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LYMPHOID NEOPLASIA
Inactivation of ribosomal protein L22 promotes transformation by induction of the
stemness factor, Lin28B
*Shuyun Rao,1,2 *Sang-Yun Lee,1,2 Alejandro Gutierrez,3 Jacqueline Perrigoue,1,2 Roshan J. Thapa,2 Zhigang Tu,4
John R. Jeffers,5 Michele Rhodes,1,2 Stephen Anderson,6 Tamas Oravecz,6 Stephen P. Hunger,7 Roman A. Timakhov,8
Rugang Zhang,4 Siddharth Balachandran,2 Gerard P. Zambetti,5 Joseph R. Testa,1,8 A. Thomas Look,3 and David L. Wiest1,2
1Blood
Cell Development and Cancer Keystone, and 2Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Philadelphia, PA;
of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital Boston, Boston, MA; 4Women’s Cancer Program, Fox Chase Cancer
Center, Philadelphia, PA; 5Department of Biochemistry, St Jude Children’s Research Hospital, Memphis, TN; 6Department of Immunology, Lexicon
Pharmaceuticals Inc, The Woodlands, TX; 7Section of Pediatric Hematology/Oncology/Bone Marrow Transplantation, University of Colorado Denver School of
Medicine, Aurora, CO; and 8Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA
3Department
Ribosomal protein (RP) mutations in diseases such as 5qⴚ syndrome both disrupt hematopoiesis and increase the risk
of developing hematologic malignancy.
However, the mechanism by which RP
mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein
L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22
was found to be inactivated in ⬃ 10% of
human T-acute lymphoblastic leukemias.
Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma
in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactiva-
tion enhances transformation potential
through induction of the stemness factor,
Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the
first insight into the mechanistic basis by
which mutations in Rpl22, and perhaps
some other RP genes, increases cancer
risk. (Blood. 2012;120(18):3764-3773)
Introduction
In addition to their role as structural components of ribosomes,
ribosomal proteins (RPs) are increasingly understood to play
critical roles in development and disease, in some cases from
outside of the ribosome. These include roles in regulation of
cell-cycle progression, apoptosis,1 and translation, through direct
interactions with mRNA.2 Mutations in RPs cause diseases collectively termed ribosomopathies, which include myelodysplastic
syndromes (MDS) and diamond blackfan anemia (DBA). DBA is
caused by mutations in a variety of RPs, with approximately
one-half of all cases resulting from mutations in RPS19, RPS26,
RPL5, and RPL11, whereas a type of myelodysplastic syndrome
known as 5q⫺ syndrome has been attributed to the monoallelic loss
of RPS14.3,4 RPS14 haploinsufficiency in 5q⫺ syndrome, as well
as the ribosome dysfunction observed in other bone marrow failure
syndromes, is associated with increased risk in patients for the
development of hematologic malignancies.5 Observations in animal models have similarly linked RP gene mutations with alterations in cancer risk because loss of one copy of numerous, essential
RP genes increased susceptibility to tumor formation in zebrafish,6
suggesting that some RPs may serve as haploinsufficient tumor
suppressors. Nevertheless, neither the basis by which RP function
as tumor suppressors nor the way RP mutations predispose to
malignancy has been explained.
The ribosomal protein L22 (Rpl22) is an RNA-binding component of the 60S ribosomal subunit that is not thought to be required
for global cap-dependent translation, but its normal physiologic
role is poorly understood. We have determined that despite the
ubiquitous expression of Rpl22, its germline ablation in mouse is
not lethal, unlike ablation of most RP genes.7,8 Instead, mice in
which the Rpl22 gene is biallelically inactivated in the germline are
viable, fertile, and grossly normal, with the only striking defect
being an exquisitely specific block in the development of ␣␤
lineage T cells.9 Because genes that are required for the normal
development of a particular cell or tissue often regulate its
transformation10 and because Rpl22 is essential for the development of T lymphocytes, we address here whether Rpl22 regulates
T-cell transformation. We present evidence that Rpl22 functions as
a haploinsufficient tumor suppressor and provide the first mechanistic insights into how mutations in an RP gene predispose cells to
transformation.
Submitted March 3, 2012; accepted September 2, 2012. Prepublished online
as Blood First Edition paper, September 13, 2012; DOI 10.1182/blood-201203-415349.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
Methods
Patient samples
Patient samples were collected with informed consent in accordance with
the Declaration of Helsinki and Institutional Review Board approval from
children with T-acute lymphoblastic leukemia (T-ALL) treated in clinical
trials at the Children’s Oncology Group or Dana-Farber Cancer Institute.
Microarray-based comparative genomic hybridization (aCGH) was performed with the use of genomic DNA on Agilent Human Genome CGH
244A Microarrays (Agilent Technologies), and circular binary segmentation was performed with the DNAcopy package of BioConductor (http://
*S.R. and S.Y.-L. contributed equally to this work.
The online version of this article contains a data supplement.
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© 2012 by The American Society of Hematology
BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
www.bioconductor.org/packages/2.2/bioc/html/DNAcopy.html), as described.11 Color plots of the segmented Log2 copy number data were
generated with dChip software (http://biosun1.harvard.edu/complab/
dchip). aCGH data discussed in this publication have been deposited in
NCBI’s Gene Expression Omnibus and are accessible through GEO series
accession no. GSE14959 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi
?acc⫽GSE14959) and no. GSE7615 (http://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi?acc⫽GSE7615). Sequencing of the RPL22 coding exons in
primary T-ALL, T-ALL cell lines, and T-ALL isolates from relapsed
patients was performed by Agencourt Inc.
Animal studies
Mice were maintained in the Association for Assessment and Accreditation
of Laboratory Animal Care–accredited Laboratory Animal Facility at Fox
Chase Cancer Center and were handled in compliance with guidelines
established by the Institutional Animal Care and Use Committees. Transgenic myristoylated Akt2 (MyrAkt2 Tg) Rpl22⫹/⫹ and Rpl22⫹/⫺ littermate
mice bearing a mixed 129-C57BL/6 background were used in all experiments. To evaluate the effect of Rpl22 inactivation on development of
thymic lymphoma, Myr-Akt2;Rpl22⫹/⫹ and ⫹/⫺ littermates were monitored
daily and killed when they began to manifest signs of disease, after which
the thymic lymphomas were excised for further analysis. All analysis of
premalignant phenotypes was performed on mice when they were 4-6 weeks
of age.
ROLE OF Rpl22 IN CANCER DEVELOPMENT
3765
Sigma-Aldrich; (4) Rpl22 and control shRNA were expressed in murine
stem cell virus–based retroviral vectors LMS or LMP, obtained from
Dr Scott Lowe (Cold Spring Harbor), as described.9 Retrovirus was
produced by transient calcium phosphate transfection of phoenix-ecotropic
packaging cells, as described.16 Lentivirus was produced by transfection of
HEK293T with both packaging (delta8.2 and VSV-G) and pLKO.1 shRNA
vectors using FuGENE 6 (Roche). Virus infected cells were drug selected
for at least 5 days before the experiments. For soft agar transformation
assays, immortalized MEFs were infected with Ras virus alone, and
primary MEFs were coinfected with both E1A and Ras, after which drug
selected cells were mixed with an equal volume of 0.7% agar (Difco;
BD Biosciences), plated on the top of 0.5% agar layer, and cultured for the
indicated time.
Measurement of protein and RNA
NP-40 detergent extracts were resolved by SDS-PAGE and immunoblotted
with antibodies reactive with the following proteins: (1) Rpl229; (2) GAPDH
(Abcam); (3) Lin28 (Abcam); (4) Lin28B (Cell Signaling Technology); (5) Ras
(BD Biosciences); (6) c-myc (Cell Signaling Technology); (7) NF-␬B
p65 (Santa Cruz Biotechnology Inc); (8) Rpl24 (Sigma-Aldrich); and
(9) Rpl11 (Sigma-Aldrich). For measurements of RNA, total RNA was
extracted from cells with the RNeasy Mini Kit (QIAGEN) and reverse
transcribed to cDNA before real-time PCR quantification on the ABI
7500 system with the use of TaqMan FAM-probes from ABI. Probe
numbers will be supplied on request.
