2 signaling and SOX2, which we could
conrm to be an important regulator of colon TIC self-renewal
by performing sphere formation assays (Supplementary Figs. S6C
and S6D for knockdown efciency). Although the direct repres-
sion of TGFBR2 inhibited SOX2 mRNA and protein expression
(Supplementary Fig. S6E and S6F), we could not observe such a
regulatory link between the miR-371373 cluster and SOX2
Figure 6.
TGFBR2 regulates ID1 in different colon TIC cultures. Relative ID1 mRNA expression (left) as well as TGFBR2 and ID1 protein expression (right) in SW480 and
SW620 SCs (A) and after stable knockdown of TGFBR2 in different SCs (B); qPCR data presented as mean SD; representativ e gures of three independent
experiments. Effect of TGFBR2 siRNA (25 nmol/L, 72 hours) and/or TGFb1 stimulation (400 pmol/L, 1 hour) on the relative expression of ID1 mRNA (C) and on
SMAD2 activation (pSMAD2) as well as on SMAD2 and ID1 protein expression of different adherent cultures (D). Representative gures of two independent
experiments; qPCR data shown as mean SD. E, Effect of the TGFBR1 inhibitor A-83-01 (10 mmol/L, 4 hours) and/or TGFb1 stimulation (400 pmol/L, 1 hour)
on the relative expression of ID1 mRNA (left) and on SMAD2 activation (pSMAD2) as well as on SMAD2 and ID1 protein expression (right) of different
adherent cultures. Representative gure of two (left) or three (right) independent experiments, respectively; qPCR data shown as mean SD. Statistical
signicance was assessed with an unpaired Student ttest for A,B,C,andE; ns, not signicant; ,P<0.05; ,P<0.01; and ,P<0.001.
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(Supplementary Fig. S6G), suggesting that neither ALDH1A1 nor
SOX2 qualify as downstream effectors of miR-371373/TGFBR2
signaling in colon TICs.
The self-renewal regulator ID1 acts downstream of TGFBR2 in
colon TICs
While looking for other miR-371373/TGFBR2 target can-
didates, our attention was drawn to ID1,whichisknowntobe
TGFb-responsive (12) and which has been described as an
important regulator of colon TIC self-renewal (11). Interest-
ingly, we could observe increased ID1 gene and protein expres-
sion, correlating with higher levels of TGFBR2, in SW620
compared with SW480 SCs (Fig. 6A). As ID1 is also a bona
de downstream target of the TGFbsuperfamily member bone
morphogenic protein (BMP; ref. 10), we rst investigated a
potential link between canonical BMP signaling and ID1 in our
SC setup (Supplementary Fig. S6H and S6I). However, micro-
array gene expression (Supplementary Fig. S6H; GSE89523)
and pSMAD1/5/9 Western blot (Supplementary Fig. S6I) anal-
ysis indicated decreased BMP activity in SW620 compared with
SW480 SCs, suggesting that alternative ID1-regulating mechan-
isms, such as TGFb-SMAD2/3 signaling, must be at work in
metastatic TICs. The potential link between TGFBR2 and ID1
was further tested by generating stable TGFBR2 knockdown SCs
(Supplementary Figs. S2 and S7A for knockdown efciency).
Indeed, stable repression of TGFBR2 resulted in reduced ID1
gene and protein expression (Fig. 6B). Similarly, we were able
to trigger ID1 expression via TGFb1stimulations(Fig.6CE)
and we could show that this effect is reversible by means of
both siRNA-mediated (Fig. 6C and D; Supplementary Fig. S6A
and S6B for knockdown efciency) and pharmacologic inhi-
bition (Fig. 6E) of TGFbsignaling.
Anti-TIC effect of the miR-371373 cluster is mediated by
TGFBR2/ID1 signaling
In a next step, further investigating the mechanisms of colon
TIC activity and colorectal cancer progression, we examined the
potential link between miR-371373/TGFBR2 signaling and ID1
expression. Strikingly, stable overexpression of miR-371373 did
not only result in a signicant repression of TGFBR2, but was also
accompanied by strongly reduced ID1 gene and protein levels
(Fig. 7A and T18 microarrays, GSE102001), highlighting a clear
molecular connection between the miR-371373 cluster and
ID1. Similarly, both TGFBR2 and ID1 were signicantly repressed
after miR-371373 overexpression in different extracted xeno-
grafts (Fig. 7B; Supplementary Fig. S7B), highlighting the rele-
vance of miR-371373/TGFBR2/ID1 signaling as a regulatory
mechanism of in vivo tumor initiation. Regarding the functional
regulation of TIC properties, ID1 is thought to drive breast cancer
metastasis by facilitating the colonization of the lung parenchyma
(38) and is known to promote tumor growth and in vivo self-
renewal in the context of colorectal cancer. Here we generated
stable ID1 knockdown SCs (Supplementary Fig. S7C for knock-
down efciency) to unravel the potential role of ID1 in regulating
TIC self-renewal and colorectal cancer progression. Indeed, the
injection of SC cells led to the formation of signicantly smaller
tumors after ID1 knockdown, compared with control cells (Fig.
7C; Supplementary Fig. S7D; knockdown was conrmed in
extracted xenografts; Supplementary Fig. S7E). Most importantly,
stable repression of ID1 also resulted in impaired in vivo metastatic
outgrowth activity (Fig. 7D), reecting the effect of miR-371373
cluster overexpression. Similar to the results obtained after lenti-
viral transduction of the miR-371373 cluster (Fig. 4F), stable
ID1 knockdown also led to a signicantly lower mPCI score and
reduced tumor incidence (Fig. 7E; Supplementary Table S4B;
Supplementary Fig. S7F), suggesting that miR-371373/
TGFBR2/ID1 signaling strongly affects dissemination and out-
growth activity of metastatic TICs. Taken together, our results thus
highlight ID1 as a functional downstream target of miR-371373
and present the miR-371373/TGFBR2/ID1 signaling axis as a
novel inhibitory mechanism of colon tumor initiation and met-
astatic colonization.
Discussion
Cancer progression and successful completion of the meta-
static cascade require MICs to (i) reach the secondary organ and
to (ii) achieve metastatic outgrowth into macroscopic malig-
nancies (3). In consequence, invasive and stem cell-like prop-
erties (8), epithelialmesenchymal plasticity (39, 40), as well as
extensive self-renewal activity (5) seem to be indispensable for
metastatic colonization and outgrowth into clinically relevant
Figure 7.
Potential role of the miR-371373/TGFBR2/ID1 axis in regulating metastatic colonization potent ial. A, Relative ID1 mRNA expression (left) as well as TGFBR2 and ID1
protein expression (right) in different SCs following lentiviral transduction of miR-371373 cluster or respective control vector. Representative gure of
three (left) or two (right) independent experiments, respectively; qPCR data presented as mean SD. B, Relative TGFBR2 and ID1 gene expression (left), and
TGFBR2, ID1, and SMAD2 protein expression as well as SMAD2 activation (right) in extracted xenograft tumors, 3 weeks after injection of 1 10
6
T18 SC
cells with stable overexpression of miR-371373 cluster or corresponding control vec tor. qPCR data shown as mean SD; Western blot data shown as representative
picture with quantication average of all the mice; ID1 and TGFBR2 were normalized to b-actin, pSMAD2 was normalized to total SMAD2; n¼3. C, Tumor
weight in NSG mice, 9 weeks after subcutaneous injection of 100 cells derived from primary T6 SCs, following lentiviral transduction of ID1 shRNA or respective control
vector; n¼9, data shown as mean SD. D, Quantication (left) of tail vein asays and representative picture (right) of in vivo liver and lung metastasis
formation in NSG mice. Hematoxylin and eosinstained tissue sections were performed following intravenous injection of 1 10
6
T6 cells that were transduced with an
ID1 shRNA pool or the corresponding control vector, respectively. Meta static nodes (arrows) per organ (liver) or per lobe (lung) were counted under a microscope.
Incidence of liver metastasis is indicated in brackets. Data presented as mean SD; n¼5 for control and n¼6forID1 shRNA groups. E, Tumor distribution in mice
measured by the modied PCI score after intraperitoneal engraftment of 5 10
5
T6 SC cells following stable knockdown of ID1, or respective control
cells. Data presented as mean SD; representative pictures of peritoneal cavities; n¼5 mice. Statistical signicance was assessed with an unpaired Student ttest for
A,D,andE, and with a paired Student ttest for Band C(,P<0.05; ,P<0.01; and ,P<0.001). F, Potential mechanism of action. High expression of
the miR-371373 cluster may repress TGFBR2 expression at the primary tumor site. During tumor progression, gradual loss of miR-371373 cluster expression
and the resulting upregulation of TGFBR2 might increase TGFbresponsiveness and cancer cell dissemination. Continuous cytokine cross-talk with different
microenvironmental elements, including blood platelets and cancer-associated broblasts (CAF), confers TIC proper ties on circulating tumor cells. At the metastatic
site, reduced miR-371373 cluster expression supports the upregulation of ALDH1A1 and TGFb-responsive signaling molecules (such as ID1), further promoting TIC
self-renewal, metastatic colonization potential, and secondary tumor formation.
