(Received for publication, September 15, 1995; and in revised form, February 8, 1996)
From the
Interferons (IFN) and retinoids failed to inhibit the growth of
a number of breast tumor cell lines. However, a combination of these
two biological response modifiers significantly suppressed the cell
growth at pharmacologically achievable doses. The molecular basis for
such enhancement was investigated in MCF-7, a breast tumor cell line
resistant to growth inhibition by IFN-. Pretreatment of cells with
retinoic acid (RA) for 16 h followed by IFN-
, but not the
converse, induced cytotoxic effects in the cells. Continuous presence
of RA was not necessary, although it enhanced the degree of cell death
when present. Further analyses revealed that IFN-
failed to
activate IFN-stimulated gene transcription. However, IFN-
strongly
up-regulated the gene expression in RA-pretreated cells. Both
IFN-
- and IFN-
-inducible gene expression were enhanced via a
modulation of the transcriptional factor IFN-stimulated gene factors-3
and GAF binding to respective cognate regulatory elements. STAT1 was
undetectable in these cells prior to RA treatment. RA increased the
levels of this crucial regulator, thereby restoring IFN responses.
Thus, RA augmentation of STAT1 may be an early step in the cooperative
anti-tumor effects of IFN and RA.
Interferons are a group of multifunctional cytokines that
regulate cellular antiviral, anti-tumor, and immunological
responses(1, 2) . Transcription of
IFN()-stimulated genes (ISGs) is up-regulated in a transient
manner upon treatment of cells with IFNs(1, 2) .
Products of ISGs carry out the biological effects of IFNs(2) .
Type I (
,
) and type II (
) IFNs employ distinct
receptors for eliciting cellular responses(2) . Genetic and
biochemical approaches identified molecular pathways that regulate ISG
expression ( (3) and references therein).
IFN-
/
-regulated genes are regulated through a promoter motif
called the IFN-stimulated response element (ISRE)(3) .
IFN-
stimulates ISGs using a wide variety of elements(2) .
Foremost of these is the
-IFN-activated site (GAS). A primary
regulatory factor, ISGF-3 (IFN-stimulated gene factor-3), is essential
for gene induction by IFN-
/
. ISGF-3 is a heteromeric factor
consisting of four proteins: p48, p84, p91, and p113. Cells lacking
this factor or its components fail to respond to IFNs(3) .
Binding of IFN-
/
to their receptor(s) activates two
nonreceptor protein tyrosine kinases, JAK1 and TYK2, that rapidly
phosphorylate cytoplasmic p84, p91, and p113, also known as STAT
(signal transducing activators of transcription) proteins, at specific
tyrosine residues (3) . These phosphoproteins migrate to the
nucleus and associate with p48, the major DNA binding protein, to
induce gene expression. p91, p84, and p113 are known as STAT1
,
STAT1
, and STAT2, respectively (3) . Ligand-bound
IFN-
receptor associates with two protein tyrosine kinases, JAK1
and JAK2, that subsequently phosphorylate STAT1
. This protein
migrates to the nucleus, binds to GAS or GAS-like elements, and
stimulates transcription(3) . Studies with mutant cells have
shown that in a cell mutant lacking STAT1, expression of STAT1
alone restores normal IFN-
/
and IFN-
responses. Thus,
JAK1 and STAT1 are shared components for all IFN signaling
pathways(3) .
Retinoids are a group of vitamin A-related
compounds with profound influences on cell growth and
differentiation(4) . A prototype of these is
all-trans-retinoic acid (RA), a natural metabolite. Upon entry
into cells, RA binds to nuclear retinoic acid receptors (RAR), which
then associate with RA response elements (RARE) to stimulate target
gene expression(5) . Multiple RAR isotypes such as ,
, and
and corresponding subtypes are present in the cells
that create a complex pattern of gene regulation. A novel receptor,
retinoid X receptor (RXR), is preferentially activated by
9-cis-RA. Similar to the RARs, three isotypes of RXRs and
corresponding subtypes have been described(5) . RXRs
heteromerize to serve as auxiliary activators for RARs, vitamin D
receptor, and thyroid hormone receptor and regulate
ligand-specific gene expression(5) . Retinoids suppress the
growth of several tumor cells in vitro and in
vivo(5, 6) . However, mechanisms of growth
suppression are not clearly understood.
