EGFR remains the only non-chemotherapy molecular target that has been successfully translated into a biological therapy with clinical efficacy . It is supported by high EGFR protein expression (in approximately 90% of HNSCCs). TGF-α and amphiregulin activate EGFR (a member of the ErbB/HER family of tyrosine kinases receptors) to phosphorylate and activate critical proteins, such as those in the PI3K, RAS or Scr kinase pathways, that also control proliferation, angiogenesis and metastasis and the differentiation of epidermal and mesenchymal cells.
The mechanisms employed in therapeutic targets against EGFR include monoclonal antibodies (against the extracellular domain of EGFR), such as cetuximab, panitumumab, zalutumumab and nimotuzumab, and small molecules, including tyrosine kinase inhibitors (TKI) that bind to the intracellular region of EGFR, such as gefitinib, erlotinib, lapatinib, afatinib and dacomitinib. These EGFR inhibitors have been tested in several clinical trials, including phase III trials, with discouraging results, except for cetuximab.
3.1.1. Anti-EGFR Monoclonal Antibodies: Cetuximab
Cetuximab is a monoclonal antibody that binds to EGFR and alters the TK-mediated signal transduction pathway. The drug is active in colon cancer and SCCHN patients. For locally advanced disease, the use of a combination of cetuximab and radiotherapy has shown to benefit survival compared to the use of radiotherapy alone as radical treatment. Cetuximab is an active treatment in platin-refractory patients with recurrent/metastatic disease.
The overexpression of EGFR in SCCHN and histologically normal tissue adjacent to tumour tissues implicates EGFR in SCCHN carcinogenesis. Some evidence suggests that the amplification of the EGFR gene may contribute to EGFR overexpression in malignant tissue . The most common mutated form of EGFR contains a six-exon deletion that encode a 268-amino acid section of its extracellular domain. This mutant, EGFRvIII, is expressed only in cancer cells . EGFRvIII expression has been described in cancers of the brain, lung, breast and prostate . Because mutant EGFR is expressed in cancer cells but not in normal epithelial cells, a mutant EGFR-targeting agent would not interfere with normal EGFR signalling and would therefore have great potential for use as a highly specific targeted approach to treat these cancers. In SCCHN, EGFR is overexpressed in 80% to 90% of tumours, and the clinical relevance of EGFR overexpression as an independent prognostic factor in SCCHN has been well documented . High tumour levels of EGFR are correlated with advanced stage, increased tumour size, decreased survival and decreased sensitivity to radiation treatment [14,15,16,17]. The aberrant functionality of the EGFR network observed in SCCHN provides compelling evidence for a relationship between EGFR and the development and progression of SCCHN and suggests a role for EGFR as a target for cancer therapies.
Cetuximab (IMC-225; Im-Clone Systems, Bridgewater, NJ, USA) [18,19] is a chimeric mouse-human monoclonal antibody that binds EGFR at its extracellular domain and blocks EGF-induced autophosphorylation in EGFR cell lines in vitro . It also induces the dimerization and downregulation of EGFR, perturbs cell cycle progression by inducing G1 arrest through an increase in the protein level of p27, an inhibitor of cyclin-dependent kinases, and inhibits tumour-induced angiogenesis . Cetuximab has been shown to have preclinical activity in vitro and in vivo as both a single agent and in combination with cytotoxic agents and radiotherapy in a wide range of human cancer cell lines, including colorectal, pancreatic, prostate, head and neck and ovarian cancer cells.
In phase I studies , doses from 5 to 400 mg/m2 have been explored without reaching a maximum tolerated dose (MTD). Pharmacokinetics analyses have shown non-linear behaviour for this drug, with saturation of drug clearance at doses over 200 mg/m2. Therefore, the dose regimen selected for phase II-III trials was a loading dose of 400 mg/m2 followed by a weekly maintenance dose of 250 mg/m2 . Phase I trials revealed favourable tolerability, with the most significant reported toxic effects being an acneiform rash and folliculitis involving the face and upper chest, which occurred in 80% of the patients . Hypersensitivity reactions, although uncommon (<5%), have been reported, with some of them occurring within minutes of the infusion. These were rarely life-threatening. Other adverse effects include asthenia, fever, hypomagnesia and changes in the results of liver function tests.
3.1.2. Cetuximab and Radiotherapy
In vivo experiments have demonstrated enhanced antitumour activity in mice treated with cetuximab and radiotherapy [24,25]. In a study by Milas et al., cetuximab improved the efficacy of local tumour irradiation, particularly when multiple injections of cetuximab were administered . Tumour radio-responsiveness was enhanced by a factor of 1.59 by a single dose and by a factor of 3.62 by three doses of cetuximab. Histological analyses of tumours revealed that cetuximab caused a striking increase in central tumour necrosis that was associated with haemorrhage and vascular thrombosis when combined with radiotherapy. In this report, the authors demonstrated that cetuximab greatly enhanced in vivo tumour responsiveness to radiation, and this effect was greater than the sum of growth delays caused by the individual treatments.
In another phase I study conducted by Robert et al., a total of sixteen patients were treated using five successive treatment schedules consisting of cetuximab and radiotherapy . Cetuximab was delivered as a loading dose at 100 to 500 mg/m2 and was followed by weekly infusions of 100 to 250 mg/m2 for 7 to 8 weeks with conventional radiotherapy (RT) or hyperfractionated RT. In this trial, the recommended phase II/III dose was defined as a loading dose of 400 to 500 mg/m2 and a maintenance weekly dose of 250 mg/m2. The most commonly adverse events were fever, asthenia and skin toxicity (grade 1 to 2 in most patients). Of the 16 included patients, 13 achieved a complete response. This was the first report wherein the activity and safety of cetuximab and radiotherapy were documented. However, in trials wherein cetuximab has been combined with RT and chemotherapy (CT), the results have been contradictory . Thus, in a study by Pfister et al. that included patients with SCCHN stage III/IV who were treated with a first line treatment of RT, cisplatin at 100 mg/m2 for weeks 1 and 4 and cetuximab (400 mg/m2 in week 1 followed by 250 mg/m2 during weeks 2 to 10) was closed as a result of significant adverse events, including three deaths (one from myocardial infarction, one from bacteremia and one from atrial fibrillation). There were not significant difference in survival. For this reason, the authors did not recommended this regimen except in a clinical trial setting. In our opinion, a weekly schedule of CT and cetuximab should be explored in combination with radiotherapy to allow this form of CT administration to better manage toxicity compared to the administration of a high dose of cisplatin every three weeks.
