Mother's milk provides multiple benefits to the offspring. However, xenobiotics transferred into breast milk may pose a risk to the nursing infant. The breast cancer resistance protein (BCRP) actively transports xenobiotics into breast milk. BCRP also transports nutrients, like riboflavin, and together with recently identified riboflavin transporters (RFT), may provide a mechanism for riboflavin secretion into breast milk. Expression of RFT in the mammary gland remained unknown. Our objective was to characterize Bcrp and Rft mRNA expression in the mammary gland of FVB/N mice, and investigate a strategy to decrease excretion of BCRP-transported xenobiotics into the milk using riboflavin intervention. Rft and Bcrp mRNA were upregulated in the mammary gland of lactating mice. An intravenous riboflavin administration significantly reduced the levels of BCRP-transported cimetidine in milk. This study demonstrates the use of riboflavin to exploit the function of mammary BCRP in order to reduce xenobiotic secretion into breast milk.

Canine Mammatumoren (CMT) sind eine der häufigsten Todesursachen bei Hündinnen (SIMON et al. 2001). Durch ihre periphere Lage sind zumeist nicht die Primärtumoren, sondern ihre Metastasen lebensbedrohlich, da diese häufig wichtige Organe wie Lunge Herz und Gehirn befallen (CLEMENTE et al. 2010). Aktuell angewandte Untersuchungsmethoden zur Erkennung von Metastasierung können entweder das invasive Wachstum beschreiben oder bereits bestehende Metastasen detektieren, jedoch fehlen bislang molekulare Marker, die frühzeitig und zuverlässig das metastatische Potential eines CMT anzeigen bevor Metastasen aufgetreten sind. Das Peptidhormon Relaxin ist in zahlreichen physiologischen und pathologischen Situationen beim Menschen als ein Induktor von Matrixmetalloproteinasen (MMP) bekannt (TOO et al. 1984; UNEMORI und AMENTO 1990; PALEJWALA et al. 2001; BINDER et al. 2002; KLONISCH et al. 2007; HENNEMAN et al. 2008). MMP sind Schlüsselenzyme des Bindegewebsabbaus, der jeder Metastasierung vorausgehen muss (WOODHOUSE et al. 1997). Bei der Frau ist im Blut messbares Relaxin ein Marker für metastatischen Brustkrebs (BINDER et al. 2004), für die Hündin scheint dagegen der Relaxinblutspiegel nicht aussagekräftig zu sein (SCHWEIZER 2010). Möglicherweise wird aber Relaxin lokal im caninen Mammagewebe exprimiert wie von GOLDSMITH et al. (1994) und SILVERTOWN et al. (2003) postuliert, so dass es wie in humanen Tumoren auto- oder parakrin Invasivität und Metastasierung (KLONISCH et al. 2007) fördern könnte. Daher sollte in der vorliegenden Arbeit die intratumorale Expression des Relaxins und seiner Rezeptoren sowie bekannter Faktoren des Bindegewebsabbaus untersucht und auf ihre prognostische Eignung überprüft werden. In zwei Studienabschnitten (LAMP et al. 2009; LAMP et al. 2011) wurden CMT-Gewebeproben von n=31 (LAMP et al. 2009) respektive n=59 Hündinnen (LAMP et al. 2011) mittels quantitativer Reverse-Transkriptase-Polymerasekettenreaktion (qRT-PCR) auf ihre Expression von Relaxin, seinen Rezeptoren, RXFP1 und RXFP2, sowie den Matrixmetalloproteinasen MMP-1, -2, -3, -9 und MMP-13, den Östradiolrezeptoren, ERα und ERβ, und dem Progesteronrezeptor (PR) analysiert. In beiden Studienabschnitten wurden die Plasmakonzentrationen der Hormone Relaxin, Östradiol und Progesteron auf mögliche Zusammenhänge mit der lokalen Genexpression überprüft. Im zweiten Studienabschnitt (LAMP et al. 2011) wurde darüber hinaus die Expression von Relaxin und RXFP1 auch immunhistologisch an n=9 CMT-Proben untersucht und in einer Multivarianzanalyse die prognostische Eignung aller untersuchten Parameter getestet. Die Expressionsanalyse konnte zeigen, dass CMT sowohl ein bisher unbekannter Ort der Relaxinexpression beim Hund sind als auch den Hauptrezeptor des Relaxins, RXFP1, exprimieren. Diese Ergebnisse der mRNA-Untersuchung ließen sich immunhistologisch bestätigen. Darüber hinaus ergab die immunhistologische Untersuchung, dass Relaxin vorwiegend im myoepithelialen Anteil der untersuchten CMT exprimiert wird. In den epithelialen CMT-Zellen fand sich die stärkste RXFP1-Reaktivität, so dass RXFP1 mit der von anderen Autoren beschriebenen MMP-2- und MMP-9-Expression in epithelialen Zellen kolokalisiert ist (PAPPARELLA et al. 1997; HIRAYAMA et al. 2002; PAPPARELLA et al. 2002). Die quantitativen Expressionsanalysen zeigten Korrelationen der Expressionsintensitäten von Relaxin, RXFP1 und MMP-2 auf. Die RXFP1 Expression war in dieser Studie sogar ein unabhängiger Marker für Metastasierung mit einem 15-fach höheren Risiko für Metastasierung für Patienten mit einer Expression oberhalb des studienspezifischen Cut-Offs. Alle untersuchten lokalen Genexpressionen waren von den systemischen Plasmakonzentrationen von Relaxin, Östradiol und Progesteron unabhängig. Die Resultate legen eine Bedeutung des intratumoral exprimierten Relaxins für eine auto- oder parakrine Steuerung der MMP-Expression, die für Invasivität und Metastasierung wichtig ist, nahe. Aufgrund des mRNA-Nachweises, der Kolokalisation der Proteine von RXFP1 und MMP-2 und -9 sowie der Korrelation der Genexpression von Ligand (Relaxin), Rezeptor (RXFP1) und Effektormolekül (MMP-2) ist es wahrscheinlich, dass CMT über das Relaxin eine autonome Steuerung ihrer Invasivität vornehmen können. RXFP1 scheint dabei eine Regulationsfunktion in der Relaxinsensibilität der CMT-Zelle zuzukommen, die in Zukunft durch die Messung der RXFP1-Expressionsintensität prognostisch nutzbar sein könnte. Zudem ist RXFP1 im CMT damit auch ein möglicher Ansatzpunkt für eine neue, auf Relaxinanaloga basierende, antimetastatische Therapie, die bereits an humanen Tumorzellen und in Mausmodellen erprobt wird (FENG et al. 2007; HOSSAIN et al. 2010). Durch den Nachweis von Relaxin und RXFP1 im CMT und ihre wahrscheinliche Relevanz für die Metastasierung ergeben sich somit neue Möglichkeiten für eine exaktere Prognose und verbesserte antimetastatische Therapie von CMT sowie die Chance, den Hund als Modell für die Erforschung Relaxin basierter Therapien des humanen Brustkrebses zu nutzen. / Canine mammary tumours (CMT) are one of the main reasons of death in female dogs (SIMON et al. 2001). Due to its peripheral location, it is normally not the primary tumour, but its metastases, which are life-threatening as they often impair the function of vital organs, such as lung, heart or brain (CLEMENTE et al. 2010). Currently used techniques for the detection of metastasis can either barely describe invasive growth patterns or detect already existing metastases. Molecular markers to determine the metastatic potential early and reliably, before metastatic spreading has occurred, are still lacking. The peptide hormone relaxin is well known as an inductor of matrix metalloproteinases (MMP) in numerous physiological as well as pathological situations in humans (TOO et al. 1984; UNEMORI und AMENTO 1990; PALEJWALA et al. 2001; BINDER et al. 2002; KLONISCH et al. 2007; HENNEMAN et al. 2008). MMP are key-enzymes of connective tissue remodelling which is a prerequisite for metastasis (WOODHOUSE et al. 1997). In women, the plasma relaxin concentration is a