Edith C. Kordon

Evidence for the transforming activity of a truncated Int6 gene, in vitro.

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1α (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT–PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.

Hormone dependence of a mouse mammary tumor line induced in vivo by medroxyprogesterone acetate

SummaryThe administration of MPA to virgin female BALB/c mice led to the development of mammary adenocarcinomas, which in furtherin vivo transplants gave rise to both MPA-dependent and MPA-independent lines. In this paper we chose one of the MPA-dependent lines with high contents of estrogen (ER) and progesterone (PR) receptors, and were able to demonstrate that a) the growth of these tumors could be manipulated by the administration or the withdrawal of the hormonal supply;b) PR were down-regulated in MPA-treated mice; c) progesterone had the same stimulatory effect as MPA on tumor growth; d) tumors did not grow in estrogen-treated mice; e) tumor growth was much lower in males than in females; f) the presence of the ovaries had a positive influence on tumor growth, even in the presence of MPA; g) the withdrawal of progestin pellets in ovariectomized mice usually led to complete remissions followed by regrowth of the tumors after several weeks; and h) the regrowing tumors maintained their steroid receptor pattern and (in 3 out of 4 cases) their hormone-dependent behavior in further passages.

Mechanical strain induces involution-associated events in mammary epithelial cells

BackgroundShortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture.ResultsWe have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition.ConclusionHere, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution.

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.

Selection of early-occurring mutations dictates hormone-independent progression in mouse mammary tumor lines.

ABSTRACT Mice harboring three mouse mammary tumor virus (MMTV) variants develop pregnancy-dependent (PD) tumors that progress to pregnancy-independent (PI) behavior through successive passages. Herein, we identified 10 predominant insertions in PI transplants from 8 independent tumor lines. These mutations were also detected in small cell populations in the early PD passages. In addition, we identified a new viral insertion upstream of the gene Rspo3, which is overexpressed in three of the eight independent tumor lines and codes for a protein very similar to the recently described protein encoded by Int7. This study suggests that during progression towards hormone independence, clonal expansion of cells with specific mutations might be more relevant than the occurrence of new MMTV insertions.

Glucocorticoid-Induced Impairment of Mammary Gland Involution Is Associated with STAT5 and STAT3 Signaling Modulation

The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation, and regression. During lactation, the signal transducer and activator of transcription factor (STAT)-5A and the glucocorticoid receptor (GR) synergize to induce milk protein expression and also act as survival factors. During involution, STAT3 activation mediates epithelial cell apoptosis and mammary gland remodeling. It has been shown that the administration of glucocorticoids at weaning prevents epithelial cell death, probably by extracellular matrix breakdown prevention. Our results show that the synthetic glucocorticoid dexamethasone (DEX) modulates STAT5A and STAT3 signaling and inhibits apoptosis induction in postlactating mouse mammary glands, only when administered within the first 48 h upon cessation of suckling. DEX administration right after weaning delayed STAT5A inactivation and degradation, preserving gene expression of target genes as β-casein (bcas) and prolactin induced protein (pip). Weaning-triggered GR down-regulation is also delayed by the hormone treatment. Moreover, DEX administration delayed STAT3 activation and translocation into epithelial cells nuclei. In particular, DEX treatment impaired the increment in gene expression of signal transducer subunit gp130, normally up-regulated from lactation to involution and responsible for STAT3 activation. Therefore, the data shown herein indicate that glucocorticoids are able to modulate early involution by controlling the strong cross talk that GR, STAT5, and STAT3 pathways maintains in the mammary epithelium.

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

IntroductionIt 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.MethodsThe 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.ResultsHigh 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.ConclusionLIF 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.

Impairment of mammary lobular development induced by expression of TGFβ1 under the control of WAP promoter does not suppress tumorigenesis in MMTV‐infected transgenic mice

It has previously been shown that transgenic female mice expressing TGFβ1 under control of regulatory elements of the whey‐acidic protein (WAP) gene were unable to lactate. This was due to the increased apoptosis of the cells committed to the lobular‐lactogenic phenotype. Our goal was to determine whether the expression of WAP‐TGFβ1 transgene could inhibit MMTV (mouse mammary tumor virus) tumorigenic activity in the mammary gland. It is well known that the infection with this virus produces focal hyperplastic secretory nodules (HANs) and, some variants can also induce ductal pregnancy‐dependent lesions (plaques). In either case, MMTV infection leads ultimately to the appearance of malignant mammary tumors. The results shown herein demonstrate that TGFβ1 expression in the secretory mammary epithelium does not suppress mammary tumorigenesis in MMTV infected mice. Although MMTV infected WAP‐TGFβ1 transgenic females displayed a strong impairment of lobule‐alveolar development, carcinogenesis induced by any of the four MMTV variants used herein proceeded unabated. WAP‐TGFβ1 tumors that showed a strong expression at the WAP promoter, appeared later and grew more slowly than their wild‐type counterparts. Transgenic females also had a lower incidence of HANs and plaques. Our study suggests that the epithelial target cells for tumorigenic mutations are probably progenitor cells that are not susceptible to the apoptotic effect of TGFβ1. Alternatively, their daughters cells that display the secretory phenotype and could be more involved in the formation of premalignant lesions continue to die due to the expression of the transgene.

Tumor Necrosis Factor Alpha Induces LIF Expression Through ERK1/2 Activation in Mammary Epithelial Cells

It has been reported that expression of tumor necrosis factor superfamily members occur at the onset of the mammary gland post‐lactational involution. One of these proteins, tumor necrosis factor alpha (TNFα), is a major mediator of inflammation that is able to induce expression of several cytokines. Leukemia inhibitory factor (LIF) is an inflammatory cytokine that is induced and plays a fundamental role during post‐lactational involution of the mammary gland. Therefore, our goal was to determine whether TNFα activity in the mammary epithelium might include regulation of LIF expression. This biological role would increase the significance of TNFα expression at the end of lactation. Our results show that TNFα was able to induce LIF transcription through ERK1/2 activation in a non‐tumorigenic mouse mammary epithelial cell line, SCp2. We found that activation of TNFα receptor‐2 (TNFR2) was specifically involved in triggering this signaling pathway. In addition, our data suggest the participation of AP‐1 transcription factor family members in this pathway. We determined that TNFα treatment induced c‐fos transcription, and blocking AP‐1 activity resulted in a significant inhibition of TNFα‐induced LIF expression. Finally, we found that TNFα was also able to trigger LIF expression and ERK1/2 activation in the mouse mammary gland in vivo. Therefore, our data suggest that TNFα may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation. J. Cell. Biochem. 110: 857–865, 2010.

Mammary differentiation induces expression of Tristetraprolin, a tumor suppressor AU-rich mRNA-binding protein

Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, “serial analysis of gene expression” libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.