TAp63 suppresses mammary tumorigenesis through regulation of the Hippo pathway.

Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.

Inhibition of mammary tumorigenesis by estrogen and progesterone in genetically engineered mice.

Estrogen and progesterone play a critical role in normal and neoplastic development of the mammary gland. A long duration of estrogen and progesterone exposure is associated with increased breast cancer risk, and a short duration of the same doses of these hormones is associated with a reduced breast cancer risk. The protective effects of estrogen and progesterone have been extensively studied in animal models. Several studies have demonstrated that these hormones induce persistent and long-lasting alterations in gene expression in the mammary epithelial cells. In the experiments discussed herein, the protective effect of estrogen and progesterone is shown to occur in genetically engineered mice (the p53-null mammary gland). The protective effect is associated with a decrease in cell proliferation. The effects of hormones seem to manifest as a delay in premalignant progression. In the nontumor-bearing glands of hormone-treated mice, premalignant foci are present at the time the control glands are actively developing mammary tumors. If the hormone-treated cells are transplanted from the treated host to the untreated host, the cells resume their predetermined tumorigenic potential. The protective effect reflects both host-mediated factors (either stroma-determined or systemic factors) and mammary epithelial intrinsic changes. If normal, untreated p53 cells are transplanted into a host that has been previously treated with a short dose of hormones, the cells exhibit a significant delay in tumorigenesis. The relative contributions of host-mediated factors and mammary cell intrinsic factors remain to be determined. Current studies are moving this research area from the biological to the molecular realm and from the rodent models to human studies and offer the potential for directing prevention efforts at specific molecular targets.

Expression of heregulin by mouse mammary tumor cells: role in activation of ErbB receptors.

The inappropriate activation of one or more members of the ErbB family of receptor tyrosine kinases [ErbB-1 (EGFR), ErbB-2, ErbB-3, ErbB-4] has been linked with oncogenesis. ErbB-2 is frequently coexpressed with ErbB-3 in breast cancer cells and in the presence of the ligand heregulin (HRG) the ErbB-2/ErbB-3 receptors form a signaling heterodimer that can affect cell proliferation and apoptosis. The major goal of the present study was to determine whether endogenous HRG causes autocrine/paracrine activation of ErbB-2/ErbB-3 and contributes to the proliferation of mammary epithelial tumor cells. Tyrosine-phosphorylated (activated) ErbB-2 and ErbB-3 receptors were detected in the majority of extracts from tumors that had formed spontaneously or as a result of oncogene expression. HRG-1 transcripts and protein were found in the epithelial cells of most of these mouse mammary tumors. Various mouse mammary cell lines also contained activated ErbB-2/ErbB-3 and HRG transcripts. A approximately 50 kDa C-terminal fragment of pro-HRG was detected, which indicates that the HRG-1 precursor is readily processed by these cells. It is likely that the secreted mature HRG activated the ErbB-2/3 receptors. Addition of an antiserum against HRG to the mammary epithelial tumor cell line TM-6 reduced ErbB-3 Tyr-phosphorylation. Treatment with HRG-1 siRNA oligonucleotides or infection with a retroviral construct to stably express HRG siRNA effectively reduced HRG protein levels, ErbB-2/ErbB-3 activation, and the rate of proliferation, which could be reversed by the addition of HRG. The cumulative findings from these experiments show that coexpression of the HRG ligand contributes to activation of ErbB-2/Erb-3 in mouse mammary tumor cells in an autocrine or paracrine fashion.

Modeling human breast cancer metastasis in mice: maspin as a paradigm.

