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THE ROLE OF SEX HORMONE-BINDING GLOBULIN (SHBG) IN THE ANDROGEN RESPONSE OF HUMAN PROSTATE CANCER CELLS
Yu-Hua Li1, Atif M. Nakhla 2,3, Daniel J. Hryb2,3,6, Richard A. Friedman7, Jenny Z. Xiang4, Nicholas Wagar1 William Rosner2,5, Nicholas A. Romas2,3,6, David Klatt2 and Scott M. Kahn2,3,6
Yerkes National Primate Research Center Microarray Core, Emory University, Atlanta, Ga.1; Dept. of Microbiology and Immunology, Weill Medical College, Cornell University, New York, N.Y.4
Departments of Medicine2 and Urology3, St. Luke's/Roosevelt Hospital Center, and Departments of Medicine5 and Urology6, and Biomedical Informatics7, College of Physicians and Surgeons, Columbia University, New York, N.Y
ABSTRACT
METHODOLOGY AND RESULTS
Sex Hormone Binding Globulin (SHBG) is a multifunctional protein that not only regulates the
concentration of free androgens and estrogens in plasma through its steroid binding properties, it
also regulates membrane based steroid hormone signaling in the prostate through its specific
membrane receptor (RSHBG), independent of the androgen receptor (AR). The SHBG gene locus on
human chromosome 17p13.1, is only 30kb away from the p53 gene, and is thus a target for allelic
deletions in tumors from many tissue types, including the prostate. Because it is now established that
the prostate makes its own SHBG mRNA and protein, it is of great interest to ascertain the normal
biologic function of SHBG in the prostate, and to address whether aberrant SHBG expression
contributes to the progression of prostate cancer. To ascertain the effects that SHBG exerts on
androgen signaling in prostate cells, we established an inducible cell line (L5S2) that can be
stimulated to reproducibly overexpress SHBG, and its isogenic sister vector control (L5V4).
Microarray analysis was performed on induced L5S2 cells in the absence and presence of
dihydrotestosterone (DHT) (the latter simulating conditions required for RSHBG activation), as well as
on mock treated cells. L5V4 vector control cells were similarly treated and examined. Upon
analysis, SHBG was found to modulate the response of LNCaP cells to androgens in a manner
consistent with two distinct modes of action, 1) through RSHBG signaling, and 2) by influencing
androgen receptor (AR) signaling, implying that locally expressed SHBG in the prostate has both
autocrine/paracrine and intracellular effects on androgen signaling. These studies lay the
groundwork for further detailed characterizations of the biologic pathways controlled by SHBG in
the prostate and the role of aberrant SHBG expression in prostate cancer.
Cell lines and treatment conditions:
The inducible L5S2 clonal cell line (which expresses SHBG in response to PonA) is indirectly derived from LNCaP cells, through an intermediate
cell line, L5. L5 was generated by stably transfecting LNCaP cells with the plasmid, pVgRXR which encodes a hybrid transactivator that is activated
by PonA. This transactivator recognizes and directs transcription from a promoter within a second plasmid, pINDhygro. L5S2 was generated by
stably transfecting L5 cells with a pINDhygro construct that contains the full length human SHBG cDNA coding sequence cloned directly
downstream of the inducible promoter. The L5V4 vector control cell line was generated by stably transfecting L5 cells with the empty vector,
pINDhygro.
Although detailed studies of AR function have greatly expanded our knowledge of how androgens act, new information about androgen action has
been constrained by the relative neglect of mechanisms of androgen action not mediated by AR. It is these alternative androgen signaling
pathways that could hold the key for why current prostate cancer treatment strategies are often lacking. One such pathway is based at the plasma
membrane and involves SHBG and its receptor, RSHBG. SHBG, which was originally described as a protein in plasma that bound estrogens and
androgens, is now known to participate in a membrane based steroid signaling pathway in certain hormonally responsive tissues, including the
prostate. A model of the initial steps of SHBG signaling in prostate cells is shown in Figure 1. RSHBG is a biochemically characterized, high
affinity receptor for SHBG, present on the outer cell membrane.
When RSHBG is occupied by SHBG, subsequent binding of specific
steroids such as DHT induces a signaling pathway that generates
the second messenger cAMP, and activates protein kinase A
(SHBG, when prebound to DHT, can not bind to RSHBG,). Little is
known about the downstream effects of RSHBG activation in
prostate cells and the biologic functions of androgen signaling
through RSHBG.
SHBG mRNA and protein has been detected in normal prostate
tissue, prostate tumors and prostate cancer cell lines. Given the
known properties attributed to SHBG, it is plausible to speculate
that, in the prostate, endogenously made SHBG acts in an
autocrine/paracrine manner to activate RSHBG, and that it binds
steroids intracellularly. To investigate the function of
endogenously made SHBG in prostate cells, we established an in
vitro system, consisting of L5S2, an inducible clonal cell line
derived from LNCaP cells, that overexpresses SHBG when treated
with Ponasterone A (PonA), and its isogenic noniducible sister
vector control cell line, L5V4. A series of microarray assays was
performed to examine the effects of SHBG, in the presence and
absence of androgen, on overall gene expression in LNCaP cells.
