(Received for publication, July 7, 1995; and in revised form, August 30, 1995)
From the
Transgenic mice that overexpress v-Ha-ras, c-myc, c-neu or int-2 proto-oncogenes in the mammary
epithelium develop breast tumors with morphologies that are
characteristic of each initiating oncogene. Since these morphological
differences reflect distinctive patterns of tumor-specific gene
expression, the identification of the products of these genes might
shed light on the mechanisms of transformation and/or the identity of
target cells that are transformed by specific classes of oncogenes. By
focusing on the tyrosine phosphorylation pathway, we have found that
the transmembranal protein-tyrosine phosphatase (PTP
) is
highly expressed in murine mammary tumors initiated by c-neu and v-Ha-ras, but not in mammary tumors initiated by
c-myc or int-2. This difference is striking and
occurs both in primary tumors and in epithelial cells cultured from
them. Moreover, PTP
overexpression appears to be mammary
tumor-specific in that it is not found in other ras-based
tumors and cell lines. These observations suggest that PTP
either plays a role in ras- and neu-mediated
transformation of mammary epithelium or marks mammary epithelial cells
particularly susceptible to transformation by these oncogenes. Because
of its distinctive expression in these mammary tumors, we have further
characterized murine PTP
, cloning and determining the complete
structures of its cDNAs and showing that it is a glycoprotein that is N-glycosylated in a tissue-specific manner.
Transgenic mice in which the expression of a specific oncogene
is targeted to the mammary epithelium are useful models for studying
the origin of breast cancer. In recent years we have developed several
such paradigms in which expression of various oncogenes is directed
primarily to the mammary gland by the action of a mouse mammary tumor
virus (MMTV) ()promoter/enhancer. Thus, mice transgenic for MMTV-c-myc, -v-Ha-ras, -c-neu, or
-int-2 develop spontaneous, generally stochastic,
adenocarcinomas of the mammary gland and occasionally of other tissues
as
well(1, 2, 3, 4, 5, 6) .
Close examination of the mammary tumors obtained from MMTV-myc, -ras, and -neu transgenic mice revealed significant correlation between the distinctive histologic appearance of each tumor and the identity of its transforming oncogene(6) . This correlation can be thought of as having arisen through either of two mechanisms(6, 7) . One stipulates that the mammary gland contains several cell types that differ in their susceptibility to transformation by individual oncogenes; that is, each transforming oncogene targets a particular cell type that is then expanded to give rise to a distinct tumor type. An alternative hypothesis suggests that one or few mammary cell types are susceptible to oncogenic transformation, but that each oncogene drives the transformed cell along a distinct developmental pathway, imparting to it a distinctive histologic phenotype. Both mechanisms could certainly operate in whole or in part simultaneously.
The existence of oncogene-specific
morphology also suggests that each type of mammary tumor expresses a
somewhat different cohort of genes. The characterization of these
marker genes might shed light on the details of transformation.
Accordingly, we undertook to define genes expressed in particular types
of mammary tumors. To date we have isolated several mRNA species whose
expression pattern parallels the morphological differences found
between tumor types (7) , ()In general, we have
found that tumors arising from ras- and neu-bearing
transgenic mice fall into a common class at the molecular level and are
distinguished from tumors that arise from transgenic mice bearing the
c-myc transgene. The central role tyrosine phosphorylation
plays in tumorigenesis has now prompted us to look specifically at
genes whose products might control this process.
Tyrosine phosphorylation is a reversible process that is controlled by the opposing actions of protein-tyrosine kinases and phosphatases (PTPases) (reviewed in (8) and (9) ). PTPases comprise a large and complex family of related molecules that can broadly be divided into transmembranal, receptor-like molecules and non-transmembranal, intracellular ones (reviewed in (10, 11, 12) ). Transmembranal PTPases generally contain two PTPase catalytic domains and are thought to be capable of responding to extracellular signals. Intracellular PTPases contain one PTPase domain flanked by protein sequences that regulate the activity or localization of the entire molecule. Dual specificity phosphatases, i.e. molecules that can dephosphorylate both phosphoserine/threonine and -tyrosine exist as well. The best characterized of these, MKP1, can dephosphorylate mitogen-activated protein kinase in vivo, thereby linking this phosphatase to the latter stages of signal transduction to the nucleus(13) .
Being the antithesis of tyrosine kinases, PTPases were originally
thought to suppress transformation. Indeed, inhibition of PTPase
activity was shown to result in transformation of cultured rat kidney
cells(14) . This interpretation was strengthened by the ability
of the cytoplasmic PTPases, TCPTP (15) and PTP1B(16) ,
to suppress oncogenic transformation in cultured cells. PTP1B has also
been shown to be up-regulated in a mammary cell line transformed by neu(17) . Furthermore, overexpression of the dual
specificity phosphatase MKP-1 has been shown to counteract some of the
actions of activated ras, suggesting that it can function as
an anti-oncogene (18) . Nonetheless, overexpression of PTPases
can in itself be transforming, as in the case of PTP(19) ,
suggesting that various PTPases may be linked to transformation in
different ways.
In an effort to focus on the role of tyrosine
phosphorylation in mammary cell transformation, we sought PTPases that
showed distinctive expression patterns in each tumor type. Here we
report that the transmembranal PTPase (20) is highly
expressed in mammary tumors initiated by ras or neu,
but is absent or nearly absent from mammary tumors initiated by myc or int-2. Moreover, PTP
expression appears
to be mammary tumor-specific and is not found in ras-based
tumors that develop in several other organs. In addition, the PTP
protein displays a tissue-specific pattern of N-glycosylation.
Figure 5:
The murine PTP cDNA. A, cDNA and translated protein sequence of the transmembranal
form of PTP
(Band A in Fig. 1Fig. 2Fig. 3Fig. 4). The signal
peptide and the transmembranal domain are underlined. Two N-linked glycosylation sites, the peptide used for generation
of antiserum, and the first two ATTTA motifs are boxed. Most
of the 3`-untranslated region is not shown. B, schematic
representation of the entire cDNA of the murine transmembranal isoform
of PTP
. Thick and thin rectangles denote coding and untranslated regions, respectively. Thick
black box denotes transmembranal domain; thick shaded boxes denote the PTPase catalytic domains. Thin black boxes mark ATTTA signals and thin shaded boxes denote the
polyadenylation signals.
Figure 1:
Northern analysis of PTP
mRNA levels in murine mammary tumor cell lines. Cell lines used were: 1, NFF; 2, NF639; 3, NaF; 4, NK417; 5, M158; 6, K485; 7, 8MA1A; 8,
11MB9A; 9, 13MA1A; 10, 16MB9A; 11, MBP6; 12, M1011; 13, AC816; 14, 1128. Similar
results were obtained also with mammary tumor cell lines SMF (MMTV-neu) and AC204, AC236, and AC711
(AC-ras), for a total of 18 lines analyzed. Bands A-D are alternative forms of PTP
mRNA as discussed in
the text. Arrows indicate the positions of the 28 S and 18 S
ribosomal RNA. Each lane contained 1.5 µg of poly(A)
RNA; loading was verified by probing with a 28 S ribosomal RNA
probe.
Figure 2:
Expression of PTP mRNA in
tumors and cell lines from various organs of mice transgenic for ras. Left, RNA blot analysis of PTP
expression in cell lines. Fibrosrc., fibrosarcomas; UT, uterine tumor; MT, mammary tumor. Lines used
were: 1, AC101; 2, AC216A; 3, AC280; 4, AC139; 5, AC816. Right, PTP
mRNA expression in samples of primary tumor tissue. ras MT, myc MT, mammary epithelial tumor tissue from MMTV-ras and -myc transgenic mice,
respectively. ras ST, salivary epithelial tumor tissue. ras MT and ras ST samples are from the same mouse.
Each lane contained 1.5 µg of
poly(A)
.
Figure 3:
Expression levels of PTP
mRNA in various adult mouse organs. ras MT line used is line
AC816; note that this lane is severely underloaded. 1.5 µg of
poly(A)
was loaded in each
lane.
Figure 4:
Expression levels of PTP
mRNA in murine mammary glands at various stages of development. 5w, 12w virgin, glands of virgin mice aged 5 or 12
weeks. All other mice were 12 weeks old at sacrifice. 10d, 17d preg, glands collected at day 10 or 17 of pregnancy. Lact, glands collected after 10 days of lactation. 1d, 3d,
5d regr, glands collected after 10 days of lactation followed by
1, 3, or 5 days of regression after weaning. neu MT line, myc MT line, mammary tumor cell lines NF639 and 16MB9A,
respectively. 1.5 µg of poly(A)
RNA was
loaded in each lane.
5`-RACE was performed using the 5`-amplifinder RACE Kit (Clontech).
Briefly, poly(A) RNA from the AC-ras mammary
tumor cell line AC204 was reverse-transcribed using oligomer E1225
(CTCTGCTGCTCTTGCTCTTCT, antisense to bases 613-593 in Fig. 5A) as primer. The 3` end of the first-strand cDNA
was ligated to the Amplifinder Anchor oligomer (Clontech) and two full
rounds of PCR amplification were performed using this oligomer and
oligomer PREX1 (AGGGAGGGTCGTGGGGACG, antisense to bases 181-163
in Fig. 5A). The amplification product was subcloned
and sequenced.
Figure 8:
Enzymatic deglycosylation of PTP.
Crude protein extracts were treated with the indicated enzymes as
described under ``Experimental Procedures,'' followed by
protein blot analysis with the anti-PTP
antiserum. Lanes
1-6, AC-816 ras mammary tumor cell line; Lanes
7-10, mouse brain; Lane 11, AC-816 cell line; Lane 12, mouse lung. Size markers are in kDa. The position of
the glycosylated (G) and deglycosylated (DG) forms of
the transmembranal PTP
protein are
denoted.
PTP mRNA
was relatively abundant and was expressed at similar levels in all the neu- and ras-based cell lines examined. On the other
hand, PTP
mRNA was either absent or present at very low
levels in the MMTV-myc and -int-2 cell lines (Band A, Fig. 1). This heterogeneity of expression is
characteristic of the myc-based mammary tumors and has been
noted previously(7) . The three additional bands present in the
blot depicted in Fig. 1(Bands B, C, and D)
were subsequently found to be other forms of PTP
mRNA
(results not shown). (
)Examination of primary tumor samples
revealed that PTP
mRNA was expressed preferentially in MMTV-ras and MMTV-neu mammary tumors (Fig. 2, right panel, and not shown). This last result
argues strongly that differential expression of PTP
mRNA
is a characteristic of the original tumors and did not result from
their adaptation to tissue culture conditions.
The
5`-untranslated region of the -transmembranal cDNA is 323
base pairs long and, as in the case of the human PTP
cDNA, lacks an in-frame stop codon. The authenticity of the 5` end was
verified by performing 5`-RACE on mRNA obtained from another ras-based mammary tumor. Sequencing of several independent
5`-RACE products revealed that the 5` ends of the
-transmembranal cDNA were essentially as cloned from the
cDNA libraries (not shown). No additional 5` sequences were found
despite extensive screening of both libraries used here. Both items, as
well as cDNA expression and protein deglycosylation studies (see
below), are consistent with the 5` end of the
-transmembranal cDNA described here being the true one.
The coding portion of
the -transmembranal cDNA begins at an ATG codon which is
part of a near perfect Kozak consensus sequence (36) and is
conserved in the human PTP
(Fig. 5A and (20) ). The putative product of this mRNA is a 699-amino
acid-long protein that has leader, extracellular, and transmembranal
domains characteristic of a transmembranal protein (Fig. 5A). Hydrophobicity and sequence analyses (37) suggests that the extracellular domain of murine PTP
is 28 amino acids long. Overall, 87% of the nucleic acid sequence and
93% of amino acid residues of the murine PTP
are
identical to the human form described in (20) .
Examination
of the relatively long 3`-untranslated sequence of
-transmembranal mRNA revealed the presence of seven
repeats of the sequence ATTTA which is found in short-lived mRNAs
(reviewed in (38) ). Two polyadenylation signals were also
found, the distal of which was used in the set of clones examined here.
This signal directed polyadenylation at either of two distinct sites,
located at nucleotides 5405 and 5412 (Fig. 5, A and B).
Figure 6:
A, detection of PTP protein by
immunoprecipitation with anti-PTP
polyclonal serum. COS7 cells
were either mock transfected or transfected with the expression
construct pSVK3/
-transmembranal and metabolically labeled
with [
S]methionine. Mock, Imm.,
mock-transfected cells precipitated with immune anti-PTP
serum.
+E, pre. and +E, Imm., cells expressing
PTP
precipitated with preimmune or immune sera, respectively. Size
markers are in kDa.
Figure 7:
Expression of the PTP protein in
several tissues and cell lines. Protein blots were hybridized with the
anti-PTP
immune serum either without (left panel) or with (right panel) a 200-fold excess of the immunizing peptide. neu MT, ras MT, myc MT, mammary tumor cell lines SMF, AC-816,
and 16MB9A. Size markers are in kDa. The positions of the large,
transmembranal, and the smaller, cytoplasmic isoforms of PTP
are
denoted.
