Computational approaches in cancer multidrug resistance research: Identification of potential biomarkers, drug targets and drug-target interactions.

Like physics in the 19th century, biology and molecular biology in particular, has been fertilized and enhanced like few other scientific fields, by the incorporation of mathematical methods. In the last decades, a whole new scientific field, bioinformatics, has developed with an output of over 30,000 papers a year (Pubmed search using the keyword "bioinformatics"). Huge databases of mass throughput data have been established, with ArrayExpress alone containing more than 2.7 million assays (October 2019). Computational methods have become indispensable tools in molecular biology, particularly in one of the most challenging areas of cancer research, multidrug resistance (MDR). However, confronted with a plethora of different algorithms, approaches, and methods, the average researcher faces key questions: Which methods do exist? Which methods can be used to tackle the aims of a given study? Or, more generally, how do I use computational biology/bioinformatics to bolster my research? The current review is aimed at providing guidance to existing methods with relevance to MDR research. In particular, we provide an overview on: a) the identification of potential biomarkers using expression data; b) the prediction of treatment response by machine learning methods; c) the employment of network approaches to identify gene/protein regulatory networks and potential key players; d) the identification of drug-target interactions; e) the use of bipartite networks to identify multidrug targets; f) the identification of cellular subpopulations with the MDR phenotype; and, finally, g) the use of molecular modeling methods to guide and enhance drug discovery. This review shall serve as a guide through some of the basic concepts useful in MDR research. It shall give the reader some ideas about the possibilities in MDR research by using computational tools, and, finally, it shall provide a short overview of relevant literature.

Patterns of genomic evolution in advanced melanoma.

Genomic alterations occurring during melanoma progression and the resulting genomic heterogeneity between metastatic deposits remain incompletely understood. Analyzing 86 metastatic melanoma deposits from 53 patients with whole-exome sequencing (WES), we show a low branch to trunk mutation ratio and little intermetastatic heterogeneity, with driver mutations almost completely shared between lesions. Branch mutations consistent with UV damage indicate that metastases may arise from different subclones in the primary tumor. Selective gain of mutated BRAF alleles occurs as an early event, contrasting whole-genome duplication (WGD) occurring as a late truncal event in about 40% of cases. One patient revealed elevated mutational diversity, probably related to previous chemotherapy and DNA repair defects. In another patient having received radiotherapy toward a lymph node metastasis, we detected a radiotherapy-related mutational signature in two subsequent distant relapses, consistent with secondary metastatic seeding. Our findings add to the understanding of genomic evolution in metastatic melanomas.

High number of kinome-mutations in non-small cell lung cancer is associated with reduced immune response and poor relapse-free survival.

Lung cancer is the leading cause of cancer related death, and the past years' improved insight into underlying molecular events has significantly improved outcome for specific subsets of patients. In particular, several new therapies that target protein kinases have been implemented, and many more are becoming available. We have investigated lung cancer specimens for somatic mutations in a targeted panel of 612 human genes, the majority being protein kinases. The somatic mutation profiles were correlated to profiles of immune cell infiltration as well as relapse-free survival. Targeted deep sequencing was performed on 117 tumour/normal pairs using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons and untranslated regions of all known kinases, kinase receptors and selected cancer-related genes (612 genes in total). CD8 staining was determined using Ventana Benchmark. Survival analyses were performed using SPSS. The number of mutations per sample ranged from 0 to 50 (within the 612 genes tested), with a median of nine. The prognosis was worse for patients with more than the median number of mutations. A significant correlation was found between mutations in one of selected DNA-repair genes and the total number of mutations in that tumour (p < 0.001). There was a significant inverse correlation between the number of infiltrating stromal CD8+ lymphocytes and the presence of EGFR mutations.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis.

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

Increased expression of IRF4 and ETS1 in CD4+ cells from patients with intermittent allergic rhinitis.

