Colon cancer cells produce immunoregulatory glucocorticoids
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First publ. in: Oncogene ; 30 (2011), 21. - pp. 2411-2419
Colon cancer cells produce immunoregulatory glucocorticoids
D Sidler', P Renzulli 2 ,5, C Schnoz' , B Berger' , S Schneider-Jakob', C Fluck'>, D Inderbitzin 2 ,
N Corazza', D Candinas 2 and T Brunner,,4
I Division of Experimental Pathology, Institute of Pathology, University of Bem, Bern, Switzerland; }Department of Visceral Surgery
and Medicine, Insel University Hospital, University of Bern, Bem, Switzerland; J Division of Pediatric Endocrinology, University
Children's Ho~pital, University of Bern, Bern, Switzerland and 'Division of Biochemical Pharmacology, Department of Biolo(JY,
University of Konstan z, Konstanz, Germany
Glucocorticoids (GC) have important anti-inflammatory Introduction
and pro-apoptotic activities. Initially thought to be
exclusively produced by the adrenal glands, there is Colorectal cancer (CRC) is one of the leading causes of
now increasing evidence for extra-adrenal sources of GCs. death in the Western world . It develops through a series
We have previously shown that the intestinal epithelium of genetic mutations in pluripotent stem cells in the
produces immunoregulatory GCs and that intestinal intestinal epithelial crypts (Davies el al., 2005). Many
steroidogenesis is regulated by the nuclear receptor cancer cells aberrantly overexpress cellu lar proteins or
liver receptor homolog-l (LRH-l). As LRH-l has been neo-antigens, which can be recognized by the immune
implicated in the development of colon cancer, we here system and provoke anti-tumor immune responses (van
investigated whether LRH-l regulates GC synthesis in den Broek el al., 1996; Smyth el al., 2000; Shankaran
colorectal tumors and whether tumor-produced GCs el al., 200 1). Also, CRC cells are often immunogenic and
suppress T-cell activation. Colorectal cancer cell lines can stimulate the activation of tumor-specific lympho-
and primary tumors were found to express steroidogenic cytes (Camus et al., 2009). Therefore, anti-tumor
enzymes and regulatory factors required for the de IIOVO immune responses may significantly contribute to tumor
synthesis of cortisol. Both cell lines and primary tumors immune surveillance and limit tumor development
constitutively produced readily detectable levels of and growth. Leukocytes frequently infiltrate neoplastic
cortisol, as measured by radioimmunoassay, thin-layer tissue indicating ongoing anti-tumoral immune res-
chromatography and bioassay. Whereas overexpression of ponses, yet their specific role in tumor survei llance is
LRH-l significantly increased the expression of steroido- unclear. Activation of tumor-infiltrating immune cells
genic enzymes and the synthesis of cortisol, downregula- and the release of pro-inflammatory mediators may not
tion or inhibition of LRH-l effectively suppressed only control tumor development but also, in the
these processes, indicating an important role of LRH-l contrary, stimulate angiogenesis and thereby accelerate
in colorectal tumor GC synthesis. An immunoregulatory tumor growth. However, recent studies by Pages el al.,
role of tumor-derived GCs could be further confirmed 2005 and Galon el al., 20Q6 revealed that the number of
by demonstrating a suppression of T-cell activation. This tumor-infiltrating lymphocytes in CRC directly corre-
study describes for the first time cortisol synthesis in a lates with a better prognosis and longer survival of CRC
non-endocrine tumor in humans, and suggests that the patients. This finding indicates that anti-CRC immune
synthesis of bioactive GCs in colon cancer cells may responses are likely substantially contributing to tumor
account as a novel mechanism of tumor immune escape. surveillance. Still, in most patients, the anti-tumor
immune response seems to be unable to control CRC
growth and metastasis formation in a long term.
Immune escape mechanisms have been recognized as
Keywords: extra-adrenal steroidogenesis; colon cancer; one of the hallmarks of cancer (Hanahan and Weinberg,
immune escape; nuclear receptors 2000; Finn, 2008) and may significantly limit the efficacy
of anti-tumor immune responses.
Glucocorticoids (GC) are lipid hormones that are
critically involved in the regulation of stress responses,
metabolism and immune homeostasis (Sapolsky el al. ,
2000). The adrenal glands are the major source of GC in
the body. However, different reports indicate that GC
Correspondence: Professor T Brunner, Division of Biochemical synthesis is not restricted to the adrenal glands but may
Pharmacology, Department of Biology, University of Konstanz, Box also occur at other sites of the body (Noti el al., 2009),
660, Universitiitsstrasse 10, 78457 Konstanz, Germany. for example, the thymus (Vacchio el al., 1994), the brain
E-mail: thomas.brunner@ uni-konstanz.de (MacKenzie et al., 2000; Davies and MacKenzie, 2003)
' Current address: Department of Surgery, Cantonal Hospital ,
M linsterlingen 8596, Switzerland
and the vascular endothelium (Takeda et al., 1994).
