(Received for publication, May 26, 1995; and in revised form, July 19, 1995)
From the
Escape from negative growth regulation by transforming growth
factor (TGF-
) as a result of the loss of TGF-
type II
receptor (RII) expression has been found to be associated with the
replication error (RER) colorectal cancer genotype, which is
characteristic of hereditary nonpolyposis colorectal cancers. The
RER-positive HCT 116 colon carcinoma cell line was examined for RII
mutations. A 1-base deletion was found within a sequence of 10
repeating adenines (nucleotides 709-718), which resulted in a
frameshift mutation. Although it is reasonable to predict that the loss
of RII function would be an important determinant of malignancy, the
large number of potential mutations in cells of this phenotype raises
the possibility that an RII mutation may not be a key event in the
tumorigenic phenotype of these cells. One way to test directly the
importance of RII mutations in determining the malignant phenotype
would be to restore its expression. If restoration of expression leads
to diminished tumorigenicity, it would indicate that RII mutation is an
important determinant of malignancy in the RER phenotype. To determine
whether restoration of RII would lead to reversal of malignancy in RER
colon cancers, an RII expression vector was transfected into the HCT
116 cell line. RII stable clones showed mRNA and protein expression of
transfected RII. The fibronectin mRNA level was increased by exogenous
TGF-
treatment in a dose-dependent manner in
RII-positive clones, whereas the control cells remained insensitive.
The RII transfectants showed reduced clonogenicity in both monolayer
culture and soft agarose. They were growth arrested at a lower
saturation density than control cells.
TGF-
-neutralizing antibody stimulated the
proliferation of RII-transfected but not control cells, indicating that
the alterations in the growth parameters of the transfected cells were
due to the acquisition of autocrine-negative activity. Tumorigenicity
in athymic mice was reduced and delayed in RII transfectants. These
results indicate that reconstitution of TGF-
autocrine activity by
reexpression of RII can reverse malignancy in RER colon cancers, thus
verifying that the malignancy of hereditary nonpolyposis colorectal
cancer can be directly associated with the loss of RII expression.
Transforming growth factor (TGF-
) (
)is a
multifunctional polypeptide that regulates a number of cellular
processes including growth, differentiation, deposition of the
extracellular matrix, and immunosuppression (Roberts and Sporn, 1991;
Massagué, 1990; Moses et al., 1990).
TGF-
exerts its effects through binding to specific cell surface
proteins. Three major types of TGF-
receptors have been identified
in most cells by receptor affinity labeling assays (Roberts and Sporn,
1991; Massagué, 1990). These receptors have been
termed type I (RI), type II (RII), and type III (RIII). RI and RII are
glycoproteins of 53 and 75 kDa, respectively, whereas RIII is a
proteoglycan of 280-330 kDa. Both RI and RII are transmembrane
serine/threonine kinase receptors indispensable for TGF-
signaling
(Lin et al., 1992; Wrana et al., 1992;
Franzén et al., 1993; Bassing et
al., 1994). RIII is a membrane protein lacking a cytoplasmic
protein kinase domain (Wang et al., 1991;
Morén et al., 1992). The direct
involvement of RI and RII in TGF-
signal transduction would
suggest that loss of functional RI and/or RII expression could
contribute to the loss of TGF-
responsiveness.
An important
feature of normal growth regulation is the balance of
autocrine-negative and -positive signals regulating the cell cycle.
TGF- has been shown to act as an autocrine-negative growth
regulator as evidenced by TGF-
-neutralizing antibody stimulation
of several cell lines (Arteaga et al., 1990; Hafez et
al., 1990; Singh et al., 1990). Accordingly, cells that
lose the ability to express or respond to TGF-
are more likely to
exhibit uncontrolled growth and to become tumorigenic. Previous work in
our laboratory showed that repression of endogenous TGF-
expression by antisense TGF-
RNA led to malignant
progression of colon cancer cells (Wu et al., 1992, 1993).
TGF-
antisense transfected cells retained sensitivity to exogenous
TGF-
, thus suggesting that the loss of autocrine TGF-
function was a key feature in the development of these transfectants to
a more progressed phenotype. The loss of TGF-
receptors in
association with the inability to respond to TGF-
has been
reported for some tumor cell lines (Arteaga et al., 1988;
Kimchi et al., 1988). In particular, some strains of MCF-7
cells appear to be resistant to TGF-
because of the loss of RII
expression (Arteaga et al., 1993; Sun et al., 1994).
Reconstitution of the autocrine TGF-
loop by reexpression of
TGF-
receptors in this breast cancer cell line restored TGF-
sensitivity and reversed malignancy (Sun et al., 1994).
Reexpression of RII by complementation of a bladder cancer and a colon
carcinoma cell line also led to reversal of tumorigenicity (Geiser et al., 1992). Transfection of an RII expression vector into a
human hepatoma cell line with a receptor defect restored TGF-
sensitivity, but in vivo tumorigenicity was not addressed in
this study (Inagaki et al., 1993). Taken together, these
studies indicated that TGF-
has a significant suppressive role in
malignancy.
Hereditary nonpolyposis colorectal cancer has been found
to have a high incidence of microsatellite instability (termed RER),
which is characterized by genetic alteration of simple repeated
sequences (Aaltonen et al., 1993, 1994; Thibodeau et
al., 1993; Lindbolm et al., 1993). Recently, RER was
found to be associated with mismatch repair defects, which are
responsible for markedly elevated gene mutation rates (Aaltonen et
al., 1994; Fishel et al., 1993; Leach et al.,
1993). We have shown that TGF- RII is a downstream mutation target
resulting in the disruption of growth regulation of this hereditary
form of colon cancer in both cell lines and primary tissues as 9 of 10
RER tumors showed loss of RII transcript, whereas 48 of 53 non-RER
tumors expressed the receptor mRNA (Markowitz et al., 1995).
Disruption of the RII gene has been noted in gastric cancer, which also
has a high incidence of RER (Park et al., 1994; Eshleman and
Markowitz, 1995). These studies indicate that RII is a tumor repressor
gene in gastrointestinal cancers.
RII is of particular interest as a
suppressor gene because of the negative growth regulatory activity of
TGF-. In view of the association of the RER colorectal cancer
phenotype with the loss of RII, it is important to determine whether
reconstitution of RII would lead to reversal of tumorigenicity. This
would provide direct evidence that RII mutation and/or loss of
transcript contributes to the malignancy of this hereditary form of
cancer. In this report we describe mutation of RII in the RER-positive
HCT 116 colon cancer cell line. Stable transfection of an RII
expression vector into HCT 116 cells reversed both in vitro and in vivo malignant properties, thus indicating that
the malignancy of hereditary nonpolyposis colorectal cancer is directly
associated with loss of RII expression.
To study the effect
of exogenous TGF- on fibronectin expression,
exponentially growing RII clones and the NEO pool were treated with
various concentrations of TGF-
for 24 h. Total RNA was
then extracted for detection of fibronectin mRNA using an RNase
protection assay. The fibronectin antisense riboprobe for RNase
protection assay was synthesized in vitro from a 232-bp BamHI-PvuII fragment of human fibronectin cDNA
inserted into pGEM3Z(-) plasmid using T7 RNA polymerase.
Figure 1: Mutant RII sequence in HCT 116 cells. Five µg of total RNA from HCT 116 cells was transcribed into cDNA by reverse transcriptase-polymerase chain reaction as described under ``Materials and Methods.'' The purified product was then sequenced directly with Sequenase using the antisense primer and electrophoresed on a 6% polyacrylamide, 7 M urea gel. The sequence from this primer covers the polyadenine tract (nucleotides 709-718).
Figure 2:
Expression of TGF- RII mRNA in HCT
116 transfectants. A typical HCT 116 limiting dilution clone was stably
transfected with an RII expression vector and selected in geneticin.
