[Show abstract][Hide abstract] ABSTRACT: Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.
[Show abstract][Hide abstract] ABSTRACT: Mass cytometry (MC) offers unparalleled potential for the development of highly parameterized assays for characterization of single cells within heterogeneous populations. Current reagents compatible with MC analysis employ antibody-metal-chelating polymer conjugates to report on the presence of biomarkers. Here, we expand the utility of MC by developing the first activity-based probe designed specifically for use with the technology. A compact MC-detectable telluroether is linked to a bioreductively sensitive 2-nitroimidazole scaffold, thereby generating a probe sensitive to cellular hypoxia. The probe exhibits low toxicity and is able to selectively label O2-deprived cells. A proof-of-concept experiment employing metal-bound DNA intercalators demonstrates that a heterogeneous mixture of cells with differential exposure to O2 can be effectively discriminated by the quantity of tellurium-labeling. The organotellurium reagents described herein provide a general approach to the development of a large toolkit of MC-compatible probes for activity-based profiling of single cells.
[Show abstract][Hide abstract] ABSTRACT: Regions of acute and chronic hypoxia exist within solid tumors and can lead to increased rates of mutagenesis and/or altered DNA damage and repair (DDR) protein expression. Base excision repair (BER) is responsible for resolving small, non-helix distorting lesions from the genome that potentially cause mutations by mispairing or promoting DNA breaks during replication. Germline and somatic mutations in BER genes, such as MutY Homolog (MUTYH/MYH) and DNA-directed polymerase (POLB), are associated with increased risk of colorectal cancer. However, very little is known regarding the expression and function of BER proteins under hypoxic stress. Using conditions of chronic hypoxia, decreased expression of BER proteins was observed due to a mechanism involving suppressed BER protein synthesis in multiple colorectal cancer cell lines. Functional BER was impaired as determined by MYH- and 8-oxoguanine (OGG1)-specific glycosylase assays. A formamidopyrimidine-DNA glycosylase (Fpg) Comet assay revealed elevated residual DNA base damage in hypoxic cells 24 h after H2O2 treatment as compared to normoxic controls. Similarly, HPLC analysis demonstrated that 8-oxo-2'-deoxyguanosine (8-OHdG) lesions were elevated in hypoxic cells 3 and 24 h after potassium bromate (KBrO3) treatment when compared to aerobic cells. Correspondingly, decreased clonogenic survival was observed following exposure to the DNA base damaging agents H2O2 and MMS, but not to the microtubule interfering agent paclitaxel. Thus, a persistent down-regulation of BER components by the microenvironment modifies and facilitates a mutator phenotype, driving genetic instability and cancer progression. Implications: Aberrant BER is a contributing factor for the observed genetic instability in hypoxic tumor cells.
[Show abstract][Hide abstract] ABSTRACT: The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.
[Show abstract][Hide abstract] ABSTRACT: Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.
[Show abstract][Hide abstract] ABSTRACT: The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.
PLoS ONE 01/2014; 9(2):e89997. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conditions of poor oxygenation (hypoxia) are present in the majority of solid human tumors and are associated with poor patient prognosis due to both hypoxia-mediated resistance to treatment, and to hypoxiainduced biological changes that promote increased malignancy, including metastasis. Tumor cells respond to hypoxia by activating several oxygen-sensitive signaling pathways that include the hypoxia inducible factor 1/2 (HIF1/2) signalling pathways and the unfolded protein response (UPR), which alter gene expression to promote adaptation and survival during hypoxic conditions. Furthermore, these hypoxia responsive pathways can lead to changes in gene expression and cellular phenotype that influence the potential of cancer cells to metastasize. However, the hypoxia-induced signaling events that promote tumor metastasis are still relatively poorly understood. Previous studies have largely focused on the contribution of the HIF signaling pathway to hypoxia-mediated metastasis. However, recent evidence demonstrates that hypoxic activation of the UPR is also an important mediator of metastasis.
Current Molecular Medicine 01/2014; 14(5). · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.
The Journal of Cell Biology 11/2013; · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor hypoxia is a negative prognostic factor in multiple cancers, due in part to its role in causing resistance to radiotherapy. Hypoxia arises in tumor regions distal to blood vessels as oxygen is consumed by more proximal tumor cells. Reducing the rate of oxygen consumption is therefore a potential strategy to reduce tumor hypoxia. We hypothesized that the anti-diabetic drug metformin, which reduces oxygen consumption through inhibition of mitochondrial complex I, would improve radiation response by increasing tumor oxygenation.
Tumor hypoxia was measured in xenografts before and after metformin treatment using 2-nitroimidazole hypoxia markers quantified by immunohistochemistry (IHC), flow cytometry and positron emission tomography (PET)-imaging. Radiation response was determined by tumor growth delay and clonogenic survival in xenografts with and without administration of metformin. The impact of metformin use on outcome was assessed in 504 localized prostate cancer patients treated with curative-intent, image-guided radiotherapy (IGRT) from 1996 to 2012. Three-year biochemical relapse-free rates were assessed using the Kaplan-Meier method.
Metformin treatment significantly improved tumor oxygenation in two xenograft models as measured by IHC, flow cytometry and PET imaging. Metformin also led to improved radiotherapy responses when mice were administered metformin immediately prior to irradiation. Clinically, metformin use was associated with an independent and significant decrease in early biochemical relapse rates (p=0.0106).
Our data demonstrate that metformin can improve tumor oxygenation and response to radiotherapy. Our study suggests that metformin may represent an effective and inexpensive means to improve radiotherapy outcome with an optimal therapeutic ratio.
