(Received for publication, September 11, 1995; and in revised form, October 4, 1995)
From the
The mucin gene, Muc-1, encodes a high molecular weight integral membrane glycoprotein that is present on the apical surface of most simple secretory epithelial cells. Muc-1 is highly expressed and aberrantly glycosylated by most carcinomas and metastatic lesions. Numerous functions have been proposed for this molecule, including protection of the epithelial cell surface, an involvement in epithelial organogenesis, and a role in tumor progression. Mice deficient in Muc-1 were generated using homologous recombination in embryonic stem cells. These mice appeared to develop normally and were healthy and fertile. However, the growth rate of primary breast tumors induced by polyoma middle T antigen was found to be significantly slower in Muc-1 deficient mice. This suggests that Muc-1 plays an important role in the progression of mammary carcinoma.
The MUC1 protein is a heavily glycosylated cell-associated mucin glycoprotein that is highly expressed and aberrantly glycosylated by the majority of carcinomas and, in particular, by >92% of primary and metastatic breast cancers. This protein was initially identified as a component of the human milk fat globule membrane(1) . Subsequently, it was found to be expressed by the majority of simple secretory epithelial cells in addition to being highly expressed by most human carcinomas, including those of the breast, colon, ovary, pancreas, lung, and stomach(2, 3) . This widespread expression pattern is in contrast to the other human mucin genes that have been isolated, which appear to have a more restricted spatial expression pattern (reviewed in (4) ). MUC1 is also known as PEM, episialin, H23 antigen, DF3 antigen, PUM, and epithelial membrane antigen (or EMA) and by a variety of other names.
Mucins, or mucin-type glycoproteins, can be defined as large extended molecules with a high percentage (50-90%) of their molecular mass made up of carbohydrate that is attached via O-glycosidic linkage through N-acetylgalactosamine to serine and/or threonine. They can be subdivided into the classical secretory or soluble mucins and the membrane-associated mucins. The secretory mucins constitute the viscous mucus of the tracheobronchial, gastrointestinal, and reproductive tracts and typically form extremely large complexed oligomers through linkage of protein monomers via disulfide bonds. These proteins are secreted from the cell and remain at the apical surface in the form of a mucus gel. This secretion serves as a selective physical barrier between the extracellular milieu and the plasma membrane and cell interior. The membrane-associated mucins are intimately associated with the plasma membrane through a hydrophobic transmembrane domain and have not been observed to form oligomeric complexes. However, these proteins can be secreted from the cell surface, most probably through a proteolytic cleavage mechanism(5, 6) .
Isolation of cDNA and genomic clones for the human MUC1 gene suggested that the gene coded for a typical type I membrane glycoprotein(7, 8, 9, 10, 11, 12) . At the amino terminus there was a short signal peptide, and at the carboxyl terminus there was a 28-31-amino acid transmembrane domain and 69-72-amino acid cytoplasmic tail. The majority of the external domain of the protein was made up of multiple copies of a 20-amino acid repeat (up to 125 copies) that was rich in serine, threonine, and proline. Serine and threonine residues act as the attachment sites for O-linked carbohydrate, and it is this region of the protein that is highly O-glycosylated. Isolation of the mouse Muc-1 gene indicated that the most strongly conserved regions corresponded to the serine, threonine, and proline residues within the repeat domain and to the transmembrane and cytoplasmic domains(13, 14) . This would suggest that the primary function of the repeat domain is to act as a scaffold for carbohydrate attachment. The sequence conservation of the transmembrane and cytoplasmic domains would suggest an important function for this part of the protein. In this respect, MUC1 has been demonstrated to be associated with elements of the actin cytoskeleton(15) , and presumably it is the cytoplasmic domain of MUC1 that is involved in this interaction. An investigation of the expression pattern of Muc-1 during mouse embryogenesis revealed that expression was regulated both spatially and temporally and correlated well with the onset of epithelial differentiation in each organ(16) . In addition, the pattern of Muc-1 expression in the adult mouse and in mouse mammary carcinomas correlated well with the pattern of expression observed for the human MUC1 gene(17, 18) .