Flow cytometry
EMSA
Explanted thymic lymphomas were purified for subsequent analysis by cell
sorting using the FACSVantageSE (Becton Dickinson). For phenotypic
analysis of thymocytes, single-cell suspensions were stained with the
indicated antibodies as described.9 BrdU staining was performed according
to manufacturer’s specifications after a 4-hour pulse with BrdU (intraperitoneal injection of 100 ␮g/mouse). Where applicable, dead cells were
excluded from analysis via the use of propidium iodide.
Metabolic labeling
Thymocyte single-cell suspensions were labeled with [35S] methionine at
1 mCi/mL for 30 minutes at 37°C, after which counts incorporated were
determined by TCA precipitation on detergent extracts as described.9 The
spectrum of proteins synthesized was assessed by SDS-PAGE and visualized after fluorography.
Cell culture and analysis of thymic lymphomas
Explanted thymic lymphomas were adapted to growth in vitro by serial
passage in IMDM (Mediatech) with standard supplements and 20% FBS
(Hyclone). To determine whether p53 responsiveness was disabled in the
thymic lymphomas, genomic DNA from fresh explanted lymphoma cells
was analyzed by Southern blotting, as described.12 In addition,tissue culture
adapted lymphomas were treated with 0.5 ␮g/mL doxorubicin for 4 hours,
after which expression of the p53 target, PUMA, was measured by real-time
PCR with the use of primers and probe from Applied Biosystems, as
described.13 All analyses were performed in triplicate and normalized to
␤-actin. Primary MEFs isolated from embryonic day 13.5 Rpl22⫹/⫹,
Rpl22⫹/⫺, and Rpl22⫺/⫺ embryos were maintained in IMDM supplemented
with 10% FBS and cultured no more than 8 passages. Immortalized MEF
lines were generated by use of the 3T3 method. Human T-ALL lines were
maintained in RPMI containing standard supplements and 10% FBS.
NF-␬B inhibitor IMD-0350 and nuclear export inhibitor LMB were
obtained from Sigma-Aldrich.
Viral transduction and cellular transformation assays
The following retroviral constructs were used: (1) pBabe-12S-E1A65
(Addgene); (2) pBabe-Puro-H-RASG12V, pBabe-Neo-H-RASG12V, and
pBabe-Neo were described previously14; (3) pLKO.1-puro lentitroviral
shRNA constructs targeting murine Rpl24, murine Rpl11, murine and
human Lin28B15; and green fluorescent protein were purchased from
We performed an EMSA analysis of NF-␬B activity as described previously.17 To summarize, after hypotonic lysis, high-salt nuclear extracts were
prepared and assessed for NF-␬B activity by mixing with end-labeled
oligonucleotides comprising an NF-␬B consensus sequence (5⬘-AGTTGA
GGGGACTTTCCC AGGC-3⬘; Santa Cruz Biotechnology Inc) or a mutant
oligonucleotide that abrogates p65 binding (5⬘-AGTTGA GGCGACTTTCCC AGGC-3⬘). Complexes were then subjected to 5% nondenaturing PAGE, vacuum dried, and subjected to autoradiography.
Results
RPL22 is inactivated in a subset of patients with T-ALL
Perturbations in ribosome biogenesis and mutations in RP genes
have been reported in animal models and in humans predisposed to
malignant transformation.5,6 Because Rpl22 is essential for the
development of the T-lineage progenitors from which T-ALL
derives, we sought to determine whether RPL22 inactivation
affected the development of T-ALL.9 To explore this possibility,
aCGH analysis was performed on primary human T-ALL samples
to determine whether the RPL22 gene (1p36.3-p36.2) exhibited
copy number alternations. As shown in Figure 1A, 4 of the
47 (⬃ 9%) samples exhibited deletion of 1 RPL22 allele. Among
the samples in which the RPL22 locus was deleted, 3 occurred in
patients who succumbed to disease, either through induction failure
or relapse. Two of 9 (22%) induction failure samples exhibited
focal deletions encompassing the RPL22 locus whereas 2 of
38 patients in whom induction chemotherapy was successful
harbored large deletions on the p arm of chromosome 1 (n ⫽ 2 of
38, or 5%). Because the deletions that monoallelically inactivated
RPL22 also eliminated other genes, we performed sequence
analysis of RPL22 in the T-ALL samples to determine whether
specific mutations were present. Although no specific point mutations in RPL22 were found in primary T-ALL samples collected at
the time of diagnosis, 6 of 19 (⬃ 30%) T-ALL cell lines (CEM,
Dnd41, Koptk1, Molt13, Molt16, and Supt7), and 1 of 20 primary
patient samples collected at relapse exhibited single adenine
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
the development of T-cell lymphoma in a mouse model suggest that
the loss of RPL22 in human T-ALL may also contribute to the
aggressiveness of disease in those cases. Furthermore, the remaining allele of Rpl22 was found to be intact in the MyrAkt2;Rpl22⫹/⫺
thymic lymphomas, as evidenced by both sequence analysis of 14
tumors (data not shown) and immunoblotting (supplemental Figure
1A, available at the Blood Web site; see the Supplemental Materials
link at the top of the online article). Taken together, these data
demonstrate that loss of a single Rpl22 allele is sufficient to
accelerate the development of thymic lymphoma and suggest that
Rpl22 is functioning as a haploinsufficient tumor suppressor.
Acceleration of lymphoma development in Rpl22ⴙ/ⴚ mice does
not depend on inactivation of p53
Figure 1. Deletions encompassing the RPL22 locus are observed in approximately 10% of primary T-ALL samples. (A) aCGH copy number analysis of primary
T-ALL samples. Genomic DNA from primary T-ALL samples was subjected to copy
number analysis as described.11 Vertical dark blue bars denote the position of the
deletion. Adjacent genes and their orientation relative to the RPL22 locus are
indicated on the right. (B) Sequence analysis of the RPL22 alleles of T-ALL relapse
patient samples and cell lines. Representative DNA sequencing chromatograms from
T-ALL cell lines with wild-type (left) or mutant (right) RPL22 alleles are depicted. The
loss of a single A nucleotide in the stretch of 8 consecutive A on the mutant allele
causes a shift in the translational reading frame that truncates Rpl22 after 18 amino
acids.
nucleotide deletions in the 5⬘ end of the coding region of
1 RPL22 allele, causing a frame-shift predicted to truncate Rpl22
protein at amino acid 18 (Figure 1B). These data indicate that
monoallelic, focal RPL22 genetic alterations are observed in some
patients with aggressive disease and frequently in cell lines derived
from relapsed patients. Moreover, several microarray studies have
revealed that Rpl22 mRNA levels were reduced in adult T-cell
lymphoma/leukemia,18 invasive breast carcinoma,19 and lung adenocarcinoma.20 Taken together, the mutation and down-regulation of
Rpl22 expression in various cancers is consistent with a potential
tumor suppressor role for Rpl22.
Rpl22 haploinsufficiency accelerates the development of
T lymphoma
To test whether the targeted inactivation of Rpl22 alters development of T-cell malignancy, we used a mouse model of T-cell
lymphoma driven by enforced expression of constitutively active
MyrAkt2 in T-cell progenitors.21 The MyrAkt2 Tg model was
selected because the most frequently observed mutations in T-ALL
(ie, Notch activation or PTEN loss) activate AKT.11,22 Non-Tg mice
did not develop disease during the time of the study; however,
MyrAkt2;Rpl22⫹/⫹ mice developed thymic lymphoma with a
median latency of 19 weeks (Figure 2A). Importantly,
MyrAkt2;Rpl22⫹/⫺ mice developed thymic lymphoma much more
rapidly, with a median latency of 11 weeks (Figure 2A). This
development was not accompanied by alterations in either thymic
cellularity or in thymic subsets defined by CD4/8 expression
(Figure 2B), consistent with our previous report that Rpl22⫹/⫺ mice
had no overt phenotype.9 The accelerated development of disease
in MyrAkt2;Rpl22⫹/⫺ mice was accompanied by greater proliferation among CD4⫹CD8⫹ double positive (DP) thymocytes, as
evidenced by their increased incorporation of BrdU (Figure 2C)
and increased Ki-67 staining of MyrAkt2;Rpl22⫹/⫺ lymphomas
(Figure 2D). Neither the increased proliferation nor the enhanced
transformation potential of MyrAkt2;Rpl22⫹/⫺ thymocytes was
accompanied by changes in global protein synthesis (Figure 2E).
These data demonstrating that Rpl22 haploinsufficiency accelerates
It is well documented that impaired ribosomal biosynthesis activates p53 by inducing a nucleolar stress response.23-26 Whereas we
also have observed that Rpl22 deficiency results in translational
derepression of p53 in developing ␣␤ lineage T cells, this was not
observed in Rpl22 haploinsufficiency. Nevertheless, we wished to
determine whether p53 responsiveness might be disabled during
development of T-cell lymphoma in Rpl22-haploinsufficient mice.
Southern blotting revealed that neither the Cdkn2a locus encoding
p19Arf, which can induce p53 in response to oncogene activation,27 nor the Trp53 locus itself exhibited genomic alterations in
thymic lymphomas from MyrAkt2;Rpl22⫹/⫹ or Rpl22⫹/⫺ mice
(supplemental Figure 1B). Moreover, induction of DNA damage
by doxorubicin treatment of thymic lymphomas from
MyrAkt2;Rpl22⫹/⫹ and ⫹/⫺ mice increased expression of the direct
p53 target, PUMA, indicating that the p53 pathway was intact
(supplemental Figure 1C).13,28 These data demonstrate that inactivation of the p53 pathway does not play a significant role in the
acceleration of thymic lymphoma in MyrAkt2;Rpl22⫹/⫺ mice.
Rpl22 serves as a haploinsufficient tumor suppressor in acute
transformation assays
To determine whether Rpl22 haploinsufficiency might also accelerate transformation in other cell types or disease models, we isolated
primary mouse embryonic fibroblasts (MEFs) from Rpl22⫹/⫹, ⫹/⫺,
and ⫺/⫺ mice. Interestingly, both Rpl22⫹/⫺ and ⫺/⫺ primary MEFs
exhibited a faster growth rate than MEFs from Rpl22⫹/⫹ littermates
(Figure 3A). Rpl22⫹/⫺ and ⫺/⫺ MEFs also displayed increased
transformation potential in a soft-agar colony formation assay after
transduction with oncogenic Ras (Ha-RasV12) and E1A (Figure
3B). Similar results were obtained with immortalized Rpl22⫹/⫺ and
⫺/⫺ MEFs transformed with oncogenic Ras alone (supplemental
Figure 2). Importantly, these findings were recapitulated in MEFs
in which Rpl22 expression was diminished by shRNA knockdown
because knockdown of Rpl22 both increased cell growth (Figures
3C-D) and soft agar colony formation (Figure 3E). These data
indicate that in addition to accelerating the development of thymic
lymphoma in a mouse model, Rpl22 inactivation also enhances the
transformation potential of both primary and immortalized MEFs
in response to a distinct oncogenic insult, altogether providing
strong support for Rpl22’s tumor suppressor function.
Rpl22 inactivation enhances transformation potential through
dysregulation of Lin28B
To explore the molecular mechanism by which Rpl22 inactivation
enhances growth and transformation, we performed microarray
analysis on immortalized MEF lines lacking Rpl22 as a result of
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
ROLE OF Rpl22 IN CANCER DEVELOPMENT
3767
Figure 2. Rpl22 haploinsufficiency accelerates the development of cancer in a mouse model of T-cell malignancy. (A) Kaplan-Meier curves of mice of the indicated
genotypes, which were killed on manifestation of outward signs of disease. Myr-Akt2;Rpl22⫹/⫹, n ⫽ 10; Myr-Akt2;Rpl22⫹/⫺ n ⫽ 14; (B) Distribution of CD4/8 subpopulations in
thymi of mice with the indicated genotypes. Single-cell suspensions of thymocytes from young adult mice (4-6 weeks) were stained with antibodies reactive CD4 and CD8.
Absolute numbers of thymocytes were determined and the mean ⫾ SD are depicted graphically to the right. Analysis was performed on a minimum of 3 mice per group and is
representative of 3 experiments performed. (C) Proliferation of explanted thymocytes from MyrAkt2 Tg mice measured by BrdU incorporation. Proliferation of the indicated
populations was assessed flow cytometrically by determining the extent of BrdU incorporation after a 4-hour pulse. The mean ⫾ SD of the fraction of BrdU⫹ cells for a
representative experiment is depicted graphically. Each bar represents an individual experiment involving at least 3 mice. Three experiments were performed.
*P ⬍ .05. (D) Assessment of the extent of proliferation of Rpl22⫹/⫹ and ⫹/⫺ thymic lymphoma cells by Ki-67 staining in situ. Thymic sections from the indicated mice were either
stained with hematoxylin and eosin (H&E) or with anti-Ki67 antibodies to detect the number of proliferating cells. The micrograph was generated using the ⫻20 objective
(⫻200 total magnification) of a Nikon Eclipse 50i microscope and a Digital Sight DS-Fi1 camera. Mean ⫾ SEM of the thymic organ weight relative to body weight from Rpl22⫹/⫹
(n ⫽ 6) and Rpl22⫹/⫺ (n ⫽ 9) mice at the time of sacrifice is depicted graphically below. *P ⬍ .05. Representative thymi are shown on the left. (E) Evaluation of the rate of
protein synthesis in thymocytes measured by metabolic labeling. Thymocyte suspensions from mice of the indicated genotypes were metabolic labeling for 30 minutes with
[35S]methionine after which the counts incorporated were quantified by TCA precipitation of aliquots of the detergent lysates. Data were derived from triplicate values from
2 independent experiments. In addition, extracts were resolved directly by SDS-PAGE and visualized by fluorography (right).
either gene targeting (Rpl22⫺/⫺) or shRNA knockdown (supplemental Table 1). Among the genes most differentially expressed in
Rpl22-deficient cells was Lin28B, whose expression was increased
nearly 20-fold. There are 2 genes in the Lin28 family, Lin28A and
Lin28B,29 both of which have been implicated in human malignancy.15,30 Real-time PCR analysis verified that expression of
Lin28B, but not Lin28A, was increased in cells with reduced Rpl22
expression (Figure 4A). Rpl22 knockdown increased Lin28B
mRNA levels from 20- to 30-fold relative to cells transduced with
control shRNA, whereas Lin28A expression was unchanged (Figure 4A). Likewise, Rpl22 knockdown in MEF increased Lin28B
protein levels (Figure 4B).
Because Lin28 negatively regulates the processing of Let-7
miRNAs,31 we next determined whether expression of Let-7 family
miRNA was altered. In accordance with the increased expression of
Lin28B, MEF lines in which Rpl22 was knocked down also
exhibited reduced expression of several Let-7 miRNA family
members (Figure 4C). The reduction in Let-7 miRNA expression,
in turn, was accompanied by increased expression of oncogene
targets Myc and Ras, which they negatively regulate (Figure 4D).