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macrometastases. It thus needs a pleiotropic control mecha-
nism like TGFbsignaling to simultaneously coordinate cancer
cell invasion, TIC plasticity, and tumor initiation at the sec-
ondary site (6). Besides, its dual role in controlling carcino-
genesis (33), the TGFbfamily has indeed been described to
efciently promote TIC and stem cell properties (8). Accord-
ingly, the inhibition of TGFbhas been shown to result in
signicantly decreased TIC self-renewal, along with reduced
expression of SOX2 (41). Moreover, TGFbis known to induce
mesenchymal traits in human colon organoids (42) and to
promote the self-renewal capacity of breast (43) and glioma
(44) TICs. In line with these ndings, we could demonstrate a
clear correlation between TGFBR2 expression, TGFbsignaling
activity, and self-renewal in different colorectal cancer TIC
cultures. Furthermore, patient sample analysis and stable
repression of TGFBR2 allowed us to highlight both the clinical
relevance of TGFBR2 and its key role in regulating colon TIC
properties.
Several studies have highlighted that TGFbsignaling also
regulates tumorigenicity in the context of metastasis. For
instance, TGFb-induced expression of ID1 has been reported
to promote mesenchymal-to-epithelial transition in breast can-
cer cells, resulting in enhanced self-renewal activity and lung
colonization (12). Similarly, specic inhibition of TGFbsig-
naling is thought to reduce the CD44(high)/ID1(high) cell
population and to abolish glioma tumor-initiating properties
(44). Strikingly, by modulating TGFbpathway activity, both via
TGFb1 stimulation and via inhibition of TGFBR2 expression,
we were able to establish a regulatory link between TGFb
signaling and ID1 in colon TICs. Moreover, we could demon-
strate substantial protumorigenic effects of ID1 in our SC setup,
hence corroborating the results of O'Brien and colleagues (11)
and extending them to the context of colorectal cancer metas-
tasis formation.
Most importantly, we identied the miR-371373 cluster as an
effective regulator of TGFBR2/ID1 signaling in colon TICs. Ever
since the discovery of its implication in testicular germ cell tumor
regulation (45), many studies have reported that the miR-
371373 cluster can have both oncomiR and tumor suppressor
activity, depending on the cellular context (16). Using colorectal
cancer patient-derived TIC cultures, we here show that stable
overexpression of the miR-371373 cluster efciently represses
colorectal tumor growth. Moreover, our results highlight that high
miR-371373 cluster expression diminishes both in vitro and in
vivo self-renewal activity of colon TICs, emphasizing the strong
colorectal cancer -suppressing activity of this miRNA cluster.
Most previous studies have focused on the role of the miR-
371373 cluster in regulating early metastatic events (16).
Accordingly, the proliferation of primary tumor cells (19), their
migration and invasion (35) as well as their entry into EMT (46),
are known to be controlled by the miR-371373 cluster. Fur-
thermore, miR-373 is thought to repress TGFb-induced invasion
of pancreatic cancer cells (47) and to inhibit breast cancer metas-
tasis by reducing TGFBR2 expression (35). Taken together, these
data suggest that high miR-371373 levels can restrain the
expression of TGFBR2 at the primary tumor site, thereby blocking
EMT and preventing colorectal cancer progression (Fig. 7F).
In contrast, not much is known about the role of these miRNAs
in regulating later steps of the metastatic cascade, such as cancer
cell dissemination or colonization of the secondary site. Accu-
mulating evidence shows that extensive cytokine-cross-talk
between tumor and stromal cells is essential for TIC self-renewal
(48). For instance, platelet-derived TGFbis thought to sustain the
mesenchymal traits of circulating tumor cells, enabling subse-
quent extravasation (49). Furthermore, in the context of colorectal
cancer, chemotherapy-treated cancer-associated broblasts were
shown to secrete tumorigenic cytokines (50) and TGFb-activated
stroma is known to emit prometastatic factors, such as IL11 (51).
Similarly, gradual loss of miR-371373 expression during tumor
progression might entail an increased responsiveness to micro-
environmental stimuli, due to the concomitant upregulation of
TGFBR2 (Fig. 7F). Even more importantly, this study clearly
demonstrates a novel inhibitory role of the miR-371373 cluster
during metastatic colonization. By blocking the self-renewal
regulator ALDH1A1 as well as TGFBR2/ID1 signaling, the
miR-371373 cluster prevents secondary tumor initiation and
outgrowth into clinically relevant macrometastases. Hence, the
herein described downregulation of miR-371373 and the result-
ing upregulation of ID1 during tumor progression likely result
in increased aggressiveness of colon MICs at the secondary site
(Fig. 7F). Altogether, our ndings identify the miR-371373/
TGFBR2/ID1 signaling axis as an efcient regulatory mechanism
of colon TIC self-renewal and colorectal cancer progression.
Disclosure of Potential Conicts of Interest
No potential conicts of interest were disclosed.