BACKGROUND: The transcription factor (TF) IRF4 is involved in the regulation of Th1, Th2, Th9, and Th17 cells, and animal studies have indicated an important role in allergy. However, IRF4 and its target genes have not been examined in human allergy. METHODS: IRF4 and its target genes were examined in allergen-challenged CD4(+) cells from patients with IAR, using combined gene expression microarrays and chromatin immunoprecipitation chips (ChIP-chips), computational target prediction, and RNAi knockdowns. RESULTS: IRF4 increased in allergen-challenged CD4(+) cells from patients with IAR, and functional studies supported its role in Th2 cell activation. IRF4 ChIP-chip showed that IRF4 regulated a large number of genes relevant to Th cell differentiation. However, neither Th1 nor Th2 cytokines were the direct targets of IRF4. To examine whether IRF4 induced Th2 cytokines via one or more downstream TFs, we combined gene expression microarrays, ChIP-chips, and computational target prediction and found a putative intermediary TF, namely ETS1 in allergen-challenged CD4(+) cells from allergic patients. ETS1 increased significantly in allergen-challenged CD4(+) cells from patients compared to controls. Gene expression microarrays before and after ETS1 RNAi knockdown showed that ETS1 induced Th2 cytokines as well as disease-related pathways. CONCLUSIONS: Increased expression of IRF4 in allergen-challenged CD4(+) cells from patients with intermittent allergic rhinitis leads to activation of a complex transcriptional program, including Th2 cytokines.

CLC and IFNAR1 are differentially expressed and a global immunity score is distinct between early- and late-onset colorectal cancer.

Colorectal cancer (CRC) incidence increases with age, and early onset of the disease is an indication of genetic predisposition, estimated to cause up to 30% of all cases. To identify genes associated with early-onset CRC, we investigated gene expression levels within a series of young patients with CRCs who are not known to carry any hereditary syndromes (n=24; mean 43 years at diagnosis), and compared this with a series of CRCs from patients diagnosed at an older age (n=17; mean 79 years). Two individual genes were found to be differentially expressed between the two groups, with statistical significance; CLC was higher and IFNAR1 was less expressed in early-onset CRCs. Furthermore, genes located at chromosome band 19q13 were found to be enriched significantly among the genes with higher expression in the early-onset samples, including CLC. An elevated immune content within the early-onset group was observed from the differentially expressed genes. By application of outlier statistics, H3F3A was identified as a top candidate gene for a subset of the early-onset CRCs. In conclusion, CLC and IFNAR1 were identified to be overall differentially expressed between early- and late-onset CRC, and are important in the development of early-onset CRC.

Connectivity can be used to identify key genes in DNA microarray data: a study based on gene expression in nasal polyps before and after treatment with glucocorticoids.

CONCLUSIONS: The presented analysis of nasal polyposis using connectivity based on the PubGene literature co-citation network demonstrates that this tool can be used to identify key genes in DNA microarray studies of human polygenic diseases. OBJECTIVES: DNA microarray studies of complex diseases may reveal differential expression of hundreds of genes. According to network theory and studies of yeast cells, genes that are connected with several other genes appear to have key regulatory roles. This study aimed to examine if this principle can be translated to DNA microarray studies of human disease, using nasal polyposis as a base for the analysis. MATERIALS AND METHODS: The connectivity of differentially expressed genes from a previously described microarray study of nasal polyposis before and after treatment with glucocorticoids was determined. This was done using the literature co-citation network PubGene. RESULTS: In all, 166 genes were differentially expressed; 39 of these were previously defined as inflammatory and considered important for nasal polyposis. The connectivity of all differentially expressed genes was analysed using the PubGene literature co-citation network. Seventy-four of the 166 genes were connected to other genes. By contrast, the average number of connected genes among 100 sets of 166 randomly chosen genes was 31.5. A small number of the differentially expressed genes were highly connected, while most genes had few or no connections. This indicated a scale-free network. The most connected gene was interleukin-8, an inflammatory gene of known importance for nasal polyposis. Twenty-eight of the 74 connected genes were inflammatory (38%), compared with 11 of the 92 unconnected genes (12%), p < 0.0001. Since most evidence suggests that nasal polyps are inflammatory in their nature, this supports the hypothesis that connected genes have more disease relevance than unconnected genes.

