Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

NUDT22 promotes cancer growth through pyrimidine salvage.

The NUDIX hydrolase NUDT22 converts UDP-glucose into glucose-1-phosphate and the pyrimidine nucleotide uridine monophosphate but a biological significance for this biochemical reaction has not yet been established. Glucose-1-phosphate is an important metabolite for energy and biomass production through glycolysis and nucleotides required for DNA replication are produced through energetically expensive de novo or energy-efficient salvage pathways. Here, we describe p53-regulated pyrimidine salvage through NUDT22-dependent hydrolysis of UDP-glucose to maintain cancer cell growth and to prevent replication stress. NUDT22 expression is consistently elevated in cancer tissues and high NUDT22 expression correlates with worse survival outcomes in patients indicating an increased dependency of cancer cells to NUDT22. Furthermore, we show that NUDT22 transcription is induced after inhibition of glycolysis, MYC-mediated oncogenic stress, and DNA damage directly through p53. NUDT22-deficient cancer cells suffer from growth retardation, S-phase delay, and slower DNA replication fork speed. Uridine supplementation rescues replication fork progression and alleviates replication stress and DNA damage. Conversely, NUDT22 deficiency sensitizes cells to de novo pyrimidine synthesis inhibition in vitro and reduces cancer growth in vivo. In conclusion, NUDT22 maintains pyrimidine supply in cancer cells and depletion of NUDT22 leads to genome instability. Targeting NUDT22 therefore has high potential for therapeutic applications in cancer therapy.

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer.

Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

Endogenous Production of IL1B by Breast Cancer Cells Drives Metastasis and Colonization of the Bone Microenvironment.

PURPOSE: Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone. EXPERIMENTAL DESIGN: Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell-derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control. RESULTS: In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05-3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26-3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation. CONCLUSIONS: Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells.

Human parturition is associated with many pro-inflammatory mediators which are regulated by the nuclear factor-kappaB (NF-κB) family of transcription factors. In the present study, we employed a ChIP-on-chip approach to define genomic loci within chromatin of PHM1-31 myometrial cells that were occupied by RelA-containing NF-κB dimers in response to a TNF stimulation of 1 h. In TNF-stimulated PHM1-31 cells, anti-RelA serum enriched 13 300 chromatin regions; importantly, 11 110 regions were also enriched by anti-RelA antibodies in the absence of TNF. DNA sequences in these regions, from both unstimulated or TNF-stimulated PHM1-31 cultures, were associated with genic regions including IκBα, COX-2, IL6RN, Jun and KCNMB3. TNF-induced binding events at a consensus κB site numbered 1667; these were represented by 112 different instances of the consensus κB motif. Of the 1667 consensus κB motif occurrences, 770 (46.2%) were identified within intronic regions. In unstimulated PHM1-31 cells, anti-RelA-serum-enriched regions were associated with sequences corresponding to open reading frames of ion channel subunit genes including CACNB3 and KCNB1. Moreover, in unstimulated cells, the consensus κB site was identified 2116 times, being defined by 103 different sequence instances of this motif. Of these 2116 consensus κB motifs, 1089 (51.5%) were identified within intronic regions. Parallel expression array analyses in PHM1-31 cultures demonstrated that TNF stimulated a >2-fold induction in 51 genes and a fold repression of >1.5 in 18 others. We identified 14 anti-RelA-serum-enriched genomic regions that correlated with 17 TNF-inducible genes, such as COX2, Egr-1, Jun, IκBα and IL6, as well as five regions associated with TNF-mediated gene repression, including Col1A2.

A high-throughput assay to identify modifiers of premature chromosome condensation.

Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.

Regulation of GTP-binding protein (Gαs) expression in human myometrial cells: a role for tumor necrosis factor in modulating Gαs promoter acetylation by transcriptional complexes.

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.

Circulating microRNA profiles reflect the presence of breast tumours but not the profiles of microRNAs within the tumours.