The increased expression of Lin28B was not restricted to immortalized MEFs in which Rpl22 expression had been knocked down
because Lin28B levels also were elevated in Rpl22⫹/⫺ and ⫺/⫺
primary MEFs, as well as in thymocytes from MyrAkt2;Rpl22⫹/⫺
and ⫺/⫺ mice (Figure 4E-G). To determine whether the link
between Rpl22 inactivation and Lin28B induction was unique to
Rpl22 or was perhaps observed on loss of other RP, we assessed the
level of Lin28B expression in MEF in which Rpl24 and Rpl11 had
been knocked down using shRNA. Nevertheless, despite effective
knockdown of Rpl24 and Rpl11, Lin28B mRNA levels were
unchanged (Figure 4H-I). These data indicate that the Lin28B
induction that accompanies inactivation of Rpl22 is not a general
cellular response to loss of RP and implicates Lin28B induction as
a potential mechanism by which Rpl22 haploinsufficiency and
deficiency increases transformation potential.
Enhanced growth and transformation potential accompanying
Rpl22 inactivation is dependent on Lin28B
Lin28B has been shown to increase cell proliferation and promote
tumor growth.15,30 Because Lin28B induction is correlated with the
enhanced proliferation and transformation potential of Rpl22
mutant cells, we used shRNA knockdown to determine whether
Lin28B was responsible for these behaviors. ShRNA knockdown
of Lin28B in immortalized Rpl22⫺/⫺ MEFs decreased the growth
rate of immortalized Rpl22⫺/⫺ MEFs to that observed in Rpl22⫹/⫹
MEFs (Figure 5A) and abrogated the spontaneous formation of soft
agar colonies by immortalized Rpl22⫺/⫺ MEFs (supplemental
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NF-␬B,32 and NF-␬B can be activated by Notch signaling,33 which
is frequently dysregulated in T-ALL.34 Together, these data provide
evidence that Rpl22 haploinsufficiency leads to Lin28B induction
in both MEF and T-cell malignancies. Moreover, the ability of
knockdown of Lin28B to reverse both the enhanced growth and
transformation potential of Rpl22-deficient cells strongly suggests
that these behaviors are dependent on increased Lin28B expression
in Rpl22⫹/⫺ and ⫺/⫺ cells.
Induction of Lin28B expression in Rpl22 mutant cells is
dependent on NF-␬B signaling
Figure 3. Rpl22 haploinsufficiency and deficiency promote growth and transformation in cell models in vitro. (A) Effect of Rpl22 inactivation on growth of primary
MEFs. Primary Rpl22⫹/⫹, ⫹/⫺, and ⫺/⫺ MEFs were seeded in triplicate, cultured in
3% O2 at 37°C, and counted at the indicated intervals for 8 days. Mean cell
number ⫾ SD at each time point is represented graphically. Results are representative of 3 independent experiments. (B) Effect of Rpl22 inactivation on transformation
of primary MEFs. Primary Rpl22⫹/⫹, ⫹/⫺, and ⫺/⫺ MEFs were transduced with
oncogenes E1A and H-RasV12, followed by drug selection for 1 week, and plating in
0.7% agar. After 3 weeks, colonies were stained with crystal violet and enumerated.
Images of representative wells were captured using an EPSON Perfection V700
Photo scanner and are depicted in the top panels. Mean colony number per
well ⫾ SD for each genotype is represented graphically in the bottom panel.
**P ⬍ .005. Data are representative of 3 independent experiments performed in
triplicate. (C) Knockdown of Rpl22 expression in immortalized MEFs. Immortalized
Rpl22⫹/⫹ MEFs were transduced with control or Rpl22 shRNA constructs, after which
the effect on Rpl22 mRNA and protein expression was evaluated by real-time PCR
(top) and immunoblotting (bottom). (D-E) Evaluation of immortalized MEF growth and
transformation after Rpl22 knockdown. MEFs stably expressing control or 2 Rpl22
shRNA constructs were transformed by oncogenic H-RasV12, after which their
growth rate was assessed by counting (panel D; *P ⬎ .05 vs control shRNA) and their
transformation by colony formation in soft agar (E) as in panel B.
Figure 3). Importantly, the increased Lin28B expression and
enhanced growth exhibited by Rpl22-deficient immortalized MEF
was reversed on ectopic expression of Rpl22, as was the increased
expression of the Let-7 target, c-myc (Figure 5B).
As was true for immortalized MEF, knockdown of Lin28B in
primary MEFs reversed the enhanced colony formation observed
on transduction of Rp22⫹/⫺ and ⫺/⫺ primary MEFs with E1A/Ras
(Figure 5C). Rpl22 haploinsufficiency also was linked to Lin28B
and c-myc induction in the thymic lymphomas that developed more
rapidly in MyrAkt2Tg:Rpl22⫹/⫺ mice (Figure 5D). Finally, we
determined that all of the 6 T-ALL cell lines bearing RPL22
mutations exhibited greater Lin28B mRNA levels than Jurkat
(RPL22⫹/⫹), whereas this was true for only half of RPL22⫹/⫹
T-ALL cell lines (Figure 5E). The induction of Lin28B in some of
the RPL22⫹/⫹ lines may have resulted from elevated NF-␬B
activity because Lin28B is a direct transcriptional target of
Because the enhanced predisposition of Rpl22-haploinsufficient
and deficient cells to transformation is dependent on induction of
Lin28B, we wished to determine how Rpl22 inactivation increases
Lin28B expression. Lin28B is a direct target of NF-␬B signaling.32
Accordingly, we sought to determine whether Rpl22 inactivation
induces Lin28B by up-regulation of NF-␬B signaling. To address
this possibility, we performed EMSA analysis by using equal
quantities of nuclear extract from primary Rpl22⫹/⫹ and ⫹/⫺ MEF.
We found that NF-␬B complexes comprising p65/p50 heterodimers
(defined by antibody supershift) were increased in Rpl22⫹/⫺
primary MEFs (Figure 6A). NF-␬B activity was increased further
upon treatment with leptomycin B, which traps NF-␬B in the
nucleus by blocking nuclear exit, and still further upon treatment
with TNF␣ (Figure 6A). Moreover, binding was abrogated by
mutation of the consensus p65 binding motif (Figure 6A). Increased NF␬B activity and Lin28B expression also were observed
upon knocking down Rpl22 in a thymic lymphoma line (supplemental Figure 4).
To determine whether the increased expression of Lin28B was
in fact dependent on NF-␬B activity, we used a pharmacologic
inhibitor of I␬B kinase ␤, IMD-0354, which inhibits NF-␬B
activation by blocking the phosphorylation of I␬B␣ that is normally responsible for translocation of NF-␬B to the nucleus. I␬B␣
phosphorylation is increased on knockdown of Rpl22 in immortalized MEF (supplemental Figure 5). Moreover, treatment of
Rpl22 knockdown MEFs with IMD-0354 partially reduced Lin28B
expression levels (Figure 6B). Activation of I␬B kinase ␤ as part of
canonical NF-␬B signaling leads to the translocation of
p65/p50 complexes to the nucleus and transactivation of target
genes.35
To determine whether canonical NF-␬B complexes comprising
p65 were required for the induction of Lin28B on Rpl22 knockdown, we knocked Rpl22 down in matched p65-sufficient and
p65-deficient (Rela⫺/⫺) littermate MEF.17 p65-deficiency abrogated the ability of Rpl22 knockdown to induce Lin28B expression
(Figure 6C). p65 deficiency blocked Lin28B induction more
effectively than the pharmacologic inhibitor, presumably because
IMD-0354 failed to completely block NF-␬B activation. Together,
these data indicate that the induction of Lin28B expression that
occurs on loss or inactivation of Rpl22 is dependent on NF-␬B
signaling.
