FOXO1 transcription factor and its role in Tamoxifen- resistant breast cancer. Christina Warner. Department of Biochemistry and Molecular Biophysics

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1 FOXO1 transcription factor and its role in Tamoxifen- resistant breast cancer Christina Warner Department of Biochemistry and Molecular Biophysics University of Arizona Abstract: 136 words Main Body: 1,292 Words

2 2 Abstract: The forkhead box O (FOXO) subfamily of forkhead domain transcription factors plays a key role in control of the cell cycle, cell differentiation, metabolism, oxidative stress response and apoptosis. There are four members in the subfamily: FOXO1, FOXO3, FOXO4 and FOXO6. These transcription factors consist of a winged helix DNA binding domain with a high degree of conservation. FOXO proteins play an important role in regulating a diverse range of cellular processes such as differentiation, proliferation, cell cycle regulation, apoptosis, metabolism, oxidative stress protection, and DNA repair. Increasing evidence suggests that FOXO proteins may function as tumor suppressors by regulating gene expression since their expression is frequently dysregulated in cancers (Choi et al., 2013). In particular, the deacetylation of FOXO1 is essential for the expression of multidrug resistance-associated protein 2 (MRP2) in Tamoxifen resistant breast cancer.

3 3 Introduction: Transcription factors are proteins which bind to specific sequences of DNA allowing for control of the transcription of DNA to mrna (Latchman, 1997). They can perform this function alone or form a complex with other proteins acting as activators or repressors to the recruitment of RNA polymerase (Roeder, 1996). These transcription factors are key components of signaling pathways connecting cell growth and stress signaling. There are many different groups of transcription factors include steroid receptors, resident nuclear proteins, and latent cytoplasmic factors (Darnell, 2002). It is the overall activity of one or all of these transcription factors that attributes to the metastatic behavior found in human cancers (Denhardt, 1996). The forkhead box O (FOXO) subfamily of transcription factors are often steroid receptors and regulate a variety of cellular processes such as cell cycle regulation, apoptosis, metabolism, DNA repair and oxidative stress protection (Eijkelenboon and Burgering, 2013; van der Heide et al., 2004; Kops et al., 2002). This subfamily consists of four highly conserved proteins: FOXO1, FOXO3, FOXO4, and FOXO6. These proteins were initially identified at chromosomal rearrangements in some human tumors (Galili et al., 1993). These proteins are all capable of acting redundantly since they bind at the same DNA recognition sequence (Eijkelenboon and Burgering, 2013). Increasing evidence suggests that FOXO proteins may act as tumor suppressors due to the regulation of genes involved in apoptosis and oxidative stress (Huang and Tindall, 2007; Kops et al., 2004). Often these transcription factors are targeted and

4 4 deregulated in some cancers (Schuur et al., 2001). Choi et al. has shown previously that one particular FOXO transcription factor, FOXO1, functions as a key regulator of multidrug resistance 1 (MDR1) gene transcription. Recently they have also shown that deacetylation of FOXO1 is essential for multidrug resistance-associated protein 2 (MRP2) expression in Tamoxifen resistant breast cancer. Main Body: Structure/Function of FOXO Transcription Factors The FOXO transcription factors make up a discrete family in the winged helix protein superfamily. It consists of 4 helical bundles and a 4 strand β-sheet. Each of the protein contains four domains: a highly conserved DNA binding domain (DBD), a nuclear localization signal (NLS), a nuclear export sequence (NES), and a C-terminal transactivation domain (TAD) (Obsil and Obsilova, 2008). FOXO1, FOXO3, and FOXO6 all have similar length of approximately 650 amino acid residues, whereas FOXO4 is shorter and consists of approximately 500 residues. Primary sequence comparison (Figure 1) shows several regions of FOXO proteins are conserved. The sequences with the highest conservation include the DBD, and the protein kinase B (PKB) phosphorylation site. The DBD of all FOXO proteins recognize two consensus DNA sequences 5 GTAAA(T/C)AA-3 and 5 -(C/A)(A/C)AAA(C/T)AA-3 (Obsil and Obsilova, 2011) and binds in the major groove of the DNA (Figure 3). This binding domain can range from 73 amino acid residues (FOXO1) to 95 residues (FOXO6 and FOXO3). The DBD interacts with the DNA sequence through both direct hydrogen bonding and van der

5 5 Waal contacts (Figure 4). The primary residues involved in binding to the major groove are Asn165 and His169, which are conserved among all forkhead proteins. The NLS functions to tag the transcription factors for import into the cell nucleus, while the NES functions to target the transcription factors for export from the nucleus. As with many other transcription factors, the FOXO proteins are largely unstructured, indicated by the number of loops in the tertiary structure shown in Figure 2. These loops in the DBD interact only with the phosphate backbone of the DNA in all FOXO-DNA structures, suggesting that these regions function to stabilize the proteindna complex without any significant contributions to the recognition of DNA (Obsil and Obsilova, 2011). It is not uncommon for FOXO proteins to be subject to several post translational modifications including phosphorylation, acetylation, and ubiquitination. While the exact mechanisms for these modifications remain unclear, several cases have shown to modify the DNA-binding potential. Phosphorylation by PKB is stimulated in response to growth signals and occurs at 4 different sites. The first site is in the PKB phosphorylation site shown in Figure 1, the second site is located in the DBD, and the last one is located between the NLS and NES. This phosphorylation is an important factor in cell survival, regulation of cell cycle, differentiation and intracellular traffic processes (van der Heide et al., 2004). Acetylation occurs after FOXO proteins have been phosphorylated and transported to the cytoplasm, and is achieved by histone acetyltransferases such as cyclic-amp responsive element binding-binding protein. It is still unclear what the

6 6 consequences of acetylation are as it has been speculated that they may function as a negative feedback signal (due to the suppression of FOXO activity upon acetylation) or they may function in a target gene specific context. Acetylation is a reversible process by silent information regulator two ortholog 1 (SIRT1). Once FOXO transcription factors are deacetylated they accumulate in the nucleus, affecting transactivation activity of the target genes (Choi et al., 2013). Finally ubiquitination (both poly- and monoubiquination) occurs in response to growth factor signaling as well as oxidative stress (Huang and Tindall, 2011). This ubiquitination results in relocalization and transcriptional activity enhancement of FOXO proteins. Discussion: Role of FOXO1 in Tamoxifen-Resistant Breast Cancer Recent studies by Choi et al. have shown that multidrug resistance protein 2 (MRP2) is overexpressed in tamoxifen-resistant breast cancer cells. Tamoxifen (TAM) is a selective estrogen receptor modulator. Most patients are responsive however over time they may acquire resistance to TAM. One of the mores important mechanisms in multidrug resistance is the active efflux of cytotoxic agents out of cancer cells by drug transporters such as ATP-binding cassettes (ABC). Many multidrug resistant proteins (MRPs) interact with a broad range of substrates, preventing their accumulation in the cell. Choi et al. have shown that FOXO1 is increased in Adriamycin-resistant breast cancer and plays an essential role in expression of the MDR1 gene. In a recently

7 7 published study, Choi et al. have shown that SIRT1-mediated deacetylation is essential for MRP2 expression. By locating the FOXO1 binding site in the MDR1 gene promoter, they found that it is essential for basal expression, suggesting that FOXO1 functions as a master regulator of ABC expression. To determine whether MRP2 transcription was controlled by FOXO1, Choi et al. also measured the effects of FOXO3 and found that MRP2 expression was increased in TAM-resistant cells with FOXO1 (MRP2 protein levels were seldom elevated by FOXO3). These results show that FOXO1 functions as an active transcription factor for MRP2 as well as MDR1. Choi et al. also showed that in FOXO1 knockdown experiments, FOXO 1 had marked inhibitory effects on small interfering RNA (sirna) and subsequent gene transcription and expression of MPR2. As discussed previously the transcriptional activity of FOXOs is regulated by phosphorylation-dependent ubiquitination and acetylation, each of which affects the DNA binding activity. The activity of FOXO1 is a target through which phosphoinositide 3-kinase (PI3K) can induce tumorigenicity. Choi et al. found that in TAM-resistant cells both SIRT1 and phosphorylated PKB were increased. Upon phosphorylation, the transcription activity is weakened through ubiquitination and proteosomal degradation. In their work, Choi et al. focus on the deacetylation of FOXO1 by SIRT1, a known regulator of cell life extension in response to cell stress. Frescas et al. suggested that it was this deacetylation that increases the nuclear retention of FOXO1 thereby increasing its transcriptional activity. The study by Choi et al. found that basal expression and activity were markedly enhanced in TAM-resistant

