Lkb1/Stk11 Regulation of Mtor Signaling Controls the Transition of Chondrocyte Fates and Suppresses Skeletal Tumor Formation

Lkb1/Stk11 regulation of mTOR signaling controls the transition of chondrocyte fates and suppresses skeletal tumor formation

Lick Pui Laia,b, Brendan N. Lilleyc, Joshua R. Sanesc, and Andrew P. McMahona,b,c,d,1

aDepartment of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad–California Institute for Regenerative Medicine Center for Regenerative Medicine and Stem Cell Research, University of Southern California Keck School of Medicine, Los Angeles, CA 90089; and Departments of bStem Cell and Regenerative Biology, cMolecular and Cellular Biology, and dHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved October 22, 2013 (received for review May 25, 2013) Liver kinase b1 (Lkb1) protein kinase activity regulates cell growth a Col2a1-Cre transgenic strain (11); here, skeletal Cre-activity is and cell polarity. Here, we show Lkb1 is essential for maintaining initiated in immature, mitotic, and early postmitotic chondrocytes a balance between mitotic and postmitotic cell fates in develop- (Fig. S1B). Through these crosses, mice were generated that lacked ment of the mammalian skeleton. In this process, Lkb1 activity Lkb1 activity specifically within chondrocytes of the endo- controls the progression of mitotic chondrocytes to a mature, post- chondral skeleton (Col2a1-Cre;Lkb1c/c; hereafter referred to mitotic hypertrophic fate. Loss of this Lkb1-dependent switch leads as Lkb1 mutants). In contrast to littermates that retained an + to a dramatic expansion of immature chondrocytes and formation active Lkb1 allele (Col2a1-Cre;Lkb1c/ ; hereafter referred to of enchondroma-like tumors. Pathway analysis points to a mamma- as control littermates), Lkb1 mutants displayed a prominent lian target of rapamycin complex 1-dependent mechanism that can postnatal phenotype. be partially suppressed by rapamycin treatment. These findings Lkb1 mutants were born at the expected Mendelian ratio, and highlight a critical requirement for integration of mammalian tar- appeared superficially normal at birth. However, marked growth get of rapamycin activity into developmental decision-making dur- retardation was evident by weaning, and, as a result of this growth ing mammalian skeletogenesis. deficiency, and a lethargic phenotype, mutants were euthanized BIOLOGY by postnatal day (P) 40 to satisfy institutional guidelines on hu- DEVELOPMENTAL chondrocyte differentiation | endochondral ossification | cell death | mane animal care. Histological analysis of long bones after hypoxia weaning (at P30) revealed a profound disorganization of the Lkb1 mutant skeleton (Fig. 1 and Fig. S2A). Alcian blue staining of long rowth of the endochondral skeleton is dependent on a car- bones from normal individuals highlights nonhypertrophic chon- Gtilaginous growth plate. In the growth plate, mitotic chondrocytes within the cartilaginous growth plate localized close to the drocytes transition to a postmitotic, terminal hypertrophic epiphysis (Fig. S2A). Chondrocytes organize into stratified tiers of + + chondrocyte fate (Fig. S1A). Reciprocal signaling between mitotic Col2a1 /Sox9 proliferative chondrocytes in the growth − − + prehypertrophic chondrocyte-derived Indian hedgehog (Ihh) plate before transitioning to Col2a1 /Sox9 /Runx2 postcolumnar, and epiphyseal secreted parathyroid hormone-related peptide postmitotic, hypertrophic chondrocytes that undergo cell death (Pthrp; also known as Pthlh) controls the spatial positioning of and replacement by bone-forming osteoblasts (Fig. 1A and Fig. the hypertrophic transition and the normal growth properties S2A). Hypertrophic chondrocytes and osteoblasts of the outer of the skeleton (1–3). The present study demonstrates an un- cortical and inner trabecular region of the main shaft of the long expected role for liver kinase b1 (Lkb1;alsoknownasStk11)in growth plate regulation. Significance Lkb1 is a multifunctional serine/threonine kinase inhibitor of mTOR signaling whose activity regulates cell cycle progression, The transition from a mitotic to a postmitotic, hypertrophic cellular energy homeostasis, and cell polarity (4, 5). Mouse chondrocyte is a key regulatory event in the growing verte- embryos lacking Lkb1 die at midgestation with vascular and neural brate skeleton. By using genetic approaches, cell culture, and tube defects (6), and germ-line inactivating mutations of Lkb1 in – cell transplantation models, we provide compelling evidence the human population underlie Peutz Jeghers syndrome, charac- that attenuating the energy-sensing mammalian target of terized by development of benign polyps in the gastrointestinal rapamycin complex 1 (mTORC1) pathway is critical for switch- tract, and an increased risk of various types of epithelial cancers (7, ing chondrocyte states. A failure of mTORC1 suppression in 8). Conditional ablation of Lkb1 in pancreatic, vascular, neural Lkb1 fi mutants leads to a dramatic disruption of the skeletal and cardiac tissue links Lkb1 to tissue speci c actions in a variety growth plate and the formation of cartilage tumors comprising of organ systems (9). Here, we provide evidence that Lkb1 regu- undifferentiated chondrocytes that display differential sensi- lation of mammalian target of rapamycin complex 1 (mTORC1) tivity to two key cartilage growth regulators, Indian hedgehog action is a critical step in the transition of mitotic chondrocytes to and Igf. The study highlights the interconnection between postmitotic hypertrophic fates suppressing cartilaginous tumor-like energy sensing pathways, normal growth control, and tumor- growths in the postnatal mammalian skeleton. igenesis in the skeletal program. Results Author contributions: L.P.L. and A.P.M. designed research; L.P.L. performed research; L.P.L. Removal of Lkb1 in Chondrocytes Results in Expansion of Columnar and A.P.M. analyzed data; and L.P.L., B.N.L., J.R.S., and A.P.M. wrote the paper. Mitotic Chondrocytes, Delayed Hypertrophic Development, and The authors declare no conflict of interest. Formation of Enchondroma-Like Tumors. We established a poten- This article is a PNAS Direct Submission. tial link between Lkb1 activity and mammalian skeletogenesis Data deposition: The data reported in this paper have been deposited in the Gene Ex- unexpectedly through conditional removal of Lkb1 activity in pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41898). a large region of the caudal mouse embryo. Given the pleiotropic 1To whom correspondence should be addressed. E-mail: [email protected]. activity of the original Cre-driver line, we intercrossed mice car- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. c/c rying a Cre-dependent conditional Lkb1 allele (Lkb1 ) (10) with 1073/pnas.1309001110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1309001110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 P3 and earlier (Fig. 1 and Fig. S2D). At P3, analysis of Alcian blue and Alizarin red staining revealed that skeletal growth was similar between Lkb1 mutants and control littermates, but the axial (vertebrae) and appendicular (long bone) skeleton was markedly deficient in mineralized matrix (Fig. 1 F–K). In line with expectations from the genetic model, the osteoblast program was not primarily affected (Alizarin red and von Kossa stains; Fig. S4A). In contrast, femur and vertebral sections (Fig. 1 M, N, P, Q, S, and T) revealed a dramatic expansion of the growth plate region in Lkb1 mutants reflected by an extended domain of Alcian blue-stained immature cartilage (Fig. S4A). Although less marked, this phenotype was evident before birth, at embryonic day (E) 18.5 (Fig. 1 L, O, and R and Fig. S2B). Measurement of specific cartilage domains showed similar pro- portions of round resting zone and postcolumnar chondrocytes between control littermates and Lkb1 mutants (Fig. 1U), but a grossly extended domain of immature columnar chondrocyte in mutants (Fig. 1U). At E16.5, no clear phenotype was evident in the femur, although a delay mineralization was evident in posterior vertebrae (Fig. S2 B and C). In summary, in the absence of Lkb1, mutant chondrocytes retained an immature identity whereby normal chondrocytes transition to a terminal hypertrophic fate. An extended growth state is likely the underlying event in the estab- lishment of tumor masses in the later skeleton.

Lkb1 Is Essential for Switching Between Chondrocyte States. To investigate these regulatory events further, we examined key markers of chondrocyte identity. Col2a1 [collagen (II)]-producing nonhypertrophic chondrocytes were expanded in the E18.5 Lkb1 mutant femur (Fig. 2 A and D), whereas the number of Col10a1 [collagen (X)]-expressing hypertrophic chondrocytes was markedly reduced, and Col10a1 protein was not detected (Fig. 2 B, E, I,and + L). In addition, late-stage, Mmp13 hypertrophic chondrocytes Fig. 1. Expansion of columnar mitotic chondrocytes results in formation of were entirely absent from long bones of mutants at E18.5 (Fig. 2 C + enchondroma-like structure. (A and B) P30 femur sections stained with H&E. and F;noteMmp13 osteoblasts were not affected by Lkb1 re- Immunohistochemistry was performed on adjacent sections with specific moval). Production of transcriptional regulators linked to chon- ′ ′ antibodies recognizing collagen (II) (A2 and B2) and Sox9 (A2 and B2 ). drocyte developmental programs displayed a similar temporal and Areas boxed in red in A and B are magnified in A1, A2, B1, and B2,respectively. Areas boxed in green in A2 and B2 are magnified in A2′ and B2′, spatial displacement. Mef2c and Runx2, key determinants of hy- respectively. (C–K) P3 skeletal preparations stained with Alcian blue and pertrophic differentiation, are activated together with Osx and Ihh Alizarin red, with higher-magnification views of the femur (F–H) and ver- in prehypertrophic chondrocytes. In Lkb1 mutants, expression of tebra (I–K). Colored bars indicate the length of mineralized regions within all of these genes was first observed within chondrocytes at an the skeletal element. (L–T) Histological sections through the femur and extended position relative to the periarticular surface indicative of lumbar vertebrae at E18.5 and P3, stained with H&E. (U) Bar graphs dis- a marked delay in chondrocyte differentiation (Fig. 2 G, H, J,and playing the length of the zones of round, columnar, and postcolumnar K and Fig. S4B). > < chondrocytes. Error bars indicate the SD of the means (n 3; *P 0.01 To examine cell proliferation, we visualized cyclin D1, a key between columnar regions of Lkb1c/c and Col2a1-Cre;Lkb1c/c on E18.5; **P < c/c c/c regulator of the G1-to-S phase transition, and the incorporation 0.001 between columnar regions of Lkb1 and Col2a1-Cre;Lkb1 at P3). ′ (Scale bars: A and B, 1 mm; C–E, 0.5 cm; I–K, 1 mm; L–U, 200 μm.) of exogenously supplied 5-ethynyl-2 -deoxyuridine (EdU) or BrdU, to identify chondrocytes undergoing DNA replication. Both approaches highlight an expanded domain of proliferating bone are mineralized and highlighted by Alizarin red (Fig. S2A). In undifferentiated chondrocytes (Fig. 2 M–R). However, the frac- contrast to control littermates, Lkb1 mutants displayed prominent tion of cells undergoing DNA replication within this domain was Alcian blue staining within normally bone-restricted regions of the not altered, suggesting that the excessive number of flattened endochondral skeleton (Fig. S2A). Detailed histological analyses chondrocytes likely reflects delayed hypertrophic differentiation of the Lkb1 mutant from P10 to P30 revealed a mass of pro- rather than an increased rate of division (Fig. S4D). Collagen (X) + + liferating immature Col2a1 /Sox9 chondrocytes deep within the protein was detected by P3; consequently, Lkb1 is not essential shaft of the long bone (Fig. 1B). At P10, the growth plate was for the hypertrophic transition, but rather Lkb1 activity controls markedly disorganized: ectopic hypertrophic chondrocytes were the normal developmental timing of this key cellular transition observed at the core of the growth plate and next to the groove of within the growth plate (Fig. S4C). Ranvier (Fig. S3). By P20, proliferating chondrocytes formed columns perpendicular to the normal longitudinal axis of growth. The mTOR Pathway Mediates the Effects of Lkb1 in Chondrocytes. Tumor-like cell nodules were also found close to the primary The mTOR pathway balances cell growth and proliferation with spongiosa (Fig. S3). Analysis at P30, showed that these are largely the energy level of the cell (12), and is negatively regulated when + + made up of Sox9 /Osx chondrocytes that displayed low levels of conditions are unfavorable (13, 14). To address mTOR signaling collagen (X) indicative of immature chondrocytes (Fig. 1B and in chondrocytes, and to distinguish between mTOR action within Fig. S3). mTORC1 and mTORC2 complexes, we examined phosphory- To investigate the genesis of this phenotype, we focused on the lation of two key mTORC1 substrates, ribosomal protein S6 period preceding the overt change in body size in Lkb1 mutants: (rpS6) and eukaryotic initiation factor 4e-binding protein

