int. J . Cancer: Supplement 5, 51-54 (1990)
0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I‘Union lnternationale Contre le Cancer
3. ROLE OF THE INT-GENES IN MURINE MAMMARY TUMOR DEVELOPMENT AND IMPLICATIONS FOR HUMAN BREAST CANCER C. DICKSON Viral Carcirwgenesis Laboratory, imperial Cancer Research Fund, Lincoln’s inn Fields, London WC2A 3PX, UK. Mouse mammary tumor virus (MMTV)-mediated carcinogenesis in mice has provided a long-standing service as a model for experimental breast cancer. The ability of MMTV to induce tumors is intimately connected with the normal viral life cycle. Like all retroviruses, MMTV is a single-stranded RNA virus that replicates through a double-standed DNA form which integrates into the host cell chromosome, in what seems to be a quasi-random manner. Thus, MMTV is an obligate mutagen. On rare occasions the viral DNA integrates into the genome at such a position that it is able to perturb the activity of a gene (a proto-oncogene) that can confer a growth advantage to the cell. The: powerful regulatory elements in the virus appear to act in cis to elicit transcriptional activity from a gene that is nomially silent; hence they can induce dominant mutations (Nusse and Varmus, 1982; Peters et al., 1983; Dickson et d . , 1984; Nusse et al., 1984; Garcia et al., 1986; Gallahan and Callahan, 1987; Peters et al., 1989). Physical linkage of the provirus adjacent to the activated proto-oncogene has been exploited as the practical means of identifying and isolating, using recombinant DNA technology, the suspected proto-oncogenes transcriptionally activated in this model system. To date, 6 “int” genes have been discovered by this approach as shown in Table I. The 2 genes that are frequently activated in virally induced mammary tumors, and which have been most extensively characterized, are int-1 and int-2 (Dickson et al., 1984; Nusse et al., 1984). The structure of these genes is shown sclnematically in Figure 1, together with the position of several activating proviruses, each identified in a different primary mammary tumor. It can be seen that activation of int-genes can be effected over a distance of several kilobases, which implies a large target domain for productive provirally induced cis activation. Despite the common naime prefix for these genes, they are in fact quite distinct entities, although int-2 and hst-1 are members of the same gene family. However, one general property which appears to be shared by these genes is their normal expression during embryogenesis, and very restricted expression in adult tissues. The int-1 gene is expressed as mRNA of 2.6kDa which encodes a protein of about 4lkDa. The nascent protein has a hydrophobic amino terminal signal sequence which directs synthesis into the endoplasmic reticulum where it is extensively glycosylated at several N-linked carbohydrate additional sites. Although it appears to have the properties of a secreted protein, it is not found in significant amounts extracellularly (Brown et al., 1987; Papkoff et al., 1987). Introduction of the in?-1 gene into epithelial cells in culture can cause conversion from a normal to a transformed phenotype, providing direct evidence of properties expected of an oncogene (Brown et al., 1986; Rijsewijk et al., 1987b). Znt-1 is very highly conserved through animal evolution with only 4 conservative substitutions occurring in the signal peptide region between the murine and human homologs (van Ooyen et al., 1985). Sequence conservation extends even further, with the Drosophila homolog (Dint- 1) demonstrating 54% amino acid homology (Baker, 1987; Rijsewijk el al., 1987~).Dint-1 was found to be the same as the segment polarity gene wingless. The nonautonomous character of wingless mutant cells suggests that the wingless gene product is secreted and determines the fate of surrounding cells, a conclusion consistent with the suspected properties of int-1. In the mouse embryo, int-1 is predominantly localized to regions of the 8- to 14-day nervous system, and is thought to function during neural development (Jakobovits et al., 1986; Wilkinson et al., 1987). In adult animals only post-meiotic spermatids express the gene and no clear biological function for this site of expression is apparent (Shackleford and Varmus, 1987). A comparison (of the properties and functions of both Drosophila and mouse homologs should provide an insight into the normal role of int-1 and a basis for its oncogenic potential. Znt-2 was the second proto-oncogene to be discovered at a common MMTV proviral integration site in mammary tumors (Fig. 1). The gene has revealed a complex transcription pattern, expressing 6 classes of RNA ranging in size from 2.9kb 10 1.6kb. These transcripts are generated from 3 distinct promoter domains, and terminate at either of 2 polyadenylation sites. This complexity of expression is reflected during embryogenesis, where transcripts are found at a number of different sites at various times during development (Jakobovits et al., 1986; Wilkinson et al., 1988, 1989). Using in situ hybridization, Wilkinson et al. (1988, 1989) have shown the main sites of expression to be the migrating mesoderm (7.5-8.5 days), pharyngeal pouches and hindbrain (8.5-9.5 days), otic vesicle and sensory regions of the inner ear (10.5-17.5 days), the developing retina, mesenchyme of the tooth bud and the Purkinje cells of the cerebellum (14.5-17.5 days). In adult mice only trace amounts of int-2 have been found in the brain and testis. The predicted amino acid sequence of int-2 shows it to be a member of the fibroblast growth factor family (Dickson and Peters, 1987). By exploiting an expression system based on the SV40 early promoter and transient
52
DICKSON TABLE I - INFORMATION AVAILABLE ON 6 int-GENES
Location Murine
Inr -gene
Human
Prouertieslremarkr ~
int-1 int-2 int-3 int-4 hst- 1
Chr. 15 chr. 7 Chr. 17 chr. 1 1 chr. 7
chr. 12
inr-4 1
?