Photochemically induced gene silencing using small interfering RNA molecules in combination with lipid carriers.

Novel strategies for efficient delivery of small interfering RNA (siRNA) molecules with a potential for targeting are required for development of RNA interference (RNAi) therapeutics. Here, we present a strategy that is based on delivery of siRNA molecules through the endocytic pathway, in order to develop a method for site-specific gene silencing. To achieve this, we combined the use of cationic lipids and photochemical internalization (PCI). Using the human S100A4 gene as a model system, we obtained potent gene silencing in four tested human cancer cell lines following PCI induction when using the cationic lipid jetSI-ENDO. Gene silencing was shown at both the RNA and protein levels, with no observed PCI toxicity when using the jetSI reagent and an optimized PCI protocol. This novel induction method opens for in vivo site-specific delivery of siRNA molecules toward a sequence of interest.

Photochemically induced gene silencing using PNA-peptide conjugates.

The potential for exploration of peptide nucleic acid (PNA) as an experimental and therapeutic regulator of gene expression has been hampered by a poor delivery and a lack of site-specific targeting. In the present study, we have developed an efficient strategy for nuclear delivery of PNA by combining cationically charged PNA-peptide conjugates and photochemical internalization (PCI) technology. When using the S100A4 gene as a model system, a consistent downregulation to around 10% remaining protein signal was obtained in three selected cell lines. Furthermore, a dose-dependent and time-dependent inhibition of the S100A4 protein was demonstrated. A main benefit of the strategy proposed is the possibility of site-specific targeting.

BRCA1 mutations in ovarian cancer and borderline tumours in Norway: a nested case-control study.

The aims of the present study were to find the frequency of the most common BRCA1 mutations in women with ovarian tumours identified from a population-based cancer registry and in the general population, to estimate the relative risk of ovarian tumours among the mutation carriers, and to explore the value of using CA125 as a prediagnostic test. The study was designed as a nested case-control study within a cohort mainly consisting of participants in population-based health examinations. The data files of The Cancer Registry of Norway and the Janus serum bank were linked to identify cases with ovarian cancer and borderline tumours. Hereditary BRCA1 mutations were determined using archived serum samples and capillary electrophoresis. Altogether 478 ovarian cancer patients and 190 patients with borderline tumours were identified, and 1421 and 568 matching controls were selected. Odds ratios (OR) of developing ovarian cancer and borderline tumours in the presence of BRCA1 mutations and CA125 level were derived from conditional logistic regression models. Among the 478 ovarian cancer patients, 19 BRCA1 mutations were identified (1675delA, 1135insA, 816delGT and 3347delAG), none among the patients with borderline tumours. Only two of the 1989 controls were BRCA1 mutation carriers (0.10%). The risk of ovarian cancer among the mutation carriers was strongly elevated (OR=29, 95% CI=6.6-120). CA125 was a marker for ovarian cancer, but the sensitivity was low. This study showed that BRCA1 mutation carriers have a very high risk of ovarian cancer. However, since the prevalence of BRCA1 mutations in the Norwegian population was low, the proportion of ovarian cancers due to BRCA1 mutations seemed to be low, about 4%. The sensitivity of using CA125 only as a screening test for ovarian cancer was low.

Interferon-gamma suppresses S100A4 transcription independently of apoptosis or cell cycle arrest.

The S100A4 protein has been associated with increased metastatic capacity of cancer cells, and recent studies have suggested a correlation between the expression level of S100A4 and the prognostic outcome for patients with various types of cancer. The knowledge about the mechanisms underlying the metastasis-promoting effects is still limited, and the aim of the present study was to elucidate signal transduction pathways involved in the regulation of S100A4. After treatment of human carcinoma cells with interferon-gamma (IFN-gamma), we observed downregulation of S100A4 both at mRNA and protein levels. The effect was not dependent on IFN-gamma-induced apoptosis or IFN-gamma-mediated cell cycle arrest. Moreover, IFN-gamma-mediated decrease in mRNA stability could not account for the observed decrease in S100A4 transcript level. Finally, microarray analysis suggests ISGF3G, ETV5, ZNF133 and CEBPG as possible candidate genes involved in IFN-gamma-mediated repression of S100A4.

