Regulation of the transmembrane mucin MUC4 by Wnt/β-catenin in gastrointestinal cancers

The transmembrane mucin MUC4 is a high molecular weight glycoprotein that is expressed de novo in pancreatic ductal adenocarcinoma (PDAC). MUC4 has been shown to play a tumor-promoting role in malignancies such as PDAC, ovarian cancer and breast cancer. Unlike the normal pancreas, MUC4 is ordinarily expressed by goblet and absorptive cells in the normal colonic epithelium. However, its expression/role in colorectal cancer (CRC) is not well studied. In this dissertation, the goal was to identify factor(s) that may differentially regulate MUC4 in these two disparate malignancies. Furthermore, in light of its pro-tumorigenic role in other malignancies, we analyzed the functional implications of MUC4 expression in CRC. A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites located in the proximal and distal promoters. Importantly, multiple TCF/LEF sites are typically present in the promoters of Wnt/β-catenin pathway target genes. In order to ascertain whether MUC4 was a Wnt/β-catenin target gene, we performed β-catenin knock down (KD) studies, treatment with Wnt3a ligand, as well as MUC4 promoter luciferase studies in the first section of this dissertation. In PDAC, it was observed that MUC4 transcript and protein were decreased upon β-catenin KD, WNT3a conditioned medium treatment increased MUC4 and MUC4 promoter luciferase activity was increased upon transfection with stabilized β-catenin. Furthermore, immunohistochemistry (IHC) with rapid autopsy PDAC tissues showed a positive correlation between MUC4 and β-catenin expression. Mutation of each of the three putative TCF/LEF sites showed that the sites closest to and furthest from the ATG site were critical for MUC4 promoter luciferase activity in the presence of stabilized β-catenin. A Chromatin immunoprecipitation assay (ChIP) confirmed that β-catenin associates with the MUC4 promoter at these two sites in PDAC. Functional studies with the β-catenin KD cells showed that migratory properties were decreased significantly upon KD, concomitant with altered levels of epithelial to mesenchymal transition (EMT) markers. We thus concluded that β-catenin up-regulates MUC4 in PDAC, and that the β-catenin-MUC4 axis likely contributes to the metastatic properties of PDAC cells. The second part of this dissertation deals with the regulation of MUC4 by β-catenin in CRC. Here, we observed that the KD of β-catenin induced an increase in MUC4 transcript and protein. This was corroborated by transient overexpression of stabilized β-catenin, which resulted in decreased MUC4. MUC4 promoter luciferase studies showed that KD of β-catenin resulted in increased promoter luciferase activity. The mutation of each of the three TCF/LEF sites and subsequent promoter luciferase assays showed that the second and third sites appeared to be repressive and that mutation of all three sites in combination caused an increase in MUC4. It was also observed that the Wnt/β-catenin pathway can indirectly repress MUC4 by suppression of Hath1, a Notch pathway target gene. Functional studies with MUC4 KD showed that MUC4 confers proliferative but not migratory properties to cells. The third part of this dissertation describes the generation of a mouse model aimed at delineating the role of Muc4 in CRC progression. Mice with colon-preferential heterozygous loss of Apc and mutant Kras, i.e. CDX2 P-NLS -Cre krasG12D/+ApcloxP/+ were crossed with the Muc4-/- mice generated in our lab. Preliminary results indicate the expression pattern of Muc4 is altered during the progression of CRC. Taken together, studies in this dissertation demonstrate the differential regulation of MUC4 by the Wnt/β-catenin pathway in PDAC and CRC, and that MUC4 expression may be pro-tumorigenic in CRC