?
Homolog of Drosophila wingless Member of FGF family
chr. 11 ? ? chr. 11
? ?
Member of FGF family ?
Data taken from the following references:inf-I; Nusse etal., 1984; Van’t Veer er al., 1984; Rijsewijk et a / . , 1987b: int-2; Peters et al., 1984; Casey et al., 1986; Dickson and Peters, 1987: int-3; Gallahan and Callahan, 1987; Gallahan etal., 1987: int-4; Nusse pers. c o r n . : hst-1; Yoshida er a!., 1987; Delli Bovi et al., 1987; Wada et al., 1988; Peters et al., 1989.: inr-41; Garcia et al., 1986.
transfection into COS-1 cells, 4 major products of between 27.5 and 31 SkDa have been detected. These proteins are the primary product showing partial carbohydrate addition at a single consensus site for N-linked glycosylation, and partial cleavage of the signal peptide. Hence, the features of int-2 products are reminiscent of secreted proteins, but like int-1 this has not been clearly demonstrated. However, introduction of the gene into cells in culture is able to confer morphological transformation upon some recipient cells, providing evidence for properties expected of a transforming gene. Of the other in?-genesdescribed, little is known about int-3, int-4 and int-41, other than the information presented in Table I. However, the 5th int-gene, hst- 1, was first discovered as a dominantly transforming oncogene for NIH3T3 cell in DNA preparations from a human stomach cancer and a Kaposi sarcoma (Yoshida et al., 1987; Delli Bovi er al., 1987). The hst-1 gene is located immediately adjacent to in?-2 on human chromosome 11 (Adelaide et al., 1988; Wada et al., 1988), and mouse chromosome 7 (Peters et al., 1989). In a few mouse mammary tumors hst-1 was shown to be transcriptionally activated, either together with in?-2 or alone, thus establishing it as an independent int-gene (Peters et al., 1989). Co-activation of int-genes is not restricted to linked loci since int-1 and int-2 are frequently expressed in the same tumor. This dual activation of int-genes is interpreted as supporting the concept of a concerted contribution of int-gene activity in a multi-step progression to frank neoplasia (Peters et al., 1986, 1989). A major hope in using a model system is that it will reveal something relevant to human disease. Therefore, it is clearly important to determine whether the human homologs of the int-genes are implicated in the pathology of human breast cancer. The most direct approach is to examine the status of the int-loci in breast tumor DNAs. Experimentally, this has involved examining breast tumor DNAs for amplifications and/or possible alterations of the in?-loci as an indirect indication of altered gene expression. Using this strategy, several groups have reported that the ZNT-2 locus is moderately amplified (2- to 8-fold) in up to 20% of breast tumor DNAs examined (Adelaide et al., 1988; Fantl et al., 1989; Lidereau et al., 1988; Varley et al., 1988; Zhou et al., 1988; Borg etal., 1989). More recent reports have also included the HST-1 gene and the BCI1 marker, both of which are physically linked to ZNT-2 on chromosome 11 band q13. The results from these studies show co-amplification of all 3 loci. Lidereau et al. (1988) showed an association between ZNT-2 amplification and local recurrent disease, while the report by Borg et al. (1989) supports this observation and shows a positive correlation with estrogen-receptor-positive tumors. If these observations are extended and further substantiated, then ZNT-2 amplification could prove to be a useful marker for identifying a subset of patients who have a poor prognosis, in spite of harboring tumors that are estrogenreceptor-positive. If amplification of ZNT-2 and HST-1 has a causative role in the development of breast tumors, then a change in
4 4 4 4 4 - 4 4 4 4 4
int- 1 bb
b
b b
b
b
+
ChrlS
4
4 4 4 4 t 4 4 4 4 4 t 4
n
lnt-2 b b b b b
I
I
b
Chr 7
FIGURE1 - Structures of int-1 and int-2 genes (diagram showing the position of several activating proviruses).
