Netrin-1 Expression Confers a Selective Advantage for Tumor Cell Survival in Metastatic Breast Cancer

Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer

Julien Fitamant*, Ce´ line Guenebeaud*, Marie-May Coissieux*, Catherine Guix*, Isabelle Treilleux†, Jean-Yves Scoazec‡, Thomas Bachelot†, Agne` s Bernet*, and Patrick Mehlen*§

*Apoptosis, Cancer, and Development Laboratory–Equipe Labelliseé ‘‘La Ligue,’’ Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 5238, and †Institut National de la Santeét de la Recherche Me´dicale U590, Centre LeónBe´ rard, Universite´de Lyon 69008 Lyon, France; and ‡Institut National de la Santeét de la Recherche Me´dicale, U865 and Plate-forme d’Histopathologie du Petit Animal IFR62, Faculte´Laennec, UniversiteĆlaude Bernard, 69437 Lyon, France

Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, and approved January 15, 2008 (received for review October 15, 2007) Netrin-1, an axon navigation cue was proposed to play a crucial role hence suggesting that the loss of UNC5H genes represents a during colorectal tumorigenesis by regulating apoptosis. The netrin-1 selective advantage for tumor development (12–14). Interestingly, receptors DCC and UNC5H were shown to belong to the family of in mice, both inactivation of UNC5H3 and overexpression of dependence receptors that share the ability to induce apoptosis in the netrin-1 in the gastrointestinal tract are associated with intestinal absence of their ligands. Such a trait confers on these receptors a tumor progression (13, 15), hence demonstrating per se that loss of tumor suppressor activity. Expression of one of these dependence netrin-1 dependence receptors in the human pathology is a causal receptors at the surface of a tumor cell is indeed speculated to render factor for tumor progression (16). this cell dependent on ligand availability for its survival, hence However, the model described above predicts that not only loss inhibiting uncontrolled cell proliferation or metastasis. Consequently, of netrin-1 receptors but also gain of ligand expression, i.e., auto- it is a selective advantage for a tumor cell to lose this dependence crine expression, should be observed in human cancers, because receptor activity, as previously described with losses of DCC and they should represent similar selective advantages for tumor pro- UNC5H expression in human cancers. However, the model predicts gression. This question is important not only for basic knowledge that a similar advantage may be obtained by gaining autocrine but also for putative therapy: Indeed, triggering tumor-cell death by expression of the ligand. We describe here that, unlike human inhibiting the extracellular interaction between netrin-1/receptors nonmetastatic breast tumors, a large fraction of metastatic breast could represent an appealing anticancer strategy. We show here cancers overexpress netrin-1. Moreover, we show that netrin-1- that the majority of breast tumors with metastatic propensity show expressing mammary metastatic tumor cell lines undergo apoptosis increased netrin-1 expression, a feature that we used in different when netrin-1 expression is experimentally decreased or when decoy mouse models to prevent/inhibit metastatic development. soluble receptor ectodomains are added. Such treatments prevent metastasis formation both in a syngenic mouse model of lung colo- Results nization of a mammary cancer cell line and in a model of spontaneous Netrin-1 Is a Marker for Human Metastatic Breast Cancer. We first lung metastasis of xenografted human breast tumor. Thus, netrin-1 analyzed by quantitative (Q)RT-PCR, expression of netrin-1, its expression observed in a large fraction of human metastatic breast dependence receptors, i.e., DCC, UNC5H2, and UNC5H3, and, as tumors confers a selective advantage for tumor cell survival and positive control, a known marker of metastasis, CXCR4 (17) in a potentially represents a promising target for alternative anticancer panel of 51 breast primary tumors. These tumors were from lymph therapeutic strategies. node-negative patients (N0, 16 patients), lymph node-positive patients (N ϩ M0, 19 patients) or metastatic patients at the time of apoptosis ͉ DCC ͉ dependence receptor diagnosis (Mϩ, 16 patients). As already described, CXCR4 is significantly more expressed in N ϩ M0 or Mϩ primary tumors etrin-1, a diffusible laminin-related protein, has been shown compared with N0 tumors [supporting information (SI) Fig. 5A in Nto play a major role in the control of neuronal navigation SI Appendix]. Although DCC was barely detectable and UNC5H during the development of the nervous system by interacting expression failed to display significant changes between the differ- with its main receptors, DCC (Deleted in Colorectal Cancer) ent types of tumors (data not shown, Fig. 1A and SI Fig. 5B in SI (1–3) and UNC5H (4, 5). However, more recently, netrin-1 has Appendix), netrin-1 is significantly more expressed in N ϩ M0 emerged as a completely different molecule that regulates cell tumors than in N0 tumors (median, 1.8 versus 0.5; P ϭ 0.007) with survival. Indeed, the netrin-1 receptors DCC and UNC5H, i.e., a range of netrin-1 expression higher in N ϩ M0 tumors (Fig. 1A UNC5H1, UNC5H2, and UNC5H3, belong to the so-called and SI Fig. 5B in SI Appendix). Whereas 31.5% of the N ϩ M0 dependence receptor family (6, 7). Such receptors share the tumors show at least a 5-fold increase in netrin-1, no such increase functional property of inducing cell death when disengaged from was detected in any tested N0 tumors (Fig. 1A and SI Fig. 5B in SI their ligands, whereas the presence of their ligands blocks this Appendix). An even more striking difference is observed when proapoptotic activity. Such receptors thus create cellular states comparing netrin-1 expression in Mϩ versus N0 tumors (median, of dependence on their respective ligands (8, 9). This dependence effect has been suggested to act as a mechanism

