Forkhead box (Fox) proteins are a superfamily of evolutionarily conserved transcriptional regulators, which control a wide spectrum of biological processes. As a consequence, a loss or gain of Fox function can alter cell fate and promote tumorigenesis as well as cancer progression. Here we discuss the evidence that the deregulation of Fox family transcription factors has a crucial role in the development and progression of cancer, and evaluate the emerging role of Fox proteins as direct and indirect targets for therapeutic intervention, as well as biomarkers for predicting and monitoring treatment responses.

SIRT proteins play an important role in the survival and drug resistance of tumor cells, especially during chemotherapy. In this study, we investigated the potency, specificity, and cellular targets of three SIRT inhibitors, Sirtinol, Salermide, and EX527. Cell proliferative and cell cycle analyses showed that Sirtinol and Salermide, but not EX527, were effective in inducing cell death at concentrations of 50 μmol/L or over in MCF-7 cells. Instead, EX527 caused cell cycle arrest at G 1 at comparable concentrations. In vitro SIRT assays using a p53 peptide substrate showed that all three compounds are potent SIRT1/2 inhibitors, with EX527 having the highest inhibitory activity for SIRT1. Computational docking analysis showed that Sirtinol and Salermide have high degrees of selectivity for SIRT1/2, whereas EX527 has high specificity for SIRT1 but not SIRT2. Consistently, Sirtinol and Salermide, but not EX527, treatment resulted in the in vivo acetylation of the SIRT1/2 target p53 and SIRT2 target tubulin in MCF-7 cells, suggesting that EX527 is ineffective in inhibiting SIRT2 and that p53 mediates the cytotoxic function of Sirtinol and Salermide. Studies using breast carcinoma cell lines and p53-deficient mouse fibroblasts confirmed that p53 is essential for the Sirtinol and Salermideinduced apoptosis. Further, we showed using small interfering RNA that silencing both SIRTs, but not SIRT1 and SIRT2 individually, can induce cell death in MCF-7 cells. Together, our results identify the specificity and cellular targets of these novel inhibitors and suggest that SIRT inhibitors require combined targeting of both SIRT1 and SIRT2 to induce p53 acetylation and cell death. Mol Cancer Ther; 9(4); 844-55. ©2010 AACR.

Endometrial cancer is the most common malignancy of the lower female reproductive tract. The tumor suppressor FOXO1 is downregulated in endometrial cancer compared with normal endometrium but the underlying mechanisms are not well understood. Using microRNA (miR) target prediction algorithms, we identified several miRs that potentially bind the 3′-untranslated region of FOXO1 transcripts. Expression profiling of normal and malignant endometrial samples by quantitative real-time PCR and Northern blot analysis revealed an inverse correlation between the levels of FOXO1 protein and the abundance of several of the in silico-predicted miRs, suggesting that loss of FOXO1 expression in endometrial cancer may be mediated by miRs. To determine the role of candidate miRs, we used the endometrial cancer cell lines HEC-1B and Ishikawa, which express FOXO1 at high and low levels, respectively. Expression of miR-9, miR-27, miR-96, miR-153, miR-182, miR-183, or miR-186, but not miR-29a, miR-128, miR-152, or miR-486 mimetics in HEC-1B cells was sufficient to significantly reduce the abundance of FOXO1. Conversely, FOXO1 expression was efficiently restored in the Ishikawa cell line upon simultaneous inhibition of miR-9, miR-27, miR-96, miR-153, miR-183, and miR-186. Moreover, induction of FOXO1 in Ishikawa cells by miR inhibitors was accompanied by G 1 cell cycle arrest and cell death, and was attenuated by the small interfering RNA-mediated downregulation of FOXO1 expression. Our findings identify several miRs overexpressed in endometrial cancer that function in concert to repress FOXO1 expression. Further, aberrant miR expression results in deregulated cell cycle control and impaired apoptotic responses, and thus, may be central to endometrial tumorigenesis.

Elevated expression or activity of the transcription factor forkhead box M1 (FOXM1) is associated with the development and progression of many malignancies, including breast cancer. In this study, we show that the thiazole antibiotic thiostrepton selectively induces cell cycle arrest and cell death in breast cancer cells through down-regulating FOXM1 expression. Crucially, our data show that thiostrepton treatment reduced FOXM1 expression in a time-and dose-dependent manner, independent of de novo protein synthesis and predominantly at transcriptional and gene promoter levels. Our results indicate that thiostrepton can induce cell death through caspase-dependent intrinsic and extrinsic apoptotic pathways as well as through caspase-independent death mechanisms, as observed in MCF-7 cells, which are deficient of caspase-3 and caspase-7. Cell cycle analysis showed that thiostrepton induced cell cycle arrest at G 1 and S phases and cell death, concomitant with FOXM1 repression in breast cancer cells. Furthermore, thiostrepton also shows efficacy in repressing breast cancer cell migration, metastasis, and transformation, which are all downstream functional attributes of FOXM1. We also show that overexpression of a constitutively active FOXM1 mutant, #N-FOXM1, can abrogate the antiproliferative effects of thiostrepton. Interestingly, thiostrepton has no affect on FOXM1 expression and proliferation of the untransformed MCF-10A breast epithelial cells. Collectively, our data show that FOXM1 is one of the primary cellular targets of thiostrepton in breast cancer cells and that thiostrepton may represent a novel lead compound for targeted therapy of breast cancer with minimal toxicity against noncancer cells.

The transcription factor Forkhead box M1 (FOXM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. To address the role of FOXM1 in chemoresistance, we generated a cisplatin-resistant breast cancer cell line (MCF-7-CIS R ), which had an elevated level of FOXM1 protein and mRNA expression relative to the parental MCF-7 cells. A close correlation was observed between FOXM1 and the expression of its proposed downstream targets that are involved in DNA repair; breast cancer-associated gene 2 (BRCA2) and X-ray cross-complementing group 1 (XRCC1) were expressed at higher levels in the resistant cell lines compared with the sensitive MCF-7 cells. Moreover, cisplatin treatment induced DNA damage repair in MCF-7-CIS R and not in MCF-7 cells. Furthermore, the expression of a constitutively active FOXM1 (ΔN-FOXM1) in MCF-7 cells alone was sufficient to confer cisplatin resistance. Crucially, the impairment of DNA damage repair pathways through the small interfering RNA knockdown inhibition of either FOXM1 or BRCA2/XRCC1 showed that only the silencing of FOXM1 could significantly reduce the rate of proliferation in response to cisplatin treatment in the resistant cells. This suggests that the targeting of FOXM1 is a viable strategy in circumventing acquired cisplatin resistance. Consistently, the FOXM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FOXM1 expression. Taken together, we have identified a novel mechanism of acquired cisplatin resistance in breast cancer cells through the induction of FOXM1. Mol Cancer Res; 8(1); 24-34. ©2010 AACR.

FoxO transcription factors are an evolutionary conserved subfamily of the forkhead transcription factors, characterized by the forkhead DNA-binding domain. FoxO factors regulate a number of cellular processes involved in cell-fate decisions in a cell-type-and environment-specific manner, including metabolism, differentiation, apoptosis and proliferation. A key mechanism by which FoxO determines cell fate is through regulation of the cell cycle machinery, and as such the cellular consequence of FoxO deregulation is often manifested through perturbation of the cell cycle. Consequently, the deregulation of FoxO factors is implicated in the development of numerous proliferative diseases, in particular cancer.

Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression. [Mol Cancer Ther 2008;7(3):670 -8]

Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, #N-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.