The origin of breast tumor heterogeneity.

How breast diversity is generated is a fascinating and fundamental question with important clinical implications. It is clear that the diversity of phenotypes displayed by breast cancer cells reflects the array of cell types present in the disease-free breast epithelium, including luminal, basal and stem cells. Therefore, it is hypothesized that the molecular regulators governing normal development of the breast epithelium may double as engines of breast tumor diversity. In the past few years, a deepened understanding of the mammary epithelial hierarchy has prompted the search for the cellular precursors of breast tumors. At the same time, the use of novel experimental strategies including the new technology of massively parallel sequencing has provided insight into the origin and evolution of breast tumors. Here, we review the current understanding of the basis of the intrinsic subtypes and the sources of inter-tumor heterogeneity.

The role of fibroblast Tiam1 in tumor cell invasion and metastasis.

The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.

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.

Cytoplasmic sequestration and functional repression of p53 in the mammary epithelium is reversed by hormonal treatment.

Proper function of the p53 tumor suppressor gene is critical for inhibiting tumor development in a broad spectrum of tissues. Although the mammary gland is highly susceptible to tumor formation, the functional status of p53 in the normal tissue had not been investigated. Therefore, expression, localization, and activity of p53 were examined in normal mammary tissues. High levels of p53 protein were found expressed in the cytoplasm of the ductal epithelium of the quiescent mammary gland. Ionizing radiation failed to recruit p53 to the nucleus, and p53-dependent responses were minimal. However, transient hormonal stimulation resulted in nuclear accumulation of p53, an induction of p21/WAF1, and a 5-fold increase in apoptosis after ionizing radiation. Therefore, the functional state of wild-type p53 in the mammary epithelium can be regulated by hormonal stimuli.

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.

Regulation of p53 and its targets during involution of the mammary gland.

Post-lactational involution of the mammary gland provides a system in which to study the expression and function of genes that regulate apoptosis in the context of a normal tissue. The functions of the p53 tumor suppressor gene have been extensively studied as a mediator of apoptosis in response to DNA damage, but its regulation in normal physiologic processes has been poorly characterized. Expression of p53 mRNA was shown to be among the first genes to be induced in mammary tissue following weaning of neonates. Although involution proceeds in the absence of a functional p53 gene, it is delayed compared to normal individuals. Therefore, involution can be viewed as biphasic with initial responses being sensitive to p53, whereas secondary responses being p53-independent. These observations can be exploited to determine the subset of genes that are p53-responsive and that mediate the effects of p53 in normal mammary tissue.

Delayed involution of the mammary epithelium in BALB/c-p53null mice.

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been defined. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21WAF1 mRNA. Induction of p21WAF1 mRNA was absent in BALB/c-p53null mice, and therefore, was shown to be p53-dependent. The BALB/c-p53null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53(wt) mice through 5 days post-weaning. The delay was transient with no differences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaffected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the first stage of involution initiated by the epithelium itself, but does not affect the second phase during which stromal proteases are induced.

Radiation-induced tumorigenesis in preneoplastic mouse mammary glands in vivo: significance of p53 status and apoptosis.

In mouse mammary tumorigenesis, p53 mutations facilitate tumorigenesis in concert with other oncogenic alterations. Ionizing radiation enhances tumorigenesis in preneoplastic mammary outgrowth lines and induces p53-dependent apoptosis. We asked if normal p53 function modulates radiation-induced tumorigenesis in preneoplastic mammary lesions by affecting the apoptotic pathway of cell deletion. Three different hyperplastic outgrowth lines were compared. Outgrowth line D1 overexpressed wild-type p53 and responded to irradiation with enhanced tumorigenicity but no induction of apoptosis. Outgrowth line TM12 exhibited normal wild-type p53 expression and responded to irradiation with no alteration in tumorigenicity but with a marked increase in apoptosis. Outgrowth line TM2L also exhibited normal wild-type p53 expression and responded to irradiation with a marked enhancement in both tumorigenicity and apoptosis. These results indicate that the two radiation-induced responses, apoptosis and tumorigenesis, are dissociable events in the mammary gland. Furthermore, radiation-induced tumorigenicity was not abrogated by either enhanced wild-type p53 expression or a robust apoptotic response. The radiation dose of 5 Gy most likely induces multiple genetic alterations in surviving cells, including genomic instability, and this may account for the tumorigenicity. Future experiments will examine lower doses of irradiation that still induce a significant apoptotic response but significantly less genomic instability.