for eliminating tumor cells that would develop in settings of ligand Author contributions: A.B. and P.M. contributed equally to this work; J.F., A.B., and P.M. unavailability: proliferation of tumor cells in a cell environment designed research; J.F., C. Guenebeaud, M.-M.C., C. Guix, I.T., J.-Y.S., and A.B. performed with constant and limited ligand presence or migration of meta- research; I.T. and T.B. contributed new reagents/analytic tools; J.F., I.T., J.-Y.S., T.B., A.B., static tumor cells toward tissues where the ligand is not expressed. and P.M. analyzed data; and P.M. wrote the paper. A selective advantage for a tumor cell would then be to lose the The authors declare no conﬂict of interest. proapoptotic activity of its dependence receptors. Along this line, This article is a PNAS Direct Submission. DCC was proposed in the early 1990s to be a tumor-suppressor §To whom correspondence should be addressed. E-mail: [email protected]. gene, whose expression is lost in the vast majority of human cancers This article contains supporting information online at www.pnas.org/cgi/content/full/ (10, 11). This hypothesis also fits with the observation that UNC5H 0709810105/DC1. genes are down-regulated in the vast majority of colorectal tumors, © 2008 by The National Academy of Sciences of the USA

4850–4855 ͉ PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709810105 Downloaded by guest on October 1, 2021 Table 1. Expression of netrin-1 examined by QRT-PCR using total evA r ega rpxe essi no RNA extracted from 48 human breast and 2 mouse mammary A 30 N0 +N +M0M cell lines 1-nirten 1 4.16 91 .31 25 NU C5H2 1 106.1 57. Cell line netrin-1 Cell line netrin-1 02 Human Human 184-A1 ϩ MDAMB 185 ϩϩϩ 51 184-B5 ϩϩ MDAMB 231 Ϫ BR-CA-MZ-01 ϩϩ MDAMB 361 ϩϩ 01 Gene expression Gene expression BR-CA-MZ-02 ϩ MDAMB 415 ϩ BT-20 ϩϩ MDAMB 435S Ϫ 5 BT-474 ϩϩϩ MDAMB 436 ϩϩϩ BT-549 ϩϩ MDAMB 453 Ϫ 0 N0 pati tne s +N 0M stneitap M+ stneitap BY-549 ϩϩ MDAMB 468 ϩϩ 0=p .0 70 0=p .0 90 CAMA-1 ϩϩ S68 ϩϩ p< 0.0 001 HBL-100 ϩϩ SKBR3 ϩϩϩ tS a it ts i slac ignificanc fe or n irte 1-n HCC38 Ϫ SKBR7 ϩϩϩϩϩ HCC202 ϩϩϩ SUM 52 ϩϩ 5.3

n HCC1395 ϩ SUM 102 ϩϩ

o B C i

d s 3 0.

e +N M 0 s ϩϩϩϩ Ϫ t HCC1500 SUM 149

r x 2 5. ϩϩ Ϫ

/ HCC1569 SUM 159

)

) +

N ϩ ϩϩϩ

( 2.0 HCC1806 SUM 185

(

e G HCC1937 ϩϩ SUM 190 ϩϩϩϩ d 1.5

04 X 04 y

a n ϩϩ ϩϩ r HCC1954 SUM 206

i o 1 0.

+M r

) p ϩϩ Ϫ m HCC2218 SUm 225

(

y l 0 5.

i ϩϩ Ϫ

t HME-1 SUM 229

r a 0

( e 0 0.5 .1 0 5.1 2 0. 5.2 3 0. Hs 578T ϩ T47D ϩϩϩϩ X 04 N 4RCXC erpxe oiss n MCF7 ϩϩ UACC812 ϩϩ l oitar( l ymp donh es (N )+ r/ e tal ed p ramir ty um )ro MCF-10A Ϫ ZR751 Ϫ MCF-10F Ϫ Mouse Ϫ 1T4 RN76 1T4 D E MDAMB 134 ϩ 67NR ϩϩϩϩϩ netri 1-n 4T1

Netrin-1 expression was analyzed compared with the housekeeping genes 0PLPR HMBS (Hydroxymethylbilane synthase) or RPLP0 expression in each sample. - α n– nirte 1 TBP was also used as a control here and gave similar results (data not shown). A range of netrin-1 expression is shown from ϩϩϩϩϩ ( high) to Ϫ (barely Fig. 1. Netrin-1 is overexpressed in metastatic breast tumors. (A) Expression detectable). profile of netrin-1 and UNC5H2 examined with quantitative real-time RT-PCR. QRT-PCR was performed by using total RNA extracted from 51 tumor biopsies. They were obtained from patients with tumors localized to the breast (N0), with able DNA microarrays, netrin-1 was not found associated with only axillary node involvement (NϩM0), and with distant metastases at diagnosis (Mϩ). Specific human netrin-1 primers (35) and primers corresponding to the breast metastatic cancer in the different profiling experiments human HMBS gene (hydroxymethylbilane synthase) were used. HMBS was used reported so far [(18), see www.oncomine.org]. Netrin-1 was, in most as a reference here because it shows a weak variability at the mRNA level between cases, detected only at background level, but, as shown in SI Fig. 5C normal and breast tumoral tissues, as described (36). Another reference TBP was in SI Appendix, this is probably due to a bias within the cDNA also used with similar results (data not shown). Netrin-1 expression is given as the amplification procedure used in the profiling experiments. Immu- ratio between netrin-1 expression in each sample and the average of netrin-1 nohistochemistry performed on a panel of N ϩ M0 or Mϩ breast expression in the N0 samples, shown by a horizontal bar. UNC5H2 expression is primary tumors sections revealed netrin-1 protein expression (Fig. MEDICAL SCIENCES also given as the ratio between UNC5H2 expression in each sample and the average of UNC5H2 level in N0 samples. Nonparametric statistical significance 1B and SI Fig. 5D in SI Appendix). To further analyze whether the tests (Mann–Whitney) were used; the P values for netrin-1 are indicated. The high netrin-1 expression observed in N ϩ M0 or Mϩ primary average of netrin-1 and UNC5H2 expression is indicated for each tumor stage tumors is also observed in disseminating tumors cells, we next (upper left). (B) Representative netrin-1 immunohistochemistries on sections measured netrin-1 mRNA level in pairs including both the primary from two human tumors presented in A. Enlargement is indicated. (C) Compar- tumors and the related metastatic lymph nodes. The expression ison between netrin-1 or VEGFR expression and CXCR4 expression in seven pairs ratio between lymph nodes and primary tumors is higher for of frozen human invaded lymph nodes/primary tumors. Ratio between the netrin-1 than for the other known genes involved in the metastatic mRNA level measured by QRT-PCR in invaded lymph node to the one detected in the primary tumor is shown for each pair and is represented as the ratio of process, i.e., CXCR4 and VEGFR, (Fig. 1C, six of seven pairs netrin-1 (VEGFR) to CXCR4. (D) cDNA amplification from 67NR or 4T1 cells loaded tested). Thus, netrin-1 up-regulation appears as a marker of lymph in an agarose gel. (E) Immunostaining on 4T1 cell line using netrin-1 antibody. A node involvement and distant metastatic disease in human breast control without the primary antibody is indicated. Nuclei were visualized with cancer. Hoescht staining. Disruption of the Netrin-1 Autocrine Loop Triggers Cancer Cell Death. Because netrin-1 appears to be up-regulated in a large fraction of 7.8 versus 0.5; P Ͻ 0.0001). Along this line, 62.5% of Mϩ tumors aggressive human breast cancers, we next investigated whether show at least a 5-fold increase in netrin-1 expression. A significant netrin-1 is also expressed in a panel of 50 breast cell lines. difference in netrin-1 expression also exists between N ϩ M0 and Interestingly netrin-1 is highly expressed in a sizeable fraction of Mϩ tumors (median, 1.8 versus 7.8; P ϭ 0.009). Moreover, netrin-1 human breast cancer cell lines (Table 1) and all cell lines showing overexpression is higher in Mϩ tumors than in N ϩ M0 tumors, high netrin-1 levels (greater than ϩϩϩ) have been derived from because 37.5% of Mϩ tumors display a Ͼ15-fold increase in aggressive tumors or/and have been shown to form metastases in netrin-1 levels, whereas such an increase is not detected in N ϩ M0 mice. We also studied two mouse cell lines, 4T1 and 67NR (Table tumors (Fig. 1A and SI Fig. 5B in SI Appendix). Interestingly, 1 and Fig. 1D), developed by Miller and colleagues (19) as cell lines, although netrin-1 is commonly spotted in the commercially avail- derived from a BALB/c mouse spontaneous primary mammary

Fitamant et al. PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 ͉ 4851 Downloaded by guest on October 1, 2021 Fig. 2. Induction of metastatic cell COOH death by inhibiting the netrin-1 au- A B H2N netrin-1 tocrine loop. (A) Induction of 4T1 01 0 30 25 H N cell death by reducing netrin-1 80 H N 2 1 2 2 3 level. The 4T1 cells were treated 20 4 60 DCC 5 5 6 with scramble siRNA (control) or 15 DCC-EC-Fc DCC-5Fbn 04 UNC5H netrin-1 siRNA. Twenty-four hours 10 % of cell death later, the netrin-1 mRNA level was 20 5 % of netrin-1 mRNA % of netrin-1 quantiﬁed by QRT-PCR (Left), and 0 0 COOH cell death was determined 1 day Con rt ol s Ri NA Co ortn l NRis A HOOC net -nir 1 later by trypan blue-exclusion assay ne rt in-1 (Right). (B) Scheme representing

netrin-1 and its receptors, DCC and C D 0=p .01 E p .0= 02 Co tn r lo 21 0 UNC5H. (Inset) The extracellular 25 co ortn l 0=p 0. 2 07 cF-CE-CCD -CE-CCD cF 06 1 00 part of the DCC receptor (DCC-EC- 02 1µ lm/g 1µ /g lm µ Fc) and the ﬁfth ﬁbronectin type III 05 .1 5 g m/ l 08 15 04 domain of DCC (DCC-5Fbn) used to 60 induce cell death. (C–E) Quantita- 01 03 40 % of cell death tive analysis of cell death in 67NR 02 % of cell survival 5 02 and 4T1 cells treated with DCC- 01 0 0