Breast cancer is the most common cancer detected in women, accounting for nearly one out of every three cancers diagnosed in the United States. Most cancer patients do not die from the primary tumor but die due to metastasis. Therefore, the study of metastasis is of most importance both to the clinician and patient. In the past, animal models have been used in breast cancer research and mammary gland biology. Our group has also established several animal models to address the function of a novel tumor suppressor gene maspin in breast tumor progression. Maspin was initially isolated from normal mammary epithelial cells. Its expression was down regulated in breast tumors. To test the protective role of maspin overexpression in mammary tumor progression, we crossed maspin overexpression transgenic mice (WAP-maspin) with a strain of oncogenic WAP-SV40 T antigen mice. The bitransgenic mice had reduced tumor growth rate and metastasis. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. In a separate animal experiment, maspin overexpressing mammary tumor cells (TM40D) were implanted into the fat pad of syngeneic mice. TM40D tumor cells were very invasive and metastatic. However, both primary tumor growth and metastasis were significantly blocked in TM40D cells that overexpress maspin as a consequence of plasmid or retrovirus infection. These evidences demonstrate that maspin function to inhibit primary tumor growth as well as invasion and metastasis. Elucidating the molecular mechanism of maspin action will shed light on our understanding of breast cancer invasion and metastasis.

Wnt-1 and int-2 mammary oncogene effects on the beta-catenin pathway in immortalized mouse mammary epithelial cells are not sufficient for tumorigenesis.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in all three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

Blocking tumor growth, invasion, and metastasis by maspin in a syngeneic breast cancer model.

Maspin is a unique serine protease inhibitor of which the down-regulation is associated with the development of breast cancers. In vitro, recombinant maspin inhibits tumor cell migration and invasion. Overexpression of maspin in transgenic mice is protective against tumor progression. Additionally, maspin acts as an angiogenesis inhibitor in rat cornea model and in a xenograft tumor model. To additionally prove that maspin is directly involved in the suppression of tumor growth and metastasis, we tested maspin in a new syngeneic mammary tumor model, TM40D. This model involves the implantation of TM40D mammary tumor cells orthotopically to the mammary gland; tumors grew within the gland and then become invasive and metastatic to other organs. Here we demonstrate that TM40D cells in implanted mammary glands are highly invasive. Overall, a 75% rate of invasion and metastasis was observed in this model. However, both primary tumor growth and metastasis were significantly blocked in TM40D cells that overexpress maspin as a consequence of plasmid or retrovirus infection. Maspin-transfected tumors tended to have tumor encapsulation and less necrosis, which were associated with better prognosis and lower invasiveness. Thus, maspin can block primary tumor growth as well as invasion and metastasis. These data support the concept that maspin has a strong protective role against tumor progression.

Cyclin-dependent kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse mammary preneoplasia.

The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16(INK4a) and p21(Cip1) protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27(Kip1) was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27(Kip1) prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.

Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice.

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

Nuclear factor-kappaB (NF-kappaB) regulates proliferation and branching in mouse mammary epithelium.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-kappaB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-kappaB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-kappaB during murine early postnatal mammary gland development by observing the consequences of increased NF-kappaB activity in mouse mammary epithelium lacking the gene encoding IkappaBalpha, a major inhibitor of NF-kappaB. Mammary tissue containing epithelium from inhibitor kappaBalpha (IkappaBalpha)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IkappaBalpha-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IkappaBalpha-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IkappaBalpha-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IkappaBalpha-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-kappaB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome.

Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.

Expression of Brca1 and splice variant Brca1delta11 RNA levels in mouse mammary gland during normal development and tumorigenesis.

Expression of Brca1 in mouse mammary cancer has yet to be analysed. We use a progressive model of neoplasia based on several mouse epithelial cell lines that represent distinct steps toward the fully tumorigenic state. Using RNase protection analysis because acceptable anti-Brca1 antibodies are not available we investigated the expression of Brca1 and a splice variant, Brca1Delta11, in several mammary hyperplasias and tumors that arose from them, and in normal mammary gland through pregnancy and involution. Expression of Brca1 was highest in rapidly proliferating cells. Expression of the full-length Brca1 was detectable in the virgin gland, was slightly elevated in the midpregnant gland, and decreased to levels similar to the age-matched virgin gland in the completely involuted gland. Expression of both forms of Brca1 was detectable in 9/9 paired hyperplasias and tumors, with levels of total Brca1, but not the splice variant Brca1Delta11, in tumors higher than those in the hyperplasias. While in disagreement with the observation that Brca1 levels decrease in human breast cancer progression, these patterns support the notion that Brca1 expression is associated with proliferating cells, and suggests that the link with differentiation seen in normal cells can be removed when cells become tumorigenic.

