SIRT6 enhances oxidative phosphorylation in breast cancer and promotes mammary tumorigenesis in mice.

BACKGROUND: Sirtuin 6 (SIRT6) is a NAD+-dependent deacetylase with key roles in cell metabolism. High SIRT6 expression is associated with adverse prognosis in breast cancer (BC) patients. However, the mechanisms through which SIRT6 exerts its pro-oncogenic effects in BC remain unclear. Here, we sought to define the role of SIRT6 in BC cell metabolism and in mouse polyoma middle T antigen (PyMT)-driven mammary tumors. METHODS: We evaluated the effect of a heterozygous deletion of Sirt6 on tumor latency and survival of mouse mammary tumor virus (MMTV)-PyMT mice. The effect of SIRT6 silencing on human BC cell growth was assessed in MDA-MB-231 xenografts. We also analyzed the effect of Sirt6 heterozygous deletion, of SIRT6 silencing, and of the overexpression of either wild-type (WT) or catalytically inactive (H133Y) SIRT6 on BC cell pyruvate dehydrogenase (PDH) expression and activity and oxidative phosphorylation (OXPHOS), including respiratory complex activity, ATP/AMP ratio, AMPK activation, and intracellular calcium concentration. RESULTS: The heterozygous Sirt6 deletion extended tumor latency and mouse survival in the MMTV-PyMT mouse BC model, while SIRT6 silencing slowed the growth of MDA-MB-231 BC cell xenografts. WT, but not catalytically inactive, SIRT6 enhanced PDH expression and activity, OXPHOS, and ATP/AMP ratio in MDA-MB-231 and MCF7 BC cells. Opposite effects were obtained by SIRT6 silencing, which also blunted the expression of genes encoding for respiratory chain proteins, such as UQCRFS1, COX5B, NDUFB8, and UQCRC2, and increased AMPK activation in BC cells. In addition, SIRT6 overexpression increased, while SIRT6 silencing reduced, intracellular calcium concentration in MDA-MB-231 cells. Consistent with these findings, the heterozygous Sirt6 deletion reduced the expression of OXPHOS-related genes, the activity of respiratory complexes, and the ATP/AMP ratio in tumors isolated from MMTV-PyMT mice. CONCLUSIONS: Via its enzymatic activity, SIRT6 enhances PDH expression and activity, OXPHOS, ATP/AMP ratio, and intracellular calcium concentration, while reducing AMPK activation, in BC cells. Thus, overall, SIRT6 inhibition appears as a viable strategy for preventing or treating BC.

Increased Arginase1 expression in tumor microenvironment promotes mammary carcinogenesis via multiple mechanisms.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/-;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/-;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway from nicotinamide. By controlling the biosynthesis of NAD+, NAMPT regulates the activity of NAD+-converting enzymes, such as CD38, poly-ADP-ribose polymerases, and sirtuins (SIRTs). SIRT6 is involved in the regulation of a wide number of metabolic processes. In this study, we investigated the ability of SIRT6 to regulate intracellular NAMPT activity and NAD(P)(H) levels. BxPC-3 cells and MCF-7 cells were engineered to overexpress a catalytically active or a catalytically inactive SIRT6 form or were engineered to silence endogenous SIRT6 expression. In SIRT6-overexpressing cells, NAD(H) levels were up-regulated, as a consequence of NAMPT activation. By immunopurification and incubation with recombinant SIRT6, NAMPT was found to be a direct substrate of SIRT6 deacetylation, with a mechanism that up-regulates NAMPT enzymatic activity. Extracellular NAMPT release was enhanced in SIRT6-silenced cells. Also glucose-6-phosphate dehydrogenase activity and NADPH levels were increased in SIRT6-overexpressing cells. Accordingly, increased SIRT6 levels reduced cancer cell susceptibility to H2O2-induced oxidative stress and to doxorubicin. Our data demonstrate that SIRT6 affects intracellular NAMPT activity, boosts NAD(P)(H) levels, and protects against oxidative stress. The use of SIRT6 inhibitors, together with agents inducing oxidative stress, may represent a promising treatment strategy in cancer.-Sociali, G., Grozio, A., Caffa, I., Schuster, S., Becherini, P., Damonte, P., Sturla, L., Fresia, C., Passalacqua, M., Mazzola, F., Raffaelli, N., Garten, A., Kiess, W., Cea, M., Nencioni, A., Bruzzone, S. SIRT6 deacetylase activity regulates NAMPT activity and NAD(P)(H) pools in cancer cells.