marker for metastatic breast cancer (BINDER et al. 2004). However, in dogs, the concentration of circulating relaxin seems to have no diagnostic value (SCHWEIZER 2010). But, possibly relaxin is expressed locally in the canine mammary tissue as postulated by GOLDSMITH et al. (1994) and SILVERTOWN et al. (2003) and it could therefore act as a pro-invasive and pro-metastatic factor in an auto- or paracrine manner as it does in various human tumours (KLONISCH et al. 2007). Thus, the present study should examine the intratumoural expression of relaxin and its receptors as well as factors of connective tissue remodelling and evaluate their prognostic abilities. In two sections of the study (LAMP et al. 2009; LAMP et al. 2011), CMT-tissue samples from n=31 bitches (LAMP et al. 2009) and n=59 bitches (LAMP et al. 2011), respectively, were analysed for their expression of relaxin, its receptors RXFP1 and RXFP2, MMP-1, -2, -3, -9 and MMP-13 as well as the oestradiol receptors ERα and ERβ and the progesterone receptor (PR) using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Additionally, the plasma concentrations of the hormones relaxin, oestradiol and progesterone were tested for possible connections with the local gene expression. In the second section of the study, the expression of relaxin and RXFP1 was also examined immunohistologically in n=9 CMT tissue samples (LAMP et al. 2011) and the prognostic value of all parameters examined was assessed by a multivariate analysis. The expression analysis showed that CMTs are a novel site of expression of relaxin and its main receptor RXFP1 in the dog. These results were confirmed by the immunohistological examination. Moreover, the immunohistological analysis demonstrated that relaxin seems to be expressed mainly in myoepithelial cells. However, the strongest signals for RXFP1 were located in epithelial cells of the CMT, thus RXFP1 is colocalised with the expression of MMP-2 and MMP-9 reported in epithelial CMT-cells (PAPPARELLA et al. 1997; HIRAYAMA et al. 2002; PAPPARELLA et al. 2002). The quantitative expression analysis revealed correlations of expression intensities for relaxin, RXFP1 and MMP-2. The expression of RXFP1 presented as an independent marker for metastasis with a 15-fold risk increase for patients with an expression intensity above the study-specific cut-off. All local gene expressions examined where independent from systemic plasma concentrations of relaxin, oestradiol and progesterone. The results propose an important role for intratumourally expressed relaxin as an auto- or paracrine modulator of MMP expression, which is important for invasiveness and metastasis. Due to the mRNA detection, the protein colocalisation of RXFP1 with MMP-2 and MMP-9 as well as the correlation of gene expressions of the ligand (relaxin), the receptor (RXFP1) and the effector (MMP-2) it is highly probable that CMT can autonomously regulate their invasiveness via locally expressed relaxin. RXFP1 seems to have a regulatory function in the relaxin responsiveness of CMT cells, which may be of prognostical use in the future. In addition, RXFP1 is also a possible target for a novel antimetastatic therapy based on relaxin analoga which has been tested in human tumour cells and mice (FENG et al. 2007; HOSSAIN et al. 2010). The detection of relaxin and RXFP1 in the CMT and their probable relevance for metastasis could be a basis for a more precise prognosis of CMT, improved anti-metastatic therapies in the dog and the use of the dog as a model for relaxin-based therapies of human breast cancer.