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309001110 Lai et al. Downloaded by guest on September 25, 2021 BIOLOGY DEVELOPMENTAL

Fig. 2. Lkb1 is essential for switching between chondrocyte states. (A–F) In situ hybridization of S35-labeled riboprobes speciﬁc for collagen (II), collagen (X), and Mmp13 on E18.5 femur sections. (G–L) E18.5 femur sections immunostained with antibodies speciﬁc to osterix, Mef2c, and collagen (X). Nuclei are visualized with DAPI. White arrows indicate the length from the periarticular end of the bone to the start of the chondrocyte regions demarcated by each protein. The yellow arrows indicate osterix plus chondrocyte domain. (M–R) E18.5 femur sections following in vivo EdU labeling (red double-headed arrow) and immunostaining with cyclin D1, Sox9, and osterix antibodies (green double-headed arrow). (Scale bars: 200 μm.)

(4e-bp1) and phosphorylation of serine 473 of Akt, a hallmark treatment decreased phosphorylation of mTORC1 substrates (Fig. of mTORC2 complex activity. S5), normalized proliferation and differentiation of chondrocytes − − + In control littermates, phosphorylation of rpS6 and 4e-bp1 was in Lkb1 mutants, and restored a cyclinD1 /Osx /Col10a1 hyper- evident in the proliferating columnar chondrocytes within the trophic chondrocyte zone by E18.5 (Fig. 3 I–T). Collectively, long bones, but their phosphorylation state was markedly re- these data indicate that an Lkb1-dependent attenuation of duced on commitment to the hypertrophic chondrocyte program mTORC1 action is critical for the normal progression of (Fig. 3 A–C; note that rpS6 displays a later burst of phosphory- chondrocytes to a terminal hypertrophic fate. lation within mature hypertrophic chondrocytes marked by an asterisk in Fig. 3C). In Lkb1 mutants, mTOR expression was not Loss of Lkb1 Results in Chondrocyte Apoptosis at the Core of the altered; however, mTORC1 activity, highlighted by rpS6 and Growth Plate. By P3, the cell density at the core of the Lkb1 4e-bp1 phosphorylation, extended into regions where hypertro- mutant growth plate was noticeably lower. To determine whether phic development should normally have initiated (Fig. 3 E–G). In cells are dying in this region, we performed TUNEL assay and contrast, phosphorylation of Akt (Ser473) was unaltered in Lkb1 examined activation of caspase-3 to visualize apoptotic cells. mutants, lending support for an mTORC1-speciﬁc role in the TUNEL and cleaved caspase-3–positive cells localize within the skeletal phenotype (Fig. 3 D and H). core of the extended growth plate (Fig. S6). EF5 staining (a To explore mTORC1 action, the mTORC1 inhibitor rapamycin chemical indicator of hypoxia) and Vegfa expression (a hypoxia- wasintroducedintodamsharboringLkb1 mutant embryos be- induced target gene) indicate that the region surrounding the tween 16.5 and 18.5 d of development. Interestingly, rapamycin area of cell death was markedly hypoxic, suggesting that low

Lai et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Fig. 3. The mTORC1 pathway mediates the effects of Lkb1 in chondrocytes. (A–H) E18.5 femur sections immunostained with mTOR, phosphorylated 4e-bp1, phosphorylated rpS6, and phosphorylated Akt-specific antibodies. (I–T) E18.5 femur sections immunostained with osterix-, cyclin D1-, and collagen (X)-specific antibodies. Nuclei were visualized with DAPI. Red asterisk indicates the phosphorylated rpS6 protein within the hypertrophic chondrocytes. Blue arrows indicate the distance from the periarticular surface to chondrocyte zones demarcated by each protein. Areas boxed in yellow are shown as high-magnification images (Right). (Scale bars: 200 μm.)