The stable clones (designated RII clone 17, 21, 26, and 37) were
compared with the NEO transfection control pool for RII mRNA levels by
RNase protection assay. Endogenous RI, RII, TGF-
, and
transfected RII mRNA levels are shown. Actin mRNA levels were used for
normalization.
Figure 3:
Cell surface expression of TGF-
receptors in HCT 116 transfectants. Receptor cross-linking assays were
used to verify cell surface expression of RII. Confluent monolayer
cultures of HCT 116 NEO pool and RII clones 17, 21, 26, and 37 were
incubated with 400 pM
I-TGF-
alone (from left, first, second, fourth, fifth, and sixth lanes) or in the
presence of 20 nM cold TGF-
(third
lane) as described under ``Materials and Methods.''
Electrophoresis of 150 µg of protein was performed (4-10%
gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis
under reducing conditions).
Figure 4:
Fibronectin mRNA induction by
TGF- in HCT 116 RII transfectants. HCT 116 NEO pool
and RII clone 17 were treated in log phase with 0, 1.0, and 5.0 ng/ml
TGF-
for 24 h. Total RNA was isolated, and RNase
protection assay for fibronectin was performed. Actin mRNA levels were
used for normalization.
Figure 5:
Growth curves of HCT 116 NEO pool
() and RII clones 17 (
) and 37 (
). Cells were
plated at 1,500 cells/well in 96-well plates in 0.1 ml of serum-free
medium. The relative cell number was determined using the MTT assay
(Carmichael et al., 1987). Values are means ± S.E. of
12 replicates.
Figure 6: Plating efficiency of HCT 116 NEO control and RII clones 17 and 37. Cells were plated in 24-well plates at 300 cells/well in 1 ml of McCoy's 5A serum-free medium. Cell colonies were observed by MTT staining after 2 weeks of incubation. This was followed by suspension in dimethyl sulfoxide. Relative cell numbers were then determined by the resultant absorbance at 595 nm. Values are the means ± S.E. of four replicates.
Figure 7:
Autocrine TGF- activity in HCT 116
RII transfectants. HCT 116 NEO pool and RII clone 17 cells were plated
in 24-well plates at 300 cells/well in the presence of 10 µg/ml
normal IgG or 10 µg/ml TGF-
-neutralizing antibody.
Cell colonies were stained and photographed, and the relative cell
number was determined as described in Fig. 6after a 2-week
incubation. Panel A depicts the effectiveness of the
TGF-
-neutralizing antibody in the clonogenicity of RII
clone 17. Panel B shows the quantitation of the colony
formation of the NEO pool and RII clone 17 cells. Stimulation by
TGF-
-neutralizing antibody is expressed as the percent
increase of absorbance relative to normal IgG-treated cells. The values
are the means ± S.E. of four
replicates.
Figure 8:
Anchorage-independent colony formation in
soft agarose of HCT 116 NEO control and RII transfectants.
Exponentially growing cells (3 10
) were suspended
in 1 ml of 0.4% SeaPlaque agarose in McCoy's 5A serum-free medium
and plated on top of a 1-ml underlayer of 0.8% agarose in the same
medium in a six-well plate. Cell colonies were visualized by staining
with 0.5 ml of p-iodonitrotetrazolium violet after 2 weeks of
incubation.
Figure 9:
Xenograft formation by HCT 116 NEO pool
and RII clones 17 and 37. Exponentially growing cells (5
10
) were inoculated subcutaneously in athymic nude mice.
Tumors were measured externally on the indicated days in two dimensions
using calipers. Tumor volume was determined by the equation V = (L
W
)
0.5,
where L = the length and W = the width
of the tumor. Values are the means ± S.E. of 10 xenografts.
Experiments for RII clones 17 and 37 were performed at different times;
consequently, the NEO control for each experiment is
shown.
Many cancers are believed to develop through a series of
sequential pathologic steps (Filmus and Kerbel, 1993) that reflect the
progressive accumulation of mutations (Fearon and Vogelstein, 1990).
