Cyclins in Breast Cancer

Breast cancer is the most common malignancy in women in Western countries. It is a heterogeneous disease with varying biological characteristics and aggressiveness. Family history is one of the strongest predisposing factors for breast cancer. The known susceptibility genes explain only around 25% of all familial breast cancers. At least part of the unknown familial aggregation may be caused by several low-penetrance variants that occur commonly in the general population. Cyclins are cell cycle-regulating proteins. Cyclin expression oscillates during the cell cycle and is under strict control. In cancer cells, cyclin expression often becomes deregulated, leading to uncontrolled cell division and proliferation, one of the hallmarks of cancer. In this study, we investigated the role of cyclins in breast cancer predisposition, pathogenesis, and tumor behavior. Cyclin A immunohistochemistry was evaluated both on traditional large sections and on tissue microarrays (TMA). The concordance of the results was good, indicating that TMA is a reliable method for studying cyclin expression in breast cancer. The expression of cyclins D1, E, and B1 was studied among 1348 invasive breast cancers on TMA. Familial BRCA1/2-mutation negative tumors had significantly more often low cyclin E and high cyclin D1 expression than BRCA1/2 related or sporadic tumors. Unique cyclin E and D1 expression patterns among familial non-BRCA1/2 breast cancers may reflect different predisposition and pathogenesis in these groups and help to differentiate mutation-positive from mutation-negative familial cancers. High cyclin E expression was associated with an aggressive breast cancer phenotype and was an independent marker of poor metastasis-free survival. High cyclin D1 was associated with high grade and high proliferation among estrogen receptor (ER)-positive but with low grade and low proliferation among ER-negative breast cancers. Among ER-positive cancers not treated with chemotherapy, high cyclin D1 showed a trend towards shorter metastasis-free survival. These results suggest that different mechanisms may drive proliferation in ER-negative and -positive breast cancers and that cyclin D1 has a particularly important role in tumorigenesis of hormone receptor-positive breast cancer. High cyclin B1 expression was associated with aggressive breast cancer features and had an independent impact on survival. The results suggest that cyclin B1 immunohistochemistry is a method that could easily be adapted for routine use and is an independent prognostic factor, adding specificity to prognostic evaluation conducted with traditional markers. A commonly occurring cyclin D1 gene polymorphism A870G was associated with increased breast cancer risk in a large material of Finnish and Canadian breast cancer patients. The interaction of the high-activity alleles of cyclin D1 gene and estrogen metabolism gene COMT conferred an even higher risk. These results show that cyclin D1 and COMT act synergistically to contribute to breast cancer progression and that individual risk for breast cancer can be altered by the combined effect of polymorphisms with low-penetrance alleles. By investigating critical cell cycle regulator protein cyclins, we revealed new aspects of breast cancer predisposition, pathogenesis, and clinical course.

CHEK2 in Breast and Colorectal Cancer

Breast and colorectal cancers, are common types of cancer, with over two million newly diagnosed cases annually worldwide. Cancer is a genetic disease and defects in DNA integrity restoring functions make a significant contribution to cancer risk. CHEK2 is a checkpoint kinase functioning as a regulator of cell cycle checkpoints, apoptosis, and DNA repair in response to DNA double-strand breaks. The aim of this study was to evaluate the role of CHEK2 in breast cancer predisposition in Finnish breast cancer families and in breast cancer risk at the population level. We were interested in the clinical and biological characteristics of the breast tumors associated with the CHEK2 germline mutations or aberrant CHEK2 protein expression and the effect on survival of patients with these CHEK2 defects. We also assessed the role of CHEK2 mutations, namely 1100delC and I157T, in colorectal cancer susceptibility in Finland. CHEK2 I157T was found to be a low-penetrance breast cancer susceptibility allele, conferring a 1.4-fold risk for carriers. Reduced or absent CHEK2 protein expression was observed in one-fifth of breast tumors from patients unselected for family history, implying that defective CHEK2 signaling contributes to tumorigenesis. Reduction in CHEK2 expression was more common in tumors with larger diameter and ER expression, but with regard to other tumor characteristics and prognosis of a patient no association was observed. Results from comparison of CHEK2 1100delC carrier tumors with noncarrier tumors were in line with the findings from the CHEK2 expression study. Tumors from CHEK2 1100delC carriers were more often of higher grade than tumors from noncarriers, and they also tended to be ER-positive more often, although generally 1100delC status does not seem to radically affect the tumor characteristics. Our results suggest that CHEK2 1100delC may not be a susceptibility allele for CRC, although a very small effect cannot be excluded. Furthermore, CHEK2 1100delC is equally frequent in HBCC (hereditary breast and colorectal cancer) phenotype families and in breast cancer families. Over 1000 CRC cases were screened for CHEK2 I157T, and a significantly higher frequency of I157T was observed among both familial and sporadic CRC cases. The relation of CHEK2 I157T with familial CRC has not been studied previously. CHEK2 I157T seems to be a susceptibility allele for both familial and sporadic CRC, conferring a 1.5-fold risk for carriers of this variant. CHEK2 I157T has been proposed to have a role as a multiple cancer susceptibility allele, which is supported by our results since we observed a trend towards higher frequency of the variant among cases with multiple primary tumors or those with a family history of cancer. During the last five years CHEK2 has established its role as an important cancer susceptibility gene. It has become apparent that CHEK2 is a low-penetrance susceptibility gene for several cancer types, significantly contributing to familial cancer risk as well as to cancer risk at the population level.

A Nationwide Study on Breast Cancer Risk in Postmenopausal Women Using Hormone Therapy in Finland

Since national differences exist in genes, environment, diet and life habits and also in the use of postmenopausal hormone therapy (HT), the associations between different hormone therapies and the risk for breast cancer were studied among Finnish postmenopausal women. All Finnish women over 50 years of age who used HT were identified from the national medical reimbursement register, established in 1994, and followed up for breast cancer incidence (n= 8,382 cases) until 2005 with the aid of the Finnish Cancer Registry. The risk for breast cancer in HT users was compared to that in the general female population of the same age. Among women using oral or transdermal estradiol alone (ET) (n = 110,984) during the study period 1994-2002 the standardized incidence ratio (SIR) for breast cancer in users for < 5 years was 0.93 (95% confidence interval (CI) 0.80–1.04), and in users for ≥ 5 years 1.44 (1.29–1.59). This therapy was associated with similar rises in ductal and lobular types of breast cancer. Both localized stage (1.45; 1.26–1.66) and cancers spread to regional nodes (1.35; 1.09–1.65) were associated with the use of systemic ET. Oral estriol or vaginal estrogens were not accompanied with a risk for breast cancer. The use of estrogen-progestagen therapy (EPT) in the study period 1994-2005 (n= 221,551) was accompanied with an increased incidence of breast cancer (1.31;1.20-1.42) among women using oral or transdermal EPT for 3-5 years, and the incidence increased along with the increasing duration of exposure (≥10 years, 2.07;1.84-2.30). Continuous EPT entailed a significantly higher (2.44; 2.17-2.72) breast cancer incidence compared to sequential EPT (1.78; 1.64-1.90) after 5 years of use. The use of norethisterone acetate (NETA) as a supplement to estradiol was accompanied with a higher incidence of breast cancer after 5 years of use (2.03; 1.88-2.18) than that of medroxyprogesterone acetate (MPA) (1.64; 1.49-1.79). The SIR for the lobular type of breast cancer was increased within 3 years of EPT exposure (1.35; 1.18-1.53), and the incidence of the lobular type of breast cancer (2.93; 2.33-3.64) was significantly higher than that of the ductal type (1.92; 1.67-2.18) after 10 years of exposure. To control for some confounding factors, two case control studies were performed. All Finnish women between the ages of 50-62 in 1995-2007 and diagnosed with a first invasive breast cancer (n= 9,956) were identified from the Finnish Cancer Registry, and 3 controls of similar age (n=29,868) without breast cancer were retrieved from the Finnish national population registry. Subjects were linked to the medical reimbursement register for defining the HT use. The use of ET was not associated with an increased risk for breast cancer (1.00; 0.92-1.08). Neither was progestagen-only therapy used less than 3 years. However, the use of tibolone was associated with an elevated risk for breast cancer (1.39; 1.07-1.81). The case-control study confirmed the results of EPT regarding sequential vs. continuous use of progestagen, including progestagen released continuously by an intrauterine device; the increased risk was seen already within 3 years of use (1.65;1.32-2.07). The dose of NETA was not a determinant as regards the breast cancer risk. Both systemic ET, and EPT are associated with an elevation in the risk for breast cancer. These risks resemble to a large extent those seen in several other countries. The use of an intrauterine system alone or as a complement to systemic estradiol is also associated with a breast cancer risk. These data emphasize the need for detailed information to women who are considering starting the use of HT.

X-ray scattering and diffraction enhanced imaging studies of in vitro breast tissues

