Targeting IL-8 signalling to inhibit breast cancer stem cell activity

Abstract

Although survival from breast cancer has improved significantly over the past 20 years, disease recurrence remains a significant clinical problem. The concept of stem-like cells in cancer has been gaining currency over the last decade or so, since evidence for stem cell activity in human leukaemia and solid tumours, including breast cancer, was first published. Evidence indicates that this sub-population of cells, known as cancer stem-like cells (CSCs), is responsible for driving tumour formation and disease progression. In breast cancer, there is good evidence that CSCs are intrinsically resistant to conventional chemo-, radio- and endocrine therapies. By evading the effects of these treatments, CSCs are held culpable for disease recurrence. Hence, in order to improve treatment there is a need to develop CSC-targeted therapies. Interleukin-8 (IL-8), an inflammatory cytokine, is upregulated in breast cancer and associated with poor prognostic factors. Accumulating evidence demonstrates that IL-8, through its receptors CXCR1/2, is an important regulator of breast CSC activity. Inhibiting CXCR1/2 signalling has proved efficacious in pre-clinical models of breast cancer providing a good rationale for targeting CXCR1/2 clinically. Here, we discuss the role of IL-8 in breast CSC regulation and development of novel therapies to target CXCR1/2 signalling in breast cancer.

Keywords::

• breast cancer
• cancer stem cell
• CXCR1
• CXCR2
• HER2
• IL-8
• inflammation

1. Introduction

Breast cancer is the most common cancer affecting women worldwide and although survival has improved significantly, disease recurrence remains a significant problem. Accumulating evidence indicates that tumour cells are organised in a hierarchical manner initiated by a population of self-renewing cancer stem cells (CSCs) [1]. Akin to their normal counterparts, CSCs have the capacity to self-renew and differentiate, but the mechanisms which normally strictly regulate these processes are deregulated leading to their expansion and production of aberrantly differentiated progeny [2].

CSCs are predicted to be responsible for tumour initiation, maintenance and metastases. CSCs have been identified in ductal carcinoma in situ (DCIS), a precursor to invasive disease, and invasive breast cancer. Importantly, their activity is reported to correlate with poor prognostic factors such as high tumour grade and over-expression of human epidermal growth factor receptor 2(HER2) [3-5]. Furthermore, by virtue of their intrinsic resistance to chemotherapy and radiotherapy, they can survive conventional treatments to repopulate the tumour. Consequently, they are held culpable for disease recurrence [6,7].

The first stage in developing novel therapies to target breast CSC is to identify key factors which regulate their activity. These are complex and multifaceted, and effective regimens are likely to involve a combination of approaches in tandem with existing therapies. There are at least three potential ways to target breast CSCs: i) inhibition of aberrant self-renewal signalling pathways thereby inducing differentiation or apoptosis, ii) targeting resistance mechanisms or iii) targeting factors within the tumour microenvironment which regulate CSC activity. We have previously reviewed these approaches [8]; in this article, we focus on the role of interleukin-8 (IL-8), an inflammatory cytokine within the tumour microenvironment, and discuss the rationale for developing novel therapies to target this signalling pathway.

Increasing evidence demonstrates that IL-8, via its cognate receptors CXCR1 and CXCR2, is a key regulator of breast CSC activity [9,10]. The association between inflammation and cancer is well established and deregulated expression of multiple inflammatory cytokines, including IL-8, in malignant breast disease is well recognised. Although IL-8 is known to be increased in breast cancer, the mechanisms by which IL-8 contributes to breast cancer progression have remained poorly understood. However, recent studies indicate that IL-8 may drive tumourigenesis by promoting CSC invasion, metastatic spread and treatment resistance. Targeting CXCR1/2 signalling experimentally has proved efficacious in pre-clinical models of breast cancer providing a good rationale for targeting CXCR1/2 signalling to improve treatment and outcomes in breast cancer.

2. IL-8 signalling and breast cancer

IL-8, originally described as a potent neutrophil chemoattractant [11], is a member of the CXC chemokine family. The biological effects of IL-8 are mediated by two G-protein coupled receptors (GPCRs), CXCR1 and CXCR2 [12,13]. Whereas CXCR1 is activated by IL-8 and granulocyte chemotactic protein-2 (GCP-2)/CXCL6 [14], CXCR2 is more promiscuous as in addition to IL-8, it can be activated by several other chemokines such as growth regulated oncogene (GRO)-α/CXCL1, GRO-β/CXCL2, GRO-γ/CXCL3, CXCL5, GCP-2 and neutrophil-activating protein-2 (NAP-2)/CXCL7 [14].

IL-8 is upregulated in breast cancer compared to normal breast tissue and serum IL-8 levels are elevated in breast cancer patients [15,16]. IL-8 has been implicated in disease progression since patients with metastatic breast cancer are reported to have higher serum IL-8 levels compared to those with localised disease [17], and serum IL-8 level is an independent prognostic factor for post-relapse survival in patients with metastatic breast cancer [18]. Over-expression of HER2, occurring in up to 20% of breast cancers and associated with poor survival, is reported to upregulate IL-8 and may explain the elevated levels observed in HER2-enriched tumours [19]. Increased levels of IL-8 are also associated with loss or inactivation of estrogen receptor (ER), another poor prognostic factor in breast cancer. Elevated levels of IL-8 in HER2 over-expressing and ER negative cancers may contribute to the poor outcomes of these tumour subtypes.

Constitutive activation of CXCR1/2 through ligand upregulation may serve as an adaptive response to protect breast cancer cells from the cytotoxic effect of conventional chemotherapy agents and thereby limit their clinical efficacy. Studies using breast cancer cell lines demonstrate that conventional chemotherapy can lead to the upregulation of IL-8 and other CXCR1/2 ligands. Indeed multidrug resistant breast cancer cell lines are reported to produce significantly higher IL-8 protein levels compared to non-resistant controls [20,21]. Failure of novel targeted therapies, such as PI3K/mammalian target of rapamycin inhibitors, has also been attributed to upregulation of IL-8 through alternative signalling pathways [22].

Regulation of IL-8 within the tumour micro-environment is complex, not only because of the variety of cells which can secrete it, but also the multitude of factors which can effect IL-8 expression by these different cell types. Important interactions between breast cancer cells and other cell types that comprise the tumour micro-environment, such as mesenchymal cells [23,24] and macrophages [25], which lead to upregulation of IL-8 are summarised in Figure 1.

Figure 1. Model of cytokine networks depicting the proposed interactions between cancer cells and stromal cells: production of IL-8 by cancer cells, mesenchymal cells and macrophages increases cancer stem cell activity via CXCR1/2 by triggering EGFR/HER2-dependent and -independent signalling pathways. Numbers in square brackets refer to relevant reference.

3. Regulation of breast CSC activity through CXCR1/2

The failure of standard therapies to eradicate breast cancer is postulated to be due to the intrinsic resistance of CSCs which survive to repopulate the tumour. It is becoming increasingly evident that CXCR1/2 signalling is important in regulating breast CSCs activity underscoring the need to develop CXCR1/2 targeted therapies. Gene expression studies demonstrate that CXCR1 is over-expressed in breast CSCs compared to bulk tumour cells, and IL-8 can increase their invasive capacity as well as increase the proportion of CSCs in vitro [10,26]. Importantly, in xenograft mouse models of breast cancer, inhibition of CXCR1/2 is reported to synergise with chemotherapy agents to retard tumour growth and inhibit metastases [9]. Interestingly, we recently reported a significant direct correlation between metastatic fluid IL-8 level and CSC activity when cells from such fluid were cultured ex vivo, suggesting that cancers with elevated IL-8 levels have greater CSC activity [10]. IL-8 may also promote a state of ‘stemness' by inducing epithelial-mesenchymal transition (EMT) [27], a process which is implicated in the acquisition of stem cell traits through the regulation of EMT transcription factors, such as ZEB1 [28-31].

Recent in vivo studies indicate that tumour growth can be accelerated by recruitment of bone marrow-derived mesenchymal stem cells (MSCs) to sites of developing tumours. Co-localisation of MSCs with breast CSCs is reported to increase the proportion of breast CSCs; an effect which is proposed to be initiated by cancer cell-derived IL-6 and sustained by IL-8 and other CXCR1/2 ligands released from both the cancer cells and mesenchymal cells [23]. Cancer cell derived IL-1 can induce expression of IL-8 and CXCL1/GRO-α by MSCs thereby contributing to the formation and maintenance of CSCs (Figure 1) [24].

Like other GPCRs, ligand activation of CXCR1/2 is known to activate multiple signalling cascades such as PI3K/AKT, PLC/PKC, Ras/Raf/ERK1/2, FAK, Rho and Rac [32]. We recently reported new insights into CXCR1/2 signalling in breast cancer by demonstrating transactivation of HER2 on ligand activation of CXCR1/2 (Figure 1) [10]. This was SRC-dependent and led to the activation of AKT and ERK1/2 signalling pathways which are known to be critical in regulating breast CSC activity [10,33]. Inhibition of HER2 activity abrogated the CSC promoting effect of IL-8 in both HER2-positive and -negative primary breast cancers indicating that the functional effects of CXCR1/2 are, at least in part, dependent on HER2. In HER2 positive cancers, CXCR1/2 inhibition added to the efficacy of HER2 inhibition [10]. Since HER2 is known to be important in regulating CSC activity, aberrant activation of this pathway through CXCR1/2 could have important biological consequences especially in tumours expressing high levels of IL-8 and other CXCR1/2 agonists.

4. Expert opinion: development of novel therapies to target CXCR1/2 signalling in breast cancer

Targeting CXCR1/2 signalling can be broadly approached by either interfering with ligand function/sequestration or receptor function. A summary of clinical trials utilising IL-8 and CXCR1/2 targeting agents in breast cancer and other diseases is presented in Table 1. Antibodies against IL-8 have demonstrated efficacy in preclinical models of bladder cancer [34] and melanoma [35]. Clinical trials in patients with chronic inflammatory diseases associated with increased production of IL-8, such as palmoplantar pustulosis, report that monoclonal humanised antibodies to IL-8 are clinically effective and well tolerated [36]. In breast cancer, targeting IL-8 alone may be of limited benefit since other CXCR1/2 agonists are co-regulated with IL-8, such as CXCL1/GRO-α, CXCL2/GRO-β and CXCL5 [37]. This problem can be overcome by inhibiting CXCR1/2.

Table 1. Summary of clinical trials of IL-8 and CXCR1/2 targeting agents in breast cancer and other diseases.

Agent	Pharmaceutical company	Clinical trial	Description
Anti-IL-8 antibodies
ABX-IL8	Abgenix	NCT00035828 Phase II (completed)	Evaluate safety and efficacy in patients with chronic bronchitis and COPD
HuMax-IL8 (HuMax-Inflam/MDX-018)	Genmab A/S	Phase I/II (completed) [36]	Evaluate safety and efficacy in patients with palmoplantar pustulosis
Decoy protein
PA401 (IL-8 mutant)	ProtAffin Biotechnologie AG	NCT01627002 Phase I (active, not recruiting)	Assess safety, tolerability, immunogenicity and pharmacokinetics
Small molecule antagonists of CXCR1/2
GSK1325756	GlaxoSmithKline	NCT01209052 Phase I (completed)	Assess the safety, tolerability, pharmacokinetics and pharmacodynamics
GSK1325756	GlaxoSmithKline	NCT01209104 Phase I (completed)	Evaluate the food-effect and proton-pump inhibition on the pharmacokinetics and safety
GSK1325756	GlaxoSmithKline	NCT01453478 Phase I (completed)	Evaluate the relative bioavailability of different drug formulations during suppression of gastric acid secretion
AZD5069	AstraZeneca	NCT01480739 Phase I (completed)	Assess neutrophil number and function in peripheral blood
SCH 527123	Merck	NCT00688467 Phase II (completed)	Evaluate the efficacy and safety in subjects with allergen-induced asthma
Reparixin	Dompé spa	Phase Ib (recruiting) [44]	Assess effects in tumour growth in combination with standard chemotherapy in patients with HER-2 negative metastatic breast cancer
Reparixin	Dompé spa	NCT01861054 Phase II (recruiting)	Investigate safety and effects on CSCs in early breast cancer patients in the time period between clinical diagnosis and surgery
Reparixin	Dompé spa	NCT00224406 Phase II (completed)	Investigate safety and efficacy in the prevention of primary graft dysfunction after lung transplantation
Reparixin	Dompé spa	NCT00248040 Phase II (completed)	Assess safety and efficacy in the prevention of delayed graft function after kidney transplantation
Reparixin	Dompé spa	NCT01817959 Phase III (recruiting)	Assess safety and efficacy in the prevention of graft dysfunction after pancreatic islet transplantation
DF2156A	Dompé spa	NCT01571895 Phase II (terminated)	Evaluate safety and efficacy in patients with active bullous pemphigoid

Trials in breast cancer are highlighted in bold. Numbers in square brackets refer to relevant reference.

NCT: National clinical trial number; COPD: chronic obstructive pulmonary disease.

Several orally active, small molecule, non-competitive antagonists of CXCR1 and CXCR2 are currently available. SCH563705 (Merck) has particularly high binding affinities to CXCR1 and CXCR2 and proved effective at inhibiting primary human breast CSC activity ex vivo [10]. Others such as reparixin (Dompé spa), SCH479833 (Merck) and SCH527123 (Merck) have demonstrated anti-tumour effects in xenograft models of breast cancer [9], colorectal cancer [38] and melanoma [39]. Other pharmaceutical companies, including AstraZeneca and GlaxoSmithKline, are also developing novel CXCR1/2 inhibitors (Table 1). These compounds are, however, still in the early stages of drug development. Phase I trials demonstrate that reparixin, originally developed to prevent IL-8-induced reperfusion injury, is well tolerated in healthy volunteers [40]. Similarly, SCH527123 has proved to be safe when trialled in patients with severe asthma [41]. Based on recent evidence that CXCR1/2 inhibition can inhibit breast CSC self-renewal and metastases in vivo [9], clinical trials are underway to determine the safety and efficacy of reparixin in combination with chemotherapy agents in patients with advanced breast cancer [42-44]. Due to the heterogeneous levels of IL-8 and other CXCR1/2 ligands in breast cancer, tumours with higher cytokine levels, such as ER-negative or triple negative tumours may be more responsive to CXCR1/2 inhibitors than those with lower levels. CXCR1/2 inhibitors could improve outcomes in these specific subtypes which are notoriously less responsive to standard chemotherapy regimens. Consequently, ligand burden needs be considered when recruiting and evaluating the efficacy of such inhibitors as only subgroups of patients may derive benefit. Moreover, in triple negative breast cancers, recent evidence indicates that IL-8 expression is coordinated with IL-6, another pro-inflammatory cytokine which has also been shown to induce and promote breast CSC activity [45-47]. Dual inhibition of IL-8/IL-6 was more effective at inhibiting colony formation and tumour engraftment compared to either alone [45]. Hence, combined inhibition of multiple cytokine signalling pathways important in regulating CSC activity may increase the efficacy of CXCR1/2 targeted therapies.

As described above, CXCR1/2 signalling is dependent on HER2 activity and inhibition of CXCR1/2 has been shown to add to the efficacy of inhibiting HER2 in HER2 positive breast CSCs [10]. Targeting CXCR1/2 in combination with HER2-targeted therapies has the potential to deliver a more effective treatment strategy to eradicate HER2 positive CSCs which may help to improve the survival of HER2 positive patients and warrants clinical evaluation.

Due to the pleiotropic effects of IL-8 it is possible that CXCR1/2 inhibitors could have unexpected toxicities. In addition to promoting tumourigenesis via multiple mechanisms, IL-8 can exert anti-tumour effects through neutrophil recruitment [48]. Neutrophils, cytotoxic T cells, T helper cells and natural killer cells form part of the immune surveillance system which operates to detect and eradicate cancer cells [49]. Hence, targeting the IL-8 signalling pathway could inadvertently promote tumour growth by abrogating the anti-tumour effects of neutrophil infiltration; it remains unknown whether this could have an effect on breast cancer progression. Whilst this primarily remains a theoretical concern, there is some evidence that IL-8 suppression can increase ER negative tumour growth in vivo in association with reduced tumour neutrophil recruitment [50]. Depending on the clinical context within which they are used, the anti-inflammatory effect of CXCR1/2 inhibitors may in fact help to prevent lung damage during radiotherapy which could be beneficial for some patients [51]. CXCR1/2 inhibitors have been shown to reduce circulating neutrophil counts with the potential for synergistic myelo-toxicity when combined with chemotherapeutic agents [41]. Targeted drug delivery specifically to the cancer cells using novel technologies could minimise these effects.

In conclusion, recent advances reveal that IL-8 signalling is a key regulator of breast CSC activity representing a promising therapeutic target. Results from clinical trials investigating the efficacy of CXCR1/2 inhibitors with chemotherapy agents in breast cancer are eagerly awaited and will hopefully pave the way for future trials combining CXCR1/2 inhibitors with other therapies to improve outcomes in both the adjuvant and advanced settings.

Declaration of interest

The authors state no conflict of interest and have received no payment in preparation of this manuscript.