Discussion
Although numerous reports in both model organisms and humans
indicate that monoallelic inactivation of RP genes can contribute to
the development of cancer, the mechanistic basis by which RP
mutations do so has remained unexplained.4,6 We provide here the
first insights into how haploinsufficiency of an RP, Rpl22, contributes to transformation. The RPL22 locus is monoallelically deleted
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
ROLE OF Rpl22 IN CANCER DEVELOPMENT
3769
Figure 4. Increased transformation potential associated with of Rpl22 loss or inactivation is accompanied by induction of Lin28B. Effect of Rpl22 knockdown on
Lin28 expression in immortalized MEF. MEF lines stably expressing control or 2 different Rpl22 shRNA constructs were harvested for RNA and protein, after which Lin28A and
Lin28B mRNA levels were evaluated by real-time PCR (A). **P ⬍ .005 for Lin28B expression in Rpl22 shRNA relative to controls. Protein levels were measured by blotting
(B). Data are representative of 2 independent experiments. (C) Let-7 miRNA levels in immortalized MEFs where Rpl22 expression was suppressed by shRNA. Expression of
Let-7 family miRNA was evaluated by real-time PCR in immortalized MEFs stably expressing control or Rpl22 shRNA constructs. Expression levels were normalized to sno202
RNA and to the expression level in cells transduced with control shRNA. Mean expression levels of triplicate measurements ⫾ SD are represented graphically. Data are
representative of 3 independent experiments. P ⬍ .05 for Let-7 miRNA levels in Rpl22 shRNA compared with control shRNA. (D) Effect of Rpl22 knockdown on expression of
Let-7 targets. Expression of Ras and Myc was assessed by immunoblotting in control or Rpl22 knockdown MEFs. GAPDH served as a loading control. Expression of Lin28B in
primary MEFs. Lin28B protein and mRNA levels were measured in primary MEFs of the indicated genotypes by immunoblotting (E) and real-time PCR (F), respectively.
Mean ⫾ SD of Lin28B mRNA expression levels are depicted graphically. Results are representative of at least 3 experiments performed. *P ⬍ .05. (G) Lin28B expression in
primary thymocytes. The expression of Lin28B in thymocytes from mice with the indicated genotypes was evaluated by immunoblotting. GAPDH served as a loading control.
Lin28B expression in MEFs after knockdown of Rpl11 and Rpl24. Immortalized MEFs were transduced with 2 different shRNA constructs targeting Rpl24 or Rpl11, after which
protein levels were evaluated by immunoblotting (H). GAPDH served as loading control. (I) The level of Lin28B mRNA expressed by these cells was measured by real time PCR
as in panel F. **P ⬍ .005.
in approximately 10% of primary T-ALL in humans. Moreover, the
monoallelic inactivation of Rpl22 enhanced development of disease in a mouse model of T-cell malignancy as well as in acute in
vitro models of transformation using MEFs. The predisposition to
transformation or tumor progression afforded by Rpl22 haploinsufficiency is associated with and dependent on induction of the
stemness factor Lin28B, which recently has been found to be
increased in late-stage, aggressive cancers in humans.15 Finally, the
induction of Lin28B that occurs accompanies repression or inactivation of Rpl22 is dependent on elevated canonical NF-␬B
signaling (Figure 6D). Together, these observations provide insight
into the mechanistic basis for how Rpl22, which plays a critical role
in normal T lymphocyte development, also contributes to T-cell
oncogenesis when haploinsufficiency occurs.9
Although the basis by which RP haploinsufficiency promotes
transformation is poorly understood, MacInnes et al recently
demonstrated that monoallelic inactivation of numerous RP genes
in zebrafish increased the development of malignant peripheral
nerve sheath tumors and that this was associated with failure to
activate p53 in response to DNA damage.36 Effects of RP gene
mutations on p53 activation have been known for some time
because inactivation of RPs has been shown to activate p53 by
inhibiting p53 degradation mediated by the E3 ubiquitin ligase,
MDM2.23,26,37 Interestingly, MacInnes et al found that the failure of
the resulting malignant peripheral nerve sheath tumors to induce
p53 did not result from mutations in p53 or effects on stability;
rather, it resulted from an inability to translate p53 mRNA.36
Nevertheless, it remained unclear whether the inability to translate
intact p53 mRNA was responsible for the increased susceptibility
to transformation or was instead an indirect effect of selection by
the tumor cells after formation. We have previously found that
Rpl22 deficiency, but not haploinsufficiency, blocks development
of ␣␤ T lineage progenitors because of selective translational
derepression of p53 in those progenitors9; however, mutations
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RAO et al
BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
Figure 5. Lin28B is necessary for the enhancement of
growth and transformation by Rpl22 inactivation.
(A) Effect of knockdown of Lin28B on the growth of
immortalized Rpl22⫺/⫺ MEFs. Immortalized MEFs of the
indicated genotypes were transduced with control or
Lin28B shRNA. The effect on expression of Lin28B was
assessed by immunoblotting. Growth of triplicate wells of
MEFs stably expressing control shRNA or Lin28B shRNA
was determined by counting and then plotted as the
mean cell number ⫾ SD *P ⬍ .05, for Rpl22⫺/⫺ compared with Rpl22⫺/⫺ in which Lin28B was knocked down.
(B) Effect on growth of reintroducing Rpl22 into Rpl22⫺/⫺
MEF. Immortalized Rpl22⫺/⫺ MEFs were transfected with
Rpl22 or empty vector (EV) control, after which we
assessed cell growth by counting triplicate wells and
depicting the mean ⫾ SD graphically. The expression of
Lin28B, c-myc, Rpl22, and GAPDH (loading control)
were evaluated by immunoblotting. (C) Dependence of
soft agar colony formation on expression of Lin28B.
Primary MEFs of the indicated genotypes were transduced with Lin28B shRNA followed by E1A and Ras, and
then plated in triplicate in soft agar as Figure 3B. Representative images were captured with a Digital Slight DS-Fi1
camera and NIS Element AR3.0 imaging software at 1⫻
with 3⫻ zoom (⫻30 total magnification) using a Nikon
SMZ1500 stereomicroscope and are shown in the top
panels. The mean colony number ⫾ SD is represented
graphically beneath. **P ⬍ .005 for colonies in Rpl22⫹/⫺
and ⫺/⫺ relative to Rpl22⫹/⫹. (D) Lin28B expression in
Rpl22-haploinsufficient thymic lymphomas. Explanted
thymic lymphomas from MyrAkt2;Rpl22⫹/⫹ and
MyrAkt2;Rpl22⫺/⫺ mice were evaluated for Lin28B and
c-myc expression by immunoblotting. GAPDH served as
a loading control. (E) Lin28B mRNA levels in RPL22⫹/⫹
and Rpl22⫹/⫺ human T-ALL lines. Lin28B mRNA levels in
the indicated T-ALL cell lines were quantified by real-time
PCR. Data are presented as Log2 value relative to Jurkat
cells (control).
disabling p53 do not appear to play a role in the susceptibility to
transformation associated with Rpl22 haploinsufficiency (supplemental Figure 1).