oxygen levels likely underlie the observed apoptosis (Fig. S6). broadly similar (Fig. S7B and Dataset S1), Gene Ontology (GO) TUNEL-positive cells were detected in a similar region of the Lkb1 analysis of differentially expressed gene highlighted significant mutant growth plate at P10, but, by P22, when the growth plate is differences among genes associated with skeletal system de- − highly disorganized, scattered apoptotic cells predominantly local- velopment (ID number GO:0001501; P = 1.06 × 10 11), regula- ized at the edge of the cartilaginous zone abutting bone-forming tion of cell proliferation (ID number GO:0042127; P = 1.71 × − areas (Fig. S6). 10 5), and positive regulation of mesenchymal cell proliferation − (ID number GO:0002053; P = 3.5 × 10 4). Igf1 and Igf2,which Loss of Lkb1 Results in Enchondroma-Like Tumors in the Postnatal encode broad regulators of cell growth and proliferation, and Skeleton. To investigate the tumorigenic properties of the Gli2, a transcriptional regulator of Hedgehog pathway targets, enchondroma-like mass that forms postnatally in Lkb1 mutant whose activity is linked to malignant transformation of chon- long bones, we assayed chondrocyte growth in anchorage- drosarcomas (15), displayed an elevated transcriptional profile in independent conditions in vitro, and growth following trans- Lkb1 mutant chondrocytes (Fig. S7A). plantation into immune deficient [NOD scid gamma (NSG)] Igf1r was present at the highest levels in the zone of pro- mice. Whereas control chondrocytes occasionally generated small liferating chondrocytes (Fig. S8). Phosphorylation of Tyr1161 on colonies in nonadherent agar cultures, Lkb1 mutant chondrocytes Igf1r, a site of autophosphorylation, indicated active Igf signaling consistently formed prominent colonies under identical con- in these cells (Fig. S8). This conclusion is supported further by ditions (Fig. 4 A–C). When Lkb1 mutant chondrocytes were analysis of Igf signaling dependent phosphorylation of Thr308 on transplanted to the flank of NSG mice, safranin O-stained carti- Akt (Fig. S8). The extension of this domain in the skeletal ele- lage matrix-secreting cells were recovered at the site of injection 3 ments of Lkb1 mutants is in agreement with a continued Igf mo later (six of eight experiments; Fig. 4 G, J,andM). In contrast, signaling input with the expanded domain of proliferating, im- no cartilage nodules were observed in the only tissue recovered mature chondrocytes, and may contribute the maintenance of from one of four control chondrocyte transplants (Fig. 4 F, I,and the proliferative state (Fig. S8). L). Consistent with an mTORC1 action, rapamycin inhibited To examine the potential role of Igf, we examined the effects growth of Lkb1 mutant chondrocytes in nonadherent agar culture of picropodophyllotoxin and PQ401, specific Igf pathway inhib- and following in vivo transplantation (Fig. 4 D, E, H, K,andN). itors, on anchorage-independent growth of Lkb1 mutant chon- To gain additional insights into the mechanisms of enchon- drocytes. Consistent with continued Igf-dependent control, both droma development, we compared the transcriptional profile compounds partially inhibited colony formation. In contrast, between control and Lkb1 mutant chondrocytes. Although GDC-0449 and XAV939, inhibitors of Hh and Wnt signaling

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309001110 Lai et al. Downloaded by guest on September 25, 2021 Pthrp signaling is also a critical determinant of the transition point between mitotic and postmitotic chondrocyte programs whereby Ihh governs Pthrp levels coupling chondrocyte proliferation (i.e., direct Ihh action) with chondrocyte differentiation (i.e., indirect action through Ihh control of Pthrp) (21). As with loss of Lkb1 activity, enhanced Pthrp signaling leads to a marked extension of the proliferative zone of immature chondrocytes at the expense of hypertrophic chondrocyte development. We failed to observe any change in the Ihh pathway aside from the appo- sitional activation of Ihh reﬂecting the marked delay in formation of postmitotic prehypertrophic chondrocytes. Further, the failure of a Hh pathway antagonist to block chondrocyte proliferation speciﬁcally in Lkb1 mutants suggests that loss of Lkb1 removes the dependence on Ihh signaling for normal proliferative control of chondrocytes. The absence of a direct readout of Pthrp signaling precludes an assessment of Pthrp signaling within the Lkb1 mutant model; however, the endochondroma-like end state observed in Lkb1 mutant mice is distinct from the skeletal phenotype observed on constitutive activation of Pthrp signaling in chondrocytes (22). Lkb1 is a multifunctional kinase: by activating different AMP kinase family members, Lkb1 regulates cellular polarity and coordinates cell growth and proliferation with the energy state of the cell (4, 5). Our data show elevated levels of mTORC1 activity in Lkb1 mutant chondrocytes that suggest a central role for Lkb1 in suppression of mTORC1 action in the transition between

mitotic and postmitotic hypertrophic cell states. Consistent with BIOLOGY

this view, high mTORC1 activity, as measured by phosphoryla- DEVELOPMENTAL tion of 4e-bp1 and rpS6, normally associates with proliferative columnar chondrocytes. Further, rapamycin-mediated inhibition of mTORC1 normalizes the Lkb1 mutant phenotype in vivo, and inhibits expansion of Lkb1 mutant chondrocytes in nonadherent Fig. 4. Loss of Lkb1 results in enchondroma in postnatal skeleton. (A and B) Colony assay for anchorage-independent growth of chondrocytes. (Scale culture, and on transplant into mice. Interestingly, the Igf path- bar: 1 mm.) (C) High-magniﬁcation view of an Lkb1 mutant colony. (Scale way is a critical regulator of mTOR action (23), and inhibition of bar: 10 μm.) Bar graphs comparing cell mass through a colorimetric cell de- Igf pathway activity inhibits proliferation of Lkb1 mutant chon- tection assay (D) and colony formation (E)inLkb1 mutant and control drocytes suggesting that the observed phenotype is dependent on chondrocyte cultures. Error bars indicate the SD of the means of three in- an upstream Igf input. dependent experiments. (F–N) Histological analysis of chondrocyte trans- The avascular growth plate has limited nutrient and oxygen plants recovered from NSG mice. Sections were stained with H&E to view supply, which may affect the energy balance of chondrocytes general histology and with safranin O to highlight cartilage matrix. (Scale constraining the zone of active chondrocyte proliferation and bars: 1 mm.) (O) Graphical plot displaying the size of recovered tissue masses potentially contributing to apoptosis of mature hypertrophic (*P < 0.01, mutant vs. control populations; **P < 0.01, indicated treatment vs. mutant population). cells. At P3, we observed a core of apoptotic cells within the extended growth plate surrounded by cells exhibiting molecular signatures of hypoxia. Likely, extreme hypoxia within the [Wnt signaling is linked to chondrocyte maturation (16)], re- core underlies the observed apoptotic phenotype (Fig. S6). spectively, had no effect (Fig. 4 D and E) (17–20). Furthermore, Despite the change in environment, ultimately proliferating although GDC-0449 abolished chondrocyte proliferation in chondrocytes transition to a hypertrophic cell fate. Thus, Lkb1 control skeletal elements in vivo, GDC-0449 failed to block EdU is not essential for making the hypertrophic switch, but coor- incorporation into Lkb1 mutant chondrocytes (Fig. S9). Thus, dinates the timing and position of this critical cellular transi- aberrant chondrocyte proliferation in Lkb1 mutants is dependent tion within the normal growth plate. on mTOR and Igf signaling, but independent of Hh and Wnt In conclusion, the coordination of chondrocyte proliferation signaling inputs. Further, loss of Lkb1 appears to abrogate the and hypertrophic differentiation is crucial to the longitudinal requirement for an Ihh input, suggesting that deregulation of Igf- growth, cellular organization, and appropriate mineralization of mediated proliferative control is likely a key component of the the developing endochondral skeleton. Our work demonstrates Lkb1 skeletal phenotype. that Lkb1 is critical for the normal function and organization of the growth plate, suggesting a link between the integration of Discussion basic pathways of energy balance and growth control in a key The coordination of chondrocyte proliferation and hypertrophic developmental decision-making process, and raising the possi- differentiation is crucial to the longitudinal growth, cellular or- bility that Lkb1/mTORC1 deregulation may contribute to carti- ganization, and appropriate mineralization of the developing laginous tumor formation in men. endochondral skeleton. The evidence presented here indicates Materials and Methods that Lkb1-dependent inhibition of mTORC1 promotes the transition of mitotic chondrocytes to a mature postmitotic fate. The Animal Breeding and Procedures. To generate the Lkb1 conditional KO (Col2a1- Cre; Lkb1c/c), Col2a1-Cre mice were mated with Lkb1c/c mice to obtain Col2a1-Cre; + consequence of the loss of Lkb1 action is an uncoupling of the Lkb1c/ mice, which were then mated with Lkb1c/c mice. Rapamycin was injected normal growth and differentiation program within the growth into the peritoneum of pregnant mice to block mTORC1 activity from E16.5 to plate of the endochondral skeleton that leads to the establish- E18.5. GDC-0449 was delivered by gavage 4 d before the mouse was euthanized ment of enchondroma-like tumors throughout the long bones. on P30. All experiments and procedures were approved by the animal and

Lai et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 care and use committees of Harvard University and the University of (ECM570; Millipore) was used to estimate cell mass through a spectropho- Southern California. tometric assay (OD, 490 nm), and colony number by histochemical staining. Culture media with or without chemical inhibitors were changed twice per Skeletal Staining, Histology, in Situ Hybridization, and Immunostaining. Skel- week for 4 wk. Chemicals used in this study are listed in Table S2. etons were stained with Alizarin red and Alcian blue as described previously (24). H&E staining and safranin O staining were performed according to Allotransplantation. Freshly isolated chondrocytes (500,000 cells) were sus- standard protocols. For in situ hybridization procedures, and hybridiza- pended in 200 μL of culture medium, and was mixed with equal volume of tion with 35S-labeled probes was carried out as described previously (24). Matrigel (no. 356234; BD Biosciences). The mixed suspension was injected s.c. Immunostaining was performed according to standard protocols, and into the right lower flank of the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) primary antibodies used in this study are listed in Table S1.Forvisuali- mouse (female, age 6–8 wk). Rapamycin treatment (delivered through zation, an HRP-conjugated secondary antibody, ABC Kit, and DAB sub- strate were used. For fluorescent visualization, secondary antibodies drinking water; 5 mg/kg body weight/d) of NSG mice started 1 d after in- fi conjugated with Alexa Fluor were used. jection. Three months after transplantation, tumors were dissected, xed, sectioned, and stained as described earlier. BrdU and EdU Analysis. BrdU or EdU (50 μg/g body weight) was injected 2 h before the animals were killed. BrdU analysis of cell proliferation was carried Statistical Analyses. One-way ANOVA with post hoc Bonferroni test was out as described previously (24). EdU labeling was carried out according to performed unless otherwise specified. Microarray data were normalized and the protocol from the Click-it EdU Cell Proliferation Assays Kit (C10339; analyzed with d-Chip, as well as the Database for Annotation, Visualization Life Technologies). and Integrated Discovery software tools (25, 26).

Chondrocyte Isolation. Epiphyseal ends of femurs and tibiae from P30 mice ACKNOWLEDGMENTS. We thank Dr. R. A. DePhino for sharing the Lkb1 were removed and subjected to serial digestion at 37 °C with Liberase TM conditional KO mouse; Jennifer Couget for her help and technical support research grade (no. 0540111900; Roche). The digestion duration for fraction on the microarray experiment; members of the Massachusetts General Hos- 1 was 45 min, and those for fractions 2 to 5 were 30 min each. Fractions 4 pital Endocrine Unit Histology Core for histology services; Drs. R. M. White and 5 were collected, and were used for transcription profiling, anchorage- and N. Ono for advice and suggestions on the transplantation experiment and chondrocyte isolation, respectively; Dr. C. Koch for providing EF5 and independent agar culture, and allotransplantation. the EF5 antibody; members of the laboratory of Dr. E. Schipani for their help and technical support on in situ hybridization; and members and advisors of Transcription Profiling. Total RNA was isolated with RNeasy Mini Kit (no. the Tabin, Kronenberg, and A. P. McMahon P01 group for discussions and 74104; Qiagen) from freshly isolated chondrocytes. cDNA was generated with ongoing support during the preparation of the manuscript. The type II col- the Ambion WT Expression Kit (no. 4411973; Ambion), and was ultimately lagen antibody developed by Dr. Linsenmayer was obtained from the De- hybridized with the Affymetrix Mouse 1.0 Gene ST array. velopmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and main- tained by the Department of Biology at the University of Iowa. Work in Anchorage-Independent Agar Culture. Anchorage-independent agar culture the laboratory of A.P.M. was supported by National Institutes of Health/ was carried out according to the protocol from the Cell Transformation National Institute of Diabetes and Digestive and Kidney Diseases Grant Detection Kit (ECM570; Millipore), with minor modification. The seeding P01 DK056246. L.P.L. was supported by an Arthritis Foundation Postdoctoral density was 2,000 cells per well of a 24-well plate. A cell detection kit fellowship.

1. Lanske B, et al. (1996) PTH/PTHrP receptor in early development and Indian hedge- 15. Ho L, et al. (2009) Gli2 and p53 cooperate to regulate IGFBP-3- mediated chondrocyte hog-regulated bone growth. Science 273(5275):663–666. apoptosis in the progression from benign to malignant cartilage tumors. Cancer Cell 2. St-Jacques B, Hammerschmidt M, McMahon AP (1999) Indian hedgehog signaling 16(2):126–136. regulates proliferation and differentiation of chondrocytes and is essential for bone 16. Yang Y, Topol L, Lee H, Wu J (2003) Wnt5a and Wnt5b exhibit distinct activities in formation. Genes Dev 13(16):2072–2086. coordinating chondrocyte proliferation and differentiation. Development 130(5): 3. Vortkamp A, et al. (1996) Regulation of rate of cartilage differentiation by Indian 1003–1015. – hedgehog and PTH-related protein. Science 273(5275):613 622. 17. Gable KL, et al. (2006) Diarylureas are small-molecule inhibitors of insulin-like 4. Hezel AF, Bardeesy N (2008) LKB1; linking cell structure and tumor suppression. On- growth factor I receptor signaling and breast cancer cell growth. Mol Cancer Ther – cogene 27(55):6908 6919. 5(4):1079–1086. 5. Jansen M, Ten Klooster JP, Offerhaus GJ, Clevers H (2009) LKB1 and AMPK family signaling: 18. Girnita A, et al. (2006) The insulin-like growth factor-I receptor inhibitor pic- The intimate link between cell polarity and energy metabolism. Physiol Rev 89(3):777–798. ropodophyllin causes tumor regression and attenuates mechanisms involved in 6. Ylikorkala A, et al. (2001) Vascular abnormalities and deregulation of VEGF in Lkb1- invasion of uveal melanoma cells. Clin Cancer Res 12(4):1383–1391. deﬁcient mice. Science 293(5533):1323–1326. 19. Huang SM, et al. (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt 7. Hemminki A, et al. (1998) A serine/threonine kinase gene defective in Peutz-Jeghers signalling. Nature 461(7264):614–620. syndrome. Nature 391(6663):184–187. 20. Rudin CM, et al. (2009) Treatment of medulloblastoma with hedgehog pathway in- 8. van Lier MG, et al. (2010) High cancer risk in Peutz-Jeghers syndrome: A systematic hibitor GDC-0449. N Engl J Med 361(12):1173–1178. review and surveillance recommendations. Am J Gastroenterol 105(6):1258–1264. 21. Kronenberg HM (2006) PTHrP and skeletal development. Ann N Y Acad Sci 1068: 9. Udd L, Mäkelä TP (2011) LKB1 signaling in advancing cell differentiation. Fam Cancer 1–13. 10(3):425–435. 22. Weir EC, et al. (1996) Targeted overexpression of parathyroid hormone-related 10. Bardeesy N, et al. (2002) Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419(6903):162–167. peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone – 11. Ovchinnikov DA, Deng JM, Ogunrinu G, Behringer RR (2000) Col2a1-directed ex- formation. Proc Natl Acad Sci USA 93(19):10240 10245. pression of Cre recombinase in differentiating chondrocytes in transgenic mice. 23. Levine AJ, Feng Z, Mak TW, You H, Jin S (2006) Coordination and communication between – Genesis 26(2):145–146. the p53 and IGF-1-AKT-TOR signal transduction pathways. Genes Dev 20(3):267 275. 12. Tee AR, Blenis J (2005) mTOR, translational control and human disease. Semin Cell 24. Long F, Zhang XM, Karp S, Yang Y, McMahon AP (2001) Genetic manipulation of Dev Biol 16(1):29–37. hedgehog signaling in the endochondral skeleton reveals a direct role in the regu- 13. Corradetti MN, Inoki K, Bardeesy N, DePinho RA, Guan KL (2004) Regulation of the lation of chondrocyte proliferation. Development 128(24):5099–5108. TSC pathway by LKB1: Evidence of a molecular link between tuberous sclerosis 25. Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of complex and Peutz-Jeghers syndrome. Genes Dev 18(13):1533–1538. large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57. 14. Shaw RJ, et al. (2004) The LKB1 tumor suppressor negatively regulates mTOR sig- 26. Huang W, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: Paths toward naling. Cancer Cell 6(1):91–99. the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37(1):1–13.

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