We describe two water‐soluble ruthenium complexes, 1Cl2 and 2Cl2, that photodissociate to release a cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with a low dose (21 J cm−2) of ...red light in an oxygen‐independent manner. Using a specific NAMPT activity assay, up to an 18‐fold increase in inhibition potency was measured upon red‐light activation of 2Cl2, while 1Cl2 was thermally unstable. For the first time, the dark and red‐light‐induced cytotoxicity of these photocaged compounds could be tested under hypoxia (1 % O2). In skin (A431) and lung (A549) cancer cells, a 3‐ to 4‐fold increase in cytotoxicity was found upon red‐light irradiation for 2Cl2, whether the cells were cultured and irradiated with 1 % or 21 % O2. These results demonstrate the potential of photoactivated chemotherapy for hypoxic cancer cells, in which classical photodynamic therapy, which relies on oxygen activation, is poorly efficient.
Seeing red: Under red‐light irradiation, a water‐soluble ruthenium complex 2Cl2 releases STF‐31, a cytotoxic inhibitor of the NAMPT enzyme, which leads to up to 18‐fold better NAMPT inhibition than in the dark. In cancer cells, this photorelease effect increases the cytotoxicity of 2Cl2 by a factor of 3–4, irrespective of whether the cells are cultured and irradiated under 1.0 % or 21 % O2. These results demonstrate the potential of of photoactivated chemotherapy for hypoxic cancer cells.
Marine alkaloid rigidins are cytotoxic compounds known to kill cancer cells at nanomolar concentrations by targeting the microtubule network. Here, a rigidin analogue containing a thioether group was ...“caged” by coordination of its thioether group to a photosensitive ruthenium complex. In the dark, the coordinated ruthenium fragment prevented the rigidin analogue from inhibiting tubulin polymerization and reduced its toxicity in 2D cancer cell line monolayers, 3D lung cancer tumor spheroids (A549), and a lung cancer tumor xenograft (A549) in nude mice. Photochemical activation of the prodrug upon green light irradiation led to the photosubstitution of the thioether ligand by water, thereby releasing the free rigidin analogue capable of inhibiting the polymerization of tubulin. In cancer cells, such photorelease was accompanied by a drastic reduction of cell growth, not only when the cells were grown in normoxia (21% O2) but also remarkably in hypoxic conditions (1% O2). In vivo, low toxicity was observed at a dose of 1 mg·kg–1 when the compound was injected intraperitoneally, and light activation of the compound in the tumor led to 30% tumor volume reduction, which represents the first demonstration of the safety and efficacy of ruthenium-based photoactivated chemotherapy compounds in a tumor xenograft.
To investigate the potential of tumor-targeting photoactivated chemotherapy, a chiral ruthenium-based anticancer warhead, Λ/Δ-Ru(Ph2phen)2(OH2)22+, was conjugated to the RGD-containing Ac-MRGDH-NH2 ...peptide by direct coordination of the M and H residues to the metal. This design afforded two diastereoisomers of a cyclic metallopeptide, Λ-1Cl2 and Δ-1Cl2. In the dark, the ruthenium-chelating peptide had a triple action. First, it prevented other biomolecules from coordinating with the metal center. Second, its hydrophilicity made 1Cl2 amphiphilic so that it self-assembled in culture medium into nanoparticles. Third, it acted as a tumor-targeting motif by strongly binding to the integrin (K d = 0.061 μM for the binding of Λ-1Cl2 to αIIbβ3), which resulted in the receptor-mediated uptake of the conjugate in vitro. Phototoxicity studies in two-dimensional (2D) monolayers of A549, U87MG, and PC-3 human cancer cell lines and U87MG three-dimensional (3D) tumor spheroids showed that the two isomers of 1Cl2 were strongly phototoxic, with photoindexes up to 17. Mechanistic studies indicated that such phototoxicity was due to a combination of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) effects, resulting from both reactive oxygen species generation and peptide photosubstitution. Finally, in vivo studies in a subcutaneous U87MG glioblastoma mice model showed that 1Cl2 efficiently accumulated in the tumor 12 h after injection, where green light irradiation generated a stronger tumoricidal effect than a nontargeted analogue ruthenium complex 2Cl2. Considering the absence of systemic toxicity for the treated mice, these results demonstrate the high potential of light-sensitive integrin-targeted ruthenium-based anticancer compounds for the treatment of brain cancer in vivo.
Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly ...active DUBs in TNBC may lead to novel therapies.
Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings
, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using
and
biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations.
Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling-induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression.
Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment.
Interindividual variability in DNA damage response (DDR) dynamics may evoke differences in susceptibility to cancer. However, pathway dynamics are often studied in cell lines as alternative to ...primary cells, disregarding variability. To compare DDR dynamics in the cell line HepG2 with primary human hepatocytes (PHHs), we developed a HepG2-based computational model that describes the dynamics of DDR regulator p53 and targets MDM2, p21 and BTG2. We used this model to generate simulations of virtual PHHs and compared the results to those for PHH donor samples. Correlations between baseline p53 and p21 or BTG2 mRNA expression in the absence and presence of DNA damage for HepG2-derived virtual samples matched the moderately positive correlations observed for 50 PHH donor samples, but not the negative correlations between p53 and its inhibitor MDM2. Model parameter manipulation that affected p53 or MDM2 dynamics was not sufficient to accurately explain the negative correlation between these genes. Thus, extrapolation from HepG2 to PHH can be done for some DDR elements, yet our analysis also reveals a knowledge gap within p53 pathway regulation, which makes such extrapolation inaccurate for the regulator MDM2. This illustrates the relevance of studying pathway dynamics in addition to gene expression comparisons to allow reliable translation of cellular responses from cell lines to primary cells. Overall, with our approach we show that dynamical modeling can be used to improve our understanding of the sources of interindividual variability of pathway dynamics.
Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these ...different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.
Hypoxia and HIF signaling drive cancer progression and therapy resistance and have been demonstrated in breast cancer. To what extent breast cancer subtypes differ in their response to hypoxia has ...not been resolved. Here, we show that hypoxia similarly triggers HIF1 stabilization in luminal and basal A triple negative breast cancer cells and we use high throughput targeted RNA sequencing to analyze its effects on gene expression in these subtypes. We focus on regulation of YAP/TAZ/TEAD targets and find overlapping as well as distinct target genes being modulated in luminal and basal A cells under hypoxia. We reveal a HIF1 mediated, basal A specific response to hypoxia by which TAZ, but not YAP, is phosphorylated at Ser89. While total YAP/TAZ localization is not affected by hypoxia, hypoxia drives a shift of p-TAZ(Ser89)/p-YAP(Ser127) from the nucleus to the cytoplasm in basal A but not luminal breast cancer cells. Cell fractionation and YAP knock-out experiments confirm cytoplasmic sequestration of TAZ(Ser89) in hypoxic basal A cells. Pharmacological and genetic interference experiments identify c-Src and CDK3 as kinases involved in such phosphorylation of TAZ at Ser89 in hypoxic basal A cells. Hypoxia attenuates growth of basal A cells and the effect of verteporfin, a disruptor of YAP/TAZ-TEAD–mediated transcription, is diminished under those conditions, while expression of a TAZ-S89A mutant does not confer basal A cells with a growth advantage under hypoxic conditions, indicating that other hypoxia regulated pathways suppressing cell growth are dominant.
Photoactivated chemotherapy agents form a new branch of physically targeted anticancer agents with potentially lower systemic side effects for patients. On the other hand, limited information exists ...on the intracellular interactions between the photoreleased metal cage and the photoreleased anticancer inhibitor. In this work, we report a new biological study of the known photoactivated compound Ru-STF31 in the glioblastoma cancer cell line, U87MG. Ru-STF31 targets nicotinamide phosphoribosyltransferase (NAMPT), an enzyme overexpressed in U87MG. Ru-STF31 is activated by red light irradiation and releases two photoproducts: the ruthenium cage and the cytotoxic inhibitor STF31. This study shows that Ru-STF31 can significantly decrease intracellular NAD+ levels in both normoxic (21% O2) and hypoxic (1% O2) U87MG cells. Strikingly, NAD+ depletion by light activation of Ru-STF31 in hypoxic U87MG cells could not be rescued by the addition of extracellular NAD+. Our data suggest an oxygen-dependent active role of the ruthenium photocage released by light activation.
Single-cell metabolomics (SCMs) is a powerful tool for studying cellular heterogeneity by providing insight into the differences between individual cells. With the development of a set of promising ...SCMs pipelines, this maturing technology is expected to be widely used in biomedical research. However, before SCMs is ready for primetime, there are some challenges to overcome. In this review, we summarize the trends and challenges in the development of SCMs. We also highlight the latest methodologies, applications, and sketch the perspective for integration with other omics and imaging approaches.
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•SCMs by MS is a powerful tool for studying cellular heterogeneity.•MS imaging excels at obtaining spatial information about single cells.•Live single-cell MS aims to sample cells with minimal disruption.•High-throughput methods, and standardization of data sharing and tools, are essential.
Over the last decade, the field of cancer metabolism has mainly focused on studying the role of tumorigenic metabolic rewiring in supporting cancer proliferation. Here, we perform the first ...genome‐scale computational study of the metabolic underpinnings of cancer migration. We build genome‐scale metabolic models of the NCI‐60 cell lines that capture the Warburg effect (aerobic glycolysis) typically occurring in cancer cells. The extent of the Warburg effect in each of these cell line models is quantified by the ratio of glycolytic to oxidative ATP flux (AFR), which is found to be highly positively associated with cancer cell migration. We hence predicted that targeting genes that mitigate the Warburg effect by reducing the AFR may specifically inhibit cancer migration. By testing the anti‐migratory effects of silencing such 17 top predicted genes in four breast and lung cancer cell lines, we find that up to 13 of these novel predictions significantly attenuate cell migration either in all or one cell line only, while having almost no effect on cell proliferation. Furthermore, in accordance with the predictions, a significant reduction is observed in the ratio between experimentally measured ECAR and OCR levels following these perturbations. Inhibiting anti‐migratory targets is a promising future avenue in treating cancer since it may decrease cytotoxic‐related side effects that plague current anti‐proliferative treatments. Furthermore, it may reduce cytotoxic‐related clonal selection of more aggressive cancer cells and the likelihood of emerging resistance.
Synopsis
A computational analysis based on genome‐scale metabolic models shows that the extent of the Warburg effect is highly associated with cancer cell migration across different cell lines and identifies anti‐migratory targets.
Genome‐scale metabolic models of each the NCI‐60 cell lines correctly capture the Warburg effect.
The extent of the Warburg effect, as quantified by the ratio between glycolytic and oxidative ATP flux rate (AFR), positively associates with cancer cell migration across the different cell lines.
siRNA knockdown of 13 genes predicted to reduce the AFR attenuates cell migration while having almost no effect on cell proliferation.
In agreement with the predictions, a significant reduction in the ratio of glycolytic/oxidative capacity is observed following these gene perturbations.
A computational analysis based on genome‐scale metabolic models shows that the extent of the Warburg effect is highly associated with cancer cell migration across different cell lines and identifies anti‐migratory targets.
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