miRNAs as Influencers of Cell-Cell Communication in Tumor Microenvironment

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, inducing the degradation of the target mRNA or translational repression. MiRNAs are involved in the control of a multiplicity of biological processes, and their absence or altered expression has been associated with a variety of human diseases, including cancer. Recently, extracellular miRNAs (ECmiRNAs) have been described as mediators of intercellular communication in multiple contexts, including tumor microenvironment. Cancer cells cooperate with stromal cells and elements of the extracellular matrix (ECM) to establish a comfortable niche to grow, to evade the immune system, and to expand. Within the tumor microenvironment, cells release ECmiRNAs and other factors in order to influence and hijack the physiological processes of surrounding cells, fostering tumor progression. Here, we discuss the role of miRNAs in the pathogenesis of multicomplex diseases, such as Alzheimer's disease, obesity, and cancer, focusing on the contribution of both intracellular miRNAs, and of released ECmiRNAs in the establishment and development of cancer niche. We also review growing evidence suggesting the use of miRNAs as novel targets or potential tools for therapeutic applications

Molecular Characterization of Ductal Carcinoma In Situ: Pilot Studies

Ductal carcinoma in situ (DCIS); is thought directly to precede invasive breast cancer (IBC). Screening mammography has driven the incidence of this key precursor lesion to \u3e65,000 cases per year. However, little is known about the factors controlling the natural history or risk for recurrence following treatment of a particular patients DCIS. Though the heterogeneity of the disease is well established, no histologic or demographic criteria have been able to stratify DCIS for treatment. We hypothesize that at initial diagnosis there exist biologically distinct subsets of DCIS with associated prognoses that may be recognized by molecular markers. Molecular approaches have been limited by technical design issues related to the types of tissue available for analysis, namely degraded formalin-fixed paraffin embedded (FFPE) specimens and small core biopsy samples. However, new technologies promise to overcome these issues. In the first phase of our investigation, we aimed a) to pilot feasibility studies on the use of FFPE DCIS for molecular analyses including gene expression microarray and b) to pilot feasibility study of selective, high throughput sequencing through the use of exon capture on small input material that simulated expected DCIS core biopsy amounts. The results of this work offer specific technical guidelines for the molecular study of DCIS. Moreover, they have enabled the initiation of the second phase of this study, which aims to assess molecular profiles of DCIS recurrence and progression

Role of HDAC4 in the process of oncogenesis in human cells

The resetting of Epigenetics is a common feature of cancer cells. By contrast how epigenetic regulators can trigger the oncogenic transformation is mystery. Previous studies reported that the hyper-activation of the epigenetic regulator HDAC4 transforms murine cells. However, the transformation process is more complex in human cells that are relatively resistant to oncogenesis. In the present thesis we addressed the oncogenic potential of HDAC4 in human primary fibroblasts (BJ). The over-expression of an hyper-active mutant of HDAC4 in human fibroblast triggers a permanent cell cycle arrest typical of premature senescent cells after the exposure to strong oncogenes. The contemporary inactivation of p53 and pRb/p16 pathways achieved by the transduction of the cells with SV40 LT allows the transformation of HDAC4-TM cells. Transcriptomic analysis of HDAC4-TM and RAS transformed cells evidenced that both the oncogenes require the repression of the interferon-response to transform the human cells. Further we have compared our signature with other two oncogenes like RAS and c-MYC which favour in vitro transformation in BJ-hTERT SV40 LT/ST. The tumorigenic properties driven by the three oncogenes rely on the activation of some common molecular pathways, but not on the activation and repression of the same genes. Importantly, commonly dysregulated genes, both up-regulated and down-regulated during in vitro transformation contribute to a worst survival rate in some cancer types. To gain insight on the cellular pathways supervised by HDAC4 and responsible for the transformation of human cells we investigated the genomic instability of leiomyosarcoma cells knocked out of HDAC4. In this cancer cell removal of HDAC4 results in the induction of cellular senescence through the augmentation of the DNA damage and the activation of the interferon response.Ingles

Distinct roles of BMP and LKB1/AMPK signalling impacting ovarian cancer spheroid biology

