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The Ph.D. thesis is a collection of eleven commented articles on the topic of molecular-genetic profiling of urogenital tract tumors published in impact factor journals. In the introduction the origin and development of classification units is described including a brief overview of recent genetic profiles of selected types of renal carcinomas. Reviewed are major signaling pathways exploited in targeted therapy as well as the function of the corresponding drugs. Directions in which the research of new biomarkers is aimed are mentioned and the introduction ends with a short list of molecular genetic methods used in classification of renal carcinomas. In the results section are summarized the most important morphological, immunohistochemical, and molecular genetic findings of the studied sets of renal carcinomas. The data are compared with the present characteristics of known entities and hypotheses of possible new entities or relations between them are inferred. In the conclusions the suitability and limitations of the used molecular genetic methods are discussed in the context of the difficulties of the material analyzed and the designs of the studies.

The overall aim of this project was first to investigate the genetic and epigenetic mechanisms underlying periodontal diseases and oral squamous cell carcinomas respectively, particularly in terms of competing endogenous RNA networks. Second, the aim was to explore the possible shared mechanisms between chronic periodontitis and oral squamous cell carcinoma regarding mRNAs, miRNAs, and signaling pathways. The results obtained from the first two publications may point to putative diagnostic or prognostic biomarkers and therapeutic targets for periodontal diseases and oral cancer respectively. The third and final publication aimed to reveal overlapping molecular mechanisms underlying both diseases that may bear translational value toward screening of the periodontitis patients with a high risk of oral cancer. In theory, if successful, such biomarkers based on screening may lead to the clinical application of tailored periodontal maintenance programs for high-risk individuals, including environmental factor mitigation. Such interceptive therapy could potentially reduce the risk of oral carcinogenesis.:1 Introduction ............................................................................................................................ 1 1.1 General aspects of periodontitis and oral squamous cell carcinoma.......................... 1 1.1.1 General aspects of periodontitis.......................................................................... 1 1.1.2 General aspects of oral squamous cell carcinoma .............................................. 2 1.1.3 The association of chronic periodontitis and oral squamous cell carcinoma...... 3 1.2 Genetic and epigenetic mechanisms of oral diseases ...................................................... 4 1.2.1 General aspects of various genetic and epigenetic factors........................................ 4 1.2.2 Genetic and epigenetic mechanisms of chronic periodontitis................................... 5 1.2.2.1 Alteration of mRNA gene expression in chronic periodontitis.......................... 5 1.2.2.2 Alteration of miRNA expression in chronic periodontitis ................................. 7 1.2.2.3 Alteration of lncRNA expression in chronic periodontitis................................. 7 1.2.2.4 Alteration of signaling pathway in chronic periodontitis................................... 8 1.2.3 Genetic and epigenetic mechanisms of oral squamous cell carcinoma .................... 9 1.2.3.1 Alteration of mRNA gene expression of oral squamous cell carcinoma ........... 9 1.2.3.2 Alteration of miRNA expression in oral squamous cell carcinoma................... 9 1.2.3.3 Alteration of lncRNA expression in oral squamous cell carcinoma ................ 10 1.2.3.4 Alteration of signaling pathway involved in oral squamous cell carcinoma ... 11 1.2.4 The shared genetic and epigenetic mechanisms between chronic periodontitis and oral squamous cell carcinoma.......................................................................................... 12 1.2.4.1 Genes as links between chronic periodontitis and oral squamous cell carcinoma ..................................................................................................................................... 12 1.2.4.2 miRNAs as links between chronic periodontitis and oral squamous cell carcinoma..................................................................................................................... 12 1.2.4.3 lncRNAs as links between chronic periodontitis and oral squamous cell carcinoma..................................................................................................................... 15 1.2.4.4 Signaling pathways as links between chronic periodontitis and oral squamous cell carcinoma .............................................................................................................. 15 1.3 Rationale of the current project ..................................................................................... 16 2 Objectives of the current project .......................................................................................... 18 2.1 General objective........................................................................................................... 18 2.2 Specific objectives......................................................................................................... 