Rare subclonal sequencing of breast cancers indicates putative metastatic driver mutations are predominately acquired after dissemination.

BACKGROUND: Evolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear. METHODS: To ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 "metastasis-specific" mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition. RESULTS: The ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable. CONCLUSIONS: Our results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.

B3GALT6 promotes dormant breast cancer cell survival and recurrence by enabling heparan sulfate-mediated FGF signaling.

Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. In vivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.

PAQR8 promotes breast cancer recurrence and confers resistance to multiple therapies.

BACKGROUND: Breast cancer mortality is principally due to recurrent disease that becomes resistant to therapy. We recently identified copy number (CN) gain of the putative membrane progesterone receptor PAQR8 as one of four focal CN alterations that preferentially occurred in recurrent metastatic tumors compared to primary tumors in breast cancer patients. Whether PAQR8 plays a functional role in cancer is unknown. Notably, PAQR8 CN gain in recurrent tumors was mutually exclusive with activating ESR1 mutations in patients treated with anti-estrogen therapies and occurred in > 50% of both patients treated with anti-estrogen therapies and those treated with chemotherapy or anti-Her2 agents. METHODS: We used orthotopic mouse models to determine whether PAQR8 overexpression or deletion alters breast cancer dormancy or recurrence following therapy. In vitro studies, including assays for colony formation, cell viability, and relative cell fitness, were employed to identify effects of PAQR8 in the context of therapy. Cell survival and proliferation were quantified by immunofluorescence staining for markers of apoptosis and proliferation. Sphingolipids were quantified by liquid chromatography-high resolution mass spectrometry. RESULTS: We show that PAQR8 is necessary and sufficient for efficient mammary tumor recurrence in mice, spontaneously upregulated and CN gained in recurrent tumors that arise following therapy in multiple mouse models, and associated with poor survival following recurrence as well as poor overall survival in breast cancer patients. PAQR8 promoted resistance to therapy by enhancing tumor cell survival following estrogen receptor pathway inhibition by fulvestrant or estrogen deprivation, Her2 pathway blockade by lapatinib or Her2 downregulation, and treatment with chemotherapeutic agents. Pro-survival effects of PAQR8 were mediated by a Gi protein-dependent reduction in cAMP levels, did not require progesterone, and involved a PAQR8-dependent decrease in ceramide levels and increase in sphingosine-1-phosphate levels, suggesting that PAQR8 may possess ceramidase activity. CONCLUSIONS: Our data provide in vivo evidence that PAQR8 plays a functional role in cancer, implicate PAQR8, cAMP, and ceramide metabolism in breast cancer recurrence, and identify a novel mechanism that may commonly contribute to the acquisition of treatment resistance in breast cancer patients.

Cellular dormancy in minimal residual disease following targeted therapy.

BACKGROUND: Breast cancer mortality is principally due to tumor recurrence, which can occur following extended periods of clinical remission that may last decades. While clinical latency has been postulated to reflect the ability of residual tumor cells to persist in a dormant state, this hypothesis remains unproven since little is known about the biology of these cells. Consequently, defining the properties of residual tumor cells is an essential goal with important clinical implications for preventing recurrence and improving cancer outcomes. METHODS: To identify conserved features of residual tumor cells, we modeled minimal residual disease using inducible transgenic mouse models for HER2/neu and Wnt1-driven tumorigenesis that recapitulate cardinal features of human breast cancer progression, as well as human breast cancer cell xenografts subjected to targeted therapy. Fluorescence-activated cell sorting was used to isolate tumor cells from primary tumors, residual lesions following oncogene blockade, and recurrent tumors to analyze gene expression signatures and evaluate tumor-initiating cell properties. RESULTS: We demonstrate that residual tumor cells surviving oncogenic pathway inhibition at both local and distant sites exist in a state of cellular dormancy, despite adequate vascularization and the absence of adaptive immunity, and retain the ability to re-enter the cell cycle and give rise to recurrent tumors after extended latency periods. Compared to primary or recurrent tumor cells, dormant residual tumor cells possess unique features that are conserved across mouse models for human breast cancer driven by different oncogenes, and express a gene signature that is strongly associated with recurrence-free survival in breast cancer patients and similar to that of tumor cells in which dormancy is induced by the microenvironment. Although residual tumor cells in both the HER2/neu and Wnt1 models are enriched for phenotypic features associated with tumor-initiating cells, limiting dilution experiments revealed that residual tumor cells are not enriched for cells capable of giving rise to primary tumors, but are enriched for cells capable of giving rise to recurrent tumors, suggesting that tumor-initiating populations underlying primary tumorigenesis may be distinct from those that give rise to recurrence following therapy. CONCLUSIONS: Residual cancer cells surviving targeted therapy reside in a well-vascularized, desmoplastic microenvironment at both local and distant sites. These cells exist in a state of cellular dormancy that bears little resemblance to primary or recurrent tumor cells, but shares similarities with cells in which dormancy is induced by microenvironmental cues. Our observations suggest that dormancy may be a conserved response to targeted therapy independent of the oncogenic pathway inhibited or properties of the primary tumor, that the mechanisms underlying dormancy at local and distant sites may be related, and that the dormant state represents a potential therapeutic target for preventing cancer recurrence.