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells.

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53(null) mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53(null) mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53(null) mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53(null) and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53(null) vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53(null) mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53(null) mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

Bcl-2 expression delays mammary tumor development in dimethylbenz(a)anthracene-treated transgenic mice.

Bcl-2 is known to have dual antiproliferative and antiapoptotic roles. Overexpression of Bcl-2 in the mammary gland using a whey acidic protein (WAP) promoter-driven Bcl-2 transgene inhibits apoptosis in the mammary gland during pregnancy, lactation, and involution, and also counteracts apoptosis induced by overexpression of a mutant p53 transgene (WAP-p53 172 R-L). WAP-Bcl-2 mice and nontransgenic controls were treated with the carcinogen dimethylbenz(a)anthracene (DMBA). Surprisingly, the nontransgenic mice developed mammary tumors with decreased latency. Tumors arising in WAP-Bcl-2 mice displayed substantially reduced levels of proliferation relative to those seen in nontransgenic mice (P < 0.015), perhaps resulting in the observed increase in tumor latency following carcinogen treatment. This WAP-Bcl-2 mouse tumor model reflects the situation seen in some human breast cancers overexpressing Bcl-2, where expression of Bcl-2 has been shown to correlate with a lower proliferative index in tumors.

Interactions of apoptosis, proliferation and host age in the regression of the mouse mammary preneoplasia, TM3, carrying an unusual mutation in p53.

We have developed an in vivo model system of mouse mammary preneoplasias in order to examine the cell and molecular changes that occur during tumorigenesis. Most of these preneoplasias are characterized by an alveolar hyperplasia morphologically similar to that present in normal pregnant mammary gland, but have tumor forming capabilities ranging from very low to high. One of these hyperplasias, the TM3 HOG (transformed mammary hyperplastic outgrowth), forms tumors infrequently and has the unusual characteristic of spontaneous regression. We have observed that 7-8 months post-transplantation into the cleared mammary fat pad of a BALB/c mouse, the TM3 hyperplasia will begin to regress, leaving only a sparse ductal tree with remnant alveolar structures by 10-12 months post-transplantation. We have sought to elucidate the mechanism of this regression by determining the apoptotic and proliferative rates of the alveolar cells during TM3 HOG development. Studies show that apoptotic rates in the TM3 HOG are consistently high (4-7%) at all times after transplantation. This apoptotic rate is higher than the rates found in other preneoplasias in our system and approach the rates observed in the normal involuting gland. An unusual p53 mutation, a serine insertion at codon 233, may be causally related to the high spontaneous apoptotic frequencies as well as elevated inducible apoptotic frequencies in TM3. In addition, a sudden decrease ( approximately 63%) in proliferation occurs around 8 months post-transplantation. Furthermore, transplantation experiments indicate that the ability of the 8-month-old host and/or mammary gland to support growth of preneoplastic mammary tissues is markedly diminished compared with 3- or 6-month-old hosts. The results presented here suggest that the persistent high apoptotic rates, concomitant with decreased proliferation rates, may be responsible for TM3's regression and implicate a unique mutant p53 as a causal factor. Additionally, the results suggest that host determinants can interact with specific molecular changes in the preneoplastic cells to influence growth and progression of the preneoplastic populations.

Murine mammary gland carcinogenesis is critically dependent on progesterone receptor function.

