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BAG3 is a pro-survival pro-invasion chaperone. We hypothesized that human BAG3 overexpression in murine breast would contribute to mammary tumorigenesis. We generated transgenic mice ectopically expressing hBAG3 in the mammary gland under the control of the Mouse Mammary Tumor Viral promoter (MMTV-hBAG3). hBAG3 was expressed in luminal cells throughout the ductal tree in multiple transgenic lines. Targeted expression of hBAG3 dysregulated mammary gland development by both increasing proliferation and differentiation. hBAG3 mammary glands had increased epithelial elongation at 5 weeks of age, premature glandular differentiation at 10 weeks, and features of ductal hyperplasia at week 13 with increased Ki67 and p-Histone H3 in the luminal compartment. At week 13 hBAG3 induced acinar differentiation. BAG3 pro-survival gain of function was recapitulated post-partum with delayed involution. Mammary glands of MMTV-hBAG3 had alveolar persistence three days after forced involution where non-transgenic controls demonstrated typical involution-driven apoptosis and remodeling. We crossed heterozygous MMTV-hBAG3 to WAP-Int3 mice with absent alveolar development in pregnancy. hBAG3 caused alveolar budding in the WAP-Int3 background. We hypothesized hBAG3 gain of function in the mammary gland would promote tumorigenesis. On aging, mammary glands of heterozygous MMTV-hBAG3 displayed densely crowded acinar structures with atypia, consistent with hyperplastic alveolar nodule (HAN). We subjected MMTV-hBAG3 mice to either multiparity or chemical carcinogenesis. After two pregnancies, most MMTV-hBAG3 animals had hyperplastic mammary ductal lesions (MMTV-hBAG3 6/7, 86%; FVB/N 1/7, 14%). Eighteen weeks post 7,12-dimethylbenz(a) antracene (DMBA) treatment, virgin females from five different transgenic lines had more hyperplastic lesions (MMTV-hBAG3 mean 6.3 lesions/gland, N=13, FVB/N mean 3.6 lesions/gland, N=9; p = 0.04), some with atypical hyperplasia. Transgenic MMTV-hBAG3 females developed ER and PR (+) malignant tumors (5/19), 26% while non-transgenic controls had none (0/12). WAP-Int3 mice developed tumors as early as two pregnancies. Heterozygous WAP-Int3xMMTV-BAG3 had the same tumor frequency but preliminary data suggest increased aggressiveness and metastasis. Collectively, our results indicate that MMTV-hBAG3 promotes proliferation, dysregulates mammary gland development and promotes hyperplasia and tumorigenesis and may provide a novel mouse model to represent the human ER+ luminal breast cancer subtype. [ABSTRACT FROM AUTHOR]

Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4-kb tumor specific Int3 RNA transcript that encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development were curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene, which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter, the activity of which, unlike that of the MMTV long terminal repeat, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In nonbreeding WAP/Int3 females, mammary tumor incidence also reached 100%, but only after 70 weeks. The WAP/Int3 mammary tumors were highly malignant, and most tumor-bearing females, irrespective of breeding history, developed metastatic lung lesions. These results suggest that WAP promotor-targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP-driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into nontransgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated.

The hereditary breast cancer gene BRCA1 previously has been localized to chromosome 17q21. We looked for evidence of involvement of this region of chromosome 17 in 130 sporadic breast cancers. Seventeen polymorphic sequence tagged site markers were examined in these tumors between the D17S250 and D17S579 loci to screen for deletions as measured by loss of heterozygosity. The smallest common region that was deleted occurred in the approximately 120-kilobase interval between the D17S846 and D17S746 loci within the BRCA1 region. Delineation of this commonly deleted area should accelerate attempts to identify the involved gene(s) and its relationship to BRCA1.

We have examined the long arm of chromosome 17 in sporadic breast carcinomas for the loss of heterozygosity (LOH) at 18 polymorphic loci. At least three distinct regions could be identified by the frequency of LOH and confirmed by high density deletion maps of individual tumor DNAs. A proximal region affected by LOH is located in a 22-cM region defined by D17S73 and NME1 and thus is similar in location to the region thought to contain the BRCA1 gene associated with familial breast and breast/ovarian cancer. The central region affected by LOH is bordered by the D17S86 and D17S21 loci and is estimated to be 28 cM in size. The third region is bordered by the D17S20 and D17S77 loci which are 11 cM apart. These results define three independent regions of chromosome 17q which are likely to contain tumor suppressor genes relevant to the etiology of sporadic breast carcinoma.

A polymerase chain reaction-single strand conformation polymorphism assay was used to assess p53 mutations in 148 invasive breast carcinomas, selected on the basis of their histotype. They comprised 56 lobular, 47 ductal, 19 mucinous, 18 medullary, and 8 papillary carcinomas. The distribution of p53 mutations was significantly different (P = 0.006) in the histotypes examined: mutations were frequent in medullary (39%) and ductal (26%), less common in lobular (12%), and absent in mucinous and papillary carcinomas. The frequency of mutations in the exon 5 of the p53 gene was significantly higher in medullary carcinomas than in the other histotypes: 5 (63%) of the mutations found in exon 5 were observed in medullary carcinomas (P = 0.012). One hundred twenty-two tumors from the total were also examined by immunohistochemistry for p53 overexpression using antibody PAb 1801. A specific immunostaining in neoplastic cells was present in 12 tumors. A strong correlation (P < 0.001) was observed between p53 mutations and nuclear accumulation of the p53 protein: 10 tumors were scored positive for both p53 mutation and overexpression. However, in 9 cases having a mutated p53 gene we failed to find a positive immunoreaction. A significant association (P = 0.01) was present between mutations in the p53 gene and high proliferative activity of the tumors determined by immunohistochemistry with monoclonal antibody Ki-67. Moreover, a significantly higher expression of the Ki-67 antigen was found in medullary carcinomas compared to the other histotypes. Our findings indicate that in invasive breast carcinomas structural abnormalities of the p53 gene are mainly seen in medullary and ductal tumors and that the other histological types, especially those associated with a high level of differentiation and favorable prognosis, show a very low incidence of p53 mutations.

Alterations of p53 are one of the most common molecular changes found in all types of lung tumors, suggesting a crucial role for p53 in bronchial carcinogenesis. However, the prognostic significance of p53 abnormalities in lung cancer patients is still unclear. By using genetic and immunohistochemical methods we have found p53 alterations in 40 of 53 (75%) primary, resected non-small cell lung cancer. A strong association (P = 0.0015) was found between deletions on chromosome region 17p13.3 and p53 mutations suggesting that loss of the wild-type p53 allele might be necessary for tumorigenesis. Correlations to clinicopathological parameters showed that p53 alterations (structural aberration of the gene and/or nuclear accumulation of the protein) are significantly linked with metastatic involvement of hilar and mediastinal lymph nodes (P < 0.01). Since the latter are well established prognostic factors for non-small cell lung cancer, p53 aberrations may also be a predictor of tumor aggressiveness.

Alteration of chromosome 1 is the most consistent cytogenetic abnormality found in human breast carcinoma. Cytogenetic studies have shown independent alterations on the two arms of chromosome 1, increased copy number of the long arm and loss of the short arm of chromosome 1. These deletions are thought to coincide with the location of tumor suppressor gene(s). We carried out deletion analysis of the 1p region by using restriction fragment length polymorphism markers mapping to the long (six markers) and short arm (22 markers). Thirty-five of the 74 (47.3%) human breast tumors tested showed somatic loss of heterozygosity at one or more loci on the short arm. Two commonly deleted regions, 1p13-p21 and 1p32-pter, were identified. The latter region is frequently involved in other types of tumors, suggesting that it harbors a common tumor suppressor gene. Our findings suggest that two tumor suppressor genes involved in the development of human breast carcinoma may occur on the short arm of the chromosome 1.

Twenty-six primary breast tumors were examined for mutations in the p53 tumor suppressor gene by an RNase protection assay and nucleotide sequence analysis of PCR-amplified p53 complementary DNAs. Each method detected p53 mutations in the same three tumors (12%). One tumor contained two mutations in the same allele. Single strand conformation polymorphism analysis of genomic DNA and complementary DNA proved more sensitive in the detection of mutations. Combining this technique with the other two a total of 12 mutations in the p53 gene were demonstrated in 11 tumors (46%), and a polymorphism at codon 213 was detected in another tumor. Loss of heterozygosity on chromosome 17p was detected by Southern blot analysis in 30% of the tumor DNAs. Not all of the tumors containing a point mutation in p53 also had loss of heterozygosity of the remaining allele, suggesting that loss of heterozygosity may represent a later event.

Tumor progression to the metastatic phenotype is accompanied in certain cell types by reduced expression of the nm23 gene. We have localized human nm23-H1 to chromosome 17 by somatic cell hybrid analysis. Regional localization in the CEPH database and in situ hybridization is reported. Somatic allelic deletion of nm23-H1 was observed in human breast, renal, colorectal, and lung carcinoma DNA samples, as compared to DNA from matched normal tissues. A homozygous deletion of nm23-H1 was observed in a lymph node metastasis of a colorectal carcinoma, indicating that nm23-H1 can be recessively inactivated. The data identify nm23-H1 as a novel, independent locus for allelic deletion in human cancer, a characteristic shared with previously described suppressor genes.

The human H-ras protooncogene was shown to be expressed in 16 of 22 invasive ductal carcinomas of the breast. The K- and N-ras protooncogenes were either not expressed or expressed at low levels. No amplification or rearrangement of the three ras genes was detected among the 104 breast carcinoma DNAs tested. These results indicate that the overexpression of H-ras in human breast tumors is not correlated with alteration of the protooncogene. In addition, we did not find any point mutation at the codon 12 of the H-ras or K-ras protooncogenes in 32 and 64, respectively, tumor DNAs examined. However, in tumor DNAs from 14 of 51 patients, heterozygous for H-ras-1 related BamHI restriction fragments, one allele was lost. This allele loss did not alter ras Mr 21,000 protein expression. Correlation with clinicopathological data showed, however, that the loss of one H-ras-1 allele in breast carcinoma DNAs is significantly linked to histological Grade III tumors, the lack of estrogen and/or progesterone receptors, and the subsequent occurrence of distal metastasis. Our results thus indicate that the loss of one H-ras-1 allele correlates with the most aggressive primary carcinomas of the breast.