Similar
experiments were repeated with protein extracts from lung and brain (Fig. 8, right panel). As in the case of the ras mammary tumor, removal of N-linked carbohydrates by N-glycosidase-F had the most significant effect on the
mobility of the -transmembranal protein. Note that the significant
differences in the apparent molecular mass of the glycosylated
-transmembranal proteins of lung and brain disappeared after
deglycosylation (compare Fig. 7, left panel, with Fig. 8, lanes 10 and 12)). This indicates that
the differences in the electrophoretic mobility of the
-transmembranal proteins of lung and brain reflect tissue-specific
glycosylation patterns. Note also that the apparent masses of the
deglycosylated forms of the
-transmembranal protein from lung and
brain were slightly different from that of the mammary tumor (Fig. 8, lanes 10-12). This raises the
possibility that other post-translational modifications, such as
phosphorylation, are still present on the deglycosylated
-transmembranal protein.
Our results indicate that there is a dramatic increase in
expression of PTP in ras- and neu-based
mammary tumors, both at the mRNA and protein levels. Mammary tumors
initiated by these two oncogenes in fact form a class that specifically
expresses a cohort of marker genes which are underexpressed or silent
in mammary tumors initiated by myc and int-2(7) .
This finding can be accounted
for by assuming that the ras and neu oncogenes,
acting in the context of the mammary epithelial cell, share a common
transformation mechanism and/or transform a common susceptible target
cell. Interestingly, neu and ras have been suggested
to act through a common pathway (7, 39, 40, 41) which may serve as
the basis for up-regulating PTP
and other marker genes.
The possibility that PTP is an invariant link in the ras transformation pathway appears less likely in view of the
fact that relatively low levels of PTP
mRNA are found in
several ras-based tumors that arise in non-mammary tissues,
including some of epithelial origin. Thus it seems that overexpression
of PTP
in ras-based tumors is at least partially
specific to mammary tumors and is not part of a general mechanism by
which ras and neu transform. Nonetheless, it remains
possible that PTP
is part of a more restricted
mammary-specific transformation pathway.
Bearing this point in mind,
it is important to note that the isoform of PTP up-regulated in
these mammary tumor cells is transmembranal and therefore can be
conceptually integrated into the mechanisms by which neu and
other tyrosine kinases act. A similar integration is possible in the
case of ras, whose association with the cellular membrane is
critical for its transforming ability(42, 43) .
Moreover, ras influences the expression of genes whose
products are thought to act upstream of itself in signal transduction
pathways. For example, ras-mediated transformation has
recently been shown to affect expression of the transcription factor
AP2 (44) which in turn regulates neu expression(45) .
Examination of the extracellular
domain of transmembranal PTP protein reveals two sites for N-linked glycosylation and a number of serine and threonine
residues that could be substrates for O-linked glycosylation.
Indeed, the transmembranal isoform of PTP
is present in various
cell types as an N-linked glycoprotein of 100-110 kDa
that is analogous to a 100-kDa form of the closely-related
phosphatase which is also predominantly N-glycosylated(29) .
In a way that creates an
opportunity for differential function, each tissue or tumor examined
displays a unique pattern of PTP glycosylation. Tissue-specific
glycosylation arises from differences in the glycosylating capabilities
of tissues rather than from a particular property of the glycosylated
protein(46) . Nonetheless, the precise glycosylation pattern of
PTP
could affect its function by, for example, influencing
proteins that interact with its extracellular domain. The
electrophoretic mobilities of PTP
from the mammary tumors and from
normal mammary tissue are identical (not shown), indicating that their
glycosylation patterns are similar. The precise pattern of
glycosylation of PTP
in the mammary tumors may therefore be
mammary gland- rather than tumor-specific.
Finally, it is worth
noting that the structurally related PTPase, PTP, is the only
PTPase known to have transforming ability when
overexpressed(19) . PTP
and PTP
are both type IV
PTPases and are the only known members of this
class(10, 12) . Given that PTP
displays the
highest degree of sequence and structural similarity to PTP
, it is
possible that PTP
may share some of the transforming abilities of
PTP
and that up-regulation of PTP
in mammary tumors is indeed
linked to the transformation process. Subsequent efforts, involving the
creation of transgenic mice, will be directed toward testing this
possibility.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35368[GenBank].