Traditionally, mucins have been thought of as protective molecules.
However, the fact that MUC1 is expressed early during epithelial
organogenesis and is highly expressed by tumors and metastatic lesions
suggests other functions. In general these functions may be a direct
reflection of the large size of the molecule. According to the model of
Jentoft(19) , the fully glycosylated human MUC1 protein may
extend as much as 300-500 nm above the cell surface, far above
the glycocalyx. Thus, due to its large extended conformation, one
potential function for MUC1 is as an antiadhesive protein, possibly
blocking cell-cell and cell-matrix
interactions(20, 21, 22) . Muc-1 in normal
polarized epithelia is expressed only on the apical surface. However,
in many adenocarcinomas polarization is lost and the protein can be
found on basolateral cell surfaces, where it could interfere with
cell-cell and cell-substratum adhesion. Alternatively, MUC1 may play an
adhesive role by presenting carbohydrates as ligands for selectin-like
molecules and thus aiding metastatic
dissemination(23, 24, 25) . This may be
particularly relevant as MUC1 has recently been shown to express sialyl
Lewis and sialyl Lewis
, ligands for P- and
E-selectins(26, 27) . Clearly both antiadhesive and
proadhesive functions could play significant roles in normal
development, tumor progression, and disease.
In order to investigate
the biological function of the mouse Muc-1 protein, we generated mice
deficient in Muc-1 through homologous recombination in mouse embryonic
stem (ES) ()cells. These mice appeared to develop normally
and were healthy and fertile. Muc-1-deficient mice exhibited no
differences in survival rate and appeared phenotypically normal in all
respects.
Direct evidence of a role for MUC1 in the development and/or progression of breast cancer has not been demonstrated previously. To investigate the role of Muc-1 in tumor development and/or progression, we compared the growth of mammary tumors in Muc-1 -/- and +/+ mice. To generate mammary tumors, we utilized mice transgenic for the polyoma virus middle T antigen(28) . In female mice, the middle T antigen is under the control of the mouse mammary tumor virus promoter, and expression is specific for the mammary gland and to a lesser extent the salivary gland. Virgin female mice of this strain have been shown to develop multifocal breast tumors by 2 months of age, and by 4 months of age greater than 50% of these mice will have developed lung metastases. Interestingly, the middle T antigen has been demonstrated to require the presence of the src oncogene for its ability to transform mammary cells(29) . Similarly, it has been demonstrated that the neu oncogene, implicated in up to 30% of human breast cancers(30) , binds to and activates src tyrosine kinase activity(31) . We have employed the middle T oncogene in this study due to its rapid time course of tumor induction, reliable production of spontaneous tumor metastases, and the possible commonality of signal transduction pathways with the neu protooncogene. The results indicate that Muc-1 does indeed play an important role in the progression of mammary carcinoma, as primary tumor growth rate was significantly slower in Muc-1-deficient mice as compared with their wild type counterparts.
The cloning strategy is diagrammed in Fig. 1. An Escherichia coli -galactosidase (LacZ) gene (33) lacking the first 7 codons, including
the translation initiation codon, designated plasmid 839 (kindly
provided by R. Krumlauf), was subcloned into the BamHI site of
pBluescript KSII
. The modified LacZ gene,
pBS-LacZ, was subcloned further into pBluescript
SKII
as a SmaI-EcoRI fragment. This
cloning step resulted in the loss of the
-galactosidase
termination codon and was necessary in order to place the appropriate
restriction sites at the 5` end of the gene to allow an in-frame
ligation between Muc-1 and LacZ to be made and to
place sites at the 3` end of the gene to allow further manipulations. A
2-kb SmaI fragment of the Muc-1 gene was cloned into
the SmaI site created at the 5` end of the LacZ gene
to construct the plasmid pMuc-LacZ. This step created a Muc-1/LacZ
fusion protein designed to be under the control of the Muc-1 transcription and translational machinery. This plasmid
incorporated the promoter and first three codons of the mouse Muc-1 gene ligated in-frame, at the SmaI site in the first
exon, to the eighth codon of the LacZ gene. A neomycin (neo) resistance gene under the control of the
phosphoglycerate kinase (PGK) promoter and poly(A) site (34, 35) was released from pKJ-1 (kindly provided by
M. McBurney) and cloned into the EcoRI-HindIII sites
of the plasmid pMuc-LacZ in the same transcriptional orientation as the Muc-1 and LacZ genes to create pMucLacNeo. To restore
the LacZ stop codon and also to place the poly(A) signal of
SV40 at the 3` end of the LacZ gene, a 0.8-kb EcoRI
fragment was subcloned from the plasmid pPGK (E/T)LacZ (kindly provided
by M. McBurney) into the EcoRI site between the LacZ and Pgkneo genes. This plasmid was designated
pMucLacZSVneo.
