UNIVERSIDAD AUTÓNOMA DE MADRID
PROGRAMA DE DOCTORADO EN BIOCIENCIAS MOLECULARES
CDK4/6 INHIBITION IN PANCREATIC CANCER:
EFFECTIVENESS AND MOLECULAR BASES IN COMBINATION WITH TAXANES
TESIS DOCTORAL
Beatriz Salvador Barbero
Madrid, 2018
DEPARTAMENTO DE BIOQUÍMICA FACULTAD DE MEDICINA
UNIVERSIDAD AUTÓNOMA DE MADRID
CDK4/6 INHIBITION IN PANCREATIC CANCER:
EFFECTIVENESS AND MOLECULAR BASES IN COMBINATION WITH TAXANES
Beatriz Salvador Barbero Licenciada en Biología
Co-director: Manuel Hidalgo Medina Co-director: Marcos Malumbres Martínez
Centro Nacional de Investigaciones Oncológicas (CNIO)
A mi abuela Valen,
El científico no es aquella persona que da las respuestas correctas, sino aquél quien hace las preguntas correctas.
Claude Lévi-Strauss
AGRADECIMIENTOS
13 Mucha gente ha formado parte de este trabajo, no sólo en el ámbito profesional, sino también en el personal. No puedo decir que sin cada uno de vosotros esto no hubiese salido adelante, pero sí que no hubiese sido igual, que no lo hubiese disfrutado como lo he hecho y que los resultados no habrían sido tan buenos.
A mi familia tengo que agradecerle que son la base sobre la que se soporta toda mi vida, y por tanto también en esta época que ha estado tan llena de altibajos emocionales.
Papá, sé que no querías que hiciese biología, pero espero haberte demostrado que los biólogos también hacemos cosas importantes para la sociedad. Gracias por interesante por todo lo que hago, por mostrarte interesado, incluso apasionado con lo que hago, y por acompañarme en momentos tan difíciles sin pensártelo dos veces, pese a las adversidades.
Marina, gracias por demostrarme que si luchas, cualquiera de tus objetivos se puede cumplir, y por estar a mi lado en cada uno de los pasos que doy.
Mamá, ¡qué decirte! Creo que es imposible tener un apoyo de mayor calibre en la vida, sea cual sea el problema tú estás ahí, no sólo para decirme que todo irá bien, sino para ayudarme de una manera crítica. Eres quién más me ha hecho crecer como persona, y si soy como soy es gracias a ti, eres mi modelo a seguir.
Alberto, tú sí que me has aguantado! Para lo bueno y para lo malo…has estado ahí para decirme una y otra vez que saldremos adelante, que juntos no hay obstáculo que no podamos superar y que me seguirías al fin del mundo. Gracias!
Pedrito, siempre atento a cómo estoy, y cómo me encuentro, y siempre dispuesto a darme un abrazo y ayudarme con lo que haga falta.
Miriam, por supuesto que entras dentro de este apartado de familia, porque no sólo eres mi amiga, eres mucho más que eso! Una de las personas que mejor me conoce, siempre estás cuando te necesito, no tengo palabras para decirte lo que significas para mí y el apoyo que supones.
Laboralmente tengo que agradecer a los dos grupos en los que he pasado este tiempo y que me han acompañado en este proceso de aprendizaje.
Al grupo de Tumores Digestivos, a todos vosotros y todos los que pasaron por allí durante el tiempo que yo estuve en el labo, tengo que deciros que fue un placer.
A mi co-director Manuel Hidalgo, gracias por seleccionarme entre tantos candidatos, y darme la oportunidad de llevar a cabo mi doctorado en CNIO.
Por supuesto tengo que destacar a Pedro, por sus preguntas, necesarias para hacerte reflexionar sobre lo que sabes, y lo que es más importante, sobre todo lo que no sabes. ¡Gracias por tus explicaciones interminables a cerca de cualquier tema, porque eres una biblioteca andante! Pero sobre todo gracias por luchar como nadie por que pudiese continuar con mi doctorado cuando las cosas se torcieron. Si esto es posible hoy, es gracias a ti.
Patri, te incluyo en este grupo, gracias por tantos y tantos paseos CNIO-Chamartin, Chamartin- CNIO, por tus risas y por todo lo que me enseñaste. Y por arreglar el mundo conmigo en cada descanso y cada desayuno.
Jenny, eres una persona maravillosa, sigue siendo como eres porque vales un montón. Gracias por esas charlas en barrera que hacen que el tiempo pase más rápido.
Mónica, al principio me imponías mucho, pero luego he llegado a comprender que eres una persona fantástica, con muchas ganas de ayudar. Y tenías razón en tantas cosas….
Camino, cómo agradecerte el infinito apoyo que me has dado tanto profesional como personal.
Enumerar tooodo lo que me has enseñado sería una locura, todo lo que sé sobre ratones, me has enseñado a investigar, siempre has estado para responder todas mis preguntas. ¡Eres una persona increíble, y tu mayor defecto es que no te das cuenta de lo increíble que eres y de lo muchísimo que vales! Lo más valioso que me llevo de Tumores Digestivos, es tenerte a ti como amiga.
Natalia, Francesca, Rodrigo, Spas, Begoña, encantada de haberos conocido, cada uno de vosotros ha aportado un granito de arena a esta tesis y a mí personalmente.
Al grupo de División Celular y Cáncer, sois un tesoro, como grupo creo que es imposible mejorar el equipo que formáis, es perfecto. Creo que esa genialidad se consigue porque sois todos buenísimas personas que os preocupáis más por los demás que por vosotros mismos. Desde que empecé a trabajar con vosotros, antes de cambiarme de labo, ya me di cuenta de ello, pero os puedo asegurar que desde que estoy con vosotros sois como mi segunda familia.
Para ir por orden empezaré por Marcos. Me diste una segunda oportunidad, me acogiste en tu grupo cuando mi doctorado quedó a la deriva, no puedes imaginar lo agradecida que estoy. Le diste un vuelco a mi proyecto mejorándolo infinitamente.
15 coco que ya quisiéramos muchos, y siempre estás dispuesta a ayudar dando todo lo que tienes.
¡Pero sobre todo has sido un apoyo importantísimo durante todo este tiempo, desde que me enseñaste prácticamente todo lo que sé de cultivos con aquellos primeros experimentos con palbo, hasta enseñarme a pensar y a crecer como investigadora…yo de mayor quiero ser como tú, eres un modelo a seguir!
Guille, te voy a echar de menos, eres un pozo sin fondo de sabiduría, y no sólo científica, sino personal. Eres una de las pocas personas que conozco que ama su trabajo, disfrutas investigando, y enseñándonos a investigar. Irradias felicidad cuando estás sentado en tu bench, al igual que cuando explicas ciencia. ¡De ti me llevo esa pasión por cada momento en el laboratorio, y por supuesto, también me llevo tus agravios verbales de alguna manera motivadores!
Bego, me encantaría algún día tener tu capacidad para tener siempre claro el camino a seguir, tu seguridad en todo lo que haces, tu rigurosidad. Gracias por todos esos cafés y pitis de desahogo, por todos tus consejos y todos esos tiempos muertos de conversaciones anti-estrés. Gracias porque eres una de las primeras personas que busco en el labo cuando tengo un súper resultado o una súper mala noticia, gracias por tu amistad.
María Salazar, eres la paciencia, la calidez, la paz personificada. Hablar contigo siempre me relaja y me hace sentir bien. Gracias por ayudarme con mis masivas dudas acerca del postdoc y preocuparte por mí.
Eli, eres la persona con mayor capacidad de trabajo que conozco, eres una máquina! Tengo que agradecerte el trabajo que me has sacado adelante, pero tengo que agradecerte mucho más el que me aguantes cada día. Da igual lo que te pregunte ya sea algo del labo o personal, que siempre sabes la respuesta, y si no lo sabes lo buscas y siempre lo encuentras. Resuelves cualquier problema que se te pone por delante.
Filipa, sobretodo tengo que darte las gracias por tooooodo lo que tiene que ver con la universidad, gracias por encargarte de eso! Pero más importante aún es el apoyo que nos hemos dado escribiendo, ha sido muy divertido y gratificante tenerte para los momentos de desesperación y para tomarnos una cerveza de desahogo, pero también para celebrar cuando íbamos cerrando capítulos. Creo que, gracias a esto, he comenzado a conocerte mucho más, y me alegro de ello.
María M., eres una biblioteca de técnicas, te sabes los protocolos de memoria y todas sus posibles variaciones. Sólo necesitas mejorar la seguridad en ti misma para saber ver todo tu potencial.
¡Gracias por esas conversaciones a través de la pared, por echarme una mano cuando lo necesito
y preocuparte por mi cuando tengo cualquier problema, y gracias por el apoyo brutal que me diste en Amsterdam, me sorprendiste con la seguridad que demostraste, deberías aplicarla siempre!
María S., gracias por tus nociones básicas de ImageJ, no te imaginas cuánto me han ayudado.
Pero sobre todo gracias por tu amistad, porque aún sin estar en el labo, sabes perfectamente qué nos pasa a cada una y estás pendiente.
Aisha, eres excepcionalmente genial! Cuando llegué al labo me ayudaste muchísimo a integrarme, y desde entonces has pasado ser una pieza muy importante en mi vida. ¡En el labo eres la generosidad en persona, siempre preguntándome qué puedes hacer por mí, además de tus masajes…qué masajes! Espero que siempre sigas a mi lado!
Paloma, ¡eres una pesada de campeonato! Pero me lo paso muy bien contigo, eres un desahogo cuando el labo se convierte en demasiado.
Bea, gracias por las cenas, las cervezas y las risas. Pepe, estás como una cabra y deberías revisar tu locura, pero me haces reír. Vero, no hemos coincidido demasiado, pero te agradezco tu sonrisa constante. Diego, gracias por esos conjuntitos que tanto me hacen reír y por tus “Bea eres lo peor”! Darío, siempre dispuesto a ayudar y a decir cualquier cosa que pueda hacerte sentir bien.
Carol M., siempre te interesas por lo que nos pasa a los demás. Carol V., siempre sonriendo…deberías creerte más el pedazo de coco que tienes!
En resumen, gracias a toda la gente con la que he coincidido durante estos cuatro años largos, porque cada uno de vosotros habéis aportado un granito a esta montaña de arena que es mi tesis.
RESUMEN/SUMMARY
19 El cáncer de páncreas se sitúa como uno de los cánceres con mayor tasa de mortalidad, la supervivencia media es de 4.6 meses a partir del diagnóstico, y 5 años después, la supervivencia global es únicamente un 3%. Esto se debe a la inexistencia de tratamientos eficaces que puedan aumentar significativamente la esperanza de vida de los pacientes.
Recientemente, se han desarrollado fármacos con la capacidad de inhibir las quinasas dependientes de ciclinas (CDK) 4 y 6. Estos inhibidores han sido aprobados por la agencia de Food and Drug Administration (FDA) para el tratamiento del cáncer de mama, además, están siendo testados para otros tumores sólidos dando resultados prometedores.
Un gen muy frecuentemente alterado (más del 90%) en los tumores de páncreas es CDKN2A, y como consecuencia se pierde la actividad o expresión de p16INK4a. El supresor tumoral p16INK4a es un inhibidor de CDK4/6, por tanto, es un inhibidor de la ruta del retinoblastoma. Debido a esta frecuente alteración, decidimos testar la eficacia de fármacos inhibidores de CDK4/6 (CDK4/6i) para el tratamiento del cáncer de páncreas.
Tanto los resultados que hemos obtenido in vitro, como in vivo, señalan al CDK4/6i palbociclib (PD-0332991) como un potencial tratamiento para el cáncer de páncreas, pero también dejan clara la necesidad de encontrar otro agente que, en combinación, incremente la eficacia de ambos fármacos por separado. Como agente para la combinación decidimos utilizar uno de los fármacos empleados habitualmente para el tratamiento del cáncer de páncreas, Paclitaxel (Taxol).
El tratamiento de líneas celulares con la combinación de taxol y palbociclib resultó en un mayor efecto anti-proliferativo que cuando ambos fármacos son utilizados por separado. Curiosamente, el esquema de tratamiento para la combinación de ambos fármacos es importante, ya que se obtienen mejores resultados cuando las células se pretratan con taxol antes de la administración de palbociclib. El análisis del ciclo celular ha demostrado que las células que sufren aberraciones debido al tratamiento con taxol, son más potentemente arrestadas por palbociclib. Los perfiles de expresión génica señalan una supresión de los mecanismos de reparación de daño en el ADN como principal diferencia entre los tratamientos por separado frente al tratamiento combinado.
Además, para evaluar la eficacia de este nuevo tratamiento combinado in vivo, tratamos ocho modelos de xenoinjertos derivados de paciente (PDX) de cáncer de páncreas con palbociclib y paclitaxel, siguiendo el mismo esquema de tratamiento. Es importante destacar que siete de ellos presentaron una mayor inhibición del crecimiento tumoral en la combinación con respecto a las monoterapias. Lo mismo ocurre en ratones genéticamente modificados (GEMMs), que desarrollan tumores de páncreas espontáneamente, en estos el efecto de la combinación es incluso más dramático.
21 Pancreatic cancer continues the deadliest human cancer, with a median survival of 4.6 months after diagnosis, and an overall survival at 5 years of only 3%. The main cause is the lack of effective treatments that significantly increase the life expectancy of patients.
Recently, cyclin-dependent kinases (CDK) 4 and 6 inhibitors (CDK4/6i) have been developed.
These inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of breast cancer, in addition, they are being tested for other solid tumors giving promising results.
One frequently altered gene (more than 90%) in pancreatic tumors is CDKN2A, and as a consequence the activity or expression of p16INK4a is lost. The p16INK4a tumor suppressor is an inhibitor of CDK4/6, therefore, is an inhibitor of the retinoblastoma pathway. Due to this frequent alteration, we decided to test the efficacy of CDK4/6i for the treatment of pancreatic cancer.
The results that we have obtained both in vitro and in vivo, point to the CDK4/6i palbociclib (PD- 0332991) as a potential treatment for pancreatic cancer, however, also make clear the necessity of finding a partner that, in combination, increases the effectiveness of both drugs separately. As agent for the combination we decided to use one of the drugs commonly used for the treatment of pancreatic cancer, Paclitaxel (Taxol).
The treatment of different cell lines with the combination of taxol and palbociclib resulted in a greater anti-proliferative effect than when both drugs are used separately. Interestingly, the schedule of treatment for the combination of both drugs is important, since better results are obtained when cells are pre-treated with taxol before the administration of palbociclib. Cell cycle analysis has shown that cells that have suffered aberrations due taxol treatment, are more potently arrested by palbociclib. The gene expression profiles indicate a suppression of DNA damage repair mechanisms as the main difference between each of the treatments single-agent versus the combined treatment.
In addition, to evaluate the efficacy in vivo of this new combination treatment, we treated eight models of patient-derived xenografts (PDX) of pancreatic cancer with palbociclib and paclitaxel, following the same treatment schedule. Importantly, seven of them presented an increased tumor growth inhibition in the combination with respect to the monotherapies. The same happens in genetically modified mice (GEMMs), which spontaneously develop pancreatic tumors, in which the effect of the combination is even more dramatic.
INDEX
25
AGRADECIMIENTOS ... 11
RESUMEN/SUMMARY ... 17
INDEX ... 23
ABBREVIATIONS ... 29
1. INTRODUCTION ... 33
1.1 Pancreatic cancer ... 35
1.1.1 Physiology of the pancreas ... 35
1.1.2 Types of pancreatic cancer ... 37
1.1.3 Incidence and diagnosis ... 38
1.1.4 Molecular basis of pancreatic cancer ... 39
1.1.5 The stroma relevance in PDAC ... 40
1.2 Preclinical models of pancreatic cancer ... 41
1.2.1 2D-cell cultures ... 41
1.2.2 3D-cell cultures ... 41
1.2.3 Genetically Engineered mouse models (GEMMs) ... 41
1.2.4 Patient Derived Xenografts (PDXs) ... 42
1.3 The cell cycle ... 43
1.3.1 The phases of cell cycle ... 43
1.3.2 Cell cycle regulation: CDKs ... 45
1.4 DNA damage ... 49
1.4.1 Damaging factors and consequences ... 49
1.4.2 Repair pathways ... 49
1.4.3. DNA repair throughout the cell cycle ... 50
1.5 Treatments for pancreatic cancer ... 51
1.5.1 Current therapies ... 51
1.5.2 CDK4/6 inhibitors ... 52
1.5.3 Taxanes in pancreatic cancer ... 54
2. OBJECTIVES ... 55
MATERIAL&METHODS ... 59
3.1 Cell culture ... 61
3.1.1 Pancreatic cancer cell lines establishment ... 61
3.1.2 Commercial cell lines ... 61
3.1.3 Organoid culture ... 62
3.1.4 Fluorescent ubiquitination-based cell-cycle indicator (FUCCI) system ... 63
3.1.5 Drug treatments ... 63
3.1.6 Cell cycle analysis by Flow Cytometry... 63
3.2 Biochemical and molecular biology procedures ... 64
3.2.1 Protein extraction and immunoblotting ... 64
3.2.2 RNA sequencing (RNAseq) ... 64
3.2.3 Videomicroscopy ... 64
3.2.4 Histological and immuno-histochemical analysis ... 65
3.3 Mouse models ... 66
3.3.1 Patient Derived Xenografts (PDX). ... 66
3.3.2 Genetically engineered mouse models ... 67
3.3.3 Mouse assays ... 67
3.3.4 Mouse housing ... 67
3.4 Statistical analysis ... 68
4. RESULTS ... 69
1.1 Efficacy of CDK4/6 inhibitors in PDAC. ... 71
4.1.1. CDK4/6 inhibitors efficacy in vitro. ... 71
4.1.2 Palbociclib induces PDAC growth arrest in vivo... 73
4.2 Effect of Taxol in pancreatic tumors. ... 75
4.2.1 Pre-treatment with taxol increases the efficacy of CDK4/6i in vitro. ... 76
4.2.2 The combination induces tumor growth delay in PDAC xenografts. ... 79
4.2.3 Combination efficacy is maintained during more time after removing drugs... 83
4.2.4 Effect of the combination in genetically modified mouse models of PDAC... 87
27
4.2.5 Improved efficacy of Combination vs. Monotherapies in tumoroid models. ... 89
4.3. Molecular mechanisms that determine the increased efficacy of combining CDK4/6i and taxanes. ... 90
4.3.1. Enhanced cell cycle arrest of PDAC cells treated with taxol and CDK4/6i. ... 90
4.3.2. Combining taxol and palbociclib do not increase senescence. ... 95
4.3.3. CDK4/6 inhibitors cooperate with other agents that block cell cycle. ... 97
4.3.4. RNA sequencing reveals DNA damage as a possible mechanism of action... 99
4.3.5. Confirming DNA damage as the mechanism of action for combination increased efficacy. 102 5. DISCUSSION ... 105
5.1 CDK4/6 inhibitors: a successful treatment for PDAC preclinical models ... 107
5.2 Combining Palbociclib with taxol: unexpected results ... 107
5.3 Why do taxol and CDK4/6i cooperate together? ... 109
5.3.1 Cell cycle in the combination ... 109
5.3.2 The combination in mouse models ... 110
5.3.3 Senescence is not the key ... 111
5.3.4 DNA damage repair hypothesis ... 111
5.3. Hypothetical model ... 114
5.4. Future plans: From the bench to the bedside ... 115
CONCLUSIONS/CONCLUSIONES ... 117
BIBLIOGRAPHY ... 121
ABBREVIATIONS
31 APC/C Anaphase- Promoting Complex/ Cyclosome
β-gal β-galactosidase BER Base Excision Repair CDK Cyclin-dependent kinase
CDK4/6i Cyclin Dependent Kinases 4/6 inhibitor Cre Cre Recombinase
CSC Cancer Stem Cell
DAPI 4',6-diamidino-2-phenylindole DBS Double Strand Break
DDR DNA Damage Response
DM Diabetes Mellitus
DMEM Dulbecco’s modified Eagle’s medium EdU 5-ethynyl-2'-deoxyuridine
ER Estrogen Receptor
FACS Fluorescence Activated Cell Sorting
FBS Fetal Bovine Serum
FC Log2fold
FDA Food and Drug Administration
G0 Gap phase 0
G1 Gap phase 1
G2 Gap phase 2
GEMM Genetically Engineered Mouse Model
GO Gene Ontology
HER2 Human Epidermal Growth Factor Receptor 2
HR Homologous Recombination
IF Inmunofluorescence
IFN Interferon
LSL Floxed STOP transcriptional cassette
M Mitosis
MCC Mitotic Control Point
mPDAC Metastatic Pancreatic Ductal AdenoCarcinoma MTT metabolic cell activity
NER Nucleotide Excision Repair NHEJ NonHomologous End Joining NSCLC Non-Small Cell Lung Carcinoma
MMR Mismatch Repair
OS Overall Survival
PanIN Pancreatic Intraepithelial Neoplasia PBS Phosphate-Buffered Saline
PDAC Pancreatic Ductal AdenoCarcinoma PDX Patient Derived Xenograft
Pen/Strep Penicillin/Streptomycin
PFA Paraformaldehyde
PFS Progression Free Survival
pH3 Phospho-Histone H3
PI Propidium Iodide
pRb Phospho-Retinoblastoma protein
R Restriction point
RPMI Roswell Park Memorial Institut
RT Room temperature
RB Retinoblastoma protein RNAseq RNA sequencing
ROS Reactive Oxygen Species
S Synthesis phase
SAC Spindle Assembly Checkpoint
SD Standard Deviation
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SEM Standard Error of the Mean
SSB Single Strand Break TGI Tumor Growth Inhibition Tregs Regulatory T cells
WB Western Blot
1. INTRODUCTION
35
1.1 Pancreatic cancer
1.1.1 Physiology of the pancreas
One of the most important organs for digestion is the pancreas. The pancreas is located in the abdomen at the level of the first and second lumbar vertebra next to the adrenal glands, behind the stomach, forming part of the content of the retroperitoneal space. This organ is physically divided into several regions called head, neck, body and tail (Longnecker, 2014). And it is formed by two very different sections, the exocrine and the endocrine sections (Figure 1).
The exocrine section secretes the pancreatic juice, which is formed by digestive enzymes necessary at the small intestine for digestion. Histologically, the exocrine pancreas is formed by pancreatic acini, composed by acinar and centro-acinar cells. The centro-acinar cells are in charge of the production of the aqueous component of the pancreatic juice, rich in bicarbonate; while the acinar cells synthetized the enzymatic component, containing the digestive enzymes for carbohydrates (amylase), lipids (lipase) and proteins (protease) digestion. The acini pour their contents into the pancreatic ducts, which join each other until they end in the main pancreatic duct or Wirsung Duct. This duct at the lower part of the head joins to the common bile duct ending in the hepatopancreatic ampulla or ampulla of Vater that enters the descending duodenum (Pandol, 2010).