The inactivation of Rpl22, as well as and some other RP (eg,
RPS14), predisposes cell transformation; however, it is important
to note that inactivation of still other RP genes actually antagonizes
cancer development and progression. For example, haploinsufficiency of Rpl24 was found to delay development of B and
T lymphoma in mouse models.38,39 The delay in development of
B and T lymphoma was associated with impairment of the core
ribosome function of CAP-dependent protein synthesis,38,39 presumably in a similar manner to the way inactivation of other RP that are
required for ribosome biogenesis or core function impairs erythrocyte development in DBA.3,5
Interestingly, Rpl38 haploinsufficiency also delayed the development of B lymphoma in a mouse model but apparently did so
without grossly altering ribosome biogenesis or global protein
synthesis.38 Rpl38 was recently shown to exert selective control
over the translation of a cluster of Hox genes, suggesting that Rpl38
can act either in the context of a specialized subset of ribosomes
with functions influenced by their complement of RP, or alternatively, in an extraribosomal fashion.40 It would appear that Rpl22
may also represent a RP that plays a more specialized, regulatory
role because it is not essential for core ribosome functions (Figure
2E) yet clearly influences the predisposition of cells to transforma-
tion by regulating the expression of Lin28B (Figures 2-5). Accordingly, although Rpl22 haploinsufficiency predisposes to transformation through the induction of Lin28B expression because Rpl22 is
dispensable for core ribosome function, it is possible, if not likely,
that it influences transformation by a mechanism that is distinct
from other RP whose inactivation affects global, CAP-dependent
translation (Figure 4H), and activates p53 through nucleolar
stress.3,5
The Lin28 RNA binding proteins are highly enriched in stem
cells and are capable, along with Oct4, Sox2, and Nanog, of
reprogramming somatic cells into inducible pluripotent stem
cells.41 In recent reports investigators also have revealed that both
Lin28A and Lin28B are overexpressed in approximately 15% of
human primary tumors, with elevations in Lin28B expression
being associated with advanced disease across multiple tumor
types.15,30 We found that the enhanced transformation potential of
Rpl22-haploinsufficient cells was not only associated with but was
also dependent on increased expression of Lin28B (Figures 4 and
5). Importantly, the elevation of Lin28B expression was already
evident in premalignant primary thymocytes as well as in primary
MEFs (Figure 4), indicating that Lin28B induction predisposed
these cells to transformation rather than being a consequence of
transformation.
Although the mechanism by which Lin28B promotes transformation is incompletely understood, a consensus is emerging that its
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
Figure 6. The increased Lin28B expression that results from Rpl22 loss or
inactivation is dependent on NF-␬B activity. (A) Measurement of NF-␬B activity in
Rpl22-haploinsufficient primary MEF. EMSA analysis was performed using equal
quantities of nuclear extract protein from primary MEFs of the indicated genotypes
using both an intact (wt) and p65 binding mutant (mut) NF-␬B probe. NF-␬B activity
was measured in untreated cells, cells pretreated with leptomycin B to trap NF-␬B in
the nucleus, and after TNF␣ stimulation (positive control). The composition of the
NF-␬B complexes was evaluated using supershift analysis using the indicated Abs.
Effect of NF-␬B inhibition on Lin28B expression. (B) Lin28B mRNA levels were
quantified by real-time PCR on RNA extracted from immortalized MEFs stably
expressing 2 different Rpl22 shRNAs, in which NF-␬B activity had been pharmacologially inhibited by treatment with 1␮M NF-␬B inhibitor, IMD-350. **P ⬍ .01 for
IMD-350 treated compared with control treated. (C) Rpl22 was knocked down by
shRNA in primary MEF from p65 wild-type (p65⫹) or Rela⫺/⫺, p65 knockout mice
(p65⫺). Lin28B induction was blocked in p65 knockout cells in which Rpl22 was
knocked down. Lin28B and Rpl22 mRNA levels were quantified by real-time PCR,
and data are plotted as the average of 2 experiments. (D) Model of Rpl22 function in
transformation. The model proposes that Rpl22 normally acts to restrain NF-␬B
activity by an unknown mechanism. However, when Rpl22 expression is diminished
either by shRNA knockdown or mutation, NF-␬B activity is increased, resulting in
increased expression of Lin28B. Lin28B, in turn, promotes transformation at least in
part by repressing Let-7 MiR processing, which results in derepression of oncogenic
targets such as c-myc. Rpl22 is also likely to regulate additional targets that
contribute to transformation.
function in antagonizing the processing of Let-7 miRNAs is likely
to play an important role.31,32 Consistent with this view, we found
that the induction of Lin28B that occurs on repression of Rpl22
also results in reduced expression of multiple Let-7 miRNA family
members. Let-7 family miRNAs, in turn, negatively regulate
expression of the critical oncogenes, Myc and Ras, as we observed
(Figures 4D, 5B,D).42 In addition, a recent report has indicated that
enforced expression of Lin28B in adult HSCs reprograms the adult
HSC to acquire fetal-like characteristics, which may also contribute
to the susceptibility to transformation.43
Accordingly, we hypothesize that induction of Lin28B in
Rpl22⫹/⫺ T-lineage progenitors predisposes them to transformation
at least in part through effects on Let-7 miRNA expression. In
support, enforced expression of Lin28b in HSC is also sufficient to
promote the development of T lymphoma.44 Although it is clear
ROLE OF Rpl22 IN CANCER DEVELOPMENT
3771
that Lin28B promotes transformation and this is likely to depend on
inhibition of Let-7 processing, it remains possible that Lin28B
might also have important Let-7–independent functions because
Let-7–independent roles of Lin28 in both normal development and
transformation have recently been described.30,45 Further studies
are required to distinguish these possibilities.
The induction of Lin28B that we observed on knockdown of
Rpl22 expression was not observed on knockdown of other RP
(Rpl11 and Rpl24; Figure 4H-I). That the induction of Lin28B is
selectively associated with Rpl22 loss raises 2 interesting questions. First, how does repression or inactivation of Rpl22 increase
Lin28B expression? Because the induction of Lin28B expression
was associated with changes in mRNA levels, we reasoned that
Rpl22 inactivation might increase expression by activating NF-␬B,
because NF-␬B has been shown to directly bind to and transactivate the Lin28b locus.32 We found that NF-␬B activity was
increased by Rpl22 inactivation and that the increase in Lin28B
expression was dependent on the activity of canonical p65containing NF␬B complexes (Figure 6A-C). The mechanistic basis
by which Rpl22 loss increases canonical NF-␬B signaling is
unclear but could be direct or indirect. An indirect mechanism
might include increasing the expression of a cytokine, such as TNF
or IL-6, which could trigger NF-␬B activation in either an
autocrine or paracrine fashion.32 Alternatively, Rpl22 might play a
more direct, proximal role in regulating NF-␬B activity. Indeed, a
recent report demonstrating that Rps3 associates with p65 and is an
integral part of the NF-␬B complex has provided precedent for
such a mechanism.46
Rps3 was found to directly bind p65 through its KH domain,
and this interaction appears to be required for transactivation of
target genes.46 Rpl22 lacks a KH domain and its loss does not
inhibit NF-␬B activity, suggesting that Rpl22 is likely to be
functioning by a distinct mechanism. Efforts are currently in
progress to determine at which level Rpl22 regulates NF-␬B
activity. The second question of interest is whether Rpl22 is
exerting its effect on NF-␬B activity from within or outside of the
ribosome. There are several examples of extraribosomal functions
for RP, but one the best understood is the interferon-induced release
of Rpl13a from ribosomes. On release, Rpl13a associates with
mRNAs containing GAIT elements and regulates their translation.2
Insight into whether Rpl22 functions from within or outside of the
ribosome was recently provided by the crystal structure of the
eukaryotic ribosome,47 which revealed that Rpl22 was represented
in monomeric form and was positioned well away from the
40S/60S interface as well as the mRNA entry and peptide exit
tunnels. This finding suggests that Rpl22 is unlikely to be exerting
its effect on NF-␬B activity from within the ribosome and that it,
like Rpl13a, may be acting in an extraribosomal manner.
