Abstract The cancer stem cell (CSC) hypothesis has gained significant recognition as the descriptor of tumorigenesis. The Sox-2 signaling pathway has an important role in the maintenance of CSCs. Tumor-associated macrophages (TAMs) play an ambiguous role in this process. Here, we demonstrate that the expression of Sox-2 was up-regulated in CSC of 4T07 breast cancer cells, effects induced and enhanced by co-culture of tumor cells with TAMs. In fact, after co-culture, 4T07 tumor cells develop an increased number of CSCs with up-regulated expression of stem cell specific markers (Scal-1 and ABCG2), as well as partner genes of Sox-2, i. e. Oct-4 and Nanog. Down regulation of Sox-2 effectively blocks the ability of TAMs to maintain the phenotype of CSC among 4T07 cancer cells, and enhances tumor development and its pulmonary metastasis in a syngeneic Balb/c mouse model. Most importantly, in a mechanism study, we found that the Sox-2 up-regulation was induced by EGF-R activation in CSC that was triggered by EGF, Macrophage Conditioned Medium (MCM) or by co-culture with TAMs. These effects can all be blocked by inhibitors of EGF-R. Our results suggest that TAMs in the tumor microenvironment(TME) provide a niche which is critical in the development and maintenance of CSCs in breast carcinoma. This critical effect of TAMs on CSC development was achieved by up-regulation of the Sox-2 signaling pathway in CSC. This, in turn, was induced by marked activation of EGF-R achieved through interactions between CSCs and TAMs. In summary, over expression of Sox-2 in CSC of 4T07 breast cancer cells was enhanced by activation of EGF-R, which was induced by TAMs in TME. This series of events is responsible for maintaining the phenotype of CSCs which enhances tumor development and pulmonary metastasis in vivo. 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 4232.
// Chen Zhou 1, * , Huimin Jiang 1, * , Zhen Zhang 1 , Guomin Zhang 1 , Hang Wang 1 , Quansheng Zhang 2 , Peiqing Sun 3 , Rong Xiang 1 and Shuang Yang 1 1 Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Medical School of Nankai University, Tianjin 300071, China 2 Tianjin Key Laboratory of Organ Transplantation, Tianjin First Center Hospital, Tianjin 300192, China 3 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA * These authors have contributed equally to this work Correspondence to: Shuang Yang, email: yangshuang@nankai.edu.cn Keywords: breast cancer, neurogenin-3, stemness properties, tumor initiation, ZEB1 Received: December 29, 2016 Accepted: March 20, 2017 Published: April 13, 2017 ABSTRACT Cancer stem cells (CSCs) are a subpopulation of cancer cells believed to be implicated in cancer initiation, progression, and recurrence. Here, we report that ectopic expression of zinc finger E-box binding homeobox 1 protein (ZEB1) results in the acquisition of CSC properties by breast cancer cells, leading to tumor initiation and progression in vitro and in vivo . The neurogenin 3 gene ( Ngn3 ) is a bona fide target of ZEB1, and its repression is a key factor contributing to ZEB1-induced cancer cell stemness. ZEB1 suppressed Ngn3 transcription by forming a ZEB1/DNA methyltransferase (DNMT)3B/histone deacetylase 1 (HDAC1) complex on the Ngn3 promoter, leading to promoter hypermethylation and gene silencing. The rescue of Ngn3 expression attenuated ZEB1-induced cancer stemness and symmetric CSC division. Immunohistological analysis of human breast cancer specimens revealed a strong inverse relationship between ZEB1 and NGN3 protein expression. Thus, our findings suggest ZEB1-mediated silencing of Ngn3 is required for breast tumor initiation and maintenance. Targeted therapies against the ZEB1/Ngn3 axis may be highly valuable for the prevention and treatment of breast cancer.
Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.
1018 The cancer stem cell hypothesis suggests that malignant clones in a tumor are maintained by a rare fraction of cells with stem cell properties. Such stem cell like cancer cells (SCLCC) have recently been described in solid tumors such as breast carcinoma and various brain tumors. One of the key features of such SCLCCs is their ability to repopulate entire tumors in vivo. Primarily human cancer stem cells have been isolated thus far and in vivo experiments rely on the use of immuno-compromised SCID/NOD mice. Here we demonstrate that a SCLCC population can be identified in the murine 4T1 breast cancer and NXS2 neuroblastoma cell lines and may serve as a model to study SCLCCs in immuno-competent syngeneic mouse models. To identify potential cancer stem cells murine 4T1 breast carcinoma and NXS2 neuroblastoma cells were stained with Hoechst dye 33342. We clearly distinguished in both cell lines a low staining Side Population (SP), a compartment which has been shown to be enriched for stem cells, of 2% and 0.2% respectively. Specificity of the SP population was confirmed by co-incubation with the Ca++ Blocker Verapamil, which suppressed it in both cell lines. Phenotyping showed that 4T1 SP cells have a Sca-1high c-kit- CD45- phenotype and NXS2 SP cells a Sca-1high CD24high c-kit- CD45- GD2high phenotype. RT-PCR further revealed upregulation of the ABCG2 transporter and TGF-beta in SP cells. Since stem cells were shown to be resistant to various chemotherapeutic drugs, 4T1 cells were tested for chemoresistance against cisplatin and doxorubicin. Non-sorted as well as sorted cells from the SP compartment showed higher proliferation rates and lower numbers of apoptotic cells than non-SP cells. SP cells also revealed a significant decrease in intracellular doxorubicin compared to the non-SP compartment. Because stem cells have the ability to re-populate entire cell populations, sorted SP, non-SP and 4T1 cells were cultured in vitro and the degree of repopulation was determined under normal culturing conditions at different time points. Whereas both SP and non-SP sorted cells could repopulate parts of the other compartment, which suggests a certain heterogeneity in these populations, the percentages of repopulated cells was significantly higher in SP than non-SP sorted cells. Importantly, tumorogenicity was determined by orthotopical injection of sorted SP and non SP cells in Balb/c mice. Disease occurred in 7/8 mice of the SP injected mice but only in 2/8 of non-SP injected animals, with one of the two tumors being barely palpable. Furthermore, single cell suspensions of SP-induced tumors revealed an increased amount of cells in the SP compartment when compared to the non-SP tumor. These data suggest that the SP compartments in 4T1 and NXS2 cells are enriched in cells with stem cell properties and could serve as a model system for the study of SCLCCs in immune competent mice.
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