Inhibition of TGF-β signaling in head and neck squamous cell carcinoma has anti- neoplastic and radiosensitizing effects in
vitro
Master Thesis
For the attainment of the academic degree
Master of Science
From the University of Applied Sciences FH Campus Wien
Submitted by:
Katharina Kladnik
Personal identity code 1810544018
Supervisor:
Assoc. Prof. Priv.-Doz Dr. Gregor Heiduschka Medical University of Vienna
Vienna Austria
Submitted on:
23.02.2021
Abstract
Head and Neck Squamous Cell Carcinoma (HNSCC) is diagnosed in almost 900.000 patients each year worldwide and is associated with high morbidity and a severe reduction in quality of life. Resistance to radiation therapy challenges the clinical outcome and is a major cause of treatment failure and relapse. The TGF-ß signaling pathway has been identified as a promising target for cancer therapy and in the tackling of radioresistance. This study aimed to investigate the potential antineoplastic and radiosensitizing effect of TGF-ß inhibition on HNSCC cells in vitro. Two TGF-ß inhibitors, pirfenidone and vactosertib, were investigated for their antineoplastic effect in three HNSCC cell lines, namely FaDu, SCC25 and SCC154. To test for potential sensitizing effects towards radiotherapy, drug treatment was combined with radiation exposure. Upon comparing the IC50 values of the two drugs, it was decided to continue experiments with the more promising candidate, vactosertib. We performed a resazurin reduction assay to evaluate cell viability. Cell migration was evaluated by wound-healing assay, clonogenic survival was evaluated by colony formation assay and cell death was evaluated by trypan blue exclusion assay. Since stromal cells play an important role in treatment response, the effect of vactosertib was also tested in an indirect co-culture with patient-derived CAFs. Vactosertib reduced viability of all cell lines and showed synergistic or additive effects in combination with radiation treatment. In addition, the capability of cells to migrate and form colonies was significantly reduced in all cell lines.
Incubation with conditioned medium from CAFs revealed stimulating effects of secreted factors on viability and migration of HNSCC cells, which were partly abrogated by vactosertib. Our findings indicate a strong antineoplastic and radiosensitizing effect of vactosertib on HNSCC in vitro. Further research of this potentially promising drug candidate is necessary to determine its effectiveness in vivo.
Kurzfassung
Jedes Jahr werden weltweit annähernd 900.000 Patienten mit einem Kopf- Hals-Plattenepithelkarzinom (HNSCC) diagnostiziert, welches mit einer hohen Morbidität und einer gravierenden Reduktion der Lebensqualität assoziiert ist.
Eine Herausforderung für das Behandlungsergebnis ist die Resistenz gegenüber Strahlentherapie, welche auch die Hauptursache für Therapieversagen und das Auftreten eines Rezidiv darstellt. Der TGF-ß- Signalweg wurde als vielversprechendes Ziel in der Krebstherapie sowie der Bekämpfung von Radioresistenz identifiziert. Diese Studie beabsichtigt, den potenziellen antineoplastischen und radiosensibilisierenden Effekt einer Hemmung des TGF-ß-Signalweges auf HNSCC Zellen in vitro zu untersuchen.
Die zwei TGF-ß Inhibitoren, Pirfenidon und Vactosertib, wurden auf ihren antineoplastischen Effekt in drei HNSCC Zellinien untersucht, FaDu, SCC25 und SCC154. Um die potenziell sensibilisierende Wirkung auf die Strahlentherapie zu untersuchen, wurde die Medikamentenbehandlung mit Strahlenexposition kombiniert. Nach einem Vergleich der IC50 Werte wurde entschieden, die Experimente mit dem vielversprechenderen Kandidaten, Vactosertib, fortzuführen. Ein Resazurin-Reduktionstest wurde durchgeführt, um die Proliferation der Zellen zu evaluieren. Die Zellmigration wurde mittels Migrationstest, Koloniebildung mittels Koloniebildungstest und Zelltod mittels Trypan-Blau-Test evaluiert. Da Bindegewebszellen eine wichtige Rolle in der Behandlungsantwort spielen, wurde der Effekt von Vactosertib auch in einer indirekten Co-Kultur mit CAFs aus Patiententumoren untersucht. Vactosertib reduzierte die Proliferation aller Zelllinien und zeigte synergistische oder additive Effekte in Kombination mit Strahlentherapie. Zusätzlich wurde die Fähigkeit der Zellen zu migrieren und Kolonien zu formen signifikant reduziert. Die Inkubation mit konditioniertem Medium der CAFs zeigte stimulierende Effekte von sekretierten Faktoren auf die Proliferation und Migration der HNSCC Zellen, welche teilweise durch Vactosertib aufgehoben wurden. Unsere Ergebnisse zeigen einen starken antineoplastischen und radiosensibilisierenden Effekt von Vactosertib auf HNSCC in vitro. Weitere Studien sind notwendig, um die Wirksamkeit dieses potenziell vielversprechenden Medikaments in vivo zu untersuchen.
Table of Contents
1 INTRODUCTION ... 1
1.1 HEAD AND NECK SQUAMOUS CELL CARCINOMA ... 1
1.1.1 Epidemiology ... 1
1.1.2 Risk Factors ... 2
1.1.3 Treatment ... 5
1.2 RADIATION ... 7
1.2.1 Radiation induced DNA damage and repair ... 7
1.2.2 Mechanisms of radiation resistance in HNSCC ... 8
1.3 TGF-Β PARADOX IN CANCER ... 13
1.3.1 The TGF-β signaling pathway ... 13
1.3.2 TGF-β as tumor suppressor ... 14
1.3.3 TGF-β as tumor promotor ... 16
1.4 TGF-Β:THERAPEUTIC TARGETING AND CURRENT THERAPIES ... 23
1.4.1 TGF-β inhibitors used in this study ... 24
1.5 AIM OF THIS STUDY ... 26
2 MATERIALS AND METHODS ... 27
2.1 CELL CULTURE ... 27
2.2 ISOLATION OF HNSCCCANCER ASSOCIATED FIBROBLASTS ... 27
2.2.1 Characterization of CAFs ... 29
2.3 PREPARATION OF CONDITIONED MEDIUM ... 30
2.4 RADIATION TREATMENT... 31
2.5 CHEMICALS AND REAGENTS ... 31
2.6 METABOLIC ACTIVITY ASSAY ... 32
2.7 ANALYSIS OF TREATMENT INTERACTION ... 33
2.8 MIGRATION ASSAY ... 33
2.9 MIGRATION ASSAY WITH CONDITIONED MEDIUM ... 34
2.10 COLONY FORMATION ASSAY ... 35
2.11 TRYPAN BLUE EXCLUSION ASSAY ... 36
3 RESULTS ... 37
3.1 PIRFENIDONE DECREASES VIABILITY OF HNSCC CELLS ... 37
3.2 VACTOSERTIB DECREASES VIABILITY OF HNSCC CELLS... 39
3.3 VACTOSERTIB ACTS AS A RADIOSENSITIZER IN HNSCC CELLS ... 42
3.4 VACTOSERTIB REDUCES THE MIGRATORY POTENTIAL OF HNSCC CELLS ... 44
3.5 VACTOSERTIB REDUCES CLONOGENIC SURVIVAL OF HNSCC CELLS ... 46
3.6 VACTOSERTIB CAN PROMOTE CELL DEATH IN HPV NEGATIVE HNSCC CELLS ... 47
3.7 ISOLATION AND CHARACTERIZATION OF HNSCCCAFS ... 49
3.8 CAF CONDITIONED MEDIUM INCREASES VIABILITY OF FADU AND SCC154 CELLS, WHICH CAN BE ABROGATED BY VACTOSERTIB ... 52
3.9 VACTOSERTIB ABROGATES CAF MEDIATED ENHANCED MIGRATION OF HNSCC CELLS ... 54
4 DISCUSSION ... 57
4.1 VACTOSERTIB HAS ANTINEOPLASTIC AND RADIOSENSITIZING EFFECTS ON HNSCC CELLS ... 57
4.2 VACTOSERTIB PARTLY ABROGATES CAF-MEDIATED PRO-TUMORIGENIC EFFECTS.. ... 60
5 REFERENCES ... 62
List of Abbreviations
α-SMA Alpha Smooth Muscle Actin ATM Ataxia Telangiectasia Mutated CAF Cancer Associated Fibroblast CDK Cyclin Dependent Kinase CM Conditioned Medium
FAP Fibroblast Activation Protein DDR DNA Damage Response DSB Double Strand Break ECM Extracellular Matrix
EGFR Epidermal Growth Factor Receptor EMT Epithelial to Mesenchymal Transition HNSCC Head and Neck Squamous Cell Carcinoma HPV Human Papilloma Virus
HR Homologous Recombination MMP Matrix Metalloproteinase NHEJ Non-Homologous End Joining P-CK Pan Cytokeratin
PI3K Phosphatidylinositol-3-Kinase PFD Pirfenidone
RB Retinoblastoma
ROS Reactive Oxygen Species
TGF-β Transforming Growth Factor Beta
TβRI Transforming Growth Factor Beta Receptor 1 TβRII Transforming Growth Factor Beta Receptor 2 Vacto Vactosertib
1
1 INTRODUCTION
Head and neck cancer is the sixth most common malignancy worldwide with an estimated 890,000 new cases and 450,000 deaths each year [1]. Over 90% of head and neck cancers represent squamous cell carcinomas arising in the stratified epithelium of the upper aerodigestive tract and are referred to as head and neck squamous cell carcinomas (HNSCCs). The most common risk factors include consumption of tobacco and alcohol, and infection with the human papilloma virus (HPV)[2]. Tumors mainly appear in the oral cavity, pharynx and larynx and are commonly associated with pain, disfigurement and impairment of speech, breathing and swallowing, making this disease especially distressing to patients. While early-stage disease can be cured in many patients using surgery combined with radio(chemo)therapy, treatment options are limited for patients with advanced disease. Notably, besides significant advances in the treatment for HNSCC, the 5-year survival rate has only marginally improved over the past three decades [1][3].
1.1.1 Epidemiology
Incidence rates of HNSCC continue to rise and are expected to reach over one million new cases annually by the year 2030 [1]. Incidence varies significantly, depending on the country, sex and anatomical subsite [4]. There is a high prevalence of HNSCC in Southeast Asia and Australia, which is caused by the consumption of cancerogenic products like alcohol and tobacco.
Contrarily, the rising rates of HPV infection contribute to the high prevalence in Western Europe and the USA [1]. Independent of the localization of the tumor, men are two to four times more likely to be diagnosed with HNSCC.
As an example, in the US, African American men have a 50% higher incidence compared to the rest of the population [2]. These gender differences are likely
1.1 Head and Neck Squamous Cell Carcinoma
2 due to differing rates of tobacco and alcohol consumption. Patients are diagnosed at a median age of 66 years (not virally associated) and 53 years (HPV associated) [1].
Epidemiology has shifted significantly over the past decades. While smoking related HNSCCs decrease in incidence, human papillomavirus (HPV) associated cancers are increasing, predominantly among younger people [2].
In consequence, the primary site distribution has shifted as well. HPV-driven HNSCCs mainly arise in the oropharynx, whereas tobacco associated HNSCCs mainly arise in the oral cavity, hypopharynx and larynx [1]. Hence, an increase in prevalence of oropharyngeal squamous cell carcinoma and a decline in laryngeal and hypopharyngeal cancers has been observed [5].
Unfortunately, more than 60% of patients present with advanced disease (stage III or IV), characterized by large tumors with local invasion and/or metastases to regional lymph nodes. Recurrent or metastatic disease develops in more than 65% of patients, leading to a poor prognosis with a 5- year survival rate of less than 50% in these patients [6]. In general, the 5- year survival for all HNSCC patients has improved modestly over the past decades from 55% (1992-1996) to 66% (2002-2006). However, it is believed that this development is not mainly due to improved treatment but due to emerging HPV associated cancers carrying a better prognosis. Sadly, the struggle with psychosocial issues and compromised quality of life furthermore results in high suicide rates among HNSCC survivors, reaching second highest after pancreatic cancer [1].
1.1.2 Risk Factors
Tobacco
The use of tobacco products is among the primary risk factors for HNSCC.
Smokers are 5 to 25 times more likely to develop HNSCC compared to never smokers. Duration and quantity of cigarette use is directly correlated with cancer incidence and most new diagnosis are associated with smoking and/or
3 alcohol consumption [2][7]. Over 70 carcinogens have been described in cigarette smoke, like nitrosamines, polycyclic aromatic hydrocarbons or aromatic amines [7]. The binding of a carcinogenic chemical to DNA can lead to the formation of DNA adducts, which are usually removed by cellular repair mechanisms. If these processes are overwhelmed or deficient, mutations will occur during DNA replication. Carcinogenesis proceeds if oncogenes like K-Ras or tumor suppressor genes like p53 are affected, leaving distinct mutational signatures [8][7]. However, the promotion of carcinogenesis is not the only negative effect of tobacco. Disadvantageous effects of smoking on the treatment outcome of HNSCC patients include inferior outcome of surgery or radiation therapy and wound healing disorders [7].
Alcohol
In contrast to tobacco, the mechanism by which alcohol mediates its tumorigenic effect, is not so well understood [9]. Because alcohol and tobacco products are often used together, it is further difficult to examine the effect of each individually [10]. It is thought that alcohol independently doubles the risk to develop HNSCC and many studies have proposed a more than additive effect when both substances are used concurrently [2]. Strikingly, heavy users of alcohol and tobacco have a 35 fold higher risk to develop HNSCC [1].
Alcohol is usually metabolized in the liver to acetaldehyde via the enzyme alcohol dehydrogenase and further to acetate via the enzyme aldehyde dehydrogenase. However, extrahepatic metabolism in the upper gastrointestinal tract, could allow the accumulation of mutagenic acetaldehyde in oral tissues. Rather than being a primary driver of carcinogenesis, it is believed that alcohol acts as a co-carcinogen, further initiating or promoting tumorigenesis. For example, it was proposed that alcohol may increase the solubility and thereby the penetration of carcinogens from tobacco smoke in the oral mucosa. Also, interference with DNA repair mechanisms have been suggested, which further increases vulnerability towards other carcinogens [10].
4
HPV
Infection with HPV has become one of the most important risk factors for the development of HNSCC in the last decade. Over the past 50 years, smoking related disease has globally declined while the incidence of HPV related disease has significantly increased, predominantly among younger people in North America and northern Europe [6][2]. HPV positive patients are typically middle-aged nonsmokers with higher socio-economic status and generally face a substantially better prognosis than HPV negative patients [5]. This finding has led to a new TNM classification of OPSCC in HPV positive and HPV negative tumors in the 8th edition AJCC. To gain a better understanding for why HPV positive HNSCCs are epidemiologically and clinically distinct, it is important to understand the biology behind HPV driven disease.
HPV is a small double-stranded DNA virus that targets the basal cells of the epithelial mucosa in the cervix, anogenital region and the oropharynx. It can be transmitted through anal, vaginal and oral sex. In HNSCC, especially unprotected oro-genital sex seems to be a leading source of transmission [11]. There is a number of subtypes of HPV, which can be divided into “low- risk” and “high-risk” subtypes, according to their ability to transform epithelial cells [12]. Low-risk subtypes usually cause nonmalignant lesions like common warts (HPV 2 and 27) or anogenital warts (HPV 6 and 11) [13]. High-risk subtypes, that are described to play an important role in many cancers are HPV 16, 18, 31, 33, 35, 45, 51, 52, and 56. These subtypes can cause cancers of the cervix, vagina, vulva and penis as well as the head and neck region.
The most prevalent subtype in HNSCC is HPV 16, accounting for more than 80% of HPV positive cases. Further, HPV 18, 33, 35 and 56 were reported to be involved in the emergence of many oropharyngeal cancers [14]. High-risk HPV subtypes are able to integrate into the human genome, though it is not clear whether this is required for malignant transformation in oral cancers [15]. The circular, 8 kilobase large HPV genome codes for eight genes, which can be divided into “late” (L) and “early” (E) genes [16]. The late proteins L1 and L2 are capsid proteins and are responsible for tissue specific infection [14]. The early proteins E1 and E2 control replication and expression of viral
5 oncogenes. E4 and E5 modify the cellular environment to promote genome amplification. The early proteins E6 and E7 are primarily responsible for oncogenesis because they interfere with key cell cycle regulators [17]. E6 activates the ubiquitin ligase E6AP that ubiquitinylates the tumor suppressor p53, which leads to its degradation. This causes a dysregulation of cell cycle arrest and apoptosis and ultimately leads to unrestricted proliferation of the infected cell. E7 inactivates the Retinoblastoma (Rb) protein. Rb usually binds the transcription factor E2F, which is released upon cell growth signals and mediates cell cycle entry. E7 binds to Rb, which means that E2F is constantly liberated and able to initiate unchecked cell division. E2 represses transcription of E6 and E7, but it is frequently lost during persistent HPV infection. This further leads to increased expression of the oncogenic proteins, driving the accumulation of genetic changes, clonal expansion and neoplastic transformation [15].
1.1.3 Treatment
Treatment options for HNSCC include surgical resection, radiotherapy, chemotherapy and immunotherapy [18]. The choice of therapy depends largely on the stage of disease, anatomical site and surgical accessibility of the tumor. Tumors are staged according to the tumor-node-metastasis (TNM) staging system of the Union for International Cancer Control (UICC). In general, stage I or II tumors are small primary tumors that show no prominent involvement of lymph nodes. Stage III or IV cancers display larger, locally advanced tumors, with accelerated lymph node involvement and invasion of surrounding tissue and distant metastasis. HPV status is routinely assessed by immunohistochemical detection of p16, which is a surrogate marker for HPV positivity [6][19].
Around 30-40% of patients are diagnosed at an early stage of disease (I or II), that can be treated with surgery or radiotherapy monotherapy, which result in similar survival [20][6]. Single modality treatment results in cure rates over 80% in these patients [1].
6 The prognosis, however, drastically worsens with increased disease progression [21]. More than 60% of patients present with locoregionally advanced disease (III or IV) which carries a high risk of distant metastasis and recurrence. These patients require multimodality treatment, whereby the curative goal and the preservation of function need to be carefully considered [6]. One option is surgery followed by radiation or chemoradiation (depending on presence of high-risk features). If surgery is no option, concomitant chemoradiotherapy is preferred. In patients with good performance status, the standard of care is a high dose of cisplatin given concomitantly with radiotherapy. Another option would be induction chemotherapy followed by radiation or chemoradiation [22][6]. In general, the use of multimodality treatment has led to a better treatment outcome, however, patients still face a poor prognosis with a 5-year overall survival rate below 50% [6].
Unfortunately, treatment is often ineffective and 65% of HNSCC patients develop metastatic and/or recurrent disease. If salvage surgery is no option and patients were previously treated with radiotherapy, systemic therapy is recommended. Active agents like taxanes, platinums or antifolates are used, alone or together with cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR). A combination of platinum-based chemotherapy together with cetuximab increased the overall survival from 7 to 10 months [6]. Another option for targeted immunotherapy is the recently approved immune checkpoint inhibitor pembrolizumab. Combined with chemotherapy, pembrolizumab led to superior survival outcome compared to cetuximab (13 vs 10 months, respectively) [1]. Single agent chemotherapy or best supportive care is offered for patients with poor performance status [5]. In the absence of treatment, the dismal prognosis for recurrent disease is 6 to 9 months [6].
Standard treatment, especially concurrent cisplatin therapy, often results in significant acute and long-term toxicity and life-changing consequences like xerostomia and dysphagia [23]. Although HPV related tumors show a better prognosis independent of treatment modality, current guidelines have not incorporated distinct treatment options for these patients. De-intensified treatment and new antiviral and immunomodulatory therapeutics are
7 currently being investigated to reduce toxicity and undesirable side-effects [24].
Radiotherapy represents one of the main treatment modalities in HNSCC.
There have been some therapeutic advances in the past. For example, Intensity-modulated radiation therapy (IMRT) helps to limit damage to surrounding tissue and proton therapy allows a decrease in radiation dose to neighboring anatomical structures. However, despite improved treatment techniques, locoregional relapse caused by radiation resistance remains a huge challenge in HNSCC treatment [22]. Approximately one third of patients are confronted with locoregional treatment failure within 5 years from treatment. They are facing a significantly worse prognosis and have to rely on often combined second- and third-line treatment approaches [25].
Survival outcomes have not improved significantly over the past decade, which is mainly due to limited therapy options for patients with recurrent disease [26]. Therefore, there is an urgent need for effective radiosensitizing agents, which could improve treatment outcome and reduce recurrence rates.
The first step in understanding the complex mechanism of radioresistance is, to look into the cellular and molecular principles behind it.
1.2.1 Radiation induced DNA damage and repair
When charged particles pass through a cell, they can either directly ionize DNA or ionize nearby water molecules, resulting in the generation of reactive oxygen species (ROS), which can in turn react with DNA. This frequently results in the generation of severe cytotoxic lesions like DNA double strand breaks (DSBs). DSBs are repaired via two major DNA damage response repair (DDR) pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ) [22].
1.2 Radiation
8 HR occurs in a sequence specific manner, utilizing the homologous sequence of a sister chromatid as a template to repair DSBs. At first, DSBs are recognized by the Mre11-Rad50-Nbs1 (MRN) complex, which then activates ataxia telangiectasia mutated (ATM), a serine-threonine kinase. ATM initiates DDR by phosphorylating and activating numerous DDR factors. Single strands are stabilized and Rad51 binds and facilitates homology search, strand invasion and DNA synthesis. Among others, BRCA1 and BRCA2 are required in facilitating this process. Since sister chromatids are only available after genome replication, HR can only happen during late S to G2 phase [27].
NHEJ however, unselectively ligates two broken ends of DNA and can therefore occur at any stage of the cell cycle. The first step in NHEJ is the binding of the Ku70/Ku80 heterodimer (canonical NHEJ) or PARP-1 (alternative NHEJ) to the ends of the DSB. They are then bound by DNA dependent protein kinases (DNA-PKs), which enables the recruitment of nucleases and polymerases. They trim overhangs and fill in gaps. Finally the processed ends are ligated by a ligase complex, containing DNA ligase IV [27][28]. Radiation induced DSBs are primarily repaired via NHEJ [22].
Other repair mechanisms like alternative end joining (alt-EJ) also do not rely on a homologous template for ligation of DNA ends, though the exact mechanism is poorly understood by now. These pathways generate large deletions, translocations and other chromosomal aberrations, frequently observed in cancer [29].
1.2.2 Mechanisms of radiation resistance in HNSCC
Since radiation damage occurs on a DNA level, the ability to activate DDR pathways is critical in the development of radiation resistance. If DNA damage is too severe, cells cannot divide which leads to induction of apoptosis, necrosis, senescence or autophagy. Therefore, radioresistant cancer cells increase their DDR rate to avoid cell death upon radiation [22]. In HNSCC, this can be achieved by directly altering DDR components such as overexpressing Ku80 or Rad51 [30]. Furthermore, radioresistance is acquired
9 via altering intracellular pathways involved in DNA repair, replication, cell cycle control and apoptosis [22] [31]. Frequently, alterations of the epidermal growth factor receptor (EGFR), the phosphatidylinositol-3-kinase (PI3K)/Akt or the p53 signaling cascade are observed. Additionally phenomena like hypoxia and epithelial to mesenchymal transition (EMT) contribute to the emergence of radioresistance in HNSCC [31]. To further gain an insight into the mechanisms of radioresistance in HNSCC, some of these pathways and their alterations will be discussed further. Additionally, emerging therapeutics that aim to overcome radioresistance in HNSCC will be mentioned.
EGFR
The tyrosine kinase transmembrane receptor EGFR is stimulated by extracellular ligands such as epidermal growth factors and transforming growth factors. Upon binding, the intracellular domain of the receptor is autophosphorylated resulting in the activation of downstream effectors, which induce survival and proliferation. Radiation mimics the binding of a ligand via autophosphorylation of EGFR and therefore supports proliferation and survival of irradiated tumor cells. Additionally, EGFR regulates transcription of genes involved in NHEJ. Therefore, EGFR contributes to successful DNA repair, allowing cell cycle progression of irradiated cancer cells [22]. EGFR is overexpressed in more than 90% of HNSCCs and high expression rates were shown to correlate with a high rate of locoregional failure after radiotherapy [22].
Notably, an EGFR-targeting monoclonal antibody (Cetuximab) was approved for the treatment of HNSCC. It has been shown to be effective when administered in combination with radiotherapy, however this is not the case for HPV positive patients [32][33].
P53
Another crucial protein in the development of radioresistance is the tumor suppressor p53. It regulates cell cycle progression, DNA repair and apoptosis.
10 In response to cellular stresses like DNA damage, p53 is activated by posttranslational modifications, which causes its release from its negative regulator MDM2 [31]. Activated p53 mediates transcriptional activation of p21, which induces G1 cell cycle arrest. During cell cycle arrest p53 stimulates the synthesis of DNA repair proteins. However, if DNA damage is too severe, apoptosis is induced [31]. Therefore, the consequence of defective p53 is an impaired capability to arrest the cell cycle, to restore DNA integrity and to induce apoptosis upon radiation stress [34]. About 40-70% of HNSCCs carry an inactivating mutation in the TP53 gene, though they are less frequent in HPV related disease [31].
There are several strategies to restore normal p53 signaling, including viral gene therapy that delivers the functioning gene, or small molecules disrupting p53 inhibitors. However, to date none of the investigated therapeutics have been approved for the treatment of HNSCC [31].
Hypoxia
Another factor leading to decreased efficacy of radiotherapy is the presence of low oxygen levels (hypoxia) [35]. Due to poor vessel perfusion, oxygen levels within the tumor are low. Tumor cells adapt to this lowered oxygen supply by switching from oxygen dependent tricarboxylic acid cycle to oxygen independent glycolysis. A key transcription factor in the regulation of this metabolic reprogramming is hypoxia-inducible transcription factor 1 (HIF-1) [34]. Since radiation induced DNA damage relies on the generation of ROS, hypoxic conditions directly contribute to the emergence of radioresistance.
The problem of hypoxia is especially relevant for smoking HNSCC patients.
Due to vasoconstrictive effects of nicotine and formation of carboxyhemoglobin, lowered oxygen levels lead to worse treatment response and survival compared to nonsmokers [34]. Hypoxia occurs equally pronounced in HPV positive and negative tumors [36][35].
The hypoxic radiosensitizer nimorazole has been proven effective in a phase III clinical trial, which led to its orphan designation for the treatment of HNSCC in Denmark in 2011 [35].
11
Cancer Stem Cells
Cellular heterogeneity among HNSCC tumors leads to particular challenges in treating the disease. Genomic instability results in an accumulation of mutations and therefore functional diversity within the tumor. Cancer Stem Cells (CSCs) are self-renewing and multipotent cells, which seem to play a critical role in maintaining the tumor after radiation treatment. CSCs are able to give rise to different heterogenous lineages and have been implicated in radiation resistance and recurrent disease in HNSCC [22]. It is hypothesized that CSCs support radioresistance and relapse after therapy by scavenging of ROS together with elevated cell survival and increased DNA repair [37]. In addition, CSCs can acquire invading and metastasizing abilities through a process called epithelial to mesenchymal transition (EMT). In turn, EMT can induce the acquisition of CSC characteristics [34]. In HNSCC patients, a high percentage of CSC marker (e.g. CD44, CD24, Oct4 and integrin β1) expressing cells is associated with poor clinical outcome after radiotherapy and treatment failure [34][30].
Targeting the CSCs for therapy is very difficult due to strong side effects on normal tissue stem cells. Therapies targeting CSC specific traits have however already reached the clinical trial stage [37].
Epithelial to mesenchymal transition
Another essential process involved in radioresistance of many cancers arising from epithelial tissues is EMT. An up-regulation of mesenchymal markers together with a loss of epithelial markers leads to an increase in cellular migration and motility. This results in an enhanced capacity of metastasis and treatment resistance. A complex network of signaling pathways is involved in the EMT process, including TGF-β, EGFR, Notch, Wnt, and PI3K [38][39]. The details linking EMT with radio resistance in HNSCC will be discussed later in the context of TGF-β signaling (see: 1.3.3.6 TGF-β: Implications in radio resistance).
12
The tumor microenvironment
Although intracellular processes fuel the direct acquisition of radioresistance in cancer cells, the surrounding stroma also plays a substantial role in determining the success of radiotherapy. There are several mechanisms by which the tumor stroma can mediate radioresistance, making it a legitimate therapeutic target on its own. Hypoxia and vasculogenesis, CAF-mediated extracellular matrix (ECM) remodeling and fibrosis, and immunomodulation, are closely intertwined processes, that can occur as a response to radiotherapy in solid tumors. One of the key inducers of stromal activation is the cytokine TGF-β [40]
There are many targetable sites in the microenvironment of HNSCC including immunologic components, vascularization, ECM and CAFs. One example for an immunologic agent is the recently approved PD-1 inhibitor pembrolizumab [41].
TGF-β
It was shown by multiple studies that the pleiotropic cytokine TGF-β can influence radioresistance by multiple mechanisms in various different cancers [42]. By interfering with different mediators of radioresistance and the DNA damage response, it was proposed that TGF-β contributes to the emergence of radioresistance [43][44][45][27]. Therefore, targeting these effects of TGF-β could radiosensitize cancer cells.
Since this study is focused on the antineoplastic and radiosensitizing effects of this cytokine, the TGF-β signaling pathway will be further discussed in detail in the following section.
13 TGF-β is a multipotent cytokine that plays a key role in epithelial homeostasis.
In normal and early stage cancer cells, TGF-β acts as a potent growth inhibitor by inducing cell cycle arrest and apoptosis [46]. However, at later stages of cancer, TGF-β acts as tumor promotor by promoting growth, invasion, immune evasion and metastasis [47]. This functional switch from tumor- suppression to tumor-promotion is called the TGF paradox [48]. To gain insight into the ambivalent role of TGF-β in carcinogenesis, the molecular basics of the signaling pathway will be discussed first.
1.3.1 The TGF-β signaling pathway
TGF-β belongs to a large family of growth factors that include activins, inhibins and bone morphogenetic proteins. Three isoforms are known, namely TGF-β1, -β2 and -β3, which are believed to have similar but not identical activities. TGF-β is secreted as a homodimer from cells in a latent, biologically inactive form. Additionally, it is bound by two other polypeptides, the latent TGF-β binding protein (LTBP) and the latency-associated peptide (LAP), which need to be cleaved to release the active TGF-β. Signal transduction is mediated by type I and II serine/threonine kinase receptors. Upon activation through proteolytic cleavage, integrin interaction or pH changes, TGF-β binds to the TGFβ receptor II (TβRII). Alternatively, certain isoforms of TGF-β can be presented to TβRII via the co-receptor TGFβ receptor III (TβRIII) [49].
Binding of the ligand to TβRII leads to the recruitment of TGFβ receptor I (TβRI). TβRI is then phosphorylated by the intracellular kinase domain of TβRII, facilitating its activation. Subsequent downstream signaling then occurs via two independent routes: the canonical and the noncanonical pathway [46][50].
In the canonical pathway, activation of TβRI leads to phosphorylation of receptor-associated Smad2 and Smad3 (R-Smads). They then form a complex with Smad4 (Co-Smad) and translocate to the nucleus. Together with other transcription factors, the complex binds to the promotor of TGF-β
1.3 TGF-β paradox in cancer
14 target genes and regulates transcription [51]. This route can be modulated by inhibitory Smads, Smad6 and Smad7. They compete with R-Smads for TβRI binding and can mediate degradation of TβRI and R-Smads [52].
Non canonical signaling is independent of Smad proteins. The activated TGF-β receptor complex transmits its signal through other pathways, such as MAP kinase (MAPK) signaling, Rho like GTPase signaling and PI3K/Akt signaling.
These pathways are activated by the receptor complex through phosphorylation or direct interaction [53].
1.3.2 TGF-β as tumor suppressor
The tumor suppressive effects of TGF-β are attained through several actions.
The key mechanisms to achieve tumor suppression are induction off cell cycle arrest and apoptosis, maintenance of genomic stability, promotion of cell differentiation and interactions with the tumor stroma [54].
TGF-β inhibits proliferation and promotes differentiation
By far the most important tumor suppressive effect of TGF-β is the inhibition of proliferation by induction of G1 cell cycle arrest. In epithelial cells, Smad dependent TGF-β signaling induces the cyclin dependent kinase (CDK) inhibitors p15 and p21. These CDK inhibitors prevent the interaction of CDKs with cyclins. As a result, Rb phosphorylation is prevented and cell cycle progression is arrested in G1 phase [54]. Moreover, Smad proteins also repress the expression of the oncogene Myc, which is a potent transcriptional activator of proliferation inducing genes [52][51].
Further, TGF-β signaling downregulates inhibitor of differentiation (ID) proteins, which can actively promote cell proliferation and inhibit differentiation [55]. Thereby, TGF-β signaling exerts antiproliferative and pro- differentiation effects, which counteract cancerogenic processes.
15
TGF-β regulates apoptosis
The role of TGF-β in apoptosis is strongly dependent on the cellular and environmental context as TGF-β can both induce and suppress apoptosis. In vitro, several Smad-dependent and independent mechanisms have been proposed to exhibit proapoptotic effects. For example, TGF-β induces transcription of the pro-apoptotic genes Bim, DAPK, SHIP and TIEG1.
However, TGF-β can also enhance the survival of epithelial cells. By activating the PI3K/Akt and NF-κB pathway, TGF-β can cause inhibition of pro-apoptotic proteins and induction of pro-survival genes [56].
TGF-β mediates genomic stability and regulates DNA repair
DNA damage repair and maintenance of genomic integrity are important cellular mechanisms. However, they also determine the effectiveness of radiotherapy in cancer treatment. There is growing evidence supporting the role of TGF-β in DNA repair and therefore proposing a genome protective effect of the cytokine.
For example, it was shown that TGF-β lacking mouse keratinocytes have increased gene amplifications in response to the drug PALA in comparison to wild type cells. When TGF-β expression levels were increased, cells were protected from genomic instability [57].
Furthermore, deficient TGF-β signaling can result in the accumulation of chromosomal aberrations which is followed by spontaneous malignant transformation. Exposure to exogenous TGF-β1 protected the cells from genomic damage and malignant transformation [58].
Strikingly, it was shown that aberrant downstream TGF-β signaling induces tumor formation in mice. SMAD 4 deletion in the head and neck epithelia of mice, gave rise to spontaneous HNSCC accompanied by increased genomic instability [59].
In the context of HNSCC, the contribution of TGF-β in maintaining DNA damage repair, becomes especially apparent when looking at HPV positive cancers. It was postulated that HPV mediated blockade of TGF-β signaling
16 impairs DNA repair capacity and therefore causes increased radiosensitivity of HPV positive HNSCC cells [27].
TGF-β controls stromal components
Besides directly affecting carcinoma cells, TGF-β can also control tumor development by interfering with the surrounding stroma [54]. For example, TGF-β can prevent epithelial cell proliferation by blocking paracrine signaling and growth factor secretion of stromal fibroblasts. In mouse models, it was shown that defective TGF-β signaling in fibroblasts causes hyperplasia and neoplastic transformation of surrounding epithelia [60]. Additionally, TGF-β acts as a key suppressor of damaging inflammatory reactions, which contribute to tumorigenesis. Furthermore, TGF-β inhibits the function of the innate and adaptive immune responses. It promotes immune tolerance via decreasing the activity of effector T-cells, macrophages, dendritic cells and NK cells and stimulating the generation of regulatory-T cells [47].
1.3.3 TGF-β as tumor promotor
As mentioned, TGF-β has a dual role in tumor development. In cancer cells, the tumor suppressive arm of the TGF pathway is lost and tumor growth and invasion are directly enhanced [47].
It is known that carcinoma cells can escape the tumor suppressive mechanisms of TGF-β by selectively disabling the pathway. They become unresponsive to TGF-β mediated growth inhibition via inactivating mutations of core elements of the TGF-β signaling pathway [50]. Accordingly, numerous alterations and mutations in TGF-β signaling pathway have been identified in HNSCC [61].
But TGF-β can also have direct pro-oncogenic effects. The key processes which occur during transformation are induction of EMT, evasion of immunity and indirect support of tumor proliferation through the action of stromal components [50]. Furthermore, TGF-β has been proposed to play a role in
17 the emergence of radioresistance [42]. Many of these pro-oncogenic responses are Smad-independent or require cooperation of canonical and non-canonical TGF-β signaling [62].
In the following, the mechanisms that determine the tumor promoting function of TGF-β in HNSCC will be discussed.
Evasion of tumor suppression via dysregulated TGF-β signaling
HNSCC cells evade the tumor suppressive mechanisms of TGF-β by dysregulating core components of the pathway [54].
Increased TGF-β1 expression is observed in ~80% of human HNSCCs and is also frequently observed in adjacent tissue [63][64][65]. It is believed that overexpression of TGF-β1 causes tumor promotion through epithelial hyperplasia, increased invasion, inflammation and angiogenesis in the tumor stroma [66][64].
Further, TβRII is a frequent target of genetic alterations and epigenetic silencing. Reduced expression of TβRII is observed in ~70% of HNSCC cases and mutations in the TGFΒR2 gene occur in 21% of tumors [67][68]. TGFΒR2 deletion in mouse models allowed accumulation of molecular alterations and resulted in increased TGF-β1 levels. Overabundance of TGF-β1 in turn mediates tumor promotion [67][66].
Mutations of TβRI are rare in primary human HNSCCs, however they are common in ~19% of metastatic HNSCCs [69]. In mice, TGFΒR1 deletion led to increased TGF-β1 expression and allowed tumor progression [66].
Furthermore, also the downstream mediators of canonical TGF-β signaling are lost or inactivated in HNSCC cells. Loss of chromosome 18q, which encodes for Smad2 and Smad4 is commonly seen [70][71]. Loss of heterozygosity of Smad4 occurs in ~50% of HNSCCs [70] and expression is reduced in 86% of tumors and 67% of adjacent mucosa [72]. Alterations of Smad2 vary significantly among different reports. While one study proposed loss of Smad2 in 38% of tumors, another study revealed that Smad2 expression is intact. However, both studies found a decrease in levels of phosphorylated, active Smad2 protein in HNSCC tumor tissue [73][74]. In a
18 number of mouse models, it was shown that a loss of Smad proteins increases genomic instability and inflammation and can cause tumor initiation [66].
While many studies focus on their transforming properties, the HPV proteins E5, E6 and E7 can also partly abrogate TGF-β signaling. It was shown that the viral protein E5 downregulates TβRII and decreases Smad2 phosphorylation as well as nuclear translocation of Smad4 [75]. E6 can degrade TIP-2/GIPC, which is a positive regulator of TβRIII [76]. E7 interacts with Smad 2,3 and 4 and blocks Smad3 binding to its target sequence [77].
The effect of subjugation of TGF-β signaling by HPV is very poorly studied by now. However, a role in increased radiosensitivity of HPV positive cancers has been proposed [78].
TGF-β induces EMT and metastasis
TGF-β signaling can further directly induce enhanced tumor growth and invasion. TGF-β stimulates invasive and metastatic potential of epithelial cells through induction of EMT and by driving the spread of malignant cells to distal sites [79].
EMT is a cellular process during which epithelial cells lose their typical characteristic cell junctions and polarity, and instead acquire motility and invasive properties. During this process, E-cadherin, a cell adhesion molecule is downregulated and key transcription factors such as Snail and Slug are upregulated [52]. TGF-β can control EMT via Smad dependent signaling as well as Erk, PI3K, Rho GTPase and p38 MAPK signaling [56].
The repression of E-cadherin is critical for cells to undergo EMT. TGF-β activates several transcriptional repressors of the E-cadherin gene like Snail, Slug, ZEB1, ZEB2, LEF1 and Twist. Thereby TGF-β inhibits expression of E-cadherin, which leads to dissolution of adherens junctions [80][81].
Furthermore, TβRII can phosphorylate Par6, which leads to dissolution of tight junctions [82]. Downregulation of E-cadherin further initiates cytoskeletal reorganization and promotes the assembly of focal adhesions.
Together with TGF-β mediated activation of mesenchymal genes like
19 N-cadherin, this leads to acquirement of a motile and invasive phenotype [81].
Upon loss of epithelial cell adhesions and generation of a motile phenotype, cancer cells need to be able to remodel the extracellular milieu to gain invasive and metastatic properties. Matrix metalloproteinases (MMPs) are gelatinases that can degrade ECM proteins. There is evidence that TGF-β1 can promote Snail and Slug-mediated increase of MMP9 expression, which correlates with invasive behavior of oral squamous cell carcinomas [83][84].
TGF-β regulates angiogenesis
The formation of new blood vessels, which provide the tumor with nutrients and oxygen, is crucial for tumor growth and invasion. TGF-β was shown to exert promoting as well as suppressing effects on angiogenesis.
Presumably, TGF-β induces expression of VEGF, which stimulates endothelial cells to proliferate and migrate and induces capillary formation [56]. In a transgenic mouse model, overexpression of TGF-β in the head and neck epithelia of mice resulted in increased angiogenesis [64]. However, the reconstitution of Smad4 in carcinoma cells suppressed VEGF transcription and induced anti-angiogenic protein thrombospondin [85]. Therefore, the pro- angiogenic mechanisms may be Smad independent. Overall, the mechanisms of angiogenic stimulation and inhibition by TGF-β are complex and context- dependent [86][87].
TGF-β modulates the immune response
TGF-β can suppress both innate and adaptive immune responses. Generally said, TGF-β is able to drive innate immune cells towards an alternative differentiation status [50]. The innate immune cells are polarized from a type 1 differentiated, tumor suppressing cell towards a type 2 tumor promoting phenotype by TGF-β. For example, CD4+ T helper cells are polarized towards the Th2 type, M1 macrophages towards M2 type and N1 neutrophils towards N2. The type 2 transitioned cells deliver an accelerated amount of
20 inflammatory and angiogenic cytokines and tumor-promoting growth factors to the tumor milieu [88]. In this way TGF-β can abrogate tumor-suppressing functions and support tumor-promoting functions of immune cells.
TGF-β in the TME
Besides tumor and immune cells, TGF-β signaling also affects other surrounding mesenchymal cells and the neoplastic ECM, which are collectively referred to as the tumor microenvironment (TME) [52][89].
Among many cell types, CAFs have been demonstrated to play a particularly critical role in promoting HNSCC tumor growth and progression. CAFs are the most abundant cells of the TME and can differentiate locally from normal stromal fibroblasts [89]. They are characterized by increased expression of α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) and secrete increased amounts of ECM components like fibronectin. The crosstalk between tumor cells and CAFs enhances the production of growth factors, stromal modulators, cytokines and inflammatory effectors and can therefore mediate tumor growth [89].
TGF-β1 is one of the major pro-metastatic factors secreted by CAFs, which has also been shown in HNSCC [65]. CAFs also secrete high amounts of other growth factors such as hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) which also play a critical role in CAF mediated invasion. In turn, TGF-β secreted from cancer cells can lead to activation and transformation of stromal fibroblasts into CAFs [90].
Since paracrine and autocrine communication of CAFs and cancer cells is critical for tumor progression, targeting these signaling pathways could have substantial therapeutic benefits.
TGF-β: Implications in radioresistance
TGF-β has been proposed to influence radioresistance by modulating the response to ionizing radiation [42][43][45]. The underlying mechanisms are complex and multifactorial. In the following, elemental concepts and
21 experimental findings that connect TGF-β and radioresistance in HNSCC will be discussed.
TGF-β mediates EMT and the acquisition of a CSC phenotype
As described previously, TGF-β mediates EMT, invasion and metastasis and this effect can be enhanced through crosstalk with CAFs in the tumor stroma.
There is increasing evidence that the induction of EMT and the crosstalk between cancer cells and the surrounding stroma contribute to the emergence of radioresistance in cancer.
For example, in HNSCC cells, it has already been shown that induction of EMT with CAF conditioned medium can induce radioresistance [91]. Moreover, in nasopharyngeal carcinoma cells, the blocking of TGF-β led to inhibition of EMT and enhanced radiosensitivity [92]. Furthermore, initiation of the EMT program resulted in failure of enhanced radiation response in a HNSCC mouse model [93].
Now we know that TGF-β mediates EMT, which is associated with radioresistance. But how does EMT confer radioresistance?
Most importantly, EMT can promote radioresistance by acting as key mediator in the acquisition of CSC characteristics. For example the EMT associated transcription factor Twist can promote expression of BMI-1 which plays an essential role in self-renewal of CSCs [37]. This is supported by a study that investigated the effect of TGF-β on the stemness of primary HNSCC CSCs.
TGF-β increased stemness-associated gene expression and cells exhibited elevated expression of Twist, Snail and Slug [94].
It is believed that CSCs can survive radiotherapy by dynamical adjustment to the changed environment, enhanced scavenging of ROS, increased DNA repair capacity and elevated cell survival (see 1.2.2.4 Cancer Stem Cells) [37].
Therefore, TGF-β might contribute to radioresistance via induction of EMT and upregulation of CSC characteristics [39].
22
TGF-β supports the DNA damage response
Another mechanism by which TGF-β regulates radioresistance is by modulating cellular responses to irradiation induced DNA damage [43].
Radiation damages cancer cells by generating DNA double strand breaks (see 1.2.1 Radiation induced DNA damage and repair). Thus, efficient repair of damaged DNA in cancer cells is unfavorable for treatment. Several studies have proposed a supporting role of TGF-β in DNA damage response (DDR):
In different human cell lines including carcinoma cells, it was shown that TGF-β enhances non homologous end joining upon radiation-induced DNA damage and protects the cells from apoptosis. Evidence is provided that this is in part due to upregulation of DNA ligase IV [43]. In the context of HNSCC, it was postulated that HPV positive cells are more radiosensitive due to abrogation of TGF-β signaling. It was shown that TGF-β inhibition via pharmaceutical blockade or HPV abrogation decreased homologous recombination (HR). Furthermore, inhibition of TGF-β shifted DSB repair from HR to error prone alternative end joining. Moreover, this study showed that TGF-β can suppress proteins that are required for ATM kinase activity, which is essential in HR repair. TGF-β suppressed miR-182 which inhibits the HR proteins BRCA1 and FOXO3. These findings suggest that abrogated TGF-β signaling compromises DNA repair [78]. Furthermore, also abrogation of downstream effectors of the TGF-β signaling pathway can compromise DNA repair. SMAD4 deletion in mouse oral mucosa was accompanied by a significant decrease of HR-related proteins. For example, the mRNA expression of BRCA1 was decreased by 74% and Rad51 was decreased by 93% [59]. Furthermore, in several cancer cell lines, suppression of TGF-β reduced phosphorylation of DDR proteins, H2AX, ATM and p53 [95][96].
All these studies suggest that disturbance of TGF-β signaling also disturbs the DDR. Therefore, TGF-β might contribute to the emergence of radioresistance by supporting DNA repair upon radiation induced DNA damage.
23 As a result of the various pro-tumorigenic effects of TGF-β, it provides many attractive targets for cancer therapy. Nevertheless, the paradoxical roles in tumor suppression and promotion demand careful consideration in the development of cancer therapies targeting TGF-β signaling. Therefore, therapeutics should aim to reduce the excessive TGF-β pathway activation in cancer and stromal cells. Furthermore, the option of combining TGF-β targeted therapeutics with other therapeutic modalities (chemotherapy, radiotherapy, immune therapy) must be considered [97].
Treatments targeting the TGF-β pathway can act on different levels: at (1) the ligand level, at (2) the receptor level, or (3) the intracellular level. There are several therapeutic agents that are currently investigated in preclinical and clinical stages, although only few are aimed at HNSCC [98].
An example for (1) would be fresolimumab. Fresolimumab is a human monoclonal antibody against TGF-β1 and TGF-β2, which was tested in a phase I trial in advanced melanoma and renal cell carcinoma patients and in pretreated breast cancer patients. In melanoma and renal cell carcinoma patients, fresolimumab failed due to unexpected development of reversible cutaneous lesions. In combination with radiation, fresolimumab treatment exhibited lesser side effects but low clinical efficacy in breast cancer patients [97]. Its potential has also been tested in a xenograft mouse model of HNSCC. In combination with cetuximab, treatment with fresolimumab resulted in tumor regression [99].
Another example for (1) is Bintrafusp alfa. This bifunctional drug however does not only sequester TGF-β. It targets PD-L1 and sequesters TGF-β simultaneously. It is currently under investigation in phase I, II and III trials for different solid tumors. A dose escalation study of patients with squamous cell carcinoma of the cervix, anus or head and neck revealed promising outcomes. Remarkably, 56% showed disease reduction, 4 patients showed durable partial response and one patient with cervical cancer showed complete response [100]. Another phase I study with partly pretreated HNSCC patients showed that in HPV positive patients, 50% exhibited partial
1.4 TGF-β: Therapeutic targeting and current therapies
24 response. The overall response rate was 22%, whereby PD-L1 expression was not predictive [101]. Further studies evaluating Bintrafusp as treatment for HNSCC are still ongoing (NCT04220775, NCT04428047).
An example for (2) would be galunisertib. Galunisertib is a small molecule TβRI inhibitor which is currently investigated in clinical trials for the treatment of hepatocellular carcinoma, carcinosarcoma, colorectal cancer and others.
Its effect has also been evaluated in a phase II clinical trial for the treatment of pancreatic cancer. In combination with the cytostatic gemcitabine, galunisertib treatment led to improved survival. However, cardiac toxicity is a serious concern in the long-term use of the drug and careful dosing strategies had to be identified [98][102]. In general, the results in clinical trials are modest and could not meet the expectations from preclinical models [102].
An example for (3) would be peptide aptamers which bind to Smad proteins and thereby block their function. These experimental designer proteins are still at the preclinical stage. However, it was shown that peptide aptamers can reduce TGF-β expression and inhibit EMT in murine epithelial cells [103].
1.4.1 TGF-β inhibitors used in this study
Pirfenidone
Pirfenidone (PFD) is a worldwide approved, orally administered anti- inflammatory and antifibrotic drug, that has been developed for the treatment of idiopathic pulmonary fibrosis (IPF). IPF is an interstitial lung disease that is characterized by exaggerated fibroblast and myofibroblast activity, leading to increased ECM deposition and scarring. TGF-β plays an important role as profibrotic mediator in the pathogenesis of the disease [104]. Although the exact mechanism of action is unknown, PFD was shown to inhibit the production of TGF-β by affecting TGF-β 2 mRNA expression and processing of TGF-β [105].
25 The effect of PFD was already studied in many different cancers. PFD was shown to inhibit the motility of non-small cell lung carcinoma (NSCLC) cells and revert EMT transition in adenocarcinoma cells [106][107]. Furthermore, PFD suppressed growth of prostate cancer cells, pancreatic cancer cells, mesothelioma cells, hepatocellular carcinoma cells and breast cancer CAFs [108][109][110][111].
PFD is currently under investigation in a phase I clinical trial for the treatment of advanced non-small cell lung cancer in combination with standard chemotherapy (NCT03177291).
Vactosertib (TEW-7197)
Vactosertib is an orally bioavailable small molecule TβRI inhibitor. It binds to the adenosine triphosphate binding site of TβRI, thereby inhibiting its activity and downstream signaling [112]. The anticancer activity of vactosertib was already demonstrated in preclinical models, pharmacokinetics have been determined in a phase 1 study and other clinical studies are currently ongoing.
In vitro, vactosertib attenuated growth and viability and inhibited TGF-β induced activation of SMAD 2/3 in human and murine multiple myeloma (MM) cells [113][113]. In vivo, vactosertib prolonged survival and prevented weight loss in mice bearing MM. In mouse models of breast cancer, vactosertib inhibited Smad/TGF-β signaling and migration, invasion, lung metastasis and EMT [114]. In a mouse melanoma model, vactosertib blocked R-Smad phosphorylation and induced degradation of Smad4, which suppressed cancer progression. Furthermore, anti-melanoma cytotoxic T- lymphocyte response was enhanced by TGFBR1 inhibition [115].
Pharmacokinetic properties have already been evaluated following a dose- escalation approach. The first-in-human phase 1 study revealed that upon oral administration of 30-340 mg daily with 2 days off, vactosertib is rapidly absorbed and eliminated in patients with advanced solid tumors [112].
Vactosertib was safe and well tolerated and the most common adverse effect was fatigue. Patients receiving 140mg or more achieved stable disease and a
26 The-cancer-genome-atlas (TCGA) analysis of stromal fibroblasts revealed higher TGF-β responsive signatures than those with disease progression [116]. Furthermore, the effect of vactosertib in combination with an immunomodulatory drug on patients with MM is currently investigated in an ongoing phase I clinical trial. An update showed that, MM patients receiving 60-120 mg daily and did not develop any dose limiting toxicity or needed vactosertib dose reduction. Through 6 months of therapy, patients showed clinical response and no progression of disease [113](NCT03143985).
Based on these results, which indicated its potential as anticancer therapy, vactosertib is currently investigated in various phase II trials for the treatment of different cancers (NCT04064190,NCT04515979,NCT03724851, NCT04656002).
HNSCC is an aggressive, life-threatening disease, which is associated with severe morbidity and reduction in quality of life. Its clinical outcome is challenged by the emergence of radioresistance, which causes treatment failure and recurrence.Therefore, the need for new and improved treatment strategies is significant for HNSCCs. Due to its tumor promoting effects in advanced tumors, TGF-β has been identified as promising target in the tackling of the disease. Thus, the aim of this study was to assess the anti- neoplastic activity of the two TGF-β inhibitors pirfenidone and vactosertib on HNSCC cells. To assess radiosensitizing effects, treatment was combined with radiation. Furthermore, the effect of the more promising treatment option vactosertib was investigated in context with the the tumor microenvironment using patient derived CAFs.
1.5 Aim of this study
27
2 MATERIALS AND METHODS
Human tongue squamous carcinoma cell lines Cal27 and SCC25, human hypopharyngeal squamous carcinoma cell line FaDu and human foreskin fibroblasts were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA.). HPV positive human tongue squamous carcinoma cell line SCC154 was purchased from the German Collection of Microorganisms and Cell Cultures (Leibniz, Germany). Cells were cultured in standard petri dishes in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher) supplemented with 100 U/mL Penicillin, 100 µg/mL Streptomycin (Thermo Fisher) and 10% fetal bovine serum (FBS; Thermo Fisher), henceforth referred to as culture medium. Cal27, FaDu, SCC25 and fibroblasts were harvested at a confluence of 70-90% while SCC154 were harvested at a confluence of 40-70%, since this cell line does not become confluent. Cells were subcultured twice a week via trypsinization. Cells were washed with PBS and 3 mL of 0.05% Trypsin - 0,53 mM EDTA solution (Sigma-Aldrich, USA) was added. Cells detached after 5-14 minutes and 10 mL culture medium was added to stop trypsinization. Subsequently, cells were collected by centrifugation at 1200 rpm for 5 minutes and resuspended in 1 mL culture medium. To determine cell number for seeding of experiments and subculturing, cells were mixed 1:1 with 0.4% Trypan Blue Solution (Sigma- Aldrich, USA) and counted using the Countess FL automated cell counter (Thermo Fisher). Cells were maintained in a humid atmosphere at 37°C with 5% CO2. For experiments, HNSCC cells and fibroblasts below passage 30 were used.
Fresh human tumor samples were obtained from patients diagnosed with squamous cell carcinoma of the oropharynx undergoing primary surgical treatment at the Vienna General Hospital. Inclusion criteria were (1) Age 18-
2.1 Cell culture
2.2 Isolation of HNSCC Cancer Associated Fibroblasts
28 80, (2) Willingness to participate in the study, (3) Histopathological diagnosis of squamous cell carcinoma of the oropharynx, (4) Primary surgical treatment and (5) Tumor UICC stage II or higher. The research protocol was approved by the Ethics Committee of the Medical University of Vienna and Vienna General Hospital (EK Nr.: 2313/2019). A small piece of ~125 mm3 was removed from the excised tumor and subsequently transported to the Laboratory in DMEM containing 1000 U/mL Penicillin, 1000 µg/mL Streptomycin (Thermo Fisher) and 2.5 µg/mL Amphotericin B (Sigma-Aldrich, USA). Under sterile conditions, the specimen was washed twice with 10 mL PBS and subsequently transferred to a Petri Dish. During processing, the tissue was always covered with a small amount of liquid to prevent drying.
Blood coagulates were removed and tissue was minced into ~1-3 mm3 pieces using sterile tweezers and a scalpel. The small pieces were transferred to a dry Petri Dish, covered with 1-2 drops of TrypLE Express Enzyme (Thermo Fisher) and incubated on 37°C for 15 minutes. 5-7 pieces of the partly digested pieces were then transferred to each well of a 6 well-plate (one well was left free to fill with PBS later) and allowed to attach to the plastic for 15 minutes in minimal liquid on room temperature. This step was critical, since tissue needed to stay attached to the dish, so fibroblasts could grow out and adhere to the plastic. To prevent contamination, the tissue was kept in medium containing additional antibiotics and fungicide for the first days (=primary culture medium). Therefore, one drop of primary culture medium (culture medium + 100 µg/mL Gentamycin (Thermo Fisher) and 0.25 µg/mL Amphotericin B (Sigma-Aldrich, USA)) was pipetted on top of each piece. One well was filled with PBS to prevent drying of the tissue in the other wells. On the next day, 1 mL of primary culture medium was carefully added to each well, whereby the majority of pieces stayed attached to the bottom. After 2 days, primary culture medium was replaced by normal culture medium and henceforth changed two times a week. Within the first 14 days, CAFs started to grow out of the tumor pieces and were further cultivated until around 80%
of the well was covered. Although mainly fibroblasts grew out of the tissue pieces, sometimes other cell types grew around the tumor as well. They were later separated by differential trypsinization. For subculturing, tumor pieces
29 were left attached, wells were washed with PBS, and 0.3 mL of 0.05%
Trypsin-0,53 mM EDTA solution was added. After 5-20 minutes incubation at 37°C, fibroblasts started to detach, whereas the majority of other cells remained attached. 1 mL of normal culture medium was added and multiple wells were combined for centrifugation at 1200 rpm for 5 minutes. The pellet was resuspended in culture medium and plated in a petri dish. As cells were further subcultured, no other cell types were observed under the microscope and fibroblasts formed a morphologically homogenous culture. The remaining tumor pieces were further kept in 6 well plates to continuously harvest fibroblasts, which kept growing out. Due to their finite nature, CAFs were subcultured for no longer than 10 passages and used until passage 9 for experiments.
2.2.1 Characterization of CAFs
To verify the CAF population, Immunocytochemical staining for specific CAF markers, alpha smooth muscle actin (α-SMA) and fibroblast activation protein (FAP), was performed. Absence of epithelial cells was verified by negative Pan-Cytokeratin staining.
Coverslips were placed into a 24 well plate, sterilized by UV light, and CAFs were seeded at a density of 2-4 x 104 cells/well. When cells reached approximately 70% confluency, culture medium was removed and cells were washed twice with PBS. To fix the cells, 400 µL Zinc Formal-Fixx solution (Thermo Fisher) was added and incubated for 30 minutes at room temperature. After washing twice with PBS, coverslips were covered with 400 µL wash buffer (0.1% BSA (Sigma-Aldrich, USA) in PBS) and stored at 4°C.
For fluorescent staining, cells were washed twice with 400 µL wash buffer and afterwards, 400 µL blocking buffer (10% normal goat serum (Thermo Fisher), 0.3% Triton X-100 (Sigma-Aldrich, USA)) was added and incubated for 45 minutes at room temperature. Subsequently, blocking buffer was removed and 400 µL of primary antibodies, which were prepared in dilution buffer (PBS, 1% BSA, 1% normal goat serum, 0.3% Triton X-100, 0.01% sodium azide), were added. Cells were immunostained with antibodies against α-SMA