Although IFNs and retinoids
have been shown to suppress the growth of certain tumor cells, a number
of tumor cells are not inhibited by these single
agents(6, 7) . However, a combination of these agents
produces significant additive or synergistic anti-tumor
activity(7, 8) . It is not clear how these two
different ligands, of which one uses cytoplasmic STATs (3) and
the other which employs nuclear receptor-transcriptional
factors(5) , cross-talk in the regulation of cellular
anti-tumor responses. In an effort toward this direction, we analyzed
the early events that occur in IFN-resistant tumor cells that may lead
to enhanced growth suppression by RA/IFN combination. In this
investigation, using pharmacologically achievable doses of IFN-
and RA, we show that growth of an IFN-unresponsive breast tumor cell is
significantly suppressed by such combination. Growth suppression is
correlated with an enhanced transcriptional induction of ISGs in the
presence of RA. Transcriptional enhancement is due to an increase in
the STAT1 levels in RA-treated cells.
Figure 1:
Inhibition of human estrogen receptor
positive breast tumor MCF-7 growth by IFN- (A) and a
combination of IFN-
and RA (B). A, cells were
treated with none (bar 1) or with human IFN-
; 10 units/ml (bar 2); 100 units/ml (bar 3); 1000 units/ml (bar
4). B, MCF-7 cells were treated with none (bar
1); 50 units/ml IFN-
(bar 2); 0.1 µM RA (bar 3); and combination of RA/IFN-
(bar 4).
Assay was performed for 5 days as described under ``Experimental
Procedures.'' Each bar represents mean ± S.E. of
quadruplicate measurements.
We
next examined whether continuous presence of RA was required for growth
inhibition. In these experiments, we incubated cells with 1 µM RA for 16 h and then removed it. Cells were washed with serum-free
medium and incubated in the presence of various doses of IFN-. As
shown in Fig. 2, RA pretreatment followed by IFN-
significantly suppressed the growth (Fig. 2, bars
2-4). As little as 10 units/ml IFN-
was sufficient
to inhibit growth in these cells by 70% (bar 2). At 100 units,
cell death was noticed (bar 3). These data on the negative
scale indicate the loss of originally plated cells. A higher extent of
cell death was noticed upon treatment with 1000 units of IFN-
(bar 4). Continuous presence of RA during IFN treatment had
pronounced effects on the cells compared with pretreatment and its
withdrawal during IFN treatment (bars 5-7). Under
these conditions, 10 units of IFN-
, 1 µM RA caused
cell death (bar 5). At higher doses of IFN-
with a
similar dose of RA, more cell death was observed as compared with
pretreatment controls (compare bars 2 and 3 to bars 5 and 6). Thus, continuous presence of RA was
not necessary; however, it enhanced the cytotoxic effects when present.
We performed a similar experiment in which cells were pretreated with
500 units of IFN-
for 16 h, washed, and incubated with 1 and 4
µM RA for the same length as in bars 2-7.No
significant growth inhibition was noticed (see Fig. 2, bars
8 and 9). While RA pretreatment makes cells conducive to
the growth inhibition of IFN, IFN pretreatment followed by RA did not.
Thus, a specific interaction between IFN and RA mediates the arrest of
cellular growth.
Figure 2:
RA pretreatment is essential for cell
growth inhibition in MCF-7 cells. Cells were treated with none (bar
1); 1 µM RA for 16 h first and then washed (bars
2-4); 1 µM RA without further removal (bars 5-7). IFN- treatments were 10 units/ml (bars 2 and 5), 100 units/ml (bars 3 and 6), and 1000 units/ml (bars 4 and 7). Bars 8 and 9 represent data obtained with cells that
were pretreated with 500 units/ml IFN-
for 16 h, removed, and then
treated with 1 and 4 µMRA, respectively. Growth assays
were performed for 5 days as described under ``Experimental
Procedures.''
