SUPPLEMENTARY INFORMATION GUIDE · S5 Supplementary Figure 1. LacZ activity in brain cells and lung adenocarcinoma cells in Ptc1lacZ/+ mice. a() Cerebellar granule cells strongly

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SUPPLEMENTARY INFORMATION GUIDE

A CRUCIAL REQUIREMENT FOR HEDGEHOG SIGNALING IN SMALL CELL LUNG CANCER

Kwon-Sik Park1,2§, Luciano G. Martelotto5§, Martin Peifer6, Martin L. Sos6,7, Anthony N. Karnezis9, Moe R. Mahjoub2,3, Katie Bernard1,2, Jamie Conklin1,2, Anette Szczepny5, Jing Yuan10, Ribo Guo10, Beatrice

Ospina10, Jeanette Falzon12, Samara Bennett12, Tracey J. Brown12, Ana Markovic11, Wendy L. Devereux11, Cory A. Ocasio4, James K. Chen4, Tim Stearns2,3, Roman K. Thomas6,7,8, Marion Dorsch10, Silvia

Buonamici10, D. Neil Watkins5, Craig D. Peacock11§* & Julien Sage1,2§*.

Departments of 1Pediatrics, 2Genetics, 3Biology, and 4Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA. 5Monash Institute of Medical Research, Monash University, Clayton 3168,

Victoria, Australia. 6Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, 7Department

I of Internal Medicine and Center of Integrated Oncology Köln-Bonn, and 8Laboratory of Translational Cancer Genomics, Center of Integrated Oncology Köln-Bonn, University of Cologne, 50924 Cologne, Germany.9University of California San Francisco Department of Pathology, San Francisco, CA 94143, USA. 10Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA. 11Sidney Kimmel

Cancer Centre, Johns Hopkins University School of Medicine, Baltimore, MD 21231. 12Department of Biochemistry and Molecular Biology, Monash University, Clayton 3168, Victoria, Australia

§ Equal contribution * To whom correspondence should be addressed. E-mail: [email protected] and [email protected]

Supplementary Discussion pages S2-S3

Supplementary Methods pages S4

Supplementary Figures 1 to 8 pages S5-S12

Supplementary Tables 1 to 2 + Primers list pages S13-S15

Supplementary References pages S16-S17

Nature Medicine doi:10.1038/nm.2473

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Supplementary Discussion

While the critical role of Hh signaling has been demonstrated in tumor types driven by activating

mutations in the Hh pathway, the functional role and the mode of action of the Hh pathway in solid

tumor cells without mutations in Hh pathway genes is only beginning to be understood. For a synopsis

of the roles of Hh signaling in normal and malignant blood cells (including the key role of the Hh

pathway in CML driven by the oncogenic BCR-ABL fusion protein) please refer to recent reviews 1,2.

Human colorectal cancer (CRC) cell lines were shown to be sensitive to Cyclopamine treatment in

culture, although the effects were variable 3. More recently, Varnat and colleagues confirmed that the

Hh-Gli pathway is essential for CRC stem cells and long-term propagation of colon cancer 4. Activation

of Gli signaling may be more important for tumor progression, especially as tumors start to spread 5.

Studies by the Beachy lab showed that the Hh pathway may have a cell autonomous role in the

expansion of digestive tract tumor cells (pancreas, stomach, oesophagus, biliary tract) but not CRC cells,

in contrast to the previous studies 6. Recently, the Ruiz i Altaba lab further examined the role of Hh

signaling in a mouse model of CRC driven by activation of the Wnt pathway. In this context,

widespread Smo deletion rescues the lethality of Apc-mutant mice 7. These data suggest that cell

autonomous activation of the Hh pathway in CRC cells may not be universal, as might be expected

given the diversity of CRC subtypes, but that CRC maintenance may depend on Hh signaling, especially

in advanced tumors.