Figure 1: Construction of a replacement targeting vector to inactivate the endogenous Muc-1 gene. Six separate cloning steps, indicated by numbered arrows, were required to construct the final vector, 129Muc-1GT. The cloning strategy is outlined in detail under ``Materials and Methods.'' The LacZ, SV40 poly(A), and Pgkneo cassettes are represented by the filled arcs, whereas sequence from the mouse Muc-1 locus is represented by the open arcs. In each case, restriction sites utilized in the proceeding ligation are indicated in boldface. Arrows internal of the plasmids indicate the direction of transcription of the respective genes. At step 3, the sequence of the in-frame fusion between the mouse Muc-1 gene and the E. coli LacZ gene is indicated. The initiation methionine is italicized, and the SmaI and BamHI sites are underlined.
The final 129 Muc-1-targeting vector was created as follows: the plasmid 129Muc-1E2 was digested with SmaI, and a 10-kb fragment, containing the cloning vector in addition to 7 kb of the Muc-1 gene locus, was gel-purified on DEAE (NA45) paper (Schleicher and Schuell). The 8-kb MucLacZSVneo cassette was removed from pMucLacZSVneo as a NotI-HindIII fragment. 3` overhanging ends were filled in using Klenow DNA polymerase, and the blunted fragment was gel-purified. Ligation of the 10- and 8-kb fragments and selection for plasmids containing the two fragments ligated in the correct orientation resulted in the creation of the final Muc-1-targeting vector, designated 129Muc-1GT. All ligation junctions were confirmed by sequencing by the dideoxy chain termination method (36) using Sequenase version 2.0 (U. S. Biochemical Corp.) and synthetic oligonucleotides.
Subconfluent ES cells at passage 9 were fed 2-3 h prior to
electroporation, trypsinized, spun down, and washed with
phosphate-buffered saline. Cells were resuspended in phosphate-buffered
saline to an approximate concentration of 5 10
ES
cells/ml. The 129Muc-1GT-targeting vector was linearized with NotI, extracted once with an equal volume of Tris-buffered
phenol-chloroform-isoamyl alcohol (25:24:1), precipitated, and
dissolved in sterile TE buffer, pH 8. DNA was added to the cell
suspension to a concentration of 5 nM. The cells were
electroporated in 800 µl of phosphate-buffered saline with a BTX
electroporator at a capacitance of 500 microfarads and 250 V at room
temperature. Cells were placed on ice for 10 min immediately after
electroporation and then plated onto 10 90-mm dishes of fresh STO-neo
feeders. Twenty-four hours after electroporation, G418 (400 µg/ml
active constituent) (Life Technologies, Inc.) was added to the culture
medium. After 10-11 days colonies were picked and expanded for
freezing and DNA analysis.
Genomic DNA was prepared from each clone,
digested with EcoRI, size-separated by agarose gel
electrophoresis, transferred to Hybond-N nylon membrane (Amersham), and
screened by Southern analysis utilizing a P-labeled 2-kb EcoRI-SmaI 5`-flanking probe. Conditions used for
hybridization were as recommended by the manufacturer. Homologous
recombinants were further investigated using the 5`-flanking probe and
internal probes. The chromosome constitution of the parental ES cell
line GK129 and correctly targeted clones was determined by analysis of
colcemid disrupted metaphase spreads as described(38) .
Heterozygous agouti offspring were intercrossed to generate mice homozygous for the Muc-1 mutation. Muc-1 +/+, +/-, and -/- mice were identified by PCR analysis utilizing two sets of oligonucleotide primers under the conditions described above. The first set comprised the 5` Muc-1 primer, previously mentioned, in combination with a 3` Muc-1 primer, 5`-TCCCCCCTGGCACATACTGGG-3` (corresponding to bp +268 to +248, antisense strand(13) ). Together, these primers amplified a 262-bp product from the wild type allele only. The second set of primers were identical to the pair utilized to screen for the presence of agouti F1 heterozygotes, as described. The validity of the PCR genotyping results was initially confirmed by Southern analysis of EcoRI-digested tail DNA utilizing the 5`-flanking probe described above.
Tumor weights plotted are the sum of the weights of the individual tumors. At the 124-day end point, tumors were dissected, and a portion was fixed in methacarn for histochemical analysis. Lungs were dissected, fixed in methacarn, and examined under a dissecting scope for visible metastatic lesions.
Comparison of the tumor weights at each time point between the two groups (Muc-1 +/+ and -/- mice) was made with the two-sample t test. All calculated p values were two-sided, and p values less than 0.05 were considered statistically significant.
Plasmid 129Muc-1GT was linearized prior to electroporation into the ES cell line GK129(37) . Electroporated cells were cultured in medium containing the antibiotic G418 for 10-11 days. Resistant colonies were picked and expanded for freezing and DNA analysis. DNA was isolated, and homologous recombinants were identified by Southern analysis of EcoRI-digested DNA using a 5`-flanking probe. Correctly targeted clones were obtained at a frequency of 1 in 18 G418 resistant colonies. The wild type (endogenous) Muc-1 allele displays an 11-kb EcoRI band when hybridized with the 5`-flanking probe (probe 1). In contrast, the correctly targeted allele displays a novel 7-kb EcoRI band (Fig. 2A). Clones identified in this way were further analyzed using EcoRI and BamHI digests in combination with a variety of internal and flanking probes. This was especially important considering the close proximity of genes 5` and 3` of Muc-1. In addition to clones displaying the expected novel hybridizing band at 7 kb (#56 and #132, Fig. 2A), a number of aberrantly targeted clones were obtained (#8, Fig. 2A). These aberrant clones appeared to be the result of the insertion of a concatemer of the construct into the Muc-1 locus followed by a complex rearrangement event.
Figure 2: Predicted structure of the targeted Muc-1 gene, analysis of targeted clones, and screening of mutant mice. Panel A, structure of the endogenous Muc-1 gene, before and after homologous recombination with the 129Muc-1GT replacement vector. The open boxes represent the Muc-1 exons, the gray box the LacZ-PGKneopA cassette, and the black filled boxes the thrombospondin-3 exons. Probes 1, 2, and 3 are indicated below. Asterisks indicate the 5` and 3` ends of the targeting vector arms of homology. All restriction sites for BamHI, EcoRI, HindIII, and SmaI are shown. A Southern blot of parental GK129 DNA, two independent correctly targeted clones (#56 and #132), and one aberrantly targeted clone (#8) was digested with EcoRI and hybridized with probe 1. Size markers and fragment sizes are shown. Panel B, PCR analysis to determine the genotypes of offspring from a representative litter generated from heterozygous parents. A 262-bp product amplified by the wild type primers indicates the presence of at least one wild type allele (primers flanked the inserted LacZ and Pgkneo cassette and were therefore separated by approximately 6 kb in the targeted allele), and a 261-bp product amplified by the mutant primers indicates at least one disrupted allele. C indicates negative control (buffer only) PCR reaction, and M indicates 1-kilobase pair ladder DNA. Panel C, Southern blot of EcoRI digested DNA from the same litter analyzed in panel B. DNA was hybridized with probe 1. A single hybridizing band at 11 kb is indicative of the wild type allele, whereas a band at 7 kb represents the disrupted allele.