Genomic landscape of metastatic breast cancer identifies preferentially dysregulated pathways and targets.

Nearly all breast cancer deaths result from metastatic disease. Despite this, the genomic events that drive metastatic recurrence are poorly understood. We performed whole-exome and shallow whole-genome sequencing to identify genes and pathways preferentially mutated or copy-number altered in metastases compared with the paired primary tumors from which they arose. Seven genes were preferentially mutated in metastases - MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1 - 5 of which are not significantly mutated in any type of human primary cancer. Four regions were preferentially copy-number altered: loss of STK11 and CDKN2A/B, as well as gain of PTK6 and the membrane-bound progesterone receptor, PAQR8. PAQR8 gain was mutually exclusive with mutations in the nuclear estrogen and progesterone receptors, suggesting a role in treatment resistance. Several pathways were preferentially mutated or altered in metastases, including mTOR, CDK/RB, cAMP/PKA, WNT, HKMT, and focal adhesion. Immunohistochemical analyses revealed that metastases preferentially inactivate pRB, upregulate the mTORC1 and WNT signaling pathways, and exhibit nuclear localization of activated PKA. Our findings identify multiple therapeutic targets in metastatic recurrence that are not significantly mutated in primary cancers, implicate membrane progesterone signaling and nuclear PKA in metastatic recurrence, and provide genomic bases for the efficacy of mTORC1, CDK4/6, and PARP inhibitors in metastatic breast cancer.

Impact of obesity on breast cancer recurrence and minimal residual disease.

BACKGROUND: Obesity is associated with an increased risk of breast cancer recurrence and cancer death. Recurrent cancers arise from the pool of residual tumor cells, or minimal residual disease (MRD), that survives primary treatment and persists in the host. Whether the association of obesity with recurrence risk is causal is unknown, and the impact of obesity on MRD and breast cancer recurrence has not been reported in humans or in animal models. METHODS: Doxycycline-inducible primary mammary tumors were generated in intact MMTV-rtTA;TetO-HER2/neu (MTB/TAN) mice or orthotopic recipients fed a high-fat diet (HFD; 60% kcal from fat) or a control low-fat diet (LFD; 10% kcal from fat). Following oncogene downregulation and tumor regression, mice were followed for clinical recurrence. Body weight was measured twice weekly and used to segregate HFD mice into obese (i.e., responders) and lean (i.e., nonresponders) study arms, and obesity was correlated with body fat percentage, glucose tolerance (measured using intraperitoneal glucose tolerance tests), serum biomarkers (measured by enzyme-linked immunosorbent assay), and tissue transcriptomics (assessed by RNA sequencing). MRD was quantified by droplet digital PCR. RESULTS: HFD-Obese mice weighed significantly more than HFD-Lean and LFD control mice (p < 0.001) and had increased body fat percentage (p < 0.001). Obese mice exhibited fasting hyperglycemia, hyperinsulinemia, and impaired glucose tolerance, as well as decreased serum levels of adiponectin and increased levels of leptin, resistin, and insulin-like growth factor 1. Tumor recurrence was accelerated in HFD-Obese mice compared with HFD-Lean and LFD control mice (median relapse-free survival 53.0 days vs. 87.0 days vs. 80.0 days, log-rank p < 0.001; HFD-Obese compared with HFD-Lean HR 2.52, 95% CI 1.52-4.16; HFD-Obese compared with LFD HR 2.27, 95% CI 1.42-3.63). HFD-Obese mice harbored a significantly greater number of residual tumor cells than HFD-Lean and LFD mice (12,550 ± 991 vs. 7339 ± 2182 vs. 4793 ± 1618 cells, p < 0.001). CONCLUSION: These studies provide a genetically engineered mouse model for study of the association of diet-induced obesity with breast cancer recurrence. They demonstrate that this model recapitulates physiological changes characteristic of obese patients, establish that the association between obesity and recurrence risk is causal in nature, and suggest that obesity is associated with the increased survival and persistence of residual tumor cells.

