Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia.

Hypoxia is linked to disease progression and poor prognosis in several cancers, including breast cancer. Cancer cells can encounter acute, chronic, and/or intermittent periods of oxygen deprivation and it is poorly understood how the different breast cancer subtypes respond to such hypoxia regimes. Here, we assessed the response of representative cell lines for the luminal and basal A subtype to acute (24 h) and chronic hypoxia (5 days). High throughput targeted transcriptomics analysis showed that HIF-related pathways are significantly activated in both subtypes. Indeed, HIF1⍺ nuclear accumulation and activation of the HIF1⍺ target gene CA9 were comparable. Based on the number of differentially expressed genes: (i) 5 days of exposure to hypoxia induced a more profound transcriptional reprogramming than 24 h, and (ii) basal A cells were less affected by acute and chronic hypoxia as compared to luminal cells. Hypoxia-regulated gene networks were identified of which hub genes were associated with worse survival in breast cancer patients. Notably, while chronic hypoxia altered the regulation of the cell cycle in both cell lines, it induced two distinct adaptation programs in these subtypes. Mainly genes controlling central carbon metabolism were affected in the luminal cells whereas genes controlling the cytoskeleton were affected in the basal A cells. In agreement, in response to chronic hypoxia, lactate secretion was more prominently increased in the luminal cell lines which were associated with the upregulation of the GAPDH glycolytic enzyme. This was not observed in the basal A cell lines. In contrast, basal A cells displayed enhanced cell migration associated with more F-actin stress fibers whereas luminal cells did not. Altogether, these data show distinct responses to acute and chronic hypoxia that differ considerably between luminal and basal A cells. This differential adaptation is expected to play a role in the progression of these different breast cancer subtypes.

GRHL2-controlled gene expression networks in luminal breast cancer.

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract.

High-content high-throughput imaging reveals distinct connections between mitochondrial morphology and functionality for OXPHOS complex I, III, and V inhibitors.

Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals.

GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer.

The transcription factor Grainyhead-like 2 (GRHL2) is a critical transcription factor for epithelial tissues that has been reported to promote cancer growth in some and suppress aspects of cancer progression in other studies. We investigated its role in different breast cancer subtypes. In breast cancer patients, GRHL2 expression was increased in all subtypes and inversely correlated with overall survival in basal-like breast cancer patients. In a large cell line panel, GRHL2 was expressed in luminal and basal A cells, but low or absent in basal B cells. The intersection of ChIP-Seq analysis in 3 luminal and 3 basal A cell lines identified conserved GRHL2 binding sites for both subtypes. A pathway analysis of ChIP-seq data revealed cell-cell junction regulation and epithelial migration as well as epithelial proliferation, as candidate GRHL2-regulated processes and further analysis of hub genes in these pathways showed similar regulatory networks in both subtypes. However, GRHL2 deletion in a luminal cell line caused cell cycle arrest while this was less prominent in a basal A cell line. Conversely, GRHL2 loss triggered enhanced migration in the basal A cells but failed to do so in the luminal cell line. ChIP-Seq and ChIP-qPCR demonstrated GRHL2 binding to CLDN4 and OVOL2 in both subtypes but not to other GRHL2 targets controlling cell-cell adhesion that were previously identified in other cell types, including CDH1 and ZEB1. Nevertheless, E-cadherin protein expression was decreased upon GRHL2 deletion especially in the luminal line and, in agreement with its selectively enhanced migration, only the basal A cell line showed concomitant induction of vimentin and N-cadherin. To address how the balance between growth reduction and aspects of EMT upon loss of GRHL2 affected in vivo behavior, we used a mouse basal A orthotopic transplantation model in which the GRHL2 gene was silenced. This resulted in reduced primary tumor growth and a reduction in number and size of lung colonies, indicating that growth suppression was the predominant consequence of GRHL2 loss. Altogether, these findings point to largely common but also distinct roles for GRHL2 in luminal and basal breast cancers with respect to growth and motility and indicate that, in agreement with its negative association with patient survival, growth suppression is the dominant response to GRHL2 loss.

Acute vs. chronic vs. intermittent hypoxia in breast Cancer: a review on its application in in vitro research.

Hypoxia has been linked to elevated instances of therapeutic resistance in breast cancer. The exposure of proliferating cancer cells to hypoxia has been shown to induce an aggressive phenotype conducive to invasion and metastasis. Regions of the primary tumors in the breast may be exposed to different types of hypoxia including acute, chronic or intermittent. Intermittent hypoxia (IH), also called cyclic hypoxia, is caused by exposure to cycles of hypoxia and reoxygenation (H-R cycles). Importantly, there is currently no consensus amongst the scientific community on the total duration of hypoxia, the oxygen level, and the possible presence of H-R cycles. In this review, we discuss current methods of hypoxia research, to explore how exposure regimes used in experiments are connected to signaling by different hypoxia inducible factors (HIFs) and to distinct cellular responses in the context of the hallmarks of cancer. We highlight discrepancies in the existing literature on hypoxia research within the field of breast cancer in particular and propose a clear definition of acute, chronic, and intermittent hypoxia based on HIF activation and cellular responses: (i) acute hypoxia is when the cells are exposed for no more than 24 h to an environment with 1% O2 or less; (ii) chronic hypoxia is when the cells are exposed for more than 48 h to an environment with 1% O2 or less and (iii) intermittent hypoxia is when the cells are exposed to at least two rounds of hypoxia (1% O2 or less) separated by at least one period of reoxygenation by exposure to normoxia (8.5% O2 or higher). Our review provides for the first time a guideline for definition of hypoxia related terms and a clear foundation for hypoxia related in vitro (breast) cancer research.

Substrate rigidity modulates traction forces and stoichiometry of cell-matrix adhesions.

In cell-matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell-matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative super-resolution microscopy to cell-matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application, whereas a ∼60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate with an effective stiffness of 47 kPa. On a substrate with a fourfold lower effective stiffness, the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ∼60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell-matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relationship between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation.

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

Hypoxia Triggers TAZ Phosphorylation in Basal A Triple Negative Breast Cancer Cells.

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.

Cancer-Associated Mutations of the Adenosine A2A Receptor Have Diverse Influences on Ligand Binding and Receptor Functions.

The adenosine A2A receptor (A2AAR) is a class A G-protein-coupled receptor (GPCR). It is an immune checkpoint in the tumor micro-environment and has become an emerging target for cancer treatment. In this study, we aimed to explore the effects of cancer-patient-derived A2AAR mutations on ligand binding and receptor functions. The wild-type A2AAR and 15 mutants identified by Genomic Data Commons (GDC) in human cancers were expressed in HEK293T cells. Firstly, we found that the binding affinity for agonist NECA was decreased in six mutants but increased for the V275A mutant. Mutations A165V and A265V decreased the binding affinity for antagonist ZM241385. Secondly, we found that the potency of NECA (EC50) in an impedance-based cell-morphology assay was mostly correlated with the binding affinity for the different mutants. Moreover, S132L and H278N were found to shift the A2AAR towards the inactive state. Importantly, we found that ZM241385 could not inhibit the activation of V275A and P285L stimulated by NECA. Taken together, the cancer-associated mutations of A2AAR modulated ligand binding and receptor functions. This study provides fundamental insights into the structure-activity relationship of the A2AAR and provides insights for A2AAR-related personalized treatment in cancer.

Metastasis: crosstalk between tissue mechanics and tumour cell plasticity.