CELL BIOLOGY
53
the expression of one or other of these genes would be expected to correlate with amplification of the locus. Unfortunately, there is little evidence of expression of either ZNT-2 or HST- 1 in tumors with or without amplifications of these genes, suggesting either that these genes are only involved in the early stages of tumor evolution, or alternatively, that they represent fortuitous markers for a very closely linked gene on the same amplicon. Whatever the outcome.,a new locus implicated in approximately 20% of breast tumor cases has been identified, and resolution of the alternatives should provide further insight into the events which lead to the development of overt neoplasia. REFERENCES ADELAIDE, J.. MATTEI,M., MARICS, I., RAYBAUD, F., PLANCHE, COT,C. and ALI, I.U., Amplification of the int-2 gene in primary human breast tumors. Oncogene Res., 2, 285-291 (1988).
J., DE LAPEYRIERE, 0. and BIRMBAUM, D., Chromosomal localization of the list oncogene and its co-amplification with the inf-2
oncogene in human melanoma. Oncogene, 2, 413416 (1988). BAKER,N.E., Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO J . , 6, 1765-1773 (1987). BORG,A , , SIGURDSSON, H., TANDON,A.K., CLARK,G.M., FERNO.M., KILLANDER, D. and MCGUIRE,W.L., Protooncogene amphfication in human breast cancer. Abstract, Nordic Cancer Union Symposium, Stockholm (1989)
NUSSE,R. and VARMUS, H.E., Many tumors induced by mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell, 31, 99-109 (1982). NUSSE,R.. VANOOYEN,A,, Cox, D., FUNG,Y.K.T. and VARMUS,H.E., Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature (Lond.), 307, 131-136 (1984).
PAPKOFF,J., BROWN,A.M.C. and VARMUS,H.E., The in!-1 proto-oncogene products are glycoproteins that appear to enter the BROWN,A.M.C., PAPKOFF,J., FUNG,Y.K.T., SHACKLEFORD,secretory pathway. Mol. cell. Biol., 7, 3978-3984 (1987). G.M. and VAIRMUS, H.E., Identification of protein products enPETERS,G., BROOKES,S . , SMITH,R. and DICKSON,C., Tumoricoded by the proto-oncogene int-1. Mol. cell. Biol., 7, 3971-3977 genesis by mouse mammary tumor virus: evidence for a common (1987). region for provirus integration in mammary tumors. Cell, 33,369BROWN,A.M C., WILDEN,R.S., PRENDERGAST, T.J. and VAR- 377 (1983). MUS,H.E., A retrovirus vector expressing the putative mammary S . , SMITH,R., PLACZEK,M. and DICKoncogene int-I causes partial transformation of a mammary epi- PETERS,G., BROOKES, SON,C., The mouse homolog of the hstlk-FGF gene is adjacent to thelial cell line. Cell, 46, 1001-1009 (1986). int-2 and is activated by proviral insertion in some virally induced D., PETERS,G. and DICK- mammary tumors. Proc. nut. Acad. Sci. (Wash.), 86, 5678-5682 CASEY,G., SMITH,R., MCGILLIVARY, SON,C., Characterization and chromosome assignment of the hu- (1989). man homolog of int-2, a potential proto-oncogene. Mol. cell. G., KOZAK,C. and DICKSON, C., Mouse mammary tumor PETERS, Biol., 6, 502-510 (1986). virus integration regions inr-1 and int-2 map on different mouse A.M., KERN,F.G., GRECO,A,, chromosomes. Mol. cell. Eiol., 4, 375-378 (1984). DELLIBow, P., CURATOLA. ITTMANN, M. and BASILICO,C., An oncogene isolated by transC., Activation of a cellular fection of Kaposi’s sarcoma DNA encodes a growth factor that is PETERS,G., LEE, A.E. and DICKSON, gene by mouse mammary tumor virus may occur early in mama member of the FGF family. Cell, 50, 72S737 (1987). mary tumor development. Nature (Lond.),320, 628-631 (1986). DICKSON.C. and PETERS,G . , Potential oncogenes product related RIISEWIJK, F., SCHUERMANN, M., WAGENAAR, E., PARREN, P., to growth factors. Nature (Lond.), 326, 833 (1987). WEIGEL,D. and NUSSE,R., The Drosophila homolog of the DICKSON, C., SMITH,R., BROOKES, S . and PETERS,G., Tumori- mouse mammary oncogene int-1 is identical to the segment polargenesis by mouse mammary tumor virus: proviral activation of a ity gene wingless. Cell, 50, 649-657 (1987~). cellular gene iin the common integration region int-2. Cell, 37, 529-536 (1984). F., VANDEEMTER, L., WAGENAAR, E., SONNENBERG, RIJSEWIJK, A. and NUSSE,R., Transfection of the int-1 mammary oncogene in D., cuboidal RAC mammary cell line results in morphological transFANTL,V., BROOKES,S . , SMITH,R., CASEY,G., BARNES, JOHNSTONE,GI., PETERS,G. and DICKSON, C., Characterization of formation and tumongenicity. EMBO J . , 6, 127-131 (19876). the proto-oncogene int-2 and its potential for the diagnosis of human breast cancers. Cancer Cells, 7, 283-287 (1989). SHACKLEFORD, G.M. and VARMUS, H.E., Expression of the protoD. and CALLAHAN, R., Mammary tumorigenesis in oncogene int-1 is restricted to post-meiotic male germ cells and the GALLAHAN, feral mice: identification of a new int locus in mouse mammary neural tube of mid-gestational embryos. Cell, 50, 89-95 (1987). tumor virus (Czech 11)-induced mammary tumors. J . Virol., 61, VAN OOYEN,A,, KWEE, V. and NUSSE,R., The nucleotide se6&74 (1987). quence of the human int-1 mammary oncogene: evolutionary conD., KOZAK,C. and CALLAHAN, R., A new common servation of coding and non-coding sequences. EMBO J . , 4,2905GALLAHAN, integration region (int-3) for mouse mammary tumor virus on 2909 (1985). mouse chromasome 17. J . Virol., 61, 218-220 (1987). VAN’TVEER, L.J., GEURTSVAN KESSEL,A., VAN HEERIKHUIGARCIA, M., WELLINGER, R., VESSAZ,A. and DIGGELMANN, H., ZEN,H . , VANOOYEN,A. and NUSSE,R., Molecular cloning and A new site of integration for mouse mammary tumor virus proviral chromosomal assignment of the human homolog of in!-1 , a mouse DNA common to BALB/cf (C3H) mammary and kidney adeno- gene implicated in mammary tumorigenesis. Mol. cell. Biol., 4, 2532-2534 (1984). carcinomas. EMBO J . , 5, 127-134 (1986). Pi., SHACKLEFORD, G.M., VARMUS, H.E. and MARJAKOBOVITS, G.R., Two proto-oncogenes implicated in mammary carcinogenesis, int-1 iind int-2, are independently regulated during mouse development. Proc. nut. Acad. Sci. (Wash.), 83, 7806-7810 (1986). TIN,
FL, CALLAHAN, R., DICKSON, C., PETERS,G., EsLINDEREAU,
VARLEY, J.M., WALKER, R.A., CASEY,G. and BRAMMAR, W.J., A common alteration of the in!-2 proto-oncogene in DNA from primary breast carcinomas. Oncogene, 3, 87-91 (1988). WADA,A., SAKAMOTO, H . , KATOH,O., YOSHIDA,T., YOKOTA, J., LITTLE,P.F.R., SUGIMURA, T. and TERADA,M., Two homologous oncogenes, HSTl and INT2, are closely located in the hu-
54
DICKSON
man genome. Biochern. biophys. Res. Comm., 157, 828-835 (1988). WILKINSON, D.G., BAILES,A.J. and MCMAHON,A.P., Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo. Cell, 50, 79-88 (1987). WILKINSON, D.G., BHATT,S . and MCMAHON,A.P., Expression pattern of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal develoment. Development, 105, 131-136 (1989). WILKINSON, D.G., PETERS,G . , DICKSON,C. and MCMAHON, A.P., Expression of the FGF-related proto-oncogene int-2 during
gastrulation and neurulation in the mouse. EMBO J., 7, 691-695 ( 1988).
YOSHIDA, T., MIYAGAWA, K., ODAGIRI,H., SAKAMOTO,H., LITTLE, P.F.R., TERADA,M. and SUGIMURA, T., Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein. Proc. nut. Acad. Sci. (Wash.),84, 7305-7309 (1987). ZHOU,D.I., CASEY,G. and CLINE,M.J., Amplification of human int-2 in breast cancers and squamous carcinomas. Oncogene, 2, 279-282 (1988).