Recently, we have characterized the intestinal epithe-
lium as a major source of immunoregulatory GC
Konstanzer Online-Publikations-System (KOPS)
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-1423612
(Cima el aI., 2004; Mueller el al., 2006, 2007; Atanasov a b
et aI., 2008). In mice, intestinal GC synthesis is highly 6 6
regulated and is induced upon activation of immune ",
~>
cells. In turn, intestinal GCs have been shown to exert ~ 4 J!! 4
.J!!
paracrine activities and to contribute to the regulation ~ ~
of intestinal immune cell homeostasis. Thus, inice with z
0:: ~
E 2 E 2
defective intestinal GC synthesis show enhanced anti-
gen-induced activation of intestinal T cells (Cima el al., e e
2004) and accelerated induction of experimental inflam- 0 0
matory bowel disease (Coste et al., 2007). Caco2 HT29 LS174T Caco2 HT29LS174T
The synthesis of GC from cholesterol involves a series C d
of enzymatic steps catalyzed by steroidogenic enzymes 0.5 300
til ",
of the cytochrome P450 (CYP) and the hydroxysteroid ~ 0.4 ~
dehydrogenase family (Miller, 2008). In the intestine, OIl J!! 200
steroidogenesis is critically regulated by the nuclear ;: 0.3 ~
z z
receptor and transcription factor liver receptor homo- ~ 0.2 0::
log- I (LRH-I, NR5A2; Mueller el at., 2006, 2007; Coste E 100
et at., 2007). Consequently, absence of LRH-I results in ~ 0.1 e
strongly reduced intestinal GC synthesis (Mueller el at., 0.0 0
2006) and enhanced susceptibility to intestinal inflam- Caco2 HT29LS174T Caco2 HT29 LS174T
matory processes (Coste et at., 2007). The function of Figure 1 Colon carcinoma cell lines express steroidogenic factors.
LRH- I in the intestinal crypts is, however, not limited to Detection of the steroidogenic enzymes CYP I IAI (a), CYPI 7 (b),
GC synthesis, but further extends to the control of cell CY PJI BI (e) and LRJ-/-I (d) by quantitative reverse transcripti on
(RT)- PC R in the colon cancer cell lines Caco2, HT29 and LS 174T.
cycle and tumorigenesis (Botrugno et at., 2004; Schoon- Mea n values of triplicates ± s. d. of a typical experiment out of two
jans el al., 2005). Accordingly, LRH- I haplo-insufficient are shown .
mice have reduced tumor formation in a mouse model
for intestinal cancer (Schoonjans el al., 2005) .
Here, we demonstrate that CRC cell lines and primary
tumors express steroidogenic enzymes and produce
bioactive Gc. LRH-I was found to be abundantly (Mueller el at., 2007). The expression of the steroid-
expressed in CRC cells and to critically regulate steroido- producing enzyme machinery and the synthesis of
genesis in tumor cells. Finally, we provide evidence cortisol in the adrenals are highly dependent on the
that tumor cell-produced GCs inhibit the activation of nuclear factor steroidogenic factor-I (SF-I, NR5A I;
T lymphocytes. These findings indicate that CRC cells Parker et al., 2002). In contrast, SF-I seems to be
have a strong steroidogenic potential and that tumor- dispensa ble for steroid synthesis in the intestinal
derived GC may contribute to tumor immune evasion. epithelium and is substituted by LRH- l. To assess the
role of LRH-I in steroidogenic enzyme expression in
CRC cells, Cac02 cells were transiently transfected with
Results an LRH- I expression plasmid , and the promoter activity
of the steroidogenic enzyme genes CYP11AI, CYP17
Coton cancer cell tines express steroidogenic Jactors and C YP 11 B 1 was analyzed by luciferase reporter
transcription Jactor SF-I assays (Supplementary Figure I). Interestingly, Cac02
GCs are produced from cholesterol via a series of cells demonstrated an already very high basal CYPllAI
enzymatic steps involving enzymes of the CYP and the and CYP 17 activity (fold induction over empty vector
hydroxysteroid dehydrogenase family. Most of these control), confirming the basal transcription of these
enzymes are transcriptionally regulated by tissue-specific enzymes seen in Figures la-d. Increasing amounts of
nuclear receptors, such as LRH-I in the intestine LRH - I expression plasmid dose-dependently induced
(Miller, 2008). We thus examined the expression of the reporter constructs for these steroidogenic enzymes .