Using c-Ha-, c-Ki-, and c-N-ras-specific probes in a RNA-RNA hybridization assay we found enhanced expression of c-Ha-ras protooncogene in stomach adenocarcinomas relative to nonneoplastic epithelium, whereas little or no transcription of either c-Ki- or c-N-ras was detected. Enhanced levels of c-Ha-ras RNA expression were detected in all of the adenocarcinomas examined. Hybridization with c-Ha-ras was also detected in nonneoplastic gastric epithelium adjacent to carcinoma, although the labeling was less intense than that of carcinoma cells. More extensive analysis of the c-Ha-ras p21 expression was then carried out in formalin-fixed, paraffin-embedded tissue sections and extracts from surgically resected stomach tissues using monoclonal antibodies (MAbs) RAP-5 and Y13-259. The data obtained from the immunohistochemical studies were consistent with the results of in situ hybridization assay. Adenocarcinomas were much more reactive with MAb RAP-5 than benign and normal tissues, and the majority of carcinomas demonstrated increased expression of c-Ha-ras p21. Quantitative liquid competition radioimmunoassays using MAb Y13-259 also demonstrated significantly higher levels of c-Ha-ras p21 in extracts from stomach adenocarcinomas than those from normal mucosae. No strict correlation was found between ras p21 expression and the degree of tumor differentiation or histological type. Although advanced carcinomas generally demonstrated higher levels of ras p21 than early carcinomas, no correlation among advanced carcinomas and ras p21 levels was observed in relation to depth of tumor invasion to the muscularis propria, subserosa, or serosa. Benign lesions, in comparison, were much less reactive with MAb RAP-5 than carcinomas. Among the benign lesions tested, dysplastic lesions were more reactive than nondysplastic lesions. Normal stomach mucosa was generally nonreactive with the exception of parietal cells. Our results indicate that transformation of the stomach mucosa from benign to malignant phenotype is associated with an increase in c-Ha-ras p21 expression.

We have studied the expression of the c-myc protooncogene and the cycle-dependent histone 4 gene at the cellular level by RNA:RNA in situ hybridization in 18 primary breast ductal adenocarcinomas. These tumors have previously been examined by Southern and Northern blot analysis for the genomic status of c-myc and its expression, respectively (Escot et al., Proc. Natl. Acad. Sci. USA, 83: 4834-4838, 1986). Positive c-myc hybridization signals were associated with carcinoma cells in all cases, including tumors which had no apparent alterations of the c-myc locus. Steady-state levels of c-myc mRNA appeared heterogeneous in carcinomas with similar histology. High levels of hybridization were found in four of seven tumors with strong amplification of the c-myc locus. Similarly high levels of c-myc hybridization were detected in two of nine cases which had an apparently normal c-myc locus but comparatively low cellularity. In addition to carcinoma cells, dense clusters of infiltrating lymphocytes, present in three tumors, exhibited c-myc hybridization. The expression of the histone 4 gene failed to correlate with levels of c-myc expression. We conclude that in infiltrating ductal carcinomas: (a) the c-myc protooncogene is transcriptionally activated; (b) c-myc amplification is probably underestimated due to heterogeneous cellularity; (c) high-level c-myc amplification is related to high-level expression, but other unknown factors also may play a role; (d) differences in levels of c-myc expression may not only be attributed to differences in the growth fractions; and (e) c-myc mRNA in total RNA from biopsy samples may be contributed by infiltrating lymphocytes.

High titers of oncornavirus-inactivating factor (OIF) were found previously in sera of laboratory mice. OIF is highly active against mouse xenotropic and polytropic envelope recombinant murine leukemia viruses (MuLVs) but not against ecotropic MuLVs. Of the 20 different mouse species or subspecies currently tested, that represent 4 subgenera, no OIF was found in the 3 subgenera more distant to the laboratory mouse. In the subgenus Mus, 7 of the 8 most distant species had no OIF, whereas all the ancestral species and subspecies of the laboratory mouse (Mus musculus musculus, Mus musculus domesticus), including a more distant member (Mus musculus cookii), had ample titers of OIF. A new separation technique was devised so that potential virus-neutralizing immunoglobulins could be separated by electrophoresis from OIF in small-volume serum samples. Active OIF was recovered from serum high-density lipoprotein, from very-low-density lipoprotein, as well as from chylomicron fractions. Murine sarcoma virus pseudotypes were made with several available exotic MuLV types. These pseudotype MuLVs were not susceptible to standard OIF preparations. The sera of exotic mice also had no factor analogous to OIF which would inactivate their own homologous or heterologous exotic MuLVs. It appears that, with one exception, OIF activity is limited to two subspecies of M. musculus and may be correlated in these subspecies with the presence of endogenous xenotropic MuLVs.