Research ArticleDown-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-β pathways in MCF-7 Cancer cells with PX-866 and Raloxifene
Introduction
The human telomerase reverse transcriptase (hTERT) gene is believed to have evolved with non-LTR retrotransposons and from reverse transcriptase genes present when DNA was replacing RNA for the maintenance of genomes of eukaryotes and/or their ancestor [1]. Accordingly, a TERT gene is present almost universally in eukaryotes, and few genes in eukaryotes have had as much time, opportunity or necessity to be shaped by evolution in adaptation to changing requirements [1]. Interestingly, few if any human genes have regulation as complex as that of hTERT, and the most extensive regulation of hTERT is at the level of transcription [2], [3], [4], [5].
The human TERT protein hTERT is the catalytic and limiting subunit of the telomerase ribonucleoprotein [2] and is required for protection of the ends of linear chromosomes from degradation [3], [4], [5]. In the absence of telomerase, the finite length of telomeres leads to limited numbers of cell divisions and senescence [3], [4]. In addition, a growing number of genes has been found to be regulated by hTERT as a transcription factor, and as a transcription factor it participates in an hTERT expression positive feedback loop [6], [7], [8], [9]. Different patterns of hTERT transcription are required for functions as different as tissue renewal, differentiation, immune cell proliferation and tumor prevention. Accordingly, a complex network of regulatory gene products, signaling pathways and expression overrides is required for hTERT to accommodate its diverse range of responses to a vast range of environmental input [8], [10], [11], [12]. Regulation of the cell cycle, cross-linked to regulation of hTERT expression as has been noted [8], [13], [14], controls a wide range of bodily functions and development [8], [9], [10], [11], [12], [15], and dysregulation accommodates a wide range of human diseases. hTERT is especially valuable as a target for prevention or treatment of the unlimited cell cycle progression, immortality, that sustains cancer [2], [4], [5]. Our experiments were designed to test the composite effects of two agents acting on two regulatory pathways as well as the process of selecting relevant pathways based on an understanding of their relationships within the comprehensive regulatory network.
The PI3K/Akt pathway increases hTERT expression through multiple mechanisms [8]. Activated Akt activates the cell cycle by blocking, through phosphorylation, the interaction between MDM2 and p14 (p19) that would prevent ubiquitin-mediated proteolysis of p53 [16], [17] and by the mTOR-mediated degradation of cMYC competitor MAD1 [18], [19]. Both the canonical and non-canonical NF-κB pathways are activated when Akt phosphorylates and activates IkB kinase (IKK), resulting in the phosphorylation and degradation of ΙκΒ [20], [21], [22]. The NF-κB pathway is subject to hormone-mediated suppression in estrogen receptor (ER) expressing cells, such as MCF-7, but potentially reversible by antiestrogens, aromatase inhibition, and growth factors or cytokines, including tumor necrosis factor α (TNF α) [23], [24], [25], [26]. Additional mechanisms downstream of Akt result in degradation of SMAD4, p53 and p27 [27], [28], [29], [30].
In the canonical TGF-β pathway, a complex involving TGF-β, ligand-activated TGF-β receptors, p107, SMAD3, SMAD4 and either E2F-4 or E2F-5 can bind to the cMYC promoter to block transcription [31]. The TGF-β pathway may be inhibited by p107 phosphorylation by complexes of cyclin and cyclin-dependent kinase (cdk) or sustained by cdk inhibitors p27 or p21 [31], [32], [33]. Estrogen receptor α (ERα), bound to an estrogen response element (ERE) in the upstream regulatory region of the hTERT promoter and activated by 17 β-estradiol (E2) as a ligand, blocks TGF-β pathway-mediated repression [31], [34], [35]. Estrogen has also been reported to block the TGF-β pathway by binding a receptor in the cytoplasm [36].
To inhibit the PI3K/Akt pathway, we used the wortmannin derivative PX-866, specific for PI3K component p110α and currently in Phase II clinical trials [37]. To up-regulate the activity of the TGF-β pathway, we used the selective estrogen receptor modulator (SERM) raloxifene, a competitive ligand for ERα [38], [39].
To explore, analyze and screen for potential involvement of treatment-associated and/or cell type-associated mechanisms related to fidelity or diversion from proliferation-limiting canonical TGF-β pathway processing, alternative divergent non-canonical transcription signatures were also examined by real-time PCR. Transcription signatures have proved valuable in associating disease conditions, perturbations and molecular mechanisms [40]. One reported signature of TGF-β pathway misdirection includes Interleukin 11 (IL-11), Cyclin D1 and Axin2 as genes with transcription levels most divergent from normal [41]. Another includes Leukemia inhibitory factor (LIF), HB EGF and ERα with most divergent transcription levels [42].
Section snippets
Cell cultures, reagents and procedures
Human cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and included ER(+) MCF-7 and ER(-) MDA-MB-231 breast cancer epithelial cells, control non-tumorigenic MCF10A breast epithelial cells and, for contrasting unrelated cells sensitive to proliferation regulated by distinctly different signaling, Jurkat, Clone E6-1, T cell leukemia lymphoblasts. MCF-7 and MDA-MB-231 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Mediatech, Manassas, VA) supplemented
Combined PX-866 and raloxifene treatment decreases phosphorylated MDM2 in MCF-7 cells
We used Western blot to evaluate treatment-associated differences in levels of specific proteins and modified proteins in MCF-7 ER+ breast cancer cells harvested 18 h after the last of three consecutive daily treatments with PX-866 and/or raloxifene or with only DMSO, as vehicle. Protein and modified protein values from Western blot images were quantified by densitometry using ImageJ and normalized to the reference protein β-actin. To assess their effects on MDM2, p53 and p21-mediated regulation
Discussion
We previously reported the highly significant decrease of proliferation of MCF-7 cells after three days of treatment with 1.0 μM raloxifene alone or in combination with 0.1, 0.4 or 0.8 μM PX-866 and the absence of any significant decrease of MCF10A cell proliferation following identical treatment [57]. As documented, there are few cellular or intracellular functions even remotely related to cell division, growth or cancer that are hTERT-independent, and this includes gene expression, signaling,
Conclusion
PX-866 and raloxifene down-regulate the PI3K/Akt pathway, up-regulate the TGF-β pathway and, by decreasing transcription of hTERT, Cyclin D1 and other associated genes, decrease proliferation of MCF-7 breast cancer cells. Previously undisclosed genes and mechanisms involved in protective regulation can be discovered from expression signatures associated with pathological conditions. Cell type-associated expression level differences can also forecast the importance of specific genes to
Conflict of interest
The authors have no conflict of interest.
Acknowledgements
The authors wish to thank Rishabh Kala for valuable technical support. This work was supported in part by Grants from the NCI (R01 CA178441) and the American Institute for Cancer Research (316184).
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