ROLE OF MMTV INTEGRATION LOCUS CELLULAR GENES IN BREAST CANCER

Rajeshwar Rao Tekmal, and Nagalakshmi Keshava

Department of Gynecology and Obstetrics and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322-4710, USA

Received 10/14/97 Accepted 10/17/97

3. Int/wnt GENES

3.1 Biological effects of int genes

Most of the int genes fall into two groups: the wnt-1 /int-4 family and the int-2/hst family. Although the two groups show no sequence similarity to each other, they may have similar functions: besides being implicated in mouse mammary tumorigenesis, both are thought to have essential functions in pattern formation in early embryogenesis genes (9,10). The location and gene products of all int genes are shown in table 1. The wnt-1 gene is related to the Drosophila gene wingless, and probably encodes a growth factor. Overexpression of the wnt-1/int-1 gene from the MMTV-LTR promoter/enhancer has resulted in a striking proliferation of mammary gland epithelium of both female and male transgenic mice. The int-2 protein is a member of the fibroblast growth factor (FGF) family proteins. When the int-2 gene was expressed in transgenic mice from the MMTV promoter, hyperplasia was seen after one or more pregnancies. The int-3 gene is related to the notch family of genes from Drosophila and encodes a transmembrane protein that is probably a receptor (11). The MMTV virus integrates in the middle of the gene and thereby separates the extracellular and intracellular domains. They may alleviate negative regulation of the function of the receptor by its ligand binding domain, and thereby potentiate its oncogenic function (11,12). Int-5 originally named int-H, was found to be rearranged in three chemically induced hyperplastic alveolar nodules and the tumors that arose from these precancerous mammary hyperplasias after transplantation (13, 14). The highly conserved int-6 gene, cloned as a common MMTV insertion site in Czech 2 hyperplastic outgrowth lines, encodes a protein of unkown function (15). An interesing aspect is that all proviruses in int-6, mapped thus far, have integrated in a reverse orientation in one of the introns of the gene. The presence of a cryptic transcription stop signal in the reverse sequence of the MMTV LTR leads to premature transcritpon termination, resutling in novel int-6/LTR chimeric mRNA species which encode different forms of truncated int-6. The biological significance of these aberrant int-6 proteins is still unclear.

3.2 Int/wnt/fgf genes in naturally occurring mammary tumors

Six int loci (int1/wnt1, int2/fgf3, wnt3, hst/K-fgf/fgf4, wnt10b, and fgf8) have been identified in mammary tumors of high incidence in inbred mouse strains or MMTV-infected transgenic mouse strains (16-20). Most of the int genes activated by MMTV are not involved in other types of tumors and have been discovered by using MMTV cDNA as a screening probe to clone integration loci. When multiple insertions in the same locus are found in individual tumors, there is usually an oncogene in the vicinity. At int1/wnt1 and int2/fgf3 loci, proviruses were found at each end of the gene that is usually pointing away from the gene itself. The typical orientation and the large distance of some proviruses from the int1/wnt1 or int2/fgf3 promoter indicate that the transcriptional activation of the int1/int2 genes is mediated by enhancers in the MMTV genome, acting on int1/wnt1 and int2/fgf3 promoters. The promoters of int1/wnt1 and int2/fgf3 genes have been mapped and are indeed similarly active in tumors as well as where the genes are expressed, except for rare cases of proviral insertions within the promoter. In some tumors, several different int genes are activated, either in the same clonal population of cells or in different clones that may cooperate with each other. The int2/fgf3 gene is amplified in up to 19% of human breast carcinomas. In most cases, the int2/fgf3 amplification is associated with co-amplification of a cluster of genes. Most of the oncogenes activated by MMTV proviral insertions encode secreted molecules that belong either to the wnt gene family or to the fgf growth factor family. The members of the fgf and wnt family have various characters in common. They appear to be involved in many crucial developmental decision, functioning as short-range intercellular signaling molecules, and both FGF and Wnt proteins can associate tightly with extracellular components of the extracellular matrix. Proviral insertion either upstream or downstream of the gene could simultaneously activate (MMTV) transcription from three dissimilar int2/fgf3 promoters. In some tumors, the activating provirus lies within the transcription unit and disrupts the structures of the various RNAs. Insertions in the 5' region of the gene had complex effects depending on the orientation and position of the provirus relative to three promoters and intron-exon boundaries. These data strongly implicate the normal product of the int-2/fgf-3 gene, which is related to the fibroblast growth factor family, as a contributory factor in virally induced mammary tumors (21).