In pancreas cancer, work from the Hebrok lab showed that Cyclopamine induced apoptosis and

blocked proliferation in a subset of human pancreatic cancer cell lines both in vitro and in vivo 8,

suggesting a cell autonomous role for Hh in tumor maintenance. In mice, forced expression of active

Gli2 in pancreas epithelial cells promoted cancer development initiated by oncogenic Ras or alone;

however, over-expression of Gli does not produce pancreas cancer, rather an undifferentiated carcinoma 9,10. These data suggest a cell autonomous role for Gli in pancreas cancer initiation but these effects may

be independent of canonical Hh signaling 11. It is also possible that tumors expressing oncogenic Ras

shift the role for Hh from autocrine to paracrine 12, rendering the interpretation of data in Ras-driven

tumors challenging. Clearly, there is an important function for Hh signaling in pancreas cancer via a

non-cell autonomous mechanism, including influencing the responsiveness of tumor cells to

chemotherapy 13. Similarly, De Sauvage and colleagues showed that the major mode of action of the Hh

pathway in several cancer types, including pancreas cancer, was paracrine 14. Together, these data point

to both cell-autonomous and non-cell autonomous roles for Hh signaling in pancreas cancer, the relative

contribution of each probably depends on tumor stage and the cellular context.


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Data with human prostate cancer cells have indicated a cell autonomous role for Hh signaling 15. But

this observation is not applicable to all cell lines 16 and evidence suggests that in several cases the

mechanism of action may be paracrine 17. On the other hand, correlative evidence also suggests that a

more prominent autocrine role for Hh signaling in prostate cancer may occur during tumor progression 18. Ectopic expression of Gli1 can initiate the unlimited proliferation of mouse prostate epithelial

progenitors, suggesting a role for Gli in prostate cancer initiation 19. Similar to pancreas and colorectal

cancer, some advanced prostate tumors activate the Hh pathway, which may play a cell autonomous role

for tumor maintenance in at least a fraction of cases.

In addition to these three carcinomas, emerging evidence also indicates a role for the Hh pathway and

Gli transcription factors in tumor maintenance in melanoma 20,21, breast cancer 22-24, neuroblastoma 25,

lung cancer 26,27, brain cancer 28,29, and rhabdoid tumors 30.

In conclusion, increasing evidence indicates that a least a subset of a large number of solid tumors

displays cell autonomous activation of the Hh pathway and that Hh signaling may play a critical role in

the maintenance of these tumors. Our data in genetically engineered mice demonstrate a role for Hh

signalling in SCLC initiation, suggesting that the Hh pathway could also play a critical role in cancer

initiation in more cases than initially suspected. Finally, our observations that inhibition of the Hh

pathway cooperates with chemotherapy to delay SCLC recurrence may also be applicable to all these

different tumor types.


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Supplementary Methods

Clonogenicity assays with human SCLC Cells. Cells were grown for 7 days in the presence of NVP-

LDE225 (Novartis), rShh ligand (StemRD and R&D Systems) or the anti-Shh antibody 5E1

(Developmental Studies Hybridoma Bank, University of Iowa). Control cultures contained vehicle alone

or an irrelevant mouse IgG1 isotype (eBioscience, Cat# 16-4714-82). Alternatively, LX22CL cells were

incubated for 4 days following transduction by Mission® shRNA lentiviral particles targeting human

Smo (Sigma-Aldrich), or an adenovirus encoding a constitutively active Smo mutant 31. Non-targeting

shRNA lentiviral particles (SHC002V) and an AdβGal vector served as controls, respectively. Cells

were washed after treatment, counted and plated at three different densities (500-2500/dish) in 1.1 ml of

Methocult®H4100 (Stemcell Technologies), reconstituted in supplemented Advanced RPMI plus 10%

FBS. 14-18 days after plating, SCLC colonies were scored using an inverted microscope. Dishes

containing 30-150 colonies were selected for re-passage after gentle disaggregation in Accumax

(eBioscience). All of the above experimental procedures were duplicated in the subset of LX22CL cells

surviving on day 4 of recovery, following 2 days of culture in 0.25 µg ml-1 etoposide and 0.5 µg ml-1

carboplatin (Calbiochem).

RT-PCR and quantitative PCR (qPCR). RNA was prepared using Trizol (Invitrogen). 1 µg of RNA

was used for reverse-transcription (RT), and 1/20 volume of the RT reaction mixture was used for each

PCR and qPCR reaction. qPCR was performed with an ABI Prism HT-7900 machine (Applied

Biosystems). The SYBR® Green reaction mixture was used according to the manufacturer’s protocol

(Invitrogen). Annealing and extension of PCR were performed at 60 °C. Forty cycles of PCR were

performed for the all the primers used. The sequences of primers for qPCR were obtained using the

ProbeFinder software in the Universal ProbeLibrary assay design centre (Roche Applied Science).

Primer sequences are shown in the Primers list page S15.

