Mechanisms of tumour resistance against chemotherapeutic agents in veterinary oncology

Accepted Manuscript Title: Mechanisms of tumor resistance against chemotherapeutic agents in veterinary oncology Author: R. Klopfleisch, B. Kohn, A.D. Gruber PII: DOI: Reference:

S1090-0233(15)00272-5 http://dx.doi.org/doi:10.1016/j.tvjl.2015.06.015 YTVJL 4547

To appear in:

The Veterinary Journal

Accepted date:

30-6-2015

Please cite this article as: R. Klopfleisch, B. Kohn, A.D. Gruber, Mechanisms of tumor resistance against chemotherapeutic agents in veterinary oncology, The Veterinary Journal (2015), http://dx.doi.org/doi:10.1016/j.tvjl.2015.06.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Review

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Mechanisms of tumor resistance against chemotherapeutic agents in veterinary oncology

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R. Klopfleisch a, B. Kohn b, A. D. Gruber a

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a

Institute of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Straße 15, 14163 Berlin, Germany b Small Animal Clinic, Freie Universität Berlin, Oertzenweg 19 b, 14163 Berlin, Germany * Corresponding author. Tel.: +49 30 83862460 E-mail address: [email protected] (R. Klopfleisch).

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Highlights     

Chemotherapy is a major treatment modality in veterinary oncology Efficacy of chemotherapy is often impacted by intrinsic/acquired drug resistance Chemotherapy resistance can be due to multiple mechanisms Molecular data generated in humans can be translatable to pets New therapies should account for mechanism of drug resistance Abstract

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Several classes of chemotherapy drugs are used as first line or adjuvant treatment of the

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majority of tumour types in veterinary oncology. However, some types of tumour are

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intrinsically resistant to several anti-cancer drugs and others, while initially sensitive, acquire

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resistance during treatment. Chemotherapy often significantly prolongs survival or disease

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free interval, but is not curative. The exact mechanisms behind intrinsic and acquired

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chemotherapy resistance are unknown for most animal tumours but there is increasing

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knowledge on the mechanisms of drug resistance in humans and a few reports on molecular

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changes in resistant canine tumours have emerged. In addition, approaches to overcome or

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prevent chemotherapy resistance are becoming available in humans and, given the overlaps in

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molecular alterations between human and animal tumours, these may also be relevant in

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veterinary oncology.

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This review provides an overview of the current state of research on general

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chemotherapy resistance mechanisms, including drug efflux, DNA repair, apoptosis evasion

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and tumour stem cells. The known resistance mechanisms in animal tumours and the potential

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of these findings for improving treatment efficacy in veterinary oncology are also explored.

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Keywords: Oncology; Chemotherapy; Resistance; Epigenetics; DNA repair; Cancer stem

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cells; Epithelial mesenchymal transition; Apoptosis

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Introduction

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Chemotherapy is commonly used to treat cancer in pets, and for some tumour types, such

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as haematopoietic tumours, chemotherapy is the treatment of choice. Complete remission and

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disease stability can often be achieved with currently available drugs and protocols. However,

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in a significant percentage of animals, standard protocols are ineffective due to intrinsic

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resistance of the tumour against available agents. Furthermore, tumour relapse during or after

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treatment is observed, commonly or sporadically, due to the development of acquired

47

resistance. The mechanisms of resistance and the therapeutic strategies employed to overcome

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this resistance are important and topical areas for focus in cancer research.

49 50

Chemotherapy research in veterinary oncology is constantly progressing, but many

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questions on the specific mechanisms of chemoresistance in pets are still unclear. In contrast,

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numerous studies have been published on the mechanisms of resistance against common anti-

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cancer drugs in humans and animal models. These studies have shown that the mechanisms of

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chemoresistance mainly consist of: (1) increased cellular efflux of chemotherapeutic drugs;

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(2) accelerated drug inactivation or lack of drug activation; (3) changes in drug targets (e.g.

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mutation and methylation); (4) efficient mechanisms of DNA repair; (5) deregulation of

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apoptosis, and (6) cancer stem cells as the nucleus of resistance in tumours and epithelial

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mesenchymal transition (EMT) (Table 1).

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This article provides an overview of the current knowledge of chemotherapeutic

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resistance and explores the mechanisms that may be relevant and so targeted in pet animals

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with chemotherapy-resistant tumours.

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Increased efflux of chemotherapeutic agents from tumour cells

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Efflux of drugs from cells is mostly based on membrane transporter proteins. Of these,

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the ATP-binding cassette (ABC) transporter family is considered to be the most important

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group of transmembrane proteins which drive the transport of many chemically unrelated

68

drugs across the plasma membrane (Holohan et al., 2013). Of the 49 (or more) members of

69

this family, the multi-drug resistance protein 1 (MDR1, P-glycoprotein [PGP], ABCB1), the

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MDR-associated protein 1 (MRP1, ABCC1) and the breast cancer resistance protein (BCRP,

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ABCG2) have been most thoroughly investigated in tumours of not only humans but also of

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dogs and cats (Fig. 1) (Brenn et al., 2008; Choi and Yu, 2014; Gramer et al., 2013; Honscha et

73

al., 2009; Pawlowski et al., 2013). All three of these proteins, and other members of the

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family, are capable of eliminating lipids and therefore various classes of hydrophobic

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chemotherapy drugs,

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compounds and microtubule inhibitors, as well as the tyrosine kinase inhibitors (TKIs)

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(Holohan et al., 2013).

including

topoisomerase

inhibitors,

antimetabolites,

platinum

78 79

MDR1 and resistance

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MDR1 is a membrane-bound, ATP-dependent efflux pump that is overexpressed in many

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human tumours before the start of any chemotherapy and thus contributes to intrinsic

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resistance (Goldstein et al., 1989). MDR1 expression is increased or even induced during

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chemotherapy in various human tumours and contributes to resistance against platinum-

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containing compounds, topoisomerase II inhibitors, microtubulin poisons and several TKIs

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(Table 1) (Holohan et al., 2013).

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ABC transporters in animal tumours 4 Page 4 of 36

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MDR1, MRP1 and BCRP expression has been analyzed in several canine tumours. In

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most studies the analysis was restricted to the presence of protein or mRNA, to its correlation

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with the efficacy and outcome of the chemotherapy protocol used, or to general clinical

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parameters in mammary tumours and lymphomas of dogs (Dhaliwal et al., 2013; Gramer et

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al., 2013; Tomiyasu et al., 2014a; Zandvliet et al., 2014b).

93 94

In a few studies, the desensitizing effects of MDR1, BCRP, MRP1 activities has been

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directly confirmed in canine mammary tumour or lymphoma cells (Table 2). For instance,

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Pawlowski et al. (2013) were able to identify the drug specificity for different ABC

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transporters in a canine mammary tumour cell line. By siRNA-mediated gene silencing, they

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showed that: (1) vinblastine efflux was mediated by MDR1 and MRP1; (2) cisplatin efflux

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was mediated by MDR1, BCRP, MRP1, and (3) cyclophosphamide resistance was mediated

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by BCRP. In addition, doxorubicin and vincristine resistance in canine lymphoma cell lines

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was completely reversed by an MDR1 inhibitor (Zandvliet et al., 2014a).

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Only a few studies on the expression of MDR1 in feline tumour cells have been

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published. MDR1 is constitutively expressed in feline lymphomas, mast cell tumours, lung

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tumours and squamous cell carcinomas (Hifumi et al., 2010; Van der Heyden et al., 2011).

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Feline mammary gland tumours showed strong membranous MDR1 labelling, especially in

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areas with infiltrative growth and in atypical cells, although it was not correlated with the

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general grade of malignancy of the complete tumour (Van der Heyden et al., 2011). Similarly,

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MDR1 expression was not predictive of remission or survival time in cats with lymphoma

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(Brenn et al., 2008). Nevertheless, one study has confirmed the impact of MDR1 on

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adriamycin and vincristine resistance in feline lymphoma cells (Okai et al., 2000).

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ABC transporter modulators to overcome resistance

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The observation that ABC transporter activity in cancer cells contributes to anti-cancer

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drug resistance has led to intensive research on agents that either block or inactivate these

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transporters and so increase the intracellular concentration and effect of the anti-cancer drugs

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(Fig. 1). The first-generation ABC transporter modulators were the MDR1 inhibitors

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verapamil, cyclosporine A and quinine (Kathawala et al., 2015). In preclinical studies, these

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modulators showed promising effects on chemotherapy resistance but these could not be

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confirmed in clinical trials. The second-generation ABC transporter modulators were

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designed to increase the effectiveness and decrease toxic side effects. Valspodar, a

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cyclosporine analogue, and biricodar indeed increased the efficacy of several classical

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chemotherapy drugs and had decreased toxicity preclinically, but again lacked sufficient

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efficacy in clinical trials (Nobili et al., 2012).

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The third-generation ABC transporter modulators (elacridar, laniquidar, zosuquidar and

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tariquidar) were specifically designed to overcome the problems faced by the first- and

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second-generation modulators. They inhibit MDR1, BCRP and MRP1 function at nM

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concentrations and are less toxic. In clinical trials, they too failed to affect clinical outcome

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significantly, which led to the assumption that MDR1 may have less impact on drug

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resistance or that the functional redundancy between ABC transporters impedes anti-cancer

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drug efflux inhibition (Holohan et al., 2013).

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The effectiveness of verapamil and cyclosporine A to block the ABC transporter and to

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overcome chemotherapy resistance has also been analyzed in canine tumour cells. For canine 6 Page 6 of 36

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mammary tumours, Krol et al. (2014) showed that verapamil and cyclosporine A were unable

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to reverse resistance against vinblastine in MDR1-expressing canine mammary cancer cells.

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However, verapamil blocked MDR1 efflux in dermal, but not oral or gastrointestinal mast cell

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tumour cells (Nakaichi et al., 2007).

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Tyrosine kinase inhibitors to overcome resistance

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TKIs, including masitinib and the alkylophospholipid perifosine, appear to be promising

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ABC-transporter inhibitor alternatives. They are capable of reversing ABC transporter-

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mediated chemotherapy resistance via inhibition of several growth factor signalling pathways.

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TKI efficacy has been confirmed in canine lymphomas in which masitinib reversed

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doxorubicin resistance by inhibiting MDR1 (Zandvliet et al., 2013). Likewise, perifosine, an

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AKT and PI3K inhibitor, has been shown to reverse vincristine in canine lymphoid tumour

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cells (Tomiyasu et al., 2014b).

149 150

Taken together, numerous modulators of ABC transporters are available and have been

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tested successfully in vivo, mostly in humans. It will be interesting to test these modulators in

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canine and feline tumours to evaluate their potential for overcoming chemotherapy resistance

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in these species.

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Drug inactivation in tumour cells

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All phases of metabolism of a chemotherapeutic drug in the body (i.e. absorption,

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distribution, metabolism and excretion) can influence its efficacy on tumour growth. In terms

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of chemotherapy resistance, recent research has however been mainly focused on intracellular 7 Page 7 of 36

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drug activation and inactivation in tumour cells. The most important enzymes for drug

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activation and inactivation include the cytochrome P450 (CYP) system, the glutathione-S-

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transferase (GST) superfamily and the uridine diphospho-glucuronosyltransferase (UGT)

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superfamily (Housman et al., 2014).

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Cytochrome P450 (CYP) system

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Several CYPs are important for drug resistance (Housman et al., 2014). Increased levels

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of CYP and CYPb5 have been detected in benign canine mammary tumours when compared

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to more malignant tumours (Kumaraguruparan et al., 2006). In addition, CYP3A12 was

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identified as a potent enzyme for vinblastine depletion in dogs in an in vitro model (Achanta

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and Maxwell, 2014).

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Glutathione-S-transferase (GST) superfamily

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GSTs and metallothionein are relevant for resistance to doxorubicin, alkylating agents

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and platinum drugs through direct detoxification and inhibition of the mitogen-activated

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protein kinase (MAPK) pathway (Karotki and Vasak, 2009; Meijer et al., 1992). Increased

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GST expression is also associated with increased risk of lymphoma development and

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resistance to a modified University of Wisconsin-Madison protocol in canine lymphoma

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expression (Ginn et al., 2014; Tomiyasu et al., 2010), while increased plasma GST

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concentrations have been found in dogs with relapsing lymphomas (Hahn et al., 1999).

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In canine urinary bladder transitional cell carcinoma, the expression level of GST did not

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correlate with survival time under cisplatin, doxorubicin and mitoxantrone treatment (Rocha

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et al., 2000). In vitro, GST-mediated resistance in canine osteosarcoma cells against cisplatin

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could be reversed by ethacrynic acid treatment (Shoieb et al., 1998).

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Platinum drug resistance can also occur through inactivation by metallothionein (Karotki

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and Vasak, 2009; Meijer et al., 1992). Expression of metallothionein has been demonstrated

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in canine and feline lung and melanocytic tumours, in canine cutaneous apocrine gland and in

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canine mammary tumours (Dincer et al., 2001; Erginsoy et al., 2006; Hifumi et al., 2010;

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Martano et al., 2012). The effect of metallothionein on platinum drug resistance has not been

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analyzed so far in cats and dogs.

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Uridine diphospho-glucuronosyltransferase (UGT) superfamily

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The UGTs catalyse glucuronidation and regulate the formation of inactive hydrophilic

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glucuronides of cytotoxic drugs. A widespread down-regulation of UGT1A1 transcription and

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microsomal activity occurs in some cancers partly by epigenetic regulation (Michael and

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Doherty, 2005). Studies on the relevance of UGT on canine or feline cancer and

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chemotherapy resistance are lacking.

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In summary, several studies indicate that effective detoxification is associated with

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chemotherapy resistance in canine tumours. Similar mechanisms are present in human

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tumours and have been targeted successfully using innovative approaches. A similar success

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in veterinary oncology can be expected in the mid-term range and should be explored in detail

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to increase the efficacy of some therapeutic interventions in pets.

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Changes in drug targets: Mutation and methylation

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Quantitative and qualitative changes of drug targets represent well-known mechanisms of

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chemotherapy resistance in human oncology. These changes can be quantitative (with

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decreased or lost expression of the target), or a compensation of the inhibited target by other

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proteins in the cells. Another response to the pressure of chemotherapeutic drugs is the

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selection of tumour cells expressing a qualitatively different, mutated version of the drug

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target, with, for instance, structural changes at the regular binding site of the drug. Research

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on chemotherapy resistance due to changes in human drug targets is extensive and first hints

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towards changes in drug targets in canine lymphomas and mast cell tumours have emerged.

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Mutation and tyrosine kinase inhibitors

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Loss of sensitivity of tumours towards the effects of TKIs is currently the only example

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of genetic (e.g. mutational) adaption of tumours due to selective pressure by chemotherapy in

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veterinary oncology. TKIs such as toceranib and masitinib are commonly used and are

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initially very effective anti-cancer drugs for canine mast cell tumours. They target several

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tyrosine kinases but are thought to mainly act through inhibition of the KIT receptor in mast

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cell tumour cells (London, 2013). One of the major drawbacks of these two TKIs, besides

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their toxicity, is the large fraction of dogs with relapsing TKI-resistant tumours under or after

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treatment (London, 2013).

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In vitro studies indicate that either overexpression or de novo expression of alternative

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proliferative pathways under TKI treatment, such as T- and B-cell receptor signalling, ERK

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signalling and overexpression of KIT, may contribute to TKI resistance (Klopfleisch et al.,

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2012; Kobayashi et al., 2015). However, two studies identified additional KIT mutations in 10 Page 10 of 36

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imatinib- and toceranib-resistant canine mast cell tumour cell lines, (Halsey et al., 2014;

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Kobayashi et al., 2015).

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Epigenetics and resistance

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Besides genetic alterations, epigenetic changes may also contribute to chemotherapy

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resistance (Brown et al., 2014). Epigenetic changes are defined as changes in gene expression

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that are independent of changes in DNA sequence and that persist over many cell divisions

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(Wilting and Dannenberg, 2012). These changes mainly consist of covalent modifications of

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DNA and histones which modulate gene expression levels directly by promotor methylation

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or indirectly by chromatin packaging, thereby regulating the accessibility of DNA to

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sequence-specific transcription factors (van Steensel, 2011). DNA is predominantly

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methylated at cytosines by DNA methyltransferases (DNMTs) in the CPG islands of gene

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promotor regions. Initially, the hypothesis that epigenetics may be of relevance for

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chemotherapy resistance was based on the observation that acquisition of resistance often has

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a rapid kinetic, is reversible and lacks genetic mutations (Brown et al., 2014).

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Epigenetic states favouring chemotherapy resistance have been associated with aberrant

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transcription of efflux transporters, DNA repair enzymes and apoptotic factors in human

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tumours (Wilting and Dannenberg, 2012). Only a few studies are available on methylation-

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associated changes in canine tumours and these mainly focus on the methylation status of the

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promotor regions of the tumour suppressor p16 and ABC transporters.

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Epigenetic p16 inactivation seems to be a rare in canine T cell lymphoma and affects the

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prognosis of dogs with high-grade lymphoma (Fosmire et al., 2007; Fujiwara-Igarashi et al., 11 Page 11 of 36

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2014a,b). DNA methylation and histone H3 acetylation are involved in ABCB1 gene

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expression and associated with a multi-drug resistant (MDR) phenotype in most canine

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lymphoid tumour cell lines (Tomiyasu et al., 2014a). Other mechanisms including JNK,

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MAPK/ERK and PKC signalling seem to be more relevant for ABC transporter expression

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levels in canine lymphomas (Tomiyasu et al., 2014b, d).

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Several drugs that can reverse epigenetic changes in tumours have been identified. Of

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these, DNMT inhibitors (DNMTis) and histone deacetylase (HDAC) inhibitors (HDACis)

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have been most intensively studied in vitro and in clinical trials in humans (Nebbioso et al.,

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2012). Common HDACis are hydroxamic acids (trichostatin A), cyclic tetrapeptides (trapoxin

263

A and B, romidepsin), short chain and aromatic fatty acids (butyrate, valproic acid) and

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benzamides (entinostat, mecetinostat), most of which are currently being tested in in humans

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(Nebbioso et al., 2012). DNMTis include nucleoside analogues (azacytidine, decitabine,

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zebularine), which covalently bind DNMT, and small molecule inhibitors (hydralazine,

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procainamide), which competitively bind to CPG-rich DNA regions (Nebbioso et al., 2012).

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The potential of these epigenetic drugs has been shown in clinical trials for several

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human tumours as both monotherapy or combined with other anti-cancer drugs (Nebbioso et

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al., 2012). A major drawback of the drugs is their lack of specificity for specific DNA regions

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of interest, which leads to unwanted demethylation of unrelated or unwanted genes including

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drug resistance genes (Glasspool et al., 2014).

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Epigenetics in canine cancer chemotherapy resistance

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Pilot studies indicate that epigenetic drugs may also be beneficial in dogs. Smallwood et

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al. (2014) showed that L-asparaginase sensitivity is strongly and negatively correlated with

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the level of methylation of the asparagine synthetase (ASNS) promoter in canine lymphoma

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cells. Furthermore, treatment with hypomethylating azacytidine increased resistance to L-

280

asparaginase. However, ASNS methylation and expression was not predictive for overall

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survival (OS) or progression-free survival (PFS) in dogs with lymphoma treated with L-

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asparaginase, indicating that resistance mechanisms in vivo may be more complex

283

(Smallwood et al., 2014).