It was also of interest to determine whether growth
inhibition could also be exerted in estrogen receptor negative breast
tumor cells treated with IFN/RA combination. In these experiments, we
employed the BT-20 breast tumor cell line that lacks estrogen receptor.
IFN- alone inhibited the growth in these cells by 35% (Fig. 3). RA by itself failed to inhibit the growth even at
higher doses (data not presented). However, upon cotreatment with these
agents, cell death occurred (Fig. 3, bar 4). These
doses were identical to the ones described under Fig. 1A. Since these continuous cell lines may have
genetically or epigenetically drifted from primary tumors, we tested
the effects of the drug combination in several polyoma virus-induced
primary murine breast tumor cells (Fig. 4). As observed with
human cell lines, growth of these cells was not suppressed by murine
IFN-
ranging from 10 to 1000 units/ml (data not presented). RA
itself had little effect on these cells. However, the combination
treatment of 10 units of IFN-
and 0.1 µM RA
suppressed >80% of cell growth (Fig. 4, bar 4). Cell
death was noticed upon increment of either IFN-
or RA doses in
combination or prolonging the time of treatment (data not shown).
Furthermore, we also noted synergistic growth inhibition by RA/IFN in
an MCF-7 cell line resistant to tamoxifen (
10 µM) (Fig. 5). As observed with parent MCF-7 cells (Fig. 1, A and B), neither IFN (Fig. 5, bars 2 and 3) nor RA (Fig. 5, bars 4 and 5) were effective inhibitors, but the combination strongly
suppressed its growth (bars 6 and 7).
Figure 3:
Growth inhibition of estrogen receptor
negative breast tumor cell line BT-20 by RA and IFN-. Cells were
treated as described in the legend for Fig. 1B. The
following treatments were given: none (bar 1), 50 units/ml
IFN-
(bar 2), 0.1 µM RA (bar 3),
and the combination (bar 4). Growth assay was similar to the
one described for Fig. 1.
Figure 4:
Inhibition of PTA breast tumor cells
derived from polyoma virus-induced murine breast tumor. Treatments were
as follows: none (bar 1), 10 units/ml murine IFN- (bar 2), 0.1 µM RA (bar 3), and the
combination (bar 4). Growth was monitored as described under Fig. 1.
Figure 5:
RA/IFN- combination inhibits the
growth of a tamoxifen-resistant human MCF-7 breast tumor growth. Cells
were preselected for tamoxifen resistance (5 µM). They
were then challenged with different doses of the indicated reagents:
none (bar 1), 10 units/ml IFN-
(bar 2), 100
units/ml IFN-
(bar 3), 0.1 µM RA (bar
4), 1 µM RA (bar 5), 0.1 µM RA,
10 units/ml IFN-
(bar 6), and 1 µM RA, 100
units/ml IFN-
(bar 7).
Figure 6:
Induction of ISG expression by
RA/IFN- combination in MCF-7 cells. Cells were pretreated either
with none (lanes 1 and 2) or with 1 µM RA (lanes 3 and 4) for 12 h where indicated.
IFN-
(100 units/ml) was added (lanes 2 and 4)
and incubated further for additional 6 h. Total RNA was isolated, and
15 µg of RNA from each sample was denatured, separated on 1%
formaldehyde/agarose gels, and subjected to Northern blotting. Blots
were probed with the indicated gene- specific probe labeled with
P. These blots were washed stringently and exposed to
x-ray films to detect gene expression.
We next tested whether the enhanced expression of ISGs was
due to an increase in the stability of mRNA or induction of
transcription in these cells, because a previous study in neuroblastoma
cells indicated a posttranscriptional effect of RA(19) .
