M
ARISAA
LEXANDRAA
NTUNESC
OELHOU
NIVERSITY OFI
NSUBRIACENTRE OF RESEARCH IN MEDICAL PHARMACOLOGY
PHDCOURSE IN CLINICAL AND EXPERIMENTAL MEDICINE AND MEDICAL HUMANITIES
U
NIVERSITY OFP
ORTO FACULTY OF MEDICINEPHDCOURSE IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY AND TOXICOLOGY
DOCTORAL THESIS IN CO-SUPERVISION AGREEMENT
UNIVERSITY OF INSUBRIA PHDCOURSE
CLINICAL AND EXPERIMENTAL MEDICINE AND MEDICAL HUMANITIES
AND
UNIVERSITY OF PORTO PHDCOURSE
EXPERIMENTAL AND CLINICAL PHARMACOLOGY AND TOXICOLOGY
SUPERVISORS
FROM UNIVERSITY OF INSUBRIA
PHD IN CLINICAL AND EXPERIMENTAL MEDICINE AND MEDICAL HUMANITIES
MARCO COSENTINO
ASSOCIATE PROFESSOR OF PHARMACOLOGY
AND
FROM UNIVERSITY OF PORTO
PHD IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY AND TOXICOLOGY
LAURA VIRGÍNIA PEREIRA TEIXEIRA RIBEIRO
ASSISTANT PROFESSOR
“Nothing in life is to be feared, it is only to be understood.
Now is the time to understand more, so that we may fear less.”
Given the international character of this PhD and the interdisciplinarity of the studies present in this thesis, the work hereby discussed was performed at several institutions: at Centre of Research in Medical Pharmacology, University of Insubria (Varese, Italy); at Unit of Biochemistry, Department of Biomedicine of the Faculty of Medicine of the University of Porto (Porto, Portugal) and Unit of Pathology, Department of Medicine and Surgery, University of Insubria, (Varese, Italy).
No part of this work has been submitted as application for other degree or qualification at both Universities or any other university or institution of learning.
I, hereby declare that I actively participated in the gathering and study of the material included in each
ACTH Adrenocorticotrophic hormone
AD Adrenaline
ADC Adenocarcinoma
ALK Anaplastic lymphoma kinase
AR Adrenoceptor
BARK β-adrenergic receptor kinase
BMI Body mass index
CA Catecholamines
cAMP Cyclic adenosine monophosphate
CSS Cancer specific survival
COPD Chronic obstructive pulmonary disease CREB cAMP-responsive element-binding protein
CTLA-4 Cytotoxic T-lymphocyte–associated antigen 4
DA Dopamine
DAT Dopamine transporter
DFS Disease free survival
DOPA Dihydroxyphenylalanine DβH Dopamine β-hydroxylase
EGF Epidermal growth factor
EPAC Exchange proteins directly activated by cAMP
ERK Extracellular signal-regulated kinase
FKA Focal adhesion kinase
FBS Fetal bovine serum
FDA Food and Drug Administration
FFA Free fatty acids
GPCR G-protein coupled receptor
HPA Hypothalamic pituitary adrenal axis
HR Hazard Ratio
H&E Haematoxylin and Eosin
IHC Immunohistochemistry
ISA Intrinsic sympathomimetic activity
L-DOPA L-3,4-dihydroxyphenylalanine MAO Monoamineoxidase enzyme
MAPK Mitogen activated protein kinase
MMP Matrix metalloproteinase
MSA Membrane-stabilizing activity
NA Noradrenaline
NSCLC Non-small cell lung cancer
OS Overall survival
PI3K Phosphatidylinositol 3-kinase
PKA Protein kinase A
PNMT Phenylethanolamine N-methyltransferase
R&D Research & Development
SCLC Small cell lung cancer SCC Squamous cell carcinoma
SNS Sympathetic nervous system
TH Tyrosine hydroxylase
TNM Tumour, Node, Metastasis
VEGF Vascular endothelial growth factor
WHO World Health Organization
List of Abbreviations ... vii
Abstract ... xiii
Resumo ... xv
List of Figures ... xvii
List of Tables ... xix
List of Publications ... xxi
Outline of the thesis ... xxiii
Chapter 1. ... 1
Background ... 1
Rationale and Aims ... 21
Bibliography ... 23
Chapter 2. ... 35
β-Adrenergic modulation of cancer cell proliferation: available evidences and clinical perspectives ... 35
Chapter 3. ... 55
β1- and β2-adrenoceptors expression patterns in human non-small cell lung cancer: relationship with cancer histology ... 55
Chapter 4 ... 83
β-adrenergic impact on human non-small cell lung cancer: an in vitro study using A549 cells ... 83
Chapter 5. ... 97
Effect of β-blockers on survival of lung cancer patients: A systematic review and meta- analysis ... 97
Chapter 6. ... 123
β-blockers in NSCLC: β1/ β2-AR selectivity matters but it might be not enough ... 123
Chapter 7. ... 131
General Discussion and Concluding Remarks ... 131
Future Perspectives ... 143
Bibliography ... 145
Acknowledgments ... 157
Reproduction Licenses ... 159
Introduction: With the growing amount of studies revealing the involvement of β- adrenoceptors (β-AR) in the progression of multiple types of tumours, the hypothesis that β- blockers could be potential candidates for drug repurposing in oncology setting has gaining momentum. In lung cancer, it could be particularly meaningful considering the disappointing results of the current therapeutic approaches. Aims: The compilation of studies disclosed in this thesis intended to investigate the presence β-AR in non-small cell lung cancer (NSCLC) as well as discuss the potential for repurposing b-blockers as a new therapeutic approach for lung cancer. Methodology: The studies included in this thesis comprise a narrative review of the existing literature about the involvement of β-AR on proliferation of human cancer cellular models; a clinicopathologic study evaluating the expression of β-AR on human NSCLC; and a meta-analysis exploring the effect of β-blockers on overall survival of lung cancer patients.