0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I‘Union lnternationale Contre le Cancer
3. ROLE OF THE INT-GENES IN MURINE MAMMARY TUMOR DEVELOPMENT AND IMPLICATIONS FOR HUMAN BREAST CANCER C. DICKSON Viral Carcirwgenesis Laboratory, imperial Cancer Research Fund, Lincoln’s inn Fields, London WC2A 3PX, UK. Mouse mammary tumor virus (MMTV)-mediated carcinogenesis in mice has provided a long-standing service as a model for experimental breast cancer. The ability of MMTV to induce tumors is intimately connected with the normal viral life cycle. Like all retroviruses, MMTV is a single-stranded RNA virus that replicates through a double-standed DNA form which integrates into the host cell chromosome, in what seems to be a quasi-random manner. Thus, MMTV is an obligate mutagen. On rare occasions the viral DNA integrates into the genome at such a position that it is able to perturb the activity of a gene (a proto-oncogene) that can confer a growth advantage to the cell. The: powerful regulatory elements in the virus appear to act in cis to elicit transcriptional activity from a gene that is nomially silent; hence they can induce dominant mutations (Nusse and Varmus, 1982; Peters et al., 1983; Dickson et d . , 1984; Nusse et al., 1984; Garcia et al., 1986; Gallahan and Callahan, 1987; Peters et al., 1989). Physical linkage of the provirus adjacent to the activated proto-oncogene has been exploited as the practical means of identifying and isolating, using recombinant DNA technology, the suspected proto-oncogenes transcriptionally activated in this model system. To date, 6 “int” genes have been discovered by this approach as shown in Table I. The 2 genes that are frequently activated in virally induced mammary tumors, and which have been most extensively characterized, are int-1 and int-2 (Dickson et al., 1984; Nusse et al., 1984). The structure of these genes is shown sclnematically in Figure 1, together with the position of several activating proviruses, each identified in a different primary mammary tumor. It can be seen that activation of int-genes can be effected over a distance of several kilobases, which implies a large target domain for productive provirally induced cis activation. Despite the common naime prefix for these genes, they are in fact quite distinct entities, although int-2 and hst-1 are members of the same gene family. However, one general property which appears to be shared by these genes is their normal expression during embryogenesis, and very restricted expression in adult tissues. The int-1 gene is expressed as mRNA of 2.6kDa which encodes a protein of about 4lkDa. The nascent protein has a hydrophobic amino terminal signal sequence which directs synthesis into the endoplasmic reticulum where it is extensively glycosylated at several N-linked carbohydrate additional sites. Although it appears to have the properties of a secreted protein, it is not found in significant amounts extracellularly (Brown et al., 1987; Papkoff et al., 1987). Introduction of the in?-1 gene into epithelial cells in culture can cause conversion from a normal to a transformed phenotype, providing direct evidence of properties expected of an oncogene (Brown et al., 1986; Rijsewijk et al., 1987b). Znt-1 is very highly conserved through animal evolution with only 4 conservative substitutions occurring in the signal peptide region between the murine and human homologs (van Ooyen et al., 1985). Sequence conservation extends even further, with the Drosophila homolog (Dint- 1) demonstrating 54% amino acid homology (Baker, 1987; Rijsewijk el al., 1987~).Dint-1 was found to be the same as the segment polarity gene wingless. The nonautonomous character of wingless mutant cells suggests that the wingless gene product is secreted and determines the fate of surrounding cells, a conclusion consistent with the suspected properties of int-1. In the mouse embryo, int-1 is predominantly localized to regions of the 8- to 14-day nervous system, and is thought to function during neural development (Jakobovits et al., 1986; Wilkinson et al., 1987). In adult animals only post-meiotic spermatids express the gene and no clear biological function for this site of expression is apparent (Shackleford and Varmus, 1987). A comparison (of the properties and functions of both Drosophila and mouse homologs should provide an insight into the normal role of int-1 and a basis for its oncogenic potential. Znt-2 was the second proto-oncogene to be discovered at a common MMTV proviral integration site in mammary tumors (Fig. 1). The gene has revealed a complex transcription pattern, expressing 6 classes of RNA ranging in size from 2.9kb 10 1.6kb. These transcripts are generated from 3 distinct promoter domains, and terminate at either of 2 polyadenylation sites. This complexity of expression is reflected during embryogenesis, where transcripts are found at a number of different sites at various times during development (Jakobovits et al., 1986; Wilkinson et al., 1988, 1989). Using in situ hybridization, Wilkinson et al. (1988, 1989) have shown the main sites of expression to be the migrating mesoderm (7.5-8.5 days), pharyngeal pouches and hindbrain (8.5-9.5 days), otic vesicle and sensory regions of the inner ear (10.5-17.5 days), the developing retina, mesenchyme of the tooth bud and the Purkinje cells of the cerebellum (14.5-17.5 days). In adult mice only trace amounts of int-2 have been found in the brain and testis. The predicted amino acid sequence of int-2 shows it to be a member of the fibroblast growth factor family (Dickson and Peters, 1987). By exploiting an expression system based on the SV40 early promoter and transient
52
DICKSON TABLE I - INFORMATION AVAILABLE ON 6 int-GENES
Location Murine
Inr -gene
Human
Prouertieslremarkr ~
int-1 int-2 int-3 int-4 hst- 1
Chr. 15 chr. 7 Chr. 17 chr. 1 1 chr. 7
chr. 12
inr-4 1
?
?
Homolog of Drosophila wingless Member of FGF family
chr. 11 ? ? chr. 11
? ?
Member of FGF family ?
Data taken from the following references:inf-I; Nusse etal., 1984; Van’t Veer er al., 1984; Rijsewijk et a / . , 1987b: int-2; Peters et al., 1984; Casey et al., 1986; Dickson and Peters, 1987: int-3; Gallahan and Callahan, 1987; Gallahan etal., 1987: int-4; Nusse pers. c o r n . : hst-1; Yoshida er a!., 1987; Delli Bovi et al., 1987; Wada et al., 1988; Peters et al., 1989.: inr-41; Garcia et al., 1986.
transfection into COS-1 cells, 4 major products of between 27.5 and 31 SkDa have been detected. These proteins are the primary product showing partial carbohydrate addition at a single consensus site for N-linked glycosylation, and partial cleavage of the signal peptide. Hence, the features of int-2 products are reminiscent of secreted proteins, but like int-1 this has not been clearly demonstrated. However, introduction of the gene into cells in culture is able to confer morphological transformation upon some recipient cells, providing evidence for properties expected of a transforming gene. Of the other in?-genesdescribed, little is known about int-3, int-4 and int-41, other than the information presented in Table I. However, the 5th int-gene, hst- 1, was first discovered as a dominantly transforming oncogene for NIH3T3 cell in DNA preparations from a human stomach cancer and a Kaposi sarcoma (Yoshida et al., 1987; Delli Bovi er al., 1987). The hst-1 gene is located immediately adjacent to in?-2 on human chromosome 11 (Adelaide et al., 1988; Wada et al., 1988), and mouse chromosome 7 (Peters et al., 1989). In a few mouse mammary tumors hst-1 was shown to be transcriptionally activated, either together with in?-2 or alone, thus establishing it as an independent int-gene (Peters et al., 1989). Co-activation of int-genes is not restricted to linked loci since int-1 and int-2 are frequently expressed in the same tumor. This dual activation of int-genes is interpreted as supporting the concept of a concerted contribution of int-gene activity in a multi-step progression to frank neoplasia (Peters et al., 1986, 1989). A major hope in using a model system is that it will reveal something relevant to human disease. Therefore, it is clearly important to determine whether the human homologs of the int-genes are implicated in the pathology of human breast cancer. The most direct approach is to examine the status of the int-loci in breast tumor DNAs. Experimentally, this has involved examining breast tumor DNAs for amplifications and/or possible alterations of the in?-loci as an indirect indication of altered gene expression. Using this strategy, several groups have reported that the ZNT-2 locus is moderately amplified (2- to 8-fold) in up to 20% of breast tumor DNAs examined (Adelaide et al., 1988; Fantl et al., 1989; Lidereau et al., 1988; Varley et al., 1988; Zhou et al., 1988; Borg etal., 1989). More recent reports have also included the HST-1 gene and the BCI1 marker, both of which are physically linked to ZNT-2 on chromosome 11 band q13. The results from these studies show co-amplification of all 3 loci. Lidereau et al. (1988) showed an association between ZNT-2 amplification and local recurrent disease, while the report by Borg et al. (1989) supports this observation and shows a positive correlation with estrogen-receptor-positive tumors. If these observations are extended and further substantiated, then ZNT-2 amplification could prove to be a useful marker for identifying a subset of patients who have a poor prognosis, in spite of harboring tumors that are estrogenreceptor-positive. If amplification of ZNT-2 and HST-1 has a causative role in the development of breast tumors, then a change in
4 4 4 4 4 - 4 4 4 4 4
int- 1 bb
b
b b
b
b
+
ChrlS
4
4 4 4 4 t 4 4 4 4 4 t 4
n
lnt-2 b b b b b
I
I
b
Chr 7
FIGURE1 - Structures of int-1 and int-2 genes (diagram showing the position of several activating proviruses).
CELL BIOLOGY
53
the expression of one or other of these genes would be expected to correlate with amplification of the locus. Unfortunately, there is little evidence of expression of either ZNT-2 or HST- 1 in tumors with or without amplifications of these genes, suggesting either that these genes are only involved in the early stages of tumor evolution, or alternatively, that they represent fortuitous markers for a very closely linked gene on the same amplicon. Whatever the outcome.,a new locus implicated in approximately 20% of breast tumor cases has been identified, and resolution of the alternatives should provide further insight into the events which lead to the development of overt neoplasia. REFERENCES ADELAIDE, J.. MATTEI,M., MARICS, I., RAYBAUD, F., PLANCHE, COT,C. and ALI, I.U., Amplification of the int-2 gene in primary human breast tumors. Oncogene Res., 2, 285-291 (1988).
J., DE LAPEYRIERE, 0. and BIRMBAUM, D., Chromosomal localization of the list oncogene and its co-amplification with the inf-2
oncogene in human melanoma. Oncogene, 2, 413416 (1988). BAKER,N.E., Molecular cloning of sequences from wingless, a segment polarity gene in Drosophila: the spatial distribution of a transcript in embryos. EMBO J . , 6, 1765-1773 (1987). BORG,A , , SIGURDSSON, H., TANDON,A.K., CLARK,G.M., FERNO.M., KILLANDER, D. and MCGUIRE,W.L., Protooncogene amphfication in human breast cancer. Abstract, Nordic Cancer Union Symposium, Stockholm (1989)
NUSSE,R. and VARMUS, H.E., Many tumors induced by mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell, 31, 99-109 (1982). NUSSE,R.. VANOOYEN,A,, Cox, D., FUNG,Y.K.T. and VARMUS,H.E., Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature (Lond.), 307, 131-136 (1984).
PAPKOFF,J., BROWN,A.M.C. and VARMUS,H.E., The in!-1 proto-oncogene products are glycoproteins that appear to enter the BROWN,A.M.C., PAPKOFF,J., FUNG,Y.K.T., SHACKLEFORD,secretory pathway. Mol. cell. Biol., 7, 3978-3984 (1987). G.M. and VAIRMUS, H.E., Identification of protein products enPETERS,G., BROOKES,S . , SMITH,R. and DICKSON,C., Tumoricoded by the proto-oncogene int-1. Mol. cell. Biol., 7, 3971-3977 genesis by mouse mammary tumor virus: evidence for a common (1987). region for provirus integration in mammary tumors. Cell, 33,369BROWN,A.M C., WILDEN,R.S., PRENDERGAST, T.J. and VAR- 377 (1983). MUS,H.E., A retrovirus vector expressing the putative mammary S . , SMITH,R., PLACZEK,M. and DICKoncogene int-I causes partial transformation of a mammary epi- PETERS,G., BROOKES, SON,C., The mouse homolog of the hstlk-FGF gene is adjacent to thelial cell line. Cell, 46, 1001-1009 (1986). int-2 and is activated by proviral insertion in some virally induced D., PETERS,G. and DICK- mammary tumors. Proc. nut. Acad. Sci. (Wash.), 86, 5678-5682 CASEY,G., SMITH,R., MCGILLIVARY, SON,C., Characterization and chromosome assignment of the hu- (1989). man homolog of int-2, a potential proto-oncogene. Mol. cell. G., KOZAK,C. and DICKSON, C., Mouse mammary tumor PETERS, Biol., 6, 502-510 (1986). virus integration regions inr-1 and int-2 map on different mouse A.M., KERN,F.G., GRECO,A,, chromosomes. Mol. cell. Eiol., 4, 375-378 (1984). DELLIBow, P., CURATOLA. ITTMANN, M. and BASILICO,C., An oncogene isolated by transC., Activation of a cellular fection of Kaposi’s sarcoma DNA encodes a growth factor that is PETERS,G., LEE, A.E. and DICKSON, gene by mouse mammary tumor virus may occur early in mama member of the FGF family. Cell, 50, 72S737 (1987). mary tumor development. Nature (Lond.),320, 628-631 (1986). DICKSON.C. and PETERS,G . , Potential oncogenes product related RIISEWIJK, F., SCHUERMANN, M., WAGENAAR, E., PARREN, P., to