Obesity increases breast cancer risk and progression in postmenopausal women. The Akt/mTOR signaling pathway is activated in tumors in response to increased levels of obesity-related growth factors, including insulin-like growth factor (IGF)-1. Hence, we evaluated energy balance modulation as a mechanism for breast cancer prevention through modulation of Akt/mTOR. Studies suggest that dietary calcium can decrease weight gain, although an exact mechanism is not yet identified. Therefore, we investigated the effects of low-fat (10 kcal % fat) or high-fat (45 kcal % fat) diets containing either calcium phosphate (dairy) or calcium carbonate (supplement) on body weight in ovariectomized (OVX) C57BL/6 mice to determine if dietary calcium could overcome the effects of a high-fat diet. We showed that dairy decreased body weight, with no effect on food consumption. However, it is not known if restoration of normal weight can reverse mammary tumor progression and/or Akt/mTOR pathway activation. To evaluate this, mice were fed a control diet, a calorie restricted regimen, or a diet-induced obesity (DIO) regimen for 17 weeks, after which the DIO mice were switched to the control diet, and this resulted in a 20% weight loss and mice of equal weight to control mice. MMTV-Wnt-1 mammary tumor cells were orthopically injected at week 20, following weight loss. At week 22, mice began placebo or RAD001, an mTOR inhibitor, treatment by oral gavage. Tumor growth and Akt/mTOR signaling were enhanced in formerly obese mice, despite reduction in weight, adiposity and serum hormone levels. RAD001 decreased tumor growth in the CR and control group, but was less effective in the formerly obese mice. In an additional study, we added a DIO gourp which was not switched to the control diet, and found that circulating IGF-1 levels remained significantly elevated in formerly obese mice relative to control and were comparable to levels in the DIO mice. We found that the mechanism of tumor progression was through enhanced Akt/mTOR signaling in both obese and formerly obese mice. Based on the Akt/mTOR activation in MMTV-Wnt-1 tumor growth and progression, we next investigated the anticancer effects of ursolic acid (UA), a pentacyclic triterpene. It was previously shown that UA can affect Akt signaling. Our results showed that UA was effective decreasing tumor growth and Akt/mTOR signaling. Taken together, our findings show that the growth-enhancing effects of obesity on mammary tumor may persist even after weight loss and suggest that a combination of dietary and pharmacologic interventions targeting IGF-1/Akt/mTOR may be an effective strategy in the treatment of postmenopausal breast cancer. / text

Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.

Glycogen synthase kinase-3 (GSK-3) alpha and beta are central regulators of key developmental pathways, including Wnt, Hedgehog and Notch, which control stem cell activities and cellular differentiation. Both forms of GSK-3 are also regulated by receptor tyrosine kinases via the PI3K/Akt growth-promoting pathway and are involved in feedback mechanisms that maintain signaling homeostasis. These signaling systems have critical functions in mammary gland development and aberrations in them have been implicated in breast cancer. However, the role of GSK-3 in breast oncogenesis is unclear. Here, I provide evidence that maintenance of appropriate GSK-3 activity is necessary for normal acinar morphogenesis of mammary cells in the ductal/alveolar epithelium. Inadequate GSK-3 levels result in enlarged structures that often lack hollow lumens and resemble early premalignant breast cancer lesions. A potential contribution for PI3K signaling to this phenotype was identified as a PI3K inhibitor partially reversed the observed morphological defects. Mammary epithelial cell (MEC) identity also requires modulation of signals through the Wnt/beta-catenin pathway. GSK-3-depleted mammary glands not only transdifferentiate into squamous epithelium but also develop highly proliferative adenosquamous carcinomas characterized by activated beta-catenin. Furthermore, beta-catenin appears to be required for both cell fate changes and tumorigenesis in the absence of GSK-3 function. Mammary tissues engineered to enable conditional deletion of beta-catenin in a GSK-3-null background also assumed an epidermoid cell fate with ensuing tumor formation albeit with a significantly longer latency and different histopathology. The metaplastic nature of tumors observed is similar to a rare yet aggressive form of human breast disease, metaplastic breast carcinomas (MBCs). Mammospheres (MS) generated from GSK-3 depleted MECs exhibited a less compact morphology compared to those with activated beta-catenin, which also exhibited an expansion of the CD24:CD49f double positive progenitor population and enhanced self-renewal. No MS were formed by MECs lacking GSK-3 and beta-catenin. ErbB2/Neu-mediated mammary tumor progression has been associated with Wnt/beta-catenin pathway activation. Loss of beta-catenin delayed tumor onset in a constitutively active ErbB2 mouse model but did not alter either the luminal characteristics of the ensuing tumors nor their metastatic potential. Collectively these studies indicate GSK-3 plays important roles in mammary gland function thereby suppressing mammary tumor formation.