Genomic analysis – CGH arrays. Tumor DNA and normal tissue DNA was extracted from explanted

Rb1/Trp53/Rbl2 mutant mouse tumors or adjacent non-tumoral mouse tissue respectively, using the

Gentra DNA-extraction kit (Qiagen, Gentra Puregene). The samples were digested, marked with

fluorescent dyes (Cy3 tumor; Cy5 normal), and hybridized to Agilent aCGH 4x180K mouse arrays

according to instructions of the manufacturer. Arrays were scanned using an Agilent G2565CA scanner

and raw copy number were computed by the ratio between the intensities of the Cy3 and Cy5 color-

channels of each probe. Next, the baseline was adjusted such that the genome-wide average over the raw

copy numbers yields two. Finally, raw copy numbers were segmented by using the circular-binary

segmentation algorithm 32.


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Supplementary Figure 1. LacZ activity in brain cells and lung adenocarcinoma cells in Ptc1lacZ/+ mice. (a)

Cerebellar granule cells strongly positive for lacZ expression (blue signal, black arrow) are observed along with

cells showing weak, perinuclear staining (white arrow) in sections from the brain of Ptc1lacZ/+ mice. (b) A

magnified view of the dotted rectangle in a. (c, d) No cells stain positive for X-gal in two non-small cell lung

cancer lesions growing in the lungs of K-RasLSL-G12D/Ptc1lacZ/+ mice 16 weeks after AdCre delivery. Arrowheads

point to the stromal cells that normally stain for lacZ activity in Ptc1lacZ/+ mice even in the absence of any tumors

(not shown). Sections were counterstained with nuclear fast red (pink). Tu: tumor.


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Supplementary Figure 2. Heterogeneous X-gal staining in the lung epithelium of Ptc1lacZ/+ mice. (a) The

majority of small lesions (defined as proliferating clusters of Syp+ neuroendocrine cells that cannot yet be defined

as parenchymal tumors) stain positive for X-gal (inset: Syp, green; DAPI, blue), while the majority of

neuroendocrine bodies (NEB) and isolated pulmonary neuroendocrine cells (PNEC) stain negative for X-gal. The

black and white arrows point to the same areas in the panel and the inset. (b) Dual staining for Synaptophysin

(Syp) (green in left panels) and X-gal (blue in right panels) shows that a subset of Syp+ pulmonary

neuroendocrine cells (PNEC) displays lacZ activity (upper right) while others are negative (lower right). DAPI

stains cell nuclei in blue. The arrowheads point to the same stromal cell adjacent to the neuroendocrine cells.

Dotted areas indicate the same areas. Quantification of this analysis is shown in Fig. 1b. (c) In the upper airway, a

number of tracheal epithelial cells stain positive for X-gal (arrow), while in the bronchiolar epithelium X-gal+

cells are nearly exclusively stromal cells underlying epithelial cells (arrowhead).


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Supplementary Figure 3. Expression of Hh pathway components in mSCLC cells. (a) Immunoblot analysis

for some members of the Hh pathway in primary mSCLC tumors and cell lines. Extracts from Shh-LIGHT2 cells

treated with conditioned media from 293 cells (Con) or ShhN-293 cells (ShhN) and from wild-type whole lung

are shown as controls. Gli3A, activator form of Gli3; Gli3R, repressor form of Gli3. Tubulin serves as a loading

control. (b) Representative immunohistochemistry (brown staining) for Cgrp (a neuroendocrine marker) and for

Gli2 on lung sections from Rb1/Trp53 mutant mice. Left, normal lung area. Right, tumor area. Sections were

counterstained with hematoxylin. Note that while we could detect Gli1 mRNA levels by RT-PCR (see Figs. 1 and

3), we were not able to reproducibly detect the Gli1 protein by immunoblot or immunohistochemistry.


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Supplementary Figure 4. Expression of the SmoM2-YFP transgene in the lungs of Rb1/Trp53 mutant mice.

(a) Immunostaining for Syp (green) in double and triple mutant animals shows that the tumors (Tu) examined are

neuroendocrine. Immunostaining for YFP (green, arrow) shows a signal at the membrane of tumor cells in the

triple mutant animals only (and non-specific, low cytoplasmic staining in sections from both double and triple

mutant tumors). DAPI (blue) stains cell nuclei. (b, c) RT-qPCR analysis for YFP and Gli1 levels in Rb1/Trp53

and Rb1/Trp53/SmoM2 mutant tumors. (C) The number of tumors found in the same animal from which one

tumor was tested for mRNA levels is indicated. *, the number of tumors was not determined in this animal. nd,

not detected.