The efficacy of cetuximab and radiotherapy was also analysed by Bonner et al. . In a multinational randomized phase III trial, radiotherapy alone was compared to RT plus cetuximab in the treatment of locoregionally advanced SCCHN. Patients with stage III/IV head and neck cancer of the oropharynx, hypopharynx or larynx were randomly assigned to treatment with high dose radiotherapy alone (n = 213) or high dose RT plus weekly cetuximab (n = 211). In this trial, the investigators were required to select one of three radiotherapy-fractionation regimens, including once daily, twice daily or a concomitant boost up to 70–72 Gy. In the group assigned to receive radiotherapy plus cetuximab, administration of intravenous cetuximab was initiated one week before radiotherapy at a loading dose of 400 mg/m2 followed by weekly 60-min infusions of 250 mg/m2 for the duration of radiotherapy. Patients were stratified according to Karnofsky Performance Status (60 to 80 vs. 90 to 100), nodal involvement (N0 vs. N+), tumour stage (T1–T3 vs. T4) and radiation-fractionation regimen (concomitant boost vs. once daily vs. twice daily). The primary end point of this study was the duration of control of the locoregional disease. With a median follow-up of 54 months, the median duration of locoregional control was 24.4 months among patients treated with cetuximab and 14.9 months among those that received radiotherapy alone (p = 0.005). The median duration of overall survival was 49 months among patients treated with combined therapy and 29.3 months in those treated with RT alone (p = 0.03). There was a 26% reduction in the risk of death in the group that received radiotherapy plus cetuximab compared to the group that received radiotherapy alone (hazard ratio, 0.74; 95% confidence interval, 0.57 to 0.97). We emphasize that locoregional control and overall survival were superior in the patients in whom an oropharynx primary tumour was treated with cetuximab/radiotherapy than other in those with other localizations, probably due to HPV prevalence, or in patients who received a concomitant boost of radiotherapy and cetuximab. However, the different arms were well balanced with respect to the primary site of the tumour and the type of radiotherapy, with a concomitant boost of radiotherapy and cetuximab being the best combination schedule. The toxicity profile was similar in both arms, with the exception of an acneiform rash and infusion reactions. Cetuximab did not exacerbate the common toxic effects associated with radiotherapy of the head and neck, including mucositis, xerostomia, dysphagia, pain and performance status deterioration. The results of this phase III trial demonstrated that the combination of cetuximab and RT is better than radiotherapy alone in locally advanced head and neck cancer and that it has a manageable toxicity profile. Therefore, this combination could be the standard treatment in certain settings, such as older patients or those with poor performance status or stage III/IV disease, in whom CT administration cannot be associated with radiotherapy. By other hand, probably there are a set of patients where RT alone is likely best.
Currently, there are several ongoing clinical trials that aim to compare cetuximab/RT with chemoradiotherapy to define the optimal treatment in this set of patients. These trials always take into account the excellent toxicity profile of cetuximab and radiotherapy. Until now, none of these trials has demonstrated a benefit with respect to a combination between cetuximab/chemoradiotherapy, inclusive of those in which there were increased side effects without a benefit in response rate or survival.
3.1.3. Cetuximab as a Single Agent and in Combination with Chemotherapy in Recurrent/Metastatic SCCHN
Every year, 72,000 patients in Europe are diagnosed with recurrent/metastatic SCCHN, for which treatment options are limited. Palliative chemotherapy with cisplatin regimens is the principal option, and it has a median survival time of 6–8 months . No standard second-line treatment after progression while on cisplatin chemotherapy is currently available. The results of a retrospective study by León, Hitt, et al. that analysed clinical records from 151 patients with SCCHN refractory to cisplatin chemotherapy showed that most of them had received only best supportive care and that they showed a median survival of 56 days. In this analysis, 28% of the patients received second-line chemotherapy without an objective response, and these had similar survival .
An analysis of the results of this retrospective study in combination with the problems that are frequently seen in clinical practice in this set of patients indicates that additional treatment options are urgently required for the management of this disease.
Baselga et al. reported the results of a phase II trial in which patients with recurrent/metastatic SCCHN refractory to cisplatin received cetuximab as second-line treatment. In this study, the overall response and disease control rates were 10% and 53%, respectively, and the duration of response was 153 days. Subgroup analysis revealed that there was a trend toward favouring a high response and disease control in older patients, those with higher KP, and those who did not present metastases. In regard to surrogate markers for responsiveness, although significant differences were not observed, there was a trend for patients developing grade 1 or 2 skin reactions in response to treatment (60%) to achieve slightly prolonged times for progression and overall survival compared to patients without skin reactions (39%) . It should be emphasized that there was a low frequency of grade 3/4 adverse events (<15%) in this trial, with skin reactions and acne-like rashes being those most frequently observed. The most interesting aspect of this study was that cetuximab may have reversed resistance to treatment with cisplatin with an acceptable safety profile in this population of patients with poor prognosis for whom there are no current recommended standard treatments.
Herbst et al. reported the results of another phase II clinical trial that studied the efficacy and safety of cetuximab administered with cisplatin to patients with refractory metastatic or recurrent SCCHN disease . Following treatment with cisplatin/paclitaxel or cisplatin/fluoruracil, patients with stable or progressive disease received combination therapy with cetuximab and cisplatin. In this trial, up to 20% of the patients with progressive disease (PD) and up to 18% of the patients with stable disease (SD) responded to the combination treatment. The median overall survival was 6.1 and 11.7 months (for PD and SD, respectively). The most common toxicities were anaemia, acne-like skin rash, fatigue and nausea-vomiting. Of these patients, 5% developed a grade 3 or 4 hypersensitivity reaction to cetuximab. The results of this trial confirmed the activity of cetuximab in patients with refractory disease and that prolonged survival was observed in the patients who showed no response to previous therapy with cisplatin.
A phase III, randomised, placebo-controlled clinical trial was conducted by the Eastern Cooperative Oncology Group to determine whether the addition of cetuximab to cisplatin therapy improves progression-free survival in metastatic/recurrent SCCHN compared to cisplatin alone . A total of 121 patients were included in the study, and an objective response was seen in 9.8% of the cisplatin/placebo patients compared to 26.3% of the cisplatin/cetuximab patients (p = 0.03). The median progression-free survival time for the control arm was 2.7 months, whereas it was 4.2 months for the cisplatin/cetuximab group (p = 0.09). The median overall survival was 8.0 months for patients receiving cisplatin/placebo compared to 9.2 months for patients receiving cisplatin/cetuximab treatment (p = 0.21). A significant survival advantage was seen for the development of rash (the hazard ratio for survival by skin toxicity in cetuximab-treated patients was 0.42). In this study, there was a significant benefit in response rate from the addition of cetuximab, albeit progression-free and overall survival were not significantly improved in these patients. The limitations of the study include the limited sample size, the significant drop-off rate in both arms and the fact that the median progression-free survival in the control arm was better than had been projected based on historical experience. All these factors could explain the failure of the primary end-point.
3.1.4. Actual Regimens
An Extreme study to compare cisplatin/fluouracil alone vs. the same schedule plus cetuximab was conducted in Europe. In this trial, patients with recurrent/metastatic disease were included in a front line treatment group, and the primary end-point of the study was overall survival. In this trial, 442 patients were included. The addition of cetuximab to platinum-fluouracil significantly prolonged median survival times from 7.4 months in the chemotherapy alone group to 10.1 months in the group that received chemotherapy plus cetuximab (hazard ratio for death, 0.80; 95% confidence interval, 0.64–0.99; p = 0.04). The addition of cetuximab also prolonged the median progression-free survival time from 3.3 to 5.6 months (hazard ratio for progression, 0.54; p < 0.001) and increased the response rate from 20% to 36% (p < 0.001). Moreover, a protocol-defined subgroup analysis showed that the beneficial effects that resulted from adding cetuximab to chemotherapy in overall survival and progression-free survival were evident in nearly all subgroups. This was the first time in over 30 years that the superiority of a new regimen over the standard platinum-based combination chemotherapy was observed. A combination cetuximab and platinum-based chemotherapy is now considered a new standard for the treatment of recurrent/metastatic HNC (reference EXTREME) .
