Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice.

Germline mutations in the tumor suppressor gene BRCA1 predispose women to breast cancer, however somatic mutations in the gene are rarely detected in sporadic cancers. To understand this phenomenon, we examined mouse models carrying conditional disruption of Brca1 in mammary epithelium in either p53 wild type (wt) or heterozygous backgrounds. Although a p53(+/-) mutation significantly accelerated tumorigenesis, both strains developed mammary tumors in a stochastic fashion, suggesting that multiple factors, in addition to p53 mutations, may be involved in Brca1 related tumorigenesis. A unique feature of Brca1 mammary tumors is their highly diverse histopathology accompanied by severe chromosome abnormalities. The tumors also display extensive genetic/molecular alterations, including overexpression of ErbB2, c-Myc, p27 and Cyclin D1 in the majority of tumors, while they were virtually ERalpha and p16 negative. Translocations involving p53 were also identified which lead to abnormal RNA and protein products. In addition, we generated cell lines from mammary tumors and found that the cells retained many of the genetic changes found in the primary tumors, suggesting that these genes may be players in Brca1-associated tumorigenesis. Despite their distinct morphology, all cultured tumor cells were Tamoxifen resistant but highly sensitive to Doxorubicin or gamma-irradiation, suggesting that these methods would be effective in treatment of this disease.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis.     

HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors, which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis, tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes, increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive, but not resistant, cell lines, an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis, tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore, the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors.     

Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2

Introduction

The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host.

Methods

FVB-huHER2 mice were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy.

Results

Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-γ production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells.

Conclusions

Anti-huHER2 antibodies elicited in the tolerant host exert antitumor activity.     

Inhibition of mammary tumorigenesis in GR mice with 2-bromo-alpha-ergocryptine.

A high incidence of mammary tumors is found in multiparous GR mice during the 2nd and 3rd pregnancies and in nulliparous GR mice treated with estrone/progesterone. The purpose of this study was to determine if prolactin is a contributing hormone in the genesis of these neoplasms. In one series of experiments, 238 15-week-old nulliparous GR mice were treated with estrone (drinking water, 0.5 mg/liter) plus progesterone (30 mg progesterone pellet with cholesterol, implanted SC once monthly) for a period of 13 weeks. Half of these mice were injected SC once daily with 100 micrograms of the prolactin-suppressing drug 2-bromo-alpha-ergocryptine (CB-154) for the duration of hormone treatment, and the other half were injected SC once daily with 0.9% NaCl solution (controls). In another series of experiments, 87 pregnant GR mice were divided into two groups and injected SC once daily from day 7 to 21 of pregnancy with 0.9% NaCl solution (controls) or CB-154 (100 micrograms/mouse). In the first series, the numbers of mice with mammary tumors and total number of mammary tumors were: controls, 58/119 (49%) and 73; CB-154 treatment, 34/119 (29%) and 37, respectively. In the second series, the numbers were: controls, 39/44 (89%) and 73; CB-154 treatment, 24/43 (56%) and 43, respectively. In both studies, CB-154 treatment significantly (p less than 0.05-0.005) reduced the percentage of mice with mammary tumors and total number of mammary tumors. These results provide evidence that prolactin is a contributing hormone in the genesis of estrone/progesterone and pregnancy-induced mammary tumors in female GR mice.     

The nuclear receptor coactivator amplified in breast cancer-1 is required for Neu (ErbB2/HER2) activation, signaling, and mammary tumorigenesis in mice

Overexpression of the oncogene amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) induces mammary tumorigenesis in mice. In breast cancer, high levels of AIB1/SRC-3 and the growth factor receptor HER2/neu predict resistance to endocrine therapy and poor outcome. However, a mechanistic relationship between AIB1/SRC-3 and HER2/neu in the development of breast cancer has not been shown. Here, we show that deletion of one allele of SRC-3 significantly delays Neu-induced mammary tumor development in mice. Homozygous deletion of SRC-3 in mice completely prevents Neu-induced tumor formation. By ages 3 to 4 months, Neu/SRC-3(+/-) mice exhibit a noticeable reduction in lateral side-bud formation, accompanied by reduced cellular levels of phosphorylated Neu compared with Neu/SRC-3(wt) mice. In Neu-induced tumors, high levels of SRC-3, phosphorylated Neu, cyclin D1, cyclin E, and proliferating cell nuclear antigen expression are observed, accompanied by activation of the AKT and c-Jun NH(2) kinase (JNK) signaling pathways. In comparison, phosphorylated Neu, cyclin D1, and cyclin E are significantly decreased in Neu/SRC-3(+/-) tumors, proliferation is reduced, and AKT and JNK activation is barely detectable. Our data indicate that AIB1/SRC-3 is required for HER2/neu oncogenic activity and for the phosphorylation and activation of the HER2/neu receptor. We predict that reducing AIB1/SRC-3 levels or activity in the mammary epithelium could potentiate therapies aimed at inhibiting HER2/neu signaling in breast cancer.     