18 3 Publications .......................................................................................................................... 19 4 Summary of dissertation....................................................................................................... 49 5 References ............................................................................................................................ 53 6 Presentation of my own contribution ................................................................................... 63 6.1 Publication I................................................................................................................... 636.2 Publication II ................................................................................................................. 65 6.3 Publication III................................................................................................................ 67 7 Declaration of Authorship .................................................................................................... 69 8 Curriculum Vitae.................................................................................................................. 70 9 Acknowledgement................................................................................................................ 72

Das NZK ist nach dem Prostata- und Harnblasenkarzinom der dritthäufigste urologische Tumor. Die Prognoseaussichten hängen beim NZK vom Metastasenstatus der Patienten ab. Die Heilungschancen für das lokal begrenzte NZK sind im Vergleich zum metastasierten NZK deutlich besser. Durch den Einsatz von TKI und mTOR-Inhibitoren wurde die Therapie des metastasierten NZK revolutioniert und das Überleben von Patienten signifikant verbessert. Nichtsdestotrotz profitiert ein Teil dieser Patienten aufgrund von Resistenzmechanismen nicht von solch einer anti-VEGF-Therapie. Bisher gibt es keine geeigneten Biomarker, die das Ansprechen auf eine solche Therapie vorhersagen könnten. Daher bestand das primäre Ziel dieser Arbeit darin, molekulare Marker für die Abschätzung der Prognose beim lokal begrenzten und metastasierten NZK sowie die Vorherage für das Ansprechen auf eine Sunitinib-Therapie beim metastasierten NZK zu identifizieren. Für das Prognosemodell am lokal begrenzten NZK konnten auf mRNA-Ebene signifikante Assoziationen der Marker HIF-2α, VEGFR3 und sVEGFR1 mit dem PFS, TSS und OS der Patienten identifiziert werden. Da bisher nur klinische Parameter für Prognosemodelle des lokal begrenzten NZK genutzt werden, könnten die hier untersuchten molekularen Marker nach einer unabhängigen Validierung zur Vorhersage der Prognose herangezogen werden. Patienten mit einem metastasierten NZK wiesen VHL-Mutationen (50%) und -Kopienzahlverluste (60 %) auf, die jedoch nicht mit dem Ansprechen auf Sunitinib assoziiert waren und nur geringfügige Auswirkungen auf die Proteinlevel von VHL und dessen Targetgene HIF-1α, CA9 und VEGFA zeigten. Bei den Untersuchungen zur VHL-Promotormethylierung wurde im tumorfreien Gewebe eine hohe Grundmethylierung festgestellt. Aufgrund der geringen Patientenzahl und der weitgehend unbekannten komplexen Methylierungsstruktur des VHL-Promotors konnten keine Assoziationen mit der Prognose und dem Ansprechen der metastasierten NZK-Patienten auf Sunitinib bestimmt werden. Für die Proteinlevel potenzieller prädiktiver Marker wie CA9, HIF-1α, VEGFR1 und -2, pVEGFR1, pPDGFRα und -β, CD31, pAkt sowie Ki67 wurden signifikante Assoziationen mit dem Ansprechen auf die Sunitinib-Behandlung beobachtet. Die CA9-Membranfärbung und das Ansprechen nach 9 Monaten wurden in der multivariaten Analyse als unabhängige prognostische Marker für das OS bei Patienten mit metastasiertem NZK identifiziert. In anderen Arbeiten wurde CA9 bereits mehrfach als potenzieller Biomarker beschrieben und könnte daher eine Anwendung in der Prognosevorhersage und Patienten-Selektion für eine Target-Therapie finden. Polymorphismen in Angiogenese-assoziierten Genen gelten ebenfalls als potenzielle Marker für das Ansprechen auf eine Therapie mit Sunitinib. Die Überlebensanalysen deckten signifikante Assoziationen zwischen dem VEGFA-SNP -2578 und dem PFS und für die SNPs VEGFR1 B sowie VEGFR2 +1191 mit dem OS auf. Das kombinierte Auftreten der Varianten-Allele der VEGFA-SNPs -2578, -1154 und +405 wirkte sich ebenfalls signifikant auf ein verlängertes OS der mit Sunitinib behandelten Patienten aus. Bei eindeutiger Bestätigung dieser Ergebnisse in prospektiven Studien könnten einfache SNP-Analysen an Blutproben die Therapieentscheidung und das Überleben der NZK-Patienten maßgeblich beeinflussen. Die künstlich erzeugte Sunitinib-Resistenz in den NZK-Zelllinien A498, Caki-1 und KTCTL-26 offenbarte nur begrenzt veränderte Proteinniveaus potenzieller Marker wie HIF-1α, Akt und pAkt zwischen den resistenten und sensitiven Zellen. Ein besseres Verständnis der molekularen Grundlagen der Resistenzentwicklung könnte zusammen mit einem wirkungsvollen, prädiktiven Biomarker für das Ansprechen die Therapie beim metastasierten NZK erheblich verbessern. In dieser Arbeit konnten letztlich verschiedenste Biomarker identifiziert und evaluiert sowie deren Bedeutung für die Prognosevorhersage und Prädiktion von Patienten mit metastasiertem NZK unter Sunitinib-Therapie herausgearbeitet werden. Diese Daten stellen damit einen weiteren Grundstein für mögliche prospektive klinische Studien dar, die den therapeutischen Nutzen der Biomarker eindeutiger definieren könnten.