Hitt et al. reported a phase II trial  to test a combination of paclitaxel/cetuximab. Patients received paclitaxel (80 mg/m2) and cetuximab (400/250 mg/m2) weekly until the disease progressed or unacceptable toxicity was observed. The primary endpoint was the response rate. Among the 46 patients enrolled, the overall response rate was 54% (95% CI 39–69), with 10 (22%) showing complete responses and a disease control rate of 80%. The median progression-free and overall survival times were 4.2 months (95% CI 2.9–5.5 months) and 8.1 months (95% CI 6.6–9.6 months), respectively. Common grade 3/4 adverse events included acne-like rash (24%), asthenia (17%) and neutropenia (13%). Prior chemotherapy and the development of acne-like rash were associated with tumour responsiveness but not survival. No association between tumour EGFR expression or EGFR gene copy number and response or survival was found.
The combination of cetuximab and weekly paclitaxel was active and well tolerated by these poor prognosis patients and may therefore be an option for the treatment of medically unfit patients, particularly those for whom platinum is contraindicated, however this is not a standard treatment.
3.1.5. Anti-EGFR non-Cetuximab Monoclonal Antibodies
After good results were observed for cetuximab, the development of new anti-EGFR antibodies was pursued in HNSCC [35,36,37,38]:
Panitumumab is a fully human anti-EGFR monoclonal antibody that was approved in combination with chemotherapy for the treatment of metastatic RAS wild-type colorectal cancer. In R/M HNSCC, the phase III SPECTRUM trial  randomized 657 patients to receive panitumumab plus platinum-based chemotherapy and 5-Fluorouracil, and patients could continue panitumumab until progression or unacceptable toxicity was observed. This treatment was tested vs. chemotherapy alone. The primary endpoint was overall survival (OS). The median OS was 11.1 months in the panitumumab arm and 9 months in the control arm (p = 0.14), and no statistically significant improvement was observed in the primary endpoint. Median progression-free survival (PFS) was 5.8 months in the panitumumab arm and 4.6 months in the control arm (HR 0.78 p = 0.0036), which was statistically significant but clinically modest. This trial also analysed HPV status by assessing p16 levels in the tumour samples of 443 patients. Of these, 22% were p16-positive and 78% were p16-negative. There was no difference in the median OS or PFS in patients who were p16-positive (OS: 11 months in the panitumumab arm and 12.6 months in the control arm; PFS: 5.6 months in the panitumumab arm vs. 5.5 months in the control arm). However, the p16-negative subgroup median OS (11.7 months in the panitumumab arm and 8.6 months in the control arm) and PFS (6 months vs. 5.1 months) were significantly longer, suggesting that p16 status could be a predictive biomarker for responsiveness in patients treated with panitumumab (or other anti-EGFR drugs). The most common adverse events registered in the trial that were associated with panitumumab were typical anti-EGFR side effects: skin toxicity, metabolic toxicity (including hypomagnesaemia or hypokalaemia), diarrhoea and cardiac arrhythmias.
These results are discordant in survival with those of the EXTREME trial  despite the fact that they share a similar design. Hypotheses that may explain this include: molecular differences between panitumumab and cetuximab, differences in the mechanisms of action (cetuximab can activate an antitumour immune response, but panitumumab does not), the type of population enrolled in both trials (most patients were from western Europe in the EXTREME trial, whereas in the SPECTRUM trial, most were from Eastern Europe and the Asia-Pacific region) or treatments prior to enrolment (38% received previous chemotherapy in the EXTREME trial, whereas 81% did so in the SPECTRUM trial). Although we do not know the cause of these differences, the fact is that panitumumab failed to improve OS in the phase III SPECTRUM trial.
Zalutumumab is a human monoclonal antibody against EGFR that has shown activity in preclinical models (both by blocking the EGFR signalling pathway and by stimulating an antitumour immune reaction by antibody-dependent cellular cytotoxicity (ADCC)).The results of a phase III trial  of this drug were published in 2011 to compare zalutumumab vs. best supportive care in patients with R/M HNSCC after the failure of platinum-based chemotherapy. The randomization was 2:1, with 191 patients in the zalutumumab arm and 95 in the control arm. The primary endpoint was OS, and the results were 6.7 months in the zalutumumab arm and 5.2 months in the control arm (p = 0.0648). The secondary endpoint was PFS, which was slightly better in the zalutumumab arm (9.9 vs. 8.4 weeks) (p = 0.0012). Rash was the most common toxicity (92%) in the experimental arm, followed by anaemia, pyrexia, diarrhoea and hypomagnesaemia. This study did not at all support the use of zalutumumab in R/M HNSCC after platinum progression, however new combination of zalutumumab could have been promising.
Nimotuzumab is a humanized anti-EGFR antibody that has been widely explored in local or locally advanced HNSCC alone or concurrent with radiotherapy, but there are no records indicating its use in R/M HNSCC. There is an ongoing phase II study of chemotherapy (cisplatin and 5-FU) combined With Nimotuzumab in untreated metastatic nasopharyngeal carcinoma (NCT01616849).
3.1.6. Small Molecule Tyrosine Kinase Inhibitors (TKIs)
Erlotinib and gefitinib are oral small molecules that reversibly inhibit the tyrosine kinase (TK) activity of EGFR. Lapatinib is a reversible dual inhibitor of EGFR/Her-2 TK activity, and afatinib is an irreversible pan-inhibitor of the ErbB family. The first trials of these drugs explored the activity of the reversible TKIs (erlotinib and gefitinib) after first-line treatments for R/M HNSCC.
Gefitinib was compared to methotrexate at different doses (250 or 500 mg/day) in a phase III trial for R/M HNSCC . The median OS was 5.6 months (gefitinib 250 mg), 6 months (gefitinib 500 mg) and 6.7 months (methotrexate), with no significant differences. The safety profiles were different: the most common adverse effects were stomatitis for methotrexate and skin reactions and diarrhoea for gefitinib. In conclusion, gefitinib did not improve survival in R/M HNSCC compared to methotrexate, and higher doses of gefitinib were not associated with better outcomes. Another strategy explored for gefitinib was its use in combination with chemotherapy (docetaxel) in a phase III trial by the Eastern Cooperative Oncology Group (ECOG) . The dose chosen for gefitinib was 250 mg/day. However, the study did not meet the primary endpoint (OS): 7.3 months for the combination of gefitinib and docetaxel vs. 6 months for docetaxel alone (p = 0.6). The incidence of diarrhoea was higher for the combination arm.
For Erlotinib, there are no results from phase III trials in this setting because it showed limited activity in two previous phase II trials [43,44]. In one of these, erlotinib was used as a monotherapy at a dose of 150 mg/day in patients with R/M HNSCC after primary treatment. The overall response rate was 4.3%, and the median PFS was 9.6 weeks, although 38% of patients showed disease stabilization for 16 weeks. The very low response rate in erlotinib was improved by its combination with cisplatin (21%) in the other phase II trial, which showed a median PFS for these patients of 3.3 months. Currently, there is a randomized phase II study to test docetaxel and cisplatin with or without erlotinib in patients with R/M HNSCC.
Lapatinib has shown a lack of activity in R/M HNSCC. In a phase II trial  of a monotherapy dose of 1500 mg daily, there was no objective response in 45 of the patients. The best response was stable disease, which was observed in the cohort of patients that was never exposed to an anti-EGFR therapy, and the PFS was 52 days. Half of the patients experienced diarrhoea.
Afatinib (an irreversible pan-ErbB inhibitor), by contrast, showed better activity in a phase II trial  that compared it with cetuximab using a two-stage design (the first stage was a comparison of afatinib and cetuximab, and the second stage permitted crossover to the other therapy). The primary endpoint was mean tumour shrinkage during the first stage. In all, 121 patients began stage 1, and 68 began stage 2 (the principal reason for crossover was the progression of the disease). The doses were afatinib at 40 mg/day and cetuximab at 250 mg/m2/weekly. In the afatinib arm, the mean tumour shrinkage was assessed by an investigator (IR) and independent central review (ICR). The IR/ICR was 10.4%/16.6% for afatinib and 5.4%/10.1% for cetuximab (p = 0.46/0.30). The objective response rate was 16.1% (IR) for afatinib and 6.5% (IR) for cetuximab. The median PFS was comparable: 13 weeks (afatinib) and 15 weeks (cetuximab) by ICR. In the result of stage 2, the disease control rate by ICR was 33.3% for afatinib and 18.8% for cetuximab, and the PFS was 9.29 weeks in the afatinib arm and 5.71 weeks in the cetuximab arm (p = 0.077). Side effects were more common in the afatinib arm, especially for rash/acne, diarrhoea and stomatitis, but they were manageable. These results supported the use of a sequential anti-EGFR treatment for some HNSCC patients.
After this phase II trial, a trial for afatinib was developed in a programme called LUX-H & N . Their recently published phase III trial (LUX-H & N1) included patients with R/M HNSCC after platinum-based therapy progression. A total of 483 patients were randomized to afatinib 40 mg/day (322 patients) or methotrexate 40 mg/m2 once weekly (161 patients). The primary endpoint was PFS, and the results were statistically positive: 2.6 months for afatinib vs. 1.7 months for methotrexate (HR 0.8 p = 0.03). However, we did not observe these differences in OS: 6.8 months for afatinib and 6 months for methotrexate. In the subgroup analyses, the benefit to PFS was greater in the afatinib arm for naïve EGFR inhibitor patients and for p16-negative tumours. Although this trial is positive in its primary endpoint, the lack of benefit to OS indicates that applying afatinib in routine clinical practice would be premature .
Dacomitinib (PF-00299804) is a novel irreversible pan-ErbB TK inhibitor. Its activity was explored in a phase II trial as a first line treatment in R/M HNSCC. A total of 69 patients were included, and they received dacomitinib at 45 mg/day. Only 63 patients were evaluable for ORR, which was 12.7% and therefore comparable to cetuximab as a monotherapy. Median PFS and OS were 12.1 weeks and 34.6 weeks, respectively. There were no surprises in its toxicity profile, which is similar to the results for the other anti-EGFR therapies previously mentioned [49,50].
3.2. VEGF Pathway: Targeting VEGF and PDGFR
VEGF is overexpressed is most HNSCCs, and it is related to poor outcomes in these patients. It has been shown that hypoxia stimulates proangiogenic factors and is associated with radiotherapy resistance. The therapeutic approaches against this pathway include monoclonal antibodies, such as bevacizumab (anti-VEGF), or multi-kinases inhibitors, such as sorafenib, sunitinib or vandetanib .
Bevacizumab is the monoclonal antibody with the most indications in medical oncology. This drug is approved for the treatment of metastatic breast cancer (not by the FDA), colorectal cancer, lung tumours, gynaecologic tumours (ovarian and cervix) and kidney cancer. The development of the drug in HNSCC has not been very successful due to the high risk of bleeding in these tumours. A phase II trial  was performed in combination with cetuximab in R/M HNSCC that excluded patients with tumours that invaded major vessels. The dose chosen for bevacizumab was 15 mg/kg. Among the 46 included patients, the ORR was 16% and the median PFS and OS were 2.8 months and 7.5 months, respectively. Even with the selection of patients, 12 of them suffered bleeding events of different grades. These results improved when the drug was combined with pemetrexed  (median PFS 5 months, median OS 11.3 months), but bleeding events were also worse (2 fatal events). Some questions that are pending with bevacizumab are: the optimal dose to minimize the risk of bleeding without jeopardizing efficacy and the search for a biomarker.
Sunitinib is an oral molecule that inhibits multiple TKIs (VEGFR-1, VEGFR-2, VEGFR-3, and PDGF) and that is approved for the treatment of renal carcinoma or GIST. However, its development in other tumours, such as breast cancer, has been stopped. The GORTEC group published a phase II trial of sunitinib at 37.5 mg daily in progressed HNSCC patients. The best response was stable disease (50%), and the median PFS and OS were very low (2–3.4 months). There was a high incidence (16%) of grade 3 to 5 bleeds .
Sorafenib is a multikinase inhibitor (VEGFR-2, VEGFR-3, PDGFR, and c-KIT) of tumour progression that acts through the MAPK family. The SWOG trial demonstrated a disease control rate of 51% (although PR was only 2%), and, interestingly, a median PFS of 4 months and a median OS of 9 months. Compared to sunitinib, the toxicity profile was more favourable for sorafenib (hand-food toxicity and diarrhoea) .
Here, we will briefly mention Vandetanib because a phase II trial that combined its use with docetaxel, drug with efficacy in head and neck cancer, did not show activity in R/M HNSCC patients . There are currently ongoing trials for bevacizumab, including a phase III trial of chemotherapy with or without bevacizumab (NTC00588770), and sorafenib, including a phase I/II trial of sorafenib in combination with cisplatin and docetaxel (NTC02035527).
3.3. Targeting the PI3K Pathway
Many growth factor receptors are involved in the activation of the PI3K pathway [8,57], including EGFR, insulin-like growth factor receptor (IGFR) and FGFR. This activation triggers a cascade of signals that stimulates protein kinase B (AKT) and the mammalian Target of Rapamycin (mTOR). The PI3K/Akt/mTOR pathway is involved in the development of multiple tumours. Specifically, we have mentioned that mutations in PI3K are found in more than 30% of HNSCC (both HPV-negative and HPV-positive tumours); hence, while targeting this pathway has become a challenging, it serves a useful purpose.
To outline the results of these trials, the components of PI3K are a p110 catalytic subunit (p110α, p110β, p110δ) and a p85 regulatory subunit. To attack this pathway, different PI3K inhibitors have been developed, including mTOR inhibitors (temsirolimus or everolimus), Pan-PI3K inhibitors (buparlisib or PX-866) and p110α inhibitors (BYL719).
3.3.1. mTOR Inhibitors
Temsirolimus (an analogue of rapamycin) has been explored in monotherapy and in combination with Erlotinib. The TEMHEAD trial recruited 40 patients with R/M HNSCC after platinum-based chemotherapy or cetuximab. Of those recruited, 57% of the patients achieved stable disease, and in 39% of the patients, the tumour shrunk. Both median PFS and OS were short (56 and 152 days, respectively). Toxicity is worth mentioning because almost 50% of the patients experienced fatigue, 25% experienced anaemia, and 20% experienced pneumonia .
The results of using temsirolimus in combination with erlotinib were even worse. Only 12 patients participated in this trial, which used a dose of 150 mg of erlotinib and 15 mg/weekly of temsirolimus. In total, 50% of the patients quit the treatment as a result of severe toxicity, which consisted on fatigue, diarrhoea, pneumonia and head and neck edema. The data for efficacy took a back seat because of these effects. The median PFS was 1.9 months .