LRH-I and different steroidogenic enzymes required for Stimulation of the cells with phorbol ester phorbol
GC synthesis in various established CRC cell lines by 12-myristate 13-acetate (PMA) further enhanced the
reverse transcription- PCR. Figures la- d illustrate that promoter activity of these genes, likely due to activation
the steroidogenic enzymes CYPIIAI and CYPIIBI were of LRH-I transcriptional activity (Lee et al., 2006;
expressed at relatively high levels in HT29, Cac02 and Mueller et al., 2007). The stimulatory activity of PMA
LS 174T cells, whereas CY 17 was not detected in LS 174T was confinued when endogenous expression of steroido-
cells. Similarly, LRH- I was expressed at high levels in genic enzymes was analyzed in Cac02 and HT29 cells.
Cac02 and HT29 cells, but was absent in LS174T cells. Treatment of cells with PMA induced CYP11AI and
CYPllBI expression in Cac02 cells and CYPll A I
LRH-I overexpression is sufficient 10 induce steroidogenic expression in HT29 cells, whereas LRH-I expression
enzyme expression in CRC cell lines levels remained unchanged in both cell lines , in
Regulation of steroid synthesis in the adrenal agreement with a PMA-induced activation of LRH - J
glands differs substantially from that in the intestine transcriptional activity (Supplementary Figure 2).3
Expression of steroidogenic enzymes in CRC cell lines is cell lines was thus analyzed for the presence of cortisol
dependent on endogenous LRH-J by different techniques. In a first step, Caco2 and HT29
To investigate whether endogenous LRH- l regulates the cells were cultured with '4C-labeled progesterone and the
basal expression of the steroidogenic enzyme in CRC conversion to '4C-cortisol over time was analyzed by
cell lines, LRH-l expression in HT29 and Cac02 cells thin-layer chromatography. Starting after 2 hand
was downregulated using RNA interference. Figure 2 further increasing over 24 h, a decrease in progesterone
shows that transfection of HT29 cells with LRH- I- and a gradual accumulation of steroi d intermediates was
specific small interfering RNA resulted in ~ 55% observed in both cell lines. Some of these products were
reduction of LRH-I expression compared with control identified as l70H-progesterone and deoxycortisol.
treated cells. Analysis of steroidogenic enzyme expres- Critically, already after 2 h, cortisol was detectable in
sion revealed that downregulation of LRH-l resulted in both cell lines, confirming that CRC cell lines are
reduced CYPllAJ, CYPJ7 and CYPllBJ expression, capable of producing GC (Supplementary Figure 3).
indicating that LRH-I is involved in the basal transcrip- We next analyzed cortisol production in a quantita-
tion of these steroidogenic enzymes. In contrast, the tive ma nner. Caco2 cells were cultured in the presence or
expression of an unrelated gene product, for example, absence of the GC synthesis inhibitor metyrapone,
retinoblastoma protein Rb, was not affected by LRH-I and cortisol production in the cell-free supernatant
downregulation . Similar findings were obtained with was measured by radioimmunoassay. Interestingly,
Cac02 cells (data not shown) . Caco2 cells secreted a considerable and readily detect-
able amount of cortisol into the supernatant. Cortisol
synthesis was blocked with metyrapone, confirming the
Colon cancer cel/lines metabolize steroid precursors de novo synthesis of GC (Figure 3a). Intriguingly and in
and produce cortisol agreement with the steroidogenic enzyme expression
The basal a nd inducible expression of steroidogenic pattern (Figures la- d), GC synthesis was not uniform
enzymes suggested that CRC cells are actually capable for all colon cancer cell lines. Caco2 and HT29 cells were
of producing cortisol. The culture supernatant of CRC found to produce significant amounts of cortisol
a b 1.5
1.5
LRH-1 Rb a 0.2 b 0.1 2
c: c Cortisol
0 0
n 1.0
:::I
:0
:::I
1.0 '0
III
'0en ** *
"0 "0 ~ ~ 0.08
.5 c 0 0
0.5 :0 0.5 (.) 0.1 (.)
"0
:e :e ea; :§Cl 0.04
c: c:
0.0 0.0
siControl slLRH1 siControl siLRH1 0.0 0.00 ~.t::.t::.t::.c:.c:.c:
Control MET
C d .§oo~oo~oo~
1.5 1.5
..Q -N-' -en- -t--
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u
N
t- V
,..,.
0 c III :x: ...