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Cancers of the human skin are divided into melanoma and non-melanoma. Being among the most commonly diagnosed cancer cases globally, non-melanoma skin cancers are comprised of basal and squamous cell carcinomas. In dermato-pathology, basal cell carcinomas (BCCs) are a frequently encountered diagnosis of skin cancer, and most cases are treated with surgical excisions. While sporadic BCC tumors appear primarily due to aging and ultra-violet exposure, the development of numerous BCCs from a young age is one of the main clinical signs in Gorlin syndrome patients. The critical driver of BCC tumor formation is the sonic hedgehog (SHH) pathway, a pivotal developmental signaling pathway that regulates organ development, cell proliferation, and tissue repair. The majority of all sporadic and syndromic BCCs exhibit mutations in two key components in this pathway, the tumor suppressor gene patched 1 (PTCH1) or the proto-oncogene smoothened (SMO), which result in aberrant pathway activation and continued transcription of SHH-dependent genes. In the last decade, SHH inhibitors have emerged as a novel treatment for advanced and metastatic BCCs. Systemic treatment with vismodegib, a potent SMO inhibitor, can effectively reduce BCC tumor burden in adult Gorlin syndrome patients. However, it is associated with chemotherapy-related adverse events, and treatment cessation results in cancer recurrence and formation of a subset of drug resistant BCCs. While aberrant SHH signaling is key, mechanisms that underlie epithelial–stromal crosstalk and reprograming of tumor metabolism can potentially converge with this pathway and promote BCC tumor development. In this study, we investigated the effects vismodegib on the morphology, behavior, and energy metabolism of human BCC cells and human dermal fibroblasts, in individual cultures as well as in co-cultures, that enabled the crosstalk between these two cell types. Computer-assisted bright-field microscopy was used to characterize cell morphology and behavior. Nuclear magnetic resonance (NMR) and metabolomics were used to determine the metabolic activity of these cells. We found that continuous crosstalk between the cells and different concentrations of vismodegib led to distinct changes in cell morphology and growth, as well as consumption of glucose, pyruvate, and glutamine and secretion of acetate, lactate, and glutamate by these cells. Deciphering tumor driver mechanisms that converge with SHH pathway and contribute to changes within the tumor microenvironment are important not only for better understanding of BCC pathobiology, but also for the development of new mechanism-based BCC therapies with improved clinical outcomes. / 2023-02-22T00:00:00Z

The urothelium is a crucial stratified epithelial barrier that protects the urinary tract. It consists of basal cells in the lower layers and intermediate and superficial cells in the luminal layer. These urothelial cells can be identified by their distinct gene expression patterns. Superficial cells are terminally differentiated, binucleated, post-mitotic cells that are responsible for the barrier function of the urothelium via the production of uroplakin proteins. Intermediate cells act as the progenitor cells for superficial cells during development, homeostasis, and after acute injury. Basal cells consist of two populations, K14-basal cells and K5-basal cells. K14- basal cells have been shown to be progenitors that can repopulate the urothelium after chronic injury and are the cells of origin that produce bladder cancer. Bladder cancer can be classified as basal subtype or luminal subtype. The basal subtype is generally immune infiltrated, aggressive, and invasive with a poor prognosis. The luminal subtype is generally immune poor, less aggressive, and non-invasive with a better prognosis compared to basal tumors. Pparg is a nuclear hormone receptor that has been described as a master regulator of adipogenesis and cellular differentiation that also carries out important anti-inflammatory functions (in part by antagonizing the NFKB pathway). Pparg is downregulated in basal subtype muscle invasive bladder cancer and amplified in luminal subtype bladder cancer. In vivo we find that Pparg is a master regulator of cell specification during urothelial development, homeostasis, regeneration, and cancer. When Pparg is ablated in the entire urothelium, Pparg KO mutants lack mature superficial cells and undergo squamous differentiation, with an expansion of the K14-basal cell population. These Pparg KO mutants also display persistent inflammation and squamous metaplasia after injury by urinary tract infection (UTI), due to unregulated NFKB signaling. However, the squamous differentiation in the Pparg KO mutants did not progress to bladder cancer. Constitutive activation of Pparg in basal cells using a novel VP16;Pparg transgenic mouse line crossed to an Krt5CreERT2 driver induces basal cells to undergo a luminal differentiation program towards post-mitotic S-cells during homeostasis. Not surprisingly, these cells did not progress to form bladder cancer on their own. Interestingly, expression of VP16;Pparg in basal cells only drives tumor formation when the basal cells are in an “activated state,” induced by 1 month of BBN treatment. In a BBN mouse model which produces basal subtype bladder cancer in wild type animals, expression of the VP16;Pparg transgene in activated basal cells drives the formation of luminal tumors with papillary morphology, suggesting that this transcription factor is a master regulator of urothelial luminal differentiation, as has been suggested from previous in vitro studies. Like their human counterparts, these VP16;Pparg luminal tumors are immune cold. Additionally, these VP16;Pparg luminal tumors have different domains; a top domain that is “luminal,” and a bottom domain that is “basal”, suggesting the luminal tumors produced by activation of Pparg are not homogenous and undergo a phenotypic shift that mimics what has previously been reported in patient-derived organoids. Understanding the molecular mechanism that drives luminal bladder cancer provides critical information in bettering our approach in diagnosing and treating MIBCs.

Prostate cancer is among the most common malignancies in males in the United States and adenocarcinoma accounts for 95% of all malignancies of prostate. Rarely prostate cancer can also present as small cell carcinoma. Pure small cell carcinoma is rare at time of initial diagnosis (<2%) however neuroendocrine differentiation into small cell carcinoma may emerge in men who have had previous treatment with ADT for prostate adenocarcinoma. These tumors, sometimes called treatment-related neuroendocrine prostate cancers or aggressive-variant prostate cancers, are increasingly recognized in the castration-resistant phases of disease progression. They account for less than 1% of all prostate cancers. A 73-year-old otherwise male had routine health screening in May 2018. Prostate specific antigen (PSA) level was elevated at 9.53 ng/mL. He had not had a screening PSA for at least two prior years but this was a significant change from prior levels. Patient was asymptomatic however the abnormal laboratory evaluation prompted consultation with Urology. Biopsy of prostate gland confirmed prostatic adenocarcinoma with Gleason's score of 5+ 4 = 9 with bilateral gland involvement. Imaging studies including CT scan of abdomen and pelvis, a bone scan and a PET scan showed no clear evidence of metastatic disease. Patient's clinical stage was determined to be IIIC with T2c N0 M0 disease. Patient began treatment with androgen deprivation therapy and received definitive radiation treatment with external bean radiation therapy from July to September 2018. PSA was 0.08 ng/ml at the end of radiation treatment. Patient did well for about 15 months, after which he had multiple hospital admissions for dyspnea, fluid retention and lower extremity edema. He was also found to have refractory hypokalemia. Patient underwent MRI brain which revealed numerous small enhancing calvarial and skull base lesions consistent with bony metastasis in the skull. Patient also underwent PET/CT scan which showed numerous thoracic spine bony lesions, numerous to count bony metastasis throughout the lumbar spine and pelvis, as well as multiple hepatic lesions. Patient underwent biopsy of right hepatic lobe lesion and pathology was consistent with small cell carcinoma with positive neuroendocrine markers including CD56, synaptophysin and TTF-1. Interestingly patient’s PSA was only 0.09ng/dL. Given refractory hypokalemia, paraneoplastic syndrome was suspected and further work-up was initiated. Serum cortisol levels were elevated at 119.6 mcg/dL (3.7-19.4) and ACTH level was 333 pg/mL (7.2 - 63.3). Aldosterone level was <1 ng/dL (0 - 30.0). Patient was diagnosed with paraneoplastic Cushing syndrome. Given aggressive nature of this small cell transformation, patient was started on treatment with systemic chemotherapy with Carboplatin/Etoposide during the hospital stay, with stabilization of potassium levels. Prostate small cell carcinoma poses a challenge for diagnosis and treatment. In contrast to adenocarcinoma of the prostate, serum prostate-specific antigen (PSA) is not predictive of disease severity, nor is it a useful tumor marker for monitoring progression or surveillance. Patients with prostate small cell cancer presents with more diverse symptoms than any other prostate cancer since it tends to metastasize early. Also paraneoplastic syndromes are more common in prostate small cell cancers as well.

