SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

B cells support the repair of injured tissues by adopting MyD88-dependent regulatory functions and phenotype.

Exogenously applied mature naïve B220+ /CD19+ /IgM+ /IgD+ B cells are strongly protective in the context of tissue injury. However, the mechanisms by which B cells detect tissue injury and aid repair remain elusive. Here, we show in distinct models of skin and brain injury that MyD88-dependent toll-like receptor (TLR) signaling through TLR2/6 and TLR4 is essential for the protective benefit of B cells in vivo, while B cell-specific deletion of MyD88 abrogated this effect. The B cell response to injury was multi-modal with simultaneous production of both regulatory cytokines, such as IL-10, IL-35, and transforming growth factor beta (TGFß), and inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), IL-6, and interferon gamma. Cytometry analysis showed that this response was time and environment-dependent in vivo, with 20%-30% of applied B cells adopting an immune modulatory phenotype with high co-expression of anti- and pro-inflammatory cytokines after 18-48 h at the injury site. B cell treatment reduced the expression of TNFα and increased IL-10 and TGFß in infiltrating immune cells and fibroblasts at the injury site. Proteomic analysis further showed that B cells have a complex time-dependent homeostatic effect on the injured microenvironment, reducing the expression of inflammation-associated proteins, and increasing proteins associated with proliferation, tissue remodeling, and protection from oxidative stress. These findings chart and validate a first mechanistic understanding of the effects of B cells as an immunomodulatory cell therapy in the context of tissue injury.

Integrated multi-omics analysis of RB-loss identifies widespread cellular programming and synthetic weaknesses.

Inactivation of RB is one of the hallmarks of cancer, however gaps remain in our understanding of how RB-loss changes human cells. Here we show that pRB-depletion results in cellular reprogramming, we quantitatively measured how RB-depletion altered the transcriptional, proteomic and metabolic output of non-tumorigenic RPE1 human cells. These profiles identified widespread changes in metabolic and cell stress response factors previously linked to E2F function. In addition, we find a number of additional pathways that are sensitive to RB-depletion that are not E2F-regulated that may represent compensatory mechanisms to support the growth of RB-depleted cells. To determine whether these molecular changes are also present in RB1-/- tumors, we compared these results to Retinoblastoma and Small Cell Lung Cancer data, and identified widespread conservation of alterations found in RPE1 cells. To define which of these changes contribute to the growth of cells with de-regulated E2F activity, we assayed how inhibiting or depleting these proteins affected the growth of RB1-/- cells and of Drosophila E2f1-RNAi models in vivo. From this analysis, we identify key metabolic pathways that are essential for the growth of pRB-deleted human cells.

E2F/Dp inactivation in fat body cells triggers systemic metabolic changes.

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.

Tau Contributes to Sevoflurane-induced Neurocognitive Impairment in Neonatal Mice.

BACKGROUND: Sevoflurane anesthesia induces Tau phosphorylation and cognitive impairment in neonatal but not in adult mice. This study tested the hypothesis that differences in brain Tau amounts and in the activity of mitochondria-adenosine triphosphate (ATP)-Nuak1-Tau cascade between the neonatal and adult mice contribute to the age-dependent effects of sevoflurane on cognitive function. METHODS: 6- and 60-day-old mice of both sexes received anesthesia with 3% sevoflurane for 2 h daily for 3 days. Biochemical methods were used to measure amounts of Tau, phosphorylated Tau, Nuak1, ATP concentrations, and mitochondrial metabolism in the cerebral cortex and hippocampus. The Morris water maze test was used to evaluate cognitive function in the neonatal and adult mice. RESULTS: Under baseline conditions and compared with 60-day-old mice, 6-day-old mice had higher amounts of Tau (2.6 ± 0.4 [arbitrary units, mean ± SD] vs. 1.3 ± 0.2; P < 0.001), Tau oligomer (0.3 ± 0.1 vs. 0.1 ± 0.1; P = 0.008), and Nuak1 (0.9 ± 0.3 vs. 0.3 ± 0.1; P = 0.025) but lesser amounts of ATP (0.8 ± 0.1 vs. 1.5 ± 0.1; P < 0.001) and mitochondrial metabolism (74.8 ± 14.1 [pmol/min] vs. 169.6 ± 15.3; P < 0.001) in the cerebral cortex. Compared with baseline conditions, sevoflurane anesthesia induced Tau phosphorylation at its serine 202/threonine 205 residues (1.1 ± 0.4 vs. 0.2 ± 0.1; P < 0.001) in the 6-day-old mice but not in the 60-day-old mice (0.05 ± 0.04 vs. 0.03 ± 0.01; P = 0.186). The sevoflurane-induced Tau phosphorylation and cognitive impairment in the neonatal mice were both attenuated by the inhibition of Nuak1 and the treatment of vitamin K2. CONCLUSIONS: Higher brain Tau concentrations and lower brain mitochondrial metabolism in neonatal compared with adult mice contribute to developmental stage-dependent cognitive dysfunction after sevoflurane anesthesia.