The prognostic and predictive value of selected biomarkers in colorectal cancer

Tissue-based biomarkers are studied to receive information about the pathologic processes and cancer outcome, and to enable development of patient-tailored treatments. The aim of this study was to investigate the potential prognostic and/or predictive value of selected biomarkers in colorectal cancer (CRC). Group IIA secretory phospholipase A2 (IIA PLA2) expression was assessed in 114 samples presenting different phases of human colorectal carcinogenesis. Securin, Ki-67, CD44 variant 6 (CD44v6), aldehyde dehydrogenase 1 (ALDH1) and β-catenin were studied in a material including 227 rectal carcinoma patients treated with short-course preoperative radiotherapy (RT), long-course preoperative (chemo)RT (CRT) or surgery only. Epidermal growth factor receptor (EGFR) gene copy number (GCN), its heterogeneity in CRC tissue, and association with response to EGFR-targeted antibodies cetuximab and panitumumab were analyzed in a cohort of 76 metastatic CRC. IIA PLA2 expression was decreased in invasive carcinomas compared to adenomas, but did not relate to patient survival. High securin expression after long-course (C)RT and high ALDH1 expression in node-negative rectal cancer were independent adverse prognostic factors, ALDH1 specifically in patients treated with adjuvant chemotherapy. The lack of membranous CD44v6 in the rectal cancer invasive front associated with infiltrative growth pattern and the risk of disease recurrence. Heterogeneous EGFR GCN increase predicted benefit from EGFR-targeted antibodies, also in the chemorefractory patient population. In summary, high securin and ALDH1 protein expression independently relate to poor outcome in subgroups of rectal cancer patients, potentially because of resistance to conventional chemotherapeutics. Heterogeneous increase in EGFR GCN was validated to be a promising predictive factor in the treatment of metastatic CRC.Valikoitujen merkkiaineiden ennusteellinen merkitys paksu- ja peräsuolisyövässä Kudoslähtöisten merkkiaineiden tutkimisella toivotaan voivan saavuttaa lisätietoa syövän biologisesta käyttäytymisestä ja ennusteesta, sekä mahdollistaa kullekin potilaalle optimaalisen hoidon valinnan. Väitöstutkimuksen tavoitteena oli selvittää valikoitujen merkkiaineiden ennusteellista merkitystä paksu- ja peräsuolisyövässä. Tyypin II sekretorisen fosfolipaasi A2:n (IIA PLA2) ilmentymistä tutkittiin 114:ssä syövän kehittymisen eri vaiheita edustavassa paksu- ja peräsuolikasvaimessa. Securinin, Ki-67:n, CD44 variantti 6:n (CD44v6), aldehydidehydrogenaasi 1:n (ALDH1) ja β-kateniinin ilmentymistä analysoitiin 227 peräsuolisyöpäpotilaan aineistossa, jossa oli mukana myös ennen leikkausta (kemo)sädehoidettuja potilaita. Levinnyttä paksu- tai peräsuolisyöpää sairastavien potilaiden (n=76) leikkausnäytteistä tutkittiin epidermaalisen kasvutekijäreseptorin (EGFR) geenikopiomäärää, sen kasvaimen sisäistä vaihtelua, sekä yhteyttä EGFR-vasta-ainehoidoista saavutettavaan hyötyyn. IIA PLA2:n ilmentyminen syövässä oli vähäisempää kuin syövän esiasteissa. Securinin korkea ilmentyminen pitkän (kemo)sädehoidon jälkeen, sekä ALDH1:n korkea ilmentyminen imusolmukkeisiin leviämättömässä peräsuolisyövässä olivat itsenäisiä huonon ennusteen tekijöitä, ALDH1 etenkin solunsalpaajilla hoidetuilla potilailla. Heikentynyt solukalvon CD44v6-ilmentyminen oli yhteydessä syövän aggressiiviseen kasvutapaan ja peräsuolisyövän uusiutumisriskiin. Heterogeeninen EGFR-geenin kopiomäärän lisääntyminen ennusti hyvää vastetta EGFR-vasta-ainehoidoille levinneessä paksu- ja peräsuolisyövässä, mikä oli nähtävissä myös solunsalpaajahoidoille kehittyneen vastustuskyvyn jälkeisissä hoitolinjoissa. Yhteenvetona todetaan, että korkea securinin ja ALDH1:n ilmentyminen peräsuolisyövässä ovat itsenäisiä huonon ennusteen merkkejä tietyissä alaryhmissä. Tämä voi heijastaa securin- ja ALDH1-positiivisten syöpäsolujen vastustuskykyä yleisesti käytetyille solunsalpaajille. Heterogeeninen EGFR-geenikopiomäärän lisääntyminen on lupaava tekijä ennustamaan EGFR-vasta-ainehoidoista saavutettavaa kliinistä hyötyä.Siirretty Doriast

