Molecular classification of familial colorectal and endometrial carcinoma

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common known clearly hereditary cause of colorectal and endometrial cancer (CRC and EC). Dominantly inherited mutations in one of the known mismatch repair (MMR) genes predispose to HNPCC. Defective MMR leads to an accumulation of mutations especially in repeat tracts, presenting microsatellite instability. HNPCC is clinically a very heterogeneous disease. The age at onset varies and the target tissue may vary. In addition, families that fulfill the diagnostic criteria for HNPCC but fail to show any predisposing mutation in MMR genes exist. Our aim was to evaluate the genetic background of familial CRC and EC. We performed comprehensive molecular and DNA copy number analyses of CRCs fulfilling the diagnostic criteria for HNPCC. We studied the role of five pathways (MMR, Wnt, p53, CIN, PI3K/AKT) and divided the tumors into two groups, one with MMR gene germline mutations and the other without. We observed that MMR proficient familial CRC consist of two molecularly distinct groups that differ from MMR deficient tumors. Group A shows paucity of common molecular and chromosomal alterations characteristic of colorectal carcinogenesis. Group B shows molecular features similar to classical microsatellite stable tumors with gross chromosomal alterations. Our finding of a unique tumor profile in group A suggests the involvement of novel predisposing genes and pathways in colorectal cancer cohorts not linked to MMR gene defects. We investigated the genetic background of familial ECs. Among 22 families with clustering of EC, two (9%) were due to MMR gene germline mutations. The remaining familial site-specific ECs are largely comparable with HNPCC associated ECs, the main difference between these groups being MMR proficiency vs. deficiency. We studied the role of PI3K/AKT pathway in familial ECs as well and observed that PIK3CA amplifications are characteristic of familial site-specific EC without MMR gene germline mutations. Most of the high-level amplifications occurred in tumors with stable microsatellites, suggesting that these tumors are more likely associated with chromosomal rather than microsatellite instability and MMR defect. The existence of site-specific endometrial carcinoma as a separate entity remains equivocal until predisposing genes are identified. It is possible that no single highly penetrant gene for this proposed syndrome exists, it may, for example be due to a combination of multiple low penetrance genes. Despite advances in deciphering the molecular genetic background of HNPCC, it is poorly understood why certain organs are more susceptible than others to cancer development. We found that important determinants of the HNPCC tumor spectrum are, in addition to different predisposing germline mutations, organ specific target genes and different instability profiles, loss of heterozygosity at MLH1 locus, and MLH1 promoter methylation. This study provided more precise molecular classification of families with CRC and EC. Our observations on familial CRC and EC are likely to have broader significance that extends to sporadic CRC and EC as well.

Determinants of vascular cognitive impairment : White matter lesions, brain atrophy and their neuropsychological correlates

The concept of vascular cognitive impairment (VCI) covers a wide spectrum of cognitive dysfunctions related to cerebrovascular disease. Among the pathophysiological determinants of VCI are cerebral stroke, white matter lesions and brain atrophy, which are known to be important risk factors for dementia. However, the specific mechanisms behind the brain abnormalities and cognitive decline are still poorly understood. The present study investigated the neuropsychological correlates of particular magnetic resonance imaging (MRI) findings, namely, medial temporal lobe atrophy (MTA), white matter hyperintensities (WMH), general cortical atrophy and corpus callosum (CC) atrophy in subjects with cerebrovascular disease. Furthermore, the cognitive profile of subcortical ischaemic vascular disease (SIVD) was examined. This study was conducted as part of two large multidisciplinary study projects, the Helsinki Stroke Aging Memory (SAM) Study and the multinational Leukoaraiosis and Disability (LADIS) Study. The SAM cohort consisted of 486 patients, between 55 and 85 years old, with ischaemic stroke from the Helsinki University Hospital, Helsinki, Finland. The LADIS Study included a mixed sample of subjects (n=639) with age-related WMH, between 65 and 84 years old, gathered from 11 centres around Europe. Both studies included comprehensive clinical and neuropsychological assessments and detailed brain MRI. The relationships between the MRI findings and the neuropsychological test performance were analysed by controlling for relevant confounding factors such as age, education and other coexisting brain lesions. The results revealed that in elderly patients with ischaemic stroke, moderate to severe MTA was specifically related to impairment of memory and visuospatial functions, but mild MTA had no clinical relevance. Instead, WMH were primarily associated with executive deficits and mental slowing. These deficits mediated the relationship between WMH and other, secondary cognitive deficits. Cognitive decline was best predicted by the overall degree of WMH, whereas the independent contribution of regional WMH measures was low. Executive deficits were the most prominent cognitive characteristic in SIVD. Compared to other stroke patients, the patients with SIVD also presented more severe memory deficits, which were related to MTA. The cognitive decline in SIVD occurred independently of depressive symptoms and, relative to healthy control subjects, it was substantial in severity. In stroke patients, general cortical atrophy also turned out to be a strong predictor of cognitive decline in a wide range of cognitive domains. Moreover, in elderly subjects with WMH, overall CC atrophy was related to reduction in mental speed, while anterior CC atrophy was independently associated with frontal lobe-mediated executive functions and attention. The present study provides cross-sectional evidence for the involvement of WMH, MTA, general cortical atrophy and CC atrophy in VCI. The results suggest that there are multifaceted pathophysiological mechanisms behind VCI in the elderly, including both vascular ischaemic lesions and neurodegenerative changes. The different pathological changes are highly interrelated processes and together they may produce cumulative effects on cognitive decline.

