Improving radiotherapy in immunosuppressive microenvironments by targeting complement receptor C5aR1.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Dynamic and adaptive cancer stem cell population admixture in colorectal neoplasia.

Intestinal homeostasis is underpinned by LGR5+ve crypt-base columnar stem cells (CBCs), but following injury, dedifferentiation results in the emergence of LGR5-ve regenerative stem cell populations (RSCs), characterized by fetal transcriptional profiles. Neoplasia hijacks regenerative signaling, so we assessed the distribution of CBCs and RSCs in mouse and human intestinal tumors. Using combined molecular-morphological analysis, we demonstrate variable expression of stem cell markers across a range of lesions. The degree of CBC-RSC admixture was associated with both epithelial mutation and microenvironmental signaling disruption and could be mapped across disease molecular subtypes. The CBC-RSC equilibrium was adaptive, with a dynamic response to acute selective pressure, and adaptability was associated with chemoresistance. We propose a fitness landscape model where individual tumors have equilibrated stem cell population distributions along a CBC-RSC phenotypic axis. Cellular plasticity is represented by position shift along this axis and is influenced by cell-intrinsic, extrinsic, and therapeutic selective pressures.

Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.

MicroRNAs as biomarkers for prostate cancer prognosis: a systematic review and a systematic reanalysis of public data.

BACKGROUND: Reliable prognostic biomarkers to distinguish indolent from aggressive prostate cancer (PCa) are lacking. Many studies investigated microRNAs (miRs) as PCa prognostic biomarkers, often reporting inconsistent findings. We present a systematic review of these; also systematic reanalysis of public miR-profile datasets to identify tissue-derived miRs prognostic of biochemical recurrence (BCR) in patients undergoing radical prostatectomy. METHODS: Independent PubMed searches were performed for relevant articles from January 2007 to December 2019. For the review, 128 studies were included. Pooled-hazard-ratios (HRs) for miRs in multiple studies were calculated using a random-effects model (REM). For the reanalysis, five studies were included and Cox proportional-hazard models, testing miR association with BCR, performed for miRs profiled in all. RESULTS: Systematic review identified 120 miRs as prognostic. Five (let-7b-5p, miR-145-5p, miR152-3p, miR-195-5p, miR-224-5p) were consistently associated with progression in multiple cohorts/studies. In the reanalysis, ten (let-7a-5p, miR-148a-3p, miR-203a-3p, miR-26b-5p, miR30a-3p, miR-30c-5p, miR-30e-3p, miR-374a-5p, miR-425-3p, miR-582-5p) were significantly prognostic of BCR. Of these, miR-148a-3p (HR = 0.80/95% CI = 0.68-0.94) and miR-582-5p (HR = 0.73/95% CI = 0.61-0.87) were also reported in prior publication(s) in the review. CONCLUSIONS: Fifteen miRs were consistently associated with disease progression in multiple publications or datasets. Further research into their biological roles is warranted to support investigations into their performance as prognostic PCa biomarkers.

Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration.

BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.

Tracing Nutrient Flux Following Monocarboxylate Transporter-1 Inhibition with AZD3965.

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.

The 14q32 maternally imprinted locus is a major source of longitudinally stable circulating microRNAs as measured by small RNA sequencing.

Understanding the normal temporal variation of serum molecules is a critical factor for identifying useful candidate biomarkers for the diagnosis and prognosis of chronic disease. Using small RNA sequencing in a longitudinal study of 66 women with no history of cancer, we determined the distribution and dynamics (via intraclass correlation coefficients, ICCs) of the miRNA profile over 3 time points sampled across 2-5 years in the course of the screening trial, UKCTOCS. We were able to define a subset of longitudinally stable miRNAs (ICC >0.75) that were individually discriminating of women who had no cancer over the study period. These miRNAs were dominated by those originating from the C14MC cluster that is subject to maternal imprinting. This assessment was not significantly affected by common confounders such as age, BMI or time to centrifugation nor alternative methods to data normalisation. Our analysis provides important benchmark data supporting the development of miRNA biomarkers for the impact of life-course exposure as well as diagnosis and prognostication of chronic disease.

The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation.

VCP/p97 regulates numerous cellular functions by mediating protein degradation through its segregase activity. Its key role in governing protein homoeostasis has made VCP/p97 an appealing anticancer drug target. Here, we provide evidence that VCP/p97 acts as a regulator of cellular metabolism. We found that VCP/p97 was tied to multiple metabolic processes on the gene expression level in a diverse range of cancer cell lines and in patient-derived multiple myeloma cells. Cellular VCP/p97 dependency to maintain proteostasis was increased under conditions of glucose and glutamine limitation in a range of cancer cell lines from different tissues. Moreover, glutamine depletion led to increased VCP/p97 expression, whereas VCP/p97 inhibition perturbed metabolic processes and intracellular amino acid turnover. GCN2, an amino acid-sensing kinase, attenuated stress signalling and cell death triggered by VCP/p97 inhibition and nutrient shortages and modulated ERK activation, autophagy, and glycolytic metabolite turnover. Together, our data point to an interconnected role of VCP/p97 and GCN2 in maintaining cancer cell metabolic and protein homoeostasis.

Spinal Cord Metabolic Signatures in Models of Fast- and Slow-Progressing SOD1G93A Amyotrophic Lateral Sclerosis.

The rate of disease progression in amyotrophic lateral sclerosis (ALS) is highly variable, even between patients with the same genetic mutations. Metabolic alterations may affect disease course variability in ALS patients, but challenges in identifying the preclinical and early phases of the disease limit our understanding of molecular mechanisms underlying differences in the rate of disease progression. We examined effects of SOD1G93A on thoracic and lumbar spinal cord metabolites in two mouse ALS models with different rates of disease progression: the transgenic SOD1G93A-C57BL/6JOlaHsd (C57-G93A, slow progression) and transgenic SOD1G93A-129SvHsd (129S-G93A, fast progression) strains. Samples from three timepoints (presymptomatic, disease onset, and late stage disease) were analyzed using Gas Chromatography-Mass Spectrometry metabolomics. Tissue metabolome differences in the lumbar spinal cord were driven primarily by mouse genetic background, although larger responses were observed in metabolic trajectories after the onset of symptoms. The significantly affected lumbar spinal cord metabolites were involved in energy and lipid metabolism. In the thoracic spinal cord, metabolic differences related to genetic background, background-SOD1 genotype interactions, and longitudinal SOD1G93A effects. The largest responses in thoracic spinal cord metabolic trajectories related to SOD1G93A effects before onset of visible symptoms. More metabolites were significantly affected in the thoracic segment, which were involved in energy homeostasis, neurotransmitter synthesis and utilization, and the oxidative stress response. We find evidence that initial metabolic alterations in SOD1G93A mice confer disadvantages for maintaining neuronal viability under ALS-related stressors, with slow-progressing C57-G93A mice potentially having more favorable spinal cord bioenergetic profiles than 129S-G93A. These genetic background-associated metabolic differences together with the different early metabolic responses underscore the need to better characterize the impact of germline genetic variation on cellular responses to ALS gene mutations both before and after the onset of symptoms in order to understand their impact on disease development.

Altered Metabolic Profiles Associate with Toxicity in SOD1G93A Astrocyte-Neuron Co-Cultures.

Non-cell autonomous processes involving astrocytes have been shown to contribute to motor neuron degeneration in amyotrophic lateral sclerosis. Mutant superoxide dismutase 1 (SOD1G93A) expression in astrocytes is selectively toxic to motor neurons in co-culture, even when mutant protein is expressed only in astrocytes and not in neurons. To examine metabolic changes in astrocyte-spinal neuron co-cultures, we carried out metabolomic analysis by 1H NMR spectroscopy of media from astrocyte-spinal neuron co-cultures and astrocyte-only cultures. We observed increased glucose uptake with SOD1G93A expression in all co-cultures, but while co-cultures with only SOD1G93A neurons had lower extracellular lactate, those with only SOD1G93A astrocytes exhibited the reverse. Reduced branched-chain amino acid uptake and increased accumulation of 3-methyl-2-oxovalerate were observed in co-culture with only SOD1G93A neurons while glutamate was reduced in all co-cultures expressing SOD1G93A. The shifts in these coupled processes suggest a potential block in glutamate processing that may impact motor neuron survival. We also observed metabolic alterations which may relate to oxidative stress responses. Overall, the different metabolite changes observed with the two SOD1G93A cell types highlight the role of the astrocyte-motor neuron interaction in the resulting metabolic phenotype, requiring further examination of altered met abolic pathways and their impact on motor neuron survival.

Suppression of MTHFD2 in MCF-7 Breast Cancer Cells Increases Glycolysis, Dependency on Exogenous Glycine, and Sensitivity to Folate Depletion.

Methylenetetrahydrofolate dehydrogenase (NAD(P)+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) is a mitochondrial enzyme involved in folate metabolism. A number of recent studies have highlighted this enzyme as being highly expressed in many solid tumors, including breast cancer, and to be correlated with poor survival. However, the metabolic functions of MTHFD2 in cancer cells have not been well-defined. To investigate the function of MTHFD2 in breast cancer cells, we generated and characterized MCF-7 cells with stable suppression of MTHFD2 expression using a combination of cellular assays and metabolic profiling. Loss of MTHFD2 caused MCF7 cells to become glycine auxotrophs, that is, reliant on exogenous glycine, and more sensitive to exogenous folate depletion. Another prominent metabolic alteration observed as a consequence of MTHFD2 suppression was a more glycolytic phenotype, consistent with widespread modifications of cellular metabolism. Collectively, these data suggest that targeting MTHFD2 activity is likely to influence multiple metabolic pathways in breast cancer and could be combined with a range of antimetabolite therapies.

Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis.

Defects in energy metabolism are potential pathogenic mechanisms in amyotrophic lateral sclerosis (ALS), a rapidly fatal disease with no cure. The mechanisms through which this occurs remain elusive and their understanding may prove therapeutically useful. We used metabolomics and stable isotope tracers to examine metabolic changes in a well-characterized cell model of familial ALS, the motor neuronal NSC-34 line stably expressing human wild-type Cu/Zn superoxide dismutase (wtSOD1) or mutant G93A (G93ASOD1). Our findings indicate that wt and G93ASOD1 expression both enhanced glucose metabolism under serum deprivation. However, in wtSOD1 cells, this phenotype increased supply of amino acids for protein and glutathione synthesis, while in G93ASOD1 cells it was associated with death, aerobic glycolysis, and a broad dysregulation of amino acid homeostasis. Aerobic glycolysis was mainly due to induction of pyruvate dehydrogenase kinase 1. Our study thus provides novel insight into the role of deranged energy metabolism as a cause of poor adaptation to stress and a promoter of neural cell damage in the presence of mutant SOD1. Furthermore, the metabolic alterations we report may help explain why mitochondrial dysfunction and impairment of the endoplasmic reticulum stress response are frequently seen in ALS.