0
ti 1.0 +:I 1.0 (.) ~
:::I :::I
"0
"0
.5 .E c d 0_3
"0' 0.5 :5! 0.5 1.5 lCO;::ti;O:t-:'"--,
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0.0 0.0
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o
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*
sl.C ontrol siLRH1 siControl slLRH1 (.) (.)
e :§Cl 0.5 :§ 0.1
1.5 Cl
c:
c:
c 0.0 0.0
0
:g 1.0 o 0.75 1.5 siControl sILRH·1
"D
.5 f,lg LRH-1
:5! 0.5 Figure 3 Colon cancer cells produce cortisol in an LRH-l-
.e dependent man ner. (a) Cac02 cells were cultured in the absence
or presence of the GC synthesis inhibitor metyrapone (Met) for
0.0 24 h, and the presence of cortisol in the supernatant was meas ured
siControl siLRH1 by radioimmunoassay. (b) Cac02 , HT29 a nd LSl74T cells
Figure 2 The expression of steroid ogen ic enzymes is dependent on were cultured for 8 a nd 24 h, a nd co rtisol in the supernatant was
endogenous LRH-1. HT29 cells were transfected with control small analyzed. (e) Cac02 cells were transfected with increasing a mou nts
interfering RNA (siRNA) or siRNA for LRH-l, and the expression of LRH - l ex pression plasmid , and cortisol production was
of LRH-l (a), Rb (b), C YPII A I (e), C YPI 7 (d) and CYPIIBI (e) measured . (d) Cac02 cells were treated with control siRNA
was measured by quantitative RT- PCR. Mean values of triplica- (siControl) or LRH-l-specific siRNA (siLRH - l) , and cortisol
tes ± s.d. of a typical experiment out of three are shown. • P4
although no specific corti sol synthesis was detected in Human CRC specimens express steroidogenic factors
LSI 74T cells (Figure 3b). We next aimed at confirming our data generate d in C RC
We next investigated the role of LRH-l in tumor cell lines in primary colon carcinoma, and comparing
cell-derived GC synthesis. A further increase in basal them to the normal colonic mucosa. Tissue specimens
cortisol production was observed when Cac02 cells from the center or the edge of CRC, and the normal
were transfected with a LRH - l expression plasmid mucosa were thus collected from 26 individual tumor
(Figure 3c). Furthermore, downregulation of LRH-l patients, and the expression of steroidogenic factors was
using RNA interference resulted in a substantial analyzed by quantitative reverse transcription- PCR
reduction of basal cortisol synthesis (Figure 3d), (Figure 4). Interestingly, steroidogenic enzyme genes
indicating that LRH-I is critically involved in the CYPIlAI and CYPIlBl, and regulatory factors such as
regulation of cortisol synthesis in CRC cell lines. The LRH - I and steroid acute regulatory protein (StAR)
critical role of LRH-l was further confirmed in LSI74T were detectable in the majority of the ti ssues analyzed .
cells. As shown in Figure I b, LSI74T cells do not CYP IlBl , StAR and LRH- l were found to be
express detectable levels of LRH- I and only a restricted expressed at higher levels either in the tumor edge or
pattern of steroidogenic enzymes. Overexpression the tumor center, when compared with leve ls seen in
of LRH-l in LSl74T cells led to a strong induction of normal mucosa, though differences in LRH-l and StAR
CYP17 and CYPIlBI expression and a substantial expression were not statistically significant (Figures 4b-
release of cortisol, indicating that LRH-l is a master d) . StAR is involved in the delivery of cholesterol to the
regulator of GC synthesis in CRC cells (Supplementary mitochondria and thus a prerequisite for effective
Figure 4). steroid synthesis. In contrast, CYP 11 A 1 expression
The capacity of CRC cell lines to produce cortisol was was in average significantly higher in the untransformed
also assessed in a G C bioassay. HEK 293T cells were tissue compared with individual tumor samples
transiently transfected with a GC receptor response (Figure 4a). Next to the relative expression, we also
element luciferase reporter construct and a GC receptor analyzed the frequencies of CYPIIAI, CYPl1 Bl, StAR
expression plasmid, as described elsewhere (Pazirandeh and LRH- I expression in tumor and normal tissue
e/ al., 1999). Incubation with increasing concentrations samples (Figures 4e- h) . This revealed that C YP 11 AI,
of cortisol led to a dose-dependent induction of the CYP 11 Bl and StAR were generally more frequently
reporter construct, that is, the GC receptor-dependent expressed in tumor samples than in normal tissue,
induction of luciferase activity . Similarly, supernatant whereas LRH-I was uniformly expressed in the majority
of NCI-H295R (positive control), Cac02 and HT29 of tumor and normal tissue samples.
induced luciferase activity, reflecting a considerable GC The expression pattern of LRH-I in normal colonic
bioactivity in the supernatant of CRC cells (Supple- mucosa and CRC was also analyzed by immunohisto-
mentary Figure 5). Critically, the GC bioactivity of all chemistry. As reported previously, LRH-l was found
the three cell lines was completely blocked when cells to be expressed in the nucleus and to be restricted to the
were cultured with metyrapone. These findings clearly intestinal crypts of the normal intestinal mucosa
demonstrate that CRC cell lines produce bioactive Gc. (Supplementary Figure 6A), in agreement with its
a c
........
b d
..