% of cells of % with caspase active -3 0 EC-Fc (C and D), with DCC-5Fbn (E) - 0.2 4.0 0 8. - 2.0 .0 4 0 8. 8.0 - DC -C bF5 n RN76 1T4 or with an unrelated soluble pro- 6 N7 R 4 1T + N et + 3LI R 6 N7 R tein IL3R-EC-Fc. Cell death/survival 4T1

was quantiﬁed by the trypan blue- 7RBKS 74T D BMADM 2 31 exclusion assay (C), by caspase ac- F 0=p . 10 G p= 0.0 3 ND NU 5C H 20.0=p N 2.0 l tivity measurement by ﬂow cytom- 001 o -D H 100 tr 5 100 n A C o H etry (D), or by MTT assay (E). In this c N 1.6 08 U latter test, netrin-1 was added in 80 80 α− 2.1 60 HA excess to reverse the effect of DCC- 60 06

5Fbn. (F) Cell-death induction by 0.8 04 40 04 α−actin

DCC-5Fbn in SKBR7 and T47D cell % of cell survival % of cell survival 4.0 02 % of cell survival 02 02 lines but not in MDAMB231 cells. 0 0 0 0 Cell survival was quantiﬁed by MTT 3R C R C - 1 - 5.0 IL C n L3 C n - 0.5 1 D Fb I D b Relative caspaseRelative (Index) activity µ µ 5 F assay (Right), and apoptosis was nbF5-CCD lm/g DCC-5 nbF lm/g nbF5-CCD µ lm/g - -5 measured by caspase activity assay (Left). (G) Quantitative analysis of cell survival by MTT assay in the presence of either IL3R-Fc or DCC-5Fbn in 4T1 cells or 4T1 cells stably transfected with UNC5H2-IC D412N (DN UNC5H; i.e., the intracellular domain of UNCSH2 mutated in the caspase site acts as a dominant negative for UNC5H proapoptotic activity (SI Fig. 7D in SI Appendix). (Inset) Western blot using an anti-HA-UNC5H2 antibody was performed on the mock-transfected and DN UNC5H-expressing 4T1 cell lines. Standard deviations are indicated (n Ͼ 3). In C–G, Mann–Whitney tests were performed, and a P value is indicated.

tumor, showing different metastatic potentials when injected into exclusion assay (Fig. 2C) or by a MTT assay (data not shown). In syngenic mice. Although 67NR cells form primary mammary addition, apoptosis was more specifically monitored by measuring tumors but no metastasis, 4T1 cells form primary tumors and caspase activity (Fig. 2D). As shown in Fig. 2 C and D, addition of metastasis, especially in the lung, the liver, and the bone marrow. the competing soluble protein in the culture medium triggers death Interestingly, although netrin-1 receptors (i.e., DCC, UNC5H1, of 4T1 cells, whereas it has no effect on 67NR cell death. UNC5H2, and UNC5H3) failed to show significant difference Because the complete extracellular domain of DCC appears as between 67NR and 4T1 cell lines, netrin-1 mRNA was hardly only of modest interest for use in vivo and in therapy (DCC-EC-Fc detected in 67NR cells, whereas it was expressed in 4T1 cells (Fig. is Ϸ1,100 aa large), we looked for an alternative polypeptide from 1DE and SI Fig. 6A in SI Appendix). This presence of the netrin-1 the DCC extracellular domain that could trigger apoptosis in 4T1 mRNA in 4T1 cells is associated with the detection of netrin-1 cells but not in 67NR cells. We consequently produced a flag- protein after netrin-1 immunostaining performed in the absence of tagged fifth fibronectin type III domain of DCC, DCC-5Fbn, a cell permeabilization (Fig. 1E). Therefore, metastatic 4T1 cells domain located in the ectodomain of DCC that is known to interact show autocrine production of netrin-1. with netrin-1 (20) (Fig. 2B Inset). This 100-aa protein does not We next investigated whether this netrin-1 autocrine production interfere with the binding of DCC/netrin-1 or UNC5H/netrin-1 but confers a selective advantage to 4T1 cells by inhibiting DCC/ affects the ability of netrin-1 to trigger multimerization of these UNC5H-induced cell death. We first down-regulated expression of receptors, a multimerization required for the inhibitory acti- netrin-1 in 4T1 cells using RNAi strategy. As shown in Fig. 2A, vity of netrin-1 on DCC- or UNC5H-induced cell death down-regulation of netrin-1 in 4T1 cells was associated with cell- (F. Mille, F. Llambi, C. Guix, C. Thibert, J.F., C. Guenebeaud, death induction monitored by a trypan blue-exclusion assay. As a S. Castro-Obregon, D. E. Bredesen, and P.M., unpublished work). second approach, we looked for a compound that may inhibit the As shown in Fig. 2E, addition of DCC-5Fbn triggers, in a dose- interaction between netrin-1 and its receptors. It was reported that dependent manner, death of 4T1 cells as measured by MTT assay. a domain located in the N terminus of netrin-1 (the so-called Similar results were obtained by using trypan blue-exclusion or laminin-VI domain) interacts with both DCC and UNC5H recep- caspase-activity assays (data not shown). This cell-death effect is tors [Fig. 2B; (20)]. We show that a soluble extracellular domain of directly associated with netrin-1 autocrine expression because (i) DCC (DCC-EC-Fc) is able to inhibit both DCC/netrin-1 and DCC-5Fbn has no effect on 67NR cells, (ii) addition of an excess UNC5H2/netrin-1 interaction, as measured by ELISA (SI Fig. 7A netrin-1 blocks the death effect of DCC-5Fbn, and (iii) IL3R-EC-Fc in SI Appendix). This soluble ectodomain is also able to inhibit (the extracellular domain of the IL3 receptor) fails to trigger 4T1 netrin-1-induced DCC-mediated signaling pathways [e.g., MAPK cell death (Fig. 2E). activation (3)] (SI Fig. 7B in SI Appendix). DCC-EC-Fc was then The acquired survival advantage through netrin-1 autocrine added to a 4T1 cells culture—or as a control 67NR cells culture— expression is not restricted to murine tumor cells, because it is and cell death/survival was monitored, either by a trypan blue- detected in human breast cancer cell lines. Indeed, addition of