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Supplementary Figure 5. Histopathological analysis of Rb1/Trp53 and Rb1/Trp53/SmoM2 lung tumors.

Lung sections from Rb1/Trp53 (left panels) and Rb1/Trp53/SmoM2 (right panels) mutant mice 8 months after

AdCre infection were counterstained with hematoxylin and eosin and examined by a trained pathologist (A. N.

K.). Similar patterns are present in mice from both genotypes: trabecular (a-b), nested/insular (c-d), peripheral

palisading (e-f) and solid (g-h). Dotted circles in E-F highlight ependymal and Flexner-Wintersteiner rosettes.

Tumors from both genotypes showed lymphatic invasion and tumor cell necrosis (data not shown).


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Supplementary Figure 6. Activation of Hh signaling does not bypass the requirement for loss of Rb1 in

SCLC development. Lung tumors (Tu) that develop in Rb1/Trp53/SmoM2 mutant mice (upper panels) stain

positive for the neuroendocrine marker Synaptophysin (green) and negative for the alveolar marker SP-C (green).

In contrast, lung tumors (Tu) that develop in Rb1het/Trp53/SmoM2 mutant mice (lower panels) stain negative for

Synaptophysin and positive for SP-C; these tumors have histopathological features of lung adenocarcinoma (data

not shown). DAPI (blue) stains cell nuclei.


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Supplementary Figure 7. Pharmacological inhibition of Hh signaling in mSCLC cells in culture and in vivo.

(a) Cell viability measured by the MTT assay for two independent mSCLC cell lines (derived from Rb1/Trp53

mutant mice) treated with Cyclopamine (Cyc, 10 µM), Tomatidine (10 µM), or vehicle control (Con) for 4 days

(n=3). (b) RT-qPCR analysis for Gli1, using RNA from cells treated with Cyc, Tom, and CON for 24 hours

(n=3). (c) Cell viability measured by the MTT assay for two independent mSCLC cell lines treated with GANT61

or Con for 4 days (n=3). (d) RT-qPCR analysis for Gli1, using RNA from cells treated with GANT61 and CON

for 24 hours (n=3). (e) Representative graph of the effects of NVP-LDE225 treatment on mSCLC primary cell

lines grown in 0.5% and 10% serum (advanced RPMI). The two cell lines used are derived from an Rb1/Trp53

mutant mouse (KP2) and from an Rb1/Trp53/Rbl2 mutant mouse (KP13). (f) Representative pictures of

immunostaining for PH3 and CC3 in SCLC sections in Rb1/Trp53 mutant mice treated with vehicle control or

Cyc following the protocol shown in Fig. 3c. The arrows point to positive cells, the asterisk points to a non-

specific auto-fluorescent area. Quantification of this staining is shown in Figure 3d. Similar immunostaining and

quantification were used for Fig. 2f (data not shown).


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Supplementary Figure 8. Increased Hh pathway activity in SCLC cells following chemotherapy. (a)

Expression of Shh determined by immunohistochemistry in LX22 xenografts, before and after treatment with

platinum-etoposide chemotherapy. Specific staining is shown in brown, with a hematoxylin counterstain in blue.

(b) Localisation of Gli2 in LX22 xenografts before and after the same chemotherapy treatment. Confocal analysis

of cells stained for DAPI (blue) and Gli2. (c) Primary cilia in chemoresistant LX22 xenograft cells in vivo.

Primary cilia identified by confocal analysis of cells stained for acetylated Tubulin (AcT), gamma Tubulin (gT)

and DAPI (blue) in the upper panel. A similar cell is shown in the lower panel counterstained for Synaptophysin

(Syp). (d) Representative histology (H&E) of LX22 xenografts never treated with chemotherapeutic agents

(“Naïve”) or recurring after chemotherapy (“Resistant”, as in Fig. 4d). Note the histopathological similarities

between the two tumors, composed of small round blue cells. Brown inset: immunostaining for CGRP (brown), a

neuroendocrine marker. The scale bar represents 50 µm.


