Research Article
Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-β pathways in MCF-7 Cancer cells with PX-866 and Raloxifene

https://doi.org/10.1016/j.yexcr.2016.03.022Get rights and content

Highlights

  • PX-866 and raloxifene affect the PI3K/Akt and TGF-β pathways.

  • PX-866 and raloxifene down-regulate genes up-regulated in cancer.

  • PX-866 and raloxifene decrease transcription of hTERT and Cyclin D1.

  • Pathological transcription signatures can identify new defense mechanisms.

Abstract

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.

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).

References (75)

  • L. Wu et al.

    Smad4 as a transcription corepressor for estrogen receptor α

    J. Biol. Chem.

    (2003)
  • J. Kawagoe et al.

    Raloxifene inhibits estrogen-induced up-regulation of telomerase activity in a human breast cancer cell line

    J. Biol. Chem.

    (2003)
  • G. Kerdivel et al.

    Activation of the MKL1/actin pathway induces hormonal escape in estrogen-responsive breast cancer cell lines

    Mol. Cell Endocrinol.

    (2014)
  • K. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method

    Methods

    (2001)
  • S. Lai et al.

    Evidence of extra-telomeric effects of hTERT and its regulation involving a feedback loop

    Exp. Cell Res.

    (2007)
  • Y. Li et al.

    AKT/PKB phosphorylation of p21Cip/WAF1 enhance protein stability of p21Cip/WAF1 and promotes cell survival

    J. Biol. Chem.

    (2002)
  • C.N. Johnstone et al.

    Emerging roles for IL-11 signaling in cancer development and progression: focus on breast cancer

    Cytokine Growth Factor Rev.

    (2015)
  • J. Berletch et al.

    Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

    J. Cell. Biochem.

    (2008)
  • Y. Cong et al.

    The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter

    Hum. Mol. Genet.

    (1999)
  • G. Saretzki

    Extra-telomeric functions of human telomerase: cancer, mitochondria and oxidative stress

    Curr. Pharm. Des.

    (2014)
  • A.E. Bilsland et al.

    Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

    PLoS Comput. Biol.

    (2014)
  • F. Wang et al.

    Bioinformatics analysis of exonic splicing enhancers (ESEs) for predicting potential regulatory elements of hTERT mRNA splicing

    Eur. Rev. Med. Pharm. Sci.

    (2014)
  • W. Arancio et al.

    CeRNA and interactome bioinformatic analyses on human telomerase

    Rejuvenation Res.

    (2014)
  • L.L. Smith et al.

    Tolemerase modulates expression of growth-controlling genes and enhances cell proliferation

    Nat. Cell Biol.

    (2003)
  • C. Yang et al.

    A key role for telomerase reverse transcriptase unit in modulating human embryonic stem cell proliferation, cell cycle dynamics, and in vitro differentiation

    Stem Cells

    (2008)
  • J. Testa et al.

    AKT plays a central role in tumorigenesis

    Proc. Natl. Acad. Sci.

    (2001)
  • B. Zhou et al.

    HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation

    Nat. Cell Biol.

    (2001)
  • C.-K. Chou et al.

    The suppression of MAD1 by AKT-mediated phosphorylation activates MAD1 target genes transcription

    Mol. Carcinog.

    (2009)
  • J. Zhu et al.

    Activation of PI3K/Akt and MAPK pathways regulates Myc-mediated transcription by phosphorylating and promoting the degradation of Mad1

    Proc. Natl. Acad. Sci.

    (2008)
  • D. Bai et al.

    Akt-mediated regulation of NFκB and the essentialness of NFκB for the oncogenicity of PI3K and Akt

    Int. J. Cancer

    (2009)
  • O.N. Ozes et al.

    NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase

    Nature

    (1999)
  • E. Wong et al.

    Roles of NF-κB in health and disease: mechanisms and therapeutic potential

    Clin. Sci.

    (2009)
  • W. Wang et al.

    Targeting the NFκB signaling pathways for breast cancer prevention and therapy

    Curr. Med. Chem.

    (2015)
  • G. Matsumoto et al.

    Targeting of nuclear factor κB pathways by dehydroxymethylepoxyquinomicin, a novel inhibitor of breast carcinomas: antitumor and antiangiogenic potential in vivo

    Cancer Res.

    (2005)
  • Y. Zhou et al.

    The NFκB pathway and endocrine-resistant breast cancer

    Endocr. Relat. Cancer

    (2005)
  • X. Wang et al.

    Oestrogen signaling inhibits invasive phenotype by repressing RelB and its target BCL2

    Nat. Cell Biol.

    (2007)
  • M.-C. Hsu et al.

    HER-2/neu transcriptionally activates Jab1 expression via the AKT/β-catenin pathway in breast cancer cells

    Endocr. Relat. Cancer

    (2007)
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