The mouse wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Int1/wnt1 and int4/wnt3 were originally identified as oncogenes activated by the insertion of MMTV in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. Another wnt gene, wnt2, was found to be amplified in progressed (hormone-independent) GR mouse mammary tumors (22). Three out of eight FGF growth factor family of genes have been shown to be implicated in MMTV-induced tumorigenesis (fgf-3/int-2, fgf-4/hst and fgf-8). In human and in mouse, fgf-3/int-2 and fgf-4/hst are closely linked, and a minority of the proviral insertions found in this region activate fgf-4/hst rather than fgf-3/int-2. Fgf8 has been shown to be activated by proviral insertions in MMTV-induced mammary tumors from wnt1/int1 transgenic mice. All three share a number of common structural features. They contain a signal peptide but lack a transmembrane domain, implicating that they are secreted.

Int3 is a common insertion site of MMTV which is frequently activated in MMTV induced tumors in the CZECH II mouse strain and Jyg strain. It is related to the Drosophila gene Notch. The Notch gene is best known as a gene involved in cell-cell interactions affecting neuronal differentiation. Activation of int3 in mouse mammary tumors occurs in the configuration of inserted provirus with respect to the gene. All proviral insertions map in the middle of the gene disrupting the transcriptional unit in such a way that the upstream transcription is terminated in the left LTR of the proviruses, and the downstream part of the gene is activated by promoter insertion from the right LTR (11, 23). Activation of int3 is the result of a truncation of the extracellular domain, since expression of a transgene expressing the cytoplasmic domain leads to mammary hyperplasia (24). Recent studies have shown that int6 gene can be converted into a putative dominant negative oncogene after retroviral insertion. Binding of HTLV-l tax oncoprotein to int6 caused its redistribution from the nuclear domain to the cytoplasm. Thus, int6 is a component of promyelocytic leukemia nuclear bodies and Tax disrupts its normal cellular localization by binding to it (25). Int6 gene, that is cloned as a common MMTV insertion in CZECH II has all provirus mapped have integrated in a reverse orientation in one of the introns of the gene. The presence of a cryptic transcription stop signal in the reverse sequence of the MMTV-LTR leads to premature transcription termination resulting in a novel int-6/LTR chimeric mRNA species which encode different forms of truncated int6. The biological significance of these aberrant int6 proteins is still unclear.

