Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.

Aberrant RET expression affects normal mammary gland post-lactation transition, enhancing cancer potential.

RET is a receptor tyrosine kinase with oncogenic potential in the mammary epithelium. Several receptors with oncogenic activity in the breast are known to participate in specific developmental stages. We found that RET is differentially expressed during mouse mammary gland development: RET is present in lactation and its expression dramatically decreases in involution, the period during which the lactating gland returns to a quiescent state after weaning. Based on epidemiological and pre-clinical findings, involution has been described as tumor promoting. Using the Ret/MTB doxycycline-inducible mouse transgenic system, we show that sustained expression of RET in the mammary epithelium during the post-lactation transition to involution is accompanied by alterations in tissue remodeling and an enhancement of cancer potential. Following constitutive Ret expression, we observed a significant increase in neoplastic lesions in the post-involuting versus the virgin mammary gland. Furthermore, we show that abnormal RET overexpression during lactation promotes factors that prime involution, including premature activation of Stat3 signaling and, using RNA sequencing, an acute-phase inflammatory signature. Our results demonstrate that RET overexpression negatively affects the normal post-lactation transition.

Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells.

Angiotensin (Ang) II, the main effector peptide of the renin-angiotensin system, has been implicated in multiple aspects of cancer progression such as proliferation, migration, invasion, angiogenesis and metastasis. Ang-(1-7), is a biologically active heptapeptide, generated predominantly from AngII by the enzymatic activity of angiotensin converting enzyme 2. Previous studies have shown that Ang-(1-7) counterbalances AngII actions in different pathophysiological settings. In this study, we have analysed the impact of Ang-(1-7) on AngII-induced pro-tumorigenic features on normal murine mammary epithelial cells NMuMG and breast cancer cells MDA-MB-231. AngII stimulated the activation of the survival factor AKT in NMuMG cells mainly through the AT1 receptor. This PI3K/AKT pathway activation also promoted epithelial-mesenchymal transition (EMT). Concomitant treatment of NMuMG cells with AngII and Ang-(1-7) completely abolished EMT features induced by AngII. Furthermore, Ang-(1-7) abrogated AngII induced migration and invasion of the MDA-MB-231 cells as well as pro-angiogenic events such as the stimulation of MMP-9 activity and VEGF expression. Together, these results demonstrate for the first time that Ang-(1-7) counteracts tumor aggressive signals stimulated by AngII in breast cancer cells emerging the peptide as a potential therapy to prevent breast cancer progression.

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes.

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system.

Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT(1) receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin-converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7-fold; P<0.01), while induction of angiotensinogen and AT(1) and AT(2) receptors expression was detected 96 h after weaning (6.2-, 10-, and 6.2-fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT(1) receptor blocker, during mammary involution. Mammary glands from losartan-treated mice showed activation of the survival factors AKT and BCL-(XL), significantly lower LIF and TNF-α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP-9 activity in a dose-dependent manner (80%; P<0.05; with losartan IC(50) value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle-treated mice. Furthermore, mammary glands of forced weaned AT(1A)- and/or AT(1B)-deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT(1A)/AT(1B) DKO); P<0.05] with remarkable delayed postlactational regression compared to wild-type animals. Taken together, these results strongly suggest that AngII, via the AT(1) receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS. angiotensin system.

Mouse mammary tumors display Stat3 activation dependent on leukemia inhibitory factor signaling.

INTRODUCTION: It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. METHODS: The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. RESULTS: High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. CONCLUSION: LIF is overexpressed in mouse mammary tumors, where it acts as the main Stat3 activator. Interestingly, the positive LIF effect on tumor cell viability is not dependent on Stat3 activation, which inhibits tumor cell survival as it does in normal mammary epithelium.

Tumor transition zone: a putative new morphological and functional hallmark of tumor aggressiveness.

A small primary or secondary tumor load can occasionally induce more deleterious effects than a histologically identical larger one. In the four murine models studied herein this enhanced tumor aggressiveness could not be attributed to NRAS mutations or other hereditary changes, differential vascularization of live tumor tissues, or necrosis content. Instead, the main tumor feature associated with a more aggressive behavior was the presence of a high number of vessels, sometimes filled with inflammatory cells, inside a tumor area, which we have identified and designated as the transition zone between the live and the necrotic zones. Our experiments suggest that during tumor growth, different cachectic factors are produced within the transition and necrotic zones by dying tumor cells and by tumor infiltrating macrophages only reaching the general circulation through the vessels present in the transition zone. Therefore, a small tumor displaying high vascularization of its transition area could be harmful to its host, while, in contrast, a large tumor could behave as a relatively benign one if its transition zone exhibited little or no vascularization, and in consequence its cachectic factors remained "trapped." Similar histological images to those observed in mice were seen in a significant percentage of human cancer biopsies, raising the possibility that such images might have a prognostic value.

Progression of pregnancy-dependent mouse mammary tumors after long dormancy periods. Involvement of Wnt pathway activation.

Mouse mammary tumor virus (LA) induces pregnancy-dependent mammary tumors that progress toward autonomy. Here we show that in virgin females, pregnancy-dependent tumor transplants are able to remain dormant for up to 300 days. During that period, these tumors synthesize DNA, express high levels of estrogen and progesterone receptors (ER+PR+) and are able to resume growth after hormone stimulation. Surprisingly, in a subsequent transplant generation, all these tumors are fully able to grow in virgin females, they express low levels of ER and PR (ER-PR-) and have a monoclonal origin; i.e., show all of the features we have described previously in pregnancy-independent tumors. Histologically, mouse mammary tumor virus (LA)-induced tumors are morphologically similar to genetically engineered mouse (GEM) mammary tumors that overexpress genes belonging to the Wnt pathway. Interestingly, in the virus-induced neoplasias, pregnancy-independent passages arising after a dormant phase usually display a lower level of glandular differentiation together with epithelial cell trans-differentiation, a specific feature associated to Wnt pathway activation. In addition, dormancy can lead to the specific selection of Int2/Fgf3 mutated and overexpressing cells. Therefore, our results indicate that during hormone-dependent tumor dormancy, relevant changes in cell population occur, allowing rapid progression after changes in the animal internal milieu.

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution.

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.