Approximately 50% of the agouti offspring of the chimeras were heterozygous for the designed mutation. These mice were indistinguishable from their wild type littermates. Heterozygous (+/-) animals were intercrossed to generate animals homozygous for the Muc-1 mutation. Homozygous (-/-) animals were obtained from clones 56 and 31. Homozygous animals were identified through a PCR-based screening procedure (Fig. 2B), and results were initially confirmed through Southern blotting of EcoRI-digested tail DNA utilizing the 5`-flanking probe (Fig. 2C). In all cases, animals homozygous for the disrupted Muc-1 allele (-/-) were obtained at the expected Mendelian frequency. In addition, inbred 129SV/J heterozygotes were obtained from the original chimeric animals and intercrossed to derive an inbred line homozygous for the Muc-1 mutation. Similarly, inbred C57BL/6J heterozygotes are being derived through a series of back-crosses onto C57BL/6J. These mice are currently at N8 (99% inbred with respect to C57BL/6J).
Figure 3: Northern analysis of Muc-1 expression in Muc-1-deficient mice. Approximately equivalent amounts of total RNA isolated from inbred 129SV/J +/+, +/-, and -/- mice were size-fractionated through a 1.2% formaldehyde agarose gel and transferred to nylon membrane. RNA was hybridized with the mouse Muc-1 cDNA probe, pMuc2TR. Below is an image of the membrane after staining with methylene blue to detect ribosomal RNAs prior to hybridization. The position of the 18 S ribosomal RNA is indicated.
Figure 4: Immunohistochemical investigation of Muc-1 expression in Muc-1-deficient mice. Tissues from +/+, +/-, and -/- mice were isolated, fixed, and sectioned. Sections were incubated with polyclonal antiserum to the cytoplasmic tail (CTI) or antiserum previously blocked with immunizing peptide, followed by fluorescein isothiocyanate-conjugated swine anti-rabbit immunoglobulins. Equivalent sections were viewed and photographed under identical conditions. Panels A, D, G, J, and M, +/+; panels B, E, H, K, and N, +/-; panels C, F, I, L, and O, -/-. Panels D-F represent the equivalent areas shown in panels A-C, the distinction being that the antiserum had been previously blocked with peptide. No Muc-1-associated fluorescence could be detected in -/- tissues. Bar = 200 µm.
An investigation of LacZ expression in Muc-1 +/- and -/-
mice indicated that the expression of LacZ in the context of
the endogenous Muc-1 promoter did not faithfully reflect the
previously described expression pattern of Muc-1 in the developing
embryo or adult. This was found to be due to a block in transcription
of the Muc-1/LacZ fusion gene. ()
Figure 5: A Screen for genes potentially up-regulated in Muc-1-deficient mice: slot-blot analysis to detect the expression of known genes. Equivalent amounts of total RNA were transferred to a nylon membrane utilizing a slot-blot apparatus. Duplicate membranes were hybridized with radioactively labeled probes for a large panel of mucin and mucin-like genes (Table 2). Results are shown for Muc-1, Muc-2, Muc-4, and Thbs-3.
Figure 6: Primary tumor growth rate is reduced in Muc-1 -/- mice. A, hematoxylin-eosin-stained sections of tumors taken at 124 days, showing poorly differentiated adenocarcinomas. B, expression of Muc-1 protein was assessed by immunofluorescent staining with a polyclonal antiserum directed against the Muc-1 cytoplasmic tail(17) . Tumors in Muc-1 +/+ mice expressed high levels of Muc-1 protein. Bar (panels A and B) = 100 µm. C, graph showing growth rate of polyoma middle T-induced mammary tumors in Muc-1 -/- (filled square) and Muc-1 +/+ (open circle) mice. At 104 days, Muc-1 -/- mice had significantly smaller tumors than did Muc-1 +/+ mice (p < 0.05). By the 124-day end point, differences in tumor size were highly significant (p < 0.001). Asterisks indicate statistical significance. D, graph showing the percentage of Muc-1 +/+ and -/- mice with metastatic lesions in the lung at 124 days. The trend toward decreased rates of tumor metastasis in Muc-1 -/- mice suggested that the lack of Muc-1 was showing some effects. Our sample size was not sufficiently large to reach statistical significance.
