"Widespread Requirement for Hedgehog Ligand Stimulation in Growth of Digestive Tract Tumours".

David M. Berman ^{1, 2, 3,} *, Sunil S. Karhadkar ^{1, 2,} *, Anirban Maitra ^2, *, Rocio Montes De Oca ¹, Meg R. Gerstenblith ¹, Kimberly Briggs ³, Antony R. Parker ², Yutaka Shimada ⁴, James R. Eshleman ², D. Neil Watkins ³ & Philip A. Beachy ¹

¹ Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
² Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
³ Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
⁴ Department of Surgery, Kyoto University, Kyoto 606-8507, Japan
* These authors contributed equally to this work

Correspondence and requests for materials should be addressed to P.A.B. (pbeachy@jhmi.edu)

Activation of the Hedgehog (Hh) signalling pathway by sporadic mutations or in familial conditions such as Gorlin's syndrome is associated with tumorigenesis in skin, the cerebellum and skeletal muscle. Here we show that a wide range of digestive tract tumours, including most of those originating in the oesophagus, stomach, biliary tract and pancreas, but not in the colon, display increased Hh pathway activity, which is suppressible
by cyclopamine, a Hh pathway antagonist. Cyclopamine also suppresses cell growth in vitro and causes durable regression of xenograft tumours in vivo. Unlike in Gorlin's syndrome tumours, pathway activity and cell growth in these digestive tract tumours are driven by endogenous expression of Hh ligands, as indicated by the presence of Sonic hedgehog and Indian hedgehog transcripts, by the pathway- and growth-inhibitory activity
of a Hh-neutralizing antibody, and by the dramatic growth-stimulatory activity of exogenously added Hh ligand. Our results identify a group of common lethal malignancies in which Hh pathway activity, essential for tumour growth, is activated not by mutation but by ligand expression.

The recent finding that Hh pathway activity is important for growth of a significant proportion of
small-cell lung cancers [7], a tumour type not associated with Gorlin's syndrome, suggested that other,
non-Gorlin's tumours might require Hh pathway activity for growth. We investigate here the role of
pathway activity in tumours derived from the gut, a tissue with prominent and diverse roles for Hh
signalling in developmental patterning, and in mature tissue homeostasis. A role in homeostasis is
suggested by the expression of Hh ligands and target genes in postnatal gut epithelium and
mesenchyme [8-11] (Fig. 1a).

Figure 1. Hh pathway activity in normal and neoplastic gut cells and tissues.

a, Intra-epithelial Hh pathway activation in mature murine gut tissues. Staining for b-galactosidase activity (blue) from the Ptch–lacZ gene reveals expression of the Hh target Ptch in epithelium (E) of the stomach (arrows indicate apparent parietal cells) and gall bladder (GB; arrow indicates basally oriented cell). Staining within stromal cells (S) was also seen.

b, Widespread expression of transcripts encoding Hh pathway components in digestive tract tumour cell lines. RT–PCR products showing expression of genes encoding Hh pathway ligands (SHH and IHH) and target genes (PTCH and GLI) in cell lines taken from sites shown in the diagram on the left. Red bars indicate the percentage of lines expressing detectable PTCH mRNA at each site. 

We began our examination of gut-derived tumours by testing for expression of Sonic hedgehog
(SHH) and Indian hedgehog (IHH), which encode members of the Hh ligand family that are
expressed in early endoderm and throughout gut development [10, 12]. We detected SHH and IHH
messenger RNA in 37 of 38 (97%) cell lines from oesophageal, stomach, biliary tract, pancreatic
and colon carcinomas (Fig. 1b). The Hh target genes PTCH and GLI were used as indicators of Hh
pathway activity and were co-expressed in most cell lines from oesophageal (4/6), stomach (6/6),
pancreatic (5/6) and biliary tract (5/9) tumours. By contrast, PTCH was not expressed in colon
tumour cell lines (0/11), although a few of these cell lines expressed GLI in the absence of PTCH.
The Hh pathway, however, is not active in these colon-tumour-derived cell lines, as indicated by our
reporter assays (see below).