We show evidence of genes whose expression is modulated in a
manner consistent with RSHBG activation, and evidence that SHBG
can affect the expression of androgen responsive genes.
Total Number of
Induced Genes
Total Number of
Repressed Genes
Genes Induced
> 1.5-fold
Genes Repressed
<0.66-fold expression
1250
1770
665
1068
Note: L5V4 vector control cells treated with PonA and DHT induced gene expression in a manner consistent with data in the NCBI microarray repository on DHT-treated LNCaP cells
L5V4 and L5S2 cells were each seeded into two groups of multiple six well plates for 24 h. One group was treated with the inducing agent, PonA
(10mM), and the other with an equal volume of ethanol, for 24 h. Triplicate wells from the PonA-treated cells were treated for an additional 24 h with
either carrier or 10 nM DHT, giving six treatment conditionsVector(control)
- L5V4 vector control cells treated with carrier alone (mock treated)
Vector(PonA)
- L5V4 vector control cells treated with 10uM PonA 24 hrs
Vector(PonA+DHT)
- L5V4 vector control cells treated with 10uM PonA 24 hrs, then with 10nM DHT 24 hrs
L5S2(control)
- L5S2 inducible SHBG cells treated with carrier alone (mock treated)
L5S2(PonA)
- L5S2 inducible SHBG cells treated with 10uM PonA 24 hrs
L5S2(PonA+DHT)
- L5S2 inducible SHBG cells treated with 10uM PonA 24 hrs, then with 10nM DHT 24 hrs
Total RNAs were isolated and their integrities were verified on an Agilent Bioanalyzer. Each triplicate RNA preparation was used for microarray
analysis. First-strand cDNAs were synthesized from 5 µg of each RNA sample. After RNase H-mediated second-stranded cDNA synthesis, doublestranded cDNAs were purified and biotinylated complementary RNAs (cRNAs) were generated by in vitro transcription. Biotinylated cRNAs were
cleaned up, fragmented, and hybridized to Affymetrix Human Genome U133 Plus 2.0 Array chips. Chips were processed using an Affymetrix fluidics
station and scanned on an Affymetrix scanner 3000 with workstation. Images were processed and raw data were extracted with GeneChip Operating
Software (GCOS).
Microarray analysis:
Microarray data were analyzed using the RMA (Robust Multichip Algorithm) method implemented in the Bioconductor package. All 18 CEL files
used in this study were included in the normalization. Differential expression was obtained using LIMMA (LInear Models for MicroArrays). Genes
estimated to have an odds >1 of being differentially expressed (Bayesian log odds score B >0) were chosen for further analysis. Gene lists
corresponding to the following biological effects were obtained.
Table 3. SHBG participates in DHT signaling in LNCaP cells in a manner consistent with two distinct
mechanisms- through RSHBG signaling, and by modulating the expression of DHT responsive genes.
CONDITION
Gene Set
1. [Vector(PonA)]-[Vector(control)]
2. [Vector(PonA+DHT)]-[Vector(PonA)]
3. [L5S2(PonA)]-[L5S2(control)]
4. [L5S2(PonA+DHT)]-[Vector(PonA+DHT)]
5. [L5S2(PonA+DHT)]-[L5S2(PonA)]
6. [L5S2(control)]-[Vector(control)]
7. [L5S2(PonA)]-[Vector(PonA)]
Distinguishing Parameters
Effect of PonA alone
Effect of DHT alone
Effect of SHBG and PonA together
Effects due to SHBG+DHT plus/or from SHBG alone
DHT effects in the presence of SHBG
Clonality of the L5S2 and L5V4 cell lines
Effects of SHBG alone
GENES REPRESSED
<0.66-fold expression
682
157
77
110
REPRESSED GENES
RSHBG responsive genes
927
DHT responsive genes
modulated by SHBG
121
FKBP5 Fold Change
STEAP4 Fold Change
1000000
100000
10000
1
0.1
6
5
4
3
2
1
0
S2
untrt
MOCK
TREATED
PonA trt
PonA
TREATED
V4
S2
unt rt
MOCK
TREATED
PonA
PonAt rt
TREATED
DHT responsive genes, modulated by SHBG are taken to be genes that are:
1.
Not found to be significantly modulated in Gene Set 1, AND
2.
Are found to be significantly modulated in Gene Set 2, AND
3.
Not found to be significantly modulated in Gene Set 3, AND
4.
Are found to be significantly modulated in Gene Set 4
(in all cases the criterion for modulation is B>0 for the comparison under consideration).
KEGG pathways that were overrepresented in the gene lists were found using Pathway Express ; Gene Ontology categories that were overrepresented
in the gene lists were found using Onto-express .
Table 1. PonA treatment reproducibly induces SHBG overexpression in the L5S2 cell line;
PonA and DHT have minimal effects on SHBG expression in vector control L5V4 cells.
P Value B value
1.34E-19 38.3
1.03E-19 39.8
4.73E-05 7.02
5.53E-13 26.8
N/A
N/A
N/A
PonA
rt DHT
+ DHT
PonAt +
TREATED
24hrs
GPR30 Fold Change
V4
DHT 24hrs
0.2
0
MOCK
untrt
TREATED
PonAtrt
+ DHT
PonA
+ DHT
TREATED
24hrs
V4
S2
1.5
0.8
0.6
0.5
0.4
0
0.2
0
PonAtrt
PonA
TREATED
1
1
PonA +trt
DHT
PonA
+
TREATED
0.6
c-MYC Fold Change
1.2
PonAtrt
PonA
TREATED
0.8
V4
S2
1.4
MOCK
untrt
TREATED
S2
1.4
1.2
0.4
PonA trt + DHT
PonA + DHT
24hrs
TREATED
S2
V4
1
GPR89a Fold Change
2.5
2
1.5
1
0.5
0
TIMP2 Fold Change
7
V4
100
10
RSHBG responsive genes, whose expression is modulated SHBG + DHT, are taken to be genes that are:
1.
Not found to be significantly modulated in Gene Set 1, AND
2.
Not found to be significantly modulated in Gene Set 2, AND
3.
Not found to be significantly modulated in Gene Set 3, AND
4.
Are found to be significantly modulated in Gene Set 4,
(in all cases the criterion for modulation is B>0 for the comparison under consideration).
CELL LINES AND CONDITIONS
EFFECTS TESTED
SHBG Fold change
L5S2: PonA vs. L5V4: PonA
PonA induction
83.7
L5S2: PonA + DHT vs. L5V4: PonA + DHT
PonA induction and DHT
89.3
L5V4:PonA vs. L5V4: Mock treated
PonA effects alone
1.57
L5S2: Mock treated vs. L5V4: Mock treated
Leakiness of L5S2 cells
4.82
L5V4: PonA + DHT vs. L5V4: PonA
DHT on L5V4 cells
N/D
N/A
L5S2: PonA + DHT vs. L5S2: PonA
DHT on L5S2 cells
N/D
(N/D- no difference detected; N/A- Not applicable)
927
GENES INDUCED
> 1.5-fold
449
INDUCED GENES
1000
INTRODUCTION
Figure 1. Model of the initial steps of SHBGmediated, membrane based androgen signaling in
prostate cells.
Table 2. L5S2 cells, induced to overexpress SHBG and treated with DHT, display gene expression differences
compared to L5V4 vector control cells similarly treated with PonA and DHT (analysis of gene set #4)
MOCK
unt rt
TREATED
PonA
PonA t rt
TREATED
PonA + DHT
PonA t rt + DHT 24hrs
TREATED
MOCK
untrt
TREATED
PonA
PonA
trt
TREATED
PonAtrt
+ DHT
PonA
+ DHT
TREATED
24hrs
Figure 2. Confirmation, by quantitative PCR analysis, of microarray results showing the combined
effects of SHBG + DHT on the expression of selected genes in LNCaP cells. Taqman qPCR amplifications were
performed in triplicate using primers specific for the indicated genes. cDNA templates were generated from the same RNAs used for microarray
analysis. Gene expression values were normalized to GAPDH expression for each cell line and treatment condition. Comparisons of specific
gene expression among cell lines and treatment conditions are presented as ratios to their expression in mock-treated L5V4 vector control cells.
CONCLUSION
Our results further support a biologic role for RSHBG signaling in LNCaP cells. In addition, we
provide the first evidence that endogenous SHBG may function intracellularly to directly or indirectly
regulate AR activity. We have identified putative downstream targets of SHBG + DHT signaling that
include STEAP4, FKBP5, TIMP2, GPR89a, GPR30, and c-MYC. These genes have important and
provocative cellular functions, especially with respect to androgen signaling and to prostate cancer. If
confirmed, we plan to identify the intermediaries in SHBG-mediated androgen signaling, and to
address the functional role of dysregulated SHBG expression in prostate cancer.
ACKNOWLEDGEMENTS- This work was supported by: Department of the Army Grant W81XWH-04-1-0228, and Awards from the American
Hellenic Educational Progressive Association-Daughters of Penelope Cancer Research Foundation, the St. Luke’s-Roosevelt Associate Trustees
Foundation, and Ed Randall’s Bat For the Cure Foundation to Strike Out Prostate Cancer