Development of mammary adenocarcinomas by tissue-specific knockout of Brca2 in mice

Cre-mediated deletion of exons 3 and 4 of the mouse Brca2 gene occurring specifically in mammary epithelial cells of conditional female mutants carrying a combination of loxP-modified and null Brca2 alleles resulted in a high incidence (77%) of breast tumors that were often palpable and developed in one or more glands after long latency (time for median tumor-free survival of approximately 1.4 years; total of 40 tumors in 20 animals). These invasive adenocarcinomas were histologically quite uniform, exhibiting predominantly a solid, nodular tumor pattern with very few variants, in striking contrast to the morphological heterogeneity of analogous Brca1-associated tumors. The karyotypes of tumor cells lacking Brca2 had various chromosomal aberrations and ranged from diploid to hypertetraploid, but this wide variability was incongruous with the histological appearance of carcinomas that was comparable between specimens. The implications of these observations in the context of models positing that Brca2 is involved in the maintenance of genomic stability are discussed.     

Streptozotocin-induced diabetes accelerates mammary tumorigenesis in shn and sln mice

Virgin female SHN and SLN mice, a high and a low spontaneous mammary tumor strains, respectively, were given a single intravenous injection of streptozotocin (2 mg/10 g body weight), resulting in a significant rise of blood glucose levels and of mammary tumor incidence. The growth rate of mammary tumors was also accelerated by streptozotocin-induced diabetes in SHN mice, but not in SLN mice. The incidence and progression of uterine adenomyosis and adenocarcinoma were not affected by diabetes in SHN and SLN mice. These findings indicate that spontaneous mammary tumorigenesis in SHN and SLN mice is accelerated by streptozotocin-induced diabetes.     

Prolonged lactation in mice and its effect on mammary tumorigenesis

Prolongation of lactation was induced by litter replacement in MTV-infected (♀ C3H × ♂ O20) F ₁ mice, each bearing two pituitary isografts under the kidney capsule. Measurement of litter-growth revealed that milk production occurs in cycles of 2–3 weeks' duration. Examination of intact and thelectomized mammary glands during prolonged lactation suggested that in the thelectomized glands there exists an “alveolar cycle”. Recently proliferated alveoli reached a size comparable to that of those in the intact lactating glands. Following distension without milk removal degenerative changes and pigment deposition occurred. It is believed that proliferation occurs in the thelectomized glands because of the presence of fat tissue. In the non-lactating mice, also bearing pituitary grafts, the size of the alveoli was smaller. Following a regression period it was observed that thelectomized glands contained hyperplastic nodules and pigment. In the contralateral intact glands, which had a lactation history, pigment was absent and only a few nodules were seen. Hyperplastic nodules were always devoid of pigment. Tumours appeared with the same rapidity in intact glands of non-lactating mice and in thelectomized glands of both lactating and non-lactating mice. Tumorigenesis was retarded only in the lactating glands. Thelectomy had no effect on tumorigenesis in non-lactating mice. It is postulated that lactation inhibits noduligenesis and thus tumorigenesis by protecting the gland against the growth-stimulating effect of hormones. On a observé qu'après une période de régression les glandes thélectomisées contenaient des nodules hyperplasiques et du pigment. Dans les glandes controlatérales intactes qui avaient produit du lait, le pigment était absent et on ne trouvait qu'un petit nombre de nodules. Il n'y avait jamais de pigment dans les nodules hyperplasiques. l'auteur émet L'HYPothèse que la lactation inhibe la formation des nodules et des tumeurs en protégeant la glande contre la stimulation de croissance produite par les hormones.     