Disclaimer
The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Authors' Contributions
Conception and design: P. Ullmann, K. Qureshi-Baig, E. Letellier
Development of methodology: P. Ullmann, K. Qureshi-Baig, P. Felten,
E. Letellier
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): P. Ullmann, F. Rodriguez, K. Qureshi-Baig, P. Felten,
L. Antunes, N. Z
ugel, R. Weiskirchen, E. Letellier
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): P. Ullmann, F. Rodriguez, A. Ginolhac, R. Weis-
kirchen, S. Haan, E. Letellier
Writing, review, and/or revision of the manuscript: P. Ullmann, M. Schmitz,
S.K. Meurer, K. Qureshi-Baig, L. Antunes, R. Weiskirchen, S. Haan, E. Letellier
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): F. Rodriguez, M. Schmitz, S.K. Meurer,
K. Qureshi-Baig, P. Felten, S. Frasquilho, E. Letellier
Study supervision: S. Haan, E. Letellier
Acknowledgments
We would like to thank all the contributing surgeons and nurses from the
Centre Hospitalier du Luxembourg, the Centre Hospitalier Emile Mayrisch, and
the Clinical and Epidemiological Investigation Centre of the Luxembourg
Institute of Health (LIH) for their work with the patients. The authors would
also like to thank their collaborators at the Integrated Biobank of Luxembourg
(IBBL), particularly Fay Betsou and Nikolai Goncharenko for the overall set-up
of the patient sample collection and RNA extraction as well as Yervan Kar-
apetyan and B
en
edicte Culot for their help with histopathology analysis. We are
also grateful to Djalil Coowar and Marthe Schmit for managing the animal
facility of the University of Luxembourg. We are grateful to all the members of
the Genomics Research Unit of the LIH for performing the microarray experi-
ments and for providing bioinformatics support. We would also like to thank
Arnaud Muller from the LIH for his help with IPA and GSEA. Finally, we would
like to thank Lasse Sinkkonen and Stephanie Kreis from the LSRU as well as
Christelle Bahlawane from the LSRU/IBBL for additional help and critical
discussions. This project was supported by the Fonds National de la Recherche
(FNR) Luxembourg (E. Letellier received grant C16/BM/11282028; K. Qureshi-
Baig received grant AFR/3093113; and P. Ullmann received grant AFR/
Cancer Res; 78(14) July 15, 2018 Cancer Research3806
Ullmann et al.
on August 9, 2018. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Published OnlineFirst May 10, 2018; DOI: 10.1158/0008-5472.CAN-17-3003
7855578) and by the Fondation Cancer (E. Letellier and S. Haan received grant
F1R-LSC-PAU-13HY2C). K. Qureshi-Baig and P. Ullmann were also supported
by the Fondation du P
elican de Mie and Pierre Hippert-Faber under the aegis of
the Fondation de Luxembourg. R. Weiskirchen is supported by grants from the
German Research Foundation (DFG, SFB/TRR 57, projects P13 and Q3) and
received a grant from the Interdisciplinary Centre for Clinical Research within
the Faculty of Medicine at the RWTH Aachen University (IZKF Aachen, Project
E03-1).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 29, 2017; revised March 9, 2018; accepted May 7, 2018;
published rst May 10, 2018.
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2018;78:3793-3808. Published OnlineFirst May 10, 2018.Cancer Res
Pit Ullmann, Fabien Rodriguez, Martine Schmitz, et al.
Axis
Metastatic Colonization by Inhibiting the TGFBR2/ID1 Signaling
373 Cluster Represses Colon Cancer Initiation andThe miR-371
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... By searching in ENCORI dataset [26], we found that 57 genes were mutually predicted by TargetScan, PITA, miRanda, microT and miRmap ( Figure 4B). Among these genes, we focused on TGFBR2, which is one of the receptors of TGF-β1 and associated with tumor migration and invasion [27,28]. We hypothesized that circARID1A promoted GBM invasion by modulating the miR-370-3p/ TGFBR2 pathway. ...
... In order to determine the target gene of miR-370-3p, we identified 60 genes mutually predicted by TargetScan, PITA, miRanda, microT and miRmap in the ENCORI dataset. Among these genes, we focused on TGFBR2, which is associated with tumor migration and invasion [27,28]. Further research indicated that transfection with miR-370-3p inhibitor significantly promoted U87 cell migration and invasion by increasing the expression of TGFBR2, while silencing circARID1A suppressed this behavior and expression of TGFBR2. ...
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... A molecular cluster known as miR-371 to 373, which is primarily responsible for regulating cancer metastasis in CRC patients, has been discovered, which could help researchers in developing new therapies that effectively inhibit tumor growth. [2] Fusobacterium nucleatum, a gramnegative oral anaerobe, has been shown to accelerate CRC by Rubinstein et al. [3] This study could help researchers identify and treat malignant CRC more easily, as well as explain why some patients' disease progresses much faster than others, which could be due to the presence of this bacteria in the mouth. [3] Kumaradevan et al [4] discovered a new target protein, c-Cbl, that may improve CRC patient survival. ...
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