Although a lack of Muc-1 protein would be expected
to affect the metastatic cascade, no significant differences in rates
of metastasis were observed. Overall, 58% of mice developed grossly
observable lung metastases, with 53% of Muc-1 -/- mice and
67% of Muc-1 +/+ mice developing metastases (Fig. 6D). Although this difference suggests a trend
toward decreased rates of tumor metastasis in Muc-1 -/-
mice, it was not statistically significant as assessed by -square
analysis (p > 0.12). However, based on the sample sizes in
this study, the power to statistically detect the observed difference
was only 33%. It is possible that with a large sample size, this
difference in metastatic rate would be statistically significant. It
should be noted that although the rate of metastasis observed in this
study was lower than that previously reported in MTag
mice(28) , this may be a consequence of the alternate type of
analysis used to detect metastasis, microscopic examination versus the RNase protection assay.
In an attempt to determine if a mucin-like molecule could potentially compensate for Muc-1 function, we obtained probes for a large panel of mucin and mucin-like genes. Slot-blot analyses of RNA were utilized to screen for their expression in Muc-1 +/+, +/-, and -/- mice. Muc-2 was strongly expressed in the colon, and Muc-4 appeared to be strongly expressed throughout epithelial tissues (Fig. 5). The structure of the human MUC4 and mouse Muc-4 genes and their encoded proteins has not been fully determined, and it is therefore possible that Muc-4 may be structurally and functionally similar to Muc-1. Other genes that did not show any consistent alteration in expression included CD34, CD43 (leukosialin), glycophorin, ASGP-2, GlyCAM-1, and MadCAM-1. In addition, the expression levels of genes closely flanking the Muc-1 gene (Thbs-3 and Gene Y) were unaffected by the targeted inactivation of Muc-1 (Fig. 5). We are currently employing the differential display PCR approach (57) on mRNAs isolated from inbred mice in an attempt to identify potential compensating genes.
The effect on metastasis in the Muc-1 -/- mice was less than expected. The trend toward decreased rates of tumor metastasis in Muc-1 null mice suggested that the lack of Muc-1 was showing some effects. However, our sample size was not sufficiently large to reach statistical significance. In addition, the rapid kinetics of the middle T tumor phenotype may have rendered some immune responses impotent. The middle T mice exhibit hyperplastic mammary glands as early as at 3 weeks of age and the rapid production of multifocal mammary adenocarcinomas by 35 days. In many animals tumors developed in every mammary gland. Alternatively, middle T may be modulating the immunogenicity of the tumor cells independently of Muc-1, or up-regulation of other molecules may compensate in the absence of Muc-1. Further analysis of the role of Muc-1 in metastasis may await a tumor model more relevant to human breast cancer. Such a study is in progress, utilizing mice with c-neu under control of the mouse mammary tumor virus LTR, which develop focal mammary tumors and metastasize after long latency(67) .
In conclusion, Muc-1 null mice developed normally. The lack of an obvious developmental phenotype suggests that Muc-1 is not required for mouse development or that functionally similar proteins have been recruited to compensate for Muc-1 deficiency. Studies investigating this area are currently in progress. As mouse lines specifically deficient in other mucin and mucin-like molecules are created it will be important to analyze the possible phenotypic effects in double and multiple mucin-deficient mice. Although normal development was not affected by a lack of Muc-1, mammary tumor growth was significantly reduced. This is a meaningful observation, as MUC1 was initially thought to be important through its role in breast cancer in humans. Clearly, the expression of Muc-1 by mouse mammary tumor cells confers upon them a selective advantage. Although a clear mechanism cannot be invoked at this time, Muc-1-deficient mice are expected to be critical in our future understanding of the role of Muc-1 in the development and progression of breast and other cancers. In addition to the work described herein, mucins have been postulated to be involved in adhesion and replication of viruses and bacteria, in receptivity of the uterus to blastocyst implantation, and in diseases such as asthma and cystic fibrosis. Muc-1-deficient mice will provide important models in the study of these aspects of mucin biology.