Targeting HER 2 and angiogenesis in gastric cancer

Gastric cancer is one of the most commonly diagnosed and the second leading cause of cancer death worldwide. Surgery combined with multimodal therapy remains the only curative therapy. However, local relapse or distant metastases occur in more than 50% of radically resected patients. Due to molecular therapies, targeting HER2 and angiogenesis, major advances in the treatment of gastric cancer could be achieved. Nevertheless, development of resistance to monoclonal antibodies, such as trastuzumab, is arising. Currently a number of promising new therapeutic are under investigation, combining chemotherapy with newly developed agents to overcome cancer resistance. In this review we report current clinical applications of targeted therapies and overview ongoing trials, investigating the use of monoclonal antibodies in (HER2 positive) gastric cancer.     

Effects of pituitary graft and 2-bromo-alpha-ergocryptine on mammary DNA synthesis in mice in relation to mammary tumorigenesis.

3H-thymidine incorporation into mammary DNA as an index of mammary DNA synthesis in C3H/He virgin females was significantly higher at proestrus than at estrus and diestrus. The rise in incorporation at proestrus was completely blocked by the administration of 2-bromo-alpha-ergocryptine (Bromocriptin) 1 day before proestrus. The incorporation was maintained at a higher level than that at proestrus by pituitary grafts. The results supported the view that mammary DNA synthesis, which is stimulated by prolactin, is an important factor for mammary tumorigenesis.     

Hoxb7 inhibits transgenic HER-2/neu-induced mouse mammary tumor onset but promotes progression and lung metastasis

Our previous studies have shown that HOXB7 mRNA is overexpressed in approximately 50% of invasive breast carcinomas and promotes tumor progression in breast cancer cells grown as xenografts in mice. In silico analysis of published microarray data showed that high levels of HOXB7 predict a poor outcome in HER-2-positive (P = 0.046), but not in HER-2-negative breast cancers (P = 0.94). To study the function of HOXB7 in vivo in the context of HER-2 overexpression, we generated mouse mammary tumor virus (MMTV)-Hoxb7 transgenic mice, and then crossed them with MMTV-HER-2/neu transgenic mice. In the mice carrying both Hoxb7 and HER-2/neu transgenes, Hoxb7 plays a dual role in mammary tumorigenesis. In double transgenic mice, overexpression of Hoxb7 delayed tumor onset and lowered tumor multiplicity. However, consistent with the clinical data, once the tumors appeared, their growth was faster and metastasis to the lungs occurred at a higher frequency. Our data show, for the first time, that deregulated expression of Hoxb7 in mammary tumor cells can significantly modulate HER-2/neu-oncogene induced tumorigenesis in vivo.     

ErbB2 enhances mammary tumorigenesis,oncogene-independent recurrence and metastasis in a model of IGF-IR-mediated mammary tumorigenesis

Background  

The type I insulin-like growth factor receptor (IGF-IR) and ErbB2 (Her-2) are receptor tyrosine kinases implicated in human breast cancer. Both proteins are currently the subject of targeted therapeutics that are used in the treatment of breast cancer or which are in clinical trials. The focus of this study was to utilize our inducible model of IGF-IR overexpression to explore the interaction of these two potent oncogenes.     

Linking DNA damage to medulloblastoma tumorigenesis in patched heterozygous knockout mice

Hemizygous Ptc1 mice have many features of Gorlin syndrome, including predisposition to medulloblastoma development. Ionizing radiation synergize with Ptc1 mutation to induce medulloblastoma only in neonatally exposed mice. To explore the mechanisms underlying age-dependent susceptibility, we irradiated Ptc(neo67/+) mice at postnatal day 1 (P1) or 10 (P10). We observed a dramatic difference in medulloblastoma incidence, which ranged from 81% in the cerebellum irradiated at P1 to 3% in the cerebellum irradiated at P10. A striking difference was also detected in the frequency of cerebellar preneoplastic lesions (100 versus 14%). Our data also show significantly lower induction of apoptosis in the cerebellum of medulloblastoma-susceptible (P1) compared to -resistant (P10) mice, strongly suggesting that medulloblastoma formation in Ptc1 mutants may be associated with resistance to radiation-induced cell killing. Furthermore, in marked contrast with P10 mice, cerebellum at P1 displays substantially increased activation of the cell survival-promoting Akt/Pkb protein, and markedly decreased p53 levels in response to radiation-induced genotoxic stress. Overall, these results show that developing cerebellar granule neuron precursors' (CGNPs) radiosensitivity to radiation-induced cell death increases with progressing development and inversely correlates with their ability to neoplastically transform.     