Results: The obtained results suggest that b1 and b2-AR are differently expressed in the histologic subtypes, adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We found that β1-AR expression is present at low levels in both SCC and ADC whereas b2-AR is higher expressed on both histologic subtypes but clearly higher expressed in ADC. The meta-analysis performed, including 7448 patients, showed that lung cancer patients using b-blockers had no increased overall survival when compared to non-users. Conclusion: Altogether, this work increased the knowledge on the expression pattern of b-AR on NSCLC highlighting that ADC that highly expresses β2-AR might be more sensible to non-selective β-blockers treatment.
Despite the lack of a positive effect of β-blockers on lung cancer overall survival obtained in the meta-analysis, this study should prompt the attention of the scientific community to the fact that there is still a huge margin for improving the concept of β-blockers repurposing in cancer.
Keywords: β-adrenoceptors; β-blockers, Lung cancer, Drug repurposing
Resumo
Introdução: Com o vasto número de estudos a revelar o envolvimento dos recetores β- adrenérgicos (β-RA) na progressão de múltiplos tipos de cancro, a hipótese de que os β- bloqueadores podem ser potenciais candidatos para reposicionamento em contexto oncológico, tem ganho especial ênfase. No cancro de pulmão, pode ser particularmente significativo tendo em conta os resultados insatisfatórios das atuais abordagens terapêuticas.
Objetivos: A compilação de estudos presente nesta tese pretende investigar a presença dos recetores β-AR no cancro de pulmão de não pequenas células (CPNPC) assim como discutir o potencial de reposicionamento dos b-bloqueadores como nova abordagem terapêutica para o cancro de pulmão. Metodologia: Os estudos incluídos nesta tese compreendem uma revisão narrativa da literatura existente sobre o envolvimento dos recetores β-AR na proliferação de linhas celulares tumorais humanas; um estudo clinico-patológico avaliando a expressão de β-RA no CPNPC; e uma meta-análise que explorou os efeitos dos β- bloqueadores na sobrevida global de doentes com cancro de pulmão. Resultados: Os resultados obtidos mostram que os recetores b1 e b2-RA estão expressos de forma diferente nos subtipos histológicos adenocarcinoma (ADC) e carcinoma espinocelular (CEC). Foi observado que β1-RA está sub-expresso em os ambos histotipos enquanto b2-RA está altamente expresso e claramente mais presente no ADC. A meta-análise realizada incluiu 7448 doentes e mostrou que os doentes que fazem b-bloqueadores não apresentam melhor sobrevida global quando comparado com doentes que não fazem esta terapêutica.
Conclusão: De forma geral, este trabalho permitiu ampliar o conhecimento relativo ao padrão de expressão dos recetores b-RA no CPNPC salientando que ADC por apresentar elevados níveis de b2-RA pode ser mais sensível ao tratamento com b-bloqueadores não- seletivos. Apesar da ausência de efeito positivo na sobrevida global, este estudo deve incitar a atenção da comunidade científica para o facto de ainda existir uma grande margem para aprofundar o conceito de reposicionamento de b-bloqueadores no cancro.
List of Figures
Chapter 1
Figure 1. Catecholamine biosynthesis ... 6 Figure 2. β-AR signalling mechanisms on tumour cells. ... 10 Figure 3. β–AR signalling influence on cells within tumour microenvironment. ... 11
Chapter 3
Figure 1. Expression of β1 and β2-AR in human ADC and in their matched surrounding non- tumour tissues. ... 64 Figure 2. Expression of β1 and β2-AR in human SCC and in their matched surrounding non- tumour tissues. ... 65 Figure 3. Expression of β1-AR in ADC and SCC. ... 66 Figure 4. Expression of β2-AR in ADC and SCC. ... 67
Chapter 4
Figure 1. ADRBs and TH mRNA expression on A549 cell line during the growth curve, on day 3, 5 and 7 days. ... 89 Figure 2. Representative A549 cell cycle analysis. ... 90 Figure 3. Concentration- response curves for the effect of isoprenaline and adrenaline on the proliferation of cultured A549 cells. ... 91
Chapter 5
Figure 1. Prisma flow diagram of the systematic literature search ... 105 Figure 2. Forest plot (random-effects model) of β-blockers use and overall survival of lung
Figure 5. Forest plot (random-effects model) of β-blockers use and overall survival considering all lung cancer patients (users vs non-users) for selective and non-selective β-blockers. ... 111 Figure 6. Forest plot (random-effects model) of β-blockers use and overall survival considering only advanced stages of lung cancer (stage III and IV) (users vs non-users). ... 111
Chapter 1
Table 1. Classification and physiopharmacology of β-AR ... 7 Table 2. Summary of clinical trials investigating β-blockers in oncology setting. ... 19
Chapter 3
Table 1. Patients and disease characteristics ... 63 Table 2. Tumour-related characteristics according to β1 and β2-AR expression in squamous- cell carcinoma (SCC) and adenocarcinoma (ADC). ... 69 Supplementary Table 1. Univariate analysis of overall survival in NSCLC patients ... 79 Supplementary Table 2. Univariate analysis of Overall Survival in NSCLC patients according to β1-AR expression ... 80 Supplementary Table 3. Univariate analysis of Overall Survival in NSCLC patients according to β2-AR expression ... 81
Chapter 5
Table 1. Characteristics of included studies ... 107 Table 2. Quality assessment of included studies ... 108 Supplementary Table 1 ... 121
The work present on this thesis is based on the following manuscripts:
Coelho, M., C. Soares-Silva, Brandão D., Cosentino M. and Ribeiro L." β-adrenergic modulation of cancer cells proliferation: available evidence and clinical perspectives. J Cancer Res Clin Oncol (2017),143 (2), 275–291. doi: 10.1007/s00432-016-2278-1
Coelho M., Imperatori A. Chiaravalli AM, Franzi, F., Castiglioni M., Marino F., Ribeiro L., Cosentino, M. β1- and β2-adrenoceptors expression patterns in human non-small cell lung cancer: relationship with cancer histology(Submitted)
Coelho M.,Squizzato A., Cassina A., Marino F., Ribeiro L., Cosentino M. “Effect of β-blockers on survival of lung cancer patients: A systematic review and meta-analysis “(Submitted)
Coelho, M., Imperatori A., Chiaravalli, AM, Franzi, F., Castiglioni, M, Marino, F., Ribeiro L., Cosentino, M., β-blockers in NSCLC: β1/ β2-AR selectivity matters but it might be not enough.
Sci. Transl. Med. (E-Letter, 12 February 2018) URL:
http://stm.sciencemag.org/content/9/415/eaao4307
Outline of the thesis
The current thesis is divided into 7 chapters throughout which the detailed purposes of the current work are explored:
Chapter 1: Consists in a general introduction of the thesis. A brief overview of the main concepts underlying the association between β-AR signalling and cancer, particularly lung cancer, is presented, as well as the concept of drug repurposing and the pharmacology of β- blockers.
Chapter 2: Reports and discusses the available preclinical and clinical evidences regarding β- AR- modulation of cancer cell proliferation, with a specific focus on the pharmacological properties and on the possible effects of β-blockers.
Chapter 3: Describes a retrospective clinicopathologic study aiming to evaluate the differences between resected tissue specimens from ADC and SCC in terms of the expression pattern of β1- and β2-AR in both, tumour, and adjacent surrounding non-tumour tissues. In addition, we intend to explore its clinical significance and its prognostic value.
Chapter 4: Reports the evaluation of the expression of the β-AR subtypes on the human NSCLC, A549 cells, and the effects of β-AR ligands upon cell proliferation.
Chapter 5: Provides the first systematic review and meta-analysis exploring the available clinical evidence on the possible effect of β-blockers use on overall survival of primary lung cancer patients.
Chapter 6: Highlights and discuss the implications of the complex pharmacology of β-blockers for the therapeutic use in oncologic setting, particularly in NSCLC.
Chapter 7: Presents an overall and integrated discussion of the thesis considering the main findings, strengths, and limitations. Future perspectives and suggestions for further research on
Chapter 1.
Background
OVERVIEW OF LUNG CANCER
Cancer is a major public health issue, accounting for around 15 millions of cases and for ~ 9 millions of deaths in 2015, representing the second leading cause of death worldwide [1].
Current evidences suggest that from 30% to 50% of cancer cases could be avoided by adopting healthiest lifestyles [1,2]. Nevertheless, nearly 24 million of new cases are expected by 2030 as a result of population aging and adoption of cancer-associated life-style choices [1,2]. Despite the significant scientific advances that have been made over the last few decades leading to significant improvements of patient survival, cancer-associated mortality and morbidity it is still a scourge in our time [3].
According to the last worldwide statistics published by Globocan 2018, primary lung cancer is the most commonly diagnosed cancer representing 11.6% of the total cases, and the leading cause of cancer-related deaths estimated to be responsible for 18.4% of the total cancer deaths [3–5].
Similar to other oncological diseases, lung cancer rates differ according to gender, age range, ethnicity, geographic region, and socioeconomic status [4]. The causes of lung cancer development is multifactorial and still not fully understood however, smoking habits is by far the main risk factor for its development explaining, in part, such differences [6,7]. In fact, the highest rates of incidence are observed in more developed countries where smoking habits began earliest such as in North America and Europe [5,7]. Besides smoking, other risk factors include, environment exposure to pollutants (e.g. radon, asbestos), genetic predisposition and family history [8].
Lung cancer is divided in two main groups according to their histologic subtype, prognostic, and therapeutic approach: small-cell carcinoma (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for about 85% of the total number of lung cancer cases and represents a heterogeneous disease that is further divided into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma [9,10]. ADC and SCC are the most
Currently, the knowledge of cancer biology establishes that it arises from a multistep process that comprise a myriad of molecular, cellular, morphologic and genetic alterations [11,12].
Several lung cancer biomarkers have been identified with promising perspectives for therapeutic approaches. Some examples include mutations in epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), KRAS, p53, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the phosphatidylinositol 3-kinase (PI3K) [8,12]. Nevertheless, the classical TNM classification, based on tumour size (T), nodal (N) and metastatic (M) involvement is still the most reproducible prognostic factor for this disease [13–15]. In lung cancer management, the therapeutic approaches are highly dependent of the type, histologic and molecular profile but usually include thoracic surgery, chemotherapy, radiotherapy, personalized medicine and more recently, immunotherapy [9,16–18]. Despite the constants efforts in research and clinical care have significantly advanced during the last decade, the prognosis of lung cancer is still very poor with an overall 5-year survival rate around only 18% [19].
Hence, the identification of novel signalling pathways affecting its development and progression may represent potential new therapeutic targets in lung cancer treatment.