This thesis is a study of the x-ray scattering properties of tissues and tumours of the breast. Clinical radiography is based on the absorption of the x-rays when passing right through the human body and gives information about the densities of the tissues. Besides being absorbed, x-rays may change their direction within the tissues due to elastic scattering or even to refraction. The phenomenon of scattering is a nuisance to radiography in general, and to mammography in particular, because it reduces the quality of the images. However, scattered x-rays bear very useful information about the structure of the tissues at the supra-molecular level. Some pathologies, like breast cancer, produce alterations to the structures of the tissues, being especially evident in collagen-rich tissues. On the other hand, the change of direction due to refraction of the x-rays on the tissue boundaries can be mapped. The diffraction enhanced imaging (DEI) technique uses a perfect crystal to convert the angular deviations of the x-rays into intensity variations, which can be recorded as images. This technique is of especial interest in the cases were the densities of the tissues are very similar (like in mammography) and the absorption images do not offer enough contrast. This thesis explores the structural differences existing in healthy and pathological collagen in breast tissue samples by the small-angle x-ray scattering (SAXS) technique and compares these differences with the morphological information found in the DEI images and the histo-pathology of the same samples. Several breast tissue samples were studied by SAXS technique in the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Scattering patterns of the different tissues of the breast were acquired and compared with the histology of the samples. The scattering signals from adipose tissue (fat), connective tissue (collagen) and necrotic tissue were identified. Moreover, a clear distinction could be done between the scattering signals from healthy collagen and from collagen from an invasive tumour. Scattering from collagen is very characteristic. It includes several scattering peaks and scattering features that carry information about the size and the spacing of the collagen fibrils in the tissues. It was found that the collagen fibrils in invaded tumours were thinner and had a d-spacing length 0,7% longer that fibrils from healthy tumours. The scattering signals from the breast tissues were compared with the histology by building colour-coded maps across the samples. They were also imaged with the DEI technique. There was a total agreement between the scattering maps, the morphological features seen in the images and the information of the histo- pathological examination. The thesis demonstrates that the x-ray scattering signal can be used to characterize tissues and that it carries important information about the pathological state of the breast tissues, thus showing the potential of the SAXS technique as a possible diagnostic tool for breast cancer.

Data integration from two microarray platforms identifies bi-allelic genetic inactivation of RIC8A in a breast cancer cell line

Background: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging. Methods: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations. Results: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006). Conclusion: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.

Integrative functional genomics analysis of sustained polyploidy phenotypes in breast cancer cells identifies an oncogenic profile for GINS2

Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and that aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication and possibly through polyploid intermediate states. Here, we used a novel cell spot microarray technique to identify genes with a loss-of-function effect inducing polyploidy and/or allowing maintenance of polyploid cell growth of breast cancer cells. Integrative genomics profiling of candidate genes highlighted GINS2 as a potential oncogene frequently overexpressed in clinical breast cancers as well as in several other cancer types. Multivariate analysis indicated GINS2 to be an independent prognostic factor for breast cancer outcome (p = 0.001). Suppression of GINS2 expression effectively inhibited breast cancer cell growth and induced polyploidy. In addition, protein level detection of nuclear GINS2 accurately distinguished actively proliferating cancer cells suggesting potential use as an operational biomarker.

Mass spectrometry and n-in-one analytics in early drug discovery: Combinatorial chemistry libraries, lipophilicity and absorption screening

This thesis describes current and past n-in-one methods and presents three early experimental studies using mass spectrometry and the triple quadrupole instrument on the application of n-in-one in drug discovery. N-in-one strategy pools and mix samples in drug discovery prior to measurement or analysis. This allows the most promising compounds to be rapidly identified and then analysed. Nowadays properties of drugs are characterised earlier and in parallel with pharmacological efficacy. Studies presented here use in vitro methods as caco-2 cells and immobilized artificial membrane chromatography for drug absorption and lipophilicity measurements. The high sensitivity and selectivity of liquid chromatography mass spectrometry are especially important for new analytical methods using n-in-one. In the first study, the fragmentation patterns of ten nitrophenoxy benzoate compounds, serial homology, were characterised and the presence of the compounds was determined in a combinatorial library. The influence of one or two nitro substituents and the alkyl chain length of methyl to pentyl on collision-induced fragmentation was studied, and interesting structurefragmentation relationships were detected. Two nitro group compounds increased fragmentation compared to one nitro group, whereas less fragmentation was noted in molecules with a longer alkyl chain. The most abundant product ions were nitrophenoxy ions, which were also tested in the precursor ion screening of the combinatorial library. In the second study, the immobilized artificial membrane chromatographic method was transferred from ultraviolet detection to mass spectrometric analysis and a new method was developed. Mass spectra were scanned and the chromatographic retention of compounds was analysed using extract ion chromatograms. When changing detectors and buffers and including n-in-one in the method, the results showed good correlation. Finally, the results demonstrated that mass spectrometric detection with gradient elution can provide a rapid and convenient n-in-one method for ranking the lipophilic properties of several structurally diverse compounds simultaneously. In the final study, a new method was developed for caco-2 samples. Compounds were separated by liquid chromatography and quantified by selected reaction monitoring using mass spectrometry. This method was used for caco-2 samples, where absorption of ten chemically and physiologically different compounds was screened using both single and nin- one approaches. These three studies used mass spectrometry for compound identification, method transfer and quantitation in the area of mixture analysis. Different mass spectrometric scanning modes for the triple quadrupole instrument were used in each method. Early drug discovery with n-in-one is area where mass spectrometric analysis, its possibilities and proper use, is especially important.

Screening Cycles

We demonstrate how endogenous information acquisition in credit markets creates lending cycles when competing banks undertake their screening decisions in an uncoordinated way, thereby highlighting the role of intertemporal screening externalities induced by lending market competition as a structural source of instability. We show that uncoordinated screening behavior of competing banks may be not only the source of an important financial multiplier, but also an independent source of fluctuations inducing business cycles. The screening cycle mechanism is robust to generalizations along many dimensions such as the lending market structure, the lending rate determination and the imperfections in the screening technology.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.

Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.