Proliferation and Tumorigenesis
of a Murine Sarcoma Cell Line
in the Absence of DICER1
Tyler Jacks,1,2,4and Phillip A. Sharp1,4,*
1Department of Biology
2Howard Hughes Medical Institute, Department of Biology
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3Harvard-MIT Health Sciences and Technology Program, Cambridge, MA 02139, USA
4David H. Koch Institute for Integrative Cancer Research, Cambridge, MA 02139, USA
5These authors contributed equally to this work
6Present address: Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
MicroRNAs are a class of short ?22 nucleotide RNAs predicted to regulate nearly half of all protein coding
genes, including many involved in basal cellular processes and organismal development. Although a global
reduction in miRNAs is commonly observed in various human tumors, complete loss has not been
documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding
that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the
characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null
cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence,
miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral
MicroRNAs (miRNAs) are short ?22 nucleotide RNAs that
comprise an essential class of regulators predicted to repress
over half of all genes posttranscriptionally (Bartel, 2009; Fried-
man et al., 2009). Consistent with computational predictions of
widespread targeting, they have been implicated experimentally
in a variety of fundamental cellular processes such as cell cycle
(Wang et al., 2008), apoptosis (Chivukula and Mendell, 2008),
and differentiation (Herranz and Cohen, 2010; Stefani and Slack,
2008). Given these broad roles, the relationship between
miRNAs and cancer is understandably complex. At the level of
individual miRNAs, either gains or losses may promote tumor
formation. However, analysis of global miRNA levels in tumors
suggests a surprisingly unidirectional relationship, with multiple
human tumors showing decreased miRNA content (Gaur et al.,
2007; Lu et al., 2005). In some cases, this downregulation may
be directly achieved by decreased expression of DICER1 and
DROSHA, key processing enzymes of miRNA production (Lin
et al., 2010; Martello et al., 2010; Torres et al., 2011) or mutations
in their binding partners (Melo et al., 2009).
Despite these trends toward decreased miRNA expression,
a number of observations suggest that miRNAs may in fact
be important for a variety of tumor types. For instance,
although heterozygous somatic mutations in DICER1 can be
found in tumor genotyping atlases, homozygous loss has not
been reported in these databases (Kumar et al., 2009). Simi-
larly, in rare cases of heterozygous germline DICER1 mutations,
The nearly global decrease in miRNAs observed across a range of human tumors suggests that restoration of miRNA levels
may have a valuable therapeutic role. Here we report that some tumor cells devoid of miRNA activity are viable and form
tumors under certain conditions. However, the failure to detect human tumors with complete loss suggests that the oppo-
site approach, namely a further decrease in these levels, may surprisingly also be beneficial. We explore this alternative in
a well-characterized sarcoma model, demonstrating that miRNA depletion can indeed inhibit tumor growth rates through
reduced proliferation and increased cell death. These findings suggest that the targeted inhibition of miRNA pathway
elements, particularly DICER1, may be a potential therapy for the treatment of cancer.
848 Cancer Cell 21, 848–855, June 12, 2012 ª2012 Elsevier Inc.
the pleuropulmonary blastomas to which patients are predis-
posed retain an intact DICER1 allele in tumor tissue (Hill
et al., 2009). Somatic point mutations in DICER1 associated
with nonepithelial ovarian cancers are hypomorphic, likely re-
sulting in expression of a full-length protein but in loss of
some specific miRNAs and retention of others, further suggest-
ing a requirement for DICER1 and miRNA expression in tumors
(Heravi-Moussavi et al., 2012). Mouse models of Dicer1 loss in
cancer also suggest an advantage of retaining miRNA regula-
tion. In a mouse model of Dicer1 deletion in the liver, tumors
emerge several months after deletion following a period of
hepatic repopulation by Dicer1-intact ‘‘escapers’’ (Sekine
et al., 2009). In Dicer1-conditional mouse models of either
soft tissue sarcoma or lung adenocarcinoma, haploinsuffi-
ciency of Dicer1 promotes tumor development but homozy-
gous loss of Dicer1 is not observed (Kumar et al., 2009).