4852 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709810105 Fitamant et al. Downloaded by guest on October 1, 2021 Fig. 3. Inhibition of metastasis for- co n t r ol (s c r a m b l e) si RN A ne t ri n- 1 p= 0. 00 2 (n = 14 ) 0.0=p 91 A B 2. 5 C 40 0 mation in mice by disruption of the 40.0=p 35 0 s i

netrin-1 autocrine loop. The 4T1-luc s

2 a e s

t 30 0 i c s s n a a

cells were i.v. injected into BALB/c t t e 25 0 e s c s c 1. 5 a m t f e

mice at day 0. (A–C) Scramble siRNA e 20 0 n o i m r

m 1 e b e n a e a n 15 0 or netrin-1 siRNA was i.v. injected Co l ou r ba r l u o i m

b 10 0 u daily, and after 9 (A and B)or14(C) (c o u n t s) M 0. 5 Mi n= 0 N 50 days, lung metastasis was studied by Ma x = 51 0 0 0 6 1 0 8 0 80 16 0 24 0 si RN A ol 1 0 16 0 32 0 48 0 co n t ro l t r A A 2 luminescence recording (A and B)or on N N ne t ri n- 1 c si R i R by examination of lungs under a mi- s croscope (C). (A) A representative im- B PB S DC C - 5 F bn p= 0. 00 6 age of luminescence recording of D E F 40 Av er a g e of Ra n g e of scramble siRNA- (Left) or netrin-1 T re a t m e n t Mi ce (n ) me t as t as i s me t as t as i s e s i c

s pe r mo u s e pe r mo u s e n

a 30 t siRNA- (Right) treated mice. (B) e s c s a

t PB S 10 42 . 4 0- 7 5 e e

Quantiﬁcation of the luminescent i n

m 20 m n a e a n

u DC C - 5 F bn 10 2. 6 0- 6 signal measured by the NightOwLB II l

o Co l ou r ba r o i b system. A luminescent signal for each (c o u n t s) M 10 Mi n= 0 group (control vs. netrin-1 siRNA) is Ma x = 26 0 0 0 60 1020 18 0 24 0 0 60 12 0 18 0 24 PB S DC C - 5 F bn given as a mean of the different ra- (n = 14 ) (n = 14 ) tios between summed luminescent PB S DC C - 5 F bn signal in the lung area (photons/sec) n= 8 H n= 21 and a background area deﬁned in G I 8 J 60 0 K 1. 0

e p < 0. 05

p= 0. 06 s *

10 0 i s PB S

s 0. 9 u o u 0 50 e 7 e a s 50 0 t

the animal body. Number of mice is s

u o u 5F bn l a l 0. 8 s

m * u o u

6 80 a r t 40 v i m

ϭ e 0. 7 e

indicated (n 14). (C) Number of 40 0 r v r

p 5 r m DC C - 5 F b n e p e u

s 0. 6 e p e o

i p = 0. 06 60 s r

0 30 s (n = 1 0 ) l c lung metastatic nodules in individual s 4 30 0 l a i 0. 5 s a t i a s r s s a m e a t f 0. 4 a 40 3 t t s mice were counted after day 14 un- 20 v e