Cathepsin D Deficiency - Molecular and Cellular Mechanisms of Neurodegeneration

Cathepsin D (CTSD) is a lysosomal protease, the deficiency of which is fatal and associated with neurodegeneration. CTSD knock-out mice, which die at the age of four weeks, show intestinal necrosis, loss of lymphoid cells and moderate pathological changes in the brain. An active-site mutation in the CTSD gene underlies a neurodegenerative disease in newborn sheep, characterized by brain atrophy without any changes to visceral tissues. The CTSD deficiences belong to the group of neuronal ceroid-lipofuscinoses (NCLs), severe neurodegenerative lysosomal storage disorders. The aim of this thesis was to examine the molecular and cellular mechanisms behind neurodegeneration in CTSD deficiency. We found the developmental expression pattern of CTSD to resemble that of synaptophysin and the increasing expression of CTSD to coincide with the active period of myelination in the rat brain, suggesting a role for CTSD in early rat brain development. An active-site mutation underlying the congenital ovine NCL not only affected enzymatic activity, but also changed the stability, processing and transport of the mutant protein, possibly contributing to the disease pathogenesis. We also provide CTSD deficiency as a first molecular explanation for human congenital NCL, a lysosomal storage disorder, characterized by neuronal loss and demyelination in the central nervous system. Finally, we show the first evidence for synaptic abnormalities and thalamocortical changes in CTSD-deficient mice at the molecular and ultrastructural levels. Keywords: cathepsin D, congenital, cortex, lysosomal storage disorder, lysosome, mutation, neurodegeneration, neuronal ceroid-lipofuscinosis, overexpression, synapse, thalamus

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Preadolescents and Their Mothers as Oral Health-Promoting Actors : Non-biologic Determinants of Oral Health among Turkish and Finnish Preadolescents

Is oral health becoming a part of the global health culture? Oral health seems to turn out to be part of the global health culture, according to the findings of a thesis-research, Institute of Dentistry, University of Helsinki. The thesis is entitled as “Preadolescents and Their Mothers as Oral Health-Promoting Actors: Non-biologic Determinants of Oral Health among Turkish and Finnish Preadolescents.” The research was supervised by Prof.Murtomaa and led by Dr.A.Basak Cinar. It was conducted as a cross-sectional study of 611 Turkish and 223 Finnish school preadolescents in Istanbul and Helsinki, from the fourth, fifth, and sixth grades, aged 10 to 12, based on self-administered and pre-tested health behavior questionnaires for them and their mothers as well as the youth’s oral health records. Clinically assessed dental status (DMFT) and self-reported oral health of Turkish preadolescents was significantly poorer than the Finns`. A similar association occurred for well-being measures (height and weight, self-esteem), but not for school performance. Turkish preadolescents were more dentally anxious and reported lower mean values of toothbrushing self-efficacy and dietary self-efficacy than did Finns. The Turks less frequently reported recommended oral health behaviors (twice daily or more toothbrushing, sweet consumption on 2 days or less/week, decreased between-meal sweet consumption) than did the Finns. Turkish mothers reported less frequently dental health as being above average and recommended oral health behaviors as well as regular dental visits. Their mean values for dental anxiety was higher and self-efficacy on implementation of twice-daily toothbrushing were lower than those of the Finnish. Despite these differences between the Turks and Finns, the associations found in common for all preadolescents, regardless of cultural differences and different oral health care systems, assessed for the first time in a holistic framework, were as follows: There seems to be interrelation between oral health and general-well being (body height-weight measures, school performance, and self-esteem) among preadolescents: • The body height was an explanatory factor for dental health, underlining the possible common life-course factors for dental health and general well-being. • Better school performance, high levels of self-esteem and self-efficacy were interrelated and they contributed to good oral health. • Good school performance was a common predictor for twice-daily toothbrushing. Self-efficacy and maternal modelling have significant role for maintenance and improvement of both oral- and general health- related behaviors. In addition, there is need for integration of self-efficacy based approaches to promote better oral health. • All preadolescents with high levels of self-efficacy were more likely to report more frequent twice-daily toothbrushing and less frequent sweet consumption. • All preadolescents were likely to imitate toothbrushing and sweet consumption behaviors of their mothers. • High levels of self-efficacy contributed to low dental anxiety in various patterns in both groups. As a conclusion: • Many health-detrimental behaviors arise from the school age years and are unlikely to change later. Schools have powerful influences on children’s development and well-being. Therefore, oral health promotion in schools should be integrated into general health promotion, school curricula, and other activities. • Health promotion messages should be reinforced in schools, enabling children and their families to develop lifelong sustainable positive health-related skills (self-esteem, self-efficacy) and behaviors. • Placing more emphasis on behavioral sciences, preventive approaches, and community-based education during undergraduate studies should encourage social responsibility and health-promoting roles among dentists. Attempts to increase general well-being and to reduce oral health inequalities among preadolescents will remain unsuccessful if the individual factors, as well as maternal and societal influences, are not considered by psycho-social holistic approaches.