SIRT6 inhibitors with salicylate-like structure show immunosuppressive and chemosensitizing effects.

The NAD+-dependent deacetylase SIRT6 is an emerging cancer drug target, whose inhibition sensitizes cancer cells to chemo-radiotherapy and has pro-differentiating effects. Here we report on the identification of novel SIRT6 inhibitors with a salicylate-based structure. The new SIRT6 inhibitors show improved potency and specificity compared to the hit inhibitor identified in an in silico compound screen. As predicted based on SIRT6 biological roles, the new leads increase histone 3 lysine 9 acetylation and glucose uptake in cultured cells, while blocking TNF-α production and T lymphocyte proliferation. Notably, the new SIRT6 inhibitors effectively sensitize pancreatic cancer cells to gemcitabine. Finally, studies of compound fingerprinting and pharmacokinetics defined the drug-like properties of one of the new SIRT6 inhibitors, potentially allowing for subsequent in vivo proof-of-concept studies. In conclusion, new SIRT6 inhibitors with a salicylate-like structure were identified, which are active in cells and could potentially find applications in disease conditions, including cancer and immune-mediated disorders.

Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity.

Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2',7'-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings demonstrate that MINO is heterogeneous with respect to intracellular pH regulation which may be associated with an acidified regional microenvironment. A change in the pH of the microenvironment might contribute to a tumor-permissive or tumor-promoting progression. We are not aware of any previous work showing that a sub-population of cells in in situ precancer exhibits a higher than normal proton production and export rate.

Sirt6 regulates dendritic cell differentiation, maturation, and function.

Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence.

Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics.

The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6's biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.

Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition.

Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use.

Nicotinamide phosphoribosyltransferase promotes epithelial-to-mesenchymal transition as a soluble factor independent of its enzymatic activity.

Boosting NAD(+) biosynthesis with NAD(+) intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD(+) biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFß signaling pathway via increased TGFß1 production. These findings have implications for the design of therapeutic strategies exploiting NAD(+) biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.

Discovery of novel and selective SIRT6 inhibitors.

SIRT6 is an NAD(+)-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC50s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents.

In vivo generation of decidual natural killer cells from resident hematopoietic progenitors.

Decidual natural killer cells accumulate at the fetal-maternal interface and play a key role in a successful pregnancy. However, their origin is still unknown. Do they derive from peripheral natural killer cells recruited in decidua or do they represent a distinct population that originates in situ? Here, we identified natural killer precursors in decidua and uterus of pregnant mice. These precursors underwent rapid in situ differentiation and large proportions of proliferating immature natural killer cells were present in decidua and uterus as early as gestation day 4.5. Here, we investigated the origin of decidua- and uterus-natural killer cells by performing transfer experiments of peripheral mature natural killer cells or precursors from EGFP(+) mice. Results showed that mature natural killer cells did not migrate into decidua and uterus, while precursors were recruited in these organs and differentiated towards natural killer cells. Moreover, decidua- and uterus-natural killer cells displayed unique phenotypic and functional features. They expressed high levels of the activating Ly49D receptor in spite of their immature phenotype. In addition, decidua- and uterus-natural killer cells were poorly cytolytic and produced low amounts of IFN-γ, while they released factors (GM-CSF, VEGF, IP-10) involved in neo-angiogenesis and tissue remodeling. Our data reveal in situ generation of decidual natural killer cells and provide an important correlation between mouse and human decidual natural killer cells, allowing further studies to be carried out on their role in pregnancy-related diseases.

Nicotinamide phosphoribosyltransferase as a target in inflammation- related disorders.

NAD⁺ biosynthesis through nicotinamide phosphoribosyltransferase (NAMPT) holds potential as a target for the treatment of inflammatory disorders due to NAD(+)'s role in immune cell signaling and metabolism. In addition to its activity as an enzyme, NAMPT is also secreted in the extracellular space where it acts as a pro-inflammatory and proangiogenic cytokine. NAMPT inhibition with FK866 has anti-inflammatory activity in different models of immune disorders and it prevents ischemia-reperfusion-induced heart damage by dampening the production of neutrophil chemoattractants. NAMPT blockade with a neutralizing antibody has beneficial effects in an acute lung injury model. Last, but not least, the anticancer activity of NAMPT inhibitors may also reflect, at least in part, their ability to modify the cancer microenvironment through their anti-inflammatory properties. Overall, NAMPT inhibition holds potential for the treatment of inflammation-related disorders and the development of effective and safe NAMPT inhibitors remains an area of strong interest in pharmaceutical research.

Epithelial-mesenchymal transition in mouse mammary tumorigenesis.

Epithelial-mesenchymal transition tumorigenesis in the mouse has been described for over 100 years using various terms and with little comprehension of the underlying mechanisms. Recently, epithelial-mesenchymal transition tumors have been recognized in mammary glands of genetically engineered mice. This review provides a historical perspective and the current observations in the context of some of the key molecular biology. The biology of mouse mammary epithelial-mesenchymal transition tumorigenesis is discussed with comparisons to human breast cancer.

Mammary carcinoma behavior is programmed in the precancer stem cell.

INTRODUCTION: The 'MINO' (mammary intraepithelial neoplasia outgrowth) mouse model of ductal carcinoma in situ (DCIS) consists of six lines with distinct morphologic phenotypes and behavior, each meeting experimentally defined criteria for 'precancer'. Specifically, these lines grow orthotopically in cleared mammary fat pads and consistently progress to an invasive phenotype that is capable of ectopic growth. Transition to carcinoma has a consistent latency for each line, and three of the lines also exhibit pulmonary metastatic potential. METHODS: Gland cleared orthotopic transplanted precancer MINO tissues were analyzed by bacterial artifical chromosome and oligo array comparative genomic hybridization, microsatellite PCR, and telomerase repeat amplification assay. MINO cells were dissociated and cultured in three dimensional culture and transplanted in syngeneic gland cleared mammary fat pads. RESULTS: Comparative genomic hybridization shows that the precancer and invasive tumors are genetically stable, with low level changes including whole chromosome gains in some lines. No changes are associated with progression, although spontaneous focal amplifications and deletions were detected occasionally. Microsatellite analysis shows a low frequency of alterations that are predominantly permanent within a MINO line. Telomerase activity is increased in both the MINO and the derived tumors when compared with normal mouse mammary gland. Dissociation of the precancer lesion cells and three dimensional 'spheroid' culture of single cells reveals a bipotential for myoepithelial and luminal differentiation and the formation of unique three-dimensional 'MINOspheres'. These MINOspheres exhibit features that are intermediate between spheroids that are derived from normal and carcinoma cells. Transplantation of a single cell derived MINOsphere recapitulates the outgrowth of the precancer morphology and progression to carcinoma. CONCLUSION: These data establish a precancer 'stem' cell that is capable of self-renewal and multilineage differentiation as the origin of invasive cancer. Within the context of this model, these cells have programmed potential for latency and metastasis that does not appear to require sequential genetic 'hits' for transformation.

EMT tumorigenesis in the mouse mammary gland.