To define the functional relevance of progesterone-initiated intracellular signaling in mammary gland tumorigenesis, the progesterone receptor knockout (PRKO) mouse model was used in the context of an established carcinogen-induced mammary tumorigenesis system. In carcinogen-treated, 7,12-dimethylbenz(a)anthracene (DMBA), pituitary-isografted mice, there was a marked reduction in mammary tumor incidence in PRKO mice as compared with isogenic wild types (WT). Mammary tumors arose in 12 (60%) of 20 WT mice compared with 3 (15%) of 20 PRKO mice by 44 weeks after the initial DMBA treatment. In the absence of a pituitary isograft, mammary tumors developed in 4 (20%) of 20 WT mice versus 4 (20%) of 20 PRKO mice by 47 weeks. At the time of carcinogen administration, the proliferative index of the pituitary-stimulated WT gland was at least 4-fold higher than similarly treated PRKO glands, supporting the importance of PR-mediated proliferative pathways in the genesis of this tumor type. Unlike the WT gland, the PRKO gland was unable to exhibit alveologenesis in response to pituitary isograft stimulation; thus, DMBA-initiated mammary tumors observed in the PRKO were assumed to be exclusively of ductal origin. Compared with previous tested strains, by 47 weeks, a higher incidence of DMBA-induced ovarian tumors was observed in this mouse strain: (a) 4 (20%) of 20 WT mice and 9 (45%) of 20 PRKO mice with a pituitary isograft; and (b) 10 (50%) of 20 WT mice and 10 (50%) of 20 PRKO mice without a pituitary isograft. Despite the host-strain's underlying propensity for DMBA-induced ovarian neoplasms, our studies underscore the specific importance of the PR (as distinct from the estrogen receptor) as a mandatory mediator for those intracellular signaling pathways that are essential for the initiation of the majority of murine mammary tumors induced by DMBA. Apart from providing strong support for progesterone's role in mammary gland tumorigenesis as well as furthering our fundamental understanding of breast cancer etiology, these studies may have implications for the routine use of progestins.

Stage-specific changes in SR splicing factors and alternative splicing in mammary tumorigenesis.

Using a mouse model of mammary gland development and tumorigenesis we examined changes in both alternative splicing and splicing factors in multiple stages of mammary cancer. The emphasis was on the SR family of splicing factors known to influence alternative splicing in a wide variety of genes, and on alternative splicing of the pre-mRNA encoding CD44, for which alternative splicing has been implicated as important in a number of human cancers, including breast cancer. We observed step-wise increases in expression of individual SR proteins and alternative splicing of CD44 mRNA during mammary gland tumorigenesis. Individual preneoplasias differed as to their expression patterns for SR proteins, often expressing only a sub-set of the family. In contrast, tumors demonstrated a complex pattern of SR expression. Little difference was observed between neoplasias and their metastases. Alternative splicing of CD44 also changed through the disease paradigm such that tumors produced RNA containing a mixture of variable exons, whereas preneoplasias exhibited a more restricted exon inclusion pattern. In contrast, other standard splicing factors changed little in either concentration or splicing pattern in the same cells. These data suggest alterations in relative concentrations of specific splicing factors during early preneoplasia that become more pronounced during tumor formation. Given the ability of SR proteins to affect alternative processing decisions, our results suggest that a number of pre-mRNAs may undergo changes in alternative splicing during the early and intermediate stages of mammary cancer.

A transgenic mouse model for mammary carcinogenesis.

Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and influence both the prognosis and response to chemotherapy. Amino acid 175 (equivalent to murine 172) is the second most common site of missense mutations in p53 in human breast cancer. Over 95% of these mutations are arginine-to-histidine (R-H) substitutions resulting in a gain-of-function, and not merely a dominant-negative phenotype. Transgenic mice expressing a p53 172(R-H) construct targeted to the mammary gland by means of a whey acidic protein (WAP) promoter were characterized as a model system in order to determine the specific effects of this mutation on mammary tumorigenesis. Although transgene expression alone had no apparent effect on normal mammary development, transgenic mice treated with the chemical carcinogen dimethylbenz(a)anthracene developed tumors with much shorter latency than did control littermates and had a greater tumor burden. Tumors arising in transgenic mice did not exhibit either decreased apoptosis or increased cell proliferation relative to tumors arising in nontransgenic littermates, but did display increased genomic instability. Large pleiomorphic nuclei were visible in many tumors from transgenic mice, and DNA flow analysis confirmed the presence of significant aneuploid cell populations. Since these transgenic mice develop very few spontaneous tumors, while accelerating carcinogen-and oncogene-mediated tumorigenesis, this mouse model will, therefore, be useful in the investigation of early events in mammary tumorigenesis. It may also be used as a preclinical model to test newly developed chemotherapeutic strategies.