WNT-Mediated Regulation of FOXO1 Constitutes a Critical Axis Maintaining Pubertal Mammary Stem Cell Homeostasis.

Puberty is characterized by dynamic tissue remodeling in the mammary gland involving ductal elongation, resolution into the mature epithelial bilayer, and lumen formation. To decipher the cellular mechanisms underlying these processes, we studied the fate of putative stem cells, termed cap cells, present in terminal end buds of pubertal mice. Employing a p63CreERT2-based lineage-tracing strategy, we identified a unipotent fate for proliferative cap cells that only generated cells with basal features. Furthermore, we observed that dislocated "cap-in-body" cells underwent apoptosis, which aided lumen formation during ductal development. Basal lineage-specific profiling and genetic loss-of-function experiments revealed a critical role for FOXO transcription factors in mediating these proliferative versus apoptotic fates. Importantly, these studies revealed a mode of WNT signaling-mediated FOXO1 inhibition, potentially mediated through AKT. Together, these data suggest that the WNT pathway confers proliferative and survival advantages on cap cells via regulation of FOXO1 localization.

A novel approach for next-generation sequencing of circulating tumor cells.

BACKGROUND: Next-generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low-input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching. METHODS: To address this, we have developed a novel approach combining tumor cell isolation from preserved blood with Repli-G WGA and Illumina TruSeq Amplicon Cancer Panel-based NGS. We purified cell pools ranging from 10 to 1000 cells from three different cell lines, and quantitatively demonstrate comparable quality of DNA extracted from preserved versus unpreserved samples. RESULTS: Preservation and WGA were compatible with the generation of high-quality libraries. Known point mutations and gene amplification were detected for libraries that had been prepared from amplified DNA from preserved blood. CONCLUSION: These spiking experiments provide proof of concept of a clinically applicable workflow for real-time monitoring of patient tumor using noninvasive liquid biopsies.

Notch promotes recurrence of dormant tumor cells following HER2/neu-targeted therapy.

Breast cancer mortality is principally due to recurrent tumors that arise from a reservoir of residual tumor cells that survive therapy. Remarkably, breast cancers can recur after extended periods of clinical remission, implying that at least some residual tumor cells pass through a dormant phase prior to relapse. Nevertheless, the mechanisms that contribute to breast cancer recurrence are poorly understood. Using a mouse model of recurrent mammary tumorigenesis in combination with bioinformatics analyses of breast cancer patients, we have identified a role for Notch signaling in mammary tumor dormancy and recurrence. Specifically, we found that Notch signaling is acutely upregulated in tumor cells following HER2/neu pathway inhibition, that Notch signaling remains activated in a subset of dormant residual tumor cells that persist following HER2/neu downregulation, that activation of Notch signaling accelerates tumor recurrence, and that inhibition of Notch signaling by either genetic or pharmacological approaches impairs recurrence in mice. Consistent with these findings, meta-analysis of microarray data from over 4,000 breast cancer patients revealed that elevated Notch pathway activity is independently associated with an increased rate of recurrence. Together, these results implicate Notch signaling in tumor recurrence from dormant residual tumor cells and provide evidence that dormancy is a targetable stage of breast cancer progression.