!~
~ ~
20 2.0
CYP11A1 CYP11B1
* -*--*-
~
~
.!
15 *-- 1.5
30000
~ ~
'E"
Z
a::
10
5
1,0
20000
0.5
1000:
~ 0 0.0
Center Edge. center Edge Conter Edge Center Edge
Tumor Mucosa Tumor Mucosa T'umor Mucosa Tumor Mucosa
e f 9 h
1_~
il'100% 100% 100%
....
c::
::J 75'-. 75% 15% 15%
~ 50% 50% 50% 50%
"0
'fl 25% 25% 25% 25%
...~ W. 0% 0% Q%
Center Edge
Conter Edge Center Edge Center Edge
Tumor Mucosa Tumor Mucosa Tumor Mucosa Tumor Mucosa
Figure 4 Prima ry hum an colorectal tum ors express stero idogenic factors . Tissue specimens from colorecta l tumor center, tumor edge
a nd no rmal colonic mucosa of 26 patients were iso lated and gene expressio n of C Y 11 Al (a), C YP 11 B f (b), StA R (e) and LRH-l (d)
was measured by qua ntitative p e R. The relative expression levels in tissue samples are show n. Samples from individual patients are
connected by li nes (tumor center, tumor edge and normal mucosa). • P < O.05 . (c-h) Shows the frequency of sa mples with detectable
(black bar) versus undetectable (white bar) gene expression for CY ll A f (e), C YPllBI (I), StAR (g) a nd LRH- l (1I).5
important role in crypt cell proliferation and epithelial the activation marker, which could be reversed by
cell renewal (Botrugno el al., 2004; Schoonjans el al., treatment of cells with the GC receptor antagonist
2005). Although LRH-l also had a nuclear expression RU-486 (Figure 6a; Cima el al., 2004, 2006). Culture of
pattern in primary CRC specimens, it was expressed T cells with the supernatant from Cac02 cells resulted in
uniformly throughout the tumor (Supplementary Figure a substantial inhibition of activation-induced CD69
6B). A similar finding was made in liver metastases of expression, which was blocked by RU-486, confirming
CRC (Supplementary Figure 6C). the presence of bioactive GC in the supernatant
(Figure 6b). As GCs are also potent inducers of apop-
tosis, we also tested the activity of GC in the Cac02 cell
Human colon cancer specimens produce bioaclive culture supernatant on the cell death induction in
GC ex vivo murine thymocytes . Figures 6d- e show that GC in the
To provide evidence for cortisol synthesis in primary tumor cell culture supernatant induced thymocyte
human CRC tissue, we established tissue cultures of apoptosis in a GC receptor-dependent manner.
CRC specimens and normal colonic mucosa and We next analyzed the supernatant of primary CRC
analyzed the culture supernatant for the presence of tumor culture for the presence of immunoregulatory
GC using a GC bioassay. Interestingly, we could
demonstrate that human colon cancer specimens pro-
duced bioactive GC, whereas normal mucosa lacked this
capacity under basal conditions. Importantly, treatment a c
of tissue cultures with metyrapone resulted in a clear b 50
reduction of GC bioactivity, indicating that GCs were C04+ sptenocytes
40
produced in situ. GC synthesis in normal colonic tissue
could be induced by treatment with PMA, whereas it 30 ----
* *
could not be further increased in the CRC tissue 20
(Figure 5), likely reflecting the pre-activated status of
steroidogenesis in colon cancer cells. 10
o 0
C069 (log)
+
Tumor cell-derived GCs suppress T-cell activation and
RU486 .:....::..:..:.:":"
06
a cytokine transforming growth factor-p and inhibits local
CD4+ splenocytes immune cell activation (Chen et a/., 1998; Wahl el al.,
c:::
2006). In addition , Fas ligand is abundantly expressed in
2h o the retina and cornea, and can induce apoptosis in
E
.c infiltrating leukocytes during inflammatory reactions
:c (Griffith and Ferguson, 1997). Various tumors are even
.5
'$.
better known for displaying a plethora of mechanisms
that help them to escape from the prosecution by the
immune system. These include the secretion of immu-
RU486 -=--.!..-=--.!.. nosuppressive cytokines, such as interleukin- I 0 and
CDS9 (log) transforming growth factor-p, reduced antigen presen-
Oh 2h
tation by downregulation of MHC molecules, condi-
b tioning of the environment and immune cells, and the
CD8+ splenocytes 50 CD8+ expression of pro-apoptotic molecules, such as Fas
c 40 * ligand and TRAIL (Igney and Krammer, 2002). This
2h o altogether leads to a 'stealth effect', which helps the
E
.c 30 tumor to limit or avoid destruction by cytotoxic
~ 20 lymphocytes.
In this study, we now show a novel and so far
'$. 10
unrecognized potential mechanism of immune evasion
o by colon carcinomas via the synthesis and release of
RU486 - + - + bioactive immunosuppressive Gc. We found that colon
Oh 2h carcinoma cell lines as well as primary tumors express
the enzymatic steroidogenic machinery, produce cortisol
Figure 7 Primary CRC release immunoregulatory Gc. Primary
CRC tumor tissue was cultured for 0 and 2 h, and cell-free sterile and can regulate T-cell activation. Although steroid
filtered supernatant was harvested . Splenic T cells were activated production has been previously shown in tumors derived
a nd exposed to the tumor culture supernatant, in the presence or from primary steroidogenic organs, such as adrenals,
absence of the GC receptor antagonist RU486. CD69 expression testis and ovaries, our present study demonstrates for
on activated CD4 + (a) and CD8 + (b) T cells were analyzed. The
left panels show examples for typica l CD69 expression profiles by
the first time the synthesis of cortisol by a tumor derived
flow cytometry, the right panels show quantitative data of culture from a non-endocrine tissue . Furthermore, we provide
supernatant-mediated inhibition ofCD69 expression (% inhibition evidence that CRC-derived GC can regulate the activa-
relative to stimulated splenocytes without tumor cell supernatant). tion of immune cells and thereby could potentially
Inserts show isotype control staining (solid line) and CD69 staining contribute to tumor immune evasion.
(doted line) of activated T cells.
Of interest is the fact that some but not all the tested
colon carcinoma cell lines expressed the majority of
Gc. Supernatant from tumor samples was harvested enzymes required for the de novo synthesis of cortisol.
directly or after 2 h, and exposed to stimulated murine The expression pattern directly correlated with the
T cells. CD69 expression in CD4 + and CD8 + T-cell capacity of these CRC cell lines to produce cortisol.
subsets was analyzed. Figure 7 shows that the expression This may reflect a previous selection process in vivo,
of CD69 in CD4 + (Figure 7a) and CD8 + T cells where the synthesis of immunoregulatory GC may have
(Figure 7b) was substantially reduced when cells were given the tumor a certain advantage over non-steroid-
exposed to the culture supernatant of primary CRC producing tumor cells. Although the analysis of
tumors. Importantly, this inhibition could be reversed steroidogenic factor expression in primary tumors
by blocking the GC receptor with RU486 (Cima et al., revealed a rather heterogeneous picture, with high and
2004, 2006). low expressors, the release of immunoregulatory GC
into the tumor environment could significantly limit
anti-tumor immune responses and favor tumor growth.
In line with this notion are recent findings demonstrat-
Discussion ing that the degree of tumor infiltration by memory
CD8 + T cells strongly correlates with the overall
While the orchestrated activation of cells of the innate survival of CRC patients (Pages el al., 2005; Galon
and adaptive immune system is essential for the defense el al., 2006), indicating that anti-tumor immune
of the body from invasion by potentially harmful responses are able to significantly limit tumor growth .
pathogens, their uncontrolled action often leads to Clearly, tumors that can limit or prevent anti-tumor
tissue damage and loss of vital organ functions. There- immune responses will develop more aggressively and
fore, different tissues and organs have developed will cause a more rapid death of the patient. GC
mechanisms that limit uncontrolled immune responses synthesis by CRC, as demonstrated in this study, may
and associated tissue damage, and thereby become substantially contribute to these processes.
'immunologically privileged'. This is particularly evident We have previously identified the nuclear receptor
in organs that are essential for survival or reproduction. and transcription factor LRH-l as a critical regulator
For example, the eye secretes the immunoregulatory of intestinal GC synthesis (Mueller el al., 2006, 2007;7
Coste el a/., 2007). Also, in human colon carcinoma cell Sweden; Pazirandeh et al., 1999). The expression plasmids for
lines, LRH- I was found to have an essential role in human LRH-I was kind ly provided by Kristina Schoonjans
the transcriptional regulation of steroidogenesis. Down- (Ecole Po lytechnique Federale de Lausanne EPFL, Lausanne,
regulation or inhibition of LRH-l in CRC cell lines Switzerland).