The prognostic and predictive value of selected biomarkers in colorectal cancer

Tissue-based biomarkers are studied to receive information about the pathologic processes and cancer outcome, and to enable development of patient-tailored treatments. The aim of this study was to investigate the potential prognostic and/or predictive value of selected biomarkers in colorectal cancer (CRC). Group IIA secretory phospholipase A2 (IIA PLA2) expression was assessed in 114 samples presenting different phases of human colorectal carcinogenesis. Securin, Ki-67, CD44 variant 6 (CD44v6), aldehyde dehydrogenase 1 (ALDH1) and β-catenin were studied in a material including 227 rectal carcinoma patients treated with short-course preoperative radiotherapy (RT), long-course preoperative (chemo)RT (CRT) or surgery only. Epidermal growth factor receptor (EGFR) gene copy number (GCN), its heterogeneity in CRC tissue, and association with response to EGFR-targeted antibodies cetuximab and panitumumab were analyzed in a cohort of 76 metastatic CRC. IIA PLA2 expression was decreased in invasive carcinomas compared to adenomas, but did not relate to patient survival. High securin expression after long-course (C)RT and high ALDH1 expression in node-negative rectal cancer were independent adverse prognostic factors, ALDH1 specifically in patients treated with adjuvant chemotherapy. The lack of membranous CD44v6 in the rectal cancer invasive front associated with infiltrative growth pattern and the risk of disease recurrence. Heterogeneous EGFR GCN increase predicted benefit from EGFR-targeted antibodies, also in the chemorefractory patient population. In summary, high securin and ALDH1 protein expression independently relate to poor outcome in subgroups of rectal cancer patients, potentially because of resistance to conventional chemotherapeutics. Heterogeneous increase in EGFR GCN was validated to be a promising predictive factor in the treatment of metastatic CRC.Valikoitujen merkkiaineiden ennusteellinen merkitys paksu- ja peräsuolisyövässä Kudoslähtöisten merkkiaineiden tutkimisella toivotaan voivan saavuttaa lisätietoa syövän biologisesta käyttäytymisestä ja ennusteesta, sekä mahdollistaa kullekin potilaalle optimaalisen hoidon valinnan. Väitöstutkimuksen tavoitteena oli selvittää valikoitujen merkkiaineiden ennusteellista merkitystä paksu- ja peräsuolisyövässä. Tyypin II sekretorisen fosfolipaasi A2:n (IIA PLA2) ilmentymistä tutkittiin 114:ssä syövän kehittymisen eri vaiheita edustavassa paksu- ja peräsuolikasvaimessa. Securinin, Ki-67:n, CD44 variantti 6:n (CD44v6), aldehydidehydrogenaasi 1:n (ALDH1) ja β-kateniinin ilmentymistä analysoitiin 227 peräsuolisyöpäpotilaan aineistossa, jossa oli mukana myös ennen leikkausta (kemo)sädehoidettuja potilaita. Levinnyttä paksu- tai peräsuolisyöpää sairastavien potilaiden (n=76) leikkausnäytteistä tutkittiin epidermaalisen kasvutekijäreseptorin (EGFR) geenikopiomäärää, sen kasvaimen sisäistä vaihtelua, sekä yhteyttä EGFR-vasta-ainehoidoista saavutettavaan hyötyyn. IIA PLA2:n ilmentyminen syövässä oli vähäisempää kuin syövän esiasteissa. Securinin korkea ilmentyminen pitkän (kemo)sädehoidon jälkeen, sekä ALDH1:n korkea ilmentyminen imusolmukkeisiin leviämättömässä peräsuolisyövässä olivat itsenäisiä huonon ennusteen tekijöitä, ALDH1 etenkin solunsalpaajilla hoidetuilla potilailla. Heikentynyt solukalvon CD44v6-ilmentyminen oli yhteydessä syövän aggressiiviseen kasvutapaan ja peräsuolisyövän uusiutumisriskiin. Heterogeeninen EGFR-geenin kopiomäärän lisääntyminen ennusti hyvää vastetta EGFR-vasta-ainehoidoille levinneessä paksu- ja peräsuolisyövässä, mikä oli nähtävissä myös solunsalpaajahoidoille kehittyneen vastustuskyvyn jälkeisissä hoitolinjoissa. Yhteenvetona todetaan, että korkea securinin ja ALDH1:n ilmentyminen peräsuolisyövässä ovat itsenäisiä huonon ennusteen merkkejä tietyissä alaryhmissä. Tämä voi heijastaa securin- ja ALDH1-positiivisten syöpäsolujen vastustuskykyä yleisesti käytetyille solunsalpaajille. Heterogeeninen EGFR-geenikopiomäärän lisääntyminen on lupaava tekijä ennustamaan EGFR-vasta-ainehoidoista saavutettavaa kliinistä hyötyä.Siirretty Doriast

Gastric Carcinoma

Gastric cancer is one of the most common tumors worldwide. It has a heterogeneous milieu, where the genetic background, tumor immunology, oxidative stress, and microbial infections are key players in the multiple stages of tumorigenesis. These diverse factors are linked to the prognosis of the gastric cancer and the survival of gastric cancer patients. This book is appropriate for scientists and students in the field of oncology, gastroenterology, molecular biology, immunology, cell biology, biology, biochemistry, and pathology. This authoritative text carefully explains the fundamentals, providing a general overview of the principles followed by more detailed explanations of these recent topics efficiently. The topics presented herein contain the most recent knowledge in gastric cancer concerning the oncogenic signaling, genetic instability, the epigenetic aspect, molecular features and their clinical implications, miRNAs, integrin and E-cadherin, carbohydrate-associated-transferases, free radicals, immune cell responses, mucins, Helicobacter-pylori, neoadjuvant and adjuvant therapy, prophylactic strategy for peritoneal recurrence, and hepatic metastasis

From Barrett's Esophagus towards Adenocarcinoma: Genetic and Clinical studies

Esophageal adenocarcinoma is a highly aggressive disease from which more than 80% of patients die within 5 years after diagnosis. Worldwide almost 400,000 new patients are diagnosed annually. Herewith esophageal cancer ranks eighth on the list of most common cancers, and sixth on the list of cancer mortality causes 1. More than 90% of esophageal cancers are either squamous cell carcinomas or adenocarcinomas 2. Esophageal cancer incidence has been rapidly increasing in Western Europe and the USA 3-5. This could be mainly ascribed to an increase in the rate of esophageal adenocarcinoma, which by now equals or even exceeds the rate of esophageal squamous cell carcinomas. Esophageal cancer incidence in the Netherlands is 10.2/100,000 for men and 3.2/100,000 for women with around 900 newly diagnosed patients annually 6. Adenocarcinomas of the gastric cardia and of the esophagus, commonly located in the distal esophagus, have several similarities. They show a parallel increase in incidence. Moreover, they show similarities in epidemiological and histomorphological features as well as in patterns of comorbidity 7-9. This thesis mainly deals with molecular biological aspects of adenocarcinomas of the distal esophagus and its precursor lesion, Barrett’s esophagus, but also includes work on adenocarcinomas of the gastric cardia. Adenocarcinomas from the distal third of the esophagus plus adenocarcinomas of the gastric cardia are in this thesis altogether mentioned by the description “gastro-esophageal junction (GEJ) adenocarcinomas”

Neue immunhistochemische und molekulare Entwicklungen in der Hepatokarzinogenese

We report the immunohistochemical and molecular findings from a series of primary hepatic carcinomas, aiming for novel markers which may add to their diagnosis and gain more understanding of genetically altered genes and pathways implicated in their geneses. Regarding the standard immunostains applied, our findings are consistent with published literature and do strongly support the use of a panel of immune markers in the discrimination of primary liver malignancies. We also focused on the biological significance of an activated β-Catenin pathway (identified by Wnt1 immunoreactivity and the nuclear translocated β-Catenin) in hepatocarcinogenesis that can open the door to evaluate existing inhibitors of this pathway for future therapeutic management. It remains to be determined which genomic lesions identified by aberrant Wnt1 expression in these tumors are translated into nuclear β-Catenin accumulation. The study documented the expression of SALL4 as a stem cell marker in this series. Our findings suggest that SALL4 may play a role in recognizing the primary liver cancers, especially the poorly differentiated “primitive” cases. In addition SALL4 is one of the potential treatment targets in liver malignancies, especially that its expression is absent in the healthy adult liver. A unique type of expression of FGG immunostain was also observed in fibrolamellar carcinomas, the finding that supports the utility of FGG in FLC diagnosis, especially in the cases with less typical morphology or in discriminating FLC from scirrhous HCC. This review also identified BRAF-V600E gene mutations in HCC, FLC and HCC component of HCC/CCC but not in other CCC cases. This provides a potential path toward therapeutic intervention of the disease. We failed to identify a corresponding immunoreactivity with the commercially available BRAF antibody in the mutant cases. The conclusion is that the antibody is not a useful surrogate to detect BRAF-V600E mutations in primary liver malignancies. From a statistical point of view, this is a small-sample study, but we hope that we will contribute to a meta-analysis combining results of similar across studies of those immunohistochemical and molecular findings in an adequately sized study

Functional and Mechanical Role of Splice Variant of Mucin4 (MUC4/X) and Trefoil Factors in Pancreatic Cancer Pathogenesis

Pancreatic Cancer (PC) is one of the vicious cancers as it ranks third in the race of leading cause of cancer-related death. Lack of early diagnostic marker, poor understanding of molecular mechanism of the disease and failure to conventional chemotherapy makes this disease dreadful. Mucin 4 (MUC4), a high molecular weight glycoprotein is one of the top differentially expressed molecules in PC while not expressed in normal pancreas. Accumulating evidence from our lab suggested its tumorigenic role in PC by increasing cell proliferation, invasion, chemotherapy resistance, tumor growth, and metastasis. Previously, our lab and other has identified 24 different splice variant of MUC4 among them MUC4/X is devoid of exon 2 and 3 and MUC4/Y is devoid of exon 2. Exon 2 encodes for the largest domain of MUC4 suggesting that MUC4/X is devoid of the largest domain of MUC4 which variable tandem repeat. Though lots of effort has been made to identify its role in PC, there is still a gap on understanding its splice variant in PC as splice variant has an invaluable role in tumor pathogenesis. Recently splice variant has emerged as one of the key players for tumorigenesis and MUC4 is one of the key players for PC pathogenesis, we aim to identify the functional and mechanical role of MUC4/X, a splice variant which is devoid of the largest domain of MUC4 yet contains all other functional domain, in PC pathogenesis. Thus, in this part of dissertation, we sought to identify the role of splice variant MUC4/X, a unique splice variant of wild-type MUC4 which contain all functional domain except largest tandem repeat. First, we identified that, MUC4/X in aberrantly expressed in poorly differentiated PC clinical sample. Then our invitro experimental evidence suggested overexpression of MUC4/X in PC cells is involved in increased cell proliferation, invasion and metastasis. Moreover, our orthotopic transplantation system also corroborated our in-vitro findings which showed increased volume of tumor and metastasis to distant organ. Using inducible tet-on system to overexpress MUC4/X in the presence of WT-MUC4 in CAPAN-1 cells, we identified that MUC4/X has increased cell proliferation and invasion suggesting their role as tumorigenic alone as well as in the presence of WT-MUC4. Our mechanical investigation indicate that overexpression of MUC4/X led to upregulation of integrin β1-FAK-ERK pathway which might be potential mechanism for MUC4/X mediated PC tumorigenesis. Lack of early effective diagnostic marker and resistance to chemotherapy are the major reasons for poor PC patient outcome. There is a pressing need to identify highly specific and sensitive biomarker as well as precise understanding of chemoresistance of PC. Trefoil factors (TFFs) are small secretory molecules mostly associated with mucin. Their primary role is to protect gastrointestinal tract partnering with mucin. Report on aberrant expression, potential as biomarker and role in tumorigenicity has conveyed for many cancers, however, their role in PC is still elusive. Recently they have emerged as a part of gene signature of classical subtype of PC, a subtype which showed gemcitabine resistance towards PC. As it is high time to identify effective biomarker and understanding the role of chemoresistance in PC, in this part of my thesis, we focused to evaluate TFFs diagnostic potential using a training and validation cohort of PC clinical sample. Here, we comprehensively investigated the diagnostic potential of all the member of trefoil family, i.e., TFF1, TFF2, and TFF3 (TFFs) in combination with CA19.9 for detection of PC. In silico analysis of publicly available datasets and expression analysis from human and spontaneous PC mouse model revealed a significantly increased expression of TFFs in precursor lesions and PC cases. Additionally, we performed a comprehensive analysis in the sample set (n= 377) comprising of independent training and validation set using ELISA consisted of benign controls (BC), chronic pancreatitis (CP), and various stages of PC. Our analysis revealed that TFF1 and TFF2 were significantly elevated in early stages of PC in comparison to BC (P Additionally, we also aim to identify the molecular landscape of TFFs role in gemcitabine resistance of PC which integrates analyzing publicly available cancer genome dataset, dissecting transcriptomic and signaling pathways and identification of biochemical interaction. From TCGA database analysis revealed a significant positive correlation between TFF1 and GR predictor of PC (P=0.0001). Our in vitro studies showed that SW1990-TFF1-KD cells induced apoptosis, reduced colony formation capacity and modulated many apoptotic regulators such increase of cleaved caspases and decrease of CIAP in the presence of gemcitabine. Furthermore, TFF1 was observed to be colocalized with MUC5AC, in human and mouse PC tissues suggesting their partnering are critical for PC pathogenesis. Interestingly, our chromatin immunoprecipitation indicates that 16 fold enrichment of GATA-6, an overexpressed transcription factor in classical subtype of PC, was observed on two distinct TFF1 promoter sites and GATA-6-siRNA repressed expression of TFF1. Moreover, protein-protein docking studies revealed the interaction of TFF1 with CXCR4 at Phe-172, Ser-122 and Glu-1 and TFF1 recombinant protein treatment in SW1990 cells increased CXCR4 mediated downstream signaling critical for GR. In this part, our overall data demonstrate that TFF1 may play a crucial role in gemcitabine resistance which is regulated by GATA6 and by interacting with MUC5AC

Integrated Transcriptomic and Proteomic Analysis Reveals Role of the Hexosamine Biosynthetic Pathway in Invasion and Metastasis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major global cause of mortality. The epithelial-mesenchymal transition (EMT) transcription factor TWIST1 has been implicated in the invasion and metastasis of HCC, but the mechanism is unclear. My goal was to define a cancer cell autonomous mechanism of Twist1-induced metastasis in HCC. Integrated transcriptomic and proteomic analyses on primary liver tumors and lung metastases from a spontaneous Twist1-dependent metastasis mouse model of MYC-induced HCC identified the hexosamine biosynthetic pathway (HBP) as a cancer cell autonomous mechanism for EMT-mediated invasion and metastasis. I demonstrated that the hexosamine biosynthetic pathway is required and sufficient for invasion and metastasis in vitro in HCC cell lines, ex vivo in HCC tumor-derived organoids, and in vivo in a genetically engineered mouse model of metastatic HCC. Therefore, the HBP may be a potential therapeutic target for advanced HCC patients

Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications

[ES] El cáncer de pulmón es el tipo de cáncer más frecuentemente diagnosticado y la principal causa de muerte debida a cáncer en el mundo, con sólo un 15% de pacientes con una supervivencia mayor a 5 años tras el diagnóstico. La resección quirúrgica es el tratamiento estándar para los pacientes en estadios tempranos con un buen ECOG, pero el 75% de los pacientes son diagnosticados en estadios avanzados, cuando la intervención quirúrgica no es posible y entre un 35% y un 50% de los pacientes operados recaen tras una cirugía aparentemente exitosa. En los últimos años, se han logrado importantes avances en el desarrollo de la inmunoterapia y de tratamientos contra mutaciones conductoras, pero muchos pacientes todavía desarrollan resistencia, progresan y mueren. Esta resistencia terapéutica ha sido asociada a las células madre tumorales (CMTs), una población tumoral con propiedades de célula madre capaz de sobrevivir a las terapias convencionales y regenerar el tumor incluso cuando son indetectables. En esta tesis doctoral, se establecieron cultivos primarios de pacientes de cáncer de pulmón no microcítico (CPNM) resecados, usando ensayos de formación de tumoresferas para el enriquecimiento en CMTs y condiciones de adherencia para los controles. Las tumoresferas derivadas de pacientes mostraron capacidad de autorenovación y crecimiento exponencial ilimitado, alta resistencia a agentes quimioterápicos, capacidad de invasión y diferenciación in vitro y un elevado potencial tumorigénico in vivo. Usando PCR cuantitativa, se analizaron los perfiles de expresión de los cultivos y se determinó que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 e ITGA6 eran los genes más diferencialmente expresados entre tumoresferas y células adherentes. Los análisis de inmunoblot e inmunofluorescencia confirmaron que las proteínas codificadas por estos genes se encuentran aumentadas en tumoresferas de los pacientes con adenocarcinoma y mostraron patrones de expresión y localización diferencial entre éstas y los controles en adherencia. El valor pronóstico de los genes significativamente sobreexpresados en tumoresferas fue evaluado in silico en una cohorte de 661 pacientes con CPNM procedente del TCGA. De todos ellos, CDKN1A, SNAI1 y ITGA6 mostraron estar relacionados con el pronóstico de los pacientes de acuerdo a un análisis de regresión de Cox y fueron seleccionados para construir una firma de expresión génica, denominada firma de CMTs. Los análisis de supervivencia por Kaplan-Meier mostraron que los pacientes con valores elevados de la firma tienen una supervivencia global (SG) menor para la cohorte completa de CPNM [37,7 vs. 60,40 meses, p = 0,001] y para la subcohorte de adenocarcinoma (ADC) [36,6 vs. 53,5 meses, p = 0,003], pero no para la de los epidermoides. Además, el análisis multivariante mostró que la firma de CMTs es un marcador pronóstico independiente para la SG de los pacientes en la cohorte completa [hazard ratio (HR): 1,498; intervalo de confianza (IC) 95%, 1,167-1,922; p = 0,001] y la subcohorte de ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Esta firma fue también analizada en un grupo independiente de 245 pacientes procedentes del Consorci Hospital General Universitari de València, confirmando su valor pronóstico en los pacientes con ADC [42,90 vs. no alcanzado (NA) meses, p = 0,020]. En resumen, nuestros hallazgos aportan información pronóstica relevante para los pacientes con ADC de pulmón y establecen las bases para el desarrollo de nuevos tratamientos.[CA] El càncer de pulmó és el tipus de càncer més diagnosticat i la principal causa de mort deguda a càncer en el món, amb només un 15% de pacients amb una supervivència major a 5 anys després del diagnòstic. La resecció quirúrgica és el tractament estàndard per als pacients en estadis primaris amb un bon ECOG, però el 75% dels pacients són diagnosticats en estadis avançats, quan la intervenció quirúrgica no és possible i entre un 35% i un 50% dels pacients operats recauen després d'una cirurgia aparentment satisfactòria. En els últims anys, s'han aconseguit importants avanços en el desenvolupament de la immunoteràpia i de tractaments contra mutacions conductores, però molts pacients encara desenvolupen resistència, progressen i moren. Aquesta resistència a les teràpies ha estat relacionada amb les cèl·lules mare tumorals (CMTs), una població tumoral amb propietats de cèl·lula mare capaç de sobreviure a les teràpies convencionals i regenerar el tumor fins i tot quan són indetectables. En aquesta tesi doctoral, es van establir cultius primaris de pacients de càncer de pulmó no microcític (CPNM) ressecats, usant assajos de formació de tumoresferes per a l'enriquiment en CMTs i condicions d'adherència per als controls. Les tumoresferes derivades de pacients van mostrar capacitat d'autorenovació, creixement exponencial il·limitat, alta resistència a agents quimioteràpics, capacitat d'invasió i diferenciació in vitro i un elevat potencial tumorigènic in vivo. Usant PCR quantitativa, es van analitzar els perfils d'expressió dels cultius i es va determinar que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 i ITGA6 eren els gens més diferencialment expressats entre tumoresferes i cèl·lules adherents. Les anàlisis de immunoblot i immunofluorescència van confirmar que les proteïnes codificades per aquests gens es troben augmentades en tumoresferes dels pacients amb adenocarcinoma i van mostrar patrons d'expressió i localització diferencial entre aquestes i els controls en adherència. El valor pronòstic dels gens significativament sobreexpressats en tumoresferes va ser avaluat in silico en una cohort de 661 pacients amb CPNM procedent del TCGA. De tots ells, CDKN1A, SNAI1 i ITGA6 van mostrar estar relacionats amb el pronòstic dels pacients d'acord a una anàlisi de regressió de Cox i van ser seleccionats per a construir una signatura d'expressió gènica, denominada signatura de CMTs. Les anàlisis de supervivència per Kaplan-Meier van mostrar que els pacients amb valors elevats de la signatura tenen una supervivència global (SG) menor per a la cohort completa de CPNM [37,7 vs. 60,40 mesos, p = 0,001] i per a la subcohort d'adenocarcinoma (ADC) [36,6 vs. 53,5 mesos, p = 0,003], però no per a la dels escamosos. A més, l'anàlisi multivariant va mostrar que la signatura de CMTs és un marcador pronòstic independent per a la SG dels pacients en la cohort completa [hazard ratio, (HR): 1,498; interval de confiança (IC) 95%, 1,167-1,922; p = 0,001] i la subcohort d'ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Aquesta signatura va ser també analitzada en un grup independent de 245 pacients procedents del Consorci Hospital General Universitari de València, confirmant el seu valor pronòstic en els pacients amb ADC [42,90 vs. no arribat (NA) mesos, p = 0,020]. En resum, els nostres resultats aporten informació pronòstica rellevant per als pacients amb ADC de pulmó i estableixen les bases per al desenvolupament de nous tractaments.[EN] Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable. In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in silico in a cohort of 661 NSCLC patients from TCGA. Based on a Cox regression analysis, CDKN1A, SNAI1 and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both, the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001], and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies.Herreros Pomares, A. (2020). Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036TESI