STRESS-RELATED PROCESSES ON CANCER PROGRESSION AND CATECHOLAMINERGIC SYSTEM In current modern lifestyle societies, stress is part of everyday life being associated with the pathogenesis of many diseases, including cancer [20]. Over the last three decades, clinical and epidemiological studies have recognised psychosocial factors such as stress, chronic depression and lack of social support as risk factors for cancer incidence and progression [21].
increasingly stressful societies and cancer incidence nowadays many epidemiological, clinical, and pre-clinical studies have been trying to clarify this association. Stress can be caused by multiple factors, commonly defined as “stressors” which are stimulus that can be categorized as psychological, physiological or physical that ultimately leads to the disruption of body homeostasis [20,22,23]. Stress is divided into two main types: acute or chronic. Acute is lived for a short period of time or in a single major event being beneficial for human health because it helps leading with the threats. Chronic stress it is experienced for longer periods of time resulting in multiple harmful effects for health [22–24]. The overall stress response involves the orchestration of sympathetic nervous system (SNS) and the hypothalamic pituitary adrenal axis (HPA) [24]. Whereas in the SNS the well-known “fight or flight” response results in the production and release of catecholamines (CA), adrenaline (AD) and noradrenaline (NA) from the adrenal medulla, in the HPA occurs the release of corticotropin-releasing hormone from the hypothalamus and consequent release of adrenocorticotrophic hormone (ACTH).
ACTH, in turns, stimulates the release of glucocorticoids such as cortisol from the adrenal cortex [24,25].
The synthesis of CA occurs in sympathetic nerve endings and in the chromaffin cells localized in the adrenal medulla [25]. Their synthesis starts from the conversion of the
amino acid tyrosine to
dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase (TH), the rate-limiting step. DOPA is converted to dopamine (DA) by a nonspecific enzyme L-aromatic amino-acid decarboxylase (AAAD). After its synthesis, DA is stored in large dense-core secretory granules and converted into NA by dopamine-β-hydroxylase (DβH). The last enzymatic step of CA biosynthesis pathway is catalysed by the soluble cytoplasmic enzyme phenylethanolamine N-methyltransferase (PNMT), which uses S-adenosyl-methionine as cofactor, for the conversion of NA to AD [25–
27]. The effects of AD and NA, are mediated by the adrenoceptors (AR) family, which have
been divided and classified into three main classes a1, a2 and β. Each class of receptors is further divided in a1A, a1B , a1D; a2A, a2B , a2C; and β1, β2 and β3, respectively. AR show different patterns of tissue expression and mediate distinct signalling pathways and physiologic
Tyrosine
Dopa
Dopamine
Noradrenaline
Adrenaline
Tyrosine Hydroxilase
Phenylethanolamine N-Methyltranferase
Dopamine β- Hydroxilase L-Aromatic Amino Acid Decarboxylase
Figure 1. Catecholamine biosynthesis
Adapted from Kvetnansky R. et al. (2006) [25].
In the human lung, the presence of β-AR is detected on a variety of cell-types including, but not limited to, airway smooth muscle cells, epithelial cells, submucosal glands, vascular epithelium, and inflammatory cells [30,31]. They are responsible for key-physiological functions such as smooth muscle relaxation [31]. In fact, β-AR agonists have been extensively used in clinical setting as first-line treatment for several respiratory disaeses such as asthma and Chronic Obstructive Pulmonary Disease (COPD) by promoting bronchodilatory effects via β2- AR [32,33].
Table 1. Classification and physiopharmacology of β-AR
Name Tissue distribution Physiological functions Therapeutic drugs (indications)
β1
Brain, lung, spleen, heart, kidney, liver, muscle
Increase of cardiac output (heart rate, contractility, automaticity, conduction), renin release from juxtaglomerular cells, lipolysis in adipose tissue
Agonists Dobutamine, isoprenaline, noradrenaline (bradycardia, heart failure, cardiogenic shock) Antagonists Metoprolol, atenolol, bisoprolol, propranolol, timolol, nebivolol (cardiac arrhythmia, congestive heart failure, glaucoma, myocardial infarction, migraine prophylaxis)
β2
Brain, lung,
lymphocytes, skin, liver, heart
Smooth muscle relaxation, striated muscle tremor, glycogenolysis, increased mass and contraction speed, increase of cardiac output, increase of aqueous humour production in eye, dilatation of arteries, glycogenolysis and gluconeogenesis in liver, insulin secretion, bronchodilation
Agonists: (short-acting) salbutamol (albuterol), levosalbutamol (levalbuterol), terbutaline, pirbuterol, procaterol, metaproterenol, fenoterol, bitolterol mesylate, ritodrine, isoprenaline, (long-acting) salmeterol, formoterol, bambuterol, clenbuterol, (ultra-long-acting) indacaterol (asthma, other effects: vasodilation in muscle and liver, relaxation of uterine muscle, and release of insulin) Antagonists: butoxamine, timolol, propranolol (glaucoma, heart attacks, hypertension, migraine headache;
investigational: stage fright, post-traumatic stress disorder).
β3
Adipose tissue, gall bladder > small intestine > stomach, prostate > left atrium
> bladder
Lipolysis, thermogenesis, relaxation of myometrium and colonic smooth muscle cells, vasodilatation of coronary arteries, negative cardiac inotropic effect
Agonists: amibegron (investigational:
antidepressant, anxiolytic), solabegron (overactive bladder, irritable bowel syndrome) Antagonists: SR 59230A
Adapted from Scanzano A. & Cosentino M. (2015)[27].
β-ADRENOCEPTORS (β-AR) SIGNALLING ON TUMOUR MICROENVIRONMENT
Studies addressing the effects of stress on cancer progression have demonstrated that CA, AD and NA, are the key players in this intricate liaison and that β-AR are the key receptors mediating its effects [34–36]. AD, released from the adrenal gland, reaches the tumour microenvironment through the circulation whereas NA is released from SNS fibres that are innervating and penetrating the tumour mass [37]. Once these neuroendocrine modulators have reached the peripheral tissues or the tumour microenvironment they modulate many cellular and genetic functions [38]. Aside from impairing the anti-tumour immune response they also directly influence on the major hallmarks of cancer progression established by Hanahan and Weinberg (2011) [11,34–36,39]. Nowadays, it is more than established that tumour progression is more than just cells growing out of control, it actually results from a multistep process andan active crosstalk between different cell types within the tumour and its surrounding microenvironment [40]. Among the hallmarks of cancer, the ones that presumably are more influenced by β-AR signalling are: sustaining proliferative signalling, activating invasion & metastasis, resisting cell death, inducing angiogenesis, and avoiding immune destruction. Current knowledge states that β-AR are highly expressed not only on tumour cells mediating the pro-tumour effects of CA but also on many other different cell types within tumour microenvironment [39]. Thus, CA by targeting β-AR present on the several aforementioned cellular types may actively influence cancer progression by a direct and indirect manner [37].
β-AR belong to the superfamily of G-protein coupled receptors (GPCR) and their binding by CA activates Gas protein [28,41] (Figure 2.). Consequently, it stimulates the synthesis of adenylyl cyclase resulting in the synthesis and accumulation of cyclic adenosine monophosphate
and pathologic conditions [38]. Besides that, PKA also activates β-AR kinase (BARK) leading to the β-arrestin recruitment then stopping the β-AR signalling. At same time, it activates Src kinase that in turns triggers several transcription factors, such as STAT3 and focal adhesion kinase (FAK) [43]. FAK is particularly associated with the regulation of cell trafficking and motility via cytoskeletal dynamics as well as cellular resistance to apoptosis [44]. Equally important, PKA also activates BAD (Bcl-2 family member) contributing for the resistance to cell apoptosis.
Regarding the second main effector, EPAC, it is involved in the activation of b-Raf/MAPK pathway that regulates diverse cellular functions, among them the gene transcription mediated by AP1 and Ets family transcription factors [45]. Moreover, some evidences also have suggested that CA mediated by β-AR signalling and Gs-PKA and β-arrestin concomitantly promoting DNA damage and p53 suppression [46]. Collectively, these mechanisms leads on the one hand, to the upregulation of metastasis-associated-genes inducing cell proliferation, apoptosis resistance, tissue invasion, angiogenesis, epithelial mesenchymal transition and on the other, to downregulation of genes facilitating-antitumor-immune responses [38] (Figure 2).
DNA replication
Nucleus
Inside
Gβ Gƴ
G !s
β-arrestinP
Src P
Adenylyl Cyclase
ATP cAMP
PKAEPAC BARK
P
AP1 STAT3 Ets
P GATA1P
CREBP PATF
Raf1a
P P G!s
B-RAF MEK1/2 ERK1/2 P
P
IL6 IL8 MMP9VEGF PTGS2 SNAI2 IFN9 JFNGIL12B Tumour cell membrane
Inflamation Angiogenesis Tissue Invasion Impaired celular Immune response BAD P
FAK P P
Anoikis, chemoresistance,
resistance to
apoptosis
✞
Outside
β-adrenoceptors adrenaline noradrenaline
Figure 2. β-AR signalling mechanisms on tumour cells.
β-AR activation by AR agonists leads to activation of Gas and synthesis and accumulation of cAMP. cAMP accumulation leads to activation of two main effectors: PKA and EPAC. Overall, the mechanisms induced by these two effectors leads from one hand to the upregulation of metastasis-associated genes and from another, to the downregulation of genes facilitating-antitumor-immune responses. PKA, protein kinase A;
EPAC, exchange proteins directly activated by cAMP (adapted from Cole SW (2012)[38])
Along the cancer development, in most solid tumours including in lung cancer, angiogenesis represent one of the most important rate-limiting step [47]. Tumours acquire and sustain a dense vascular networks enabling their nutrient supply and favouring its growing and tumour cells dissemination and thereby facilitating cancer metastasis [11]. Among the several Figure 3. β–AR signalling influence on cells within tumour microenvironment.
β-AR are highly expressed across multiple cellular types within tumour microenvironment mediating direct and indirect pro-tumour effects exerted by catecholamines (CA), adrenaline (AD) and noradrenaline (NA). AD reaches tumour microenvironment through vasculature while NA is released from sympathetic nervous fibbers that innervates tumour microenvironment. β-AR signalling modulates tumour biology by a myriad of mechanisms that ultimately leads, on the one hand, to the upregulation of pro-tumour pathways and one the other hand, to the suppression of the anti-tumour immune response. CSF1, macrophage colony-stimulating factor 1; CCL2, Chemokine C-C motif ligand 2; FFA, free fatty acids; TGF- β, transforming growth factor β; VEGF, Vascular endothelial growth factor; MMP, matrix metalloproteinase 9 IL-6, Interleukin 6; PTGS2, prostaglandin synthesis enzymes.
↓ DNA damage repair; ↓ p53-associated apoptosis; ↑ Cell proliferation; ↑ CSF1 and CCL2; ↑ VEGF; ↑ IL-6; MMP 2,9
↑ TGF-β; ↑ VEGF; ↑ IL-6; ↑ MMP-9; ↑ PTGS2
Effector CD8+ T cells: ↓ IL-2 and IFN-γ;
Regulatory cells : ↑ suppressive function ↑ CTLA expression
↓ activity; ↓ cell number
↑ Proliferation
↑ lipolysis (FFA) Adipocyte
Macrophage
T- Lymphocyte Tumour cell
Endothelial cell Natural killer cell
β-adrenoceptors adrenaline noradrenaline
expression of several pro-angiogenic factors such as VEGF and IL-6 which paracrinaly act on endothelial cells catalysing the growth of the vasculature and hence supporting the tumour expansion [49,51]. Besides that, β-AR are also expressed on endothelial cells and their activation results in VEGFR-2-mediated ERK signalling which in turns, stimulates their proliferation supporting the growth of blood vessels and therefore favours angiogenesis [52].
Currently, it is clearly established that adipose tissue is an endocrine organ being able of producing and release multiple biologically active compounds [53]. Dysregulated production and secretion of adipokines and free fatty acids (FFA), caused by excess adiposity or adipose tissue dysfunction, has been associated with the pathogenesis of several obesity-linked disease, including cancer [54]. Binding of CA to β-AR, particularly β3-AR, represent one of the most important pathways in the regulation of lipolysis [55]. Tumour-associated adipocytes when activate lipolysis release a significant amount of free FFA [56]. In turn, these FFA can be taken up by tumour cells and esterified to phospholipids fuelling the membrane synthesis [56].
Part of these FFA can be also internalized by other microenvironment components such as endothelial cells, thereby supporting the angiogenesis [56].
The association between chronic stress-mediated pathways and immune system impairment has been extensively studied over time [57]. Among the several pathways by which stress markedly affects immune system, adrenergic signalling is by far one of the most important [27,57–60]. β-AR, particularly β2-AR, has been found to be highly expressed on several components of both, innate (neutrophils, monocytes, macrophages, mature dendritic cells, Natural killer (NK)) and adaptive immune cells (lymphocytes T and B) playing important immunoregulatory effects [60]. On macrophages, β-AR signalling is associated with the increase of their recruitment into tumour microenvironment from the bone marrow and spleen
mechanism mediated by β-AR on macrophages [62,63]. Upon β-AR activation, macrophages release prostaglandins stimulating tumour cells production of VEGFC which in turns promotes tumour lymphatic remodelling and metastasis to lymphatic nodes [62,63].
NK cells are innate cytotoxic cells that represent one of the most important immune cells avoiding tumour development by controlling many steps on cancer progression [42]. Several studies have been reported that β-AR signalling activation promotes not only a decrease in the NK activity but also a reduction in its number and in its cytotoxicity [64].
CD8+ T lymphocytes play a tremendous important role in anti-tumour immune response [65].
Their function is also markedly influenced by β-AR signalling, particularly through β2-AR which is differently expressed on the several differentiation stages and thereby conditioning its effects [66,67]. β2-ARs are highly expressed on memory and effector CD8+ T lymphocytes when compared with naïve cells [68]. On memory cells, its activation by CA decreases the production of IL-2 and IFN-γ. IL-2 in turns enhances β2-AR expression on effector CD8+ T cells and further β2-AR activation promotes, in loop, a decrease in the production of IL-2 negatively regulating CD8+ T cells activation and its function [67].
Regulatory T cells (Tregs) are a lymphocyte lineage responsible by the control of the immune homeostasis by keeping the immunologic self-tolerance [69,70]. Being able to suppress the responses of both innate and adaptive immune cells, its role on anti-tumour immune response is of paramount importance [69,70]. β2-AR signalling activation on Tregs is associated with an increasing of CTLA-4 expression enhancing their suppressive function in a PKA-dependent manner, thereby contributing for the decrease of the anti-tumour immune response [60,71].
Furthermore, Cosentino et al. (2007) also described that Tregs express the enzymatic machinery for the CA synthesis [69]. In addition, it was also reported that endogenous CA content might play an autocrine or paracrine inhibitory functional loop in the impairment of their suppressive activity toward mitogen-induced Teff proliferation. [69]
Given the growing body of knowledge linking β-AR signalling and cancer progression and the
studies have emerged evaluating its in situ tumour expression and its potential as a novel tumour biomarker. Indeed, β-AR overexpression, particularly β2-AR, has been reported in a variety human tumours [72–77]. Moreover, tumour-associated β2-AR overexpression was also found to be highly correlated with poor clinicopathological features, tumour recurrence, metastasis, and reduced survival in the most tumours assessed [72–77].
The intense exploration of the β-AR signalling on cancer progression, has aroused much interest of the scientific community by antagonizing β-AR in order to counteract the CA-driven cancer progression. Hence, over the last years, β-blockers have been extensively proposed as a new add-on treatment for several cancer types including lung cancer [27,78–81].
β-BLOCKERS
β-blockers, also known as β-AR antagonists or β-AR blocking agents, represent one of the most widely prescribed class of drugs in clinical setting [82]. Developed by Sir James Black in 1960s, propranolol, the first β-blocker discovered, rapidly revolutionized the management of cardiovascular diseases awarding him the Nobel Prize of Physiology or Medicine in 1988 [83].
Since then, the number of different β-blockers has dramatically increased. To date, many β- blockers have been described and approved for the treatment of several and distinct conditions including hypertension, angina, myocardial infarction, arrhythmias, and heart failure [84–86].
Classically defined as competitive antagonists for CA at β-AR, β-blockers are a group of drugs highly complex and heterogenous in terms of several pharmacologic characteristics, namely, selectivity for β-AR subtypes, lipophilicity, membrane-stabilizing activity (MSA), vasodilatory
β-blockers are the ones that show equal selectivity for β1-, β2 and β3-AR (e.g. propranolol, nadolol) [28,85,87,88]. Although not being a striking drug characteristic since it depends of the dose, i.e. it decreases with the increase of the dose, selectivity definition has been useful to differentiate the β-blockers in the clinical setting.
With regards to lipophilicity, β-blockers that are highly lipophilic (e.g. timolol and pindolol) have the ability to cross the blood-brain barrier which ultimately lead to central nervous system associated side effects namely confusion, depression and lethargy [84].
MSA, also defined as local anaesthetic activity is another property showed by some β-blockers (e.g. propranolol and acebutolol). When used in concentrations above the therapeutic levels have the ability for blocking the myocyte sodium channels by a β-AR independent manner without having a relevant clinical impact [86]. Apart from its effects on β-AR, some β-blockers also display vasodilatory effects via a1-AR antagonist activity (e.g. carvedilol and labetalol) and/or nitric oxide release (e.g. nevibolol) which is useful in the treatment of several diseases such as glaucoma [85].
Certain β-blockers exhibit ISA properties (e.g. acebutolol, labetalol) meaning that they have some partial agonism activity. This means that although their ability to inhibit the stimulatory effects promoted by CA via β-AR, they incite, by their own, agonist responses making them less effective in their therapeutic effect [85].
During the last few years, the huge advances in technology, namely in BRET (Bioluminescence resonance energy transfer) allowed a deeper understanding of the complex pharmacology displayed by drugs commonly used in clinical setting [89]. Aside from the successful blocking of the canonical β-AR signalling pathway, some β-blockers also display biased agonism activity [90–93]. In the light of the classical pharmacology, agonists induce a receptor conformation which leads to its activation and antagonists stabilize the inactive conformation.
Today, it is already know that this binary categorization underestimates the complexity of receptor-ligand behaviour [82,91]. In fact, nowadays, it is recognized that there are more than
signalling [82,91]. Indeed, β-blockers are not merely antagonists for G-protein pathways but may independently modulate two-pathways and behave as partial-agonist, inverse-agonists or pure-antagonists in each pathway [89,94,95]. This means that β-blockers may potentially promote by themselves a receptor-mediated biologic response, an opposite effect to that of receptors agonist drugs or a neutral antagonist effect [89]. This result can have markedly different effects in vivo which can explain, in part, the distinct efficacy profiles of β-blockers in the treatment of cardiovascular diseases. Similarly, in cancer it is crucial to investigate which β-blockers are more effective in antagonizing CA-effects.
Although very few studies have distinguish the class of β-blockers used, some retrospective pharmacoepidemiologic studies published over the last few years, have revealed that oncological patients who take β-blockers for other concomitant ailments have improved overall survival rates and better prognosis [96]. These evidences, along with pre-clinical data have intensively point out β-blockers as an opportunity for drug repurposing in oncological setting.
DRUG REPURPOSING
Despite continuous advances in the comprehension of molecular biology behind the development and progression of cancer and in the innovation of novel target-focused drugs, mortality rate ascribed to oncological disease is still extremely high. The Drug Research &
Development (R&D) processes are extremely complex, expensive, time consuming with very low success rates for pharmaceutical companies [97]. Consequently, when approved, one single molecule comes to the market very costly to patients, promoting critical economic implications for health systems [98]. This is particularly important in the context of oncological diseases, in which the approval of novel drugs is very low and the average time until approval
Repurposing existing therapeutic options to a new disease indication, ultimately holds promise of rapid clinical impact at a lower cost than de novo drug development [98,103].
History is actually full of examples of successful drug repurposing cases [97]. Drug repurposing of thalidomide is a classic and effective example. In 1957, this drug that was originally developed to treat morning sickness in pregnant women, was withdraw because promoted severe birth defects. Some decades later, in 1998, developed by Celgene, thalidomide was approved by FDA for treating leprosy and in 2006 for treating multiple myeloma [97].
Another very well-known and famous example is Sildenafil that was initially developed to treat angina pectoris [104]. This drug proving to be completely ineffective for this condition, showed unusual erections as side effect. Pfizer then decided to repurpose it and market it as a treatment for erectile dysfunction revolutionizing the therapeutic for this condition [104].
In the context of oncology, accumulating data have providing an arsenal of new potential and promising candidates for drug repurposing [98]. Prompted by several pre-clinical, epidemiologic, clinical trials and case reports, some classical drugs already approved for non- oncological indications, have shown efficacy in treating cancer. Several examples include, aspirin, metformin, statins and β-blockers [97,99,101,105,106].
Considering that β-blockers are extensively used as cardiovascular therapeutics with favourable risk-benefit profile, well-known pharmacokinetics, dose range, tolerability and cost remarkably low, in particular in comparison to novel biopharmaceuticals, β-blockers are therefore excellent candidates for repurposing as anticancer agents. In lung cancer, it could be particularly significant bearing in mind the disappointing results provided by current therapeutic approaches.
Notably, propranolol is already an FDA and EMA-approved drug for the treatment of infantile hemangioma (noncancerous growth of blood vessels) being already, an excellent example of drug repurposing. In 2008, Labrèze et al. [107] reported that paediatric patients with infantile
pioneer finding, many pre- and clinical studies have reported the efficacy of propranolol in treating infantile haemangiomas which quickly revolutionized the therapeutic approach for this condition. As a result, since 2015 propranolol is repurposed and approved by FDA and EMA as first line therapy to treat this disease [108]. Despite the exact mechanisms underlying its effects remain unclear, studies indicate that is ascribed to its ability as non-selective β-blocker by down-regulating the expression of VEGF. Therefore, it promotes inhibition of angiogenesis, vasoconstriction and apoptosis of endothelial cells [52,109–111].
As previously mentioned, a growing body of epidemiological evidences have been published, suggesting β-blockers as a new potential candidate for drug repurposing in oncology setting [101,105,106,112]. In particular, because such evidences indicate that they may have antitumor activity, reducing metastasis, tumour recurrence, and cancer-specific mortality in different cancer types, including but not limited to breast [113,114], melanoma [115,116], colon [117] and prostate [118,119]. Such findings have leveraged further high-quality research leading to implementation of the first clinical trials. According to the Table 2, the list of tumours in which β-blockers are being studied by clinical trials is already quite extensive accounting so far for 12 different tumors under study. Unsurprisingly, the great majority is assessing the effect of propranolol upon the most frequently diagnosed types of tumours: breast, melanoma, and prostate cancer. Remarkably, in lung cancer, there are currently no ongoing clinical trials assessing the therapeutic potential of β-blockers.
Cancer types β-blocker Study Phase
Registration Date
Status at time of
search Clinical Trial ID
Breast
Propranolol II 06.05.2013 Active, not recruiting NCT01847001 Propranolol II 04.11.2015 Terminated NCT02596867 Propranolol NA 18.07.2007 Unknown status NCT00502684 Cervical Propranolol Feasibility 18.07.2013 Active, not recruiting NCT01902966 Colorectal Propranolol III 28.04.2009 Unknown status NCT00888797 Gastrointestinal Propranolol Early I 10.08.2017 Not yet recruiting NCT03245554 Hepatocellular
carcinoma
Carvedilol NA 07.08.2012 Withdrawn NCT01659346
VT-1221 II 23.10.2010 Unknown status NCT01265576
Melanoma
Propranolol II 20.11.2013 Suspended NCT0198883
Propranolol II / III 15.11.2016 Not yet recruiting NCT02962947 Propranolol II / III 27.12.2017 Not yet recruiting NCT03384836 Multiple myeloma Propranolol II 17.04.2015 Active, not recruiting NCT02420223 Paediatric Propranolol2 I 13.09.2016 Not yet recruiting NCT02897986
Ovarian Propranolol I 04.03.2011 Completed NCT01308944
Propranolol Feasibility 05.01.2012 Active, not recruiting NCT01504126
Prostate
Propranolol II 20.05.2013 Unknown status NCT01857817 Propranolol II 25.10.2016 Recruiting NCT02944201 Propranolol II 15.05.2017 Not yet recruiting NCT03152786 Renal cell carcinoma Propranolol II 27.10.2017 Not yet recruiting NCT03323710 Soft Tissue sarcoma Propranolol II 11.04.2017 Not yet recruiting NCT03108300 Source: https://clinicaltrials.gov/ [accessed on 10.02.2018] Abbreviation: NA: not applicable; Excluding Hemangioma; 1 VT-122 – Propranolol plus etodolac; 2HEMANGIOL®
Although huge steps have been made in recent years in the field of β-AR signalling and β- blockers on cancer, there remains several open questions, particularly in lung cancer.
Therefore, the lack of studies exploring this issue reinforce the need for investigating this association and to understand if this particular cancer might benefit from β-blockers repurposing in oncology.
Table 2. Summary of clinical trials investigating β-blockers in oncology setting.
Rationale and Aims
Current knowledge supports the notion that β-AR system influences cancer progression by multiple mechanisms. In lung cancer, this link and how drug repurposing of β-blockers could play a protective effect is not fully elucidated. Over the last years, much attention has been paid to the concept of drug repurposing in oncology setting ascribed to the lack effective treatments, costly and highly time-consuming processes in the R&D of novel therapies. With the increasing incidence of cancer, particularly lung cancer, it is imperative to point out new ways of research and find new therapeutic approaches. In line with this, the rationale of this thesis is based on three main evidences from the literature: 1. Epidemiologic data postulates that stress-associated mechanisms enhance the progression of several types of cancer; 2. Pre- clinical studies show that CA influence cancer progression by affecting multiple pro-tumour mechanisms through β-AR activation; 3. Pharmaco-epidemiologic data indicates an association between incidental β-blockers use and improved overall survival in several oncological diseases.
With this in mind, the general aim of this thesis was to investigate the presence of β-AR in Lung cancer (in particular, non-small cell lung cancer (NSCLC)) as well as explore the potential for repurposing b-blockers as a new therapeutic approach for this oncological disease. In order to accomplish that, the following studies were performed to assess specific aims:
1. To discuss the available preclinical and clinical evidences regarding β-AR- modulation of cancer cell proliferation, with a specific focus on the pharmacological properties and on the possible effects of β-blockers.
2. To perform a retrospective clinicopathologic study aiming to evaluate the differences between resected tissue specimens from ADC and SCC in terms of the expression pattern of β1- and β2-AR in both, tumour and adjacent surrounding non-tumour tissues.
In addition, we intend to explore its clinical significance and its prognostic value.
3. To evaluate the expression of the β-AR subtypes on the human non-small cell lung cancer cell line, A549 cells, and the effects of β-AR ligands upon cell proliferation, metabolic activity, and cell growth.
4. To perform a systematic review and meta-analysis exploring the available clinical evidence on the possible effect of β-blockers use on overall survival of primary lung cancer patients.
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