Expression of PTCH and GLI within cells from several types of digestive tract tumours that also
express Hh ligands suggests the autonomous operation of an active signalling process. Autonomous
pathway activity was confirmed by the high level of luciferase activity produced by an exogenously
introduced Hh-inducible GLI–luciferase reporter [13] in all cell lines producing detectable PTCH
mRNA (Fig. 2a). Furthermore, Hh pathway activity in these cell lines was inhibited in a
dose-dependent manner by the Hh-pathway-specific antagonist cyclopamine, but not by tomatidine,
an inactive but structurally related compound [14] (Fig. 2a). These results suggest that high levels of Hh
pathway activity are a common feature of digestive tract tumours and prompted us to further
investigate the role of the pathway in tumour growth. We found that cyclopamine treatment reduced
the growth of tumour cell lines from the oesophagus, stomach, biliary tract and pancreas by 75–95%
compared with tomatidine controls (Fig. 2b). Strikingly, significant growth inhibition was observed
only in tumour lines expressing PTCH mRNA, confirming that the effects of cyclopamine treatment
are pathway specific rather than generally cytotoxic.

Figure 2. Cyclopamine suppression of Hh pathway activity and growth in digestive tract tumour cell lines correlates with expression of PTCH mRNA.

a, Normalized activity of transiently transfected Hh-responsive luciferase reporter and dose-dependent suppression by the Hh pathway antagonist cyclopamine.

b, Change in tumour cell viability measured by MTS (soluble tetrazolium salt) assay after culture in 3.0 µM cyclopamine or tomatidine (control). Bil, biliary tract. Note that the viability of PTCH mRNA-negative cells is not affected by cyclopamine. 

Figure 3. Hh pathway activity and requirement for growth of tumour cells in vivo.

a, Elevated PTCH mRNA in surgically resected pancreatic and gastric carcinomas was detected by quantitative RT–PCR and normalized to adjacent normal stomach (n = 10) and pancreas (n = 1) levels.

b, Normalized Hh-responsive reporter activity and suppression by 3.0 µM cyclopamine in first-passage pancreas carcinoma xenografts.

c, Corresponding reduction in viable tumour cells when cultured with 3.0 µM cyclopamine. Note that reduced viability is observed exclusively in lines with elevated Hh pathway activity.

d, Change in HUCCT1 human cholangiocarcinoma xenograft volume in mice treated for 22 days with vehicle (control; n = 9) or cyclopamine (n = 9). A durable response was observed for the full 98-day observation period after cessation of therapy.

e, Representative photographs of control and cyclopamine-treated mice. Note the full regression of the tumour on the lower right. 

To examine the role of Hh pathway activity in growth, we analysed pancreatic carcinomas that had
been passaged once as xenografts in nude mice then cultured and immediately assayed in vitro. Of
six such xenografts, four expressed PTCH mRNA (data not shown); two of these were a matched
pair of primary and metastatic tumours from a single patient. All four of these PTCH-expressing
primary xenografts expressed the GLI–luciferase reporter in a cyclopamine-sensitive manner (Fig.
3b). Cyclopamine treatment of these PTCH mRNA-expressing xenografts also resulted in
decreased viable cell mass (Fig. 3c), demonstrating more extreme cell-killing effects of Hh pathway
blockade than those observed in established tumour cell lines (Fig. 2b). By contrast, single-passage
xenografts lacking PTCH mRNA grew equally well in control and cyclopamine-containing media
(Fig. 3c), again confirming that cyclopamine effects are pathway specific rather than generally
cytotoxic.