Oncogene pathway activation in mammary tumors dictates FDG-PET uptake.

Increased glucose utilization is a hallmark of human cancer that is used to image tumors clinically. In this widely used application, glucose uptake by tumors is monitored by positron emission tomography of the labeled glucose analogue 2[(18)F]fluoro-2-deoxy-D-glucose (FDG). Despite its widespread clinical use, the cellular and molecular mechanisms that determine FDG uptake--and that underlie the heterogeneity observed across cancers-remain poorly understood. In this study, we compared FDG uptake in mammary tumors driven by the Akt1, c-MYC, HER2/neu, Wnt1, or H-Ras oncogenes in genetically engineered mice, correlating it to tumor growth, cell proliferation, and expression levels of gene involved in key steps of glycolytic metabolism. We found that FDG uptake by tumors was dictated principally by the driver oncogene and was not independently associated with tumor growth or cellular proliferation. Oncogene downregulation resulted in a rapid decrease in FDG uptake, preceding effects on tumor regression, irrespective of the baseline level of uptake. FDG uptake correlated positively with expression of hexokinase-2 (HK2) and hypoxia-inducible factor-1α (HIF1α) and associated negatively with PFK-2b expression and p-AMPK. The correlation between HK2 and FDG uptake was independent of all variables tested, including the initiating oncogene, suggesting that HK2 is an independent predictor of FDG uptake. In contrast, expression of Glut1 was correlated with FDG uptake only in tumors driven by Akt or HER2/neu. Together, these results demonstrate that the oncogenic pathway activated within a tumor is a primary determinant of its FDG uptake, mediated by key glycolytic enzymes, and provide a framework to interpret effects on this key parameter in clinical imaging.

Hunk negatively regulates c-myc to promote Akt-mediated cell survival and mammary tumorigenesis induced by loss of Pten.

The protooncogenes Akt and c-myc each positively regulate cell growth and proliferation, but have opposing effects on cell survival. These oncogenes cooperate to promote tumorigenesis, in part because the prosurvival effects of Akt offset the proapoptotic effects of c-myc. Akt's ability to counterbalance c-myc's proapoptotic effects has primarily been attributed to Akt-induced stimulation of prosurvival pathways that indirectly antagonize the effects of c-myc. We report a more direct mechanism by which Akt modulates the proapoptotic effects of c-myc. Specifically, we demonstrate that Akt up-regulates the adenosine monophosphate-associated kinase (AMPK)-related protein kinase, Hormonally up-regulated neu-associated kinase (Hunk), which serves as an effector of Akt prosurvival signaling by suppressing c-myc expression in a kinase-dependent manner to levels that are compatible with cell survival. Consequently, Akt pathway activation in the mammary glands of Hunk(-/-) mice results in induction of c-myc expression to levels that induce apoptosis. c-myc knockdown rescues the increase in apoptosis induced by Hunk deletion in cells in which Akt has been activated, indicating that repression of c-myc is a principal mechanism by which Hunk mediates the prosurvival effects of Akt. Consistent with this mechanism of action, we find that Hunk is required for c-myc suppression and mammary tumorigenesis induced by phosphatase and tensin homolog (Pten) deletion in mice. Together, our findings establish a prosurvival function for Hunk in tumorigenesis, define an essential mechanism by which Akt suppresses c-myc-induced apoptosis, and identify Hunk as a previously unrecognized link between the Akt and c-myc oncogenic pathways.

Autocrine prolactin induced by the Pten-Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways.

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten-PI3K-Akt pathway. Conditional activation of the PI3K-Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K-Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)-Jak-Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl(-/-), Prlr(-/-), and Stat5(-/-) mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1(-/-);Akt2(+/-) mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K-Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K-Akt pathway.

PET imaging of glutaminolysis in tumors by 18F-(2S,4R)4-fluoroglutamine.

UNLABELLED: Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled (18)F-FDG PET tumor imaging. However, of the millions of (18)F-FDG PET scans conducted per year, a significant number of malignant tumors are (18)F-FDG PET-negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, (18)F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. METHODS: A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of (18)F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. RESULTS: In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, ≈ 16% dose/100 µg of protein). The cell uptake of (18)F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of (3)H-L-glutamine but higher than that of (18)F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that (18)F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. CONCLUSION: The results support that (18)F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.

Isoform-specific requirement for Akt1 in the developmental regulation of cellular metabolism during lactation.

The metabolic demands and synthetic capacity of the lactating mammary gland exceed that of any other tissue, thereby providing a useful paradigm for understanding the developmental regulation of cellular metabolism. By evaluating mice bearing targeted deletions in Akt1 or Akt2, we demonstrate that Akt1 is specifically required for lactating mice to synthesize sufficient quantities of milk to support their offspring. Whereas cellular proliferation, differentiation, and apoptosis are unaffected, loss of Akt1 disrupts the coordinate regulation of metabolic pathways that normally occurs at the onset of lactation. This results in a failure to upregulate glucose uptake, Glut1 surface localization, lipid synthesis, and multiple lipogenic enzymes, as well as a failure to downregulate lipid catabolic enzymes. These findings demonstrate that Akt1 is required in an isoform-specific manner for orchestrating many of the developmental changes in cellular metabolism that occur at the onset of lactation and establish a role for Akt1 in glucose metabolism.

Hormone-induced protection against mammary tumorigenesis is conserved in multiple rat strains and identifies a core gene expression signature induced by pregnancy.

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.

Developmental stage determines the effects of MYC in the mammary epithelium.

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.

Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis.

Aberrant activation of Wnt signaling is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible Wnt1 transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued Wnt signaling. Using this model we show that targeted down-regulation of the Wnt pathway results in the rapid disappearance of essentially all Wnt-initiated invasive primary tumors as well as pulmonary metastases. Tumor regression does not require p53 and occurs even in highly aneuploid tumors. However, despite the dependence of primary mammary tumors and metastases on continued Wnt signaling and the dispensability of p53 for tumor regression, we find that a substantial fraction of tumors progress to a Wnt-independent state and that p53 suppresses this process. Specifically, loss of one p53 allele dramatically facilitates the progression of mammary tumors to a Wnt1-independent state both by impairing the regression of primary tumors following doxycycline withdrawal and by promoting the recurrence of fully regressed tumors in the absence of doxycycline. Thus, although p53 itself is dispensable for tumor regression, it nevertheless plays a critical role in the suppression of tumor recurrence. Our findings demonstrate that although even advanced stages of epithelial malignancy remain dependent upon continued Wnt signaling for maintenance and growth, loss of p53 facilitates tumor escape and the acquisition of oncogene independence.

Functional microarray analysis of mammary organogenesis reveals a developmental role in adaptive thermogenesis.

The use of DNA microarrays to study vertebrate organogenesis presents unique analytical challenges compared with expression profiling of homogeneous cell populations. We have used a general approach that permits the automated, unbiased identification of biologically relevant patterns of gene expression to study murine mammary gland development. Our studies confirm the utility of this approach by demonstrating the ready identification of cellular processes and pathways of known functional importance in mammary development. Additionally, this approach permitted the identification of genetic pathways with unpredicted patterns of developmental regulation, including those involved in angiogenesis, extracellular matrix synthesis, and the beta-oxidation of fatty acids. Surprisingly, our findings demonstrate that the coordinate regulation of genes involved in the beta-oxidation of fatty acids reflects the presence of an abundant, yet previously unrecognized stromal compartment within the mammary gland that is composed of brown adipose tissue. Our data demonstrate that the amount of brown adipose tissue present in the mammary gland is developmentally regulated; that PPARalpha, Ucp1, and genes involved in fatty acid oxidation are spatially and temporally coregulated during development; that the mammary gland plays a functional role in adaptive thermogenesis; and that the transcriptional control of this adaptive response to cold is itself developmentally regulated.

A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology.

Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, constitutive transgenic mouse models that rely on mammary-specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV-LTR to drive expression of the reverse tetracycline-dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator-controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary-specific, doxycycline-inducible transgenic mouse model for studying the effect of mammary gland development on transgene-mediated phenotypes. Unlike other mammary-specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.