o 20 0 s a O t 0. 3 PB S a r m t 2 e e b e o e

der a dissection microscope in three 20 c r (n = 11 ) 0 10 M 0. 2

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u 0. 1 treated populations [scramble (con- M 0 0 0 N 0 0. 0 trol), ﬁrst set of siRNA also used in A F aD D DC C - 5 F bn 0 0. 15 0. 3 0. 6/ 1. 25 µg / g DC C - PB S 0 5 10 15 20 25 0 2 10 12 14 16 18 20 -G ST -G ST DC C - 5 F bn co n c e n t ra t i on 5F bn Da y s af t er t re a t m en t Da y s af t er t re a t m en t and B, and second set of siRNA]. (D–F) As in A–C except than PBS or DCC- 5Fbn were injected once daily i.v. or once i.p. starting at day 0. After 13 days, metastasis development was studied by luminescence recording (D and E) or by examination of lungs under a microscope (F). (F) A representative macroscopic photograph of lungs from PBS- (Left) or DCC-5Fbn- (Right) treated mice (Lower). Total number of lung metastatic nodules in individual mice were counted in the two treated populations (ϩPBS, ϩDCC-5Fbn) (Upper). (G)AsinF except that GST-DCC-5Fbn (instead of Flag-DCC-5Fbn) or an unrelated protein GST-FaDD was injected daily. (H) Dose effect of DCC-5Fbn. As in F except that different concentrations of DCC-5Fbn were injected daily; n ϭ 21. (I) The 4T1 cells (1 ϫ 103) were i.v. injected at day 0. At day 7 (at this day from one to eight micrometastasis are visible, data not shown), the mice were treated daily with PBS or DCC-5Fbn, and enumeration of metastasis was performed at day 11. (J) The 4T1 cells (5 ϫ 105) were i.v. injected at day 0. At day 8, when at least 90 macrometastasis are visible, the mice were treated daily with PBS or DCC-5Fbn, and enumeration of metastasis was performed from day 8 to day 18 for PBS-treated mice, as we were required ethically to euthanize them, to day 20 for DCC-5Fbn-treated mice (each point represents three mice). (K) Overall survival of mice, which were i.v. injected with 106 4T1 cells, treated daily with PBS (black) or DCC-5Fbn (gray). Pvalues were calculated by using the Kaplan–Meier test. Standard deviations are indicated. In B, C, E, G, and I, Mann–Whitney tests were performed, and a P value is indicated.

DCC-5Fbn to naturally netrin-1-expressing human breast adeno- metastasis, netrin-1 siRNA injection is associated with a significant carcinoma T47D or SKBR7 cell cultures triggers cell-death induc- reduction of lung metastasis measured by luminescence recording. tion measured either by caspase-3 activity assay or MTT assay, Similar results were obtained by direct anatomopathological exam- whereas it had no effect on netrin-1-negative cells like MDAMB- ination of lungs after 14 days of injection (Fig. 3C). To bring the in 231 (Fig. 2F). Similar results were obtained by netrin-1 siRNA vivo experiment closer to therapeutic relevance, we then performed MEDICAL SCIENCES approach (SI Fig. 7C in SI Appendix and data not shown). a similar set of experiments using DCC-5Fbn injection rather than This cell-death induction upon inhibition of the netrin-1 auto- siRNA. Mice were then i.p. and i.v. injected (once i.v., once i.p.) crine loop, i.e., via inhibition of netrin-1 level, via inhibition of from day 0 to day 13 with either PBS buffer or Flag-tagged-DCC- netrin-1/receptors interaction, or via inhibition of netrin-1 receptors 5Fbn (1.25 ␮g per gram of body weight per injection). Metastasis multimerization, appears to occur through UNC5H receptors. formation was again analyzed by using luminescence recording. As Indeed, DCC is hardly detected in 4T1, T47D, or SKBR7 cells (SI shown in Fig. 3 D and E, although in PBS-treated mice 4T1-luc cells Fig. 6A in SI Appendix and data not shown). Moreover, 4T1 cells efficiently colonize lungs, mice treated with DCC-5Fbn show a stably expressing a dominant negative mutant of UNC5H receptors dramatic reduction of lung metastasis. This inhibition of metastasis inhibiting UNC5H-induced apoptosis (SI Fig. 7D in SI Appendix) formation was then confirmed by anatomopathological examina- are no longer sensitive to DCC-5Fbn (Fig. 2G). tion of lungs (data not shown and Fig. 3F). Similar results were obtained when we performed daily i.p. injection of GST-tagged- Disruption of the Netrin-1 Autocrine Loop Prevents Metastasis For- DCC-5Fbn instead of Flag-tagged-DCC-5Fbn and when an unre- mation. We next investigated whether the cell death effect observed lated protein (GST-FaDD) was used as a negative control (Fig. 3G). in vitro may be extended in vivo. As a first approach, 4T1 cells stably A dose-dependence experiment with DCC-5Fbn was then per- transfected with a luciferase-based vector (4T1-luc) were i.v. in- formed, and, as shown in Fig. 3H,a0.15␮g per gram of body weight jected into syngenic BALB/c mice. Metastasis formation was then is sufficient to decrease metastasis formation. To further analyze analyzed by using luminescence recording. As shown in Fig. 3, when whether this treatment may not only prevent metastatic formation i.v. injected, 4T1-luc cells efficiently colonize lungs. To first assay by triggering tumor-cell death before extravazation but also could the survival role of netrin-1 in promoting 4T1 cell-mediated lung erase metastatic lesions already established in the secondary organ, metastasis, netrin-1 was down-regulated in vivo by daily i.p. injection the DCC-5Fbn treatment was administered once micrometastases of netrin-1 siRNA. I.p. injection allows the detection by QRT-PCR were detected in the lung. As shown in Fig. 3I, DCC-5Fbn treatment of netrin-1 siRNA molecules in different organs including lungs (SI was associated with a decrease in metastasis compared with PBS Fig. 8A in SI Appendix). As shown in Fig. 3 A and B, after 9 days treatment. To address whether DCC-5Fbn treatment could also of injection, whereas scramble siRNA injected mice showed lung decrease macrometastasis, DCC-5Fbn was administered once mac-

