Targeting nuclear factor-kappa B to overcome resistance to chemotherapy

Intrinsic or acquired resistance to chemotherapeutic agents is a common phenomenon and a major challenge in the treatment of cancer patients. Chemoresistance is defined by a complex network of factors including multi-drug resistance proteins, reduced cellular uptake of the drug, enhanced DNA repair, intracellular drug inactivation, and evasion of apoptosis. Pre-clinical models have demonstrated that many chemotherapy drugs, such as platinum-based agents, antracyclines, and taxanes, promote the activation of the NF-κB pathway. NF-κB is a key transcription factor, playing a role in the development and progression of cancer and chemoresistance through the activation of a multitude of mediators including anti-apoptotic genes. Consequently, NF-κB has emerged as a promising anti-cancer target. Here, we describe the role of NF-κB in cancer and in the development of resistance, particularly cisplatin. Additionally, the potential benefits and disadvantages of targeting NF-κB signaling by pharmacological intervention will be addressed.

Polysubstance use in cannabis users referred for treatment: drug use profiles, psychiatric comorbidity and cannabis-related beliefs

Background: Population-based surveys demonstrate cannabis users are more likely to use both illicit and licit substances, compared with non-cannabis users. Few studies have examined the substance use profiles of cannabis users referred for treatment. Co-existing mental health symptoms and underlying cannabis-related beliefs associated with these profiles remains unexplored. Methods: Comprehensive drug use and dependence severity (Severity of Dependence Scale-Cannabis) data were collected on a sample of 826 cannabis users referred for treatment. Patients completed the General Health Questionnaire, Cannabis Expectancy Questionnaire, Cannabis Refusal Self-Efficacy Questionnaire, and Positive Symptoms and Manic-Excitement subscales of the Brief Psychiatric Rating Scale. Latent class analysis was performed on last month use of drugs to identify patterns of multiple drug use. Mental health comorbidity and cannabis beliefs were examined by identified drug use pattern. Results: A three-class solution provided the best fit to the data: (1) cannabis and tobacco users (n = 176), (2) cannabis, tobacco, and alcohol users (n = 498), and (3) wide-ranging sub- stance users (n = 132). Wide-ranging substance users (3) reported higher levels of cannabis dependence severity, negative cannabis expectancies, lower opportunistic, and emotional relief self-efficacy, higher levels of depression and anxiety and higher manic-excitement and positive psychotic symptoms. Conclusion: In a sample of cannabis users referred for treatment, wide-ranging substance use was associated with elevated risk on measures of cannabis dependence, co-morbid psychopathology, and dysfunctional cannabis cognitions. These findings have implications for cognitive-behavioral assessment and treatment.

Proteins, drug targets and the mechanisms they control : the simple truth about complex networks

Realizing the promise of molecularly targeted inhibitors for cancer therapy will require a new level of knowledge about how a drug target is wired into the control circuitry of a complex cellular network. Here we review general homeostatic principles of cellular networks that enable the cell to be resilient in the face of molecular perturbations, while at the same time being sensitive to subtle input signals. Insights into such mechanisms may facilitate the development of combination therapies that take advantage of the cellular control circuitry, with the aim of achieving higher efficacy at a lower drug dosage and with a reduced probability of drug-resistance development.

Artemisinin resistance in Plasmodium falciparum : a process linked to dormancy?

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria in over 100 countries and is the cornerstone of malaria control and elimination programs in these areas. However, despite the high potency and rapid parasite killing action of ART derivatives there is a high rate of recrudescence associated with ART monotherapy and recrudescence is not uncommon even when ACT is used. Compounding this problem are reports that some parasites in Cambodia, a known foci of drug resistance, have decreased in vivo sensitivity to ART. This raises serious concerns for the development of ART resistance in the field even though no major phenotypic and genotypic changes have yet been identified in these parasites. In this article we review available data on the characteristics of ART, its effects on Plasmodium falciparum parasites and present a hypothesis to explain the high rate of recrudescence associated with this potent class of drugs and the current enigma surrounding ART resistance.

No genetic bottleneck in Plasmodium falciparum wild-type Pfcrt alleles reemerging in Hainan Island, China, following high-level chloroquine resistance

Chloroquine-resistant Plasmodium falciparum was highly prevalent in Hainan, China, in the 1970s. Twenty-five years after cessation of chloroquine therapy, the prevalence of P. falciparum wild-type Pfcrt alleles has risen to 36% (95% confidence interval, 22.1 to 52.4%). The diverse origins of wild-type alleles indicate that there was no genetic bottleneck caused by high chloroquine resistance.

Slaving and release in co-infection control

Background Animal and human infection with multiple parasite species is the norm rather than the exception, and empirical studies and animal models have provided evidence for a diverse range of interactions among parasites. We demonstrate how an optimal control strategy should be tailored to the pathogen community and tempered by species-level knowledge of drug sensitivity with use of a simple epidemiological model of gastro-intestinal nematodes. Methods We construct a fully mechanistic model of macroparasite co-infection and use it to explore a range of control scenarios involving chemotherapy as well as improvements to sanitation. Results Scenarios are presented whereby control not only releases a more resistant parasite from antagonistic interactions, but risks increasing co-infection rates, exacerbating the burden of disease. In contrast, synergisms between species result in their becoming epidemiologically slaved within hosts, presenting a novel opportunity for controlling drug resistant parasites by targeting co-circulating species. Conclusions Understanding the effects on control of multi-parasite species interactions, and vice versa, is of increasing urgency in the advent of integrated mass intervention programmes.

Targeting drug-resistant prostate cancer with dual PI3K/mTOR inhibition

The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is one of the most frequently activated signaling pathways in prostate cancer cells, and loss of the tumor suppressor PTEN and amplification of PIK3CA are the two most commonly detected mechanisms for the activation of these pathways. Aberrant activation of PI3K/Akt/mTOR has been implicated not only in the survival and metastasis of prostate cancer cells but also in the development of drug resistance. As such, selective inactivation of this pathway may provide opportunities to attack prostate cancer from all fronts. However, while preclinical studies examining specific inhibitors of PI3K or mTOR have yielded promising results, the evidence from clinical trials is less convincing. Emerging evidence from the analyses of some solid tumors suggests that a class of dual PI3K/mTOR inhibitors, which bind to and inactivate both PI3K and mTOR, may achieve better anti-cancer outcomes. In this review, we will summarize the mechanisms of action of these inhibitors, their effectiveness when used alone or in combination with other chemotherapeutic compounds, and their potential to serve as the next generation therapies for prostate cancer patients, particularly those who are resistant to the frontline chemotherapeutic drugs.

Comparative study of coagulase negative Staphylococci (CoNS) from clinical isolates, skin and nasal sources

Staphylococci are important pathogenic bacteria responsible for a range of diseases in humans. The most frequently isolated microorganisms in a hospital microbiology laboratory are staphylococci. The general classification of staphylococci divides them into two major groups; Coagulase-positive staphylococci (e.g. Staphylococcus aureus) and Coagulase-negative staphylococci (e.g. Staphylococcus epidermidis). Coagulase-negative staphylococcal (CoNS) isolates include a variety of species and many different strains but are often dominated by the most important organism of this group, S. epidermidis. Currently, these organisms are regarded as important pathogenic organisms causing infections related to prosthetic materials and surgical wounds. A significant number of S. epidermidis isolates are also resistant to different antimicrobial agents. Virulence factors in CoNS are not very clearly established and not well documented. S. epidermidis is evolving as a resistant and powerful microbe related to nosocomial infections because it has different properties which independently, and in combination, make it a successful infectious agent, especially in the hospital environment. Such characteristics include biofilm formation, drug resistance and the evolution of genetic variables. The purpose of this project was to develop a novel SNP genotyping method to genotype S. epidermidis strains originating from hospital patients and healthy individuals. High-Resolution Melt Analysis was used to assign binary typing profiles to both clinical and commensal strains using a new bioinformatics approach. The presence of antibiotic resistance genes and biofilm coding genes were also interrogated in these isolates.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11–19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.

Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

The identification of therapeutic targets in metastatic melanoma

Metastatic melanoma, a cancer historically refractory to chemotherapeutic strategies, has a poor prognosis and accounts for the majority of skin cancer related mortality. Although the recent approval of two new drugs combating this disease, Ipilimumab and Vemurafenib (PLX4032), has demonstrated for the first time in decades an improvement in overall survival; the clinical efficacy of these drugs has been marred by severe adverse immune reactions and acquired drug resistance in patients, respectively. Thus, understanding the etiology of metastatic melanoma will contribute to the improvement of current therapeutic strategies while leading to the development of novel drug approaches. In order to identify recurrently mutated genes of therapeutic relevance in metastatic melanoma, a panel of stage III local lymph node melanomas were extensively characterised using high-throughput genomic technologies. This led to the identification of mutations in TFG in 5% of melanomas from a candidate gene sequencing approach using SNP array analysis, 24% of melanomas with mutations in MAP3K5 or MAP3K9 though unbiased whole-exome sequencing strategies, and inactivating mutations in NF1 in BRAF/NRAS wild type tumours though pathway analysis. Lastly, this thesis describes the development of a melanoma specific mutation panel that can rapidly identify clinically relevant mutation profiles that could guide effective treatment strategies through a personalised therapeutic approach. These findings are discussed in respect to a number of important issues raised by this study including the current limitation of next-generation sequencing technology, the difficulty in identifying ‘driver’ mutations critical to the development of melanoma due to high carcinogenic exposure by UV radiation, and the ultimate application of mutation screening in a personalised therapeutic setting. In summary, a number novel genes involved in metastatic melanoma have been identified that may have relevance for current therapeutic strategies in treating this disease.

A high-throughput panel for identifying clinically relevant mutation profiles in melanoma

Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient's tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n = 154, 57%), NRAS (n = 55, 20%), CDK4 (n = 8, 3%), PTK2B (n = 7, 2.5%), and ERBB4 (n = 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs

Management of endocrine resistant breast cancer

Metastatic breast cancer (MBC) may present de novo but more commonly develops in women initially presenting with early breast cancer despite the widespread use of adjuvant hormonal and cytotoxic chemotherapy. MBC is incurable. Hormone sensitive MBC eventually becomes resistant to endocrine therapy in most women. Anthracyclines are the agents of choice in the treatment of endocrine resistant MBC. With the widespread use of anthracyclines in the adjuvant setting, taxanes have become the agents of choice for many patients. Recently capecitabine has become established as a standard of care for patients pretreated with anthracyclines and taxanes. However, a range of agents have activity as third line treatment. These include gemcitabine, vinorelbine and platinum analogues. The sequential use of non-cross resistant single agents rather than combination therapy is preferable in most women with MBC. Even though combination therapy can improve response rates and increase progression free interval, there is no robust evidence to indicate an advantage in terms of overall survival. Moreover, combination therapy is associated with a higher toxicity rate and poor quality of life. There is no role for dose-intense therapy, high dose therapy or maintenance chemotherapy outside the context of a clinical trial. The introduction of trastuzumab, monoclonal antibody targeting growth factor receptors, has improved the therapeutic options for women with tumours overexpressing HER2/neu. DNA micro-array profiles of tumours can potentially help to individualise therapy in future. Molecular targeted therapy has the potential to revolutionise the management of MBC.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.