Everolimus was tested in a phase II trial that followed the same design, with a dose of 5 mg/day in combination with erlotinib at 150 mg/day (R/M HNSCC) after platinum-based chemotherapy. Tolerance was better in this trial, and only one patient withdrew from treatment. ORR at 12 weeks was 2.8%, but 31% of the patients achieved stable disease. Median PFR was 11.9 weeks, and median OS was 10.25 months .
These three trials included a broad biomarker analysis (aimed at analysing EGFR, PI3KCA, PTEN, AKT, cKIT, and RAS, among others), but results were inconclusive because a predictive biomarker has not yet been identified.
3.3.2. Pan-PI3K Inhibitors
Buparlisib (BKM-120): The results of a Korean trial were presented at ASCO 2015. This trial included a clinical study (tolerance and efficacy of buparlisib) and a preclinical study (to enhance buparlisib activity in combination with other drugs). A total of 37 patients were enrolled, but the efficacy was modest: RR was 2.7%, median PFS was 7.4 weeks and OS was 19.2 weeks. Common toxicities included anorexia (62%) and hyperglycaemia (59%). The preclinical data showed inhibited cell growth when BKM120 was combined with cetuximab. This combination will be explored in an ongoing clinical trial (NTC01816984).
PX-866 is another oral Pan-PI3K inhibitor that was explored in combination with cetuximab in a phase II randomized trial in patients after platinum-based progression (R/M HNSCC). A total of 83 patients were enrolled to receive weekly cetuximab or cetuximab in combination with PX-866 (8 mg/day). The combination did not improve ORR, PFS (80 days in both groups) or OS (211 days vs. 256 days), and toxicity was greater in the combination arm, especially gastrointestinal toxicity.
The same design was used in another phase II randomized trial that tested the drug combined with docetaxel (docetaxel plus/minus PX-866) in 85 patients, and the same results were observed. In summary: the efficacy results in terms of ORR, PFS or OS were not better for the combination, but toxicity was worse in that arm and included higher rates of febrile neutropenia [61,62].
In conclusion, although the role of the PI3K pathway is a promising target for drug development (proof of this hypothesis is the number of related ongoing clinical trials), the lack of biomarkers to select patients for these kind of treatments causes the results found in the clinical trials to be disappointing. We also cannot discount the high rates of associated toxicity. Perhaps with selected p110α-inhibitors, toxicity profiles will be improved.
3.4. Targeting Src
Src is a cytoplasmic tyrosine kinase that is associated with the proliferation or migration of tumour cells. Many growth factor receptors or G-protein-coupled receptors located in the plasma membrane can activate Src, including EGFR, VEGFR, PDGFD or FGFR. Consistent with the overexpression of EGFR in HNSCC, Src activity is also increased in these tumours. There is therefore a reason to develop inhibitors of Src tyrosine kinase to treat HNSCC.
Saracatinib (AZD0530) is an oral small molecule inhibitor of Src TK and Bcr-Abl. A total of nine patients were enrolled in a phase II trial after at least one prior therapy. The study was closed because eight of these patients progressed early (within the first 60 days). Toxicity was manageable, and fatigue was the most common adverse event .
Dasatinib (BMS-354825) is a multikinase inhibitor (Scr, PDGFR, BCR-ABL and c-Kit). A lack of activity for this drug was demonstrated in a phase II trial in a group of 15 patients. Only two of the patients showed disease stabilization, while seven patients progressed, and four patients withdrew because of toxicity (gastrointestinal effects and pleural effusion) . Because of these results, blocking only Src in HNSCC is not the best strategy to explore in further clinical trials.
3.5. Promising Pathways
Alterations in NOTCH1 have been reported in more than 10% of HNSCCs. There are four transmembrane receptors (Notch1-4), and their ligands are Delta (δ1-3) and Jagged (Jag1-2). The ligand-receptor interaction stimulates proteolytic processes, including the following: ADAM protease splits the Notch extracellular domain, γ-secretase breaks the intracellular domain, and Notch is translocated to the nucleus, where it activates transcription factors such as HES, HERP, p21 or CDK-1. This pathway appears has been related to cell differentiation. There are currently no available drugs that work against this pathway [65,66].
The hedgehog (Hh) pathway is related to cell adhesion and epithelial-mesenchymal transition (EMT). Hh activates Gli1, which regulates many cancer genes (e.g., myc, VEGF or PTCH). Overexpression of the Gli1 protein can promote tumour growth and metastasis. A Hh inhibitor (IPI-926) is currently being explored in a clinical trial in R/M HNSCC (NTC01255800).
Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells
1Breast Biology, Institute of Cancer Sciences, University of Manchester, Paterson Building, Wilmslow Road, Manchester, M20 4BX, UK
2Department of Medical Oncology, The Christie NHS Foundation Trust, Wilmslow Road, Manchester, M20 4BX, UK
3Cancer Stem Cell Research, Institute of Cancer Sciences, University of Manchester, Paterson Building, Wilmslow Road, Manchester, M20 4BX, UK
Jagdeep K Singh: moc.liamtoh@iarkj; Bruno M Simões: ku.ca.nam.rcip@seomisb; Sacha J Howell: ku.shn.eitsirhc@llewoH.ahcaS; Gillian Farnie: ku.ca.nam.rcip@einrafg; Robert B Clarke: firstname.lastname@example.org
Author information ►Copyright and License information ►
Copyright © 2013 BioMed Central Ltd.
Breast Cancer Res. 2013; 15(4): 210.
Published online 2013 Aug 30. doi: 10.1186/bcr3436
This article has been cited by other articles in PMC.
Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.
The cancer stem cell model of carcinogenesis posits that cancers arise from, and are sustained by, a rare sub-population of cells that possess stem-like characteristics . Akin to normal tissue, cancer tissue is proposed to be organized in a hierarchical manner, which may underpin the cellular heterogeneity of cancers. At the apex lies the tumor-initiating or cancer stem-like cell (CSC), so called because these cells share key stem cell properties of their normal tissue counterparts . CSCs have the capacity to self-renew and differentiate, but the mechanisms that strictly regulate these processes under normal conditions are deregulated, leading to their expansion and production of aberrantly differentiated progeny .
CSCs are defined functionally by their capacity to initiate a human tumor in immunocompromised mice and to self-renew, giving rise to a new tumor when passaged into another mouse, and their ability to differentiate into non-self-renewing cells which constitute the bulk of the tumor . Breast cancer was the first solid tumor in which CSCs were identified . By sorting cells derived from metastatic pleural effusions, Al-Hajj and colleagues demonstrated that cells expressing high levels of CD44 but low or absent CD24 and lineage markers (CD44+/CD24−/low/Lin−) were highly enriched for tumor-forming capacity in non-obese diabetic/severe combined immunodeficient mice. Other cell surface markers such as CD133 and CD49f and intracellular cytokeratin 5 and ALDH1 (aldehyde dehydrogenase)/ ALDEFLUOR have subsequently been used to enrich for or identify human breast CSCs [5-8]. This subpopulation of cells is also characterized by their capacity to survive in anchorage-independent conditions and be cultivated in vitro as mammospheres [9,10].
There is evidence that breast CSCs are relatively resistant to chemo-, radio-, and endocrine therapies [6,11,12]. By evading the effects of these treatments, CSCs can survive to repopulate the tumor, leading to disease recurrence. Hence, to halt disease progression, there is a need to develop novel CSC-targeted therapies. Multiple intrinsic factors - such as drug efflux, more efficient DNA repair mechanisms, masking of receptors, quiescence, inactivation of phosphatase and tensin homolog (PTEN), and overexpression of HER2 - are reported to confer resistance of breast CSCs to conventional therapies.
