Distinct E2F-Mediated Transcriptional Mechanisms in Cell Proliferation, Endoreplication and Apoptosis

E2F and DP family proteins are evolutionally conserved transcription factors among higher eukaryotes. E2F and DP proteins typically form a heterodimeric complex, which controls cell proliferation by regulating expression of growth-related genes. In addition, E2F family proteins have roles in various cellular events that require the expression of context-specific genes. E2F proteins use distinct mechanisms to regulate context-specific genes in different circumstances. The primary goal of this chapter is to compare three distinct mechanisms of mammalian E2F-mediated transcriptional regulation that control cell proliferation, endoreplication and apoptosis. Briefly, E2F7 and E2F8 control endoreplication by suppressing the expression of their target genes. They do not require DP or pRb. In control of apoptosis, E2F1 regulates the expression of the tumor suppressor gene Arf by binding to a non-canonical E2F binding site, within the Arf promoter, in a DP-independent manner. Furthermore, we examine the functions of E2F and DP in Drosophila melanogaster (fruit fly) to identify those mechanisms of E2F-mediated transcriptional regulation that have been evolutionarily conserved. The detailed mechanisms of how E2F protein regulates the expression of context-specific target genes will be instrumental in understanding how a single family of transcription factor regulates diverse pleiotropic cellular processes in an organism

The Key Role of E2F in Tumor Suppression through Specific Regulation of Tumor Suppressor Genes in Response to Oncogenic Changes

E2F, the principal target of the tumor suppressor pRB, plays crucial roles in tumor suppression. Upon dysfunction of pRB, E2F activates tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, resulting in the induction of apoptosis and tumor suppression. The E2F activity that activates the tumor suppressor genes is detected only in cancer cells and not in normal growing cells. The E2F activity can drive selective suicide gene expression and induce apoptosis specifically in cancer cells. Thus, the E2F activity provides a beneficial tool to specifically target cancer cells in cancer treatment

Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3

Abstract Introduction Triple-negative breast cancers, particularly the claudin-low subtype, are highly aggressive and exhibit increased tumor-initiating cell (TIC) characteristics. In this study, we demonstrate that vascular endothelial growth factor C (VEGF-C) is highly expressed in the claudin-low breast cancer subtype and also that it mediates tumor progression, not only through its role in lymphangiogenesis but also through regulating TIC characteristics and the response to reactive oxygen species (ROS). Methods VEGF C expression was examined in breast cancer subtypes, and a VEGF C expression signature was derived. VEGF C expression and/or its associated signature was correlated with TIC and chemoresistance signatures. In vitro and in vivo assays were performed to determine whether VEGF-C expression alters TIC characteristics and the response of breast cancer cells to chemotherapy and oxidative stress. Array analysis was used to identify a downstream effector of VEGF-C, superoxide dismutase 3 (Sod3), which was tested for its involvement in VEGF-C-mediated resistance to oxidative stress and enhancement of in vivo metastasis. The VEGF-C-associated receptor neuropilin 2 (Nrp2) was knocked down to determine whether it is required for the observed effects of VEGF-C. Expression of VEGF C and Sod3 was assessed in human breast cancers. Results VEGF C is highly expressed in claudin-low breast cancers, and VEGF C and the VEGF C signature are associated with TIC-related gene signatures. VEGF-C-knockdown in mammary carcinoma cells decreases TIC properties in vitro and in vivo, sensitizing cells to oxidative stress and chemotherapy. We identified Sod3 as a target of VEGF-C in breast cancer cells by demonstrating that it is required for VEGF-C-mediated cell survival in response to oxidative stress and for VEGF-C-mediated metastasis. We demonstrate that Nrp2 is the VEGF-C-associated receptor that mediates alterations in Sod3 expression and the response of tumor cells to oxidative stress. We show that VEGF C and Sod3 are positively associated in human breast cancer. Conclusions We describe a novel mechanism by which VEGF-C contributes to metastasis via its ability to enhance TIC-associated characteristics, particularly the response to ROS. We identified Sod3 as a critical mediator of VEGF-C-induced metastasis, and we provide evidence that the VEGF-C-Sod3 axis plays a role in human breast cancers

Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways

Abstract Introduction Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. Methods We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. Results High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers. Conclusions Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells

Distinct E2F-mediated transcriptional program regulates p14(ARF) gene expression

The tumor suppressor p14(ARF) gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14(ARF) gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14(ARF) gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin-dependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14(ARF) function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals

Histopathology of skin lesions.

<p>A biopsied specimen was stained with hematoxylin-eosin (A, B), Elastica van Gieson (C), von Kossa (D, G–I), and picrosirius red (E, F). The swollen collagen bundles were colored yellow−orange in polarizing microscopy (F). In the deeper dermis, the internal elastic lamina and a part of the arteriole walls were positive for calcification (G–I). The bar depicts 500 μm in (A–F) and 50 μm in (G–I).</p

Clinical features and fundoscopy.

<p>Loose and sagging skin around the neck (A), legs (B), and back and hips (C). Peripapillary atrophy of the retina (arrow, D).</p