Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that begins in the epithelial cells that line the mucosal surfaces of the head and neck, including the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity, and salivary glands. Head and neck cancer is the sixth most common type of cancer with a 5-year survival rate of 60% for all cases. Over the past few years, a subset of cells with stem-like properties, called cancer stem cells, are believed to have tumor-initiation capabilities and are responsible for maintaining on-going tumor growth. Previous data from our lab suggested that cells grown in suspension, called spheroids, may have stem cell like properties. We employed a model system where a primary HNSCC cell line, HN4, was used to set up spheroids. We found that expression of EPS8 and its downstream targets, FOXM1 and CXCL5, was increased in HN4 spheroids. In addition, we measured the expression of Nanog, as it is a transcription factor involved in the self-renewal of human embryonic stem cells. We also used a metastatic HNSCC cell line, HN12, to see how it compared to spheroids. We wanted to investigate the hypothesis that activation of Src potentiates EPS8 function to deregulate downstream signaling pathways. We used a small molecule tyrosine kinase inhibitor, Dasatinib, on HN4 spheroids and HN12 cells. We found that when Src is inhibited, EPS8 expression is decreased in HN4 spheroids and it also interferes with spheroid formation. The results of the current study were also able to show that the proliferation capability of HN12 cells is greatly diminished when treated with Dasatinib, due to G1 arrest in the cell cycle. When we measured for FOXM1, which is a cell cycle regulator, we found the levels were reduced in Dasatinib treated cells, preventing the cells from completing mitosis. With all of the data taken together, it suggests that Src does in fact play a role in regulating the downstream signaling pathways of EPS8, and its inhibition leads to the loss of cell proliferation. Additional studies need to be performed to discover whether Src inhibition will stop the proliferation of cancer stem cells, which are believed to be more resistant to cytotoxic therapies.

The p300 transcriptional coactivator has been implicated in the development of a large number of malignancies; however, the precise mechanism of p300-associated tumorigenesis remains unclear. Here, we demonstrate the functional impact of p300 in human melanomas using both genetic and chemical approach. Depletion of p300 in human melanoma cells was associated with cellular growth arrest and senescence. Microarray analysis identified the Microphthalmia-associated transcription factor (MITF), a critical lineage-specific transcription factor in melanocytes and melanomas, as a major downstream target of p300 in human melanoma. Ectopic expression of MITF in p300-depleted melanoma cells allowed rescue of the p300-silencing phenotype, suggesting a critical regulatory axis involving p300 and MITF. Chromatin immunoprecipitation studies revealed direct regulation of MITF transcription through p300 acetylation of proximal regulatory domains. Critically, we identified that Forkhead Box M1 (FOXM1), a potent pro-proliferation transcription factor, is a target of the p300-MITF signaling axis. Further evaluation of p300 regulation of melanoma cell growth was performed using a highly selective p300/CBP HAT inhibitor, 228-1. Inhibition of p300/CBP histone acetyltransferase (HAT) activity was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion. Together, these data support the role of p300 as a promising therapeutic target in human melanoma and suggest particular therapeutic efficacy of small molecule inhibitors of p300 HAT activity in tumors expressing high levels of MITF. / 2018-12-14T00:00:00Z

The healthy development of a living cell requires precise spatial-temporal gene expression. The code that dictates when and where genes are expressed is stored in a pattern of specific sequence motifs, which can be recognised by transcription factors. Understanding the interaction between these DNA sequence motifs and transcription factors will help to elucidate how genomic sequences build transcriptional control networks. However, the DNA-binding specificities of ~1400 human transcription factors are largely unknown. The in vivo DNA-binding events of transcription factors involve great subtlety, because most transcription factors recognise degenerate sequence motifs and related transcription factors often prefer similar or even identical sequences. Forkhead transcription factors exemplify these challenges. To understand how members within the Forkhead transcription factor family gain their binding and functional specificities, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) to interrogate the genome-wide chromatin binding events of three Forkhead transcription factors: FOXM1, FOXO3 and FOXK2. We find that FOXM1 specifically binds to the promoters of a large array of genes whose activities peak at the G2 and M phases of the cell cycle. The canonical Forkhead consensus GTAAACA is not enriched within the FOXM1 cistrome. It gains its own specific binding events and biological functions by interacting and cooperating with the MMB complex. FOXO3 and FOXK2 are recruited to chromatin by the canonical Forkhead consensus GTAAACA, and they bind both shared and specific regions in the genome. FOXO3 mostly binds to the regions which are also bound by FOXK2, but no competitive or assisted binding between FOXO3 and FOXK2 is detected within those regions. Overall, these results help explain how individual members of the Forkhead transcription factor family gain binding specificity within the genome yet raises new questions of how functional specificity is achieved by other family members.

Background: Oesophagogastric cancers generally present late in life with advanced disease and carry a poor prognosis. Few patients receive curative treatment. Polo-like Kinase 1 (PLK1) is a mitotic kinase with regulatory functions at the G2/M cell cycle phase transition. In mammalian cells, PLK1 phosphorylates and activates FOXM1, a forkhead transcription factor at the G2/M cell cycle phase transition. FOXM1 then promotes transcription of multiple gene products, including PLK1 and CCNB1, which then act individually or in complexes to further phosphorylate FOXM1 generating a positive feedback loop driving the cell into M phase. Aims: We aimed to assess the expression of PLK1 and FOXM1 in oesophageal and gastric cancer patients. Secondly we aimed to investigate the expression and inter- relationship of PLK1 and FOXM1 in oesophageal cell lines during the cell cycle. Results: FOXM1 and PLK1 are expressed in oesophageal cell lines and demonstrate cross-regulatory interactions. Inhibition of PLK1 leads to the decreased expression of FOXM1 and it’s target gene in oesophageal cell lines. FOXM1 and PLK1 are also concomitantly overexpressed in a large proportion of oesophageal and gastric carcinoma’s at both the protein and mRNA level. Other FOXM1 target genes including, CCBN1, AURKB and CKS1 are co-expressed in a similar manner. In a homogenous cohort of patients who underwent surgery, the expression of PLK1 and AURKB was prognostic for overall survival. Conclusions: This study has demonstrated that FOXM1 and a number of target genes including PLK1 are coordinately expressed in a proportion of oesophageal and gastric carcinomas. This suggests that chemotherapeutic treatments that target this pathway may be of clinical utility.

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Radiation is a common mode of cancer therapy that is well known to generate reactive oxygen species leading to cell damage and death. However, there are many limitations to radiation therapy including normal tissue toxicity and the presence of quiescent cancer cells that are radio-resistant. There are many factors that regulate normal and cancer cell radiation response including the cellular redox environment which includes a complex network of antioxidants. In this study, two specific objectives will be explored: (A) SEPP1 regulation of normal cell toxicity; and (B) FoxM1 regulation of quiescence-associated radiation resistance in human oral squamous carcinoma cells. Results from DHE-oxidation analysis show that in irradiated proliferating normal cells there is a late ROS accumulation that occurs independent of cell cycle checkpoint activation and precedes cell death. Additionally, Q-RT-PCR and immunoblot analysis show an increase in Selenoprotein P (SEPP1) expression following radiation. SEPP1 is an extracellular glycoprotein with proposed selenium transport and antioxidant functions. However, pretreatment of normal cells with sodium selenite or overexpression of sepp1 is able to mitigate radiation-induced normal cell toxicity. It is well-accepted that quiescent populations exist in most solid tumors and are often the reason for tumor recurrence. In this study, we see that quiescent head and neck cancer cells that are resistant to radiation have low basal expression of Forkhead box M1 (FoxM1) compared to proliferating cancer cells. FoxM1 is a transcription factor that has recently been implicated in the cellular response to oxidative stress. Results indicate that although basal expression is low in quiescent cells, following irradiation FoxM1 is increased in quiescent cancer cells but not in proliferating cancer cells. Additionally, pharmacological and genetic knockdown of FoxM1 led to sensitization of quiescent cancer cells indicating that FoxM1 inhibitors could be useful radiation sensitizers. Together, these objectives will help to identify possible treatment options to use in addition to radiation therapy to better target quiescence-associated resistant tumors and induce less normal cell toxicity.

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