Elinor Ng Eaton

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.

hSIR2SIRT1 Functions as an NAD-Dependent p53 Deacetylase

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. We propose that hSir2 is involved in the regulation of p53 function via deacetylation.

hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.

Inhibition of telomerase limits the growth of human cancer cells

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

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.

Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase

The expression of telomerase, the enzyme that synthesizes telomeric DNA de novo, is suppressed in normal somatic human cells but is reactivated during tumorigenesis. This reactivation appears to arrest the normal loss of telomeric DNA incurred as human cells divide. Since continual loss of telomeric DNA is predicted to eventually limit cell proliferation, activation of telomerase in cancer cells may represent an important step in the acquisition of the cell immortalization which occurs during tumor progression. The telomerase holoenzyme is composed of both RNA and protein subunits. In humans, mRNA expression of hTERT (hEST2), the candidate telomerase catalytic subunit gene, appears to parallel the levels of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase activity in tumor cells. To test this model directly, we ectopically expressed an epitope-tagged version of hTERT in telomerase-negative cells and show that telomerase activity was induced to levels comparable to those seen in immortal telomerase-positive cells and that the expressed hTERT protein was physically associated with the cellular telomerase activity. We conclude that synthesis of the hTERT telomerase subunit represents the rate-limiting determinant of telomerase activity in these cells and that this protein, once expressed, becomes part of the functional telomerase holoenzyme.

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.

Interaction of the Ski Oncoprotein with Smad3 Regulates TGF-β Signaling

Abstract TGF-β treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-β induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-β signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-β. The transcriptional repression as well as the growth resistance to TGF-β by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-β signaling pathway and shed light on the mechanism of action of the Ski oncoprotein.

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.