growth factors. Nature (Lond.), 326, 833 (1987). WEIGEL,D. and NUSSE,R., The Drosophila homolog of the DICKSON, C., SMITH,R., BROOKES, S . and PETERS,G., Tumori- mouse mammary oncogene int-1 is identical to the segment polargenesis by mouse mammary tumor virus: proviral activation of a ity gene wingless. Cell, 50, 649-657 (1987~). cellular gene iin the common integration region int-2. Cell, 37, 529-536 (1984). F., VANDEEMTER, L., WAGENAAR, E., SONNENBERG, RIJSEWIJK, A. and NUSSE,R., Transfection of the int-1 mammary oncogene in D., cuboidal RAC mammary cell line results in morphological transFANTL,V., BROOKES,S . , SMITH,R., CASEY,G., BARNES, JOHNSTONE,GI., PETERS,G. and DICKSON, C., Characterization of formation and tumongenicity. EMBO J . , 6, 127-131 (19876). the proto-oncogene int-2 and its potential for the diagnosis of human breast cancers. Cancer Cells, 7, 283-287 (1989). SHACKLEFORD, G.M. and VARMUS, H.E., Expression of the protoD. and CALLAHAN, R., Mammary tumorigenesis in oncogene int-1 is restricted to post-meiotic male germ cells and the GALLAHAN, feral mice: identification of a new int locus in mouse mammary neural tube of mid-gestational embryos. Cell, 50, 89-95 (1987). tumor virus (Czech 11)-induced mammary tumors. J . Virol., 61, VAN OOYEN,A,, KWEE, V. and NUSSE,R., The nucleotide se6&74 (1987). quence of the human int-1 mammary oncogene: evolutionary conD., KOZAK,C. and CALLAHAN, R., A new common servation of coding and non-coding sequences. EMBO J . , 4,2905GALLAHAN, integration region (int-3) for mouse mammary tumor virus on 2909 (1985). mouse chromasome 17. J . Virol., 61, 218-220 (1987). VAN’TVEER, L.J., GEURTSVAN KESSEL,A., VAN HEERIKHUIGARCIA, M., WELLINGER, R., VESSAZ,A. and DIGGELMANN, H., ZEN,H . , VANOOYEN,A. and NUSSE,R., Molecular cloning and A new site of integration for mouse mammary tumor virus proviral chromosomal assignment of the human homolog of in!-1 , a mouse DNA common to BALB/cf (C3H) mammary and kidney adeno- gene implicated in mammary tumorigenesis. Mol. cell. Biol., 4, 2532-2534 (1984). carcinomas. EMBO J . , 5, 127-134 (1986). Pi., SHACKLEFORD, G.M., VARMUS, H.E. and MARJAKOBOVITS, G.R., Two proto-oncogenes implicated in mammary carcinogenesis, int-1 iind int-2, are independently regulated during mouse development. Proc. nut. Acad. Sci. (Wash.), 83, 7806-7810 (1986). TIN,
FL, CALLAHAN, R., DICKSON, C., PETERS,G., EsLINDEREAU,
VARLEY, J.M., WALKER, R.A., CASEY,G. and BRAMMAR, W.J., A common alteration of the in!-2 proto-oncogene in DNA from primary breast carcinomas. Oncogene, 3, 87-91 (1988). WADA,A., SAKAMOTO, H . , KATOH,O., YOSHIDA,T., YOKOTA, J., LITTLE,P.F.R., SUGIMURA, T. and TERADA,M., Two homologous oncogenes, HSTl and INT2, are closely located in the hu-
54
DICKSON
man genome. Biochern. biophys. Res. Comm., 157, 828-835 (1988). WILKINSON, D.G., BAILES,A.J. and MCMAHON,A.P., Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo. Cell, 50, 79-88 (1987). WILKINSON, D.G., BHATT,S . and MCMAHON,A.P., Expression pattern of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal develoment. Development, 105, 131-136 (1989). WILKINSON, D.G., PETERS,G . , DICKSON,C. and MCMAHON, A.P., Expression of the FGF-related proto-oncogene int-2 during
gastrulation and neurulation in the mouse. EMBO J., 7, 691-695 ( 1988).
YOSHIDA, T., MIYAGAWA, K., ODAGIRI,H., SAKAMOTO,H., LITTLE, P.F.R., TERADA,M. and SUGIMURA, T., Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein. Proc. nut. Acad. Sci. (Wash.),84, 7305-7309 (1987). ZHOU,D.I., CASEY,G. and CLINE,M.J., Amplification of human int-2 in breast cancers and squamous carcinomas. Oncogene, 2, 279-282 (1988).