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Primers list: primers used for quantitative RT-PCR analysis of gene expression

Forward Reverse

Shh 5’-TTAAATGCCTTGGCCATCTC-3’ 5’-CCACGGAGTTCTCTGCTTTC-3’

Smo 5’-GCCCAAGTGTGAGAATGACCG-3’ 5’-GGTTGTCTGTTCGCACCAAGG-3’

Gli1 5’-CCTGGTGGCTTTCATCAACT-3’ 5’-GCTAGACATGTCCCCTTCCA-3’

Ptc1 5’-TACGTGGAGGTGGTTCATCA-3’ 5’-CCTGAGTTGTCGCAGCATTA-3’

Cgrp 5’-GCATGGCCACTCTCAGTGAAG-3’ 5’-TTATCTGTTCAGGCCTGAAGG-3’

Gapdh 5’-CTTTACCACCATGGAGAAGGC-3’ 5’-GGCATGGACTGTGGTCATGAG-3’

ARPP P0 5’-GATGCCCAGGGAAGACAG-3’ 5’-ACAATGAAGCATTTTGGATAATCA-3’

Gli1 5’-TGGAGGTCTGCGTGGTAGA-3’ 5’-TTGAACATGGCGTCTCAGG-3’

YFP 5’-ACTACAACAGCCACAACGTCTATATCA-3’ 5’-GGCGGATCTTGAAGTTCACC-3’


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Supplementary References

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3. Qualtrough, D., Buda, A., Gaffield, W., Williams, A.C. & Paraskeva, C. Hedgehog signalling in colorectal tumour cells: induction of apoptosis with cyclopamine treatment. Int J Cancer 110, 831-837 (2004).

4. Varnat, F., et al. Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion. EMBO Mol Med 1, 338-351 (2009).

5. Varnat, F., Siegl-Cachedenier, I., Malerba, M., Gervaz, P. & Ruiz i Altaba, A. Loss of WNT-TCF addiction and enhancement of HH-GLI1 signalling define the metastatic transition of human colon carcinomas. EMBO Mol Med 2, 440-457 (2010).

6. Berman, D.M., et al. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425, 846-851 (2003).

7. Varnat, F., Zacchetti, G. & Ruiz i Altaba, A. Hedgehog pathway activity is required for the lethality and intestinal phenotypes of mice with hyperactive Wnt signaling. Mech Dev 127, 73-81 (2010).

8. Thayer, S.P., et al. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851-856 (2003).

9. Pasca di Magliano, M., et al. Hedgehog/Ras interactions regulate early stages of pancreatic cancer. Genes Dev 20, 3161-3173 (2006).

10. Lau, J., Kawahira, H. & Hebrok, M. Hedgehog signaling in pancreas development and disease. Cell Mol Life Sci 63, 642-652 (2006).

11. Nolan-Stevaux, O., et al. GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation. Genes Dev 23, 24-36 (2009).

12. Lauth, M., et al. DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS. Nat Struct Mol Biol 17, 718-725 (2010).

13. Olive, K.P., et al. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 1457-1461 (2009).

14. Yauch, R.L., et al. A paracrine requirement for hedgehog signalling in cancer. Nature 455, 406-410 (2008).

15. Sanchez, P., et al. Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc Natl Acad Sci U S A 101, 12561-12566 (2004).

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17. Shaw, A., Gipp, J. & Bushman, W. The Sonic Hedgehog pathway stimulates prostate tumor growth by paracrine signaling and recapitulates embryonic gene expression in tumor myofibroblasts. Oncogene 28, 4480-4490 (2009).

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19. Karhadkar, S.S., et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431, 707-712 (2004).


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20. Stecca, B., et al. Melanomas require HEDGEHOG-GLI signaling regulated by interactions between GLI1 and the RAS-MEK/AKT pathways. Proc Natl Acad Sci U S A 104, 5895-5900 (2007).

21. Alexaki, V.I., et al. GLI2-mediated melanoma invasion and metastasis. J Natl Cancer Inst 102, 1148-1159 (2010).

22. Mukherjee, S., et al. Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer. Cancer Biol Ther 5, 674-683 (2006).

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25. Mao, L., et al. A critical role of Sonic Hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells. Cancer Sci 100, 1848-1855 (2009).

26. Yuan, Z., et al. Frequent requirement of hedgehog signaling in non-small cell lung carcinoma. Oncogene 26, 1046-1055 (2007).

27. Singh, S., et al. Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity. Cancer Res (2011).

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Documents

SUPPLEMENTARY INFORMATION GUIDE · S5 Supplementary Figure 1. LacZ activity in brain cells and lung adenocarcinoma cells in Ptc1lacZ/+ mice. a() Cerebellar granule cells strongly