Early malignant models of colorectal carcinoma, which are unaggressive
and well differentiated, retain some responsiveness to TGF- growth
inhibition, whereas their highly aggressive counterparts do not
(Hoosein et al., 1989). Several studies have shown that the
progression from adenomas to carcinomas is accompanied by a reduced
responsiveness to the growth inhibitory effects to TGF-
(Hoosein et al., 1989; Manning et al., 1991; Markowitz et
al., 1994). Since malignant progression in cancer is thought to be
related to an accumulation of genetic defects, it was of interest to
correlate TGF-
resistance to specific gene mutations or
alterations of gene expression. An obvious mechanism that can be
proposed when cells develop resistance to TGF-
effects is the loss
or significant reduction of TGF-
receptor expression. Our recent
studies showed that TGF-
RII, but not RI, can be a downstream
mutation target in hereditary nonpolyposis colon cancers which are
characterized by high incidence of RER. Although inactivation of
TGF-
RII correlated with DNA repair-defective RER colon tumors
(Markowitz et al., 1995), it had not been shown that the loss
of the receptor had a direct impact on the malignant phenotype of the
cells. To show that the loss of RII expression is directly associated
with malignancy of hereditary nonpolyposis colorectal cancer, we
restored RII expression in HCT 116 cells. Our results showed that RII
expression led to reconstitution of a TGF-
autocrine-negative loop
in HCT 116 cells and reversed both its in vitro and in
vivo malignant properties.
In addition to restoration of
autocrine-negative activity, the RII-transfected cells displayed
sensitivity to exogenous TGF- for induction of the ECM pathway.
However, RII-transfected cells were insensitive to growth inhibition by
exogenous TGF-
(data not shown). Segregation of growth inhibition
and ECM protein induction in response to exogenous TGF-
has been
observed in other colon carcinoma cells (Geiser et al., 1992)
as well as other model systems (Ebner et al., 1993). As such,
the TGF-
signal transduction pathways for these two types of
TGF-
responses may diverge downstream of receptor binding, and the
effectors of ECM induction may be more sensitive than growth inhibition
effectors. The ECM response is completely absent from untransfected HCT
116 cells, whereas RII clone 26 with a low level of exogenous RII
expression showed only 20% as much induction of fibronectin as RII
clones 17 and 37 following TGF-
treatment. Thus, the ECM response
appears to be a function of available receptors in this model system.
Exogenous TGF-
may not be inhibitory because the pathways capable
of transducing the inhibitory pathway are saturated by the endogenous
TGF-
produced by HCT 116 RII-transfected cells, whereas pathways
capable of transducing the ECM pathway are not saturated by these
levels of TGF-
and hence are capable of responding to exogenous
TGF-
. For example, HCT 116 cells may have a low
potential for induction of cdk inhibitors such as p15 or p27
(Ewen et al., 1993; Hannon and Beach, 1994; Polyak et
al., 1994a; Slingerland et al., 1994) which allows for
saturation of the pathway by autocrine-negative TGF-
activity.
Presumably, the ECM pathway would not be dependent upon the induction
of these inhibitors but rather upon specific transactivation factors
associated with ECM molecule transcription (Polyak et al.,
1994b).
Our finding that the tumorigenicity of HCT 116 is reversible upon restoration of wild type RII expression establishes that RII is a tumor suppressor gene in RER colon cancer. Thus the inactivation of RII in HCT 116 by a 1-base truncation of a polyadenylate sequence is an event that directly promotes tumor progression. Deletions and insertions in repetitive DNA sequences are characteristic of RER tumors (Ionov et al., 1993; Aaltonen et al., 1993; Kim et al., 1994), and the shortening of polyadenylate sequences in RER tumors is particularly common (Chen et al., 1995). The occurrence of a polyadenylate tract within the RII coding region thus renders it particularly vulnerable to mutation in a cell with the RER mutator phenotype. RII inactivation is thus both a consequence of the RER mutator defect and a mechanism by which the RER defect is able to drive tumor progression forward.