The endocrine portion secretes hormones that pass into the blood, such as insulin, glucagon, pancreatic polypeptide and somatostatin. The histological units of this region are the islets of Langerhans, which consist of clusters of hormone-secreting cells. There are several types of cells in the islets and each of them produces a different hormone type. Alpha cells synthesize glucagon, which increases the level of blood glucose; while beta cells produce insulin, the hormone that regulates the level of glucose in the blood. Delta cells produce somatostatin, which inhibits insulin Figure 1. Anatomy of the pancreas.
The healthy human pancreas contains both an endocrine (Langerhans islets) and an exocrine (acini and ducts) compartment.
and glucagon secretion, and vasoactive intestinal polypeptide, that has several functions involving digestion muscle activation; and epsilon cells, produce ghrelin, the hormone that stimulates appetite and fat accumulation. Finally, PP cells produce the pancreatic polypeptide that controls and regulates the exocrine secretion of the pancreas (Ionescu-Tirgoviste et al., 2015).
The pancreas can be affected by a wide range of diseases as summarized below.
One of the most common diseases is pancreatitis, characterized by inflammation of the pancreas.
The signs and symptoms include pain in the upper abdomen, nausea and vomiting (Hammad et al., 2018). There are two main types, acute pancreatitis and chronic pancreatitis. Acute pancreatitis is a sudden inflammation of the pancreas, in which fever may occur and symptoms usually disappear in a few days, however, it can be fatal if not treated immediately (Sand and Nordback, 2009). Chronic pancreatitis is an inflammatory process resulting from the release of active pancreatic enzymes within the glandular parenchyma, and in these disease weight loss, fatty stools and diarrhoea may occur (Kleeff et al., 2017).
Cystic fibrosis is a genetic disorder that affects the mucous and sweat glands, mainly the lungs, but also the pancreas, liver, kidneys and intestine. The thick mucus seen in the lungs has a counterpart in the thick secretions of the pancreas. These secretions block the exocrine movement of digestive enzymes in the duodenum and produce irreversible damage to the pancreas, often with pancreatitis. The pancreatic ducts are completely covered in the most advanced cases, which causes atrophy of the exocrine glands and progressive fibrosis (Ooi and Durie, 2016).
Diabetes mellitus (DM) is a group of metabolic disorders, related to the pancreas, in which there are high levels of sugar in the blood over a prolonged period. Glucose accumulates because either the pancreas does not produce enough insulin or cells do not respond adequately to the insulin produced. Symptoms include frequent urination, increased thirst and increased hunger. Acute complications can include diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death.
Severe long-term complications include cardiovascular disease, stroke, chronic kidney disease, foot ulcers, and eye damage (Poudel et al., 2018).
There are three main types of diabetes mellitus. DM type 1 results from the inability of the pancreas to produce enough insulin due to the loss of beta cells by autoimmune destruction. The cause of DM type 1 is unknown, however, it is thought to involve a combination of genetic and environmental factors (Gromada et al., 2018). DM type 2 starts with insulin resistance, cells do not respond properly to insulin. As the disease progresses, a defect in insulin production may also develop. The most common cause is excessive body weight and insufficient exercise (Hackett and
37 Steptoe, 2017). Gestational diabetes is the third major form, and occurs when pregnant women develop high blood sugar levels due to insulin resistance and poor insulin production (Buchanan et al., 2012).
Finally, the most aggressive of pancreas disorders is pancreatic cancer.
1.1.2 Types of pancreatic cancer
Pancreatic tumors can be mainly of two types depending on their origin, exocrine or neuroendocrine (endocrine origin) (Figure 2).
Exocrine pancreatic cancers are the most common type of pancreatic cancers. Around 85% of these exocrine cancers are pancreatic ductal adenocarcinomas (PDAC), which are originated from the ductal cells of the pancreas (Kleeff et al., 2016). Less common are other carcinomas such as the acinar cell carcinomas, very rare and heterogeneous pancreatic neoplasms, accounting for 5%
of exocrine cancers, which can cause an overproduction of digestive enzymes (La Rosa et al., 2015). Cystadenocarcinoma is a malignant form of a cystadenoma and derives from the glandular epithelium, and represent 1% of pancreatic cancers (King et al., 2009). Adenosquamous carcinoma contains two types of cells: squamous and glandular cells (Rao, 2014). The signet-ring cell carcinoma is an epithelial neoplasm characterized by the histological appearance of mucus- producing signet ring cells (Radojkovic et al., 2017). Other rare exocrine carcinomas are squamous cell carcinomas, undifferentiated carcinomas with or without giant cells, and carcinoma of the ampulla of Vater.
Neuroendocrine tumors are a diverse group of benign or malignant tumors that arise from neuroendocrine cells, which are responsible for integrating the nervous and endocrine systems.
Neuroendocrine tumors are uncommon, making up less than 5% of all pancreatic cancers, and can be divided into "functional "and" non-functional ", according to the degree to which they produce hormones (Rindi and Wiedenmann, 2012). The functional type secretes hormones such as insulin, gastrin and glucagon into the bloodstream, often in large quantities, which leads to severe symptoms. They are garstrinomas, insulinomas, glucagonomas, somatostatinomas, VIPomas, and PPomas among others (Öberg, 2018). The non-functional type does not secrete hormones in a sufficient amount to give rise to overt clinical symptoms (Cloyd and Poultsides, 2015).
In the present study, we will focus on PDAC, the most common of the pancreatic cancers, and we will refer to it as pancreatic cancer.
Figure 2. Types of pancreatic cancer.
Graph showing the percentage of each type among the total of pancreatic cancers.
1.1.3 Incidence and diagnosis
Pancreatic cancer is one of the most aggressive cancers. Actually, its incidence is not very high;
of the total of cancers diagnosed worldwide in 2018 only 2.5% (458,918 patients) of them were pancreatic cancer. But its mortality reaches alarming figures, during the same year 432,242 people died from this disease (Bray et al., 2018). Therefore, pancreatic cancer reaches the seventh position in the ranking of cancer related deaths worldwide, with PDAC representing the majority of the cases. In fact, is expected that in the future, pancreatic cancer will overtake breast cancer as the third cause of death in the European Union (Bray et al., 2018).
Pancreatic cancer is a very aggressive disease due to different factors, such as aggressive tumor dissemination, a desmoplasic microenvironment, the lack of effective systemic therapies, and the rapid development of resistance to these existing therapies. However, the most important factor, that determines in most cases the fate of the patient, is definitely the late diagnosis of the disease.
Studies on the genetic basis of pancreatic cancer suggest that is needed more than ten years for the initial mutation in a cell to progress to metastatic PDAC (mPDAC). Thus, there is a large window opportunity for early detection. However, PDAC is an asymptomatic disease, and there are not reliable biomarkers for detection. Therefore, the majority of patients are diagnosed with advanced- stage disease (Haeno et al., 2012). As consequence, less than 20% of patients present resectable tumors at the time of diagnosis, because local invasion or distal metastasis. Early disease has good prognosis, while the remaining 80% of patients is treated with palliative therapies based on chemotherapeutic agents such as FOLFIRINOX, gemcitabine, gemcitabine/nab-paclitaxel, 5-FU, nal-irinotecan (O’Reilly et al., 2018). Even more, at the time of diagnosis many pancreatic cancers
39 are in an exponential growth phase (Haeno et al., 2012). Sadly, these patients have very poor prognosis, with very poor overall survival (OS) and quality of life.
Lifestyle factors markedly affect the risk of pancreatic cancer, including tobacco smoking, alcohol consumption, the quality of the diet, weight, and physical activity. The combined healthy lifestyle factors may have direct implications for pancreatic cancer prevention (Jiao et al., 2009). It was estimated that more than a quarter of pancreatic cancer cases may have been prevented if all people in the general population were non-smokers and had limited alcohol consumption, healthy diet, normal weight, and regular physical activity (Korc et al., 2017). Other risk factors for pancreatic cancer are the age (the average age at the time of diagnosis is 71), family history, inherited genetic syndromes, diabetes, liver cirrhosis, stomach problems, and one of the most relevant, chronic pancreatitis.
Taking into account all this information, it seems clear the necessity of looking for new therapies that are capable of increasing the life expectancy of these patients. Several efforts have been made to find drugs that meet these characteristics, but today the median survival in Europe is 4.6 months since diagnosis, and overall survival at 5 years is only the 3% of the patients (Carrato et al., 2015).
1.1.4 Molecular basis of pancreatic cancer
PDAC is characterized by a progressive accumulation of genetic alterations during the development of the disease (Figure 3). It is considered that pancreatic cancer is caused by a chronic pancreatitis that progresses to intraepithelial neoplasms that have been classified in different degrees according to tissue disorganization. At the beginning, the pancreatic intraepithelial neoplasia (PanIN) type 1 is related to the activation of the KRAS oncogene. Next, the loss of the tumor suppressor gene CDKN2A is associated with the PanIN-2 status. The more advanced stages of the disease, such as PanIN-3, are accompanied of alterations in important genes such as TP53 and SMAD4 (Bardeesy and DePinho, 2002; Hidalgo, 2010; Iacobuzio-Donahue et al., 2012) (Figure 3). This accumulation of genetic errors is clearly reflected in the final tumor that concurs with activation of KRAS in 90-95% of cases, loss of CDKN2A in 85-90% of patients, mutation of TP53 in 75% and SMAD4 in 50% of the tumors diagnosed (Hidalgo, 2010). Inactivation of SMAD4 may contribute to the development of metastases, but much of the genetic heterogeneity found in metastases is already present in the primary carcinoma (Iacobuzio- Donahue et al., 2009).
Figure 3. From normal pancreatic tissue to mPDAC and genetic alterations related with each stage.
Point mutations in the KRAS gene occur early, inactivation of the p16 gene at an intermediate stage, and the inactivation of p53, and SMAD4 occur relatively late. Adapted from Hidalgo, 2010.
Beside these alterations, which are relatively common to all pancreatic tumors, PDAC is characterized by being very diverse in gene alterations with marked differences between patients.
Pancreatic cancer has been classified using different methods based on molecular alterations, and also on the histology of the tumors, but few or none of these have finally resulted as biomarkers of response to treatments (Krantz and O’Reilly, 2018).
1.1.5 The stroma relevance in PDAC
Another important characteristic of PDAC is the intense desmoplasia, or abundant stroma, that consists of activated stellate cells, myofibroblasts, immune cells, inflammatory cells, blood and lymphatic vessels and complex extracellular matrix. Desmoplastic response can be initiated at the PanIN stage, and increases with the progression of the disease. The implications of this desmoplasia are related to autocrine and paracrine signalling interactions between tumor cells and stroma, leading to acceleration of pancreatic cancer initiation, progression and metastasis (Erkan et al., 2012; Vennin et al., 2018). Tumor cells secrete factors to the microenvironment that mainly activate pancreatic stellate cells, which in response secrete excess extracellular matrix proteins and a number of factors including matrix metalloproteases, promoting pancreatic cancer progression, and acting as a barrier for drug delivery (Erkan et al., 2012; Vennin et al., 2018).
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1.2 Preclinical models of pancreatic cancer
1.2.1 2D-cell cultures
In vitro propagation of cells from pancreatic cancer was first described in the 1960s (Dobrynin, 1963). In the following years, more than twenty human PDAC cell lines have been developed, among them Panc-1, MiaPaCa-2, AsPC-1, BxPC-3, which have been very useful in the study of some pathways, validation of biomarkers, identification of possible targets and drug screenings (Deer et al., 2010). In addition to these, new cell lines, both of human and murine origin, are being established and characterized. Although this model is widely used, cell lines do not accurately reflect the genetic and epigenetic complexity of the tumor, neither the influence of tumor microenvironment (Behrens et al., 2017). They serve as a basis for research, but they should be complemented with other preclinical models before making a translation to the clinic.
1.2.2 3D-cell cultures
The maintenance of stem or progenitor cells in advanced extra-cellular matrix using carefully selected specific factors is an interesting characteristic of three-dimensional organoids. 3D organoids have been established from human and murine cells, using both healthy tissue and cancers (Boj et al., 2015; Hohwieler et al., 2017; Huang et al., 2015; Huch et al., 2013). Through this approach the different stages of pancreatic organogenesis, carcinogenesis and interaction with the desmoplastic surroundings have been reproduced. The xenografts obtained from these cultures represent more accurately the architecture and morphology of the tumor than the 2D cultures xenografts (Boj et al., 2015; Hohwieler et al., 2017; Huang et al., 2015; Huch et al., 2013).
Otherwise, these models also have some disadvantages, such as the strong selection of some specific cell types during this process; or the limited use of this model in the study of metastatic and immunological aspects of PDAC (Behrens et al., 2017).
1.2.3 Genetically Engineered mouse models (GEMMs)
To reproduce the development of pancreatic cancer, GEMMs have been used to develop tumors of an aggressive, heterogeneous and stromal nature. These animals eventually suffer the typical symptoms of PDAC and distal metastases in lung and liver.
One of the most representative models includes the expression of K-RasG12V specifically activated in pancreas using the LSL (lox-STOP-lox transcriptional cassette) targeting construct under Cre
recombinase, which is under the control of the Elastase promoter in a tetracycline-inducible system (Guerra et al., 2003). Elastase is expressed in embryonic cells of acinar/centro-acinar linage. This activation results, at 12 months of age, in 80% of the mice containing multiple high- grade PanIN lesions, and about half of these mice developing at least one PDAC per pancreas (Guerra et al., 2007). Another highly used promoter for these GEMMs is Pdx-1 (Gannon et al., 2000) which is expressed during the development of the exocrine pancreas, endocrine pancreas and ductal cell population.
Other alterations in the genes most commonly altered in PDAC have been added to K-Ras. Loss of p16Ink4a/p19Arf encoding gene (Krimpenfort et al., 2001), also known as CDKN2A, in early pancreatic precursors during embryonic development efficiently cooperates with K-Ras oncogenes to induce invasive and metastatic PDAC. At 6 months of age, all mice have developed high-grade PanIN lessions and PDAC (Guerra et al., 2011). Also the conditional expression of p53 in a K-Ras activated context decreases the latency of PDAC (Hingorani et al., 2003).
1.2.4 Patient Derived Xenografts (PDXs)
Cell-line derived xenografts frequently lack the cellular and structural interactions of the tissues from which they originate, resulting in differences in spatial organization and intra-tumoral heterogeneity, as well as discrepancies in genetic expression profiles and response read-outs (Cree et al., 2010). To overcome these disadvantages, PDXs of several solid tumors have been established. A tumor fragment obtained by surgery or biopsy (usually chemotherapy-naïve) is transplanted directly into immunocompromised mice, either subcutaneously or orthotopically. The success of the engraftment depends on several factors, including the type of tumor and its aggressiveness; the worse the patient's outcome, the better engraftment. For all this, the growth of the xenograft in the mouse can take between one and four months, with a success of 20 to 80% (Tentler et al., 2012).
Once the PDX has been established, it can be transplanted serially into larger cohorts of animals, maintaining the genetic and phenotypic characteristics of the original tumor (Perales-Patón et al., 2017). The PDXs obtained from PDAC have served to study the complexity of this cancer and the efficacy of novel treatment strategies (Rajeshkumar et al., 2017). These grafts have also demonstrated their genetic and phenotypic homology with the tumor of origin and its correlation between engraftment rates and patient survival (Walters et al., 2013), with an engraftment rate of 72-84% (Davies et al., 1981; Jung et al., 2016). An important characteristic of PDAC is the
43 abundant desmoplasia, and in the PDXs derived from these tumors, the human microenvironment is rapidly replaced by the murine stroma.
As disadvantages of this model, it should be noted that the PDXs are obtained from a tumor fraction, therefore only represents a part of the tumor. In addition, immunocompromised mice are used for the engraftment, leading to basic differences in the immunological context of the tumors.
Nevertheless, PDAC PDX models are strongly recommended as a complementary tool for addressing specific translational questions.
1.3 The cell cycle
Cell proliferation is an essential mechanism for growth, development and regeneration of eukaryotic organisms; however, its deregulation is also the cause of one of the most devastating diseases: cancer.
1.3.1 The phases of cell cycle
The cell cycle is a sequence of meticulously organized and monitored processes responsible for the division of a cell into two daughter cells. This process requires a series of events that occur in an encompassed and orderly manner, and guarantee the correct duplication of the genetic material and its adequate segregation. The cell cycle is divided into four phases (Figure 4). The G1
phase precedes the S phase, and during this stage the cell grows and prepares for DNA synthesis.
Then, during the S phase the cell replicates its genetic material, and in the G2 phase the cell prepares for its division (Morgan, 2007). The distribution of the previously duplicated material between the two daughter cells occurs during mitosis (M) (Figure 4).
G1 is a period when many signals intervene to influence cell division and the deployment of a cell's developmental programme. Diverse metabolic, stress and environmental cues are integrated and interpreted during this period. On the basis of these inputs, the cell decides whether to enter S phase or pause. Moreover, in multicellular organisms the behaviour of a cell must obey dictums from its neighbours. To this end, during G1 the cell makes further decisions regarding whether to self-renew, differentiate or die (Massagué, 2004).
During S phase the entire DNA content of the nucleus must be replicated completely and precisely, in a period of few hours. This is achieved by initiating bidirectional replication at multiple sites along each chromosome. Importantly, chromosome replication involves more than the replication of DNA, the complex architecture of the chromosome must be duplicated too. This must involve
assembly of nucleosomes and chromosome scaffolds. Furthermore, in some cells it also involves copying specific patters of gene activity and inactivity (Laskey et al., 1989).
G2 phase is a period of rapid cell growth and protein synthesis during which the cell prepares itself for mitosis.
Figure 4. The cell cycle.
Cells enter into G1 to prepare for S-phase. Once the genome is duplicated, cells prepare to divide in G2. During Mitosis, chromosomes bind to microtubules in prometaphase, get aligned in metaphase and separated in anaphase. Finally, in telophase two nuclei are formed and cell is divided by cytokinesis in two daughter cells.
45 Mitosis (M) is the process by which cells ensures the proper segregation of the two copies of the genome between the two daughter cells. This process consists of five phases (Figure 4). During prophase, interphasic chromosomes start to condense moving to the poles of the cell where the spindle structure will be formed. Nuclear envelope breakdown signals the transition between prophase and prometaphase and is an essential step for the mitotic spindle formation. The spindle microtubules rapidly assemble and disassemble searching for attachment sites at chromosome kinetochores, which are complex structures that assemble during prometaphase on one face of each sister chromatid at its centromere. Microtubules from opposite poles interact with chromosomes and make them to become bioriented and congressed. Congression of the last chromosome marks the transition to the next stage of mitosis, metaphase, in which all chromosomes reach the equator of the spindle forming the “metaphase plate”. The progression of cells into anaphase is marked by the abrupt separation of sister chromatids. Early in anaphase, chromosomes lose their cohesion and each chromatid moves apart towards one spindle pole. At late anaphase, the spindle is elongated and separates further the two set of chromatids. Mitosis ends with telophase, the stage at which the chromosomes reach the poles, decondense into their interphase conformation, and the nuclear envelope is reformed around the two daughter nuclei.
Then, the cytoplasm division, also called cytokinesis, and whose regulation is precisely linked to mitosis, occurs. A contractile ring is formed at the cortex of the cell giving rise to the midbody that marks the abscission site. Finally, abscission of the midbody results in the complete physically separation of the two daughter cells which have identical genetic composition (McIntosh and Koonce, 1989; Morgan, 2007).
1.3.2 Cell cycle regulation: CDKs
As we have already mentioned, progression through the cell cycle in mammals requires a precise orchestration of the sequence of events. Among the numerous regulatory elements that participate in this process, the sequential activation of complexes formed by cyclin-dependent kinases (CDK)- cyclin have been described as key regulators. Cyclins are expressed in an oscillatory way throughout the cell cycle (Figure 6), thus regulating the activity of the CDKs at each moment of the cycle. CDKs are inhibited by two families of proteins: the INK4 (p16INK4a, p15INK4b, p18INK4c and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1 and p57Kip2) CDK inhibitors.
One of the most commonly altered regions in human cancers is the chromosomal region 9p21, where the INK4a and b locus resides (Ortega et al., 2002). These alterations lead to loss of p16INK4a and p15INK4b as well as p19ARF, an additional protein expressed from the p16INK4a
locus (also known as CDKN2A). While p16INK4a acts as a phospho-retinoblastoma (pRb) activator by inhibiting the activity of the CDK4 and CDK6 kinases (Figure 5), p19ARF blocks the degradation of the p53 tumor suppressor by Mdm2 (Ortega et al., 2002). Most of the deletions found in tumors affect these three proteins, which makes it difficult to evaluate their role in tumorigenesis. Other members of this regulatory family are p18INK4c, which has tumor suppressor properties in a variety of tissues (Latres et al., 2000), and p19INK4d, whose role still remains unknown.
Figure 5. Rb/E2F pathway.
Mitogenic signals give rise to inhibition of p16 and increased levels of cyclin D and the consequent formation of active cyclin D- CDK4/CDK6 complexes leads to the phosphorylation of pRb facilitating the transcription of E2F-regulated genes required for the S-phase.
The Cip/Kip family consists of three proteins: p21cip1/waf1, p27kip1, p57kip2. p21Cip1 is a key mediator of the growth suppression dependent on p53 and is involved in cellular processes such as senescence and differentiation. p27Kip1 is strongly expressed in non-proliferative cells and plays an important role in the regulation of quiescence and G1 progression (Fero et al., 1996). The role of p57Kip2 in the development of tumors is not clear; it is the only inhibitor of CDKs whose interruption causes abnormalities in development, mainly due to late differentiation and increased apoptosis (Zhang et al., 1997).
The progression through each phase of the cycle and the transition from one phase to the next is meticulously regulated by checkpoints, which guarantee the precise sequence of events and avoid the transition to the next phase if the previous one has not been completed. The ultimate goal is to ensure the detection and repair of genetic damage, if it occurs, by ensuring that the two daughter cells will receive an exact copy of the genome of the cell from which they come. If a problem is detected, the checkpoints are activated. At this time, two things can happen: the
47 problem is solved by allowing the checkpoint to be disabled, or the repair of the damage is not successful so that the cell is driven to a state of senescence or apoptosis. The cancellation of the control points of the cell cycle can cause many diseases, including cancer.
Figure 6. Cyclin flow along the cell cycle.
The concentrations of cyclin proteins change throughout the cell cycle. Adapted from CuboCube.
One of the first effects produced by the activation of mitogenic signals is the expression of D-type cyclins (D1, D2 and D3). These cyclins are mainly involved in activating the Rb/E2F pathway (Figure 5). In G0 and initial G1, phospho-Rb (pRb) is hypophosphorilated and sequestrates E2F preventing its interaction with promoters of genes involved in proliferation. When the levels of D-type cyclins increase, CDK4 and CDK6 form a complex with them, increasing the phosphorylation of pRb and other pocket proteins as p107 and p130, at the end of G1 (Johnson et al., 2016). The inactivation of pocked proteins leaves E2F free, which promotes gene expression. Some of these genes whose expression is activated are the E-type cyclins (E1 and E2), which bind to and activate CDK2. The complex formed by CDK2-cyclin E phosphorylates to a greater degree the pocket proteins that are now completely inactivated. These events allow cell cycle progression from G1 to S phase (Malumbres and Barbacid, 2009). During the last stages of DNA replication, CDK2 binds to cyclin A2 which drives CDK2 activation to monitor the transition from S phase to M, passing through G2
phase. Once G2 is completed, cyclin A joins CDK1, activating it and facilitating the mitosis progression. Finally, when the nuclear membrane is broken, type A cyclins are degraded, and CDK1 is free to form complexes with type B cyclins that will guide the cell throughout mitosis (Figure 6) (Malumbres and Barbacid, 2009).
Later on, during M, the spindle assembly checkpoint (SAC) monitors the proper attachment of microtubules to chromosomes (Figure 7). During prometaphase unbound kinetochores catalyze the formation of the mitotic control point (MCC) complex composed of BubR1, Bub3, Mad2 and Cdc20, leading to the inhibition of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that has as some of its targets the cyclins, marking them for degradation. Once all the chromosomes are aligned with their kinetochores attached to the spindle,
in the metaphase, the generation of MCC ceases, which allows Cdc20 to activate the APC/C, leading to the ubiquitylation and degradation of securin and cyclin B1. Degradation of securin liberates the separase which, in turn, cleaves the scle1 kleisin subunit of the cohesin ring structure;
this opens the ring, allowing the sister chromatids to separate into the anaphase. Meanwhile, the degradation of cyclin B1 inactivates CDK1, leading to the mitotic exit (Morgan, 1999; Wittmann et al., 2001). If this process of deactivating CDK1 does not occur, the chromosomes become condensed and the cell is not able to exit from mitosis. Once the anaphase has been overcome, the APC/C function is continued by CDH1, which prevents the activation of CDK1 during the mitosis exit and the G1 phase of the next cell cycle (Malumbres and Barbacid, 2009).
Figure 7. The Spindle Assembly Checkpoint (SAC).
Unattached or improperly attached kinetochores are sensed by the SAC inducing MCC binding and inhibition if the APC/C, which is required for the metaphase–anaphase transition. Once SAC is satisfied on all kinetochores (at metaphase), activation of APC/Cpromotes Cyclin B and securin ubiquitination and proteolysis. This promotes mitotic exit and sister chromatid separation, the latter through activation of separase.
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1.4 DNA damage
The most important molecule and the largest one that we can find in any living being is DNA. This molecule is susceptible to injury due to different causes and, therefore, it is understandable that there are mechanisms to protect DNA and repair it in case it suffers any damage, especially in the germline and cells that need to proliferate.
1.4.1 Damaging factors and consequences
DNA can be damaged mainly due to three factors, spontaneous reactions because of the chemical nature of this molecule, some of the molecules that our metabolism generates as reactive oxygen species (ROS) or nitrogen species, and exogenous physical and chemical agents (ultraviolet radiation, tobacco) (Bartek et al., 2007; Lindahl, 1993). As consequence, it is estimated that each cell suffers 105 injuries per day. The impact of the damage suffered by the DNA, if is not repaired, generates mutations that lead the cell to decide between entering senescence or dying, which contributes to aging. In the worst case, the cell does not make any of these decisions and this leads to cancer, usually due to the loss of tumor suppressors or the activation of oncogenes due to these mutations (Bartek et al., 2007).
1.4.2 Repair pathways
The maintenance of genome integrity is controlled by a compendium of multiple repair pathways, at least five, each dedicated to repairing a different category of DNA lesion (Figure 8). For the types of damage that occur in only one of the two DNA strands, there are three main repair systems. Base-excision repair (BER) consists of removing the damaged base and filling the gap by DNA synthesis, which means replacing the flanking sequences. It serves to repair subtle alterations in the DNA, including oxidative injuries, small alkylation products and some types of single-strand breaks (SSB) (Caldecott, 2008; Lindahl, 1993). Nucleotide-excision repair (NER) eliminates the distortion created in one of the DNA helixes (Sugasawa, 2016). Mismatch repair (MMR) recognizes and removes the flawed stretch of DNA, and then novel DNA synthesis fills in the gap (Groothuizen and Sixma, 2016).
When the damage generates double-strand breaks (DSB) there are two main repair mechanisms.
Non-homologous end joining (NHEJ) simply joins the two ends that have been separated by the breakage of the DNA. The NHEJ usually occurs before the replication, so due to the absence of an identical copy of DNA, certain bases may be lost or added during the process. In contrast,
homologous recombination (HR) occurs after replication, and takes advantage of the sister chromatid to use the appropriate template and insert the appropriate information (Hoeijmakers, 2009). Finally, there is another DNA damage repair pathway less common, interstrand cross-links repair which solves the cross-links that covalently connect the two strands (Räschle et al., 2008).
Figure 8. Type of DNA damage, repair pathways and repair enzymes.
Most common repair pathways and enzymes implicated in single and double strand break repair.
1.4.3. DNA repair throughout the cell cycle
Cells in G1 phase need to repair accidental damage, which has to be repaired preferentially before the onset of replication, when the primary DNA lesions can stall replication or can be converted into other types of DNA damage, with hazardous consequences for the cell. The most common repair pathways during this phase are NER for SSBs and NHEJ for DSBs due to high compaction of chromatin and the absence of sister chromatids. DNA synthesis is frequently associated with nucleotide misincorporation, accumulation of nicks and gaps, slippage at repetitive sequences, fork collapse at DNA breaks and aberrant transitions at collapsed forks that cause reversed and/or resected forks. These aberrant replication-fork transitions can endanger the stability of the chromosomes if they are not promptly repaired. Moreover, the torsional stress that is generated when the replication fork advances, when two replicons fuse together at termination or when the forks encounter transcription bubbles causes topological modifications that lead to the accumulation of supercoils and/or precatenanes. DSBs can often occur during S phase as a result of replication-fork collapse, and are commonly solved by HR in the majority of the cases. If gaps and DSBs that occur during replication left unrepaired by the end of the S phase, need to be
51 repaired before mitosis. For HR to occur during S and G2 phases using the sister chromatid as a template, it is important that the sister chromatids are in proximity to one another. When the DSBs occur during chromosome segregation repair is likely to occur by NHEJ in the subsequent G1
phase if checkpoints or caretaker genes had not caused cell-cycle arrest during G2 and M phases (Branzei and Foiani, 2008).
1.5 Treatments for pancreatic cancer 1.5.1 Current therapies
As we previously mentioned, only the 20% of patients have tumors that can be surgically resected at the time of diagnosis, and most patients are diagnosed at advanced stages of the disease.
Tumor-resectable patients are treated with neo-adjuvant therapies and post-surgical treatments (Álvarez et al., 2017). Patients with advanced disease are treated with palliative treatments that usually develop fast resistance to the regular drugs administered. In this study we will focus on these patients without surgical options, which are usually mPDAC diagnosed (Macarulla et al., 2018).
The treatments currently available for mPDAC are based on the administration of gemcitabine, a chemotherapeutic agent that was a revolution for pancreatic cancer when it was tested for these patients in 1997 compared to the, at that moment, standard of care 5-fluorouracil (5-FU).
Treatment with gemcitabine was superior in terms of survival and clinical benefit. Taking into account the results of this study, gemcitabine has become the reference drug in the treatment of advanced pancreatic cancer (Burris et al., 1997).
In 2011 Conroy et al. compared FOLFIRINOX over gemcitabine, in the PRODIGE 4/ ACCORD 11 clinical trial in mPDAC patients. FOLFIRINOX is a combination of cancer drugs that includes: FOL – folinic acid (also called leucovorin, calcium folinate or FA); F – fluorouracil (also called 5-FU);
IRIN – irinotecan; and OX – oxaliplatin). In the clinical trial the median overall survival (OS) was increased from 6.8 (gemcitabine) to 11.1 (FOLFIRINOX) months (Conroy et al., 2011).
A couple of years later, in 2013, the MPACT clinical trial was published comparing the combination of gemcitabine plus nab-paclitaxel versus gemcitabine alone. MPACT demonstrated an improved response rate of 23%, an increased progression-free survival (PFS) of 5.5 months and an OS of 8.5 months compared to 6.7 months (Von Hoff et al., 2011).
Although both trials generally included younger patients with good performance status, the patient populations in the PRODIGE 4/ACCORD 11 and the MPACT trials showed distinct differences in
mean patient age, metastatic sites, serum CA19-9 levels and patient status; these differences preclude any head-to-head comparison between the trial results, although the gemcitabine control arm performed similarly in both trials, suggesting broadly similar patient demographics. Both available combination regimens for first-line therapy of metastatic pancreatic cancer, FOLFIRINOX and nab-paclitaxel– gemcitabine, are associated with a substantial toxicity profile (Martín et al., 2018). Figure 9 represents a suggested treatment algorithm for the treatment of patients with PDAC in different stages.
Figure 9. Schematic representation of current treatment for pancreatic cancer.
Suggested treatment algorithm for patients with pancreatic cancer. Adapted from Neoptolemos, 2018.
Having in mind all this data, is clear the necessity of new drugs that could increase the OS and life quality of pancreatic cancer patients. Currently, no target therapy is approved for the treatment of pancreatic cancer. Based on genetic alterations, the most attractive drugs are those able to target KRAS oncogene. However, the available drugs that target mutant KRAS or the pathways in which it is involved, have failed in increasing efficacy versus standards of care, or are still in development (Zhang et al., 2018). The second gene most altered in pancreatic cancers is CDKN2A, which offers a huge potential for the treatment of PDAC, and this is the reason why we decided to test CDK4/6 inhibitors.
1.5.2 CDK4/6 inhibitors
CDKs have long been regarded as promising targets for cancer therapies, although many of the early, first-generation CDK inhibitors failed in clinical development, at least in part because nonselective pan-CDK inhibition was found to be toxic to non-cancer cells. These issues of effectiveness and toxicity seem to have been overcome by more selective targeting of CDKs 4 and 6, which have a number of potential advantages over the use of less-selective inhibitors. Many
53 types of somatic cells might be capable of initiating the cell cycle despite CDK4/6 inhibition.
Additionally, in contrast to the cytotoxic effects of less selective CDK inhibitors, CDK4/6 inhibitors (CDK4/6i) are usually found to have cytostatic effects, which might further limit the potential of these agents to cause clinical toxicity (O’Leary et al., 2016).
Three of these new CDK4/6i, abemaciclib, palbociclib and ribociclib, have emerged, following the findings of early phase trials, as agents with promising anticancer activity and manageable toxicity.
These orally-administered compounds are of similar structure, bind within the ATP-binding pocket of CDK4 and CDK6, and all have a high degree of selectivity for CDK4 and CDK6, compared with CDK1 and CDK2 (Asghar et al., 2015).
Palbocicliib, also named as Ibrance® or PD-0332991 is an inhibitor of CDKs 4 and 6 at low nanomolar concentrations, but has limited inhibitory effects on other CDKs or tyrosine kinases and is currently the most clinically developed. Studies in Rb-positive breast cancer cell lines showed that palbociclib inhibited the phosphorylation of pRb and reduced the expression of E2F- dependent genes, which led to a strong inhibition of cancer cell proliferation (Leonard et al., 2012).
In the clinic, palbociclib received the accelerate approval in 2015 and regular approval in 2017 from the Food and Drug Administration (FDA) for first-line treatment in combination with letrozole for postmenopausal women with hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. In 2016, the FDA expanded the approval of palbociclib to be used in combination with fulvestrant in patients who had received prior endocrine therapy (Bardia and Hurvitz, 2018a).
Palbociclib has been also tested for other tumor types. The NCT01209598 clinical trial tested palbociclib single-agent in well-differentiated or differentiated liposarcoma (Dickson et al., 2016) and the NCT00420056 clinical trial in mantle cell lymphoma. Both trials gave promising results, however the number of patients included and the design of the study and the absence of randomized data, make it difficult to compare the results with the standards of care (Dickson et al., 2016; Leonard et al., 2012).
Although CDK4/6i are well known to induce cell cycle arrest, recent studies have identified novel effects of these therapies on tumor growth, especially an indirect effect resulting from the activation of immune surveillance. Goel et al. showed that CDK4/6 inhibition promotes antitumor immunity through multiple mechanisms: increased antigen presentation due to Rb response to interferon (IFN)-gamma; induction of endogenous retroviral genes due to the downregulation of DNA methyltransferase, an E2F target gene; and reduction of immunosuppressive regulatory T
cells (Tregs) in the tumor microenvironment probably due to cell cycle inhibition (Chaikovsky and Sage, 2018; Goel et al., 2017).
1.5.3 Taxanes in pancreatic cancer
Taxanes were discovered in 1968 due to a project conducted by the Research Triangle Institute to isolate natural compounds with potential therapeutic effect. Monroe E. Wall and Mansukh C. Wani isolated the taxanes from the bark of the Pacific yew (Taxus brevifolia), and studied their anti- tumoral activity in several types of tumors. Finally, in 1970, the structure of paclitaxel, a cyclic diterpene derived from the taxane nucleus, was isolated. It has a four-membered oxetane ring and an amide chain, which, when modified, gives rise to docetaxel.
Paclitaxel or Taxol® main function is to stabilize the microtubules. Taxanes inhibit cell proliferation by the induction of mitotic blockade at the metaphase/anaphase boundary, as well as the incomplete metaphase chromosome plate formation and the abnormal microtubule spindle organization. Therefore, the inhibitory effect of these drugs lies on the final aberrant mitotic spindles and the mitotic blockade due to the microtubule stabilization (Rowinsky, 1997).
Paclitaxel is widely used for the treatment of solid tumors, however, the solvent applied in the commercial formulation is associated with severe hypersensitivity reactions. Therefore, paclitaxel was reformulated as nab-paclitaxel. Nab-paclitaxel, commercially known as Abraxane®, is a solvent-free albumin-bound form of paclitaxel, which allows the ability of deliver significantly higher doses of paclitaxel preventing the hypersensitivity reactions. Other advantage of nab- paclitaxel is the enhanced transport of paclitaxel across endothelial cell and greater delivery of paclitaxel to tumors, due to the usage albumin transport pathways, in some way bypassing the stroma delivery problems. Nab-paclitaxel is now approved by the FDA for the treatment of metastatic breast cancer, advanced or metastatic non-small cell lung carcinoma (NSCLC), and metastatic pancreatic cancer, with successful results. In addition, other clinical trials in other solid tumors as urothelial, squamous-cell carcinoma of the head and neck, gastric cancer, and colorectal cancer and small bowel carcinoma, are ongoing (Giordano et al., 2017).
For the treatment of metastatic pancreatic cancer, the introduction of nab-paclitaxel in the clinic has represented a very important advance. The combination of nab-paclitaxel plus gemcitabine is suitable for a wide spectrum of mPDAC patients, with different characteristics and clinical presentations. Further studies are suggesting nab-paclitaxel as a good candidate for the treatment of early PDAC patients and a “re-challenge” in metastatic disease (Giordano et al., 2017).
2. OBJECTIVES
57 Pancreatic cancer is the cancer with worst life expectancy for its patients. Available treatments fail to improve median survival beyond 4.6 months, and new treatments to increase the survival of these patients are urgently needed. CDK4/6i have already been approved in the clinic for the treatment of hormone-positive breast cancers. Due to the genetic background of pancreatic cancer, CDK4/6 are attractive therapeutic targets in this tumor type. Therefore, the major aim of the project is to assess the effectiveness of CDK4/6i in pancreatic cancer.
1. To determine the efficacy of CDK4/6i in the treatment of pancreatic adenocarcinoma.
2. To establish if the combination of taxanes and CDK4/6i is effective for the treatment of pancreatic cancer.
3. To clarify the mechanism of action through which the combination of taxanes and CDK4/6i increase the efficacy when compared to monotherapies.