Clinical Cancer Research 10/2013; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: Tumor hypoxia is a negative prognostic factor in multiple cancers, due in part to its role in causing resistance to radiotherapy. Hypoxia arises in tumor regions distal to blood vessels as oxygen is consumed by more proximal tumor cells. Reducing the rate of oxygen consumption is therefore a potential strategy to reduce tumor hypoxia. We hypothesized that the anti-diabetic drug metformin, which reduces oxygen consumption through inhibition of mitochondrial complex I, would improve radiation response by increasing tumor oxygenation. Experimental Design: Tumor hypoxia was measured in xenografts before and after metformin treatment using 2-nitroimidazole hypoxia markers quantified by immunohistochemistry (IHC), flow cytometry and positron emission tomography (PET)-imaging. Radiation response was determined by tumor growth delay and clonogenic survival in xenografts with and without administration of metformin. The impact of metformin use on outcome was assessed in 504 localized prostate cancer patients treated with curative-intent, image-guided radiotherapy (IGRT) from 1996 to 2012. Three-year biochemical relapse-free rates were assessed using the Kaplan-Meier method. Results: Metformin treatment significantly improved tumor oxygenation in two xenograft models as measured by IHC, flow cytometry and PET imaging. Metformin also led to improved radiotherapy responses when mice were administered metformin immediately prior to irradiation. Clinically, metformin use was associated with an independent and significant decrease in early biochemical relapse rates (p=0.0106). Conclusion: Our data demonstrate that metformin can improve tumor oxygenation and response to radiotherapy. Our study suggests that metformin may represent an effective and inexpensive means to improve radiotherapy outcome with an optimal therapeutic ratio.
Clinical Cancer Research 10/2013; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC)-TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumor staging summarizes data on tumor burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status, and gene expression-based stratification. These parameters rely on tumor-cell characteristics. Extensive literature investigated the host-immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumors. A methodology named "Immunoscore" has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM-classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).
The Journal of Pathology 10/2013; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor hypoxia (low oxygenation) causes treatment resistance and poor patient outcome. A substantial fraction of tumor cells experience cycling hypoxia, characterized by transient episodes of hypoxia and reoxygenation. These cells are under a unique burden of stress, mediated by excessive production of reactive oxygen species (ROS). Cellular components damaged by ROS can be cleared by autophagy, rendering cycling hypoxic tumor cells particularly vulnerable to inhibition of autophagy and its upstream regulatory pathways. Activation of the PERK-mediated signaling arm of the unfolded protein response during hypoxia plays a critical role in the defense against ROS, both by stimulating glutathione synthesis pathways and through promoting autophagy.
Seminars in radiation oncology 10/2013; 23(4):252-61. · 4.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conditions of poor oxygenation (hypoxia) are present in many human tumors including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated.
Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis.
We demonstrate that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations, and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due in part to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration.
These data suggest that the association between hypoxia, metastasis and poor prognosis is due in part to hypoxic activation of the UPR and expression of LAMP3.
Clinical Cancer Research 09/2013; · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Radiotherapy with concurrent cisplatin (CRT) is standard treatment for locally advanced cervical cancer. However, not all patients benefit from the addition of cisplatin to RT alone. This study explored the value of pre-treatment tumor interstitial fluid pressure (IFP) and hypoxia measurements as predictors of cisplatin response in 291 patients who were treated with RT (1994-1998) or RT plus concurrent cisplatin (1999-2009). Clinical characteristics were similar between the two groups, apart from a greater proportion of patients with pelvic lymph node metastases and hypoxic tumors in the CRT cohort. Patients were followed for a median duration of 5.6 years. Information about recurrence and survival was recorded prospectively. The addition of cisplatin to RT improved survival compared to treatment with RT alone (HR 0.61, p=0.0097). This improvement was confined to patients with high-IFP tumors at diagnosis (HR 0.40, p=0.00091). There was no benefit of adding cisplatin in those with low-IFP tumors (HR 1.05, p=0.87). There was no difference in the effectiveness of cisplatin in patients with more or less hypoxic tumors. In conclusion, patients with locally advanced cervical cancer and high tumor IFP at diagnosis have greater benefit from the addition of cisplatin to RT than those with low IFP. This may reflect high tumor cell proliferation, which is known to influence IFP, local tumor control and patient survival.
International Journal of Cancer 07/2013; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoxia can promote tumor metastasis by mechanisms that are believed to result from changes in gene expression. The current study examined the role of putative metastatic genes regulated by cyclic hypoxia in relation to metastasis formation in orthotopic models of cervix cancer.
Orthotopic tumors derived from ME180 human cervix cancer cells or from early generation human cervix cancer xenografts were exposed to cyclic hypoxic conditions during growth in vivo and tumor growth and lymphnode metastases were monitored. Expression of the chemokine receptor CXCR4 and various genes in the Hedgehog (Hh) pathway were inhibited using genetic (inducible shRNA vs CXCR4) small molecule (AMD3100) or antibody (5E1) treatment (CXCR4 and Hh genes, respectively) during tumor growth.
As reported previously, exposure of tumor bearing mice to cyclic hypoxia caused a reduction of tumor growth but a large increase in metastasis. Inhibition of CXCR4 or Hh gene activity during tumor growth further reduced primary tumor size and reduced lymphatic metastasis to levels below those seen in control mice exposed to normoxic conditions.
Blocking CXCR4 or Hh gene expression are potential therapeutic pathways for improving cervix cancer treatment.
Radiotherapy and Oncology 07/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment.
Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients.
ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia.
We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.
Radiotherapy and Oncology 07/2013; · 4.52 Impact Factor