The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells.

Stem Cell Antigen-1 ( Sca-1/Ly6A ) was the first identified member of the Lymphocyte antigen-6 ( Ly6 ) gene family. Sca-1 serves as a marker of cancer stem cells and tissue resident stem cells in mice. The Sca-1 gene is located on mouse chromosome 15. While a direct homolog of Sca-1 in humans is missing, human chromosome 8—the syntenic region to mouse chromosome 15—harbors several genes containing the characteristic domain known as LU domain. The function of the LU domain in human LY6 gene family is not yet defined. The LY6 gene family proteins are present on human chromosome 6, 8, 11, and 19. The most interesting of these genes are located on chromosome 8q24.3, a frequently amplified locus in human cancer. Human LY6 genes represent novel biomarkers for poor cancer prognosis and are required for cancer progression in addition to playing an important role in immune escape. Although the mechanism associated with these phenotype is not yet clear, it is timely to review the current literature in order to address the critical need for future advancements in this field. This review will summarize recent findings which describe the role of human LY6 genes— LY6D, LY6E, LY6H, LY6K, PSCA, LYPD2, SLURP1, GML, GPIHBP1 , and LYNX1 ; and their orthologs in mice at chromosome 15.

Stem cell antigen (Sca)-1/Ly6A, a glycerophosphatidylinositollinked surface protein, was found to be associated with murine stem cell-and progenitor cell-enriched populations, and also has been linked to the capacity of tumor-initiating cells. Despite these interesting associations, this protein's functional role in these processes remains largely unknown. To identify the mechanism underlying the protein's possible role in mammary tumorigenesis, Sca-1 expression was examined in Sca-1 +/EGFP mice during carcinogenesis. Mammary tumor cells derived from these mice readily engrafted in syngeneic mice, and tumor growth was markedly inhibited on down-regulation of Sca-1 expression. The latter effect was associated with significantly elevated expression of the TGF-β ligand growth differentiation factor-10 (GDF10), which was found to selectively activate TGF-β receptor (TβRI/II)-dependent Smad3 phosphorylation. Overexpression of GDF10 attenuated tumor formation; conversely, silencing of GDF10 expression reversed these effects. Sca-1 attenuated GDF10-dependent TGF-β signaling by disrupting the heterodimerization of TβRI and TβRII receptors. These findings suggest a new functional role for Sca-1 in maintaining tumorigenicity, in part by acting as a potent suppressor of TGF-β signaling.S tem cell antigen (Sca)-1 is a member of the Ly6A superfamily of glycerophosphatidylinositol (GPI)-anchored membrane proteins (1), which is associated with murine stem and progenitor cell populations in several tissues. In the mammary gland, Sca-1-positive cells are able to reconstitute the cleared fat pad (2, 3) and give rise to alveolar and ductal structures (4). In addition to Sca-1's normal role in stem cell self-renewal, Sca-1 expression is elevated in malignant tissues, such as retinoblastomas (5), prostate tumors (6), mammary tumors (7, 8) and chronic myeloid leukemia (9), which generally reflects a more aggressive phenotype (10). Despite these associations, the role of Sca-1 in these processes remains largely unknown. To address this question, we used Sca-1 +/EGFP mice, in which EGFP is under the control of the Sca-1 locus (11), to study mammary tumorigenesis. We found that Sca-1 suppresses TGF-β signaling by inhibiting expression of the TGF-β family ligand GDF10 and binding to the type I TGF-β receptor (TβRI), thereby inhibiting ligand-induced TGF-β receptor complex formation and Smad3 phosphorylation. This study is the first to demonstrate specific functions for Sca-1 and GDF10 in tumorigenesis. ResultsSca-1 Is Increased Early in Mammary Tumorigenesis. To explore the role of Sca-1 in tumorigenesis, we induced mammary tumors in heterozygous Sca-1 +/EGFP mice (11) using medroxyprogesterone (MPA) and 7,12-dimethylbenz[a]anthracene (DMBA) (8,12,13). Cultures of primary mammary epithelial cells exhibited enhanced EGFP fluorescence coincident with increased Sca-1 expression immediately after treatment with MPA and DMBA (( Fig. S1 A and B ), suggesting that the Sca-1 locus is activated at the onset of carcinogenesis. Both Sca-1 +/EGFP a...

Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologues Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E were required for TGF-β signaling and proliferation in breast cancer cells where they contributed to phosphorylation of Smad1/5 and Smad2/3. Further, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Lastly, we found that Ly6K/E promoted drug resistance and facilitate immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGF-β in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention.