Research Pillar: Biomedical

Dosage CIN genes: A comprehensive analysis of gene dosage effects on genome stability

Chemotherapy is one of our strongest weapons for treating cancer, but it also harms healthy cells and causes serious side effects in patients. Researchers at the Hieter Laboratory at the University of British Columbia in Vancouver hope to develop a more targeted approach, one that takes advantage of the genetic changes that exists in cancer. Their approach identifies which combination of genetic changes will selectively kill cancer cells. Answering that question will be key to developing new targeted drugs to fight cancer.   

Cancer cells often contain multiple gene mutations or changes which affect the stability of the genome, but whether this instability is a cause or consequence of cancer remains to be understood. A project led by Dr. Supipi Kaluarachchi Duffy is using high throughput genetic screens, overproducing one gene at a time in yeast, to identify which genes lead to genome stability. She will then identify genetic changes that are common in both yeast and human cancers and leverage these to find secondary genetic targets. Yeast is a great model organism for this work because it shares many of the fundamental biological pathways that are essential for life.

“The first step is distinguishing between a gene whose overproduction contributes to genome instability and a gene that has no effect,” says Duffy. “The end result would be more targeted chemotherapy at lower doses and with fewer side effects.”

Fight flu with mechanism-based covalent neuraminidase inhibitors

The development of resistance to commonly used antiviral drugs Relenza and Tamiflu has become a serious problem facing the world. It is reported that 98 percent of influenza A/H1N1 strains in North America are resistant to Tamiflu. The Withers group has designed a series of sialic acid analogs in which the C-2 OH group was replaced with fluoride to trap the virus by the formation of a covalent intermediate. To slow down the reactivation rates of neuraminidase, an electronegative flurorine was introduced at C-3 position to destabilize the positively charged transition state en-route to the formation and hydrolysis of the covalent intermediate. In addition, to confer specificity for the influenza neuraminidase over human sialidases, a positively charged nitrogen substituent was incorporated by analogy with the design of Relenza and Tamiflu. Those compounds showed very promising properties against the influenza virus.

Dr. Zhizeng Gao proposes to take advantage of the mechanism of action of this class of inhibitors, and develop a set of fluorescent and fluorogenic neuraminidase probes. Such probes share structural similarities with the inhibitor but the fluorine at C-2 was replaced with latent fluorophores, or C-7-OH was linked with fluorophores. Such probes are useful for investigation of the presence and role of neuraminidases in biological systems, especially if their presence or activity can be detected, quantitated and in some cases localized.

Placental proteomics: Gaining a system-wide understanding of the dynamic protease networks in normal placental tissue and upon inflammation to identify diagnostic signatures as biomarkers for preterm labor

Preterm birth affects approximately 12 percent of all deliveries. Prematurity is the leading cause of neo-natal mortality in Canada and is a major risk factor for impaired growth and development. There is a pressing need for tests to predict the risk of premature delivery accurately enough to provide the best treatment to prevent pre-term delivery and avoid unnecessary interventions.

It is thought that preterm labour can result from infection and inflammation of the placenta. Fetal and/or maternal inflammatory proteins in threatened preterm labour may form a diagnostic signature that can be used to predict whether preterm delivery is imminent or not. The Overall lab has developed several techniques to identify diagnostic inflammatory signatures in tissues. Using these methods, Dr. Eckhard aims to establish a functional, system-wide understanding of infection-induced inflammation in preterm labour using human placentas as a model of infection and inflammation.

Dr. Eckhard will elucidate placental molecular pathways that are activated in response to escalating infection resulting from the rupture of placental membranes. Collection of placentas from various documented times following membrane rupture to delivery will capture a range of inflammatory responses to infection — these “timed infection” placentas will be compared to non-inflamed placentas from full-term caesarean section deliveries and to placentas from defined pre-term deliveries to establish biomarkers and determine how infection-induced inflammation leads to pre-term labour.  

The role of histone methyltransferase SETD2 in the development of acute myeloid leukemia

Acute myeloid leukemia (AML) results from genetic defects. Recurrent  variations in chromosomal structures are common in AML, and several genes have been identified to be recurrently mutated in AML. Identification of these genetic defects in AML patients has improved diagnosis and treatment. However, more than twenty-five percent of AML patients carry no mutations in the known leukemia-associated genes, and the heterogeneity of AML and various survival outcomes suggest that as yet, undiscovered genes and pathways contribute to AML.

Dr. Gerben Duns performed high-throughput RNA sequencing and resequenced whole exomes, a portion of the genome, on 92 AML clinical samples to discover novel genes involved in AML. Mutations were identified within a gene called SETD2 in 7.6 percent of samples, suggesting a role for SETD2 in a subset of AML samples. The nature of the identified mutations suggests that these mutations are inactivating, which is in concordance with the recent identification of inactivating SETD2 mutations in several other cancer types.

Through in vitro and in vivo studies, Dr. Duns will examine the effects of the inactivating and mutating gene SETD2 as it contributes to AML development. Bioinformatic approaches are also used to investigate the potential association between the presence of SETD2 mutations and the response to therapy and disease outcome.

This study will provide insights into the mechanisms of AML pathogenesis, and will potentially reveal novel diagnostic and prognostic markers, as well as therapeutical targets.

Harnessing natural product biosynthetic machinery

Cladoniamide, a new alkaloid originally isolated from a bacterial strain found on the surface of a British Columbia lichen, has attracted attention as a potential new colorectal cancer drug.

The objective of this proposal is to harness the cladoniamide biosynthetic pathway to produce cladoniamide and new derivatives at high levels and generate new “hybrid” compounds by genetic manipulation of cladoniamide and related indolocarbazole biosynthetic pathway

This proposal aims to use biological and chemical tools to access new therapeutic molecules, specifically targeting colon cancer. The approach uses biosynthetic machinery from microorganisms to make complex natural products and their “unnatural” derivatives. Dr. Du will develop engineered biological platforms for the production of molecules that cannot be acquired in sufficient quantities through either organic synthesis or cultivation of a natural producer.

This project represents a unique approach that combines genomic and chemical tools to develop new drug leads from the cladoniamide scaffold. Results from this work will lay the groundwork for future, collaborative efforts in drug research, development, formulation, testing and commercialization.

Targeting androgen receptor dimerization as a potential therapy for prostate cancer

Prostate cancer is a leading cause of death in men. Treatment involves reducing production of di-hydro-testosterone (DHT) or blocking the interaction of this hormone with the androgen receptor (AR), a transcription factor responsible to drive expression of genes responsible for tumour growth. This treatment is unfortunately temporary and tumours eventually undergo genetic changes to become castration-resistant and able to grow in the absence of DHT.

This research project aims to use drugs to block AR activity in castration-resistant prostate cancer. Within the nucleus, two androgen receptor molecules must self-associate (“dimerize”) before binding DNA and initiating transcription. Dr. Dalal hypothesizes that slowing tumour growth can be achieved by preventing the AR-DNA interaction directly or by interfering with AR dimerization.

Cell biology and biochemistry approaches will allow Dr. Dalal to study the molecular mechanism of DNA-blocking or dimer-interfering compounds. Several compounds targeting both processes have been optimized to show potent and specific inhibition of the androgen receptor in cultured prostate cancer cells. Gel shift assays, calorimetry methods and confocal microscopy are now being used to gauge the effects of drugs on both DNA binding and AR dimerization. Site-directed mutagenesis of amino acids on the AR protein surface will validate the binding location of inhibitors. Promising compounds will be tested in mice to show effects on the size of prostate tumours.

Targeting transcription factor dimerization and DNA binding is a novel strategy that holds great promise to treat advanced forms of prostate and other cancers.

Beyond the known genome: long non-coding RNAs as novel therapeutic targets and biomarkers for metastatic prostate cancer

Recent evidence indicates that non-coding RNAs (NC-RNAs) play crucial functions in physiological and pathological cellular processes. Long non-coding RNAs (lncRNAs) are the most abundant NC-RNA class, accounting for 10–20,000 genes. Despite this, the role of only a few of them (approxim. 40) has been characterized. Many lncRNAs show a tissue-specific expression pattern and are altered in cancer cells. For this reason, it has been suggested that they may be useful as biomarkers in oncology.

We performed RNA-Seq. on non-metastatic and metastatic prostate cancer (PCa) tumor tissue xenografts. Our analysis revealed 159 up- and 77 down-regulated lncRNAs in the metastatic samples. We validated the differential expression of 7 up-regulated lncRNas in metastatic xenografts (QPCR). Using pooled plasma samples from 3 three patient groups (normal, localized PCa and metastatic PCa) one lncRNA (JUPITER) assayed to date differentiates amongst the three groups. We hypothesize that lncRNAs play critical roles in PCa progression and can be exploited as biomarkers and therapy targets. To address these hypotheses we will: 1: Characterize the function of selected lncRNAs. We will select the most up-regulated lncRNAs in metastatic vs. primary PCa xenografts and assay their expression in a panel of PCa cell lines. Once we identify 2-3 cell lines expressing the highest levels of a transcript, we will silence it using siRNAs. Silenced and control-treated cells will be assayed for proliferation, migration, invasion, apoptosis, and cell cycle progression. 2: Measure by QPCR the expression of selected lncRNas on RNA extracted from freshly frozen prostate samples (normal prostate, prostate intraepithelial neoplasia, local and metastatic PCa). For each gene, we will statistically compute correlations with clinico-pathological variables (grade, stage, PSA level). 3: Further analyze lncRNAs as biomarkers. The expression of JUPITER (and other differentially expressed lncRNAs) will be assayed in individual plasma samples from patients with different PCa stages, in order to estimate the optimal threshold values for early detection of metastatic PCa (ROC curve).

While localized PCa is a treatable disease, progression to a metastatic and drug-resistant cancer accounts for 4000 deaths annually in Canada. Understanding the mechanisms of Pca progression and identifying new molecular markers and therapeutic targets will allow better disease management and ultimately reduce deaths. In brief, we discovered a long non-coding RNA (PCAT18) that is expressed exclusively by prostate cancer cells and is required for prostate cancer cell growth and motility. This gene can be used as a biomarker and as a therapeutic target for metastatic prostate cancer.