significantly reduced the expression of steroidogenic
enzymes and the ability to produce cortisol. Interest- Detection ofsteroidogellic enzyme genes alld LRH-J mRNA by
ingly, LRH-I seems to have at least a dual role in CRe. quantitative reverse trallscriptioll- PCR
While we here clearly show that LRH-l has a prominent Tissue specimens were obtained from patients undergoing
function in the regulation of GC synthesis, previous surgery for primary CRC at the Department of Visceral
Surgery and Medicine, Insel University Hospital, University of
findings also demonstrated that LRH-l regulates the Bern, Switzerland. All patients provided informed consent and
expression of cell-cycle proteins (Botrugno el at., 2004) experiments were reviewed by an institutional review board.
and thereby might be involved in the development of Tissue samples from the tumor center, the tumor edge and
intestinal tumors (Schoonjans et al. , 2005). As LRH-I non-cancerous norma l mucosa, ~ 10 cm from the tumor edge,
haplo-insufficient mice have reduced tumor develop- were collected. Tissues and CRC cell lines were homogenized
ment in two different models of intestinal tumor in RNA isolation reagent, RNA was isola ted and complemen-
formation, it is likely that LRH-l contributes to CRC tary DNA using a HighCapacity RT reaction kit (Qiagen
development via the regulation of cell-cycle progression, (Hombrechtikon, Switzerland). The expression of C YP 11 A J,
in addition to the here-proposed function in tumor CYPI1BJ, as well as StAR and LRH-I was analyzed by
immune escape via the synthesis of Ge. In support of quantitative PCR on an Applied Biosystems Real-time PCR
7500 machine using SYBR green and Quantitect primer assays
this notion is a recent finding that the LRH-l antagonist (Qiagen) . Gene expression was normalized by GAPOH RNA
small heterodimer partner (SHP) is frequently down- levels and plotted as relative mRNA levels.
regulated in hepatocellular carcinoma by epigenetic
silencing, which is associated with increased tumor
Lucilerase reporter assays
growth (He el at., 2008). Also, in CRC uniform LRH-I Luciferase reporter constructs and ~-ga l actosidase expression
expression was frequently observed, whereas in the vector for transfection control were transiently transfected into
normal mucosa it remained restricted to the proliferat- Cac02 cells by lipofection. The amount of total transfected
ing cells of the crypts. DNA was normalized with the corresponding empty vector
In summary, we demonstrated for the first time that plasmid. At 24 h after transfection, cell Iysates were assessed
CRC cell lines and primary tumor specimens express for luciferase and ~-galactosidase activity as previously
steroidogenic factors and produce immunoregulatory described (Mueller et 01., 2007; Atanasov et al., 2008). In
Ge. Our findings further suggest that tumor-derived some experiments, cells were stimulated with PMA (Calbio-
GC may modulate or suppress anti-tumor immune chern) as indicated for 16 h before luciferase assay. Values were
responses and thereby contribute to tumor immune calculated as fold induction over activity of the corresponding
control vector and plotted as mean ± s.d.
escape. Given the important role of LRH-l in both
tumor cell-cycle progression and GC synthesis, the
specific inhibition of this nuclear receptor and transcrip- RNA intelj"erence
tion factor may become an interesting target in CRC Cac02 and HT29 cells were plated in six-well plates. Cells were
transfected with 10nM small interfering RNA for LRH-I
therapy. (siLRH - I) or control, small interfering RNA (siControl;
Ohm'macon ON-TARGET plus SMARTpool, Thermo Fisher
Scientific, Lafayette, CO, USA) using HiPerfect transfection
Materials and methods reagents (Qiagen). This treatment was repeated after 24 h.
After 48 h, cells were harvested and mRNA expression levels
Cells and reagents for LRH - I and steroidogenic enzymes were assessed by
The human embryonic kidney fibrob last cell line 293T (CRL- quantitative PCR.
11268), the human colon cancer cell lines Cac02 (A TCC HTB-
37), HT29 (ATCC HTB-38), T84 (ATCC CCL-248), Col0205 Ex vivo tissue culture of tumor and mucosal tissue
(ATCC CCL-222) and LSI74T (ATCC CCL-188), and Cancerous and non-malignant human colon tissue samples
the human adrenal cancer cell line NCI-H295R (ATCC were cut into small pieces « 2 mm diameter), washed in
CRL-2 128) were obtained from the American Type Culture
phosphate-buffered saline, distributed equally in 24-well plates
Collection (Manassas, VA). Cells were cultured in Isocove's and incubated with Isocove's modified Oulbecco's medium
modified Oulbecco's medium containing 10% fetal calf serum, containing 10% steroid-free fetal calf serum at 37 °C. In some
2 mM glutamine and 50 Ilg/ml gentamycin at 37 °C and 5% experiments, GC synthesis was either stimulated by adding
CO 2 , All reagents were from Sigma-Aldrich (Buchs, Switzer- PMA or blocked by addition of 200 Ilg/ml metyrapone to the
land), unless stated otherwise.