Accumulating evidence indicates that extrinsic factors and other cells that form part of the tumor microenvironment and CSC niche are also responsible for regulating and promoting CSC activity. The association between inflammation and cancer is well established, and deregulated expression of multiple inflammatory cytokines, including interleukin-8 (IL-8), in malignant breast disease has been recognized for more than 15 years. Although there is substantial evidence that IL-8 is increased in breast cancer, the mechanisms by which IL-8 contributes to breast cancer progression have remained virtually unknown. However, recent studies indicate that IL-8 can promote CSC invasion, metastases, and treatment resistance. Targeting CXCR1/2 signaling has proven efficacious in in vivo models of breast cancer as well as primary invasive and metastatic breast cancers, catalyzing the initiation of clinical trials evaluating CXCR1/2 inhibitors. Here, we review the key components of the IL-8 signaling pathway, evidence implicating IL-8 in breast cancer, regulation of CSC activity via CXCR1/2, and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments to improve outcomes in breast cancer.
The IL-8 signaling pathway
IL-8, also known as C-X-C motif ligand (CXCL) 8 (CXCL8), is a small soluble protein and belongs to the CXC chemokine family, which is one of four chemokine families . The CXC family is characterized by a single amino acid, named 'X', between the first two cysteines and is derived from a gene cluster mapped to chromosome 4 between q12 and q21 . IL-8 was originally identified as a potent neutrophil activator and chemotactic factor secreted by activated monocytes and macrophages [15-17]. Many other cell types - including fibroblasts , lymphocytes , neutrophils , endothelial cells , and a variety of normal and malignant epithelial cells [22-27] - have since been shown to secrete IL-8. IL-8 is pro-angiogenic and this property is conferred by a Glu-Leu-Arg motif that precedes the first N-terminal cysteine residue [28,29].
The biological effects of IL-8 are mediated via two class A, rhodopsin-like guanine-protein-coupled receptors (GPCRs): CXCR1 (IL-8RA) and CXCR2 (IL-8RB) [30,31].The genes encoding CXCR1 and CXCR2 are located in close proximity to each other on chromosome 2 (2q34-35) . The CXCR1 gene consists of two exons separated by an intron , whereas CXCR2 consists of three exons separated by two introns . The receptors share 77% amino acid homology and retain common structural motifs, raising the possibility that they may have derived from gene duplication [31,35].
Like other GPCRs, CXCR1 and CXCR2 are characterized by 7-transmembrane-spanning regions, an extra-cellular N-terminus, and an intracellular C-terminus . CXCR1 is activated by IL-8 and granulocyte chemotactic protein-2 (GCP-2)/CXCL6 . CXCR2 is more promiscuous as it can be activated not only by IL-8 but by many other CXC 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 . Studies in phospholipid bilayers indicate that the cell membrane is important in mediating IL-8 binding to the N-terminal residues of CXCR1 . Ligand receptor interactions are complex and exactly how these interactions mediate ligand-binding affinity and receptor activation remains largely unknown . However, studies demonstrate that signaling requires interaction between the N-terminal region of IL-8 and the N-terminal extracellular domain of the receptors [40,41].
As with other GPCRs, CXCR1 and CXCR2 are characterized by their association with heterotrimeric G proteins, which consist of α, β, and γ subunits . Ligand binding catalyzes the exchange of guanosine diphosphate for guanosine triphosphate on the Gα subunit, which triggers the release of this subunit from the receptor and the Gβγ subunits . The Gα and Gβγ subunits subsequently activate a variety of signaling pathways that can have different effects depending on the cell type. The three principal pathways are phosphatidylinositol 3' kinase/Akt (PI3K/Akt), phospholipase C/ protein kinase C (PLC/PKC), and Ras/Raf/extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) [44-46]. Other signaling pathways include focal adhesion kinase, Rho, Rac, and janus kinase/signal transducers and activators of transcription (JAK/STAT) [47-49].
IL-8 and breast cancer
IL-8 is upregulated in a wide variety of solid cancers, such as prostate, gastric, bladder, ovarian, lung, and melanoma, and is reported to contribute to multiple hallmarks of cancer, such as increased proliferation, angiogenesis, invasion, and metastases [25,26,50-53]. IL-8 is overexpressed in breast cancer compared with normal breast tissue, and although there is substantial evidence that IL-8 may promote breast cancer initiation and progression via the above mechanisms, more recent evidence indicates that this cytokine is a key regulator of CSC activity. Novel therapeutics aimed at inhibiting CXCR1/2 signaling may halt disease progression in tumors driven by IL-8 and other CXCR1/2 ligands.
Breast cancer cells are reported to express CXCR1/2  and secrete IL-8 [27,55], and rare genetic polymorphisms of CXCR2 and IL-8 are associated with an increased risk of developing large, high-grade, lymph node-positive breast cancers [56,57]. Regulation of IL-8 within the tumor microenvironment is complex, not only because of the variety of cells that can secrete it but also because of the multitude of factors that can affect IL-8 expression by these different cell types (Figure 1). Other cytokines such as IL-1β, tumor necrosis factor-alpha, and IL-6; growth factors such as epidermal growth factor; and hormones such as estrogen and progesterone are all reported to upregulate IL-8 expression in breast cancer cells [58-60]. Recent co-culture studies indicate that activation of fibroblasts growth factor receptor in mammary tumor cells induces upregulation of IL-8 and other CXCR1/2 ligands by macrophages through decreased activity of the transforming growth factor-beta/Smad3 pathway, resulting in increased tumor cell migration and invasion . Cross-talk between breast cancer cells and mesenchymal cells, which form an integral part of the tumor stroma, can also induce secretion of several CXCR1/2 ligands, including IL-8, CXCL1/GRO-α, CXCL6/GCP-2, and CXCL7/NAP-2 .
Model of cytokine networks depicting the proposed interactions between breast cancer cells and stromal cells. (a) Interleukin-8 (IL-8) is secreted by cancer cells, mesenchymal cells, and macrophages via multiple signaling pathways. (b) IL-8 promotes breast...
In order for cells to metastasize, they must invade surrounding structures. Several studies have demonstrated a positive correlation between ectopic IL-8 expression and the invasive potential of breast cancer cells. In breast cancer cell lines, invasion is directly proportional to IL-8 expression, and overexpression or treatment with recombinant IL-8 promotes invasion [27,63]. Conversely, short-interfering RNA knockdown of IL-8 in breast cancer cell lines with relatively high constitutive expression, such as MDA-MB 231, inhibits invasion .
Further evidence implicating IL-8 in the metastatic process comes from in vivo studies. Using an in vivo selection method to generate cells with a high or low metastatic phenotype from the parental MDA-MB 231 cell line, Bendre and colleagues  (2002) demonstrated significant upregulation of IL-8 and invasion in the highly metastatic cells compared with the poorly metastatic cells. Furthermore, IL-8 expression in cells cultured from experimental lung metastases is substantially greater than in cells cultured from primary tumors . Consistent with these findings, clinical studies report significantly higher IL-8 levels at metastatic sites compared with primary sites . More recent studies demonstrate that knockdown of CXCR2 or inhibition of CXCR1/2 activity decreases spontaneous metastases in vivo [67,68]. Together, these studies demonstrate that IL-8 and other CXCR1/2 ligands are important in facilitating metastatic colonization in breast cancer.
Chemo-resistance resulting in incurable disease remains a significant problem in managing patients with breast cancer, especially in the metastatic setting, in which cure is not achievable. Several mechanisms - such as drug efflux via breast cancer resistance proteins, more efficient DNA repair mechanisms, masking of receptors, and inactivation of PTEN - have been implicated in conferring this phenotype. Accumulating evidence indicates that increased activity of CXCR1/2 via ligand upregulation may serve as an adaptive response to protect cancer cells from the cytotoxic effect of conventional chemotherapy agents, thereby limiting their clinical efficacy. Certainly, patients with metastatic disease treated with multiple cycles of chemotherapy have elevated serum IL-8 levels compared with those with localized cancers . Moreover, serum IL-8 level is an independent prognostic factor for post-relapse survival in patients with metastatic breast cancer . In vitro upregulation of IL-8 and other CXCR1/2 ligands by breast cancer cells is observed in response to conventional chemotherapy, and multidrug-resistant breast cancer cell lines produce significantly higher IL-8 protein levels compared with non-resistant controls [71,72]. Furthermore, failure of novel targeted therapies, such as PI3K/mammalian target of rapamycin (mTOR) inhibitors, has been attributed to upregulation of IL-8 via alternative signaling pathways involving JAK2/STAT5 .
Pharmacological inhibition or genetic knockdown of IL-8, however, has been shown to sensitize breast cancer cells to the cytotoxic effects of conventional chemotherapy agents . Importantly, in human breast cancer xenograft models, CXCR1 inhibition is reported to add to the efficacy of docetaxel, resulting in greater inhibition of tumor growth and reduction in systemic metastases . Given these promising results, combining CXCR1/2 inhibitors with standard chemotherapy agents and emerging targeted therapies, which may directly or indirectly increase IL-8 as part of the adaptive response, could help to overcome treatment resistance, resulting in improved outcomes.
Regulation of breast cancer stem cell activity via CXCR1/2
The failure of current therapies to eradicate breast cancer is hypothesized to be due to the existence of a subpopulation of cells that can evade the effects of these treatments. Substantial evidence indicates that breast CSCs are intrinsically resistant to chemo-, radio-, and endocrine therapies and, as such, can survive to repopulate the tumor, resulting in disease recurrence. It is becoming evident that, in addition to intrinsic resistance mechanisms, complex cytokine networks within the tumor microenvironment can rescue and promote CSC activity while more differentiated cells within the tumor succumb to the effects of conventional therapies. Recent studies indicate that CXCR1/2 signaling forms an important defense mechanism in regulating breast CSC activity. CXCR1 is overexpressed in the CSC subpopulation compared with bulk tumor cells, and ligand activation promotes their invasive capacity [68,73]. More recent studies indicate that IL-8 can promote a state of 'stemness' by inducing epithelial-mesenchymal transition , a process that is implicated in regulating invasion and metastasis and in the acquisition of stem cell characteristics [75-77].
Activation of CXCR1/2 in breast cancer cell lines with recombinant IL-8 is reported to expand the pool of CSCs and increase self-renewal [62,73]. We recently validated these findings by using a different panel of breast cancer cell lines and, importantly, found the same effect on self-renewal in primary samples isolated both from invasive breast tumors and from metastatic cells from ascites and pleural effusions . Interestingly, we demonstrated a significant direct correlation between metastatic fluid IL-8 level and mammosphere formation when cells from such fluid were cultured ex vivo, suggesting that cancers with higher IL-8 levels have greater CSC activity .
Recent in vivo studies report that bone marrow-derived mesenchymal stem cells (MSCs) are recruited to sites of developing tumors, co-localize with breast CSCs within the CSC niche, and are responsible for accelerating tumor growth by increasing the population of breast CSCs through cytokine networks . Mechanistically, Liu and colleagues  propose that these effects are initiated by cancer cell-derived IL-6 and sustained by IL-8 and other CXCR1/2 ligands, namely CXCL6/GCP-2 and CXCL7/ NAP-2, released from both the cancer cells and mesenchymal cells. Furthermore, 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 .
A recent study reports coordinated regulation of IL-8 with IL-6 and CXCL1/GRO-α in triple-negative breast cancers (TNBCs) which may contribute to the poor prognosis of this subgroup . In that study, Hartman and colleagues demonstrated that stimulation of TNBC cells with lysophosphatidic acid (LPA), an inducer of inflammatory signaling, resulted in coordinated secretion of IL-8, IL-6, and CXCL1/GRO-α via an EZH2/nuclear factor κ-light-chain-enhancer of activated B cells (NFκB)-dependent pathway. These effects were abrogated by pharmacological or genetic inhibition of the LPA receptor, EZH2 (a critical regulator of NFκB-induced inflammatory gene expression), or direct inhibition of NFκB .
Inhibition of CXCR1/2 signaling by using repertaxin, a non-competitive inhibitor, is reported to decrease the proportion and activity of CSCs in vitro, as measured by a decrease in the fraction of ALDEFLUOR-positive cells and mammosphere formation, respectively . In vivo, treatment of mouse xenografts with repertaxin decreased tumor growth and increased the efficacy of docetaxel. This was associated with a reduction in the tumor-initiating and self-renewal properties of the remaining CSCs as demonstrated by a reduction of tumor formation following re-implantation of cells into recipient mice . More recently, Hartman and colleagues  (2013) reported similar effects on anchorage-independent colony formation in TNBC cells by using short-hairpin RNA (shRNA) to knock down IL-8 and CXCR1. More-over, concurrent inhibition of IL-8 and IL-6 had a synergistic effect on reducing colony formation and tumor initiation in vivo as well as increasing apoptosis and sensitizing cells to paclitaxel . Together, the above studies demonstrate that the CXCR1/2 signaling axis forms an integral component of a complex inflammatory cytokine response which is critical in maintaining breast CSC activity via autocrine or paracrine routes or both.
IL-8 inflammatory feedback loop in breast cancer: the role of HER2
Up to 25% of breast cancers overexpress HER2, conferring a higher rate of recurrence and mortality . Studies suggest that HER2 overexpression promotes tumor formation and metastasis by increasing the proportion of CSCs and their self-renewal and invasive properties [82,83]. More recently, it has been proposed that HER2 may promote breast cancer initiation and progression by activating multiple pro-inflammatory cytokine feedback loops. Overexpression of HER2 is reported to increase IL-6 expression, generating an autocrine-positive feedback loop via STAT3/Akt/NFκB signaling pathways, resulting in enhanced breast CSC activity and HER2 treatment resistance [60,84]. Accumulating evidence indicates that HER2 modulates IL-8, and in light of recent findings discussed below, we propose that HER2 forms an integral component of an IL-8-dependent positive feedback loop leading to increased breast CSC activity. Mechanistically, this may contribute to the poor prognosis of HER2-enriched tumors.
Overexpression of HER2 in breast cancer cell lines is reported to induce a 'cytokine signature' characterized by upregulation of IL-8 and other CXCR1/2 agonists such as CXCL1/GRO-α [85,86]. This effect is potentiated by co-expression of HER2 and HER3 via increased autoactivation of HER2 . Conversely, inhibition of HER2 activity by using pharmacological inhibitors or genetic knockdown reduces IL-8 expression in HER2-over-expressing breast cancer cell lines through inhibition of the PI3K-Akt signaling pathway [85,86]. In vivo, inhibition of tumor growth with trastuzumab is associated with downregulation of IL-8 in HER2-positive xenografts .
