FOXM1 Inhibition by Thiazole Antibiotic Thiostrepton as a Novel Cancer Therapeutic Agent

Introduction

Forkhead Box 1 (FOXM1) is an oncogenic transcription factor possessing a winged-helix DNA-binding domain whose inhibition has potential as a novel cancer therapeutic agent. It is directly involved in cell-cycle progression and stress-induced apoptosis but has also been associated with DNA damage repair and cellular transportation^[1]. Thiazole antibiotics such as thiostrepton have been identified as inhibitors of FOXM1. This is a crucial discovery, as it has been observed that malignant tumors often exhibit overexpression of FOXM1 since it allows for the transcription of needed genes for cell cycle completion (Fig. 1). Research is being done to assess the use of thiazole antibiotics in targeted therapy for different cancers by inducing apoptotic pathways with the ultimate goal of reducing cancer cell proliferation.

Overview of Forkhead Transcription Factors

Forkhead transcription factors are composed of 17 subfamilies with functions varying from cell cycle regulation to insulin action targets. This transcription factor is upregulated in cells with high proliferation such as epithelial cells, but high levels of it can also be stimulated in less rapidly produced cells by tissue damage^[2]. One of the subgroups referred to as FOXO was initially studied because of its apparent connection to tumor development. They directly lead to the formation of a hyperactive transcriptional fusion protein which can ultimately contribute to uncontrolled cell proliferation when mutations appear in FOXO^[3]. For example, 13q14 chromosomal mutation leading to the absence of FOXO1A was commonly seen as a preceding marker to prostate cancer^[4]. Mutations in other FOX genes such as FOXC1 and FOXF1 have been shown to cause a wide range of congenital disorders from cleft palate abnormalities to Alveolar Capillary Dysplasia.

In addition to the characteristic winged-helix helix domain (FKH), the FOXM1 protein itself has two more general domains: the N-terminal repressor domain (NRD) and the transactivation domain (TAD). These two regions are found on either side of FKH, and the NRD has the ability to regulate the function of the transactivation domain^[5].

FOXM1 in Cell Cycle and DNA Damage Repair

FOXM1 is no exception to the previously outlined diverse nature of forkhead transcription factors. Recent studies of FOXM1 revealed the gene’s role in cell replication (proliferation), maintaining internal stability (homeostasis), and DNA damage repair. Although light has been shed on the multifunctionality of it, FOXM1 was initially studied because of its activity in the cell cycle as a transcription factor. Post-translational modifications (PTMs) of FOXM1 such as phosphorylation, ubiquitination, and methylation play a critical role in its transcriptional activity during cellular replication^[6].

As interphase progresses, FOXM1 is increasingly phosphorylated. The transactivation domain (TAD) is initially suppressed by the NRD, so the transcription rate in the G1 phase is low (Fig. 1). For this reason, target genes are not expressed before the G2 phase. Prior to this, the transcription level is very low despite FOXM1 expression. To enter the S phase, FOXM1 is positively regulated via phosphorylation by Cdk-cyclin complexes 4/6 on roughly 6 sites on the C-terminus such as T611, T627, and S704 (Fig.1)⁷. During the S phase, the transcriptional activity of FOXM1 increases via hypo-phosphorylation by the Raf/MEK/MAPK pathway. At the end of this phase, cyclin A/cdk must phosphorylate site T611 on FOXM1 to stop the NRD from suppressing TAD, allowing transcription. MicroRNAs (miRNAs) in the M phase attach to FOXM1 mRNA at 3’UTRs and have been shown to increase drug sensitivity of cervical cancer, introducing the idea that abnormal FOXM1 expression in cancer tissues can be be targeted for downregulation by miR-214^[8].

Figure 1. FOXM1 regulation in cell cycle^[6] Figure 2. FOXM1 DNA damage response^[5]

Within the cell cycle, DNA damage checkpoints serve a crucial role in preventing the uncontrolled proliferation of damaged genetic information. FOXM1 regulates the transcription of many crucial proteins in this process. For example, double-strand DNA breaks are identified by the FOXM1-regulated MRN complex that activates ATM and later effectors such as BRCA 1 and p53 (Fig. 2). This supports the notion that FOXM1’s function is not limited to cell cycle progression, as it is also an integral role in DNA damage response (DDR). It is responsible for the transcription of genes with information for DNA damage recognition, excision, mediators, and effectors. DNA breaks have been demonstrated to be much more prevalent in mice lacking FOXM1, supporting the connection between these two elements^[9].

In eukaryotes, there are various DDR pathways, including nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end-joining (NHEJ). NER depends on excising incorrect base lesions as a single-stranded DNA (ssDNA) to be repaired, and the resulting gap is filled by DNA pol δ or ε, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). FOXM1 indirectly regulates this process since it allows for the expression of the gene that encodes DNA pol ε and PolE^[5]. Long-patch BER involves FOXM1-regulated X-ray cross-complementing group 1 (XRCC1) and is driven to completion with DNA ligase III. Unlike in NER, HR relies on the homologous chromosome to guide repairs, ultimately resulting in less mistakes. The MRN complex, composed of subunits MRE11, RAD50, and NBS1, is responsible for the first step in this pathway, identifying the DNA damage (Fig. 2). In the NHEJ process, FOXM1 causes the transcription of Csk1 and Skp2 before they are phosphorylated and ubiquitinated and passed to NBS1^[10]. DNA mismatch repair (MMR) relies on direct exonuclease 1 (Exo1) to excise the mispaired bases. The transcriptional activity of Exo1is regulated by FOXM1and has been linked to drug sensitivity in breast cancer^[11]. FOXM1’s influence at various steps of DDR has been supported by the fact that it regulates proteins involved in DNA error identification, excision, synthesis, and ligation.

Thiostrepton as FOXM1 Inhibitor

Thiazole antibiotics such as siomycin A and thiostrepton have been shown to inhibit FOXM1 through 23S rRNA. They are recognized by a main pyridine, tetrapyridine, or dihydropyridine ring surrounded by thiazole substituents^[12]. Thiazole antibiotic thiostrepton binds to the previously mentioned 23S rRNA at 1067, which is referred to as the L11 binding domain (L11BD).

Thiostrepton and siomycin A, another studied thiazole antibiotic, suppress FOXM1 by binding within this general FKH domain so that it is not able to regulate transcriptional activity of genes¹³. These two possible anticancer agents act very similarly; their only structural difference is two residues at R1-R2 ( thiostrepton contains isoleucine-alanine, while siomycin A has valine-dehydroalanine). When breast cancer cells are treated with thiostrepton there is a drastic decrease in FOXM1 expression after four hours. However, regulators of FOXM1 as p14^ARF, ERK1/2, and FOXO3a are not affected by the introduction of thiostrepton. The idea that FOXM1 is directly targeted by thiostrepton is also supported by the fact that FOXM1-regulated Polo-kinase-1 and Cyclin D1 also decreased^[14]. Although the expression of FOXM1 mRNA is suppressed, it appears as though thiostrepton mainly targets FOXM1 at gene promoter level.

The down-regulation of FOXM1 expression by thiostrepton suppresses the migration of cancer cells and targets gene expression^[14]. There is specific interest in using thiostrepton as a chemotherapy strategy since it reduces the proliferation of cancer cells but does not affect non transformed cells. Likewise, the inhibition of FOXM1 has been shown to increase cancer drug sensitivity. By repressing the expression of FOXM1, thiostrepton prevents cells from entering the S and M phases in the cell cycle. Intrinsic apoptosis is common among cells with upregulated FOXM1, for this is demonstrated by an increase in caspase-3 cleavage among thiostrepton-treated cell lines^[15].

It has been concluded that thiostrepton specifically targets FOXM1, as levels of other Forkead transcription factors, including FOXA1 and FOXO1, are not altered. Previously, thiostrepton was believed to have not directly interacted with FOXM1, and instead believed to have repressed the proteasome degradation pathway^[16]. However, more recent studies have shown it can also directly bind to a FOXM1 monomer at the DNA-binding domain (DBD) at three locations.

The hydrogen bond stability which thiostrepton causes can be linked to a decrease in FOXM1 expression^[17]. This is valuable information, which sheds light on the process by which thiostrepton causes transcriptional inhibition. The specificity of thiostrepton to FOXM1 and their exact interaction is still a subject of great interest in research being conducted. With a deeper understanding of that relationship, FOXM1 inhibitory drugs can be structurally modified to produce the best results.

Expectations for Future Studies

Studies are currently being conducted to evaluate the use of thiostrepton as an anticancer agent with the ability to inhibit the FOXM1 transcription factor which has been shown to play a crucial role in the cell cycle, migration, and DNA damage repair. The downregulation of FOXM1 should theoretically put a halt to the uncontrollable proliferation of cells, a hallmark characteristic of cancer, by apoptosis. The use of thiostrepton as an inhibitor of tumorigenesis in vivo has also been explored with promising results. There is much ongoing research in this field, with the ultimate goal of designing an anticancer compound with the ability to enhance or replace current chemotherapy treatments. Contributions from studies focusing on thiostrepton’s impact on different types of malignant cell lines serve as a foundation for trends future research can expect.

The results of thiostrepton inhibition of FOXM1 in a wide array of subtypes of breast cancer have been studied. Early on, scientists observed that human breast cancer MCF-7 cells reacted in an antiproliferative manner to thiostrepton^[14]. After 24 hours of being treated with doses ranging from 0 to 20μmol/L of thiostrepton, FOXM1 expression in MCF-7 cells dramatically decreased proportionally to the dose. A crucial observation in this specific study was that while thiostrepton does have antiproliferative effects on breast cancer cells, it does not affect the replication of nontransformed MCF-10A cells^[14]. This is a major concern in current chemotherapy options, and has prompted more interest in thiostrepton as an alternate anticancer agent with minimal adverse effects.

The effect of this thiazole antibiotic on triple-negative breast cancer cell lines correlated with previous results and serves as a promising foundation for future research^[18]. All cell lines tested demonstrated a drastic decrease in proliferation when treated with an average of 10 or more μM of thiostrepton. This type of cellular differentiation activity has proved to be a beneficial anticancer tactic. Overall, this specific study analyzed the decrease in cell viability among a variety of TNBC cell lines when treated with thiostrepton over extended periods of time. With this data, researchers are one step closer to finding a possible alternative treatment for breast cancer.

Earlier this year, research was published for the first time regarding thiostrepton’s impact on non-small-cell lung cancer (NSCLC) to evaluate the extent of its antiproliferative activity on cancer stem cells (CSC)^[19]. Cell viability was considerably reduced in A549, CL141, CL152, and H1299 cells in a thiostrepton dose-dependent trend (Fig. 3). The half maximal inhibitory concentration (IC₅₀) was calculated to be 0.05 μmol/L, alternatively much lower than that of the previously discussed breast cancer cells. Interestingly, it was observed that thistrepton’s antiproliferative activity proved most effective when this thiazole antibiotic was combined with gemcitabine, a common anticancer drug. The cells demonstrated significantly increased rates of apoptosis when treated with this combination of anticancer drugs, in comparison to their individual use. By reducing the expression of Sox2, β‐catenin, and EMT transcription factor Slug, these drugs have the potential to serve as metastasis suppressors on CSC^[19].

 

Figure 3. Thiostrepton decreases cell viability of NSCLC^[19]

The future of thiostrepton as a tumorigenesis suppressor likewise appears bright. Mice with H441 tumors have served to demonstrate this, treating them with either thiostrepton or a vehicle control 5 mg/kg/d 5 days a week. After five weeks, there was a significant difference in NSCLC tumor growth, as the tumors in mice treated with thiostrepton had only a 7-fold increase from their original size while the control group had a 16-fold increase in tumor growth. The data presented supported the activity of thiostrepton as an inhibitor of NSCLC tumorigenesis, for the vehicle control tumors were 2-fold larger than the average thiostrepton-treated tumor. This study was able to show how thiostrepton plays a prominent role in suppressing EMT by inducing the expression of E-cadherin^[19]. The research conducted provides the groundwork needed for future studies regarding the use of thiostrepton to target NSCLC in combination with previously successful chemotherapy treatments or individually.

Conclusion

Thiostrepton has emerged as a promising anticancer agent over the past few years for its direct inhibition of FOXM1. The FOXM1 transcription factor plays a crucial role in various cellular functions such as proliferation and DNA damage repair. Likewise, it is overexpressed in cancer cells and is thought to contribute to metastasis and drug resistance. The use of thiostrepton to inhibit FOXM1 has been shown to cause cell cycle arrest, caspase-induced apoptosis, and increased drug sensitivity. For these reasons, thiostrepton presents itself as a favorable anticancer agent with the ability to suppress various subtypes of cancer and enhance current chemotherapeutic agents in the future.

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About the Author

Camila Gutierrez is a rising senior at Paul VI Catholic High School in Virginia. She is specifically passionate about research in molecular biology, veterinary medicine, and genetics. In her free time, she enjoys volunteering at her local animal shelter, dancing, and watching foreign movies.