Obtusaquinone: A Cysteine-Modifying Compound That Targets Keap1 for Degradation.

We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[18F]fluoro-2-deoxy-d-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

Author Correction: LKB1 loss links serine metabolism to DNA methylation and tumorigenesis.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

SETD1A protects from senescence through regulation of the mitotic gene expression program.

SETD1A, a Set1/COMPASS family member maintaining histone-H3-lysine-4 (H3K4) methylation on transcriptionally active promoters, is overexpressed in breast cancer. Here, we show that SETD1A supports mitotic processes and consequentially, its knockdown induces senescence. SETD1A, through promoter H3K4 methylation, regulates several genes orchestrating mitosis and DNA-damage responses, and its depletion causes chromosome misalignment and segregation defects. Cell cycle arrest in SETD1A knockdown senescent cells is independent of mutations in p53, RB and p16, known senescence mediators; instead, it is sustained through transcriptional suppression of SKP2, which degrades p27 and p21. Rare cells escaping senescence by restoring SKP2 expression display genomic instability. In > 200 cancer cell lines and in primary circulating tumor cells, SETD1A expression correlates with genes promoting mitosis and cell cycle suggesting a broad role in suppressing senescence induced by aberrant mitosis. Thus, SETD1A is essential to maintain mitosis and proliferation and its suppression unleashes the tumor suppressive effects of senescence.

A Code of Mono-phosphorylation Modulates the Function of RB.

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism.

G protein αs (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.

Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation.

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

TOX Regulates Growth, DNA Repair, and Genomic Instability in T-cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Using a transgenic screen in zebrafish, thymocyte selection-associated high mobility group box protein (TOX) was uncovered as a collaborating oncogenic driver that accelerated T-ALL onset by expanding the initiating pool of transformed clones and elevating genomic instability. TOX is highly expressed in a majority of human T-ALL and is required for proliferation and continued xenograft growth in mice. Using a wide array of functional analyses, we uncovered that TOX binds directly to KU70/80 and suppresses recruitment of this complex to DNA breaks to inhibit nonhomologous end joining (NHEJ) repair. Impaired NHEJ is well known to cause genomic instability, including development of T-cell malignancies in KU70- and KU80-deficient mice. Collectively, our work has uncovered important roles for TOX in regulating NHEJ by elevating genomic instability during leukemia initiation and sustaining leukemic cell proliferation following transformation.Significance: TOX is an HMG box-containing protein that has important roles in T-ALL initiation and maintenance. TOX inhibits the recruitment of KU70/KU80 to DNA breaks, thereby inhibiting NHEJ repair. Thus, TOX is likely a dominant oncogenic driver in a large fraction of human T-ALL and enhances genomic instability. Cancer Discov; 7(11); 1336-53. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein.

Germline mutations in the Folliculin (FLCN) tumour suppressor gene result in fibrofolliculomas, lung cysts and renal cancers, but the precise mechanisms of tumour suppression by FLCN remain elusive. Here we identify Rab7A, a small GTPase important for endocytic trafficking, as a novel FLCN interacting protein and demonstrate that FLCN acts as a Rab7A GTPase-activating protein. FLCN-/- cells display slower trafficking of epidermal growth factor receptors (EGFR) from early to late endosomes and enhanced activation of EGFR signalling upon ligand stimulation. Reintroduction of wild-type FLCN, but not tumour-associated FLCN mutants, suppresses EGFR signalling in a Rab7A-dependent manner. EGFR signalling is elevated in FLCN-/- tumours and the EGFR inhibitor afatinib suppresses the growth of human FLCN-/- cells as tumour xenografts. The functional interaction between FLCN and Rab7A appears conserved across species. Our work highlights a mechanism explaining, at least in part, the tumour suppressor function of FLCN.

Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis.

The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr308, but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined.

Genomic Instability Is Induced by Persistent Proliferation of Cells Undergoing Epithelial-to-Mesenchymal Transition.

TGF-ß secreted by tumor stroma induces epithelial-to-mesenchymal transition (EMT) in cancer cells, a reversible phenotype linked to cancer progression and drug resistance. However, exposure to stromal signals may also lead to heritable changes in cancer cells, which are poorly understood. We show that epithelial cells failing to undergo proliferation arrest during TGF-ß-induced EMT sustain mitotic abnormalities due to failed cytokinesis, resulting in aneuploidy. This genomic instability is associated with the suppression of multiple nuclear envelope proteins implicated in mitotic regulation and is phenocopied by modulating the expression of LaminB1. While TGF-ß-induced mitotic defects in proliferating cells are reversible upon its withdrawal, the acquired genomic abnormalities persist, leading to increased tumorigenic phenotypes. In metastatic breast cancer patients, increased mesenchymal marker expression within single circulating tumor cells is correlated with genomic instability. These observations identify a mechanism whereby microenvironment-derived signals trigger heritable genetic changes within cancer cells, contributing to tumor evolution.

LKB1 loss links serine metabolism to DNA methylation and tumorigenesis.

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.