Our observation that germline ablation of the Rpl22 locus was
not lethal, as most RP knockouts are,7,8 but instead caused a
selective block in development of ␣␤ T-lineage progenitors,
prompted us to investigate whether Rpl22 mutations might predispose T cells to transformation. To do so, we focused on the human
cancer that arises from those cells, T-ALL. We found that Rpl22haploinsufficiency in a mouse model of T-cell malignancy accelerated the development of thymic lymphoma. Rpl22 haploinsufficiency was also observed in approximately10% of primary T-ALL
isolates and one-third of T-ALL cell lines. All of the T-ALL cell
lines bearing RPL22 mutations exhibited Lin28B mRNA levels in
excess of those observed in Jurkat (RPL22⫹/⫹), compared with
approximately one-half of the lines with intact RPL22 alleles
(Figure 5E). Finally, meta-analysis of gene expression data from
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BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
RAO et al
primary T-ALL revealed that of the 10 isolates with reduced Rpl22
mRNA levels, 70% exhibited elevated Lin28B expression levels.48
If, as our in vitro analysis suggests, increased NF-␬B activity
underlies the increased Lin28B expression observed in T-ALL
either bearing RPL22 mutations or in which Rpl22 expression is
reduced, then the NF-␬B pathway may represent an opportunity for
therapeutic intervention in aggressive, Rpl22-haploinsufficient
T-ALL (Figure 1), as the targeting NF-␬B has shown some efficacy
in T-ALL.49,50
Our identification of the link between Rpl22 haploinsufficiency
and Lin28B induction represents the first insight into how inactivation of an RP can predispose to transformation and contribute to
tumorigenesis. Although in this initial analysis we focused on the
role of RPL22 inactivation in pediatric T-ALL, it is possible that
Rpl22 may play a role in transformation of other tissues as well.
Indeed, in a human breast cancer study, mRNA encoding Rpl22
was found to be substantially down-regulated in invasive breast
carcinoma compared with normal breast.19 Down-regulation of
Rpl22 mRNA also has been observed in other cancer types,
including lung adenocarcinoma, small squamous cell lung carcinoma,20 and adult T-cell leukemia.18 Finally, it will be particularly
important to determine whether RPL22 inactivation is observed in
other diseases and whether the link between RPL22 inactivation
and Lin28B induction is unique to RPL22 or if it might also
observed on inactivation of other RP that appear to be dispensable
for global, CAP-dependent translation and instead appear to
function more selectively in controlling target gene expression.
Acknowledgments
The authors thank Drs Dietmar Kappes and Maureen Murphy for
helpful discussion and critical review of the manuscript. They
gratefully acknowledge the assistance of the following core
facilities of the Fox Chase Cancer Center: Cell Culture, DNA
Sequencing, Flow Cytometry, Genomics, Histopathology, and
Laboratory Animal.
This work was supported by National Institutes of Health grants
R01-AI073920, R01-CA77429, and R21-CA141194; National In-
stitutes of Health core grant P01CA06927; Center grant P30-DK50306; an appropriation from the Commonwealth of Pennsylvania;
and support from the Blood Cell Development and Cancer
Keystone. This work was supported in part by grants to the COG,
including U10 CA98543 (COG Chair’s grant), U10 CA98413
(COG Statistical Center), and U24 CA114766 (COG Specimen
Banking), as well as by grant 5P01CA068484 (to the Dana-Farber
Cancer Institute and A.T.L.). S.Y.L. was supported by both the
Greenwald and Plain & Fancy Fellowships. J.P. is a Merck fellow
of the Life Sciences Research Fellowship and was a fellow of the
National Institutes of HealthT32 CA009035 training grant. A.G. is
a scholar of the American Society of Hematology–Amos Faculty
Development Program and is supported by National Institutes of
Health grant 5K08CA133103.
Authorship
Contribution: S.R. performed all in vitro transformation analysis
and investigation of the role of Lin28 in transformation; S.-Y.L.
performed all analysis of the murine T-cell malignancy model;
A.G. and A.T.L. assembled and analyzed primary T-ALL samples;
J.P. produced primary MEF lines and performed growth analysis;
R.J.T. and S.B. performed EMSA studies on NF-␬B; Z.T. and R.Z.
generated oncogenic Ras and E1A retrovirus; J.R.J. and G.P.Z.
performed Southern blot analysis on the Tp53 and Cdkn2a loci;
M.R. evaluated the p53 pathway in thymic lymphoma lines; S.A.
and T.O. generated the Rpl22 mutant mice; S.P.H. provided relapse
patient samples; R.A.T. and J.R.T. produced and maintained
MyrAkt2 Tg mice; and S.-Y.L., S.R., and D.L.W. conceived of the
study, analyzed data, and wrote the manuscript.
Conflict-of-interest disclosure: S.A. and T.O. are or have been
employees of, and received stock options from, Lexicon Pharmaceuticals Inc. The remaining authors declare no competing financial
interests.
Correspondence: David L. Wiest, Fox Chase Cancer Center,
333 Cottman Ave, Philadelphia, PA 19111; e-mail: david.wiest@
fccc.edu.
References
1. Neumann F, Krawinkel U. Constitutive expression
of human ribosomal protein L7 arrests the cell
cycle in G1 and induces apoptosis in Jurkat
T-lymphoma cells. Exp Cell Res. 1997;230(2):
252-261.
2. Mazumder B, Sampath P, Seshadri V, Maitra RK,
DiCorleto PE, Fox PL. Regulated release of L13a
from the 60S ribosomal subunit as a mechanism
of transcript-specific translational control. Cell.
2003;115(2):187-198.
3. Ball S. Diamond Blackfan anemia. Hematology
Am Soc Hematol Educ Program. 2011;2011:487491.
4. Ebert BL, Pretz J, Bosco J, et al. Identification of
RPS14 as a 5q-syndrome gene by RNA interference screen. Nature. 2008;451(7176):335-339.
5. Narla A, Ebert BL. Ribosomopathies: human disorders of ribosome dysfunction. Blood. 2010;
115(16):3196-3205.
6. Amsterdam A, Hopkins N. Retroviral-mediated
insertional mutagenesis in zebrafish. Methods
Cell Biol. 2004;77:3-20.
7. Matsson H, Davey EJ, Draptchinskaia N, et al.
Targeted disruption of the ribosomal protein S19
gene is lethal prior to implantation. Mol Cell Biol.
2004;24(9):4032-4037.
8. Oliver ER, Saunders TL, Tarle SA, Glaser T. Ribo-
somal protein L24 defect in belly spot and tail
(Bst), a mouse Minute. Development. 2004;
131(16):3907-3920.
9. Anderson SJ, Lauritsen JP, Hartman MG, et al.
Ablation of ribosomal protein L22 selectively impairs alphabeta T cell development by activation
of a p53-dependent checkpoint. Immunity. 2007;
26(6):759-772.
10. Amsterdam A, Sadler KC, Lai K, et al. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol. 2004;2(5):E139.
11. Gutierrez A, Sanda T, Grebliunaite R, et al. High
frequency of PTEN, PI3K and AKT abnormalities
in T cell acute lymphoblastic leukemia. Blood.
2009;114(3):647-650.
12. Eischen CM, Weber JD, Roussel MF, Sherr CJ,
Cleveland JL. Disruption of the ARF-Mdm2-p53
tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev. 1999;13(20):26582669.
13. Stadanlick JE, Zhang Z, Lee SY, et al. Developmental arrest of T cells in Rpl22-deficient mice is
dependent upon multiple p53 effectors. J Immunol. 2011;187(2):664-675.
14. Kennedy AL, Morton JP, Manoharan I, et al. Activation of the PIK3CA/AKT pathway suppresses
senescence induced by an activated RAS onco-
gene to promote tumorigenesis. Mol Cell. 2011;
42(1):36-49.
15. Viswanathan SR, Powers JT, Einhorn W, et al.
Lin28 promotes transformation and is associated
with advanced human malignancies. Nat Genet.
2009;41(7):843-848.
16. Haks MC, Lefebvre JM, Lauritsen JP, et al. Attenuation of gammadeltaTCR signaling efficiently
diverts thymocytes to the alphabeta lineage. Immunity. 2005;22(5):595-606.