3.3 Int-5:MMTV integrations in hormone/carcinogen-induced HAN model

Gray et al (26) and others (27,28) have shown that mammary tumors induced by 7,12-dimethylbenz (a)-anthracene (DMBA) or mammotrophic hormones (MTH) also contain one or more newly integrated MMTV proviral DNA copies. Not only the tumors, but also preneoplastic lesions, the HAN, contain MMTV provirus. HAN are at a greater risk for tumor development than are corresponding normal mammary alveolar cells and serve as a source of pre-neoplastic or pre-malignant changes in cancer. Three different high cancer-incidence hyperplasias (D2, C4, C5) had provirus integrated at the same genomic locus, designated as int5 (formerly called int-H). Integration of the MMTV proviral DNA at the int5 locus is associated with tumorigenicity and "turn off" of casein, which is a marker of terminal differentiation. These data indicate an insertional mechanism for chemical carcinogenesis and implicate int5 as a cellular proto-oncogene. Recently, we have cloned int5 both from mouse pre-cancerous D2 HAN genomic DNA and its normal allele from BALB/c genome (14). The localization of int5 on chromosome 9 unequivocally distinguishes this gene from any other int genes. Unlike other int genes, int-5 was identified and cloned from a chemically induced HAN model. The MMTV provirus integrated at int5 in D2 neoplasms was clearly a novel integration and the activation of latent endogenous virus in BALB/c mice as a result of hormonal or chemical treatment might have led to these novel integrations. Int5 is unique, and also different from other oncogenes implicated in breast cancer, because of its possible role in early neoplastic changes leading to HAN formation. Our studies have shown that the cellular gene at the MMTV integration site in the int5 locus is identical to the gene encoding aromatase (CYP 19), a member of the cytochrome P450 gene superfamily. MMTV is integrated within the 3' untranslated region of the aromatase gene and this integration is responsible for the overexpression of this gene (hereafter called as int5/aromatase). Aromatase catalyzes the conversion of androgens to estrogen, which is the rate-limiting step in estrogen biosynthesis. Int5/aromatase is expressed in normal mammary gland and overexpressed in mammary tumors. Using a cell line derived from the D2 tumor (generated using D2 HAN that carried novel MMTV integration in the int5 locus), we have demonstrated the effect of the aromatase substrate, androstenedione, on the proliferation of tumor cells. Proliferative effects of androstenedione were blocked by aromatase inhibitors, such as fadrozole hydrochloride, aminoglutethimide, arimidex, providing evidence for the role of int5/aromatase in this process. Furthermore, the androstenedione-mediated proliferation was inhibited by the addition of anti-estrogen ICI 164, 384, suggesting that the estrogen formed from the conversion of androstenedione by int5/aromatase acts like a mitogen to stimulate the growth of D2 tumor cells. We have also demonstrated the formation of estrogens in these cells using androstenedione as a substrate. These results suggested that the overexpression of this gene may be responsible for mammary tumorigenesis. This was the first demonstration of integration of MMTV in a cellular gene that plays a role in hormone-dependent breast cancer (14, 24-31). Estrogens are the most important hormones involved in supporting the growth of hormone-dependent breast cancers. Breast tumors from postmenopausal women maintain a high estrogen content, even though the plasma levels of estradiol fall to low levels following menopause. Maintenance of high tumor estrogen concentrations reflects the in situ estradiol production from plasma estrogen precursors. One pathway for in situ synthesis involves the conversion of androstenedione to estrone/estradiol catalyzed by aromatase. Several workers have demonstrated the presence of intra-tumoral aromatase activity in breast carcinomas and have also shown a significant correlation between the aromatase activity and clinical response to endocrine therapy with aromatase inhibitors (32-34).

3.4 Characterization of int genes using in vivo breast cancer transgenic models

Transgenic animal models have been used to study breast transformation for a number of years and have yielded valuable information on the subject. The int genes were first discovered because of adjacent integration of the MMTV proviral DNA. As described earlier, this virus does not carry a transduced cellular oncogene like many of the well known oncogenic retroviruses, but rather acts as an insertional mutagen. Overexpression of the int1/wnt1 gene from the MMTV-LTR promoter/enhancer resulted in a striking proliferation of mammary gland epithelium of both female and male transgenic mice. Mean tumor onset time was around 5 months for transgenic females, and by 7 months of age more than 80% had developed tumors. Transgenic male animals develop tumors less frequently and later in life (35). Transcriptional activation of int1/wnt1 gene by proviral insertion mutations is thought to be a key step in mammary tumor induction by MMTV. Int1/wnt1 gene appears not to be expressed in normal mammary glands from pregnant or lactating mice (17,36). Studies by Tsukamoto et al (35) have shown that transgenic mice with MMTV-LTR on 5' end of int1/wnt1 gene had hyperplasia compared to non-transgenic littermates. It is suggested that transcriptional activation of int1/wnt1 and associated hyperplasia are initiating events in multi step carcinogenesis.

Overexpression of wnt1/int1 in the mammary gland of MMTV-wnt1 transgenic mice caused massive proliferation of mammary epithelial cells, resulted in hyperplastic glands (35). Also it has been shown that mammary glands of wnt1/int1 transgenic virgins are similar to hormonally-stimulated glands in pregnant animals with increased number of terminal buds and alveoli showing hyperplasia. MMTV-wnt1 or MMTV-fgf transgenic mice develop mammary tumors after a variable latency period indicating that activation of either wnt1/int1 or fgf alone is not sufficient for complete malignancy (3). Tumors develop faster in bitransgenic mice for wnt1/int1 and fgf-3/int2, compared to either gene alone. This clearly shows that co-operation of these two growth factors in tumorigenesis (37). Int-2/fgf-3 transgenic mice infected with MMTV showed activation of wnt1/int1 and a new wnt gene family member, wnt10b. On the contrary, wnt1/int1 transgenic mice infected with MMTV developed many tumors with proviral insertions in members of the Fgf family. The virus associated with int2/fgf3 insertions is from the RIII strain. When int2/fgf3 gene was expressed in transgenic mice from the MMTV promoter, hyperplasia was seen after one or more pregnancies. About one half of transgenic females developed tumors before the age of one year. Some virgin females also developed tumors, but at a lower frequency than multiparous females (38). While transgenic females display a pronounced mammary gland hyperplasia, expression of MMTV fgf3/int2 in the prostate gland of male carriers results in a benign epithelial hyperplasia, indicating that fgf3/int2 can act as a growth factor in different epithelial tissues (3). In this case, microscopic ductal hyperplasia was observed in gland of virgin transgenic females. Ductal hyperplasia, papillocystic forms and nodular solid aggregates of cells were observed during pregnancy. Some lesions regress and some remained static and one's that remained static become more pronounced in subsequent pregnancies.

Integration of MMTV provirus into the int3/notch locus promotes the transcription and translation of flanking cellular int3/notch sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila. Transgenic mice were generated (24) with the DNA fragment consisting of the MMTV-LTR and flanking cellular int3/notch sequences. Poor differentiation of mammary and salivary adenocarcinoma were prominent in transgenic mice between 2 and 7 months of age. All females int3/notch mice were lactational deficient and mammary glands were arrested in development. Jhappan et al (24) have also shown that all male int3/notch transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. Thus overexpression of int3/notch gene in in vivo may cause deregulation of normal developmental controls and hyperproliferation of glandular epithelia. These findings also suggest that the activated int3/notch gene product may have been a contributing factor in the development of Czech II mouse mammary tumors in which the MMTV-induced rearrangement in the int3/notch locus (39).

Our recent studies (40) have shown that the cellular gene at the mouse mammary tumor virus integration site in the int5 locus is aromatase. We have generated transgenic mice that overexpress int5/aromatase under the control of mouse mammary tumor virus enhancer/promoter, and demonstrated for the first time that increased mammary estrogens due to the overexpression of int5/aromatase in mammary glands of virgin and postlactational females leads to the induction of various preneoplastic and neoplastic changes that are similar to early breast cancer, that may, in turn, increase the risk of developing breast cancer. The preneoplastic/neoplastic changes continues to progress in postlactational females that overexpress int5/aromatase. In fact when these animals are exposed to carcinogens like DMBA, there is increased incidence of mammary tumorigenesis suggesting that indeed increased mammary estrogen increases the risk of developing breast cancer due to carcinogen exposure (unpublished data). Overexpression of int5/aromatase in males leads to the increased mammary growth and hyperplastic and dysplastic changes that are similar to gynecomastia (early male breast cancer). These observations indicate that increased mammary estrogen alone without the influence of circulating ovarian estrogen may be sufficient to induce early breast cancer.