This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Background: Metastasis accounts for 90% of cancer-associated mortality in patients with renal cell carcinoma (RCC). However, the clinical management of RCC metastasis is challenging. Lactate export is known to play an important role in cancer cell migration. This study investigated the role of heat shock protein A12A (HSPA12A) in RCC migration. Methods: HSPA12A expression was examined in 82 pairs of matched RCC tumors and corresponding normal kidney tissues from patients by immunoblotting and immunofluorescence analyses. The proliferation of RCC cells was analyzed using MTT and EdU incorporation assays. The migration of RCC cells was evaluated by wound healing and Transwell migration assays. Extracellular acidification was examined using Seahorse technology. Protein stability was determined following treatment with protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132. Mass spectrometry, immunoprecipitation, and immunoblotting were employed to examine protein-protein interactions. Results: RCC tumors from patients showed downregulation of HSPA12A, which was associated with advanced tumor node metastasis stage. Intriguingly, overexpression of HSPA12A in RCC cells inhibited migration, whereas HSPA12A knockdown had the opposite effect. Lactate export, glycolysis rate, and CD147 protein abundance were also inhibited by HSPA12A overexpression but promoted by HSPA12A knockdown. An interaction of HSPA12A with HRD1 ubiquitin E3 ligase was detected in RCC cells. Further studies demonstrated that CD147 ubiquitination and proteasomal degradation were promoted by HSPA12A overexpression whereas inhibited by HSPA12A knockdown. Notably, the HSPA12A overexpression-induced inhibition of lactate export and migration were abolished by CD147 overexpression. Conclusion: Human RCC shows downregulation of HSPA12A. Overexpression of HSPA12A in RCC cells unstabilizes CD147 through increasing its ubiquitin-proteasome degradation, thereby inhibits lactate export and glycolysis, and ultimately suppresses RCC cell migration. Our results demonstrate that overexpression of HSPA12A might represent a viable strategy for managing RCC metastasis.

Accurate diagnosis of small‐cell carcinoma of the lung (SCLC) is clinically important because of the therapeutic implications. SCLC must be distinguished from non‐small‐cell carcinoma (NSCLC) and lymphoma. Paranuclear blue inclusions (PBIs) were recently described as a feature of metastatic SCLC on air‐dried Wright‐stained bone marrow aspirate smears. To determine the utility of PBIs in distinguishing SCLC from NSCLC and lymphoma, we evaluated air‐dried Diff‐Quik‐stained smears from 103 fine‐needle aspiration (FNA) specimens and 14 touch imprint specimens. PBIs were identified in 24 (89%) of 27 cases of SCLC, in 6 (9%) of 64 non‐small‐cell carcinomas (P < 0.00001), and in two (8%) of the 26 lymphoma cases (P < 0.00001). No PBIs were seen on any of the alcohol‐fixed Papanicolaou or hematoxylin‐eosin (HandE) stained smears examined. In conclusion, PBIs appear to be a feature of SCLC on air‐dried cytologic material stained with Romanowsky type stains. In the presence of cytologic features of SCLC, the identification of PBIs provides a useful diagnostic feature for diferentiating between SCLC and NSCLC carcinomas, and between SCLC and lymphomas in FNA specimens and touch imprints from surgical specimens.