Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.

Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.

Methodological Advancements for Investigating Intra-tumoral Heterogeneity in Breast Cancer at the Bench and Bedside.

There is a major need to overcome therapeutic resistance and metastasis that eventually arises in many breast cancer patients. Therapy resistant and metastatic tumors are increasingly recognized to possess intra-tumoral heterogeneity (ITH), a diversity of cells within an individual tumor. First hypothesized in the 1970s, the possibility that this complex ITH may endow tumors with adaptability and evolvability to metastasize and evade therapies is now supported by multiple lines of evidence. Our understanding of ITH has been driven by recent methodological advances including next-generation sequencing, computational modeling, lineage tracing, single-cell technologies, and multiplexed in situ approaches. These have been applied across a range of specimens, including patient tumor biopsies, liquid biopsies, cultured cell lines, and mouse models. In this review, we discuss these approaches and how they have deepened our understanding of the mechanistic origins of ITH amongst tumor cells, including stem cell-like differentiation hierarchies and Darwinian evolution, and the functional role for ITH in breast cancer progression. While ITH presents a challenge for combating tumor evolution, in-depth analyses of ITH in clinical biopsies and laboratory models hold promise to elucidate therapeutic strategies that should ultimately improve outcomes for breast cancer patients.

Circadian clock regulation of the glycogen synthase (gsn) gene by WCC is critical for rhythmic glycogen metabolism in Neurospora crassa.

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.

Mathematical modeling and validation of glucose compensation of the neurospora circadian clock.

Autonomous circadian oscillations arise from transcriptional-translational feedback loops of core clock components. The period of a circadian oscillator is relatively insensitive to changes in nutrients (e.g., glucose), which is referred to as "nutrient compensation". Recently, a transcription repressor, CSP-1, was identified as a component of the circadian system in Neurospora crassa. The transcription of csp-1 is under the circadian regulation. Intriguingly, CSP-1 represses the circadian transcription factor, WC-1, forming a negative feedback loop that can influence the core oscillator. This feedback mechanism is suggested to maintain the circadian period in a wide range of glucose concentrations. In this report, we constructed a mathematical model of the Neurospora circadian clock incorporating the above WC-1/CSP-1 feedback loop, and investigated molecular mechanisms of glucose compensation. Our model shows that glucose compensation exists within a narrow range of parameter space where the activation rates of csp-1 and wc-1 are balanced with each other, and simulates loss of glucose compensation in csp-1 mutants. More importantly, we experimentally validated rhythmic oscillations of the wc-1 gene expression and loss of glucose compensation in the wc-1(ov) mutant as predicted in the model. Furthermore, our stochastic simulations demonstrate that the CSP-1-dependent negative feedback loop functions in glucose compensation, but does not enhance the overall robustness of oscillations against molecular noise. Our work highlights predictive modeling of circadian clock machinery and experimental validations employing Neurospora and brings a deeper understanding of molecular mechanisms of glucose compensation.

Efficient gene editing in Neurospora crassa with CRISPR technology.

BACKGROUND: Efficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, Neurospora crassa. FINDINGS: In this report, we demonstrate efficient gene replacement in a model filamentous fungus, Neurospora crassa, with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of clr-2 with the ß-tubulin promoter, and (2) introduce a codon optimized fire fly luciferase under the control of the gsy-1 promoter at the csr-1 locus. CLR-2 is one of the core transcription factors that regulate the expression of cellulases, and GSY-1 regulates the conversion of glucose into glycogen. We show that the ß-tubulin promoter driven clr-2 strain shows increased expression of cellulases, and gsy-1-luciferase reporter strain can be easily screened with a bioluminescence assay. CONCLUSION: CRISPR/Cas9 system works efficiently in Neurospora crassa, which may be adapted to Neurospora natural isolates and other filamentous fungi. It will be beneficial for the filamentous fungal research community to take advantage of CRISPR/Cas9 tool kits that enable genetic perturbations including gene replacement and insertions.

Circadian rhythms synchronize mitosis in Neurospora crassa.

The cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. Alterations in this network may lead to diseases such as cancer. Therefore, it is critical to identify molecular components that connect these two oscillators. However, molecular mechanisms between the clock and the cell cycle remain largely unknown. A model filamentous fungus, Neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhythms, but not used to investigate the molecular coupling between these two oscillators. In this report, we show that a conserved coupling between the circadian clock and the cell cycle exists via serine/threonine protein kinase-29 (STK-29), the Neurospora homolog of mammalian WEE1 kinase. Based on this finding, we established a mathematical model that predicts circadian oscillations of cell cycle components and circadian clock-dependent synchronized nuclear divisions. We experimentally demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, oscillate in a circadian manner with bioluminescence reporters. The oscillations of clb-1 and stk-29 gene expression are abolished in a circadian arrhythmic frq(ko) mutant. Additionally, we show the light-induced phase shifts of a core circadian component, frq, as well as the gene expression of the cell cycle components clb-1 and stk-29, which may alter the timing of divisions. We then used a histone hH1-GFP reporter to observe nuclear divisions over time, and show that a large number of nuclear divisions occur in the evening. Our findings demonstrate the circadian clock-dependent molecular dynamics of cell cycle components that result in synchronized nuclear divisions in Neurospora.