Ayyakkannu Ayyanan

The epithelial-mesenchymal transition generates cells with properties of stem cells

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland

The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor α (ERα) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(−) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-κB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR−/− phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.

Progesterone/RANKL Is a Major Regulatory Axis in the Human Breast

Ex vivo model identifies the progesterone/RANKL axis as an important proliferative stimulus in the human breast. 3D Without the Glasses Biomedical research has the lofty goal of adding to our understanding of biology in the hope that we can improve human health. However, there are difficulties—both ethical and logistic—of performing experiments in humans. One way researchers have overcome these hurdles is through the use of cell culture or animal models. Yet, these models sometimes don’t accurately represent human biology, especially in complex tissues. Now, Tanos et al. develop an ex vivo three-dimensional model using fresh breast tissue microstructures to examine the role of the progesterone-mediator RANKL in human breast. They found that although progesterone failed to induce RANKL in cell lines and dissociated breast tissue, in their microstructures, RANKL expression responded to progesterone and was required for progesterone-induced breast tissue proliferation. They validated these findings in samples from human breast epithelium. These studies could have clinical relevance: The RANKL inhibitor denosumab is currently used in the clinic to treat bone disease and could be repurposed to block breast epithelial proliferation in breast cancer. Estrogens and progesterones are major drivers of breast development but also promote carcinogenesis in this organ. Yet, their respective roles and the mechanisms underlying their action in the human breast are unclear. Receptor activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal paracrine mediator of progesterone function in mouse mammary gland development and mammary carcinogenesis. Whether the factor has the same role in humans is of clinical interest because an inhibitor for RANKL, denosumab, is already used for the treatment of bone disease and might benefit breast cancer patients. We show that progesterone receptor (PR) signaling failed to induce RANKL in PR+ breast cancer cell lines and in dissociated, cultured breast epithelial cells. In clinical specimens from healthy donors and intact breast tissue microstructures, hormone response was maintained and RANKL expression was under progesterone control, which increased RNA stability. RANKL was sufficient to trigger cell proliferation and was required for progesterone-induced proliferation. The findings were validated in vivo where RANKL protein expression in the breast epithelium correlated with serum progesterone levels and the protein was expressed in a subset of luminal cells that express PR. Thus, important hormonal control mechanisms are conserved across species, making RANKL a potential target in breast cancer treatment and prevention.

Perinatal Exposure to Bisphenol A Increases Adult Mammary Gland Progesterone Response and Cell Number

Bisphenol A [BPA, 2,2,-bis (hydroxyphenyl) propane] is one of the highest-volume chemicals produced worldwide. It is detected in body fluids of more than 90% of the human population. Originally synthesized as an estrogenic compound, it is currently utilized to manufacture food and beverage containers resulting in uptake with food and drinks. There is concern that exposure to low doses of BPA, defined as less than or equal to 5 mg/kg body weight /d, may have developmental effects on various hormone-responsive organs including the mammary gland. Here, we asked whether perinatal exposure to a range of low doses of BPA is sufficient to alter mammary gland hormone response later on in life, with a possible impact on breast cancer risk. To mimic human exposure, we added BPA to the drinking water of C57/Bl6 breeding pairs. Analysis of the mammary glands of their daughters at puberty showed that estrogen-dependent transcriptional events were perturbed and the number of terminal end buds, estrogen-induced proliferative structures, was altered in a dose-dependent fashion. Importantly, adult females showed an increase in mammary epithelial cell numbers comparable to that seen in females exposed to diethylbestrol, a compound exposure to which was previously linked to increased breast cancer risk. Molecularly, the mRNAs encoding Wnt-4 and receptor activator of nuclear factor κB ligand, two key mediators of hormone function implicated in control of mammary stem cell proliferation and carcinogenesis, showed increased induction by progesterone in the mammary tissue of exposed mice. Thus, perinatal exposure to environmentally relevant doses of BPA alters long-term hormone response that may increase the propensity to develop breast cancer.

A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response

Seventy-five percent of breast cancers are estrogen receptor α positive (ER⁺). Research on these tumors is hampered by lack of adequate in vivo models; cell line xenografts require non-physiological hormone supplements, and patient-derived xenografts (PDXs) are hard to establish. We show that the traditional grafting of ER⁺ tumor cells into mammary fat pads induces TGFβ/SLUG signaling and basal differentiation when they require low SLUG levels to grow in vivo. Grafting into the milk ducts suppresses SLUG; ER⁺ tumor cells develop, like their clinical counterparts, in the presence of physiological hormone levels. Intraductal ER⁺ PDXs are retransplantable, predictive, and appear genomically stable. The model provides opportunities for translational research and the study of physiologically relevant hormone action in breast carcinogenesis.

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf‐κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR+ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal–myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.

Prolactin signaling and Stat5: going their own separate ways?

Miyoshi et al. compared the role of the prolactin receptor (PrlR) and its downstream mediator, the signal transducer and activator of transcription 5 (Stat5), in mammary epithelial cells in vivo by studying PrlR-/- and Stat5ab-/- mouse mammary epithelial transplants during pregnancy. At first glance, the two mutant epithelia appear to have similar defects in the differentiation of the alveolar epithelium. However, a closer examination by Miyoshi et al. revealed defects in the epithelial architecture of the smallest ducts of Stat5ab-/- transplants not apparent in the PrlR-/- transplants, suggesting that Stat5 is more than a simple mediator of PrlR action.

Intraductal patient derived xenografts of estrogen receptor positive breast cancer recapitulate the histopathological spectrum and metastatic potential of human lesions

Estrogen receptor α positive (ER+) or “luminal” breast cancers were notoriously difficult to establish as patient-derived xenografts (PDXs). We and others recently demonstrated that the microenvironment is critical for ER+ tumor cells; by grafting them into milk ducts >90% take rates are achieved and many features of the human disease are recapitulated. This intra-ductal (ID) approach holds promise for personalized medicine, yet human and murine stroma are organized differently and this and other species specificities may limit the value of this model. Here, we analyzed 21 ER+ ID-PDXs histopathologically. We find that ID-PDXs vary in extent and define four histopathological patterns: flat, lobular, in situ, and invasive, which occur in pure and combined forms. The ID-PDXs replicate earlier stages of tumor development than their clinical counterparts. Micrometastases are already detected when lesions appear in situ. Tumor extent, histopathological patterns, and metastatic load correlate with biological properties of their tumors of origin. Our findings add evidence to the validity of the intraductal model for in vivo studies of ER+ breast cancer and raise the intriguing possibility that tumor cell dissemination may occur earlier than currently thought. Conflict of interest statement: The authors declare no conflict of interest.

S16 Breast stem cells and hormonal mechanisms in carcinogenesis

Reference EPFL-CONF-171520View record in Web of Science Record created on 2011-12-16, modified on 2017-05-12

Abstract 2314: Epithelial-mesenchymal transition derived cells exhibit multi-lineage differentiation potential similar to mesenchymal stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows stem cell traits to breast epithelial cells. Mesenchymal stem cells (MSC) have the capacity to self-renew and differentiate into multiple tissue lineages. We hypothesized that the activation of EMT by ectopic expression of Twist, Snail or TGF-β in immortalized human mammary epithelial cells (HMEC) will result in the generation of cells with a phenotype and functionality similar to MSC. We found that the EMT-derived cells not only showed similar morphology but also displayed the typical MSC phenotype i.e. CD44+, CD24− and CD45−. Alternatively, MSC expressed EMT inducing genes such as Twist, Snail and FOXC2. Interestingly, CD140b (PDGFR-β), a marker for naive MSC, was exclusively expressed in EMT-derived cells compared to their epithelial counterparts. Moreover, functional analysis revealed that EMT-derived but not the control cells differentiate into Alizarin Red S-positive mature osteoblasts, Oil Red O-positive adipocytes and Alcian Blue-positive chondrocytes similar to MSC. We also observed that EMT-derived but not control cells invade and migrate towards MDA-MB-231 tumor cells in-vitro similar to MSC, displaying the characteristic tropism of MSC for tumor cells as previously reported by us. In-vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSC. In conclusion, we demonstrated that the EMT-derived cells are similar to MSC in gene-expression, multi-lineage differentiation, migration towards tumor cells and their ability to home to wounds. These results also suggest that EMT-derived MSC are active participants in cancer growth and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2314.