organ culture as previously described (Cima et al., 2004). After
2- 16h, supernatant was harvested , heat inactivated and ex vivo
Plasm ids
The luciferase reporter constructs for the genomic promoter GC synthesis was analyzed .
sequences of CYPI1AJ, CYPJ7, CYPI1BJ, as well as the
expression plasm ids for ~-galactosidase, have been described Detection of steroidogenesis by thin-layer chromatography
previously (Mueller et al., 2006, 2007; Kempna et al., 2007; Cac02 and HT29 cells were cultured in six-well plates and
Atanasov et al., 2008). The reporter p lasm id GRE2tk and the 200000 c.p.m. I·C-Iabeled progesterone ([4- '4C]-progesterone,
expression plasmid for GC receptor SVG R I were kindly 55 mCi/mmo l, American Radiolabeled Chemicals Inc. ,
provided by Sam Okret (Karolinska Institute, Stockholm, St Louis, MO, USA) was added per well. Cell supernatant8
was harves ted at various time points and steroids were Apoptosis induc/ion thymocytes
extracted as described elsewhere (Dardis and Miller, 2003). Thymocytes were isolated from 7-week-o ld CL57/BL6 mice
Samples were then separated by thin-thin- Iayer chroma- and cultured in 96-well plates in the presence of control
tography using methylene chloride:methano le:H 20 (200:30: I) medium, cortisol standard or supernatant from 8 h Cac02
as solvent system. Steroids were visualized on a Storm tumor cell culture. In some experiments, GC action was
PhosphorImager (GE Healthcare, Little Chalfont, UK). an tagonized by adding 30 ng/ml RU486. Cells were harvested
The identity of the radioactive steroid spots was confirmed after 16 hand apop tosis was measured by Annexin V binding
by co-chromatography, wit h steroid standards as indicated. and flow cytometry.
GC bioassay S/atistical analysis
HEK 293T cells were transiently transfected with GRE2kt GC Statistical analyses were done using Prism 5 software.
receptor reporter plasmid and the SVG R I GC receptor Student's t-test wa s used for the comparison of two
expression plasmid using the calci um phosphate precipitation independent gro ups, analysis of variance test with Bonferroni
method. Then, heat-inactivated supernatant from CRC cell correction for the comparison of three and more independent
lines o r ex vivo tumor cultures was added as indicated for 8 h. groups. Values < 0.05 were cons idered significant.
Cortisol titration was used as standard. Reporter cells were
lysed and luciferase activities were determined. Values were
calculated as fold induction over basal promoter activity Abbreviations
and were plotted as mea n ± s. d .
CRC, colorecta l carcinoma; CYP, cytochrome P450; GC,
Cortisol radioimmunoassay gl ucocorticoids; LRH- I, li ver receptor homolog- I/NR5A2;
Colon cancer cell lines were plated in six-well plates a nd were SF-I, steroid ogenic factor-I /NR5AI; StAR , steroid acute
a ll owed to ad here. In some experiments, LRH-I was either regulatory pro tein.
si lenced by small interfering RNA-mediated gene knock down
or overexpressed as described a bove. Then, medium was
changed and incubated for the indicated time points. Cortisol
concentrations in culture supernatan t from cell lines were Conflict of interest
measured by radioimmunoassay using an adapted commercial
cortisol assay (C8409, Sigma-Aldrich). Cortisol concentration The authors declare no conflict of interest.
was ca lculated from a cortisol standard curve and plotted as
mean ± s.d.
Acknowledgements
T- cell activation assay
Murine splenocytes from a C57BI/6 mouse were incubated We thank Mario Noti, Pamela Bianchi, Andrea Hirsch,
with standard concen trations of cortisol or heat-inactivated Gabi Hofer and Andreas Kappeler for technical help and
supern atant from Cac02, HT29 or NCI-H295R cells. In advice, the members of the Division of C linical Histopathol-
some experiments, splenocytes were pre-incubated with the ogy for preparation of tissue sections, and Sam Okret and
GC receptor antagonist 100nM RU486 (Brunner et al., 200 1). Kristina Schoo nj ans for reagents. This work was supported by
After 2 h, cells were stimulated with Il!g/m l concanavalin A research grants from Oncosuisse (OCS-02025-02-2007) and the
for 20 h, and CD69 expression on CD4 + T cells (antibodies Swiss National Science Fo und ation (310000-121854) to TB.
from eBiosciences, San Diego, CA, USA) was detected by flow DS was supported by an MD-PhD fellowship from the Swiss
cytometry. National Science Foundation .
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