Microarray expression profiling in melanoma reveals a BRAF mutation signature

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Quality of life and genetics in men with prostate cancer

As family history has been established as a risk factor for prostate cancer, attempts have been made to isolate predisposing genetic variants that are related to hereditary prostate cancer. With many genetic variants still to be identified and investigated, it is not yet possible to fully understand the impact of genetic variants on prostate cancer development. The high survival rates among men with prostate cancer have meant that other issues, such as quality of life (QoL), have also become important. Through their effect on a person’s health, a range of inherited genetic variants may potentially influence QoL in men with prostate cancer, even prior to treatment. Until now, limited research has been conducted on the relationship between genetics and QoL. Thus, this study contributes to an emerging field by aiming to identify certain genetic variants related to the QoL found in men with prostate cancer. It is hoped that this study may lead to future research that will identify men who have an increased risk of a poor QoL following prostate cancer treatment, which will aid in developing treatments that are individually tailored to support them. Previous studies have established that genetic variants of Vascular Endothelial Growth Factor (VEGF) and Insulin-like Growth Factor 1 (IGF-1) may play a role in prostate cancer development. VEGF and IGF-1 have also been reported to be associated with QoL in people with ovarian cancer and colorectal cancer, respectively. This study completed a series of secondary analyses using two major data-sets (from 850 men newly diagnosed with prostate cancer, and approximately 550 men from the general Queensland population), in which genetic variants of VEGF and IGF-1 were investigated for associations with prostate cancer susceptibility and QoL. The first aim of this research was to investigate genetic variants in the VEGF and IGF-I gene for an association with the risk of prostate cancer. It was found that one IGF-1 genetic variant (rs35765) had a statistically significant association with prostate cancer (p = 0.04), and one VEGF genetic variant (rs2146323) had a statistically significant association with advanced prostate cancer (p = 0.02). The estimates suggest that carriers of the CA and AA genotype for rs35765 may have a reduced risk of developing prostate cancer (Odds Ratio (OR) = 0.72, 95% Confidence Interval (CI) = 0.55, 0.95, OR = 0.60, 95% CI = 0.26, 1.39, respectively). Meanwhile, carriers of the CA and AA genotype for rs2146323 may be at increased risk of advanced prostate cancer, which was determined by a Gleason score of above 7 (OR = 1.72, 95% CI = 1.12, 2.63, OR = 1.90, 95% CI = 1.08, 3.34, respectively). Utilising the widely used short-form health survey, the SF-36v2, the second aim of this study was to investigate the relationship between prostate cancer and QoL prior to treatment. Assessing QoL at this time-point was important as little research has been conducted to evaluate if prostate cancer affects QoL regardless of treatment. The analyses found that mean SF-36v2 scale scores related to physical health were higher by at least 0.3 Standard Deviations (SD) among men with prostate cancer than the general population comparison group. This difference was considered clinically significant (defined by group differences in mean SF-36v2 scores by at least 0.3 SD). These differences were also statistically significant (p<0.05). Mean QoL scale scores related to mental health were similar between men with prostate cancer and those from the general population comparison group. The third aim of this study was to investigate genetic variants in the VEGF and IGF-1 gene for an association with QoL in prostate cancer patients prior to their treatment. It was essential to evaluate these relationships prior to treatment, before the involvement of these genes was potentially interrupted by treatment. The analyses found that some genetic variants had a small clinically significant association (0.3 SD) to some QoL domains experienced by these men. However, most relationships were not statistically significant (p>0.05). Most of the associations found identified that a small sub-group of men with prostate cancer (approximately 2%) reported, on average, a slightly better QoL than the majority of the prostate cancer patients. The fourth aim of this research was to investigate whether associations between genetic variants in VEGF and IGF-1 and QoL were specific to men with prostate cancer, or were also applicable to the general male population. It was found that twenty out of one-hundred relationships between the genetic variants of VEGF and IGF-1 and QoL health-measures and scales examined differed between these groups. In the majority of the relationships involving VEGF SNPs that differed, a clinically significant difference (0.3 or more SD) between mean scores among the genotype groups in prostate cancer patients was found, while mean scores among men from the general-population comparison group were similar. For example, prostate cancer participants who carried at least one T allele (CT or TT genotype) for rs3024994 had a clinically significant higher (0.3 SD) mean QoL score in terms of the role-physical scale, than participants who carried the CC genotype. This was not seen among men from the general population sample, as the mean score was similar between genotype groups. The opposite was seen in regards to the IGF-1 SNPs examined. Overall, these relationships were not considered to directly impact on the clinical options for men with prostate cancer. As this study utilised secondary data from two separate studies, there are a number of important limitations that should be acknowledged including issues of multiple comparisons, power, and missing or unavailable data. It is recommended that this study be replicated as a better-designed study that takes greater consideration of the many factors involved in prostate cancer and QoL. Investigation into other genetic variants of VEGF or IGF-1 is also warranted, as is consideration of other genes and their relationship with QoL. Through identifying certain genetic variants that have a modest association to prostate cancer, this project adds to the knowledge surrounding VEGF and IGF-1 and their role in prostate cancer susceptibility. Importantly, this project has also introduced the potential role genetics plays in QoL, through investigating the relationships between genetic variants of VEGF and IGF-1 and QoL.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

Background: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. Methods: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. Results: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. Conclusions: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target. © 2005 Lee et al., licensee BioMed Central Ltd.

Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G1-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G1-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G 1 into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

Cellular settings mediating Src Substrate switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

Targeting aurora kinases : a novel approach to curb the growth and chemoresistance of androgen refractory prostate cancer

Aurora Kinase (AK) based therapy targeting AK-A & B is effective against some cancers. We have explored its potential against previously unreported incurable, metastatic androgen depletion independent Prostate Cancer (ADIPC). We used androgen sensitive (AS) and ADI lines derived from Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice. The relevance of this model was unequivocally established through focussed array, quantitative PCR and western blotting studies; significantly greater alteration of genes (fold change and number) representing major cancer pathways was shown in ADI cells compared to AS lines. A marked enhancement of in vivo growth of the ADI subline showing the greatest degree of gene modulations [TRAMP C1 (TC1)-T5: TC1-T5] reflected this. In contrast to the parental AS TC1 line, TC1-T5 cells grew with 100% incidence in the prostate, as lung pseudometastases and migrated to the bone and other soft tissues. The potential involvement of AKs in this transition was indicated by the significant upregulation of AK-A/B and their downstream regulators, survivin and phosphorylated-histone H3 in TC1-T5 cells compared to TC1 cells. This led to enhanced sensitivity of TC1-T5 cells to the pan-AK inhibitor, VX680 and to significant reduction in in vivo tumour growth rates when AK-A and/or B were downregulated in TC1-T5 cells. This cell growth inhibition was markedly enhanced when both AKs were downregulated and also led to substantially greater sensitivity of these cells to docetaxel, the only chemotherapeutic with activity against ADI PC. Finally, use of VX680 with docetaxel led to impressive synergies suggesting promise for treating clinical ADI metastatic PC.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

Signaling of receptor tyrosine kinases in the nucleus

Since the discovery of the first receptor tyrosine kinase (RTK) proteins in the late 1970s and early 1980s, many scientists have explored the functions of these important cell signaling molecules. The finding that these proteins are often deregulated or mutated in diseases such as cancers and diabetes, together with their potential as clinical therapeutic targets, has further highlighted the necessity for understanding the signaling functions of these important proteins. The mechanisms of RTK regulation and function have been recently reviewed by Lemmon & Schlessinger (2010) but in this review we instead focus on the results of several recent studies that show receptor tyrosine kinases can function from subcellular localisations, including in particular the nucleus, in addition to their classical plasma membrane location. Nuclear localisation of receptor tyrosine kinases has been demonstrated to be important for normal cell function but is also believed to contribute to the pathogenesis of several human diseases.

A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib and ponatinib ATP-competitive inhibitors

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified fourteen dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (EC). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors. Whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, 3/7 showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R) conferred resistance (~5 fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also shows ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Evolutionary genetics and human assisted movement of a globally invasive pest (Russian wheat aphid : Diuraphis noxia)

This PhD study has examined the population genetics of the Russian wheat aphid (RWA, Diuraphis noxia), one of the world’s most invasive agricultural pests, throughout its native and introduced global range. Firstly, this study investigated the geographic distribution of genetic diversity within and among RWA populations in western China. Analysis of mitochondrial data from 18 sites provided evidence for the long-term existence and expansion of RWAs in western China. The results refute the hypothesis that RWA is an exotic species only present in China since 1975. The estimated date of RWA expansion throughout western China coincides with the debut of wheat domestication and cultivation practices in western Asia in the Holocene. It is concluded that western China represents the limit of the far eastern native range of this species. Analysis of microsatellite data indicated high contemporary gene flow among northern populations in western China, while clear geographic isolation between northern and southern populations was identified across the Tianshan mountain range and extensive desert regions. Secondly, this study analyzed the worldwide pathway of invasion using both microsatellite and endosymbiont genetic data. Individual RWAs were obtained from native populations in Central Asia and the Middle East and invasive populations in Africa and the Americas. Results indicated two pathways of RWA invasion from 1) Syria in the Middle East to North Africa and 2) Turkey to South Africa, Mexico and then North and South America. Very little clone diversity was identified among invasive populations suggesting that a limited founder event occurred together with predominantly asexual reproduction and rapid population expansion. The most likely explanation for the rapid spread (within two years) from South Africa to the New World is by human movement, probably as a result of the transfer of wheat breeding material. Furthermore, the mitochondrial data revealed the presence of a universal haplotype and it is proposed that this haplotype is representative of a wheat associated super-clone that has gained dominance worldwide as a result of the widespread planting of domesticated wheat. Finally, this study examined salivary gland gene diversity to determine whether a functional basis for RWA invasiveness could be identified. Peroxidase DNA sequence data were obtained for a selection of worldwide RWA samples. Results demonstrated that most native populations were polymorphic while invasive populations were monomorphic, supporting previous conclusions relating to demographic founder effects in invasive populations. Purifying selection most likely explains the existence of a universal allele present in Middle Eastern populations, while balancing selection was evident in East Asian populations. Selection acting on the peroxidase gene may provide an allele-dependent advantage linked to the successful establishment of RWAs on wheat, and ultimately their invasion potential. In conclusion, this study is the most comprehensive molecular genetic investigation of RWA population genetics undertaken to date and provides significant insights into the source and pathway of global invasion and the potential existence of a wheat-adapted genotype that has colonised major wheat growing countries worldwide except for Australia. This research has major biosecurity implications for Australia’s grain industry.

Molecular genetics of migraine

Migraine is a common neurological disorder with a significantly heritable component. It is a complex disease and despite numerous molecular genetic studies, the exact pathogenesis causing the neurological disturbance remains poorly understood. Although several known molecular mechanisms have been associated with an increased risk for developing migraine, there remains significant scope for future studies. The majority of studies have investigated the most plausible candidate genes involved in common migraine pathogenesis utilising criteria that takes into account a combination of physiological functionality in conjunction with regions of genomic association. Thus, far genes involved in neurological, vascular or hormonal pathways have been identified and investigated on this basis. Genome-wide association studies (GWAS) studies have helped to identify novel regions that may be associated with migraine and have aided in providing the basis for further molecular investigations. However, further studies utilising sequencing technologies are required to characterise the genetic basis for migraine.