The term EMT (epithelial-mesenchymal transition) is used in many settings. This term is used to describe the mechanisms facilitating cellular repositioning and redeployment during embryonic development and tissue reconstruction after injury. Recently, EMT has also been applied to potential mechanisms for malignant progression and has appeared as a specific diagnostic category of tumors. In mice, most 'EMT' tumors have a spindle cell phenotype. The definition of EMT is controversial because spindle cell tumors are not common in humans, especially in human breast cancers. Spindle cell tumors of the mouse mammary gland have been observed for many years where they are usually classified as sarcomas or carcinosarcomas. Genetically engineered mice develop mammary spindle cell tumors that appear to arise in the epithelium and undergo EMT. To better understand the origin and evolution of these spindle cell tumors in progression and metastases, seven cohorts of spindle cell tumors from the archives of the University of California, Davis Mutant Mouse Pathology Laboratory were studied. This study provides experimental and immunohistochemical evidence of EMT showing that dual epithelial and mesenchymal staining of tumor spindle cells identifies some, but not all, EMT-type tumors in the mouse. This suggests that potential EMT tumors are best designated EMT-phenotype tumors.

Rapamycin inhibits growth of premalignant and malignant mammary lesions in a mouse model of ductal carcinoma in situ.

PURPOSE: Rapamycin has been shown to have antitumor effects in various tumor models. To study the effect of rapamycin at different stages of breast cancer development, we used two unique mouse models of breast cancer with activated phosphatidylinositol 3-kinase (PI3K) pathway. Met-1 tumors are highly invasive and metastatic, and mammary intraepithelial neoplasia-outgrowths (MIN-O), a model for human ductal carcinoma in situ, are transplantable premalignant mammary lesions that develop invasive carcinoma with predictable latencies. Both of these models were derived from mammary lesions in Tg(MMTV-PyV-mT) mice. EXPERIMENTAL DESIGN: Met-1 tumors were used to study the effect of rapamycin treatment on invasive disease. Transplanted MIN-O model was used to study the effect of rapamycin on premalignant mammary lesions. Animals were in vivo micro-positron emission tomography imaged to follow the lesion growth and transformation to tumor during the treatment. Cell proliferation, angiogenesis, and apoptosis was assayed by immunohistochemistry. RESULTS: Rapamycin inhibited in vitro tumor cell proliferation and in vivo Met-1 tumor growth. The growth inhibition was correlated with dephosphorylation of mammalian target of rapamycin (mTOR) targets. Rapamycin treatment significantly reduced the growth of the premalignant MIN-O lesion, as well as tumor incidence and tumor burden. Growth inhibition was associated with reduced cell proliferation and angiogenesis and increased apoptosis. CONCLUSIONS: In PyV-mT mouse mammary models, rapamycin inhibits the growth of premalignant lesions and invasive tumors. Although the inhibitory effect of rapamycin was striking, rapamycin treatment did not completely obliterate the lesions.

Fate of embryonal carcinoma cells injected into postimplantation mouse embryos.

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.

Inhibition of ductal morphogenesis in the mammary gland of WAP -fgf4 transgenic mice.

We previously demonstrated that expression of human FGF4 in the epithelial compartment of murine mammary glands caused hyperplasia during lactation and a dramatic delay in gland involution due to inhibition of cellular apoptosis. We now analyse the effects of transgene expression during development of the organ. Expression of WAP- Fgf4 initiate with the onset of sexual hormones (4 weeks of age), and defects in morphogenesis of the organ were already apparent at 5 weeks of age and persisted throughout all stages of post-natal development. These defects involved ductal development, but not lobuloalveolar morphogenesis, and were due to a decrease in the level of apoptosis within the terminal end buds. We also show that regulation of apoptosis by FGF4 in the mammary gland, both during development and involution, could occur via inhibition of Bcl2 expression. Overall our data demonstrate that FGF4 is a regulator of mammary epithelial cells apoptosis during all stages in which programmed cell death is an important mechanism of development, namely morphogenesis and involution. We also suggest that this growth factor could act by interfering with the Bcl2 pathway.