17. Thapa RJ, Basagoudanavar SH, Nogusa S, et al.
NF-kappaB protects cells from gamma interferoninduced RIP1-dependent necroptosis. Mol Cell
Biol. 2011;31(14):2934-2946.
18. Choi YL, Tsukasaki K, O’Neill MC, et al. A
genomic analysis of adult T-cell leukemia. Oncogene. 2007;26(8):1245-1255.
19. Finak G, Bertos N, Pepin F, et al. Stromal gene
expression predicts clinical outcome in breast
cancer. Nat Med. 2008;14(5):518-527.
20. Bhattacharjee A, Richards WG, Staunton J, et al.
Classification of human lung carcinomas by
mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci
U S A. 2001;98(24):13790-13795.
21. Timakhov RA, Tan Y, Rao M, et al. Recurrent
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 1 NOVEMBER 2012 䡠 VOLUME 120, NUMBER 18
chromosomal rearrangements implicate oncogenes contributing to T-cell lymphomagenesis in
Lck-MyrAkt2 transgenic mice. Genes Chromosomes Cancer. 2009;48(9):786-794.
22. Palomero T, Sulis ML, Cortina M, et al. Mutational
loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia. Nat Med. 2007;13(10):
1203-1210.
23. Bhat KP, Itahana K, Jin A, Zhang Y. Essential role
of ribosomal protein L11 in mediating growth
inhibition-induced p53 activation. EMBO J. 2004;
23(12):2402-2412.
24. Dai MS, Lu H. Inhibition of MDM2-mediated p53
ubiquitination and degradation by ribosomal protein L5. J Biol Chem. 2004;279(43):44475-44482.
25. Dai MS, Zeng SX, Jin Y, Sun XX, David L, Lu H.
Ribosomal protein L23 activates p53 by inhibiting
MDM2 function in response to ribosomal perturbation but not to translation inhibition. Mol Cell
Biol. 2004;24(17):7654-7668.
26. Zhu Y, Poyurovsky MV, Li Y, et al. Ribosomal protein S7 is both a regulator and a substrate of
MDM2. Mol Cell. 2009;35(3):316-326.
27. Zindy F, Eischen CM, Randle DH, et al. Myc signaling via the ARF tumor suppressor regulates
p53-dependent apoptosis and immortalization.
Genes Dev. 1998;12(15):2424-2433.
28. Jeffers JR, Parganas E, Lee Y, et al. Puma is an
essential mediator of p53-dependent and
-independent apoptotic pathways. Cancer Cell.
2003;4(4):321-328.
ROLE OF Rpl22 IN CANCER DEVELOPMENT
31. Viswanathan SR, Daley GQ, Gregory RI. Selective blockade of microRNA processing by Lin28.
Science. 2008;320(5872):97-100.
32. Iliopoulos D, Hirsch HA, Struhl K. An epigenetic
switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell. 2009;139(4):693-706.
42.
33. Espinosa L, Cathelin S, D’Altri T, et al. The Notch/
Hes1 pathway sustains NF-kappaB activation
through CYLD repression in T cell leukemia. Cancer Cell. 2010;18(3):268-281.
43.
34. Weng AP, Ferrando AA, Lee W, et al. Activating
mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306(5694):
269-271.
44.
35. Oeckinghaus A, Hayden MS, Ghosh S. Crosstalk
in NF-kappaB signaling pathways. Nat Immunol.
2011;12(8):695-708.
45.
36. MacInnes AW, Amsterdam A, Whittaker CA,
Hopkins N, Lees JA. Loss of p53 synthesis in
zebrafish tumors with ribosomal protein gene mutations. Proc Natl Acad Sci U S A. 2008;105(30):
10408-10413.
46.
37. Dai MS, Chao TY, Kao WY, Shyu RY, Liu TM. Delayed hepatitis B virus reactivation after cessation
of preemptive lamivudine in lymphoma patients
treated with rituximab plus CHOP. Ann Hematol.
2004;83(12):769-774.
38. Barna M, Pusic A, Zollo O, et al. Suppression of
Myc oncogenic activity by ribosomal protein haploinsufficiency. Nature. 2008;456(7224):971-975.
29. Guo Y, Chen Y, Ito H, et al. Identification and
characterization of lin-28 homolog B (LIN28B) in
human hepatocellular carcinoma. Gene. 2006;
384:51-61.
39. Hsieh AC, Costa M, Zollo O, et al. Genetic dissection of the oncogenic mROR pathway reveals
druggable addiction to translational control via
4EBP-eIF4E. Cancer Cell. 2010;17(3):249-261.
30. King CE, Wang L, Winograd R, et al. LIN28B fosters colon cancer migration, invasion and transformation through let-7–dependent and
-independent mechanisms. Oncogene. 2011;
30(40):4185-4193.
40. Kondrashov N, Pusic A, Stumpf CR, et al.
Ribosome-mediated specificity in Hox mRNA
translation and vertebrate tissue patterning. Cell.
2011;145(3):383-397.
41. Wang Y, Mah N, Prigione A, Wolfrum K,
47.
48.
49.
50.
3773
Andrade-Navarro MA, Adjaye J. A transcriptional
roadmap to the induction of pluripotency in somatic cells. Stem Cell Rev. 2010;6(2):282-296.
Rybak A, Fuchs H, Smirnova L, et al. A feedback
loop comprising lin-28 and let-7 controls pre-let-7
maturation during neural stem-cell commitment.
Nat Cell Biol. 2008;10(8):987-993.
Yuan J, Nguyen CK, Liu X, Kanellopoulou C,
Muljo SA. Lin28b reprograms adult bone marrow
hematopoietic progenitors to mediate fetal-like
lymphopoiesis. Science. 2012;335(6073):11951200.
Beachy SH, Onozawa M, Chung YJ, et al. Enforced expression of Lin28b leads to impaired
T-cell development, release of inflammatory cytokines and peripheral T-cell lymphoma. Blood.
2012;120(5):1048-1059.
Balzer E, Heine C, Jiang Q, Lee VM, Moss EG.
LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro. Development. 2010;137(6):891900.
Wan F, Anderson DE, Barnitz RA, et al. Ribosomal protein S3: a KH domain subunit in NFkappaB complexes that mediates selective gene
regulation. Cell. 2007;131(5):927-939.
Ben-Shem A, Garreau de Loubresse N, et al. The
structure of the eukaryotic ribosome at 3.0 A resolution. Science. 2011;334(6062):1524-1529.
Fine BM, Stanulla M, Schrappe M, et al. Gene
expression patterns associated with recurrent
chromosomal translocations in acute lymphoblastic leukemia. Blood. 2004;103(3):1043-1049.
Lee CH, Jeon YT, Kim SH, Song YS. NF-kappaB
as a potential molecular target for cancer therapy.
BioFactors. 2007;29(1):19-35.
Hu X, Xu J, Sun A, Shen Y, He G, Guo F. Successful T-cell acute lymphoblastic leukemia treatment with proteasome inhibitor bortezomib based
on evaluation of nuclear factor-kappaB activity.
Leuk Lymphoma. 2011;52(12):2393-2395.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2012 120: 3764-3773
doi:10.1182/blood-2012-03-415349 originally published
online September 13, 2012
Inactivation of ribosomal protein L22 promotes transformation by
induction of the stemness factor, Lin28B
Shuyun Rao, Sang-Yun Lee, Alejandro Gutierrez, Jacqueline Perrigoue, Roshan J. Thapa, Zhigang
Tu, John R. Jeffers, Michele Rhodes, Stephen Anderson, Tamas Oravecz, Stephen P. Hunger,
Roman A. Timakhov, Rugang Zhang, Siddharth Balachandran, Gerard P. Zambetti, Joseph R. Testa,
A. Thomas Look and David L. Wiest
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