BACKGROUND: Extra-cellular microRNAs have been identified within blood and their profiles reflect various pathologies; therefore they have potential as disease biomarkers. Our aim was to investigate how circulating microRNA profiles change during cancer treatment. Our hypothesis was that tumour-related profiles are lost after tumour resection and therefore that comparison of profiles before and after surgery would allow identification of biomarker microRNAs. We aimed to examine whether these microRNAs were directly derived from tumours, and whether longitudinal expression monitoring could provide recurrence diagnoses. METHODS: Plasma was obtained from ten breast cancer patients before and at two time-points after resection. Tumour tissue was also obtained. Quantitative PCR were used to determine levels of 367 miRNAs. Relative expressions were determined after normalisation to miR-16, as is typical in the field, or to the mean microRNA level. RESULTS: 210 microRNAs were detected in at least one plasma sample. Using miR-16 normalisation, we found few consistent changes in circulating microRNAs after resection, and statistical analyses indicated that this normalisation was not justifiable. However, using data normalised to mean microRNA expression we found a significant bias for levels of individual circulating microRNAs to be reduced after resection. Potential biomarker microRNAs were identified, including let-7b, let-7g and miR-18b, with higher levels associated with tumours. These microRNAs were over-represented within the more highly expressed microRNAs in matched tumours, suggesting that circulating populations are tumour-derived in part. Longitudinal monitoring did not allow early recurrence detection. CONCLUSIONS: We concluded that specific circulating microRNAs may act as breast cancer biomarkers but methodological issues are critical.

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells.

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.

Epithelial-mesenchymal interactions in breast cancer: evidence for a role of nuclear localized ß-catenin in carcinoma-associated fibroblasts.

AIMS: Characteristics of the stroma around tumours are critical in defining the behaviour of cancers. ß-Catenin is well established as a critical regulator of carcinogenesis, acting as a transcriptional co-activator in the nuclei of epithelial cancer cells. We have examined the prevalence and influence of nuclear ß-catenin within the stromal fibroblasts of breast cancer. METHODS AND RESULTS: We examined ß-catenin expression in 201 breast cancers and adjacent normal tissue. Fibroblasts expressing nuclear ß-catenin were present in a significantly greater proportion of tumour tissues than normal tissues. The presence of fibroblasts with nuclear ß-catenin in tumours correlated with survival; tumours with prevalent positive fibroblasts were associated significantly with relatively good prognoses. Functional studies to examine influences of fibroblasts with nuclear ß-catenin, showed fibroblasts transfected to allow overexpression of ß-catenin were capable of inducing increases in both proliferation and invasion of breast cancer cell lines. CONCLUSION: The presence of fibroblasts with nuclear ß-catenin in tumours is a good prognostic indicator, although in the context of tissue culture models these cells can increase the growth and metastatic potential of cancer cells. These apparently paradoxical observations underline the complexity of epithelial-stromal signalling within tumours and highlight an area for further study.

Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer.

BACKGROUND: Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours. RESULTS: We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition. CONCLUSIONS: Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.

NF-kappaB function in the human myometrium during pregnancy and parturition.

Interactions between the nuclear factor kappaB (NF-kappaB) family of proteins (RelA, RelB, c-Rel, p50 and p52) and DNA are vital for cells to function normally; for example, in the human myometrium, NF-kappaB-regulated pro-inflammatory mediators, including TNFalpha, IL-1beta, IL-8 and COX-2 are associated with the onset of labour. NF-kappaB, however, regulates the expression of over 400 genes, although it is unlikely these would all be activated in concert by a single inducer. At present, defining the role of the NF-kappaB RelA:p50 dimer, which governs a number of inflammatory promoters, is at the forefront of the parturition research field. However, to over-look the function of other family members and how they may regulate alternative signalling networks within reproductive tissues, only serves to ensure we will never fully understand the molecular circuitry influenced by this family of transcription factors. Consequently this review highlights other mechanisms by which the NF-kappaB family of regulators have been shown to function in other systems and how they may readily translate to understanding the regulation underpinning human parturition.