Similarly, in both an Em-myc lymphoma model and a retinoblas-
toma model, viable tumors could not be identified following
homozygous Dicer1 deletion (Arrate et al., 2010; Lambertz
et al., 2010). These studies suggest that complete Dicer1 loss
and the subsequent misregulation of gene expression are
highly deleterious to tumor development.
KrasG12D; Trp53?/?;Dicer1f/?tumors. Clones iso-
lated following Cre-ER integration and tamoxifen
treatment were genotyped by PCR to identify
(B) miRNA expression (copies per cell). Per cell
calculations are based on relative representation
of each miRNA in Dicer1f/?and Dicer1?/?small
RNA-seq libraries, normalized to quantitative
northern blot of miR-22 in Dicer1f/?cells (shown in
Figure S1C). miR-21, miR-22, and let-7 family
members are indicated.
(C) Northern analysis for precursor and mature
miRNAs. Glutamine tRNA was used to control for
loading, and a dilution series of Dicer1f/?RNA (1:1
to 1:16) is provided for quantitation.
(D) Luciferase reporter assays for abundant miR-
NAs. The Renilla luciferase reporter contains six
bulged sites for the let-7 family, and two perfect
sites for miR-16 and miR-17. Targeted Renilla
luciferase reporters were normalized to non-
targeted firefly luciferase reporters. Renilla/firefly
luciferase expression was normalized to expres-
sion in the Dicer1f/?sarcoma cell line.
(E) Proliferation assay.
(F) Cell cycle distribution determined by BrdU
(G) Apoptosis determined by caspase-3 cleavage
All error bars represent the SEM (D–G). See also
Figure S1 and Table S1.
1. Characterizationof KrasG12D;
To better understand how cancer cells
respond to loss of miRNA expression,
we characterized the effects of homo-
zygous deletion of Dicer1-conditional
alleles on the tumorigenicity of an estab-
lished line of murine sarcoma cells and on the cellular phenotype
of immortalized murine mesenchymal stem cells (MSCs).
Dicer1 Null Cells Derived from a Mouse Sarcoma
Proliferate Indefinitely In Vitro
Previously, we generated Dicer1-heterozygous tumors by
injection of Adeno-Cre virus into the hindlimbs of KrasLSL-G12D;
Trp53f/f;Dicer1f/fmice. The resultant tumors always retained at
least one conditional Dicer1 allele (Kumar et al., 2009). From
sarcoma cell lines and deleted the remaining allele of Dicer1
by transducing the cells with a retroviral construct encoding
MSCV.CreERT2.puro and then activating recombination in vitro
with tamoxifen treatment (Figure 1A). A genotyping time course
indicated efficient homozygous recombination (Figure S1A
available online). After multiple passages, however, genotyping
PCR indicated the outgrowth of heterozygous cells, consistent
with previous findings in both this sarcoma model and an
Em-myc/Dicer1 lymphoma model (Arrate et al., 2010; Kumar
et al., 2009).
tumors, we established
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To prevent the preferential outgrowth of DICER1-expressing
cells, we isolated monoclonal populations by plating low-density
cultures immediately after a 24 hr treatment with tamoxifen.
The resulting clones appeared at comparable frequencies in
tamoxifen-treated and control cultures, and were also morpho-
logically similar to the parental cell lines. Genotyping PCR
indicated that the majority of isolated clones had deleted the
second allele of Dicer1 (Figure 1A). We also confirmed recom-
bination of the conditional Dicer1 allele at the protein level by
western blot against DICER1 (Figure S1B). Once a KrasG12D;
Trp53?/?;Dicer1?/?clonal line was established, we did not
observe outgrowth of KrasG12D;Trp53?/?;Dicer1f/?cells, even
after several months of continual passage. Hereafter, we will
refer to the monoclonal homozygous
Dicer1?/?line as Dicer1?/?cells and the parental heterozygous
KrasG12D;Trp53?/?;Dicer1f/?cell line as Dicer1f/?cells. These
results suggest that sarcoma cells survive after homozygous
Dicer1 deletion but have a growth disadvantage relative to cells
retaining Dicer1 expression. To prevent outgrowth of Dicer1f/?
sarcoma cells, all subsequent experiments were carried out
with monoclonal Dicer1?/?sarcoma cell lines.
To determine whether Dicer1?/?clones lacked miRNAs, we
carried out massively parallel sequencing of small RNAs (small
RNA-seq), ?15–50 nucleotides in length, from Dicer1f/?and
Dicer1?/?sarcoma cells. Both libraries contained comparable
sequencing depths at 9.3 and 9.6 million reads, respectively.
However, due to miRNA loss, sequence complexity was greater
in Dicer1?/?cells, which contained 830,000 unique sequences,
relative to 190,000 unique sequences in Dicer1f/?cells. Of
all reads mapping to the genome with 0 or 1 mismatch, 58%
correspond to mature miRNAs in Dicer1f/?cells in comparison
to 0.8% in Dicer1?/?cells. Approximately 48% of mature
miRNAs detected in Dicer1f/?cells became undetectable in
Dicer1?/?cells, whereas the remainder of miRNAs underwent
a median decrease of 111-fold, confirming the global loss of
mature miRNAs with homozygous Dicer1 loss. By quantitative
northern blot, miR-22 was present at ?4,000 copies per cell in
Dicer1f/?sarcoma cells (Figure S1C). Based on the ratio of
miRNA reads in the Dicer1f/?to Dicer1?/?small RNA-seq
libraries normalized to the copy number of miR-22 in Dicer1f/?
sarcoma cells, miR-22 is present at fewer than 10 copies per
Dicer1?/?sarcoma cell. Similarly, based on normalization to
miR-22, other abundant miRNAs such as individual let-7 family
members are also expressed at fewer than ten copies per cell
in Dicer1?/?cells, as compared to several thousand in Dicer1f/?
cells (Figure 1B). miR-451, a DICER1-independent miRNA pro-
cessed by Ago2 and expressed abundantly in red blood cells,
is not detectable in Dicer1f/?sarcoma cells and is present at
extremely low levels (0.4 copies/cell) in Dicer1?/?sarcoma cells
(Cheloufi et al., 2010; Cifuentes et al., 2010). These results indi-
cate near-complete loss of miRNAs upon Dicer1 deletion.
(13%), let-7f (7%), let-7a (7%), let-7c (7%), and miR-21 (6%)
seed sequence corresponds to let-7, accounting for 31.6% of
all miRNA reads. Let-7 dominance has been observed in other
somatic tissues, such as embryonic fibroblasts and neural
precursors (Marson et al., 2008). In addition, we observed
miRNAs associated with tissue-specific expression and func-
tion, such as kidney-specific miR-196a and -196b (1.2% com-
bined) (Landgraf et al., 2007) and miR-96 (2.2%), implicated in
progressive hearing loss (Lewis et al., 2009), suggesting broader
regulatory roles for these short RNAs. Reads from the miR-290-
295 cluster, specific to embryonic stem cells, were negligible in
number, distinguishing these cells as somatic. In total, our
results establish let-7 as the dominant seed in Dicer1f/?sarcoma
cells and confirm the loss of mature miRNAs following Dicer1
As a confirmation of these sequencing results, we performed
northern analysis for let-7g, miR-16, and miR-17, all detected
abundantly in our Dicer1f/?sequencing library. In contrast to
Dicer1f/?cells, Dicer1?/?cells showed an absence of mature
miRNAs and concomitant accumulation of precursors (Fig-
ure 1C). Luciferase reporters containing six bulged sites for
let-7g or one perfect site for either miR-16 or miR-17 were also
derepressed 3- to 6-fold, consistent with functional loss (Fig-
ure 1D). To evaluate proliferative differences, we measured
doubling times for each genotype (Figure 1E). Dicer1?/?cells
divided more slowly (?15 hr) than the Dicer1f/?controls
(?12 hr), but without obvious senescence or onset of crisis.
Dicer1?/?sarcoma cells exhibited a delay in G1 phase relative
sarcoma cells (Figure 1F). Additionally, the
Dicer1?/?sarcoma cells exhibited elevated levels of apoptosis
Dicer1–/–Sarcoma Cells Retain Tumorigenicity In Vivo
Our findings indicate that genetic ablation of Dicer1 is tolerated
in mouse sarcoma cells in vitro. However, in vivo mouse models
and human patient data suggest that homozygous deletion of
Dicer1 is not tolerated in tumors. To test whether proliferative
defects in homozygous Dicer1-deleted tumors, and subsequent
loss through competition in vivo by DICER1-expressing cells,
account for these differences, we carried out tumor formation
assays. Upon subcutaneous injection of 1 3 106cells into the
flanks of immune-compromised mice, Dicer1?/?cells were
indeed tumorigenic, forming tumors at 7/18 sites within
24 days, as compared to 4/8 sites by day 14 for the original
Dicer1f/?strain. To better evaluate the difference in tumor for-
mation kinetics, we repeated this injection experiment with
2.5 3 104cells. At this lower cell number, Dicer1?/?sarcoma
cells began to develop tumors in ?45 days, as compared to
22 days for the parental Dicer1f/?sarcoma cell line (Figure 2A).
Pathologic analysis of either Dicer1?/?or Dicer1f/?tumors iden-
tified both as high grade sarcomas with pleomorphic nuclei and
abnormal mitoses, consistent with previous reports of KRAS-
driven sarcoma models (Figure 2B) (Kirsch et al., 2007; Kumar
et al., 2007). Sample genotypes could not be readily distin-
guished in a blinded analysis.
We also performed syngeneic injections into immunocompe-
tent C57Bl6/SV129 F1 mice. As before, the rate was slower
than the parental Dicer1f/?line, with the first tumors appearing
7 days after injection of the Dicer1f/?cells and 21 days after
injection of the Dicer1?/?cells (Figure S2). Thus, the absence
of DICER1 impairs but does not preclude tumor formation,
even in an immunocompetent background.
Although the sarcoma cells at the time of the injection were
Dicer1?/?, it is possible that in vivo selection resulted in
outgrowth of contaminating Dicer1f/?cells. Therefore, we
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850 Cancer Cell 21, 848–855, June 12, 2012 ª2012 Elsevier Inc.
genotyped DNA prepared from primary tumor tissue and
confirmed a significant recombined band corresponding to the
injected Dicer1?/?cells with an accompanying background
wild-type band contributed by contaminating host tissue (Fig-
ure 2C). Northern analysis of primary tumorsrevealed accumula-
tion of precursors as well as significant but incomplete depletion
of mature miRNAs (Figure 2D).
To test if the residual miRNAs were a result of contaminating
wild-type tissue, we generated cell lines from these tumors.
PCR genotyping confirmed a depletion of the wild-type tissue
during this process (Figure 2E). By northern blotting, the level
of mature miRNAs was lower than the detection limit of the
blot, and this was again accompanied by enrichment in the
pre-miRNA (Figure 2F). The residual mature miRNA observed
is likely due to host tissue contamination, as evidenced by the
greatest miRNA signal in the tumor sample showing the greatest
wild-type contaminant band by PCR (Figures 2E and 2F). Thus,
injected Dicer1?/?cells survived and proliferated in vivo without
recovery of miRNA processing. The earlier in vitro results extend
to an in vivo setting, with sarcoma cells retaining the capacity to
form phenotypically similar tumors, albeit more slowly, in the
absence of DICER1 and miRNAs.
Trp53–/–;Dicer1–/–Sarcoma Cells in Trans-
(A) Injection of 2.5 3 104Dicer1f/?and Dicer1?/?
sarcoma cells into the flanks of nude mice. Error
bars represent the SEM.
(B) Hematoxylin and eosin section of Dicer1f/?and
independent tumor derived from one injection of
the indicated Dicer1?/?sarcoma cell line. Scale
bar represents 100 mm.
(C) PCR genotyping of Dicer1?/?tumors. Re-
combined and floxed bands are derived from the
injected tumor cells, whereas wild-type bands
derive from host tissue.
(D) Northern analysis of tumor tissue derived from
(E and F) PCR (E) and northern (F) analysis
following one round of in vitro passage of
secondary tumors. In (C–F), each sample ID
contains a prefix identifying the injected sarcoma
cell clone followed by a suffix identifying the tumor
replicate (e.g., sample 1-3 corresponds to clone 1
and tumor replicate 3).
See also Figure S2.
2. Tumorigenesis of KrasG12D;
Mesenchymal Stem Cells Were
Generated as an Alternative Model
of Somatic Dicer1 Deletion
The viability of Dicer1 null sarcoma cells,
which lack TRP53 and express onco-
genic KRAS, may be a function of the
strong oncogenic background required
for rapid in vivo growth or may require
additional genetic alterations that occur
during tumor formation. Therefore, we
tested whether Dicer1 loss could be
tolerated in a defined immortalized cell
model. Because sarcomas are thought to be mesenchymal in
origin (Clark et al., 2005), we turned to mesenchymal stem
cells (MSCs), a multipotent population of cells that can differen-
tiate into osteoblasts, chondrocytes, adipocytes, or myocytes
(Pittenger et al., 1999).
From a 1-year-old adult Dicer1f/fmouse, we prepared
a primary culture of MSCs that was then immortalized with
a retroviral vector encoding SV40 large T-antigen (Figure 3A).
Individual clones were isolated and analyzed by flow cytometry
to confirm the expression of CD49e and CD106 (Figure 3B, left
panels), surface markers associated with MSCs (Pittenger,
2008), and the absence of CD31, specific to endothelial cells,
and CD45, a marker of hematopoietic stem cells (data not
To delete Dicer1, we carried out Adeno-Cre-GFP infection
and sorted the infected cells by GFP. This protocol enriched
for Dicer1?/?cells, as seen by the predominance of the dele-
tion-specific PCR product 6 days after sorting (Figure S3A).
This signal was accompanied by loss of DICER1 protein (Fig-
ure S3B), as well as a decrease in mature miRNA levels by
qPCR (Figure S3C) and northern blot at day 7 (Figure S3D).
However, as observed for the sarcoma cells, additional passage
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Cancer Cell 21, 848–855, June 12, 2012 ª2012 Elsevier Inc. 851
Figure 3. Derivation and Characterization of Dicer1–/–Mesenchymal Stem Cells
(A) Schematic of MSC preparation. Primary MSC cultures were prepared from the tibia, femur, and pelvicbones of a1-year-old Dicer1f/fmouse.The primary cells
were then infected with retrovirus encoding SV40 large T-antigen. Monoclonal cultures were then isolated, infected with Adeno-Cre-GFP, sorted by FACS for
GFP-positive cells, and plated at low density to isolate Dicer1-recombined clones.
(B) Cell surface marker expression in Dicer1f/f(left)and Dicer1?/?(right) MSCs.Cells were analyzed by flow cytometrywithantibodies against CD49e and CD106.
(C) PCR genotyping of clonally isolated Dicer1f/for Dicer1?/?MSCs. Clones 6.8 and 6.9 (lanes 3, 4) were derived from parental clone 6 (lane 2), and clones 12.2
and 12.4 (lanes 6, 7) were derived from parental clone 12 (lane 5). PCR genotyping of a Dicer1f/?sarcoma cell line was used as a heterozygous control (lane 1).
(D) Expression of miRNAs in Dicer1f/fand Dicer1?/?MSCs. Total RNA was analyzed with a QIAGEN miScript qPCR assay for let-7a, miR-24, -26, and -31. A
representative qPCR experiment is shown. Error bars represent standard deviation.
(E) Luciferase reporter assay for let-7g. The reporter contains six bulged sites. Targeted Renilla luciferase reporters were normalized to nontargeted firefly
luciferase reporters. Renilla/firefly luciferase expression was normalized to expression in the Dicer1f/fMSC line.
(F) Proliferation assay.
(G) Cell cycle distribution determined by BrdU labeling.
(H) Apoptosis determined by caspase-3 cleavage assay.
Error bars represent SEM (E–H). See also Figure S3.
Tumor Formation in the Absence of MicroRNAs
852 Cancer Cell 21, 848–855, June 12, 2012 ª2012 Elsevier Inc.