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.
Recurrent somatic DICER1 mutations in nonepithelial ovarian cancers.
N. Engl. J. Med. 366, 234–242.
Hermeking, H. (2007). p53 enters the microRNA world. Cancer Cell 12,
Herranz, H., and Cohen, S.M. (2010). MicroRNAs and gene regulatory
networks: managing the impact of noise in biological systems. Genes Dev.
Hill, D.A., Ivanovich, J., Priest, J.R., Gurnett, C.A., Dehner, L.P., Desruisseau,
D., Jarzembowski, J.A., Wikenheiser-Brokamp, K.A., Suarez, B.K., Whelan,
A.J., et al. (2009). DICER1 mutations in familial pleuropulmonary blastoma.
Science 325, 965.
Kirsch, D.G., Dinulescu, D.M., Miller, J.B., Grimm, J., Santiago, P.M., Young,
N.P., Nielsen, G.P., Quade, B.J., Chaber, C.J., Schultz, C.P., et al. (2007).
A spatially and temporally restricted mouse model of soft tissue sarcoma.
Nat. Med. 13, 992–997.
Kumar, M.S., Lu, J., Mercer, K.L., Golub, T.R., and Jacks, T. (2007). Impaired
microRNA processing enhances cellular transformation and tumorigenesis.
Nat. Genet. 39, 673–677.
Kumar, M.S., Pester, R.E., Chen, C.Y., Lane, K., Chin, C., Lu, J., Kirsch, D.G.,
Golub, T.R., and Jacks,T. (2009). Dicer1 functions as a haploinsufficient tumor
suppressor. Genes Dev. 23, 2700–2704.
Lambertz, I., Nittner, D., Mestdagh, P., Denecker, G., Vandesompele, J., Dyer,
M.A., and Marine, J.C. (2010). Monoallelic but not Biallelic loss of Dicer1
promotes tumorigenesis in vivo. Cell Death Differ. 17, 633–641.
Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer,
S., Rice, A., Kamphorst, A.O., Landthaler, M., et al. (2007). A mammalian
microRNA expression atlas based on small RNA library sequencing. Cell
Leung, A.K., and Sharp, P.A. (2010). MicroRNA functions in stress responses.
Mol. Cell 40, 205–215.
Leung, A.K., Vyas, S., Rood, J.E., Bhutkar, A., Sharp, P.A., and Chang, P.
(2011a). Poly(ADP-ribose) regulates stress responses and microRNA activity
in the cytoplasm. Mol. Cell 42, 489–499.
Leung, A.K., Young, A.G., Bhutkar, A., Zheng, G.X., Bosson, A.D., Nielsen,
C.B., and Sharp, P.A. (2011b). Genome-wide identification of Ago2 binding
sites from mouse embryonic stem cells with and without mature microRNAs.
Nat. Struct. Mol. Biol. 18, 237–244.
Lewis,M.A., Quint, E.,Glazier, A.M.,Fuchs,H., DeAngelis, M.H.,Langford, C.,
van Dongen, S., Abreu-Goodger, C., Piipari, M., Redshaw, N., et al. (2009). An
ENU-induced mutation of miR-96 associated with progressive hearing loss in
mice. Nat. Genet. 41, 614–618.
Lin, R.J., Lin, Y.C., Chen, J., Kuo, H.H., Chen, Y.Y., Diccianni, M.B., London,
W.B., Chang, C.H., and Yu, A.L. (2010). microRNA signature and expression
of Dicer and Drosha can predict prognosis and delineate risk groups in
neuroblastoma. Cancer Res. 70, 7841–7850.
Lu, J., Getz, G., Miska, E.A., Alvarez-Saavedra, E., Lamb, J., Peck, D., Sweet-
Cordero, A., Ebert, B.L., Mak, R.H., Ferrando, A.A., et al. (2005). MicroRNA
expression profiles classify human cancers. Nature 435, 834–838.
Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T.,
Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J.,
circuitry of embryonic stem cells. Cell 134, 521–533.
Martello, G., Rosato, A., Ferrari, F., Manfrin, A., Cordenonsi, M., Dupont, S.,
Enzo, E., Guzzardo, V., Rondina, M., Spruce, T., et al. (2010). A MicroRNA
targeting dicer for metastasis control. Cell 141, 1195–1207.
Melo, S.A., Ropero, S., Moutinho, C., Aaltonen, L.A., Yamamoto, H., Calin,
G.A., Rossi, S., Fernandez, A.F., Carneiro, F., Oliveira, C., et al. (2009). A
TARBP2 mutation in human cancer impairs microRNA processing and
DICER1 function. Nat. Genet. 41, 365–370.
Mudhasani, R., Zhu, Z., Hutvagner, G., Eischen, C.M., Lyle, S., Hall, L.L.,
Lawrence, J.B., Imbalzano, A.N., and Jones, S.N. (2008). Loss of miRNA
biogenesis induces p19Arf-p53 signaling and senescence in primary cells.
J. Cell Biol. 181, 1055–1063.
Mukherjee, S., Raje, N., Schoonmaker, J.A., Liu, J.C., Hideshima, T., Wein,
M.N., Jones, D.C., Vallet, S., Bouxsein, M.L., Pozzi, S., et al. (2008).
Pharmacologic targeting of a stem/progenitor population in vivo is associated
with enhanced bone regeneration in mice. J. Clin. Invest. 118, 491–504.
Pittenger, M.F. (2008). Mesenchymal stem cells from adult bone marrow.
Methods Mol. Biol. 449, 27–44.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca,
J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999).
Multilineage potential of adult human mesenchymal stem cells. Science 284,
Sage, J., Mulligan, G.J., Attardi, L.D., Miller, A., Chen, S., Williams, B.,
Theodorou, E., and Jacks, T. (2000). Targeted disruption of the three
Rb-related genes leads to loss of G(1) control and immortalization. Genes
Dev. 14, 3037–3050.
Sekine, S., Ogawa, R., Ito, R., Hiraoka, N., McManus, M.T., Kanai, Y., and
Hebrok, M. (2009). Disruption of Dicer1 induces dysregulated fetal gene
expression and promotes hepatocarcinogenesis. Gastroenterology 136,
Stefani, G., and Slack, F.J. (2008). Small non-coding RNAs in animal develop-
ment. Nat. Rev. Mol. Cell Biol. 9, 219–230.
Torres, A., Torres, K., Paszkowski, T., Jod1owska-Je ˛drych, B., Radoma? nski,
T., Ksia ˛ _ zek, A., and Maciejewski, R. (2011). Major regulators of microRNAs
biogenesis Dicer and Drosha are down-regulated in endometrial cancer.
Tumour Biol. 32, 769–776.
Wang, Y., Baskerville, S., Shenoy, A., Babiarz, J.E., Baehner, L., and Blelloch,
R. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transi-
tion and promote rapid proliferation. Nat. Genet. 40, 1478–1483.
Zheng, G.X., Ravi, A., Calabrese, J.M., Medeiros, L.A., Kirak, O., Dennis, L.M.,
for the mir-290-295 cluster in mouse embryonic stem cells. PLoS Genet. 7,
Tumor Formation in the Absence of MicroRNAs
Cancer Cell 21, 848–855, June 12, 2012 ª2012 Elsevier Inc. 855