Roland K. Chiu

Hypoxia as a target for combined modality treatments

There is overwhelming evidence that solid human tumours grow within a unique micro-environment. This environment is characterised by an abnormal vasculature, which leads to an insufficient supply of oxygen and nutrients to the tumour cells. These characteristics of the environment limit the effectiveness of both radiotherapy and chemotherapy. Measurement of the oxygenation status of human tumours has unequivocally demonstrated the importance of this parameter on patient prognosis. Tumour hypoxia has been shown to be an independent prognostic indicator of poor outcome in prostate, head and neck and cervical cancers. Recent laboratory and clinical data have shown that hypoxia is also associated with a more malignant phenotype, affecting genomic stability, apoptosis, angiogenesis and metastasis. Several years ago, scientists realised that the unique properties within the tumour micro-environment could provide the basis for tumour-specific therapies. Efforts that are underway to develop therapies that exploit the tumour micro-environment can be categorised into three groups. The first includes agents that exploit the environmental changes that occur within the micro-environment such as hypoxia and reduced pH. This includes bioreductive drugs that are specifically toxic to hypoxic cells, as well as hypoxia-specific gene delivery systems. The second category includes therapies designed to exploit the unique properties of the tumour vasculature and include both angiogenesis inhibitors and vascular targeting agents. The final category includes agents that exploit the molecular and cellular responses to hypoxia. For example, many genes are induced by hypoxia and promoter elements from these genes can be used for the selective expression of therapeutic proteins in hypoxic tumour cells. An overview of the various properties ascribed to tumour hypoxia and the current efforts underway to exploit hypoxia for improving cancer treatment will be discussed.

Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated salmonella

To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells that are most resistant to conventional therapies. We demonstrate that the exponential growth of the attenuated bacteria is identical under normoxia and hypoxia. A hypoxia-inducible promoter (HIP-1) was created from a portion of the endogenous Salmonella pepT promoter and was shown to drive reporter gene expression under both acute and chronic hypoxia, but not under normoxia. Genetic engineering of the TATA- and FNR-box within HIP-1 allowed fine tuning of gene induction, resulting in hypoxic induction factors of up to 200-fold. Finally, we demonstrate that HIP-1 can drive hypoxia-mediated gene expression in bacteria which have colonized human tumor xenografts in mouse models. Expression of both GFP and RFP under control of HIP-1 demonstrated an ~15-fold increase relative to a constitutive promoter when tumors were made hypoxic. Moreover, the use of a constitutive promoter resulted in reporter gene expression in both tumors and normal tissues, whereas reporter gene expressing using HIP-1 was confined to the tumor.

Diminished Carcinogen Detoxification Is a Novel Mechanism for Hypoxia-inducible Factor 1-mediated Genetic Instability

The hypoxia-inducible factor 1 (HIF-1) pathway is induced in many tumors and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1α dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). We hypothesized that HIF-1α activation attenuates BaP-induced AhR-mediated gene expression, which may lead to increased genetic instability and malignant progression. Human lung carcinoma cells (A549) were simultaneously stimulated with CoCl2, which leads to HIF-1α stabilization and varying concentrations of BaP. Both quantitative PCR and immunoblot analysis indicated that induction of the hypoxia response pathway significantly reduced the levels of AhR downstream targets CYP1A1 and CYP1B1 and AhR protein binding to ARNT. We further demonstrate that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and γ-H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaP-DNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability.

Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils

BackgroundLung cancer often develops in association with chronic pulmonary inflammatory diseases with an influx of neutrophils. More detailed information on inflammatory pathways and the role of neutrophils herein is a prerequisite for understanding the mechanism of inflammation associated cancer.MethodsIn the present study, we used microarrays in order to obtain a global view of the transcriptional responses of the lung to LPS in mice, which mimics an acute lung inflammation. To investigate the influence of neutrophils in this process, we depleted mice from circulating neutrophils by treatment with anti-PMN antibodies prior to LPS exposure.ResultsA total of 514 genes was greater than 1.5-fold differentially expressed in the LPS induced lung inflammation model. 394 of the 514 were up regulated genes mostly involved in cell cycle and immune/inflammation related processes, such as cytokine/chemokine activity and signalling. Down regulated genes represented nonimmune processes, such as development, metabolism and transport. Notably, the number of genes and pathways that were differentially expressed, was reduced when animals were depleted from circulating neutrophils, confirming the central role of neutrophils in the inflammatory response. Furthermore, there was a significant correlation between the differentially expressed gene list and the promutagenic DNA lesion M1dG, suggesting that it is the extent of the immune response which drives genetic instability in the inflamed lung. Several genes that were specifically regulated by the presence of activated neutrophils could be identified and these were mostly involved in interferon signalling, oxidative stress response and cell cycle progression. The latter possibly refers to a higher rate of cell turnover in the inflamed lung with neutrophils, suggesting that the neutrophil influx is associated with a higher risk for the accumulation and fixation of mutations.ConclusionGene expression profiling identified specific genes and pathways that are related to neutrophilic inflammation and could be associated to cancer development and indicate an active role of neutrophils in mediating the LPS induced inflammatory response in the mouse lung.

Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS) (20 microg), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by approximately 50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo(-/-) or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

Potential and limitations of bacterial-mediated cancer therapy

Bacterial-based tumor-targeted therapy is an area of growing interest and holds promise for the treatment of solid tumors. Upon systemic administration, various types of non-pathogenic obligate anaerobes and facultative anaerobes have been shown to infiltrate and selectively replicate within solid tumors. The tumor specificity is based upon the unique physiology of solid tumors, which is often characterized by regions of hypoxia and necrosis. Prokaryotic vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models. Although the amount of clinical experience with bacterial vectors is limited to date, the available data clearly demonstrated the feasibility of bacterial-mediated therapy in humans. There are several issues however that are still unknown and remain major challenges. In this review, using Clostridium and modified Salmonella as prototypical agents, we will discuss the major advantages, challenges and shortcomings of bacterial systems for tumor-specific therapy. In addition, we will highlight the requirements needed to advance the approach into clinical trials.

The deletion mutant EGFRvIII significantly contributes to stress resistance typical for the tumour microenvironment

BACKGROUND AND PURPOSE The epidermal growth factor receptor (EGFR) is overexpressed or mutated in many tumour types. The truncated, constitutively active EGFRvIII variant has not been detected in normal tissues but is found in many malignancies. In the current study, we have investigated the hypothesis that EGFRvIII contributes to a growth and survival advantage under tumour microenvironment-related stress conditions. MATERIALS AND METHODS U373MG doxycycline-regulated isogenic cells expressing EGFRwt or EGFRvIII were created and validated using Western blot, FACS and qRT-PCR. In vitro proliferation was evaluated with standard growth assays. Cell survival was assayed using clonogenic survival. Animal experiments were performed using NMRI-nu-xenografted mice. RESULTS Inducible isogenic cell lines were created and showed high induction of EGFRwt and EGFRvIII upon doxycycline addition. Overexpression of EGFRvIII but not of EGFRwt in this model resulted in a growth and survival advantage upon different tumour microenvironment-related stress conditions in vitro. Induction of EGFRvIII increased tumour growth in vivo, which was reversible upon loss of expression. CONCLUSIONS Under conditions where nutrients are limited and stress is apparent, as in the tumour microenvironment, expression of EGFRvIII leads to a growth and survival advantage. These data indicate a potential selection of EGFRvIII-expressing tumour cells under such stress conditions.

Regulation of PCNA polyubiquitination in human cells

BackgroundThe ubiquitin-based molecular switch dictating error free versus error prone repair has been conserved throughout eukaryotic evolution. A central component of this switch is the homotrimeric clamp PCNA, which is ubiquitinated in response to genotoxic stress allowing recovery of replication forks blocked at sites of DNA damage. The particulars of PCNA ubiquitination have been elucidated in yeast and to a further extent recently in human cells. However, gaps in the detailed mechanism and regulation of PCNA polyubiquitination still persist in human cells.FindingsWe expand upon several studies and show that PCNA is polyubiquitnated in normal skin fibroblasts, and that this ubiquitination is dependant on RAD18. Furthermore we define the types of DNA damage that induce ubiquitination on PCNA. Cisplatin, methylmethane sulphonate and benzo(a)pyrene-diol-epoxide induce the polyubiquitination of PCNA to the same extent as UV while polyubiquitination is not detected after X-ray treatment. Moreover, we show that ubiquitination of PCNA is not regulated by cell cycle checkpoint kinases ATM-Chk2 or ATR-Chk1. Significantly, we report that PCNA polyubiquitination is negatively regulated by USP1.ConclusionsOur results demonstrate the importance of PCNA polyubiquitination in human cells and define the key regulator of this ubiquitination.

hMMS2 serves a redundant role in human PCNA polyubiquitination

BackgroundIn yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.ResultsIn this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.ConclusionOur results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.

Rad18 and Rnf8 facilitate homologous recombination by two distinct mechanisms, promoting Rad51 focus formation and suppressing the toxic effect of nonhomologous end-joining

The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18−/−, RNF8−/− and RAD18−/−/RNF8−/− clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13−/−cells, they are more sensitive to camptothecin and olaparib than UBC13−/−cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18−/− and RNF8−/− cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18−/−, RNF8−/−, RAD18−/−/RNF8−/− and UBC13−/−cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy-induced DSB sites.