High-grade serous (HGS) carcinoma, the most prevalent and most deadly subtype of epithelial ovarian cancer (EOC), presents unique therapeutic challenges since the majority of cases are diagnosed at advanced, metastatic stage. At this point widespread intraperitoneal metastatic lesions are numerous, which is why models that recapitulate disease dissemination are critical to uncover novel therapeutic targets. One of the initiating events in ovarian cancer metastasis is shedding from the primary tumour into the peritoneal cavity where cells must survive in suspension in order to seed secondary tumours. This non-adherent population of cells exists as multicellular aggregates, or spheroids; data from our lab has demonstrated that cells within spheroids are dormant, yet are readily alter their phenotype upon reattachment to an adherent substratum. To further explore the pathobiology of ovarian cancer spheroids, my thesis work describes the functional characterization of two different signalling pathways— bone morphogenetic protein (BMP), and the liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK)—which mediate distinct and important aspects of spheroid formation and reattachment. Activated BMP signalling resulted in smaller, loosely-aggregated spheroids, which were more readily able to reattach and disperse. These phenotypic alterations observed as a result of active BMP signalling were mediated, at least in part, by cooperation with the AKT signalling pathway. These studies implicate inhibition of BMP and AKT signalling as potential strategies for therapeutic targeting of reattaching spheroids, which is critical for the formation of secondary metastatic lesions. Other work in our lab implicated the downregulation of AKT signalling in spheroid formation-induced dormancy. In an attempt to uncover additional pathways promoting the dormant phenotype of ovarian cancer spheroids, I investigated the LKB1/AMPK signalling cascade given its ability to alter cellular metabolism in response to nutrient and energy availability. Despite a dramatic enhancement in AMPK activity observed in ovarian cancer spheroids, targeted knockdown had no effect on viability of cells in this context. However, knockdown of its upstream kinase, LKB1, revealed a dramatic decrease in ovarian cancer spheroid viability, suggesting a role for this kinase in mediating anoikis-resistance in an AMPK-independent manner. Taken together, my results have uncovered two distinct and important signalling pathways that regulate unique aspects of spheroid formation, cell survival, and reattachment. By understanding the molecular mechanisms used by ovarian cancer spheroids to survive during dissemination and promote secondary metastasis, my work has uncovered additional therapeutic targets for the potential treatment of advanced-stage ovarian cancer

Analysis of Kinase Gene Expression Patterns across 5681 Human Tissue Samples Reveals Functional Genomic Taxonomy of the Kinome

Peer reviewe

Extracellular Vesicles: Biology and Potentials in Cancer Therapeutics

Extracellular vesicles (EVs) are particles wrapped in a lipid bilayer membrane and are naturally released from cells. This kind of cargo vessel is a nanostructure that mainly transfers lipids, proteins, various nucleic acid fragments, and metabolic components to neighboring cells or distant parts of the body through the circulatory system. EVs are of great significance to the communication mechanism between cells. This book collects feature articles to enhance our understanding of the biological characteristics of EVs and their potential applications

Aberrant Gene Expression: Diagnostic Markers and Therapeutic Targets for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer and the fourth leading cause of cancer-related death in the United States. The overall median survival for patients diagnosed with PDAC is five to eight months. The poor outcome is due, in part, to a lack of disease-specific symptoms that can be used for early detection, and as such, most patients present with locally advanced or metastatic disease at the time of diagnosis. Therefore, the need for diagnostic tools is both great and urgent. Furthermore, current chemotherapies have low response rates and high toxicity, limiting their use, and there are currently no effective targeted therapies for PDAC. Therefore, a greater understanding of the underlying biology of pancreatic cancer is needed to identify tumor-specific vulnerabilities that can be therapeutically exploited. Pancreatic cancer development is driven by genomic changes that alter gene expression. Aberrant gene expression produces changes in protein expression, which in turn may confer growth advantages to the tumor; often the tumor then develops a dependency on continued aberrant gene and protein expression. Determining how aberrant genome-wide gene expression changes affect the biology of the tumor is of paramount importance in our continued efforts to improve patient therapy. By identifying and establishing a role for the overexpressed genes in PDAC we can discover new avenues for therapeutic intervention and potentially predict the most effective therapy for each individual and avoid therapies that may have little clinical efficacy. My research aimed to identify aberrantly expressed genes in primary tumor samples from PDAC patients and characterize the diagnostic and therapeutic value of the identified genes. The work outlined in this dissertation focuses first on identifying a prognostic signature of genes with the ability to stratify patients into high and low risk groups, and second on assessing the biological importance of overexpression of the dual-specificity protein kinase TTK for pancreatic cancer oncogenesis. Together these two investigations provide a basis for translating molecular changes in tumor biology into improved therapy for pancreatic cancer patients.Doctor of Philosoph

Prognostic Implications of Cellular Senescence in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease for which biologically grounded predictors of outcome remain a clinical need. In addition to tumor cell death, antineoplastic drugs can mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence, but structured investigations into its prognostic and predictive power are lacking. Besides its occurrence in response to chemotherapeutic treatment, cellular senescence can be evoked by replicative stress or activation of oncogenes and serves as an initial barrier to cancer development. Yet, long-term effects of senescent cancer cells on tumor growth are unclear as they are known to mediate inflammation via a senescence-associated secretory phenotype (SASP) and are subjected to epigenetic remodeling, thereby acquiring cancer stemness characteristics. In an ex vivo analysis of AML blast samples from patients at diagnosis, I aimed to characterize basal as well as treatment-evoked senescence and determine its role as a prognostic and predictive biomarker. I established assays to detect and therapeutically induce senescence in a primary AML culture setting. Senescence was assessed by senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers. Gene expression analyses validated my experimental characterization of AML samples as “senescent”, as evidenced by upregulation of senescence-associated gene expression signatures. For prognostic analysis, clinical outcomes and molecular genetics of AML sample donors were retrieved. I found the intra-individual changes of senescence levels in response to the standard anti-leukemic agent daunorubicin to be positively correlated with better disease-free- and overall patient survival. In line with this, a more favorable molecular risk group, normal karyotype, and NPM1 as well as DNMT3AR882 mutations were associated with higher therapy-induced senescence levels. Other therapeutic AML agents, namely hydroxyurea, decitabine and gemtuzumab ozogamicin were also shown to induce senescence. Finally, in a consecutive ex vivo treatment with daunorubicin (to induce senescence), followed by the “senolytic” (i.e., selectively cytotoxic to senescent cells) BCL2 inhibitors venetoclax and navitoclax, both growth and viability of AML blasts were additionally reduced compared to single-agent treatments only in senescence-capable samples. To the best of my knowledge, this is the first study providing direct evidence that cellular senescence, induced ex vivo in patient-derived AML blasts by chemotherapeutic drugs, could serve as a predictive biomarker of long-term response to standard therapy. I believe that therapy-induced senescence might explain, at least in part, the underlying biology of current paraclinical risk indicators, and, as an outlook, might serve as a guidance for future personalized treatment of AML.Die akute myeloische Leukämie (AML) ist eine heterogene Erkrankung, für die die Entwicklung neuer pathophysiologisch fundierter prädiktiver Biomarker von großer klinischer Notwendigkeit ist. Zusätzlich zu apoptotischem Zelltod von Tumorzellen können antineoplastische Medikamente zu einem dauerhaften Zellzyklusarrest viabler, metabolisch aktiver Tumorzellen führen, welches Phänomen als Therapie-induzierte Seneszenz in verschiedenen Tumorentitäten charakterisiert wurde. Die prognostische und prädiktive Relevanz Therapie-induzierter Seneszenz für den Verlauf von Tumorerkrankungen ist derzeit unklar. Außer einer Induktion durch Chemotherapeutika kann Seneszenz u.a. durch replikativen Stress oder Onkogen-Expression hervorgerufen werden und dient dadurch als initiale zelluläre Barriere gegen maligne Entartung. Die langfristige Bedeutung von im Organismus persistierenden seneszenten Tumorzellen bleibt jedoch unklar, da diese durch ihren Seneszenz-assoziierten sekretorischen Phänotyp (SASP) auch proinflammatorisch wirken und durch epigenetische Veränderungen Krebsstammzelleigenschaften aufweisen können. In ex vivo-Untersuchungen an aus Patient:innenproben zum Zeitpunkt der Diagnosestellung gewonnenen AML-Blasten konnte ich zunächst „basale“ und Therapie-bedingte Seneszenz in der AML charakterisieren um daraufhin Seneszenz als prädiktiven Biomarker zu analysieren. Nach Etablierung von Primärkulturbedingungen für die zytostatische Behandlung (und somit mögliche Seneszenzinduktion) aufgereinigter AML-Blasten konnte ich mit zytochemischen und Fluoreszenz-basierten Assays die Zunahme der Seneszenz-assoziierten-β-Galaktosidase (SA-β-gal)-Aktivität und anderer Seneszenzmarker nachweisen. Durch RNA-Sequenzierung konnte meine experimentelle Klassifikation individueller AML-Proben als „Seneszenz-fähig“ anhand Seneszenz-assoziierter Genexpressionssignaturen bestätigt und weiter charakterisiert werden. Zur Analyse der prädiktiven Bedeutung Therapie-bedingter Seneszenz wurden die einzelnen AML Proben weiter molekulargenetisch untersucht und experimentelle Ergebnisse mit dem jeweiligen klinischen Verlauf individueller Patient:innen korreliert. Ich konnte zeigen, dass die intraindividuelle Induzierbarkeit von Seneszenz durch ex vivo-Behandlung mit dem AML-Standardchemotherapeutikum Daunorubicin positiv mit einem verbesserten erkrankungsfreien Überleben und Gesamtüberleben korrelierte. Zudem waren eine günstigere molekulare Risikogruppe, ein normaler Karyotyp sowie NPM1- und DNMT3AR882-Mutationen mit höheren Leveln Therapie-induzierter Seneszenz assoziiert. Durch die Behandlung mit anderen AML-Therapeutika wie Hydroxyurea, Decitabin oder Gemtuzumab-Ozogamicin konnte ebenfalls Seneszenz ausgelöst werden. Schließlich konnten ich durch eine konsekutive ex vivo-Behandlung mit zunächst Daunorubicin (zur Seneszenzinduktion) und darauffolgend mit den „senolytisch“ wirkenden (d.h. selektiv zytotoxisch gegenüber seneszenten Zellen) BCL2-Inhibitoren Venetoclax und Navitoclax sowohl Zellzahl als auch Viabilität seneszenzfähiger AML-Proben im Vergleich zu einer Therapie mit den Einzelsubstanzen oder zu AML-Proben, welche nicht seneszenzfähig waren, zusätzlich reduzieren. Nach meinem Kenntnisstand konnte im Rahmen dieses Promotionsprojektes erstmals nachgewiesen werden, dass durch ex vivo-Chemotherapie in aus Patient:innen gewonnenen AML-Blasten induzierte zelluläre Seneszenz als prädiktiver Biomarker für das langzeitige Therapieansprechen auf die Standard-Induktionstherapie dienen kann. Möglicherweise erklärt Therapie-induzierte Seneszenz Teilaspekte der etablierten paraklinischen Risikofaktoren zugrundliegenden Tumorbiologie und kann perspektivisch als Marker für personalisierte Behandlungskonzepte in der AML verwendet werden

Dissecting Tumor Heterogeneity in Lung Cancer

Lung cancer is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells as well as a heterogeneous microenvironment consisting of non-cancer cells and extracellular matrix. Constant interactions among these components ultimately leads to a complex tumor tissue that is ever evolving and poses a therapeutic challenge for sustained benefit. Strategies for targeting lung cancers are largely guided by the genetic alterations identified in the tumor specimens. However, in order to gain a better understanding of lung cancer progression and develop effective treatment modalities, studying tumor in context of its microenvironment is crucial. The first aim of this project was to establish an experimental model to capture tumor heterogeneity. We developed an Ex Vivo Tumor system that preserved tumor composition and allowed the introduction of specific modifications in the tumor microenvironment to investigate their role in tumor progression. We utilized this system to demonstrate the role of extrinsic as well as intrinsic alterations that modify tumor cell behavior. Next, we explored the biological phenomenon epithelial-to-mesenchymal transition as a source of tumor cell heterogeneity and therapeutic resistance. Genetically identical KRAS mutant lung cancer cells displayed different phenotypic states that were associated with distinct survival pathways that allowed cancer cells to escape therapeutic targeting. With the use of extensive in vitro, ex vivo and in vivo models, we identified that a combinatorial approach of utilizing CDK4 and MEK inhibitors to effectively control tumor growth by targeting distinct tumor subpopulations within lung cancer and prevented emergent resistance to either single agent