Associations between gene expressions in breast cancer and patient survival.

We analyzed associations between gene expression in breast cancer and patient survival for 8024 genes from a previously published microarray data set. Analysis of survival, by using the logrank test, was performed automatically for each gene. After correcting for multiple testing, we identified 95 genes whose expression was significantly associated with patient survival. The independent prognostic value of the genes ranking the highest in univariate analysis, together with clinical parameters, was assessed by Cox multivariate regression analysis. The P-values from these logrank tests were also mapped to chromosomal positions and compared with previously reported amplicon regions. We used PubGene web tools to identify groups of genes that had co-occurred in the literature and whose expression patterns were associated with survival. Our analyses demonstrate the comprehensiveness of the microarray technology with respect to measuring gene expression and indicate that the technology may be used to screen for potential clinical markers.

Genetic epidemiology of BRCA1 mutations in Norway.

Familial breast-ovarian cancer has been demonstrated to be frequent but unevenly distributed in Norway. This was assumed to be caused by the reduced population size created by the medieval Bubonic plagues 25 generations ago, and by the following rapid expansion. We have previously reported that four mutations account for 68% of the BRCA1 mutation carriers. Subsequent analysis has resulted in a total of 100 separate families carrying one of these founder mutations. The four mutations occurred on one specific BRCA1 haplotype each. The 1675delA, 816delGT and 3347delAG families originated from the South-West coast of Norway with a few families in the north, while the traceable ancestors of the 1135insA families clustered along the historical inland road from the South-East to mid-Norway. The carriers of each of the four mutations today are descendants of one or a few individuals surviving the plagues. We may identify the majority of BRCA1 mutation carriers in Norway by screening for local founder mutations.

Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA.

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.

The BRCA1 syndrome and other inherited breast or breast-ovarian cancers in a Norwegian prospective series.

Inherited breast cancer is a heterogenous group of diseases. We examined this heterogeneity in a prospective series of inherited breast and ovarian cancers, previously demonstrated to include 84% of inherited cancers. Ninety-two tumours (65 breast and 27 ovarian) in 82 patients from 70 kindreds were prospectively diagnosed. Fifteen of the breast cancers were in situ, 50 were infiltrating. 40 (49%) of the 82 women carried a BRCA1 mutation, whereas no mutation in BRCA2 was found. Approximately, two-thirds of the BRCA1 mutation carriers had one of the four most frequent Norwegian founder mutations. Ninety-five per cent of the epithelial ovarian cancers occurred in BRCA1 mutation carrying women versus 38% of infiltrating breast cancers and 7% of carcinoma in situ of the breast. The BRCA1 syndrome was phenotypically distinct with invasive, high grade, oestrogen receptor-negative breast cancers and epithelial ovarian cancers. Non-BRCA1/2 inherited breast cancers included carcinoma in situ and lobular carcinoma and were frequently bilateral. Non-BRCA1/2 inherited breast cancer is not associated with epithelial ovarian cancer and in breast cancers has distinct biological characteristics, indicating that the different subgroups of inherited breast cancer may need different healthcare services.

[Gene therapy in cancer]. / Genterapi ved kreft.

BACKGROUND: There has been a substantial increase in our understanding of the pathogenesis of cancer over the past decades, and new treatment modalities are being developed on the basis of this knowledge. MATERIAL AND METHODS: The literature is reviewed, and the status of gene therapy protocols approved in Norway is presented. RESULTS: About 70% of the more than 400 clinical gene therapy studies started are targeted at cancer. Several different principles are used, including gene therapy targeted at tumour suppressor genes, oncogenes, or central signaling molecules, as well as "suicide gene" therapy. In addition, various gene therapy protocols aim at strengthening immune responses. Most studies have been early clinical trials primarily designed to study safety, applicability and toxicity. Several of these phase I and II studies have, however, shown partial remission of tumours and, in rare cases, complete remission, although curation has not yet been shown. In some trials, including TP53 gene therapy trials, effects on tumour size have been observed in up to 50% of the patients. Up until now, only two phase III and one phase II/III studies have been initiated, but results from these studies have not yet been published. The two first gene therapy protocols approved in Norway are also targeted at cancer. So far, six patients in Norway have undergone gene therapy. INTERPRETATION: As of today, the results should be seen as promising for some of the principles which are being tried out; their clinical importance must, however, be documented in larger controlled clinical trials.

CA 125: the end of the beginning.

CA 125, a high-molecular-weight mucin, was first defined in 1981 by the monoclonal antibody OC125. Until recently, it has defied many attempts to purify it from a variety of sources, although many research groups have successfully raised antibodies that bind to CA 125. Nevertheless, CA 125 has demonstrated its considerable value as a marker in monitoring patients with ovarian cancer. This year, two research groups have succeeded in cloning the high-molecular-weight mucin CA 125. Their findings are summarized and the significance discussed in light of existing data from the human genome.

BRCA1 1675delA and 1135insA account for one third of Norwegian familial breast-ovarian cancer and are associated with later disease onset than less frequent mutations.

A total of 845 women from breast-ovarian cancer kindreds were enrolled in a clinical follow-up program for early disease diagnosis; 35 women were prospectively identified with cancer. In order to estimate the role of genetic factors for cancer predisposition in this well-defined set of patients, considered as representative for familial breast-ovarian cancer in the Norwegian population, the BRCA1 gene was investigated for germline mutations. The entire coding region of BRCA1 was analysed using a protein truncation test, direct sequencing and a screen for known large genomic deletions and insertions. Twenty one (60%) of the 35 patients were identified as carriers of 11 distinct BRCA1 mutations. Two previously described founder mutations, 1675delA and 1135insA, were found to account for more than half (11/21) of all BRCA1 cases and for almost one third (11/35) of all breast and ovarian cancers. Supported by a previous population-based analysis of these founder mutations in ovarian cancer, our findings suggest that a significant proportion of women at risk for developing inherited breast and ovarian cancer can be identified. This is particularly obvious in certain geographic regions where these founder mutations are prevalent. Women carrying the two founder mutations had a significantly older age of disease onset as compared to women with other BRCA1 mutations. This observation indicates that BRCA mutation penetrance estimates from populations with strong founder effects may be biased. One reason why some deleterious mutations are allowed to prevail in a population may be coupled to penetrance and the fact that they seldom induce disease in women in child-bearing ages. Eleven out of 12 (92%) breast cancers in BRCA1 mutation carriers were estrogen receptor negative, versus 4 out of 9 (44%) in mutation negative patients (p = 0.03). Histopathological characteristics of the prospectively detected cancers indicated an unfavourable prognosis in mutation carriers.

Three per cent of Norwegian ovarian cancers are caused by BRCA1 1675delA or 1135insA.

Our aim was to determine the prevalence of two Norwegian BRCA1 founder mutations in ovarian cancer patients, to identify carriers and their families for medical follow-up, and to study histopathological factors. Of a cohort of 727 ovarian cancer patients, 615 gave informed consent to testing. 2.9% (18/615) of the tested patients were found to be carriers of BRCA1 1675delA (n = 13) or 1135insA (n = 5). The total frequency of the mutations was 4.7% (8/171) in patients below 50 years of age, and zero (0/144) in patients above 70 years of age. In patients below 70 years of age, the frequency of 1675delA and 1135insA mutations was 2.8% and 1.0%, respectively. Out of 13 patients with 1675delA mutation, 4 had breast cancer. 14/16 (87.5%) families fulfilled clinical criteria for familial breast-ovarian cancer. Median age of onset of ovarian and breast cancer was 51 years and 37 years, respectively. Mutation carriers tended to have tumours with unfavourable prognostic factors. This is, to our knowledge, the highest reported frequency of founder mutations in a national ovarian cancer cohort (less than in the Ashkenazis). It seems justified to offer such testing to ovarian cancer patients below 70 years of age in Norway, identify their risk of breast cancer and offer medical follow-up to the families.

Expression of a novel factor in human breast cancer cells with metastatic potential.

Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.