284 285

The efficacy of azacytidine and trichostatin A on chemotherapy reversal has also been

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shown for MDR1-based vincristine resistance in canine lymphoma cells (Tomiyasu et al.,

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2014c) and in dogs with invasive urothelial carcinoma treated with azacitidine (Hahn et al.,

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2012). Finally, hydroxamic acid and zebularine treatment of canine osteosarcoma cells

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restored microRNA expression at the 14q32 locus and stabilized expression of cMYC,

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suggesting a potential benefit as an adjuvant treatment of rapidly progressive osteosarcomas

291

(Thayanithy et al., 2012)

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Increased DNA damage repair

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Inefficient DNA damage repair systems are a major mechanism of carcinogenesis. For

295

instance, dysfunctional p53 activity or inherited mutations in the BRCA1 or -2 genes, both

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important regulators of genomic integrity, are important risk factors for the development of

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several cancer types in humans and are also thought to be relevant for canine and feline

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tumours (Grosse et al., 2014).

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DNA damage response and resistance

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A more differentiated view on the desirability of an intact DNA damage response (DDR)

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is however necessary in terms of chemotherapy resistance (Fig. 2). Direct or indirect

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induction of DNA damage is the main mechanism of action for platinum drugs, alkylating

304

agents and topoisomerase inhibitors (Holohan et al., 2013). The favoured result of this

305

treatment is tumour cell death due to overwhelming DNA damage. This requires either direct

306

breakdown of cellular metabolism due to massive DNA damage or apoptosis induction via

307

p53 and p21. Loss of function mutations of p53 therefore leads to insensitivity of the tumour

308

against induced DNA-damage, since the DNA damage signal is not submitted to the intrinsic

309

apoptosis pathway to induce apoptosis (Enoch and Norbury, 1995). Highly effective DDR is

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nevertheless also a cause of resistance because it allows tumour cells to repair and survive

311

induced DNA damage (Bouwman and Jonkers, 2012).

312 313

Inhibition of DNA damage response

314

Inhibition of DDR together with the administration of DNA damaging agents is therefore

315

an accepted, albeit somewhat counterintuitive, therapeutic strategy. This therapeutic approach

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takes advantage of the fact that although DNA damage may drive carcinogenesis, tumour

317

cells nevertheless require some level of genetic stability to perform basic metabolism.

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Fortunately, tumour cells often depend on only one remaining DDR pathway, while in normal

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cells all six DDR pathways (i.e. mismatch repair [MMR], nucleotide excision repair [NER],

320

base excision repair [BER], homologous recombination [HR], inter-strand cross-link repair

321

[ICL] and the non-homologous end-joining or NHEJ) redundantly provide optimal DDR.

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Inhibition of the remaining pathway in tumour cells thus markedly increases the sensitivity of

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tumour cells against DNA-damaging agents (Bouwman and Jonkers, 2012).

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The most prominent examples of DDR targeting are inhibitors of the single strand-break

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DNA repair enzyme poly-ADP-ribose polymerase (PARP) such as olaparib. PARP inhibitors

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cause lethality in tumour cells with mutations in the BRCA1 and BRCA2 genes. Both genes

328

are essential for effective homologous recombination DNA repair. Loss of homologous

329

recombination is normally compensated by single strand repair mechanisms, which in turn are

330

dependent on PARP function. Blocking PARP leads to cell death due to overwhelming

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genetic instability in rapidly dividing tumour cells (Farmer et al., 2005). Ironically, treatment

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of BRCA2-mutated breast cancer with PARP inhibitors may also lead to resistance to PARP

333

inhibitors due to BRCA2 mutations, which restore the function of the protein and thus the

334

primary genetic starting point of the tumour (Edwards et al., 2008).

335 336

Data on the influence of PARP and its inhibition in pet cancers are not available.

337

However, several studies have focused on BRCA expression and sequence mainly in canine

338

mammary tumours. Several BRCA1 and BRCA2 single nucleotide polymorphisms (SNPs)

339

have been detected and proposed to be relevant for canine mammary tumour development,

340

but the actual biological relevance for tumour development (as in hereditary human breast

341

cancer) has not been proven (Klopfleisch et al., 2011b). BRCA1 expression levels however

342

correlate with malignancy for canine mammary tumours, but not with chemotherapeutic

343

outcome (Klopfleisch and Gruber, 2009; Klopfleisch et al., 2010; Nieto et al., 2003).

344 345

A defective activation of the p53 DNA damage signalling has been described as the cause

346

of doxorubicin, mitoxantrone and vincristine resistance in feline mammary carcinoma stem

347

cells (Pang et al., 2013) and DNA damage repair deficiency may be responsible for

348

lymphoma development in Golden Retrievers (Thamm et al., 2013).

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Mismatch repair system and resistance

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Treatment of mismatch repair (MMR) system-deficient tumours with methotrexate is

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another promising approach currently tested in humans. MMR genes including MutL homolog

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1 (MLH1) and MutS protein homolog 2 and 6 (MSH2, MSH6) correct DNA damage caused by

354

DNA polymerase errors. Deficiency of these genes is associated with resistance to DNA

355

damaging agents like cisplatin and carboplatin, which can be reversed with methotrexate

356

(Martin et al., 2009). MLH1, MSH2, and MSH6 expression is strong in canine mast cell

357

tumours and feline intestinal lymphoma (Aberdein et al., 2012; Munday et al., 2009). Of note,

358

the effect of methotrexate as rescue drug has not been evaluated in pets, but may be

359

unknowingly used since it is part of an established multi-agent chemotherapeutic protocol for

360

canine lymphoma (Simon et al., 2008).

361 362

Taken together, although several similarities with human tumours in terms of protein

363

expression exist, only few studies using canine tumour samples have addressed the

364

importance of altered DDR pathways in tumour progression and none in chemotherapy

365

resistance, thus leaving many questions to be addressed in the future (Grosse et al., 2014;

366

Klopfleisch et al., 2011a; Klose et al., 2011).

367 368

Apoptosis deregulation

369

If a chemotherapeutic agent has reached sufficient concentration in the tumour cell and

370

induced DNA damage, the success of treatment depends on the downstream reaction of the

371

cells. This reaction is expected to be cell death, usually inflicted by energy-dependent

372

apoptosis rather than necrosis. One of the classic hallmarks of cancer cells is however 16 Page 16 of 36

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apoptosis deregulation, and chemotherapy may thus fail despite unchanged efflux and

374

sensitive drug targets in the tumour cell (Hanahan and Weinberg, 2000).

375 376

The accentuated anti-apoptotic status of tumour cells is often based on only few

377

dysregulated genes, which make the development of drugs targeting their gene products an

378

attractive goal. Bcl-2 and tumour necrosis factor (TNF)-related apoptosis-inducing ligand

379

(TRAIL) are the most intensely studied proteins of the numerous pro- and anti-apoptotic

380

proteins known (Holohan et al., 2013). Overexpression of the anti-apoptotic BCL-2 is able to

381

block the intrinsic mitochondria-associated pathway of apoptosis and induces resistance to

382

cytotoxic agents (Miyashita and Reed, 1992). Compounds like navitoclax, which antagonizes

383

pro-apoptotic Bcl-2 and promotes the pro-apoptotic BAX and BAK, have been developed to

384

shift tumour cells towards a more pro-apoptotic state when treated with cytotoxic agents

385

(Konopleva et al., 2006). Another clinically promising approach is recombinant TRAIL and

386

TRAIL receptor-activating antibodies to stimulate the extrinsic apoptosis pathway (Pavet et

387

al., 2011).

388 389

Apoptosis with chemotherapy in animals

390

The apoptosis-inducing effect has been confirmed for nearly all cytotoxic agents

391

currently used in veterinary oncology (Pawlak et al., 2014). In addition, the induction of

392

apoptosis is a standard read out for the evaluation of new anti-cancer compounds in in vitro

393

assays of canine and feline tumours (Pang et al., 2014). Available data on apoptosis

394

dysfunction as a basis of chemotherapy resistance in veterinary oncology are however sparse.

395

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396

One study on prognostic factors for radiotherapy success for canine lymphoma found

397

increased levels of the anti-apoptotic protein survivin before treatment as associated with an

398

unfavourable prognosis (Fu et al., 2014). Survivin was also identified as a negative prognostic

399

factor for early treatment failure of canine lymphoma treated with a CHOP-based protocol

400

(Rebhun et al., 2008). Furthermore, expression level of myeloid cell leucaemia sequence 1

401

(MCL1), a potent anti-apoptotic protein associated with drug resistance in various human

402

cancers, is increased when canine mast cells are exposed to specific inhibitors of mitogen-

403

activated protein kinase (MAPK) or Janus kinase-signalling pathways, but not when KIT is

404

inhibited. MCL1 expression may therefore contribute to mast cell tumour survival and drug

405

resistance at least in some chemotherapy protocols (Amagai et al., 2013).

406 407

Together these three studies indirectly indicate that an accentuated anti-apoptotic status

408

may also be an inherited or acquired mechanism of chemotherapy resistance in canine

409

tumours and that therapies involving direct targeting of canine tumours may be promising.

410 411

Cancer stem cells and epithelial mesenchymal transition (EMT)

412

Increasing evidence points towards cancer stem cells (CSC) to be of central relevance for

413

chemoresistance of tumours. CSC or tumour-initiating cells (TICs) resemble in many aspects

414

‘normal’ stem cells but are not necessarily derived from them. Normal stem cells have

415

multiple mechanisms to protect them from cytotoxic insults. These include highly active drug-

416

efflux pumps, increased detoxification enzyme levels, enhanced DNA repair efficacy,

417

apoptosis resistance and quiescence. CSCs also have all of these features, which make them

418

an area of focus for the understanding and treatment of chemoresistance (Fig. 3) (Colak and

419

Medema, 2014; Pang and Argyle, 2014). 18 Page 18 of 36

420 421

Cancer stem cell-specific therapy targets

422

The phenotypic features and growth pattern of CSCs are based on the activity of few

423

signalling pathways that regulate the balance between self-renewal and differentiation. The

424

Wnt, Notch and Hedgehog (HH) signalling pathways have been identified as essential for the

425

CSC phenotype of several solid and haematopoietic tumours (Dawood et al., 2014; Duncan et

426

al., 2005; Lai et al., 2003; Yoshimoto et al., 2011). In contrast, bone morphogenetic protein

427

(BMP) signalling inhibits stem cell expansion by suppression of Wnt signalling, induces CSC

428

differentiation, increases sensitivity to chemotherapy in vivo, and therefore seems an

429

promising therapeutic approach for CSC-based resistance (He et al., 2004; Lombardo et al.,

430

2011).

431 432

Targeting the Wnt, Notch and HHS pathways is an intensely studied approach to

433

overcome chemotherapy resistance due to CSCs (Fig. 3). Cyclopamine, an HHS pathway

434

inhibitor, has been shown to increase sensitivity against TKIs and to increase survival of mice

435

in a chronic myeloid leucaemia model (Qiu et al., 2013; Zhao et al., 2009). Furthermore,

436

inhibition of the Notch pathway with antibodies against its ligands and receptor or gamma-

437

secretase inhibitors has also been successfully applied to reduce the population of breast and

438

glioblastoma CSCs and to increase the sensitivity against of taxanes (Fan et al., 2010; Hoey et

439

al., 2009). Common Wnt signalling inhibitors are non-steroidal anti-inflammatory drugs

440

(NSAIDs), the beta-catenin antagonist ICG-001 and biological inhibitors, such as antibodies,

441

RNA interference, and recombinant proteins (Takahashi-Yanaga and Kahn, 2010). So far

442

those have not been tested in canine or feline tumours.

443 444

Cancer stem cells and epithelial-mesenchymal transition 19 Page 19 of 36

445

The interleukin 8 (IL-8) signalling and transforming growth factor‑β receptor (TGFβR)

446

pathways are stem cell-associated pathways (Liu et al., 2011). Recent evidence indicates that

447

IL-8 can induce a state of ‘stemness’ by effecting EMT, which is needed to acquire stem cell

448

characteristics at least for epithelial tumour cells (Fernando et al., 2011). EMT in general may

449

be one potential mechanism for explaining how CSCs develop from epithelial cells. During

450

EMT, epithelial cells develop a mesenchymal phenotype, which is characterized by a loss of

451

polarization and tight cell–cell junctions and a switch to fibroblast-like cell shape.

452

Chemotherapy is assumed to force EMT of tumour cells. For instance, therapy with and

453

resistance against EGFR inhibitors is tightly associated with EMT in vitro and in vivo (Byers

454

et al., 2013; Fuchs et al., 2008). Mutations in the downstream effectors of the receptor with

455

consecutive activation of TGFβR signalling were identified as potential molecular mediators

456

of both EMT and chemotherapy resistance (Huang et al., 2012).

457 458

Drugs inhibiting IL-8 signalling are thought to halt EMT and thus to prevent development

459

of stem cell characteristics by differentiated tumour cells (Fernando et al., 2011). For instance,

460

repertaxin, a non-competitive inhibitor of IL-8 signalling, reduces the number and activity of

461

cancer stem cells and increases the efficacy of docetaxel in breast cancer cells (Ginestier et

462

al., 2010) but has not been tested in veterinary oncology.

463 464

Stem cells are also characterized by a high DNA repair activity and resistance to

465

apoptosis induction by anti-cancer drugs and radiation (Baumann et al., 2008; Bertolini et al.,

466

2009; Colak et al., 2014). This efficient DNA repair may also be their Achilles heel and an

467

interesting approach to target CSCs. For example, inhibition of the activity DNA repair genes

468

with the cell cycle checkpoint inhibitor staurosporin drastically increases the sensitivity of

469

lung and glioblastoma CSCs towards irradiation and chemotherapy (Bao et al., 2006; Signore

470

et al., 2014). 20 Page 20 of 36

471 472

Immunotoxins directly targeting surface markers is another approach of directly targeting

473

CSCs (Fig. 3). Antibodies against the stem cell marker CD133 conjugated to paclitaxel and

474

oncolytic CD133-specific measles viruses have been successfully tested in vitro and in mouse

475

models (Bach et al., 2013; Swaminathan et al., 2013). Finally, direct apoptosis induction via

476

caspase 9 activation efficiently kills colon CSCs, while inhibition of the anti-apoptotic

477

proteins BCL2, BCLXL and BCLW by small molecule inhibitors shifts the balance a more

478

pro-apoptotic state in tumour cells to overcome the anti-apoptotic state in dasatinib-resistant

479

chronic myeloic CSCs in vivo (Goff et al., 2013; Kemper et al., 2012).

480 481

One of the major challenges of targeting CSCs is their ability to switch into a state of

482

non-proliferative quiescence. Uncontrolled and rapid proliferation is a common feature of

483

tumour cells, but of only few non-neoplastic cells (Colak and Medema, 2014). Classic

484

chemotherapeutic agents were developed to target highly proliferative tumour cells but not

485

quiescent cells. Quiescent CSCs are thus believed to be a major cause for tumour relapse after

486

treatment with microtubule inhibitors or DNA damaging drugs.

487 488

Besides their lack of proliferation, quiescent CSCs also have an increased potential to

489

repair DNA damage induced by DNA-damaging agents or radiotherapy (Bao et al., 2006).

490

The rationale to target quiescent CSCs is to force them back into a proliferative state and to

491

overcome their efficient DNA damage repair systems. Initial clinical trials have shown that

492

administration of granulocyte colony-stimulating factor (G-CSF) induces proliferation of

493

quiescent CSCs and reverses imatinib and cytarabine resistance in acute myeloid leucaemia

494

(Fig. 3) (Lowenberg et al., 2003; Pabst et al., 2012).

495

21 Page 21 of 36

496

Research on CSCs is still a mostly uncharted territory in veterinary oncology. Moreover,

497

many questions remain on the characteristics of these cells in non-human species. The

498

relevance of these cells for chemotherapy resistance, however, is undoubtedly an important

499

area to address in order to increase success of chemotherapies in veterinary oncology.

500 501

Conclusions

502

There is significant progress in the understanding of the general mechanisms of

503

chemotherapy resistance of tumours. Strategies and drugs to overcome or prevent these

504

mechanisms are already available or in development and should be of use in veterinary

505

medicine. Increasing the efficacy of existing classic drugs remains of particular relevance in

506

veterinary medicine, since slow progress in elucidating the basic molecular mechanisms of

507

carcinogenesis of animal tumours is hampering the development of new tumour therapies.

508 509 510 511

Conflict of interest The authors of this paper have no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

512 513 514

Acknowledgment The review was supported by the Deutsche Forschungsgemeinschaft DFG KL2240-1.

515 516

References

517 518 519

Aberdein, D., Munday, J.S., Howe, L., French, A.F., Gibson, I.R., 2012. Widespread mismatch repair expression in feline small intestinal lymphomas. Journal of Comparative Pathology 147, 24-30.

520 521

Achanta, S., Maxwell, L.K., 2014. Reaction phenotyping of vinblastine metabolism in dogs. Veterinary and Comparative Oncology DOI: 10.1111/vco.12084

22 Page 22 of 36

522 523 524

Alagoz, M., Gilbert, D.C., El-Khamisy, S., Chalmers, A.J., 2012. DNA repair and resistance to topoisomerase I inhibitors: mechanisms, biomarkers and therapeutic targets. Current Medicinal Chemistry 19, 3874-3885.

525 526 527

Amagai, Y., Tanaka, A., Matsuda, A., Oida, K., Jung, K., Nishikawa, S., Jang, H., Ishizaka, S., Matsuda, H., 2013. Increased expression of the antiapoptotic protein MCL1 in canine mast cell tumors. The Journal of Veterinary Medical Science 75, 971-974.

528 529 530

Arlt, A., Gehrz, A., Muerkoster, S., Vorndamm, J., Kruse, M.L., Folsch, U.R., Schafer, H., 2003. Role of NF-kappaB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death. Oncogene 22, 3243-3251.

531 532 533

Bach, P., Abel, T., Hoffmann, C., Gal, Z., Braun, G., Voelker, I., Ball, C.R., Johnston, I.C., Lauer, U.M., Herold-Mende, C., et al., 2013. Specific elimination of CD133+ tumor cells with targeted oncolytic measles virus. Cancer Research 73, 865-874.

534 535 536

Bao, S., Wu, Q., McLendon, R.E., Hao, Y., Shi, Q., Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., Rich, J.N., 2006. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756-760.

537 538 539 540

Bardenheuer, W., Lehmberg, K., Rattmann, I., Brueckner, A., Schneider, A., Sorg, U.R., Seeber, S., Moritz, T., Flasshove, M., 2005. Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells. Leukemia 19, 2281-2288.

541 542

Baumann, M., Krause, M., Hill, R., 2008. Exploring the role of cancer stem cells in radioresistance. Nature reviews. Cancer 8, 545-554.

543 544 545

Beretta, G.L., Gatti, L., Perego, P., Zaffaroni, N., 2013. Camptothecin resistance in cancer: insights into the molecular mechanisms of a DNA-damaging drug. Current Medicinal Chemistry 20, 1541-1565.

546 547 548 549

Bertolini, G., Roz, L., Perego, P., Tortoreto, M., Fontanella, E., Gatti, L., Pratesi, G., Fabbri, A., Andriani, F., Tinelli, S., et al., 2009. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proceedings of the National Academy of Sciences of the United States of America 106, 16281-16286.

550 551

Bouwman, P., Jonkers, J., 2012. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nature Reviews Cancer 12, 587-598.

552 553 554

Brenn, S.H., Couto, S.S., Craft, D.M., Leung, C., Bergman, P.J., 2008. Evaluation of Pglycoprotein expression in feline lymphoma and correlation with clinical outcome. Veterinary and Comparative Oncology 6, 201-211.

555 556

Brown, R., Curry, E., Magnani, L., Wilhelm-Benartzi, C.S., Borley, J., 2014. Poised epigenetic states and acquired drug resistance in cancer. Nature Reviews Cancer 14, 747-753.

557 558 559 560

Byers, L.A., Diao, L., Wang, J., Saintigny, P., Girard, L., Peyton, M., Shen, L., Fan, Y., Giri, U., Tumula, P.K., et al., 2013. An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clinical Cancer Research 19, 279-290. 23 Page 23 of 36

561 562 563

Cai, J., Damaraju, V.L., Groulx, N., Mowles, D., Peng, Y., Robins, M.J., Cass, C.E., Gros, P., 2008. Two distinct molecular mechanisms underlying cytarabine resistance in human leukemic cells. Cancer Research 68, 2349-2357.

564 565 566 567

Chien, W.W., Le Beux, C., Rachinel, N., Julien, M., Lacroix, C.E., Allas, S., Sahakian, P., Cornut-Thibaut, A., Lionnard, L., Kucharczak, J., Aouacheria, A., Abribat, T., Salles, G., 2015. Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines. Scientific Reports 5, 8068.

568 569

Choi, Y.H., Yu, A.M., 2014. ABC transporters in multidrug resistance and pharmacokinetics, and strategies for drug development. Current Pharmaceutical Design 20, 793-807.

570 571 572 573

Chu, X.Y., Kato, Y., Niinuma, K., Sudo, K.I., Hakusui, H., Sugiyama, Y., 1997. Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats. The Journal of Pharmacology and Experimental Therapeutics 281, 304-314.

574 575

Colak, S., Medema, J.P., 2014. Cancer stem cells--important players in tumor therapy resistance. The FEBS Journal 281, 4779-4791.

576 577 578

Colak, S., Zimberlin, C.D., Fessler, E., Hogdal, L., Prasetyanti, P.R., Grandela, C.M., Letai, A., Medema, J.P., 2014. Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells. Cell Death and Differentiation 21, 1170-1177.

579 580

Dawood, S., Austin, L., Cristofanilli, M., 2014. Cancer Stem Cells: Implications for Cancer Therapy. Oncology 28.

581 582 583

Dhaliwal, R.S., Kitchell, B.E., Ehrhart, E., Valli, V.E., Dervisis, N.G., 2013. Clinicopathologic significance of histologic grade, pgp, and p53 expression in canine lymphoma. Journal of the American Animal Hospital Association 49, 175-184.

584 585 586

Dincer, Z., Jasani, B., Haywood, S., Mullins, J.E., Fuentealba, I.C., 2001. Metallothionein expression in canine and feline mammary and melanotic tumours. Journal of Comparative Pathology 125, 130-136.

587 588 589

Duncan, A.W., Rattis, F.M., DiMascio, L.N., Congdon, K.L., Pazianos, G., Zhao, C., Yoon, K., Cook, J.M., Willert, K., Gaiano, N., Reya, T., 2005. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nature Immunology 6, 314-322.

590 591 592

Edwards, S.L., Brough, R., Lord, C.J., Natrajan, R., Vatcheva, R., Levine, D.A., Boyd, J., Reis-Filho, J.S., Ashworth, A., 2008. Resistance to therapy caused by intragenic deletion in BRCA2. Nature 451, 1111-1115.

593 594

Enoch, T., Norbury, C., 1995. Cellular responses to DNA damage: cell-cycle checkpoints, apoptosis and the roles of p53 and ATM. Trends in Biochemical Sciences 20, 426-430.

595 596 597

Erginsoy, S.D., Sozmen, M., Caldin, M., Furlanello, T., 2006. Metallothionein expression in benign and malignant canine mammary gland tumours. Research in Veterinary Science 81, 46-50.

598 599

Fan, X., Khaki, L., Zhu, T.S., Soules, M.E., Talsma, C.E., Gul, N., Koh, C., Zhang, J., Li, Y.M., Maciaczyk, J., et al., 2010. NOTCH pathway blockade depletes CD133-positive 24 Page 24 of 36

600 601

glioblastoma cells and inhibits growth of tumor neurospheres and xenografts. Stem Cells 28, 5-16.

602 603 604

Farmer, H., McCabe, N., Lord, C.J., Tutt, A.N., Johnson, D.A., Richardson, T.B., Santarosa, M., Dillon, K.J., Hickson, I., Knights, C., et al., 2005. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917-921.

605 606 607

Fernando, R.I., Castillo, M.D., Litzinger, M., Hamilton, D.H., Palena, C., 2011. IL-8 signaling plays a critical role in the epithelial-mesenchymal transition of human carcinoma cells. Cancer Research 71, 5296-5306.

608 609

Fink, D., Aebi, S., Howell, S.B., 1998. The role of DNA mismatch repair in drug resistance. Clinical Cancer Research 4, 1-6.

610 611 612 613

Fosmire, S.P., Thomas, R., Jubala, C.M., Wojcieszyn, J.W., Valli, V.E., Getzy, D.M., Smith, T.L., Gardner, L.A., Ritt, M.G., Bell, J.S., et al., 2007. Inactivation of the p16 cyclindependent kinase inhibitor in high-grade canine non-Hodgkin's T-cell lymphoma. Veterinary Pathology 44, 467-478.

614 615 616

Fu, D.R., Kato, D., Watabe, A., Endo, Y., Kadosawa, T., 2014. Prognostic utility of apoptosis index, Ki-67 and survivin expression in dogs with nasal carcinoma treated with orthovoltage radiation therapy. The Journal of Veterinary Medical Science 76, 1505-1512.

617 618 619 620

Fuchs, B.C., Fujii, T., Dorfman, J.D., Goodwin, J.M., Zhu, A.X., Lanuti, M., Tanabe, K.K., 2008. Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. Cancer Research 68, 2391-2399.

621 622 623

Fujiwara-Igarashi, A., Goto-Koshino, Y., Mochizuki, H., Sato, M., Fujino, Y., Ohno, K., Tsujimoto, H., 2014a. Inhibition of p16 tumor suppressor gene expression via promoter hypermethylation in canine lymphoid tumor cells. Research in Veterinary Science 97, 60-63.

624 625 626 627

Fujiwara-Igarashi, A., Goto-Koshino, Y., Sato, M., Maeda, S., Igarashi, H., Takahashi, M., Fujino, Y., Ohno, K., Tsujimoto, H., 2014b. Prognostic significance of the expression levels of the p16, p15, and p14 genes in dogs with high-grade lymphoma. The Veterinary Journal 199, 236-244.

628 629 630 631

Ginestier, C., Liu, S., Diebel, M.E., Korkaya, H., Luo, M., Brown, M., Wicinski, J., Cabaud, O., Charafe-Jauffret, E., Birnbaum, D., et al., 2010. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. The Journal of Clinical Investigation 120, 485-497.

632 633 634

Ginn, J., Sacco, J., Wong, Y.Y., Motsinger-Reif, A., Chun, R., Trepanier, L.A., 2014. Positive association between a glutathione-S-transferase polymorphism and lymphoma in dogs. Veterinary and Comparative Oncology 12, 227-236.

635 636 637 638 639

Glasspool, R.M., Brown, R., Gore, M.E., Rustin, G.J., McNeish, I.A., Wilson, R.H., Pledge, S., Paul, J., Mackean, M., Hall, G.D., et al., 2014. A randomised, phase II trial of the DNAhypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer. British Journal of Cancer 110, 1923-1929. 25 Page 25 of 36

640 641 642 643

Goff, D.J., Court Recart, A., Sadarangani, A., Chun, H.J., Barrett, C.L., Krajewska, M., Leu, H., Low-Marchelli, J., Ma, W., Shih, A.Y., et al., 2013. A Pan-BCL2 inhibitor renders bonemarrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell 12, 316-328.

644 645 646

Goldstein, L.J., Galski, H., Fojo, A., Willingham, M., Lai, S.L., Gazdar, A., Pirker, R., Green, A., Crist, W., Brodeur, G.M., et al., 1989. Expression of a multidrug resistance gene in human cancers. Journal of the National Cancer Institute 81, 116-124.

647 648 649

Gramer, I., Kessler, M., Geyer, J., 2013. Determination of MDR1 gene expression for prediction of chemotherapy tolerance and treatment outcome in dogs with lymphoma. Veterinary and Comparative Oncology DOI: 10.1111/vco.12051

650 651

Grosse, N., van Loon, B., Rohrer Bley, C., 2014. DNA damage response and DNA repair dog as a model? BMC Cancer 14, 203.

652 653 654

Hahn, K.A., Barnhill, M.A., Freeman, K.P., Shoieb, A.M., 1999. Detection and clinical significance of plasma glutathione-S-transferases in dogs with lymphoma. In Vivo 13, 173175.

655 656 657 658

Hahn, N.M., Bonney, P.L., Dhawan, D., Jones, D.R., Balch, C., Guo, Z., Hartman-Frey, C., Fang, F., Parker, H.G., Kwon, E.M., et al., 2012. Subcutaneous 5-azacitidine treatment of naturally occurring canine urothelial carcinoma: a novel epigenetic approach to human urothelial carcinoma drug development. The Journal of Urology 187, 302-309.

659 660 661 662

Halsey, C.H., Gustafson, D.L., Rose, B.J., Wolf-Ringwall, A., Burnett, R.C., Duval, D.L., Avery, A.C., Thamm, D.H., 2014. Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia(R)) in canine mast cell tumor. BMC Veterinary Research 10, 105.

663

Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 57-70.

664 665 666

He, X.C., Zhang, J., Tong, W.G., Tawfik, O., Ross, J., Scoville, D.H., Tian, Q., Zeng, X., He, X., Wiedemann, L.M., et al., 2004. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nature Genetics 36, 1117-1121.

667 668 669 670

Hifumi, T., Miyoshi, N., Kawaguchi, H., Nomura, K., Yasuda, N., 2010. Immunohistochemical detection of proteins associated with multidrug resistance to anticancer drugs in canine and feline primary pulmonary carcinoma. The Journal of Veterinary Medical Science 72, 665-668.

671 672 673

Hoey, T., Yen, W.C., Axelrod, F., Basi, J., Donigian, L., Dylla, S., Fitch-Bruhns, M., Lazetic, S., Park, I.K., Sato, A., et al., 2009. DLL4 blockade inhibits tumor growth and reduces tumorinitiating cell frequency. Cell Stem Cell 5, 168-177.

674 675

Holohan, C., Van Schaeybroeck, S., Longley, D.B., Johnston, P.G., 2013. Cancer drug resistance: an evolving paradigm. Nature Reviews Cancer 13, 714-726.

676 677 678

Honscha, K.U., Schirmer, A., Reischauer, A., Schoon, H.A., Einspanier, A., Gabel, G., 2009. Expression of ABC-transport proteins in canine mammary cancer: consequences for chemotherapy. Reproduction in Domestic Animals 44 Suppl. 2, 218-223. 26 Page 26 of 36

679 680

Housman, G., Byler, S., Heerboth, S., Lapinska, K., Longacre, M., Snyder, N., Sarkar, S., 2014. Drug resistance in cancer: an overview. Cancers 6, 1769-1792.

681 682 683

Huang, S., Holzel, M., Knijnenburg, T., Schlicker, A., Roepman, P., McDermott, U., Garnett, M., Grernrum, W., Sun, C., Prahallad, A., et al., 2012. MED12 controls the response to multiple cancer drugs through regulation of TGF-beta receptor signaling. Cell 151, 937-950.

684 685

Karotki, A.V., Vasak, M., 2009. Reaction of human metallothionein-3 with cisplatin and transplatin. Journal of Biological Inorganic Chemistry 14, 1129-1138.

686 687 688

Kathawala, R.J., Gupta, P., Ashby, C.R., Jr., Chen, Z.S., 2015. The modulation of ABC transporter-mediated multidrug resistance in cancer: A review of the past decade. Drug Resistance Updates 18C, 1-17.

689 690 691

Kavallaris, M., Tait, A.S., Walsh, B.J., He, L., Horwitz, S.B., Norris, M.D., Haber, M., 2001. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Research 61, 5803-5809.

692 693

Kemper, K., Rodermond, H., Colak, S., Grandela, C., Medema, J.P., 2012. Targeting colorectal cancer stem cells with inducible caspase-9. Apoptosis 17, 528-537.

694 695

Klopfleisch, R., 2015. Personalised medicine in veterinary oncology: One to cure just one. The Veterinary Journal doi.org/10.1016/j.tvjl.2015.01.004

696 697

Klopfleisch, R., Gruber, A.D., 2009. Increased expression of BRCA2 and RAD51 in lymph node metastases of canine mammary adenocarcinomas. Veterinary Pathology 46, 416-422.

698 699 700

Klopfleisch, R., Klose, P., Gruber, A.D., 2010. The combined expression pattern of BMP2, LTBP4, and DERL1 discriminates malignant from benign canine mammary tumors. Veterinary Pathology 47, 446-454.

701 702 703

Klopfleisch, R., Lenze, D., Hummel, M., Gruber, A.D., 2011a. The metastatic cascade is reflected in the transcriptome of metastatic canine mammary carcinomas. The Veterinary Journal 190, 236-243.

704 705 706 707

Klopfleisch, R., Meyer, A., Schlieben, P., Bondzio, A., Weise, C., Lenze, D., Hummel, M., Einspanier, R., Gruber, A.D., 2012. Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes. BMC Veterinary Research 8, 96.

708 709 710

Klopfleisch, R., von Euler, H., Sarli, G., Pinho, S.S., Gartner, F., Gruber, A.D., 2011b. Molecular carcinogenesis of canine mammary tumors: news from an old disease. Veterinary Pathology 48, 98-116.

711 712 713 714

Klose, P., Weise, C., Bondzio, A., Multhaup, G., Einspanier, R., Gruber, A.D., Klopfleisch, R., 2011. Is there a malignant progression associated with a linear change in protein expression levels from normal canine mammary gland to metastatic mammary tumors? Journal of Proteome Research 10, 4405-4415.

715 716 717

Kobayashi, M., Kuroki, S., Tanaka, Y., Moriya, Y., Kozutumi, Y., Uehara, Y., Ono, K., Tamura, K., Washizu, T., Bonkobara, M., 2015. Molecular changes associated with the development of resistance to imatinib in an imatinib-sensitive canine neoplastic mast cell line 27 Page 27 of 36

718 719

carrying a KIT c.1523A>T mutation. European Journal of Haematology doi: 10.1111/ejh.12526

720 721 722

Konopleva, M., Contractor, R., Tsao, T., Samudio, I., Ruvolo, P.P., Kitada, S., Deng, X., Zhai, D., Shi, Y.X., Sneed, T., et al., 2006. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 10, 375-388.

723 724 725

Krol, M., Pawlowski, K.M., Majchrzak, K., Mucha, J., Motyl, T., 2014. Canine mammary carcinoma cell line are resistant to chemosensitizers: verapamil and cyclosporin A. Polish Journal of Veterinary Sciences 17, 9-17.

726 727 728

Kumaraguruparan, R., Subapriya, R., Balachandran, C., Manohar, B.M., Thangadurai, A., Nagini, S., 2006. Xenobiotic-metabolizing enzymes in canine mammary tumours. The Veterinary Journal 172, 364-368.

729 730 731 732

Kwon, H.C., Roh, M.S., Oh, S.Y., Kim, S.H., Kim, M.C., Kim, J.S., Kim, H.J., 2007. Prognostic value of expression of ERCC1, thymidylate synthase, and glutathione Stransferase P1 for 5-fluorouracil/oxaliplatin chemotherapy in advanced gastric cancer. Annals of Oncology 18, 504-509.

733 734

Lai, K., Kaspar, B.K., Gage, F.H., Schaffer, D.V., 2003. Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nature Neuroscience 6, 21-27.

735 736 737

Liu, S., Ginestier, C., Ou, S.J., Clouthier, S.G., Patel, S.H., Monville, F., Korkaya, H., Heath, A., Dutcher, J., Kleer, C.G., et al., 2011. Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks. Cancer Research 71, 614-624.

738 739 740 741

Lombardo, Y., Scopelliti, A., Cammareri, P., Todaro, M., Iovino, F., Ricci-Vitiani, L., Gulotta, G., Dieli, F., de Maria, R., Stassi, G., 2011. Bone morphogenetic protein 4 induces differentiation of colorectal cancer stem cells and increases their response to chemotherapy in mice. Gastroenterology 140, 297-309.

742 743

London, C.A., 2013. Kinase dysfunction and kinase inhibitors. Veterinary Dermatology 24, 181-187 e139-140.

744 745 746 747

Lowenberg, B., van Putten, W., Theobald, M., Gmur, J., Verdonck, L., Sonneveld, P., Fey, M., Schouten, H., de Greef, G., Ferrant, A., et al., 2003. Effect of priming with granulocyte colony-stimulating factor on the outcome of chemotherapy for acute myeloid leukemia. The New England Journal of Medicine 349, 743-752.

748 749 750

Martano, M., Carella, F., Squillacioti, C., Restucci, B., Mazzotta, M., Lo Muzio, L., Maiolino, P., 2012. Metallothionein expression in canine cutaneous apocrine gland tumors. Anticancer Research 32, 747-752.

751 752 753 754

Martin, S.A., McCarthy, A., Barber, L.J., Burgess, D.J., Parry, S., Lord, C.J., Ashworth, A., 2009. Methotrexate induces oxidative DNA damage and is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2. EMBO Molecular Medicine 1, 323337.

755 756 757

Meijer, C., Mulder, N.H., Timmer-Bosscha, H., Sluiter, W.J., Meersma, G.J., de Vries, E.G., 1992. Relationship of cellular glutathione to the cytotoxicity and resistance of seven platinum compounds. Cancer Research 52, 6885-6889. 28 Page 28 of 36

758 759 760

Mhaidat, N.M., Alali, F.Q., Matalqah, S.M., Matalka, II, Jaradat, S.A., Al-Sawalha, N.A., Thorne, R.F., 2009. Inhibition of MEK sensitizes paclitaxel-induced apoptosis of human colorectal cancer cells by downregulation of GRP78. Anti-Cancer Drugs 20, 601-606.

761 762

Michael, M., Doherty, M.M., 2005. Tumoral drug metabolism: overview and its implications for cancer therapy. Journal of Clinical Oncology 23, 205-229.

763 764 765 766

Mirmohammadsadegh, A., Mota, R., Gustrau, A., Hassan, M., Nambiar, S., Marini, A., Bojar, H., Tannapfel, A., Hengge, U.R., 2007. ERK1/2 is highly phosphorylated in melanoma metastases and protects melanoma cells from cisplatin-mediated apoptosis. The Journal of Investigative Dermatology 127, 2207-2215.

767 768 769

Miyashita, T., Reed, J.C., 1992. bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Research 52, 5407-5411.

770 771

Munday, J.S., French, A.F., Gibson, I.R., Gwynne, K., 2009. Widespread mismatch repair protein expression in canine cutaneous mast cell tumors. Veterinary Pathology 46, 227-232.

772 773 774 775

Nakaichi, M., Takeshita, Y., Okuda, M., Nakamoto, Y., Itamoto, K., Une, S., Sasaki, N., Kadosawa, T., Takahashi, T., Taura, Y., 2007. Expression of the MDR1 gene and Pglycoprotein in canine mast cell tumor cell lines. The Journal of Veterinary Medical Science 69, 111-115.

776 777

Nebbioso, A., Carafa, V., Benedetti, R., Altucci, L., 2012. Trials with 'epigenetic' drugs: an update. Molecular Oncology 6, 657-682.

778 779 780

Nieto, A., Perez-Alenza, M.D., Del Castillo, N., Tabanera, E., Castano, M., Pena, L., 2003. BRCA1 expression in canine mammary dysplasias and tumours: relationship with prognostic variables. Journal of Comparative Pathology 128, 260-268.

781 782 783

Nobili, S., Landini, I., Mazzei, T., Mini, E., 2012. Overcoming tumor multidrug resistance using drugs able to evade P-glycoprotein or to exploit its expression. Medicinal Research Reviews 32, 1220-1262.

784 785 786

Noguchi, K., Katayama, K., Sugimoto, Y., 2014. Human ABC transporter ABCG2/BCRP expression in chemoresistance: basic and clinical perspectives for molecular cancer therapeutics. Pharmacogenomics and Personalized Medicine 7, 53-64.

787 788 789

Okai, Y., Nakamura, N., Matsushiro, H., Kato, H., Setoguchi, A., Yazawa, M., Okuda, M., Watari, T., Hasegawa, A., Tsujimoto, H., 2000. Molecular analysis of multidrug resistance in feline lymphoma cells. American Journal of Veterinary Research 61, 1122-1127.

790 791 792 793

Pabst, T., Vellenga, E., van Putten, W., Schouten, H.C., Graux, C., Vekemans, M.C., Biemond, B., Sonneveld, P., Passweg, J., Verdonck, L., et al., 2012. Favorable effect of priming with granulocyte colony-stimulating factor in remission induction of acute myeloid leukemia restricted to dose escalation of cytarabine. Blood 119, 5367-5373.

794 795

Pang, L.Y., Argyle, D.J., 2014. The evolving cancer stem cell paradigm: Implications in veterinary oncology. The VeterinaryJournal doi.org/10.1016/j.tvjl.2014.12.029

29 Page 29 of 36

796 797 798

Pang, L.Y., Argyle, S.A., Kamida, A., Morrison, K.O., Argyle, D.J., 2014. The long-acting COX-2 inhibitor mavacoxib (Trocoxil) has anti-proliferative and pro-apoptotic effects on canine cancer cell lines and cancer stem cells in vitro. BMC Veterinary Research 10, 184.

799 800 801 802

Pang, L.Y., Blacking, T.M., Else, R.W., Sherman, A., Sang, H.M., Whitelaw, B.A., Hupp, T.R., Argyle, D.J., 2013. Feline mammary carcinoma stem cells are tumorigenic, radioresistant, chemoresistant and defective in activation of the ATM/p53 DNA damage pathway. The Veterinary Journal 196, 414-423.

803 804

Pavet, V., Portal, M.M., Moulin, J.C., Herbrecht, R., Gronemeyer, H., 2011. Towards novel paradigms for cancer therapy. Oncogene 30, 1-20.

805 806 807

Pawlak, A., Rapak, A., Zbyryt, I., Obminska-Mrukowicz, B., 2014. The effect of common antineoplastic agents on induction of apoptosis in canine lymphoma and leukemia cell lines. In Vivo 28, 843-850.

808 809 810

Pawlowski, K.M., Mucha, J., Majchrzak, K., Motyl, T., Krol, M., 2013. Expression and role of PGP, BCRP, MRP1 and MRP3 in multidrug resistance of canine mammary cancer cells. BMC Veterinary Research 9, 119.

811 812 813

Perez, E.A., 2009. Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Molecular Cancer Therapeutics 8, 2086-2095.

814 815 816 817

Piovan, E., Yu, J., Tosello, V., Herranz, D., Ambesi-Impiombato, A., Da Silva, A.C., Sanchez-Martin, M., Perez-Garcia, A., Rigo, I., Castillo, M., et al., 2013. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia. Cancer Cell 24, 766-776.

818 819 820 821

Qiu, M., Peng, Q., Jiang, I., Carroll, C., Han, G., Rymer, I., Lippincott, J., Zachwieja, J., Gajiwala, K., Kraynov, E., et al., 2013. Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells. Cancer Letters 328, 261-270.

822 823

Rabik, C.A., Dolan, M.E., 2007. Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treatment Reviews 33, 9-23.

824 825 826

Rebhun, R.B., Lana, S.E., Ehrhart, E.J., Charles, J.B., Thamm, D.H., 2008. Comparative analysis of survivin expression in untreated and relapsed canine lymphoma. Journal of Veterinary Internal Medicine 22, 989-995.

827 828

Rocha, T.A., Mauldin, G.N., Patnaik, A.K., Bergman, P.J., 2000. Prognostic factors in dogs with urinary bladder carcinoma. Journal of Veterinary Internal Medicine 14, 486-490.

829 830 831 832

Saez-Ayala, M., Fernandez-Perez, M.P., Montenegro, M.F., Sanchez-del-Campo, L., Chazarra, S., Pinero-Madrona, A., Cabezas-Herrera, J., Rodriguez-Lopez, J.N., 2012. Melanoma coordinates general and cell-specific mechanisms to promote methotrexate resistance. Experimental Cell Research 318, 1146-1159.

833 834 835

Sarkaria, J.N., Kitange, G.J., James, C.D., Plummer, R., Calvert, H., Weller, M., Wick, W., 2008. Mechanisms of chemoresistance to alkylating agents in malignant glioma. Clinical Cancer Research 14, 2900-2908. 30 Page 30 of 36

836 837

Schlossmacher, G., Stevens, A., White, A., 2011. Glucocorticoid receptor-mediated apoptosis: mechanisms of resistance in cancer cells. The Journal of Endocrinology 211, 17-25.

838 839 840

Shoieb, A.M., Hahn, K.A., Van Laack, R.L., Barnhill, M.A., 1998. In vitro reversal of glutathione-S-transferase-mediated resistance in canine osteosarcoma (COS31) cells. In Vivo 12, 455-462.

841 842 843 844

Signore, M., Pelacchi, F., di Martino, S., Runci, D., Biffoni, M., Giannetti, S., Morgante, L., De Majo, M., Petricoin, E.F., Stancato, L.,et al., 2014. Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo. Cell Death and Disease 5, e1223.

845 846 847 848

Simon, D., Moreno, S.N., Hirschberger, J., Moritz, A., Kohn, B., Neumann, S., Jurina, K., Scharvogel, S., Schwedes, C., Reinacher, M., et al., 2008. Efficacy of a continuous, multiagent chemotherapeutic protocol versus a short-term single-agent protocol in dogs with lymphoma. Journal of the American Veterinary Medical Association 232, 879-885.

849 850 851

Smallwood, T.L., Small, G.W., Suter, S.E., Richards, K.L., 2014. Expression of asparagine synthetase predicts in vitro response to L-asparaginase in canine lymphoid cell lines. Leukemia and Lymphoma 55, 1357-1365.

852 853 854

Sugimoto, Y., Tsukahara, S., Oh-hara, T., Isoe, T., Tsuruo, T., 1990. Decreased expression of DNA topoisomerase I in camptothecin-resistant tumor cell lines as determined by a monoclonal antibody. Cancer Research 50, 6925-6930.

855 856 857

Swaminathan, S.K., Roger, E., Toti, U., Niu, L., Ohlfest, J.R., Panyam, J., 2013. CD133targeted paclitaxel delivery inhibits local tumor recurrence in a mouse model of breast cancer. Journal of Controlled Release 171, 280-287.

858 859

Takahashi-Yanaga, F., Kahn, M., 2010. Targeting Wnt signaling: can we safely eradicate cancer stem cells? Clinical Cancer Research 16, 3153-3162.

860 861 862

Tang, J., Xie, X., Zhang, X., Qiao, X., Jiang, S., Shi, W., Shao, Y., Zhou, X., 2012. Long term cultured HL-60 cells are intrinsically resistant to Ara-C through high CDA activity. Frontiers in Bioscience 17, 569-574.

863 864 865

Thamm, D.H., Grunerud, K.K., Rose, B.J., Vail, D.M., Bailey, S.M., 2013. DNA repair deficiency as a susceptibility marker for spontaneous lymphoma in golden retriever dogs: a case-control study. PloS One 8, e69192.

866 867 868 869

Thayanithy, V., Park, C., Sarver, A.L., Kartha, R.V., Korpela, D.M., Graef, A.J., Steer, C.J., Modiano, J.F., Subramanian, S., 2012. Combinatorial treatment of DNA and chromatinmodifying drugs cause cell death in human and canine osteosarcoma cell lines. PloS One 7, e43720.

870 871 872

Tomiyasu, H., Fujiwara-Igarashi, A., Goto-Koshino, Y., Fujino, Y., Ohno, K., Tsujimoto, H., 2014a. Evaluation of DNA methylation profiles of the CpG island of the ABCB1 gene in dogs with lymphoma. American Journal of Veterinary Research 75, 835-841.

873 874 875

Tomiyasu, H., Goto-Koshino, Y., Fujino, Y., Ohno, K., Tsujimoto, H., 2014b. Antitumour effect and modulation of expression of the ABCB1 gene by perifosine in canine lymphoid tumour cell lines. The Veterinary Journal 201, 83-90. 31 Page 31 of 36

876 877 878

Tomiyasu, H., Goto-Koshino, Y., Fujino, Y., Ohno, K., Tsujimoto, H., 2014c. Epigenetic regulation of the ABCB1 gene in drug-sensitive and drug-resistant lymphoid tumour cell lines obtained from canine patients. The Veterinary Journal 199, 103-109.

879 880 881

Tomiyasu, H., Goto-Koshino, Y., Fujino, Y., Ohno, K., Tsujimoto, H., 2014d. The regulation of the expression of ABCG2 gene through mitogen-activated protein kinase pathways in canine lymphoid tumor cell lines. The Journal of Veterinary Medical Science 76, 237-242.

882 883 884

Tomiyasu, H., Goto-Koshino, Y., Takahashi, M., Fujino, Y., Ohno, K., Tsujimoto, H., 2010. Quantitative analysis of mRNA for 10 different drug resistance factors in dogs with lymphoma. The Journal of Veterinary Medical Science 72, 1165-1172.

885 886 887

Van der Heyden, S., Chiers, K., Vercauteren, G., Daminet, S., Wegge, B., Paepe, D., Ducatelle, R., 2011. Expression of multidrug resistance-associated P-glycoprotein in feline tumours. Journal of Comparative Pathology 144, 164-169.

888 889

van Steensel, B., 2011. Chromatin: constructing the big picture. The EMBO Journal 30, 18851895.

890 891

Wilting, R.H., Dannenberg, J.H., 2012. Epigenetic mechanisms in tumorigenesis, tumor cell heterogeneity and drug resistance. Drug Resistance Updates 15, 21-38.

892 893 894

Yang, Z., Zhu, W., Gao, S., Yin, T., Jiang, W., Hu, M., 2012. Breast cancer resistance protein (ABCG2) determines distribution of genistein phase II metabolites: reevaluation of the roles of ABCG2 in the disposition of genistein. Drug Metabolism and Disposition 40, 1883-1893.

895 896 897

Yoshimoto, K., Ma, X., Guan, Y., Mizoguchi, M., Nakamizo, A., Amano, T., Hata, N., Kuga, D., Sasaki, T., 2011. Expression of stem cell marker and receptor kinase genes in glioblastoma tissue quantified by real-time RT-PCR. Brain Tumor Pathology 28, 291-296.

898 899 900

Zandvliet, M., Teske, E., Chapuis, T., Fink-Gremmels, J., Schrickx, J.A., 2013. Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of Pglycoprotein. Journal of Veterinary Pharmacology and Therapeutics 36, 583-587.

901 902 903

Zandvliet, M., Teske, E., Schrickx, J.A., 2014a. Multi-drug resistance in a canine lymphoid cell line due to increased P-glycoprotein expression, a potential model for drug-resistant canine lymphoma. Toxicology In Vitro 28, 1498-1506.

904 905

Zandvliet, M., Teske, E., Schrickx, J.A., Mol, J.A., 2014b. A longitudinal study of ABC transporter expression in canine multicentric lymphoma. Veterinary journal.

906 907

Zhang, N., Yin, Y., Xu, S.J., Chen, W.S., 2008. 5-Fluorouracil: mechanisms of resistance and reversal strategies. Molecules 13, 1551-1569.

908 909 910

Zhao, C., Chen, A., Jamieson, C.H., Fereshteh, M., Abrahamsson, A., Blum, J., Kwon, H.Y., Kim, J., Chute, J.P., Rizzieri, D., et al., 2009. Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 458, 776-779.

911 912 32 Page 32 of 36

913

Table 1

914 915

Specific mechanisms of resistance to chemotherapeutic agents commonly used in veterinary oncology Agent Alkylating agents Cyclophosphamide Chlorambucil Lomustine Procarbazine

Target/mechanism

Resistance mechanisms (References)

DNA-crosslinking, inhibition of DNA repair/synthesis

Increased efflux (Pawlowski et al., 2013) Increased DNA repair (Fink et al., 1998) MGMT activity (Sarkaria et al., 2008)

Unclear, maybe DNA break induction, t-RNA inhibition

Platinum containing drugs Cisplatin DNA-crosslinking, prevention of DNA-uncoiling/strand Carboplatin separation Antimetabolites Methotrexate Cytarabine

Dihydrofolate reductase DNA-Synthesis inhibition, DNA-chain termination 5-Fluorouracil (5-FU) Thymidylate synthase inhibition, replacement of cytosine, thymidine, uracil in DNA/RNA strands Gemcitabine Replaces cytidine DNAsynthesis inhibition Topoisomerase I (TOPO1) inhibitors Camptothecins Stabilization of the cleavable (Irinotecan, Topotecan) DNA-enzyme complex and thereby inducing DNA damage Indenoisoquinoline Complexing with TOPO1 Topoisomerase II (TOPO2) inhibitors Doxorubicin Inhibition of TOPO2 by DNA intercalation Mitoxantrone Microtubule poisons Paclitaxel Vinblastine Vincristine Vinorelbine Miscellaneous Agents Prednisone

L-Asparaginase

Increased efflux (Pawlowski et al., 2013), ERK activity (Mirmohammadsadegh et al., 2007) DNA repair (Kwon et al., 2007) Replicative bypass (Rabik and Dolan, 2007) Melanosomal efflux (Saez-Ayala et al., 2012) Decreased uptake (Cai et al., 2008) Decreased activation by DCK, DPD (Cai et al., 2008; Zhang et al., 2008) Detoxification by CDD, CDA, NT5C2 (Bardenheuer et al., 2005; Tang et al., 2012) Increased DNA repair (Zhang et al., 2008) Induction of anti-apoptotic molecules (Zhang et al., 2008) Activation of survival pathways (Arlt et al., 2003) TOPO1 mutations (Sugimoto et al., 1990) Reduced TOPO expression (Meijer et al., 1992) Improved DNA repair (Alagoz et al., 2012) Lack of apoptosis induction (Beretta et al., 2013) Increase efflux by MDR and BCRP (Chu et al., 1997) Unknown so far (Yang et al., 2012) Increased efflux (Zandvliet et al., 2014b) Amplification of TOPO2 and ERBB2 (Noguchi et al., 2014)

Microtubules stabilization Microtubule Depolymerisation/stabilization

Apoptosis inhibition (Mhaidat et al., 2009) Increase efflux (Pawlowski et al., 2013) Tubulin mutation (Kavallaris et al., 2001) Stathmin, MAP4, y-actin overexpression of (Perez, 2009)

Unclear, apoptosis induction

Increased efflux (Dhaliwal et al., 2013) STAT3, pSTAT3, KIT overexpression (Dhaliwal et al., 2013) PTEN loss, AKT1 activity (Piovan et al., 2013) Decreased receptor expression (Schlossmacher et al., 2011) Increased asparagine synthetase (Chien et al., 2015) Decreased cellular efflux of asparagine (Chien et al., 2015) Increased L-glutaminase activity (Chien et al., 2015) Aspartic acid synthesis (Chien et al., 2015) Activation of glutamine uptake (Chien et al., 2015)

Asparagine deprivation

Tyrosine kinase inhibitors Toceranib KIT, VEGFR2, PDGFRB Imatinib KIT, PDGFR Masitinib KIT, PDGFRA

Target mutation (Halsey et al., 2014; Kobayashi et al., 2015) Increased (MCL1) expression (Amagai et al., 2013)

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916 917 918 919 920

Genistein Diverse tyrosine kinases CDA, cytidine-deaminase; CDD, cytidine deaminase; DPD, dihydropyrimidine dehydrogenase; ERK, extracellular signalregulated kinase; DCK, deoxycytidine kinase; HR, homologous recombination; MCL1, Myeloid cell leucaemia sequence 1; MMR, mismatch repair; MGMT, O6-methylguanine methyltransferase; NER, nucleotide excision repair; NT5C2, cytoplasmic 5′nucleotidase; PDGFR, platelet-derived growth factor; Replicative bypass, ability of DNA polymerase to bypass DNA cross-links; VGEFR, vascular endothelial growth factor

921

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922

Table 2

923

Studies confirming MDR1, BCRP, MRP1-mediated chemotherapy resistance in animal

924

tumours ABCtransporter MDR1

Cancer type Canine mammary tumour

Confirmed contribution chemoresistance (agent) Cisplatin Vinblastine No effect on vinblastine No effect on cyclophosphamide Doxorubicin

to

Reference

No effect on prednisone

(Pawlowski et al., 2013) (Pawlowski et al., 2013) (Krol et al., 2014) (Pawlowski et al., 2013) (Zandvliet et al., 2013; Zandvliet et al., 2014a) (Tomiyasu et al., 2014b; Zandvliet et al., 2014a) (Zandvliet et al., 2014a)

Canine mast cells

General effect on efflux

(Nakaichi et al., 2007)

BCRP

Canine mammary tumour

Cyclophosphamide Cisplatin No effect on vinblastine Doxorubicin No effect on methotrexate

(Pawlowski et al., 2013) (Pawlowski et al., 2013) (Pawlowski et al., 2013) (Honscha et al., 2009) (Honscha et al., 2009)

MRP1

Canine mammary tumour

Cisplatin Vinblastine No effect on cyclophosphamide

(Pawlowski et al., 2013) (Pawlowski et al., 2013) (Pawlowski et al., 2013)

Canine lymphoma

Vincristine

925 926

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927

Figure legends

928 929 930 931 932 933 934 935

Figure 1. Mechanisms of ABC-protein mediated resistance and corresponding therapies. Each of the almost 50 ABC-transporters identified so far is able to transport a specific set of anticancer drugs out of tumour cells. These sets are however overlapping for many transporters. Specific modulators of single or few transporters therefore often lack efficacy in overcoming chemotherapy resistance. Numerous modulators of ABC transporters have been developed and tested with variable success in human clinical trials and few in vivo studies in veterinary oncology. TKI, tyrosine kinase inhibitors; MDR, multi-drug resistance gene; MRP1, MDRassociated protein 1; BCRP, breast cancer resistance protein.

936 937

Figure 2. The ambivalence of effective DNA-repair in chemotherapy resistance

938 939 940 941

Figure 3. Potential targets for the effective treatment of cancer stem cells. Chk, checkpoint kinase; G-CSF, granulocyte-colony stimulating factor; HH, hedgehog

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