Delay of dimethylbenz[a]anthracene-induced mammary tumorigenesis in transgenic mice by apoptosis induced by an unusual mutant p53 protein.

Murine p53 containing an Arg-->Leu substitution at amino acid 172 possesses many properties characteristic of wild-type p53, including the ability to induce p21/WAF/Cip1 and apoptosis. To determine if p53-dependent apoptosis plays a critical role in mammary tumorigenesis, transgenic mice were generated in which the expression of this mutant p53 protein was targeted to the mammary gland by using the rat whey acidic protein gene promoter. Mice bearing pituitary isografts were treated with 7,12-dimethylbenz[a]anthracene (DMBA) and examined for mammary tumor development. Mice overexpressing the p53 transgene exhibited a statistically significant increase in apoptosis in the mammary gland and a statistically significant decrease in the incidence of DMBA-induced mammary tumors. No difference in tumor incidence was observed in mice without pituitary isografts who were treated with DMBA, because the transgene is not overexpressed in the absence of hormone stimulation provided by the pituitary isograft. The unexpected wild-type properties of the 172Arg-->Leu mutant p53, including its ability to stimulate apoptosis, make it a possible candidate for use in gene therapy protocols.

Mutations in p53 are frequent in the preneoplastic stage of mouse mammary tumor development.

Preneoplastic lesions in the mammary gland represent a population of cells at increased risk of progression to tumors. Because p53 is the most commonly mutated gene in human breast cancer, we sought to determine whether mutations in p53 were present in preneoplastic lesions or were acquired during progression to overt tumors. In the mouse mammary gland, hyperplastic alveolar nodules (HAN) are the most common preneoplastic lesion. Analysis of the TM series of transplantable murine HAN outgrowths and tumors allowed the status of p53 to be determined at distinct stages of mammary tumorigenesis. Alterations in the p53 gene or in the pattern of p53 protein expression were observed in all five HAN outgrowth lines examined. Altered expression of p53 protein was detected in 3 of 5 TM HAN outgrowth lines as determined by immunohistochemistry. Overexpression of nuclear p53 was detected in only a fraction of the cells (10-50%) in TM3 and TM4 HAN outgrowths, whereas in tumors that arose from TM4 HAN outgrowths, the proportion of cells overexpressing p53 protein approached approximately 100%. Despite overexpression of p53 in TM3 HAN outgrowths, no tumors have developed in this line. The TM9 outgrowth line exhibited a different pattern of p53 expression by immunohistochemistry: p53 protein was overexpressed in the cytoplasm of virtually all cells in the HAN outgrowths but was localized to the nuclei of TM9 tumor cells. Direct sequencing of p53 transcripts from tumors and cell lines revealed various genetic changes: point mutations in exons 4 and 5 (TM2H, nonsense; TM4, missense); a deletion in exon 5 (TM4); and an insertion in exon 7 (TM3). Although p53 protein was overexpressed in TM9 tumors, it was shown to be wild-type both by immunoprecipitation and direct sequencing of the entire coding region of the cDNA. TM4 cells were homogeneous with respect to mutant p53 genotype and uniformly expressed p53 by immunohistochemical staining in vitro, but transplantation of TM4 cells to fat pads of BALB/c hosts resulted in HAN outgrowths in situ in which < 50% of the cells expressed the mutant p53 at detectable levels. In summary, mutation of the p53 gene and overexpression of p53 protein can occur in preneoplastic mammary epithelial cells, and those mutations are maintained in tumors that arise from the HAN. Conversely, expression of mutant p53 was decreased when cells were grown in situ, implicating the presence of cellular factors that can suppress p53 expression in vivo. These observations demonstrate that the p53 pathway may be a common target for mutation in murine mammary tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)