To examine the effects of cyclopamine treatment in vivo, subcutaneous xenografts from HUCCT1
cells, a metastatic cholangiocarcinoma cell line, were established in athymic mice. After the tumours
had grown to an average size of 180 mm³, mice bearing these tumours were injected daily with
cyclopamine or vehicle, and control tumours continued to grow until animals were euthanized at 22
days with an average tumour volume exceeding 800 mm³ (Fig. 3d, e). Tumours in cyclopamine-treated
animals, by contrast, regressed completely by 12 days. Treatment continued for a further 10 days,
followed by an observation period of 98 days. Remarkably, all treatedtumours were grossly and
histologically undetectable at the end of the 3 month observation period, indicating a complete and durable tumoricidal effect of blocking the Hh pathway with cyclopamine (Fig. 3d, e). As previously reported for shorter treatment courses [7, 15], all mice survived cyclopamine treatment and the observation period with no obvious adverse effects.

These findings establish that the Hh pathway is widely activated in gut-derived tumours, and further
demonstrate a role for pathway activity in tumour cell growth in vitro and in vivo. Yet Gorlin's
syndrome is not associated with a higher incidence of gut-derived tumours, and PTCH mutations in
these tumours have not been reported, which suggests a distinct mechanism for Hh pathway
activation that does not involve mutation of pathway components. Given that SHH and IHH mRNA
are expressed in nearly all gut-derived tumours examined, we investigated the role of Hh ligand
binding in pathway activity. We measured Hh-inducible reporter activity in cells from tumours of the
oesophagus, stomach, pancreas and biliary tract treated with 5E1 monoclonal antibody [16], which
binds to Shh and Ihh ligands [17] and blocks signalling by disrupting ligand binding to Ptch [18].
Autonomous activation of the transfected reporter was not affected by the control antibody, but was
markedly reduced by incubation with 5E1 at 0.1 or 10 µg ml-1 (Fig. 4a and Supplementary Fig. 1a).
By contrast, reporter activity was augmented by around eightfold by the addition of purified Shh
ligand to a concentration of 25 nM. Addition of 5E1 in combination with Shh ligand reduced
reporter activity to a level intermediate between those produced with either reagent alone (Fig. 4a
and Supplementary Fig. 1a), which indicates a mutual antagonism between 5E1 antibody and Hh
ligand in activating the pathway.

Figure 4. Ligand dependence of Hh pathway activity and growth in digestive tract tumours.

a, Mutually antagonistic effects of the Hh ligand and blocking antibody on activity of the Hh reporter. The Hh-neutralizing 5E1 monoclonal antibody suppresses and Shh ligand increases reporter activity in HUCCT1 cells. Combined addition of antibody and ligand produces intermediate effects, depending on their relative concentrations.

b, Hh reporter activity in first-passage pancreatic carcinoma xenografts and dose-dependent suppression with 5E1 monoclonal antibody.

c, MTS assay showing reduced viability corresponding to Hh pathway suppression by 5E1 monoclonal antibody.

d, MTS assay showing growth (in arbitrary units) of PX184 first-passage PTCH-mRNA-expressing pancreas
xenograft cells cultured with the control antibody (dashed line) or with 5E1 monoclonal antibody at a level just sufficient to suppress growth (0.1 µg ml^-1; solid lines) and with the indicated concentrations of added Shh ligand.

e, f, Modulation of cell growth rate (in arbitrary units) by 5E1 antibody, Shh ligand and cyclopamine in single-passage pancreatic xenografts and in medulloblastoma cells (PZp53^MED1).

e, Shh ligand concentrations control growth rates of PTCH mRNA-positive pancreatic xenograft lines (average; n = 4) but not of PTCH mRNA-negative lines (n = 2) or of PZp53^MED1 cells.

f, Opposite responses to ligand and antibody of PX-184 cells, which express PTCH mRNA, and the lack of response of PX-169 and PZp53^MED1 cells, which respectively lack detectable Hh pathway activation or display constitutive pathway activation owing to lack of functional PTCH [15]. Cyclopamine, by contrast, blocks growth in cells with pathway activation because of either PTCH mutation or ligand stimulation. 

Reporter activity in cells from single-passage pancreatic cancer xenografts was also antagonized by
5E1 (Fig. 4b). In addition, treatment with the 5E1 antibody markedly reduced viable cell mass (Fig.4c).
This cell-killing effect and the reporter effect were observed exclusively in cells from tumours
that expressed endogenous PTCH mRNA. We further investigated the relationship between ligand
concentration and growth by adding 5E1 antibody to cells from a single-passage pancreatic tumour
xenograft at a level that was just sufficient to block growth. We then added Shh protein and found
that growth correlated positively with increasing concentrations (Fig. 4d). Rates of growth from this
experiment plotted as a function of Shh concentration (Fig. 4e) indicate that ligand-induced pathway
activation is rate limiting and that unperturbed growth of these cells is sub-maximal. Assays of
tumour cell lines derived from the oesophagus, stomach, pancreas and biliary tract yielded similar
results (Supplementary Fig. 1a–c), confirming a widespread requirement for Hh ligand in the growth
of these tumours.

The Hh ligand and 5E1 antibody are mutually antagonistic in their effects on reporter activity and
produce opposite effects on the growth of cells from these gut-derived tumours (Fig. 4a–e and
Supplementary Fig. 1a–c). Thus, pathway activation and cell growth must be dependent on Hh
ligand. By contrast, addition of neither Hh ligand nor 5E1 blocking antibody significantly affected the
growth of cells from a single-passage pancreatic tumour xenograft that did not express PTCH
mRNA (Fig. 4f), which demonstrates the specificity of antibody and ligand effects. We also
observed no significant ligand- or antibody-induced change in growth of medulloblastoma cells
derived from a mouse model of Gorlin's syndrome (Fig. 4f) in which the Hh pathway is activated
through loss of Ptch function [15, 19]. In contrast to antibody-resistant xenograft cells,
medulloblastoma-derived cells require pathway activity for growth and can be killed by cyclopamine
treatment [15] (Fig. 4f).

Ligand-independent mutational activation of the Hh pathway has been linked to the formation of
tumours, such as medulloblastoma, associated with Gorlin's syndrome. Despite a widespread
activation of and dependence on the Hh pathway for medulloblastoma growth [15], only a fraction of
sporadic tumours can be assigned to pathway-activating mutations, suggesting that other mechanisms
of pathway activation may be at play. Here we establish such a mechanism by showing that pathway
activation and growth of cells from a group of commonly lethal gut-derived malignancies is ligand
dependent. Small-cell lung cancer, also arising from endodermally derived epithelium and associated
with Hh ligand expression, has recently been linked to transient reactivation of the Hh pathway within
the airway epithelium, where it regulates progenitor cell fates during injury repair [7]. A similar role for
Hh signalling in renewal of mature digestive tract epithelium is suggested by expression of the Hh
pathway targets Ptch and Gli [9, 10] (Fig. 1a) and by the requirement for Hh signalling for proliferation
of gut progenitor cells [10]. It is not known whether renewal of injured gut epithelium is associated with
transient Hh pathway reactivation. However, increased rates of oesophageal, gastric and pancreatic
carcinomas occur in association with acid injury in Barrett's oesophagus, in Helicobacter pylori
infection, and with exposure to alcohol, cigarette smoke and certain dietary components [20-22].
Exposure to such factors probably causes injury to the gut epithelium, eliciting a chronic state of
injury repair and a consequent increase in proliferative stem or progenitor cells that may arise
through ligand-dependent reactivation of the Hh pathway. Many of these agents are also mutagenic,
thus potentially enhancing tumour formation by subjecting an enlarged pool of stem or stem-like
target cells to potentially oncogenic mutations. Our results identify a group of common and frequently
lethal gut-derived tumours, readily diagnosed by their expression of endogenous pathway targets
such as PTCH, which may respond to antagonist- or antibody-mediated pathway blockade, even at
advanced stages of metastatic disease.

Methods

Detection of -galactosidase expression Ptch–lacZ mice were killed at 4–6 weeks of age.
Staining was performed as described previously [7].