Epigenetics plays an important role in carcinogenesis with heritable changes in DNA methylation and histone modifications intricately linked to the initiation, promotion, and progression of cancer. Evidence shows that a number of chemical and physical agents can induce epigenetic changes during carcinogenesis. Two such agents, estrogen and ionizing radiation, are generally recognized as being carcinogenic. Yet the epigenetic repercussions of these carcinogens remain relatively unknown. More importantly, the combined effect of these carcinogens has never been addressed in vivo from an epigenetic standpoint. Therefore, we focused on the effect of estrogen and ionizing radiation applied separately or in conjunction. We have found that the exposure to estrogen, either alone or in combination with radiation, induced pronounced morphological alterations, which was paralleled by modifications to the epigenomic landscape in the mammary gland. The results obtained from these rodent models can potentially be extrapolated to humans. / xiv, 190 leaves : ill. (chiefly col.) ; 29 cm

The mammary gland is a dynamic organ that undergoes the majority of its development in the postnatal period in four stages; mature virgin, pregnancy, lactation, and involution. Every stage relies on tightly regulated cellular proliferation, programmed cell death, and tissue remodeling mechanisms. Misregulation of autophagy, an intracellular catabolic process to maintain energy stores, has long been associated with mammary tumorigenesis and other pathologies. We hypothesize that appropriate regulation and execution of autophagy are necessary for proper development of the mammary ductal tree and maintenance of the secretory epithelia during late pregnancy and lactation. To test this hypothesis we examined the role of two genes during development of the mammary gland. Beclin1 (Becn1) is an essential autophagy gene. Since the Becn1 knockout model is embryonic lethal, we have generated a Becn1 conditional knockout (cKO). We used two discrete mammary gland-specific Cre transgenic lines to interrogate the role of BECN1 during development. We report that MMTV-CreD; Becn1fl/fl mice have a hyper-branching phenotype and WAP-Cre; Becn1fl/- mice are unable to sustain a lactation phase. Becn1 mutants exhibit abnormal glandular morphology during pregnancy and after parturition. Moreover, when autophagy is chemically inhibited in vitro, mammary epithelial cells have an increased mean number of lipid droplets per cell. MTOR inhibits autophagy upstream of BECN1; we looked higher in the regulatory pathway for regulatory candidates. It has been well characterized that Tuberous sclerosis complex 1 (TSC1), in a heterodimer with its primary binding partner TSC2, inhibits MTOR signaling via inhibition of RHEB. Using the Tsc1 floxed model we generated a mammary gland specific Tsc1 cKO and found that these mice phenocopy the Becn1 cKO mice, including a gross lactation failure. Tsc1 cKO glands have altered morphology, retained lipid droplets in secretory epithelia, and an overall increase in MTOR signaling. We show that TSC1 and BECN1 are interacting partners, and that the interaction is nutrient responsive. These results suggest that Becn1 and Tsc1 are necessary for proper mammary gland development and differentiation. Furthermore, we have demonstrated a novel murine protein-protein interaction and an important link between regulation of MTOR pathway and regulation of autophagy in a developmental context.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

Made available in DSpace on 2014-10-09T12:44:41Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:10Z (GMT). No. of bitstreams: 1 07150.pdf: 9130636 bytes, checksum: cb7e14964ddd9719ff5fe8392bed1d90 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP