Christina Scheel

Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.

Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.

The epithelial-mesenchymal transition (EMT) has been associated with the acquisition of motility, invasiveness, and self-renewal traits. During both normal development and tumor pathogenesis, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment. The signals that are responsible for inducing an EMT and maintaining the resulting cellular state have been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-β and canonical and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal state. Downregulation of endogenously synthesized inhibitors of autocrine signals in epithelial cells enables the induction of the EMT program. Conversely, disruption of autocrine signaling by added inhibitors of these pathways inhibits migration and self-renewal in primary mammary epithelial cells and reduces tumorigenicity and metastasis by their transformed derivatives.

Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice

Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+ cKit- hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+ cKit- BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets.

Phenotypic plasticity and epithelial-mesenchymal transitions in cancer and normal stem cells?

Cancer stem cells (CSCs) are similar to normal stem cells in their ability to self‐renew and to generate large populations of more differentiated descendants. In contrast to the hierarchical organization that is presumed to be the prevalent mode of normal tissue homeostasis, phenotypic plasticity allows cancer cells to dynamically enter into and exit from stem‐cell states. The epithelial–mesenchymal transition (EMT) has been closely associated with the acquisition of both invasive and stem‐cell properties in cancer cells. Thereby, EMT programs emerge as important regulators of phenotypic plasticity in cancer cells including their entrance into stem‐cell states. Much is still to be learned about the regulation of EMTs through epigenetic mechanisms in cancer cells and the contributions that EMT programs make to normal tissue homeostasis.

Adaptation versus Selection: The Origins of Metastatic Behavior

During the formation of a primary tumor, premalignant cells acquire a series of genetic and epigenetic changes that enable them to proliferate in the absence of growth factors, to resist proapoptotic stimuli, and to stimulate angiogenesis ( [1][1]). Each of these acquired traits confers a distinct

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells

BackgroundThe miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors.FindingsWe show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus.ConclusionsThe present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.

Abstract B25: Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells

The epithelial-mesenchymal transition (EMT), a pleiotropic cellular program, has been associated with the acquisition of metastatic ability, self-renewal traits and resistance to chemotherapeutic drugs in breast cancer and other carcinomas. During normal development and tumor progression, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment, thereby promoting cellular heterogeneity. The signaling context responsible for inducing an EMT and maintaining the resulting cellular states has been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-beta and canonical/beta-catenin dependent and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal and stem cell-like state. Importantly, the induction of the EMT program and associated autocrine signaling is partly enabled by the downregulation of endogenous inhibitors of autocrine signals secreted by epithelial cells prior EMT, indicating a homeostatic mechanism. These inhibitors include secreted frizzled-related protein 1 (SFRP1), which has been shown by others to be a rate-limiting determinant of Wnt signaling, and Bone Morphogenetic Proteins, which appear to inhibit TGF-beta signaling. Conversely, disruption of autocrine signaling by adding back endogenous inhibitors of autocrine signaling as recombinant proteins inhibits migration and self-renewal of breast cancer cells in vitro and reduces tumorigenicity and spontaneous metastasis in vivo. Together, our results indicate that ongoing autocrine signaling is required for the maintenance of mesenchymal and stem cell traits in breast cancer cells. In the longer term, we intent to develop a cocktail small-molecule inhibitors to therapeutically interrupt multiple autocrine pathways, thereby forcing tumor-initiating cells of breast and other carcinomas to exit the mesenchymal, stem cell-like state. Since this cellular state is associated with resistance to chemotherapeutic drugs, we predict that such a treatment protocol might render breast cancer cells that have passed through an EMT more sensitive to standard chemotherapy, thereby serving as a therapeutic strategy to overcome tumor heterogeneity and relapse. Citation Format: Christina Scheel, Diana Dragoi, Elinor Ng Eaton, Sophia Hsin-Jung Li, Ferenc Reinhardt, Robert A. Weinberg. Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B25.

Abstract LB-108: Formation and maintenance of mesenchymal and stem-cell states in the breast by paracrine and autocrine signals

Passage through an epithelial-mesenchymal transition (EMT) is associated with the acquisition of migratory and self-renewal abilities in human mammary epithelial cells (MECs). The signaling mechanisms that induce and then maintain these properties have remained unclear. We describe three signaling pathways, involving Transforming Growth Factor (TGF)-beta as well as canonical and non-canonical Wnt signaling, that collaborate to induce epithelial MECs to enter into a mesenchymal and SC-like state. Acting as autocrine signaling loops, these pathways then maintain migratory and self-renewal abilities in normal MECs and control tumorigenicity and metastasis in their transformed derivatives. Autocrine signaling is enabled, at least in part, through downregulation of secreted Wnt antagonists and Bone Morphogenetic Proteins. Like immortalized and transformed MECs, the maintenance of migratory and self-renewal abilities in primary human MECs is dependent on these autocrine loops, suggesting that similar mechanisms regulate biological properties of normal tissue stem cells and tumor-initiating cells in the breast Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2011-LB-108

Abstract C75: Granulin‐expressing bone marrow cells promote the outgrowth of indolent tumors

We previously demonstrated that certain vigorously growing xenografted human carcinomas (“instigators”) stimulate the growth of otherwise‐indolent carcinoma cells and metastases (“responders”) implanted at distant anatomical sites — a process we termed “systemic instigation” (S. McAllister, et al.; Cell, 2008). We showed that systemic instigation is largely the result of activation of bone marrow cells (BMCs) that subsequently contribute to the stroma of the responding tumors; however, the identity of the activated BMCs and their contribution to outgrowth of the once‐indolent cells was unknown. Here, we demonstrate that Sca1+/cKit− BMCs of hosts bearing instigating tumors are activated prior to their mobilization into the circulation and are very potent in their ability to promote outgrowth of the responding tumors. Strikingly, responding tumors, which result from either the presence of a contralateral instigating tumor or the admixture of Sca1+/cKit− BMCs harvested from instigator‐bearing mice, form with a desmoplastic stroma. Stromal desmoplasia, the accumulation of a myofibroblast‐rich reactive connective tissue, is almost always observed in malignant human adenocarcinomas. The Sca1+/cKit− BMCs do not directly give rise to stromal myofibroblasts; instead, we found that factors secreted by these BMCs induce fibroblasts to express alpha‐smooth muscle actin (ACTA2), a myofibroblast marker. Gene expression profiling of the Sca1+/cKit− BMCs from instigator‐ and non‐instigator‐bearing mice identified granulin, a pluripotent secreted growth factor, as the most highly upregulated gene in the activated BMCs. We found: i) that granulin is indeed expressed by bone marrow‐derived cells that are recruited into the responding tumor stroma; ii) that treatment of human mammary fibroblasts with granulin induces ACTA2 expression; iii) that granulin treatment is sufficient to enhance responding tumor growth in vivo . Mining of existing microarray datasets revealed that high granulin expression is correlated with the expression of ACTA2 in human breast cancers and is associated with shorter disease‐free survival of breast cancer patients. Our results indicate that the formation of desmoplastic stroma, a condition that is associated with invasive carcinomas, can be instigated systemically by certain tumors via activation of granulin‐expressing Sca1+/cKit− in the bone marrow. Citation Information: Cancer Res 2009;69(23 Suppl):C75.