Despite the fact that different genetic programmes drive metastasis of solid tumours, the ultimate outcome is the same: tumour cells are empowered to pass a series of physical hurdles to escape the primary tumour and disseminate to other organs. Epithelial-to-mesenchymal transition (EMT) has been proposed to drive the detachment of individual cells from primary tumour masses and facilitate the subsequent establishment of metastases in distant organs. However, this concept has been challenged by observations from pathologists and from studies in animal models, in which partial and transient acquisition of mesenchymal traits is seen but tumour cells travel collectively rather than as individuals. In this review, we discuss how crosstalk between a hybrid E/M state and variations in the mechanical aspects of the tumour microenvironment can provide tumour cells with the plasticity required for strategies to navigate surrounding tissues en route to dissemination. Targeting such plasticity provides therapeutic opportunities to combat metastasis.

Mechanical interplay between cell shape and actin cytoskeleton organization.

We investigate the mechanical interplay between the spatial organization of the actin cytoskeleton and the shape of animal cells adhering on micropillar arrays. Using a combination of analytical work, computer simulations and in vitro experiments, we demonstrate that the orientation of the stress fibers strongly influences the geometry of the cell edge. In the presence of a uniformly aligned cytoskeleton, the cell edge can be well approximated by elliptical arcs, whose eccentricity reflects the degree of anisotropy of the cell's internal stresses. Upon modeling the actin cytoskeleton as a nematic liquid crystal, we further show that the geometry of the cell edge feeds back on the organization of the stress fibers by altering the length scale at which these are confined. This feedback mechanism is controlled by a dimensionless number, the anchoring number, representing the relative weight of surface-anchoring and bulk-aligning torques. Our model allows to predict both cellular shape and the internal structure of the actin cytoskeleton and is in good quantitative agreement with experiments on fibroblastoid (GDß1, GDß3) and epithelioid (GEß1, GEß3) cells.

Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

In the original publication of the article.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Cytoskeletal Anisotropy Controls Geometry and Forces of Adherent Cells.

We investigate the geometrical and mechanical properties of adherent cells characterized by a highly anisotropic actin cytoskeleton. Using a combination of theoretical work and experiments on micropillar arrays, we demonstrate that the shape of the cell edge is accurately described by elliptical arcs, whose eccentricity expresses the degree of anisotropy of the internal cell stresses. This results in a spatially varying tension along the cell edge, that significantly affects the traction forces exerted by the cell on the substrate. Our work highlights the strong interplay between cell mechanics and geometry and paves the way towards the reconstruction of cellular forces from geometrical data.

A mechanopharmacology approach to overcome chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly chemoresistant malignancy. This chemoresistant phenotype has been historically associated with genetic factors. Major biomedical research efforts were concentrated that resulted in the identification of subtypes characterized by specific genetic lesions and gene expression signatures that suggest important biological differences. However, to date, these distinct differences could not be exploited for therapeutic interventions. Apart from these genetic factors, desmoplasia and tumor microenvironment have been recognized as key contributors to PDAC chemoresistance. However, while several strategies targeting tumor-stroma have been explored including drugs against members of the Hedgehog family, they failed to meet the expectations in the clinical setting. These unsatisfactory clinical results suggest that, an important link between genetics and the influence of tumor microenvironment on PDAC chemoresistance remains to be elucidated. In this respect, mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology as well as biophysics and bioengineering. Herein we provide a comprehensive overview of the key players in pancreatic cancer chemoresistance from the perspective of mechanobiology, and discuss novel experimental avenues such as elastic micropillar arrays that could provide fresh insights for the development of mechanobiology-targeted therapeutic approaches (know as mechanopharmacology) to overcome anticancer drug resistance in pancreatic cancer.

The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5ß1 and αvß3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvß3 expression can fully compensate for loss of α5ß1 and other ß1 integrins to support outside-in and inside-out force transmission. α5ß1 and αvß3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5ß1 and αvß3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvß3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5ß1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

A guide to mechanobiology: Where biology and physics meet.

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates. This article is part of a Special Issue entitled: Mechanobiology.

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone.

BACKGROUND: Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study. METHODS: Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines. RESULTS: Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression. CONCLUSIONS: The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone.

Aven-mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in conventional osteosarcoma.

Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy.