Clinical and Translational Implications of Centrosome Amplification and Clustering in Multiple Malignancies

Cancer initiation and progression are multistep processes that rely on the generation and accumulation of non-lethal mutations, which deregulate function of tumor suppressor genes and activate oncogenic pathways. Evolving through a landscape of heterogeneous somatic mutations, mutated cells undergo subsequent selection pressures and the one endowed with the greatest fitness advantage survives giving rise to genetically diverse cell populations resulting in intratumor heterogeneity (ITH). Presence of the abnormal number of centrosomes is one of the key factors contributing towards ITH. Clustering of amplified centrosomes allows cancer cells to avoid mitotic spindle multipolarity that could otherwise result in cell death either by mitotic catastrophe or a high-grade multipolar division yielding intolerably severe aneuploidy. Thus, centrosome clustering enables low-grade chromosomal missegregation and their unequal distribution to daughter cells resulting in chromosomal instability (CIN), thus contributing to neoplastic transformation. Owing to the presence of genetically different cells in a tumor, monotargeted therapy spares clones lacking therapy-specific targets giving them the opportunity to repopulate the tumor with immunity toward the applied therapy and propensity to recur. Therefore, ITH poses major challenges to both clinicians and drug developers as it precludes detection of low-level clones, prediction of tumor evolution, development of drugs to target specific clones and evaluation of effective, yet non-toxic combinatorial regimens to combat ITH. I envision that a comprehensive quantitative analysis of centrosome amplification (CA), which is a bonafide driver of ITH might help better understand clinical behavior and improve therapeutic management of tumors. To this end, my research, presented here, primarily focuses on testing i) the impact of centrosome amplification and centrosome clustering protein (KIFC1) on clinical outcomes in multiple malignancies and ii) the role of tumor hypoxia in inducing centrosome amplification in cancer. Collectively, my findings reveal that CA and KIFC1 are prognostic and predictive in multiple malignancies and that tumor hypoxia plays a crucial role in inducing CA in tumors. This body of work expands our knowledge in causes and clinical implications of CA to help guide treatment decisions and development of precision medicine for multiple malignancies

Illuminating Actionable Biology in Breast Cancer: Novel Predictive and Prognostic Biomarkers

Assessing hormone receptors (the estrogen and progesterone receptors) and the human epidermal growth factor receptor 2 (HER2) to guide clinical decision making revolutionized treatment for breast cancer patients. However, in the years since these biomarkers were first incorporated into routine clinical care, only a few others have been validated as clinically useful in guiding adjuvant chemotherapy decisions and are recommended by the American Society of Clinical Oncology (ASCO) for patients with hormone-positive breast cancer. For patients with triple-negative breast cancer (TNBC), which lacks hormone and HER2 receptors, not any of these biomarkers are recommended by ASCO due to insufficient evidence that they meaningfully improve clinical outcomes. Breast cancer is the second-leading cause of cancer-related death among women in the US, indicating an unmet need to improve treatments, which can be accomplished in part by identifying and validating novel predictive and prognostic biomarkers that yield actionable information about the clinical course of breast cancers, especially TNBCs. A major obstacle to improving outcomes for breast cancer patients is intratumor heterogeneity (ITH), which can be extensive in breast cancer and drives treatment resistance and relapse. I envision that assaying drivers of ITH can inform clinicians about which breast tumors may be intrinsically more aggressive and carry a greater risk of breast cancer-related morbidity and mortality. My research, presented here, primarily focuses on testing the impact of drivers of ITH (namely, centrosome amplification [CA], the clustering protein KIFC1, and mitotic propensity and its drivers) on clinical outcomes in breast cancer in multivariable models as well as the correlates of in vitro efficacy of centrosome declustering drugs (which can selectively eliminate cancer cells with CA). Collectively, these studies reveal gene signatures and immunohistochemical biomarkers that are independent predictors of aggressive breast cancer course and rational strategies to optimize targeted therapy to combat cancer cells exhibiting CA, thereby contributing to the literature on the development of precision medicine for breast cancer patients, including TNBC patients

Understanding the disease and supporting clinical decisions

Barrett’s esophagus (BE) is a recognized premalignant condition of the distal esophagus that constitutes the major risk factor for the development of esophageal adenocarcinoma (EA). Despite the known low rates of BE progression to EA, the incidence of both has increased profoundly over the last decades and esophageal malignancy remains to be a deadly cancer with high morbidity and mortality unless diagnosed at early stages. Current BE clinical management has revealed unsuccessful in reverting this worrisome epidemiological picture and a major hurdle has been the incapacity to discriminate among BE patients those who have a higher risk of malignant progression. In fact, to this date, besides dysplasia none of the existing clinical and histologic criteria could anticipate malignant progression. It is therefore imperative to find reliable molecular biomarkers to guide medical practice and improve the standard of care for BE patients. The global aim of this thesis was to better understand the pathways underlying BE malignant progression and thereby identify reliable biomarkers relevant for the diagnosis, prognosis and management of BE patients thus contributing to an improved understanding of BE biology and an optimized support for clinical decisions. To accomplish these challenging goals an unbiased and a hypothesis-driven strategies were followed. Through the unbiased approach, a meta-analysis of transcriptome datasets and subsequent experimental validation in a cohort BE patients in follow-up was used to define a gene set associated with BE cancer development and, therefore, identify early biomarkers predictive of BE malignant progression. In silico analysis singled out two genes, CYR61 and TAZ as candidate predictive markers for BE malignant progression and experimental validation using quantitative PCR and immunohistochemistry revealed that both genes are upregulated and overexpressed in non-dysplastic BE index biopsies from progressors years before cancer development when compared with index biopsies from BE patients that did not progressed. We also found that EMT and stemness-related genes were also significantly over represented in BE associated with progression. Together, these results support that CYR61 and TAZ are promising early biomarkers to stratify BE patients according to their cancer risk and suggest a novel mechanist route for BE neoplastic progression. Using an hypothesis-driven approach, we explored when and how centrosome abnormalities arise along BE malignant pathway, from the early premalignant condition stage to metastatic disease, by establishing an accurate method to identify and score centrosomes, at the single-cell level, in patient samples and cell lines. We found that centrosome amplification arises as early as the premalignant condition of patients that progress to malignancy and significantly expands at dysplasia stage, which is dependent of p53 loss of function, being then present along cancer progression, namely in EA and metastasis. So, these finding suggest that centrosome amplification could contribute to BE initiation and malignant progression. Considering that centrosome amplification is specific of patients that progress to cancer, this could be further explored to be translated into useful tools to be used in the clinical setting and potentially improve its diagnosis, prognosis and treatment. Moreover, given widespread occurrence of both p53 mutations and centrosome abnormalities in human tumors, our findings are likely to be extended to other cancers. Collectively, both research avenues suggest the existence of different cellular and molecular abnormalities dictating different pathological propensity for malignant progression in BE, right from the beginning, and this could be further explored to trace a cancer risk profile for every patient and guide medical decisions and improve patient care.O esófago de Barrett (EB) é uma reconhecida condição pré-maligna que surge no esófago distal associada ao refluxo gastroesofágico crónico e constitui o maior factor de risco para o desenvolvimento do adenocarcinoma esofágico (AE). Apesar da taxa de progressão maligna do EB ser baixa, a incidência de ambos tem aumentado drasticamente nas últimas décadas. Como agravante, esta neoplasia em estadios avançados está associada a taxas elevadas de morbilidade e mortalidade a menos que diagnosticada e tratada em estadios iniciais, pelo que todos os doentes com EB são integrados em programas de vigilância com vista à detecção precoce de progressão neoplásica. Contudo, esta prática clínica tem-se mostrado ineficaz para reverter este cenário epidemiológico e um dos factores limitantes relaciona-se com o facto da displasia continuar a ser o único marcador de risco de progressão maligna e não haver marcadores moleculares e clínicos que possam predizer e estratificar o potencial maligno do EB. É assim urgente encontrar biomarcadores sensíveis e específicos capazes de guiar a prática médica, melhorar a relação custo-benefício dos programas de vigilância, mas principalmente a qualidade de vida dos doentes com EB. O objectivo principal desta tese foi descobrir novas vias moleculares subjacentes à progressão maligna do EB de forma a identificar biomarcadores fidedignos passíveis de serem aplicados à clínica apoiando o diagnóstico, prognóstico e manejo destes doentes, e assim contribuir para aprofundar o conhecimento relativo ao processo de cancerigénese desta doença e potencialmente melhorar a abordagem clínica aos doentes com EB. Para tal foram seguidas duas linhas de investigação diferentes. De modo a encontrar biomarcadores com potencial preditivo para progressão maligna em doentes com EB foi realizada uma meta-análise de dados de transcriptomas previamente publicados, seguida de uma validação experimental utilizando amostras de EB de doentes acompanhados no programa de vigilância do Instituto Português de Oncologia Francisco Gentil. Esta estratégia permitiu identificar dois promissores biomarcadores, o CYR61 e o TAZ, capazes de estratificar o risco de progressão maligna do EB, não só pela sua expressão diferencial ser a mais significativa na análise bioinformática mas porque a validação experimental revelou que estes dois genes se encontravam sobreexpressos logo na primeira biopsia (vários anos antes do desenvolvimento de cancro) onde foi feito o diagnóstico de EB dos doentes que progrediram comparativamente com a expressão detetada no EB dos não progressores. Adicionalmente, foram ainda identificados outros genes diferencialmente expressos em EB associados a progressão maligna, cujas funções estão associadas a fenótipos de células estaminais e fenómenos de transição epitélio-mesenquimal (TEM) em cancro. Assim, foi também descoberto um potencial novo processo molecular associado ao desenvolvimento de cancro em EB. Paralelamente, e considerando que alterações numéricas dos centrossomas podem estar presentes ao longo da progressão maligna em EB e assim contribuir para o seu processo de cancerigénese, decidimos explorar quando e como surgem as alterações numéricas dos centrossomas ao longo da progressão do EB, desde a condição prémaligna até às metástases ganglionares, tanto em amostras de doentes como em linhas celulares, usando um método de dupla marcação por imunofluorescência para identificar e quantificar fidedignamente o número de centrossomas por célula. Esta análise revelou a existência de células com centrossomas supranumerários logo na fase de metaplasia dos doentes que progrediram para cancro, e que a sua incidência aumenta significativamente na fase de displasia, a qual é dependente da perda de função do gene supressor tumoral p53, estando também depois presentes ao longo das restantes fases de progressão. Estes resultados sugerem assim que a desregulação dos centrossomas pode contribuir para a iniciação e progressão neoplásica do EB. No futuro será importante aprofundar o contributo dos centrossomas na cancerigénese do EB e tentar perceber qual o seu impacto no diagnóstico, prognóstico e tratamento destes doentes. Uma vez que tanto as alterações numéricas dos centrossomas como a perda de função do p53 são achados prevalentes em cancro, os resultados deste estudo poderão ser relevantes para outros modelos tumorais. Em conjunto, os resultados obtidos sugerem que a propensão maligna não é igual em todos os EB. Logo muito cedo no processo, aqueles cujo risco de virem a desenvolver cancro é maior sofrem alterações moleculares e celulares passíveis de serem detetadas e utilizadas como biomarcadores preditivos estratificando o risco de progressão maligna, e desta forma orientar as decisões clínicas, adequar tempos de vigilância e melhorar a abordagem terapêutica

LMW-E MEDIATES MAMMARY TUMORIGENESIS BY DEREGULATING ACINAR MORPHOGENESIS & GENERATING CANCER STEM CELLS

Cyclin E is the regulatory subunit of the cyclin E/CDK2 complex that mediates the G1-S phase transition. N-terminal cleavage of cyclin E by elastase in breast cancer generates two low molecular weight (LMW) isoforms that exhibit both enhanced kinase activity and resistance to p21 and p27 inhibition compared to fulllength cyclin E. Clinically, approximately 27% of breast cancer patients overexpress LMW-E and associate with poor survival. Therefore, we hypothesize that LMW-E disrupts normal mammary acinar morphogenesis and serves as the initial route into breast tumor development. We first demonstrate that LMW-E overexpression in non-tumorigenic hMECs is sufficient to induce tumor formation in athymic mice significantly more than overexpression of full-length cyclin E and requires CDK2- associated kinase activity. Further in vivo passaging of these tumors augments LMW-E expression and tumorigenic potential. When subjected to acinar morphogenesis in vitro, LMW-E mediates significant morphological disruption by generating hyperproliferative and multi-acinar complexes. Proteomic analysis of patient tissues and tumor cells with high LMW-E expression reveals that the activation of the b-Raf-ERK1/2-mTOR pathway in concert with high LMW-E expression predicts poor patient survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (b-raf inhibitor) effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing the G1/S cell cycle arrest. In addition, the LMW-E-expressing tumor cells exhibit phenotypes characteristic of the EMT and enhanced cellular invasiveness. These tumor cells also enrich for cells with CSC phenotypes such as increased CD44hi/CD24lo population, enhanced mammosphere formation, and upregulation of ALDH expression and enzymatic activity. Furthermore, the CD44hi/CD24lo population also shows positive correlation with LMW-E expression in both the tumor cell line model and breast cancer patient samples (p\u3c0.0001 & p=0.0435, respectively). Combination treatment using doxorubicin and salinomycin demonstrates synergistic cytotoxic effects in cells with LMW-E expression but not in those with full-length cyclin E expression. Finally, ProtoArray microarray identifies Hbo1 as a novel substrate of the cyclin E/CDK2 complex and its overexpression results in enrichment for CSCs. Collectively, these data emphasize the strong oncogenic potential of LMW-E in mammary tumorigenesis and suggest possible therapeutic strategies to treat breast cancer patients with high LMW-E expression

Diagnostic, Prognostic and Therapeutic Value of Gene Signatures

Gene expression studies have revealed diagnostic profiles and upregulation of specific pathways in many solid tumors. Some gene-expression signatures are already used as predictors of relapse in early breast cancer patients. The explosion of new information in gene expression profiling could potentially lead to the development of tailored treatments in many solid tumors. In addition, many studies are ongoing to validate these signatures also in predicting response to hormonal, chemotherapeutic, and targeted agents in breast cancer as well as in other tumors. This book has been carried out with the aim of providing readers a useful and comprehensive resource about the range of applications of microarray technology on oncological diseases. The book is principally addressed to resident and fellow physicians, medical oncologists, molecular biologists, biotechnologists, and those who study oncological diseases. The chapters have been written by leading international researchers on these topics who have prepared their manuscripts according to current literature and field experience with microarray technology

DNA damage in paediatric obesity: a promoter and predictor of cancer in adulthood

Obesity in children is one of the most serious, global, public health challenges of the 21st century. The accumulation of adipose tissue is associated with a range of metabolic complications including diabetes, cardiovascular disease and dyslipidaemia. Epidemiological evidence links obesity in childhood with developing certain types of cancer later in life. It is postulated that excess adipose tissue and consequent inflammation derived oxidative stress may inflict an accumulation of deleterious DNA mutations and promote genome instability and drive carcinogenesis. Furthermore, a deficiency in micronutrients that are essential for DNA repair may exacerbate this pathological state. This research combined the assessment of anthropometric, inflammatory, micro-nutritional and DNA damage biomarkers via non-invasive techniques. In total, 112 children were recruited from schools and NHS obesity clinics. Anthropometric markers assessed were waist to hip ratio, body fat percentage via bioelectrical impedance, and body mass index standard deviation scores (BMI-SDS). These markers were used to classify participants as obese or nonobese and used for correlational analysis. Inflammation and micronutrient status were determined via C-reactive protein and vitamin D Enzyme Immune Assay (EIA) in saliva. DNA damage assessments include a microscopic assessment of nuclear anomalies via the buccal cytome assay, salivary telomere length via quantitative Polymerase Chain Reaction (qPCR) and urinary 8- hydroxyguanosine (8-OHdG) via EIA. The results from this study indicate obesity to be concurrent with increased inflammation and vitamin D deficiency in this cohort of participants. In addition, obesity was associated with increased oxidative DNA damage (8-OHdG) in urine and DNA damage events in the buccal mucosa. Salivary telomere length was positively correlated with obesity and the total frequency of nuclear anomalies found in buccal epithelial cells. Furthermore, there was a negative correlation between vitamin D and the frequency of nuclear anomalies in the oral cavity. Importantly, odds ratio analysis indicates a high BMI Z-score, waist circumference, body fat percentage, salivary CRP and low salivary vitamin D to be independent risk factors for increased nuclear anomalies in the buccal mucosa. This research is the first to accrue evidence for acquired DNA damage in multiple tissues obtained non-invasively from children with obesity. Our findings instigate that biomonitoring of ‘genome health’ for pre-cancerous molecular and morphological markers in obese patients may inform prioritization and severity of clinical intervention measures to prevent malignancy

Genotoxicity assessment of andrographolide.

In this thesis, the genotoxic potential of andrographolide, which is a primary phytochemical of an annual herb Andrographis paniculata, was assessed and the possible mechanisms of action were elucidated. In silico predictions showed that andrographolide may be capable of inducing skin sensitisation and chromosome damage in mammals in vitro and the identification of possible metabolites. Andrographolide was demonstrated to induce micronuclei formation in vitro in three cell lines tested namely AHH-1, MCL-5 and V-79. In AHH-1 and V-79 cell lines, the highest frequency of micronuclei was recorded at 30 muM whereas in MCL-5 cell line, 50 p,M of the compound was necessary to elicit similar damage, which may be due differences in cellular metabolism capacity. The phytochemical was shown to cause dose-dependent cellular cytotoxicity in all the cell lines tested with cells dying primarily via necrosis compared to apoptosis and effectively reduced cell number. Kinetochore labelling on MCL-5 cells challenged with increasing doses of andrographolide revealed that the phytochemical acts in aneugenic manner evident from the increment in kinetochore-positive micronuclei. The compound of interest was also found to disrupt segregation fidelity via centrosome amplification resulting in chromosomal aberration. The presence of extra microtubule organising centres may play a role in the promotion of aberrant mitoses. Disruption to normal cell division was observed even after the removal of the compound in vitro. Mammalian HFRT point mutation assay using AHH-1 cell line revealed that andrographolide induced mutation evident from HPRT- mutant colonies formation between 5muM and 30muM. The phytochemical was also found to exert cytotoxicity in a concentration-dependent manner between 1muM and 50muM. Thus, andrographolide is capable of disrupting normal mammalian cell division and causing dose-dependent cytotoxicity in vitro

Tumor Suppressor Genes

Functional evidence obtained from somatic cell fusion studies indicated that a group of genes from normal cells might replace or correct a defective function of cancer cells. Tumorigenesis that could be initiated by two mutations was established by the analysis of hereditary retinoblastoma, which led to the eventual cloning of RB1 gene. The two-hit hypothesis helped isolate many tumor suppressor genes (TSG) since then. More recently, the roles of haploinsufficiency, epigenetic control, and gene dosage effects in some TSGs, such as P53, P16 and PTEN, have been studied extensively. It is now widely recognized that deregulation of growth control is one of the major hallmarks of cancer biological capabilities, and TSGs play critical roles in many cellular activities through signaling transduction networks. This book is an excellent review of current understanding of TSGs, and indicates that the accumulated TSG knowledge has opened a new frontier for cancer therapies

Investigating the role of centrosome amplification in extracellular vesicle secretion in pancreatic ductal adenocarcinoma

PhD thesisPancreatic Ductal Adenocarcinoma (PDAC) is characterised by a dense desmoplastic reaction that is attributed to the activation of pancreatic stellate cells (PSCs) in the stroma. This alteration of the tumour microenvironment is thought to contribute to PDAC aggressiveness and resistance to therapy. Recent studies have shown that exosomes (a subgroup of secreted extracellular vesicles) secreted by cancer cells facilitate cross talk between tumour cells and the microenvironment. However, the mechanisms that lead to the secretion of these vesicles remains elusive. Here, we report for the first time, a novel role for centrosome amplification, a common feature of human tumours, in the secretion of small extracellular vesicles (sEVs). We show that centrosome amplification significantly correlates with and is sufficient to induce the elevated secretion of sEVs in PDAC cell lines. Furthermore, we demonstrate that oxidative stress in cells with supernumerary centrosomes is the driving force behind this altered sEV secretion. An analysis of centrosome amplification-associated increases in cellular reactive oxygen species (ROS) demonstrated an impaired lysosome function and the prevention of MVB/lysosome fusion events. The results indicate that centrosome amplification induced ROS induces sEV secretion by preventing MVB degradation by the lysosome, shifting their fate to fusion with the plasma membrane and subsequent secretion of their intraluminal vesicles (ILVs) as exosomes. To understand if exosomes secreted from cells with amplified centrosomes could impact the tumour microenvironment, we subsequently investigated the role of these sEVs on the activation of PSCs, as measured by the formation of fibres containing alpha-smooth muscle actin (α-SMA). We found that sEVs isolated from cells with supernumerary centrosomes elicit significantly stronger activation of PSCs compared to sEVs isolated from cells with a normal centrosome number, suggesting a difference in their biological cargo. SILAC based-proteomic analysis revealed the gain or loss of 6 EV protein in sEVs isolated from cells upon the induction of centrosome amplification, that may have a role in the activation of PSCs. We hypothesise, that further understanding the role of centrosome amplification in sEV-mediated PSC activation may help us to identify innovative ways to block PSC activation and prevent the progression of PDAC, which could have major clinical implications for patients with this devastating disease

Myeloid Leukemia

The current book comprises a series of chapters from experts in the field of myeloid cell biology and myeloid leukemia pathogenesis. It is meant to provide reviews about current knowledge in the area of basic science of acute (AML) and chronic myeloid leukemia (CML) as well as original publications covering specific aspects of these important diseases. Covering the specifics of leukemia biology and pathogenesis by authors from different parts of the World, including America, Europe, Africa, and Asia, this book provides a colorful view on research activities in this field around the globe