8 8 cells, as well as finding the SIRT1 inhibitor nicotinamide reduced the FOXO1 protein and promoter activity levels. This demonstrates that increased SIRT1 deacetylation is a key mechanism responsible for the upregulation of MRP2 in TAM-resistant cells. These findings are significant in that the SIRT1/FOXO1 pathway could pose a new therapeutic target for overcoming acquisitional chemoresistance in Tam-resistant breast cancer

9 9 References Choi, H., Cho, K., Phuong, N., Han, C., Han, H., Hien, T., Choi, H. and Kang, K SIRT1-mediated FoxO1 deacetylation is essential for multidrug resistanceassociated protein 2 expression in tamoxifen-resistant breast cancer cells. Molecular Pharmaceutics. Darnell, J Transcription factors as targets for cancer therapy. Nature Reviews Cancer, 2 (10), pp Denhardt, D Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.. Biochem. J, 318 pp Eijkelenboom, A. and Burgering, B FOXOs: signalling integrators for homeostasis maintenance.nature Reviews Molecular Cell Biology, 14 (2), pp Frescas,D., Valenti, L. and Accili, D Nuclear trapping of the forkhead transcription factor FoxO1 vis SIRT-dependent deacetylation promotes expression of glucogenetic genes. J. Biol. Chem., 280, pp Galili, N., Davis, R., Fredericks, W., Mukhopadhyay, S., Rauscher, F., Emanuel, B., Rovera, G. and Barr, F Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nature genetics, 5 (3), pp

10 10 Huang, H. and Tindall, D Regulation of FOXO protein stability via ubiquitination and proteasome degradation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1813 (11), pp Kops, G., Dansen, T., Polderman, P., Saarloos, I., Wirtz, K., Coffer, P., Huang, T., Bos, J., Medema, R. and Burgering, B Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature, 419 (6904), pp Latchman, D Transcription factors: an overview. Int. J. Biochem. Cell Biol., 29 (12), pp Obsil, T. and Obsilova, V Structure/function relationships underlying regulation of FOXO transcription factors. Oncogene, 27 (16), pp Obsil, T. and Obsilova, V Structural basis for DNA recognition by FOXO proteins. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1813 (11), pp Roeder, R Role of general initiation facotrs in transcription by RNA polymerase II. Trends Biochem. Sci., 21 (9), pp Schuur, E., Loktev, A., Sharma, M., Sun, Z., Roth, R. and Weigel, R Liganddependent Interaction of Estrogen Receptor- (alpha) with Members of the Forkhead Transcription Factor Family. The Journal of Biological Chemistry, 276 (36), pp

11 11 Van Der HEIDE, L., Hoekman, M. and Smidt, M The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J, 380 pp

12 12 Figure Legend Figure 1. Primary sequence alignment of FOXO subfamily of proteins. Highlighted in red is the protein kinase B (PKB) phosphorylation site. Highlighted in yellow is the DNABinding domain. The nuclear localization signal motif is located under the red box. The nuclear export signal is highlighted in magenta. Finally, the C-terminal translocation domain is highlighted in gray. Sequence alignment was achieved using CLUSTAL O from expasy.org. Exact matches are marked * and shown in blue text. Matches with strong similarity are marked : and shown in red text. Matches with weak similarity are marked. and shown in green text. Figure 2. Tertiary structure of FOXO subfamily of proteins. All images were rendered using PyMOL. Structure A in purple illustrates FOXO1 (3CO6), Structure B in green illustrates FOXO3 (2UZK), Structure C in blue illustrates FOXO4 (3L2C). All molecular graphics were created using PyMOL ( Figure 3. Superimposition of the FOXO subfamily and position of DNA binding domain (DBD). FOXO1 is shown in purple, FOXO3 is shown in green and FOXO4 is shown in blue. It can be seen in this illustration that the DBD is helical and binds in the major groove of the DNA. Figure 4. Key interactions between the forkhead domain and DNA. Illustrated in this figure are the hydrogen bonding interactions that are undergone by the binding of FOXO1 to DNA sequence (5 -GTAAACAA-3 ). FOXO1 protein is shown in purple.

13 13 Figure 1.

14 14 Figure 2. A. C. B.

15 15 Figure 3.

16 16 Figure 4.