Fitamant et al. PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 ͉ 4853 Downloaded by guest on October 1, 2021 rometastases were detected. As shown in Fig. 3J, DCC-5Fbn expression of the dependence receptor can be down-regulated, as significantly decreases lung macrometastasis. The antimetastatic extensively described for DCC and more recently for UNC5H (10, effect of DCC-5Fbn is, moreover, associated with increased mouse 12–14, 21). Second, the downstream death signaling can be shut survival as shown in Fig. 3K. down. We have recently shown that UNC5H2 proapoptotic activity As a second approach to evaluate the antimetastatic effect of relies on the binding of UNC5H2 to the serine/threonine DAPK DCC-5Fbn, we investigated whether spontaneous development of (22), a protein that was demonstrated to be involved in metastasis metastases in lung from a primary breast cancer could also be regulation and down-regulated in human malignancy (23). Here, we inhibited by DCC-5Fbn. We took advantage of a fresh human show that a third selective advantage for the tumor cell is the breast tumor, which was characterized as expressing a high level of self-production of the dependency ligand. One intriguing question netrin-1, and that, when xenografted in nude mice, was giving rise remains as to why breast tumors with metastatic propensity seem to spontaneous development of metastases in the lung. Once to have preferably selected netrin-1 self-production rather than tumors reached an optimal size after xenograft in nude mice, [i.e., receptor loss, whereas colorectal tumors have mostly selected loss lung colonization was detected at this stage (data not shown)], mice of the receptors rather than gain of netrin-1 expression—indeed, were treated daily with either PBS or DCC-5Fbn. As shown in Fig. only 7% of colorectal cancers show an increase of netrin-1 expres- 4, DCC-5Fbn treatment is associated with a significant decrease in sion (13, 15). A possible explanation is that netrin-1 expression not the number of aggressive metastases in lungs. Interestingly, this only confers a gain in survival to the disseminating cells, but also reduction in the number of metastases is associated with the possibly a gain in the nonapoptotic/positive signaling of netrin-1 detection of metastatic lesions that are much smaller and associated receptors. It is important to note that netrin-1 was originally with dense inflammatory reactions (Fig. 4 A and B), hence further described as a guidance cue (24), which, even though completely supporting the view that, in mice, the inhibition by DCC-5Fbn of the unproven, could play a role in the tropism of metastatic cells. Other prosurvival activity conferred by netrin-1 autocrine expression is proposed roles of netrin-1 include adhesion and morphogenesis associated with antimetastatic effect. regulation (25–27), both mechanisms that may be of importance for Discussion metastasis development. Similarly, netrin-1 was recently proposed Here, we show that netrin-1 expression may be considered as a to play a role during embryonic angiogenesis, and, even though marker of a breast tumor’s ability to disseminate. Most of the breast conflicting results have been published (28–32), we cannot, at this primary tumors with metastasis abilities showed elevated netrin-1 stage, rule out the role of netrin-1 as an angiogenic factor that expression. Both the human pathology and tumor cell line data and somehow could favor metastasis development at the secondary site. the mouse models described above support the view that this However, the gain of ‘‘positive’’ signaling by netrin-1 autocrine elevated netrin-1 level is a selective advantage acquired by the expression is probably not sufficient per se to promote metastasis. cancer cell to escape netrin-1-dependence receptor-induced apo- Indeed, when netrin-1 was forced in the nonmetastatic 67NR cells, ptosis and, consequently, to survive independently of netrin-1 we failed to observe any significant increase of lung metastasis after availability. From a mechanistic point of view, in human pathology, 67NR injection into mammary glands (SI Fig. 6 in SI Appendix). this autocrine expression of netrin-1 probably inhibits UNC5H- Thus, netrin-1 expression in tumor cells is probably not sufficient to induced cell death. Along this line, the antimetastatic effect of enable metastasis formation from the primary site but mainly netrin-1 siRNA in the 4T1 model is inhibited by injection of confers survival to metastatic cells. UNC5H1 and UNC5H2 siRNA (SI Fig. 8B in SI Appendix). Thus, These observations not only provide evidence for the importance DCC, which is barely detectable in the different groups (N0, N ϩ of ligand/dependence receptor pairs in the regulation of tumor M0, Mϩ) of breast cancers studied, is probably of relatively modest development, but also suggest a putative therapeutic strategy. interest in metastatic breast cancers. Thus, as predicted by the Indeed, as of today, there is no efficient treatment for patients with dependence receptor model, we have now shown that a tumor cell metastatic breast cancer, a lack of treatment that leads to the death can escape receptor dependency in at least three ways. First, of 400,000 women worldwide per year (33). Here, we propose that a treatment based on inhibition of the interaction between netrin-1 and its dependence receptors could positively effect a large fraction A 4 5. B of the patients suffering from metastatic breast cancer, i.e., patients

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N .0 5 Methods 0 SBP CCD - A detailed methods section is provided in SI Materials and Methods in SI nbF5 Appendix. Fig. 4. Disruption of the netrin-1 autocrine loop inhibits spontaneous lung Cell Line, Transfection, Immunoblotting, Immunoprecipitation, Plasmid Con- metastasis from a xenografted human breast tumor. (A) Antitumor effect of DCC-5Fbn in nude mice xenografted with a fresh human breast tumor. A human structs, siRNA, and Reagents. Human breast and mouse mammary cell lines listed breast tumor showing a high netrin-1 level was xenografted into nude mice, and were cultured by using standard procedures and were obtained from D. Birn- when the tumor reached a palpable size, the mice were i.v. treated daily with PBS baum (Institut National de la Santeét de la Recherche Scientifque U 599, Centre or DCC-5Fbn. Metastases were analyzed and enumerated by a pathologist. (B) de Recherche, Marseille, France) and F. Miller (Wayne State University, Detroit). Comparative low-power magnifications of lung metastases in control (a) and Transfection, immunoblotting, and immunoprecipitation were performed as DCC-5Fbn-treated (b) animals; tumor cells, characterized by large size, abundant described in SI Materials and Methods in SI Appendix and essentially as described cytoplasm, and irregular nucleus, are arranged in small masses of variable diam- (22). The different constructs are described in SI Materials and Methods in SI eter, scattered within the lung parenchyma. (c and d) Higher magnifications of Appendix. For cell culture use, scramble and netrin-1 siRNAs were designed by lung metastases in a DCC-5Fbn-treated animal. Metastatic cells, revealed by an Santa Cruz Biotechnology as a pool of three target-specific 20- to 25-nt siRNAs. anti-human cytokeratin 7 antibody, form a small mass surrounded by a dense For the in vivo approach, two different single netrin-1, DCC, UNC5H1, or UNC5H2 inflammatory reaction (c); at an advanced stage, tumor cells are dissociated by siRNAs were produced (Sigma–Proligo). DCC-5Fbn was produced upon expres- infiltrative inflammatory cells (d). (a and b) H&E staining, original magnifications sion in BL21 lysate and upon affinity chromatography using Flag-Sepharose ϫ200. (c and d) Immunoperoxidase, followed by nuclear counterstaining with (Sigma). DCC-EC-Fc was obtained fromR&DSystems and Netrin-1, UNC5H2-EC- Mayer’s hematoxylin, original magnification ϫ400. Fc, GST-DCC-5Fbn, and GST-FaDD from Apotech/Axxora.

4854 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709810105 Fitamant et al. Downloaded by guest on October 1, 2021 Human Breast Tumor Samples, QRT-PCR, and Immunohistology. Fifty-one human performed in a blinded fashion and according to standard procedures. Daily i.p breast cancer samples were provided by the tumor bank of the Centre León injection of scramble siRNA or netrin-1 siRNA (4 ␮g per mouse) (or DCC, UNC5H1, Be´rard. Fresh tissue of the tumor was obtained during breast surgery before any or UNC5H2) were performed according the procedure established by SeleXel systemic therapy and snap-frozen in liquid nitrogen. QRT-PCR was performed as Corp. DCC-5Fbn treatments were performed according the Fig. 3 legend. Xeno- described in SI Materials and Methods in SI Appendix and essentially as described grafts of fresh human breast tumors in nude mice were performed according the (13). The sequences of the primers used are available upon request. Paraffin- protocol developed by Xentech. Among 20 fresh breast tumors, the tumor BC174 embedded tumors tissues fixed in formalin were used for analysis. The patholo- expressing high levels of netrin-1 and of UNC5H receptors was selected. For each gist selected representative areas from breast carcinomas (N ϩ M0, Mϩ) or liver xenograft, when tumors reached 62 mm3, PBS or 1.25 ␮g per gram of body weight or uterine metastases. Immunostaining was performed as described in SI Mate- DCC-5Fbn were administered i.v. every day. rials and Methods in SI Appendix and essentially as described (13). ACKNOWLEDGMENTS. We thank O. Donze (Apotech, Lausanne, Switzerland) Cell Death Assays. The different cell death assays (trypan blue exclusion, MTT, for advice and reagents; F. Cabon, I. Puisieux, and C. Delloye for technical advice; caspase-3 activity assay) were performed as described in the supplementary J. Blachier, A. Potemski, I. Durand, N. Gadot, and S. Goddard-Leon for excellent materials and essentially as described (22, 34). technical help; F. Miller, J. Ade´laı¨de[Institut National de la Santeét de la Recher- che Scientifique (INSERM) U 599, Marsielle, France] C. Caux, D. Birnbaum (INSERM U 590, Lyon, France), and G. Grelier (INSERM U 590, Lyon, France) for materials; H. Tumor Mouse Models. A complete description of the mouse models is provided Bilak for text correction; A. Puisieux for helpful discussion; Xentech for human in SI Materials and Methods in SI Appendix. Briefly, for mammary gland injection breast tumor xenografts; and SeleXel for in vivo siRNA. This work was supported 6 5 of 67NR cells, 10 cells were injected. For i.v. injection, 10 (unless indicated) by an institutional grant from the Centre National de la Recherche Scientifique, 4T1-luc cells were injected into a tail vein, and mice were either killed or analyzed Centre LeónBe´ rard, University of Lyon, and from the Ligue Contre le Cancer, by using luminescence recording. Lungs were removed, and metastatic nodules Institut National du Cancer, Agence Nationale de la Recherche Blanche, and were counted and histological classification and grading of neoplastic lesions was European Union Specific Targeted Research Project Hermione.

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