Tumour cells and tissues Origins and sources of our cells and tissues are described in the
Supplementary Table. First-passage pancreatic cancer xenografts were derived from freshly
harvested pancreaticoduodenectomy specimens as described previously [15]. In previous experiments,
approximately 65% of specimens yielded xenografts (data not shown), so we inferred that these
xenografts represent pancreatic tumours in the general population. The diagnosis of frozen samples
from gastric and pancreatic adenocarcinoma resections and adjacent normal stomach and pancreas
was microscopically confirmed by two pathologists (D.M.B. and A.M.), and RNA was prepared as
described previously [15].

RT–PCR Templates were prepared and amplified as described elsewhere15. For all primer pairs,
specificity was confirmed by sequencing of PCR products. For quantitative RT–PCR, 10% of the
first-strand reaction was amplified using IQ-SYBR Green Supermix, an i-cyclerIQ real-time
detection system (Bio-Rad) and specific oligonucleotide primers for PTCH or PGK
(phosphoglycerate kinase). Amplification was performed at 95 °C for 5 min followed by 40 cycles
of 10, 15 and 30 s at 95 °C, 55 °C and 75 °C, respectively. Bio-Rad software was used to
calculate threshold cycle (C_T) values for PTCH and for the housekeeping gene PGK. For each
sample, PTCH expression was derived from the ratio of PTCH to PGK levels using the formula
2^-deltaCT, where deltaC_T = C_T-PTCH–C_T-PGK. PTCH levels in tumours were presented as a ratio to
levels detected in adjacent normal tissue (Fig. 3a).

Hh-responsive reporter assays Hh-responsive firefly luciferase and control SV40 Renilla
luciferase reporter assays were performed on subconfluent triplicate cultures as described
previously [23]. Two days after transfection, culture medium was replaced for a 2-day culture period
with assay medium: RPMI-1640 (Bio-Whittaker) supplemented with 0.5% (established cell lines) or
20% (first-passage xenografts) FBS and containing combinations of 5E1 anti-Hh monoclonal
antibody, recombinant doubly lipid-modified Sonic hedgehog (ShhNp) peptide [13], cyclopamine
purified from Veratrum extract or tomatidine (ICN Pharmaceuticals) at the concentrations indicated
in the main text. Lysates were prepared and analysed as described elsewhere [13].

Proliferation assays Cells were cultured in triplicate in 96-well plates in assay media to which 5E1
monoclonal antibody, ShhNp and/or cyclopamine were added at 0 h at concentrations indicated in
the main text. Viable cell mass was determined by optical density measurements at 490 nm (OD₄₉₀)
at 2 and 4 days using the CellTiter96 (Promega) colorimetric assay. Relative growth was calculated
as OD (day 4) - OD (day 2)/OD (day 2).

Xenograft treatment In accordance with approved Johns Hopkins University Animal Care and
Use Committee protocols, HUCCT1 tumours (n = 18) were grown in athymic (nude) mice and
treated with cyclopamine (50 mg kg^-1 d^-1, subcutaneous injection) or control vehicle as described
previously [15].

Supplementary information accompanies this paper.

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Acknowledgements. We thank E. Traband and K. Young for technical assistance, E.
Montgomery, K. Miyazaki and J. Harmon for cell lines, J. Chen for help with cyclopamine
purification and P. Fussell for help with figures. This work was supported by the family of Margaret
Lee and grants from the National Institutes of Health (D.M.B., A.M., J.R.E. and P.A.B.). P.A.B. is
an investigator and M.R.G. a medical fellow of the Howard Hughes Medical Institute.

Competing interests statement. The authors declare competing financial interests.
Declaration of competing financial interests

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

Declaration: Under a licensing agreement between Curis, Inc. and the Johns Hopkins University, P. A. Beachy and the University hold equity in Curis and are entitled to a share of royalties from sales of the products described in this article. P. A. Beachy also receives payment and equity for service as a consultant to Curis, Inc. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

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