The cyclooxygenase-2 inhibitor, celecoxib, prevents the development of mammary tumors in Her-2/neu mice.

Evidence is now available showing that cyclooxygenase (COX)-2, which is involved in prostaglandin production, is overexpressed in many types of tumors including breast. Several reports have indicated that HER-2/neu-positive breast tumors are associated with an increased amount of COX-2 protein. In this study, we evaluated the effectiveness of the select COX-1 and COX-2 inhibitors in preventing mammary tumor development in HER-2/neu transgenic mice. At 4 weeks of age, female HER-2/neu mice were fed a #5020 rodent diet supplemented with 900 ppm celecoxib, a COX-2 inhibitor, 64 ppm of SC560, a COX-1 inhibitor, or the unsupplemented #5001 diet (control). The incidence of mammary tumors was significantly lower in the celecoxib-fed mice (71%; P = 0.001 versus control) than in the control mice (95%) or in the SC560-fed mice (91%). Celecoxib-treated mice also developed fewer tumors (1.3 +/- 1.1 SD; P = 0.039 versus control) than the control mice (2.2 +/- 1.2) or the SC560 treated mice (2.3 +/- 1.3). The median time to tumor development was 266 days in the control group versus 291 days in the celecoxib-treated group (P = 0.003 versus control). Lung metastasis was also reduced by treatment with celecoxib. The COX-1 inhibitor SC560 had no protective effect. The protection offered by celecoxib was associated with significantly lower concentrations of prostacyclin and prostaglandin E(2) in mammary tumors and their adjacent mammary glands. Our findings provide additional preclinical evidence to support the clinical studies to investigate the potential effectiveness of COX-2 inhibitors in protecting woman who are at high risk for breast cancer.     

Inhibition by indomethacin of spontaneous mammary tumorigenesis in SHN mice

This experiment was designed to examine the effects of indomethacin, a potent prostaglandin synthesis inhibitor, on spontaneous mammary tumors in mice. The growth of established mammary tumors and the appearance of new tumors in multiparous SHN mice were significantly suppressed by the subcutaneous implantation of pellets of indomethacin diluted to 1/12 with cholesterol. Furthermore, the same treatment inhibited normal and preneoplastic mammary gland growth in virgin SHN mice. The pattern of estrous cycles, ovarian structure, and plasma prolactin levels were not affected significantly by the treatment. All results have demonstrated that indomethacin inhibits mammary tumorigenesis of mice primarily by route(s) other than the endocrine system under the present experimental conditions. Indomethacin would be the first agent that appears to inhibit the growth of spontaneous mammary tumors of palpable size in mice.     

Parity-induced mammary epithelial cells facilitate tumorigenesis in MMTV-neu transgenic mice

Using a Cre-lox-based genetic labeling technique, we have recently discovered a parity-induced mammary epithelial subtype that is abundant in nonlactating and nonpregnant, parous females. These mammary epithelial cells serve as alveolar progenitors in subsequent pregnancies, and transplantation studies revealed that they possess features of multipotent progenitors such as self-renewal and the capability to contribute to ductal and alveolar morphogenesis. Here, we report that these cells are the cellular targets for transformation in MMTV-neu transgenic mice that exhibit accelerated mammary tumorigenesis in multiparous animals. The selective ablation of this epithelial subtype reduces the onset of tumorigenesis in multiparous MMTV-neu transgenics. There is, however, experimental evidence to suggest that parity-induced mammary epithelial cells may not be the only cellular targets in other MMTV-promoter-based transgenic strains. In particular, the heterogeneous MMTV-wnt1 lesions predominantly express the ductal differentiation marker Nkcc1 that is absent in MMTV-neu-derived tumors. Our observations support the idea that tumors originate from distinctly different epithelial subtypes in selected MMTV-promoter-driven cancer models and that diverse oncogenes might exert discrete effects on particular mammary epithelial subtypes.