The correlation between HER2 overexpression and increased IL-8 is supported by clinical evidence. A series of early small studies reported higher serum IL-8 levels in patients with metastatic HER2-positive disease compared with HER2-negative disease and higher IL-8 protein level in HER2-positive cancers compared with HER2-negative cancers [86,88]. These findings have recently been corroborated by using bioinformatic analysis of 1,881 primary breast cancer samples derived from multiple large datasets . Using gene set analysis, Aceto and colleagues reported significantly higher levels of IL-8 transcripts in tumors displaying a HER2-enriched phenotype compared with normal-like and luminal subtypes.
We recently reported fresh insights into HER2 signaling in breast cancer by demonstrating transactivation of HER2 upon ligand activation of CXCR1/2 . We found that transactivation of HER2 via CXCR1/2 is Srcdependent and leads to the activation of AKT and ERK1/2 signaling pathways, which are known to be critical in regulating breast CSC activity [78,89]. Inhibition of HER2 activity with lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor, abrogated the mammosphere-promoting effect of IL-8 in both HER2-positive and -negative primary breast cancers. These findings demonstrate that the functional effects of CXCR1/2 activation are dependent, at least in part, on HER2. Importantly, in HER2-positive cancers, CXCR1/2 inhibition was found to add to the efficacy of HER2 inhibition . Given the importance of HER2 in regulating CSC activity, activation of this pathway could have important biological consequences especially in tumors expressing high levels of CXCR1/2 ligands.
There is evidence that IL-8 shows an inverse association with estrogen receptor (ER) expression, with ER-negative breast cancers expressing higher levels of IL-8 compared with ER-positive cancers [27,88]. In light of recent evidence implicating IL-8 in driving disease progression, it is possible that elevated levels of IL-8 are responsible for selectively driving disease progression in ER-negative/ triple-negative tumors compared with ER-positive tumors; however, this remains to be determined.
Together, the above studies support the existence of an IL-8-positive feedback loop, as summarized in Figure 1. Once activated, this can initiate a self-perpetuating cycle leading to continuous stimulation of breast CSC activity and thereby hastening disease progression. Production of IL-8 by cancer cells, mesenchymal cells, and macrophages promotes breast CSC activity via CXCR1/2 by activating EGFR/HER2-dependent and -independent signaling pathways. Direct activation or auto-activation of HER2 can increase IL-8 expression which can further activate CXCR1/2 signaling via a paracrine or autocrine route or both. Other cytokine feedback loops, such as IL-6, could potentially feed into the above cycle by upregulating IL-8 production via NFκB signaling . Transcriptional activation of IL-8 is controlled primarily by NFκB; however, other transcription factors such as activating protein-1 and CAAT/enhancer-binding proteins can also regulate IL-8 expression and act synergistically with NFκB to augment gene expression [90,91]. NFκB is also responsible for regulating expression of IL-6, although more recent evidence indicates that this transcription factor is involved in regulating the coordinated expression of IL-6 and IL-8 in TNBCs. Mechanistically, this may be responsible for conferring the synergistic effect of these cytokines in regulating breast CSC activity and contribute to the poor prognosis of this subtype . Thus, dual inhibition of IL-6 and IL-8 or inhibition of NFκB may be required to derive therapeutic benefit in tumors in which cytokine expression is coordinated.
Targeting CXCR1/2 signaling in breast cancer
Increasing evidence indicates that IL-8 is a key extrinsic regulator of breast CSC activity. This may contribute to the poorer prognosis of tumors that express high levels of IL-8 or other CXCR1/2 ligands and contribute to treatment resistance. Consequently, targeting CXCR1/2 signaling may abrogate disease progression in a subset of breast cancers. Pharmacologically, this can be approached by interfering with either ligand activation or receptor function. Antibodies against IL-8 have demonstrated anti-tumor effects in xenograft models of bladder cancer  and melanoma . Trials in patients with chronic inflammatory diseases associated with increased production of IL-8, such as palmoplantar pustulosis, report that monoclonal humanized antibodies to IL-8 are clinically effective and well tolerated . Although IL-8 is the most well-studied CXCR1/2 ligand in breast cancer, targeting IL-8 alone may be of limited benefit since other CXCR1/2 agonists - such as CXCL1/GRO-α, CXCL2/ GRO-β CXCL3/GRO-γ, and CXCL5 - are co-regulated with IL-8 . This problem can be circumvented by inhibiting CXCR1/2.
Various orally active small-molecule non-competitive antagonists of CXCR1 and CXCR2 - such as repertaxin (Dompé, Milan, Italy), SCH479833 (Merck, Whitehouse Station, NJ, USA), and SCH527123 (Merck) - have demonstrated anti-tumor effects in xenograft models of breast cancer , colorectal cancer , and melanoma , including the inhibition of spontaneous colon cancer liver metastasis . SCH563705 (Merck) has demonstrated particularly high binding affinities to CXCR1 and CXCR2 and has proven effective at inhibiting primary human breast CSC activity .
Owing to the pleiotropic effects of IL-8, caution must be exercised when trialling CXCR1/2 inhibitors as they could have unexpected toxicities. In addition to promoting tumorigenesis by increasing angiogenesis and invasion, IL-8 is reported to exert anti-tumor effects through neutrophil recruitment . Neutrophils and other cells such as 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 . Hence, CXCR1/2 inhibitors could inadvertently promote tumor growth by blocking the anti-tumor effects of neutrophil infiltration. Novel technologies aimed at delivering drugs specifically to the cancer cells could help minimize these effects. Similarly, CXCR1/2 inhibitors have been shown to reduce circulating neutrophil counts with the potential for synergistic myelo-toxicity when combined with chemotherapeutic agents .
Although the above CXCR1/2 inhibitors have shown efficacy in preclinical studies, they are still in the early stages of drug development. Repertaxin, originally developed to prevent IL-8-induced reperfusion injury, is the only CXCR1/2 inhibitor that has undergone clinical testing . Phase I trials demonstrate that repertaxin  and SCH527123  are well tolerated in healthy volunteers and patients with severe asthma, respectively. Based on recent evidence that CXCR1/2 inhibition can inhibit breast CSC self-renewal and metastases in vivo , clinical trials are under way to determine the safety and efficacy of repertaxin in combination with docetaxel chemotherapy in patients with advanced breast cancer [102-104].
Ultimately, combining CXCR1/2 inhibitors with existing chemotherapy and endocrine therapy agents or HER2-targeted therapies (or both) may be more effective at eliminating both the CSC and non-CSC populations, leading to improved outcomes in both the adjuvant and advanced settings.
CSC: cancer stem-like cell; CXCL: C-X-C motif ligand; EGFR: epidermal growth factor receptor; ER: estrogen receptor; ERK1/2: extracellular signal-regulated protein kinase 1 and 2; GCP-2: granulocyte chemotactic protein-2; GPCR: guanine-protein-coupled receptor; GRO: growth-regulated oncogene; HER2: human growth factor receptor 2; IL: interleukin; JAK/STAT: Janus kinase/ signal transducers and activators of transcription; LPA: lysophosphatidic acid; MSC: mesenchymal stem cell; NAP-2: neutrophil-activating protein-2; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K: phosphatidylinositol 3' kinase; PTEN: phosphatase and tensin homolog; TNBC: triple-negative breast cancer.
The authors declare that they have no competing interests.
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