Nuclear run-off transcription assays were performed to identify the
level of regulation (Fig. 7). MCF-7 cells were treated with
indicated agents as described above, except that IFN- treatment
was for 45 min. Nuclei were isolated, and run-off transcription assays
were performed as described earlier(10) . As shown in Fig. 7, no transcription of three ISGs was detected in control
as well as IFN-
-treated cells (lanes 1 and 2).
RA alone did not induce the gene expression (lane 3). However,
a dramatic induction of ISG transcription by IFN-
was noted in the
RA-pretreated cells (Fig. 7, lane 4). Lack of gene
expression in these cells was not global because a housekeeping gene,
-actin, was transcribed normally under all these conditions. Thus,
transcriptional up-regulation by IFN-
was stronger in
RA-pretreated cells.
Figure 7:
Induction of ISG transcription by
RA/IFN- combination. Cells were pretreated either with none (lanes 1 and 2) or with 1 µM RA (lanes 3 and 4) for 12 h where indicated. IFN-
(100 units/ml) was added (lanes 2 and 4) and
incubated further for an additional 45 min. Nuclei (5
10
) were isolated, and transcripts were extended at room
temperature for 30 min. Total nuclear RNA was extracted and hybridized
to gene-specific probes immobilized on nylon filters. Autoradiography
was performed after washing.
Figure 8:
Induction of IFN-/
-regulated
reporter genes in MCF-7 cells. Cells were transfected with
pRM-luciferase (A) and ISG6-16-CAT (B)
plasmids. Cytomegalovirus enhaner driven
-galactosidase reporter
gene was also co-transfected to monitor for variations in transfection
efficiency. Forty hours later, they were treated either with none (bars/lanes 1 and 2) or with 1 µM RA (bars/lanes 3 and 4) for 12 h. IFN-
(100
units/ml) was added to the cells in bars/lanes 2 and 4 and incubated for 16 h. Cell extracts were prepared luciferase (A) and CAT (B) assays were performed using equal
amounts of proteins from each sample. Triplicate sample measurements
were taken. Enzyme activity was normalized to
-galactosidase
activity.
Figure 9:
Induction of IFN--regulated reporter
genes in MCF-7 cells. This experiment was conducted in a manner similar
to the one described under Fig. 8, except that
IFN-
-regulated pIRE-luciferase (A) and guanylate binding
protein-CAT (B) plasmids were transfected into the cells.
Treatments were similar to Fig. 8except that IFN-
(100
units/ml) was used instead of IFN-
. Bars/lanes correspond
to the following treatments: 1, none; 2, IFN-
; 3, RA; and 4,
RA/IFN-
.
Figure 10:
Binding of trans-acting factors
to ISRE. MCF-7 cells were treated with none (lane 1),
IFN- (100 units/ml) for 30 min (lane 2), 1 µM RA (lane 3) for 12 h, and 1 µM RA for 12 h
followed by IFN-
for 30 min (lane 4). Nuclei were
isolated, extracts were prepared, and an equal amount of protein
extracts (2.5 µg) were employed for EMSA.
P-Labeled
ISRE probe (30,000 cpm) was added to the proteins that were
preincubated with 2 µg of sonicated salmon sperm DNA. Samples were
incubated for 30 min and then separated on 6% polyacrylamide gels. Gels
were dried and autoradiographed.
We next determined the binding of
transacting factors to GAS and pIRE elements. Similar to ISGF-3
binding, no transacting factor binding to GAS or pIRE element was
observed in control, IFN-, or RA-treated cells (Fig. 11, A and B, lanes 1-3). However, in
RA-pretreated cells, IFN-
efficiently activated the binding of
factors to these elements (see lanes 4 in Fig. 11, A and B). In RA-treated cells a slightly enhanced
binding of STAT1
to pIRE element, but not to GAS element, was
observed. These complexes were supershifted with an anti-STAT1
antibody indicating their authenticity (data not presented). Thus,
transcriptional activation by IFNs was augmented by RA at the level of trans-acting factor binding. Mixing of RA/IFN-treated extracts
with control cell extracts in vitro did not alter binding of
ISGF-3 or STAT1 to their cognate elements (data not shown). Thus a
direct inhibitor of ISRE/GAS binding is absent in the untreated cell
extracts.
