Site specific gene expression patterns in oral cancer

Gesche Frohwitter, Dr.med. [MD] 2017

Abstract
Squamous cell carcinomas (SCCs) are the most prevalent malignant tumours within the head and neck. Evidence exists that distinct genes are differentially regulated in SCCs of the oral cavity compared to other head and neck regions. Given this background, the aim of this study was to investigate whether such tumour site-specific gene expression can also be observed in different localizations within the oral cavity.
Using tissue microarrays (TMAs), we investigated 76 SCCs of the floor of the mouth, 49 SCCs of the tongue and 68 SCCs of other anatomic regions within the oral cavity. The expression of 17 genes involved in cell cycle and growth control (p16, p21, p27, p53, cyclin D1, EGFR, c-kit, bcl-6), cell adhesion (alpha-, beta-, and gamma-catenin), and apoptosis/stress response genes (Hif-1-alpha, Glut 1, CA IX, caspase, hsp70, XIAP) were investigated by means of immunohistochemistry. The data were subjected to chi 2 , interdependency and Kaplan-Meier analysis.
Our study suggests a remote difference in the site-specific gene expression patterns of oral cancer. X-linked inhibitor of apoptosis (XIAP) showed a significantly higher expression (p <0.05) in SCCs of the floor of the mouth compared to SCCs of the tongue and other locations within the oral cavity. The increased XIAP expression was further associated with significantly decreased overall survival in all cases of SCCs of the oral cavity (p <0.05). Expression levels of p53, CA IX, beta-catenin, Hif-1-alpha, and c-kit were also observed to be inversely related between SCCs of the floor of the mouth and those of the tongue respectively, although these differences did not reach statistical significance. Overall and event-free survival did not differ in patients with T1/T2/N0 SCCs according to tumour localization.
In summary, the protein expression patterns of SCCs of the oral cavity suggest the existence of a molecular and morphological spectrum of SCCs in the oral cavity. In particular the expression pattern of XIAP indicates distinct gene expression patterns between carcinomas of the floor of the mouth and oral tongue cancer. Further studies are needed to identify possible tumour site-specific factors that influence patient prognosis and management.
Thesis is published in Head Face Med. 2017 May 10;13(1):6.
Supervisors: PD.Dr.E.Korsching, PD.Dr.T.Fillies, Prof.Dr.H.Bürger

Cytokeratin and protein expression patterns in squamous cell carcinoma of the oral cavity provide evidence for two distinct pathogenetic pathways

Gesche Frohwitter, Dr.med.dent. [DMD] 2016

Abstract
Squamous cell carcinoma (SCC) of the oral cavity is a morphological heterogeneous disease. Various cytokeratin (CK) expression patterns with different prognostic values have been described, but little is known concerning the underlying biological cell mechanisms.
Therefore, the present study investigated 193 cases of oral SCCs using immunohistochemistry for α/β/γ ‐catenin, glucose transporter 1, caspase‐3, X‐linked inhibitor of apoptosis protein, hypoxia inducible factor‐1 α, carbonic anhydrase 9, heat shock protein (hsp) 70, mast/stem cell growth factor receptor, p21, p27, p16, p53, B‐cell lymphoma 6, epidermal growth factor receptor, cyclin D1 and CK1, 5/6, 8/18, 10, 14 and 19. Expression patterns were analyzed with biomathematical permutation analysis.
The present results revealed a significant association between the expression of low‐molecular weight CK8/18 and 19 and a high‐tumor grade, β and γ ‐catenin expression, deregulated cell cycle proteins and a predominant localization of the tumor on the floor of the mouth. By contrast, expression of high‐molecular weight CK1, 5/6, 10 and 14 was significantly associated with the expression of p21 and hsp70.
In conclusion, the current study presents evidence for the existence of two parallel pathogenetic pathways in oral SCCs, characterized by the expression of low‐ and high‐molecular weight CKs. Additional studies are required to demonstrate the extent that these results may be used to improve therapeutic regimens.
Thesis is published in Oncol Lett. 2016 Jul;12(1):107-113.
Supervisors: PD.Dr.E.Korsching, PD.Dr.T.Fillies, Prof.Dr.H.Bürger

The pathogenesis of osteosarcoma - Systems biological approaches to explain cancer development and progression

Kathrin Poos (now Haeffs), Dr.rer.nat. [PhD] 2014

Abstract
Osteosarcomas (OS) constitute the most frequent pediatric bone cancers with vast amounts of molecular heterogeneity. This molecular heterogeneity hampers the identification of common genetic causes. To address this issue, systems biological approaches are promising to generate new models that explain the pathogenesis of OS by seeking for common molecular networks and/or pathways. These molecular networks and/or pathways are the basis to improve diagnostic and therapeutic procedures.
In the scope of this thesis, systems biological approaches are applied to an in-vitro model of OS cell proliferation and to genomic alteration patterns obtained from pre-treatment OS biopsies. In-vitro, the deregulation of microRNA and transcription factor (TF) co-regulatory networks was analyzed in OS cell lines with different proliferative capabilities. According to their location within the co-regulatory networks, the microRNA-9-5p, -138, and -214 and the TFs SP1 and MYC have been identified as important players of the networks. Together, they act in NFKB1 and RB1 signaling as well as in focal adhesion processes, known cellular pathways that influence cell proliferation. Therefrom, a new model of OS cell proliferation was proposed, which is primarily based on the interactions of the identified microRNAs and TF combinations and their target genes (e.g. CDK6, CTNNB1, E2F4, HES1, ITGA6, NFKB1, NOTCH1, and SIN3A). In pre-treatment OS biopsies, genomic alterations affected a huge number of heterogeneous genes between individual tumors. By examining their network neighborhood, 26 densely connected network modules were obtained that function in cancer-related processes. Even well-characterized genes in OS such as TP53 and CDKN1A that were not affected by genomic alterations, appeared in the resulting network modules. This indicates that the molecular heterogeneity of OS is visible on the gene level but becomes homogenous on the system level. Additionally, certain modules uncovered genes that alteration patterns reflect an adverse disease outcome, e.g. losses of CDKN2A mark a poor survival. Noticeably, 50% of these genes are located on chromosome 6q indicating an influence of chromosome 6q on OS progression.
Furthermore, an OS-gene association database was implemented dedicated to the ever increasing biomedical literature. The database provides structured and annotated knowledge of OS-related genes and microRNAs. It supports and simplifies the ongoing OS research by quickly extracting information about experimental settings, type of samples, and prognostic or therapeutic impact of genes and microRNAs. This database serves as a unique and helpful information and hypothesis generation resource and supports the interpretaion of complex results in OS research.
Summarizing, the applied systems biological approaches facilitate the formulation of new models implicated in OS development and progression. These models are based on the integration of several independent data types and supported by current literature of cancer in general and of OS. They represent a useful basis for further studies that can be assisted by the OS database.
Thesis is published in PLoS One. 2015 Apr 7;10(4):e0123082, Database (Oxford). 2014 May 27;2014. pii:bau042, PLoS Comput Biol. 2013;9(8):e1003210.
Supervisors: PD.Dr.E.Korsching, Prof.Dr.E.Bornberg-Bauer (Faculty of Biology)

Morphological and immunohistochemical analysis of progression pathways in invasive breast cancer

Barbara Schymik, Dr.med. [MD] 2013

Abstract
Recent molecular data pointed towards the possibility of a stepwise dedifferentiation in a subgroup of invasive breast cancer (BC) cases. It was hypothesized that oestrogen receptor positive (ER+) grade 3 (G3) ductal invasive BCs are the end stage of a dedifferentiation process of luminal BC. A progression of luminal A towards luminal B BCs associated with a ‘progression through grade’ and an increased cell proliferation seemed the obvious explanation.
In order to verify this hypothesis on a morphological and immunohistochemical level, we investigated 865 invasive BC cases. All cases were reviewed for the presence of intratumoural heterogeneity in grade of the invasive cancer and the presence of associated ductal carcinoma in situ (DCIS). With the use of tissue microarrays, the molecular subtype was determined and correlated with clinico-pathological features. In addition, all cases were stained for p21, p27, Ki-67, Cyclin D1, bcl-2, p53, and p16 and the results subjected to a biomathematical dependency analysis.
The frequency of ER-positivity decreased with tumour size. The frequency of luminal A BC decreased as well, whereas the number of luminal B BCs remained constant. A gradual increase of the frequency of basal-like, HER2-driven and non-expressor BCs with tumour size was seen. In only 1 out of 865 BC cases, both a G1 and a G3 invasive cancer component was seen within the same BC. In two cases, a ductal invasive G1 carcinoma was associated with a poorly-differentiated DCIS. The frequency of columnar cell lesions was evenly distributed over ER+ and ER- ductal invasive G3 carcinomas. The biomathematical analysis gave striking hints against an obligate progression of BC trough grade.
In conclusion, our results show that a morphological recognizable striking ‘progression through grade’ at least in its extreme form from G1 towards G3 is a very rare event in the natural course of invasive BC, including luminal BC.
Thesis is published in Breast Cancer Res Treat. 2012 Oct;135(3):693-703.
Supervisors: PD.Dr.E.Korsching, Prof.Dr.H.Bürger

Evaluation of quality parameters for the breast cancer screening region Ostwestfalen

Axel Graewingholt, Dr.med. [MD] 2011

Abstract
The launch of the nationwide mammography screening in Germany in 2005 is based on international recommendations and the implicit objective to reduce the breast cancer mortality by up to 30%. This objective is based on the consideration, that the proportion of the undetected, good differentiated, very small and nodal-negative carcinomas, need to be decreased. In comparison to the expert knowledge of other European countries, the starting conditions in Germany are different from the surrounding countries, because of an known opportunistic screening behavior, which hardly can be quantitatively estimated.
The scope of the work was, to monitor all the German wide defined quality parameters of the mammography screening in the screening unit Paderborn / Hoexter from the implementation on in a continuous way for the time period 2005 to 2010. The evaluation was performed separately for each screening sub-unit. In parallel, the histopathological findings for the sub-collective of patients treated in the St.Vinzenz gynecological clinic were evaluated and for more than 400 of these patients also the long-term follow up was considered for the rating. These breast cancer patients span a time frame from 1997 to 2010 - so this cohort also includes patients from the non screening phase and the screening phase.
The results show, that with the establishment of the mammography screening in the region of Paderborn / Hoexter an increased amount of asymptomatic tumors with a diameter of less than 15mm, with a degree of G1 and in 70% of cases without axillary lymph node involvement, were diagnosed and treated. Especially for the degree of malignancy, a significant difference exists compared to the symptomatic control group of the same age. Moreover the analysis by screening sub-units showed, that the results are differing according to location. Unfortunately, the underlying causes could not be uncovered based on the set of the collected parameters.
In summary, the preliminary data collection which was available for this study, supports the a priory assumption, that the implementation of the mammography screening might lead to a long term reduction of the breast cancer mortality. As a consequence thereof the treatment spectrum of invasive breast cancer will change too over time.
Thesis is published at the University and State Library Muenster - ULB.
Supervisors: PD.Dr.E.Korsching, Prof.Dr.H.Bürger

Identifying candidate genes triggering osteosarcoma invasiveness and dissecting their functional role

Anna Neumann, Dr.rer.nat. [PhD] 2011

Abstract
Osteosarcoma (OS) is the most common primary malignancy of bone, with up to 80% of all patients suffering from metastatic or micrometastatic disease at the time of diagnosis. These metastases are the main cause of death in OS patients. OS is very well characterized in regard to incidence, localization or aggressive behavior. But unfortunately there is a lack of knowledge of the exact molecular mechanism of OS development, invasion and metastasis. Therefore, a strong focus of this thesis was placed on the identification of molecular markers triggering these processes.
OS development of seven OS cell lines (HOS, MG63, MNNG, SJSA1, SAOS, U2OS, ZK58) was analyzed using an in vivo model, which was established in this thesis as an adequate model not only for OS development in vivo but also to support and reflect the in vitro results of OS invasiveness. Those in vitro results of invasiveness were determined for the same seven cell lines using a modified invasion assay. Doing so, the cell lines were classified into three groups’ high, moderate and low invasive. Moreover, invasive and non invasive fractions were separated to perform genome wide expression profiling to close the gap of genes affecting the different invasive behavior. The profiles were performed separately for the invasive and the non-invasive fraction. Via pairwise comparison 48 candidate genes holding a higher invasive potential could be detected. Two hot spots affecting OS cell invasion could be found within this set of candidate genes, in detail: 17.5 % of all genes were regulated directly or indirectly (via NGFR) by p53 and 7.5 % were located on chromosome 11q13. All of these genes were selected for further analysis. This set of genes was extended by three candidate genes already associated with metastasis in other cancer entities. To investigate their functional relevance in the process of cancer cell invasion a combination of reverse siRNA transfection and a modified Boyden-Chamber assay was performed. The knockdown efficiency was verified on mRNA level as well as on protein level after knockdown via TaqMan assays respectively Immunofluorescence.
11 out of the 13 candidate genes proved to be functionally relevant. These genes are supposed to be a first draft of reliable molecular markers for OS metastasis. Most promising ones seem to be GABBR1 and HNRNPA2B1, both decreases OS invasiveness after knockdown. HNRNPA2B1 is actually associated with lung cancer and therefore it might be also essential for OS metastases, which are noteworthy located in the lung in 90 % of all cases.
To determine the clinical relevance of the verified molecular markers it would be good to test their expression status in human OS samples by comparing patients suffering of metastasis with those without a metastatic process. These results provide a solid basis for a more detailed functional analysis in mammalian in vivo models.
Thesis is published by the German University Library network.
Supervisors: PD.Dr.E.Korsching, Prof.Dr.A.Pueschel (Faculty of Biology)

Morphological and immunohistochemical characterization of colorectal carcinoma concerning the expression of microsatellite instability (MSI) repair genes

Bettina Andrea Renate Wirth (now Scheffer), Dr.med. [MD] 2011

Abstract
MSS and MSI tumors differ significantly in the mechanisms of their carcinogenesis. MSI tumors appear to have a more aggressive phenotype than MSS tumors, with CDX2 and CK20 losses and an increased EGFR and CA19-9 expression, while in MSS tumors, the dysregulation of the Wnt signaling pathway and p53 defects are the main observable alterations.
The mechanisms of carcinogenesis seem to differ depending on the MMR protein loss within the MSI tumors: the present study exemplifies that, especially in sporadic MSI tumors with immune histochemically detectable loss of MLH1 and / or PMS2, an increasingly proximal localization, poorer differentiation, lymph node and lymphatic vessel infiltration, a loss of cytokeratin CK20, an increased CA19-9 expression, and an E-cadherin-related stabilization of the membrane β-catenin can be observed.
To get more insight into further mechanistic details, it must be examined whether the individual MMR protein loss is associated with a hypermethylation of the corresponding promoter or a (germline) mutation in the corresponding MMR gene. This would also pave the way for the identification of important molecular targets for a (neo) adjuvant therapy option.
Thesis is published by the German University Library network.
Supervisors: PD.Dr.E.Korsching, Prof.Dr.H.Bürger

New intronic Sp1-transcription factor binding sites and their role in the regulation of the egfr oncogene

Ronaldo Schuch, Dr.rer.nat. [PhD] 2011

Abstract
Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSsdue to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs.
The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue.
The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme digestion enables a reliable and individual detection option for densely arranged binding motifs in vivo not provided by e.g. EMSA or in vivo footprinting. Given the important function of transcription factors in neoplastic mechanism, our method enables a broad diversity of application options for clinical studies.
Thesis is published in BMC Res Notes. 2012 Feb 20;5:109.
Supervisors:, PD.Dr.E.Korsching, Prof.Dr.KH.Klempnauer (Faculty of Chemistry)