Figure 11:
IFN--induced binding of trans-acting factors to GAS (A) and pIRE (B)
elements. Cells were treated with RA as described under Fig. 10.
They were treated with IFN-
(100 units/ml) for 30 min. Nuclear
extracts were prepared and the EMSA was performed using 6 µg (A) and 3 µg (B) of total nuclear extract from
each sample. EMSA was performed as described under Fig. 10,
except that either a
P-labeled GAS (A) probe
(80,000 cpm) or pIRE (B) probe (80,000 cpm) were employed. Arrow indicates the STAT1 (GAF)
complex.
Figure 12:
Levels of JAK-STAT pathway components in
MCF-7 cells. Cell extracts were prepared from different treatment
groups, and Western blotting was performed using indicated specific
antibodies. Lane 1, none; lane 2, IFN- (100
units/ml); lane 3, RA (1 µM); and lane
4, RA/IFN-
. All treatments were performed for 18 h, and equal
amounts of cell extracts (70 µg) were
analyzed.
Figure 13:
A, over-expression of STAT1 gene promotes
the growth inhibition by IFN-. MCF-7 cells, stably transfected
with either a pCXN2 control vector (bars 1-3) or the
same vector that carries the STAT1 cDNA (bars 4-6), were
assayed for growth inhibition by IFN-
for 4 days. Treatments were
as follows: none (bars 1 and 4), 10 units/ml
IFN-
(bars 2 and 5), and 100 units/ml IFN-
(bars 3 and 6). B shows the over-expression
of STAT1 protein in the transfected cells. Cell extracts (50 µg)
were Western-blotted as described under ``Experimental
Procedures.'' Rabbit anti-STAT1 was used as a primary antibody. Lane 1, cells transfected with vector alone; lane 2,
cells transfected with expression vector carrying the STAT1 cDNA. C shows the functional activity of the transfected STAT1 protein.
Cells were transfected with ISG 6-16-CAT (as in Fig. 8).
They were either untreated or treated with IFN-
and the CAT
activity was assayed. Lanes 1 and 2, cells that
transfected with STAT1 gene. Lanes 3 and 4, cell that
carried the expression vector alone. Treatments were as follows: none (lanes 1 and 3), 100 units/ml IFN-
(lanes 2 and 4).
Our studies have identified an early molecular event that
results in enhanced growth suppression. In IFN--resistant MCF-7
cells (Fig. 1), pretreatment of cells with RA was necessary to
mediate the growth inhibitory action of IFN (Fig. 2). RA alone
could not mediate growth-suppressive actions in IFN-pretreated cells.
These results indicate that RA modulates IFN effects rather than the
converse. Continuous presence of RA during IFN treatment induced
cytocidal action. These data indicate an additional level of RA/IFN
interaction. This is evident from the data obtained with
STAT1-transfected cells where IFN inhibited the growth but failed to
induce cytotoxicity. In addition, cytotoxic and cytostatic effects may
be mediated by different gene products. Based on these observations, we
suggest that conversion to an IFN-responsive state is an early event in
IFN/RA synergy. Consistent with this notion, IFN-
readily induced
ISG transcription in RA-pretreated cells. Specifically, RA modulates
the activity of transcriptional factor ISGF-3 which in turn activates
ISGs. RA also enhances IFN-
-regulated gene expression. Therefore,
RA modulates a common step in the regulatory cascade induced by these
ligands. STAT1, a shared component of IFN-
/
and IFN-
signaling pathways(3) , was undetectable in these cells. RA
treatment increased the level of this crucial molecule thus restoring
cellular IFN responses.
A number of extracellular ligands that
include cytokines and growth factors use the JAK-STAT pathway for
transducing signals that regulate cell growth(3) . Several of
these ligands activate STAT1, in addition to other specific STAT
proteins(3) . Unlike these ligands, RA does not affect the
functional activity of these STAT1 proteins, rather it appears to
increase its physical levels. With the availability of STAT1, IFN could
efficiently activate the signal transduction cascade that leads to
growth inhibition. Subversion of normal growth regulatory pathways
either by pathogenic agents due to mutations in the signaling
components or over-expression of oncogenes may lead to sustenance of
cell growth in the absence of ligands. Examples for these mechanisms
are presented by recent studies that indicated that in both human
T-cell leukemia virus-1 and Abelson virus-transformed
cells(20, 21) and in v-src-transformed
cells(22) , the JAK-STAT pathway is operated in a
ligand-independent manner. Defective IFN signaling has been documented
in oncogene-transfected cells. For example, the adenoviral oncogene E1A
and hepatitis B viral terminal protein shut off IFN-induced gene
expression by interfering with the formation of ISGF-3 (11, 23, 24, 25) . Inhibition of
anticellular activity of IFN- was also observed in Epstein-Barr
viral nuclear antigen-2 transfected cells (26) . A protein
factor that interferes with binding of IRF-1 and p48 to ISRE in certain
human tumor cells has also been reported(27) . Mutations in JAK
gene homologue of Drosophila resulted in leukemia-like
disease(28) . Thus, either through an interference of their
activation or by expression of inhibitors of signal transduction
components of IFN pathway, certain tumor cells may evade the action of
cytokines that mediate growth arrest.
Down-regulation of signaling
component(s) in some tumor cells may be a novel mechanism of resistance
to the growth inhibition by IFNs. Consistent with such a notion, in
normal breast epithelium and cell lines STAT proteins are not
down-regulated (data not shown). At a supra-pharmacological dose of
IFN- (1500 units/ml), we observed a faint ISGF-3 band formation in
MCF-7 cells (data not presented) which indicates a weaker activation of
STAT1. RA may restore growth control in association with IFNs, by
increasing the STAT1 levels in IFN-resistant cells. The mechanism of
such augmentation is being investigated. Lastly, augmentation of IFN
action by RA may not be due to enhancement of IFN receptors because
both in breast tumor cells and embryonic tumor cells, which
differentiate in response to RA, no increase in the affinity or number
of IFN receptors was noted in RA-treated
cells(29, 30) .
Our investigations have identified a mechanism of RA/IFN synergy and illustrate how disparate extracellular growth regulatory molecules cross-talk and cooperate in mediating these effects. To our knowledge this is the first report on the defective STAT activation in tumor cells and its correction by a simple molecule such as RA. It will be interesting to examine whether other tumor cell types also have reduced STAT levels and what other small molecules can induce their levels. Interestingly, in a melanoma (31) and an acute promyelocytic leukemia cell line(32) , STAT1 expression was undetectable. In the case of acute promyelocytic leukemia cell line, RA treatment enhanced STAT1 levels (32) . Although our studies identified the early events of IFN/RA interaction in the anti-tumor action, they did not identify the gene product(s) that mediates the growth suppression. An important next step is to characterize these terminal factors that inhibit growth. IFN-inducible gene products 2`-5` A-dependent RNase (33) and protein kinase R (34) may be two such candidates.
An important finding that emerges from our studies is
that RA/IFN combination effectively suppresses the growth of breast and
other tumors in vitro and in vivo (this paper) ()in a manner independent of their type, etiology, species,
and estrogen receptor status. Potent antiproliferative activity,
irrespective of their estrogen receptor status, of the IFN/RA
combination makes it an attractive regimen for the therapy of human
breast cancer. Furthermore, this combination also inhibits the growth
of drug-resistant cells. Recent studies have shown that IFN-
and
13-cis-RA produced substantial therapeutic benefits in
patients with advanced squamous cell carcinomas of the cervix and
skin(7) . It remains to be seen if this combination provides a
new strategy for breast cancer therapy where IFNs were marginally
effective as single agents(35) .