Targeting and Sensitization of Breast Cancer Cells to Killing with a Novel Interleukin-13 Receptor α2-Specific Hybrid Cytolytic Peptide

Simple Summary Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer deaths among women globally. Due to its biological heterogeneity, breast cancer treatment and prognosis are highly variable among patients. In particular, the triple-negative subtype (TNBC) has a poorer prognosis with a lack of targeted therapies. With this in mind, we sought to develop a potentially more effective, novel receptor-targeting strategy. This study describes measuring the expression of the putative biomarker, interleukin-13 receptor (IL-13R)α2, in breast cancer (including TNBC) and its therapeutic targeting using a novel hybrid cytolytic peptide (Pep-1-Phor21) approach. We have shown in this manuscript that IL-13Rα2 exhibits potential as a therapeutic target, particularly for TNBC types of breast cancer. Importantly, drug-induced breast cancer cell lysis could be enhanced by treatment with epigenetically active anti-cancer compounds, suggesting that a combination adjuvant therapy of Pep-1-Phor21 with such compounds may be a particularly productive strategy for TNBC. Abstract Highly metastatic breast cancers, such as triple-negative subtypes (TNBC), require the most effective treatments. Since interleukin-13 receptor (IL-13R)α2 is reportedly over-expressed in some cancers, we investigated here its expression and the feasibility of therapeutically targeting this receptor in breast cancer using a novel hybrid cytolytic peptide (Pep-1-Phor21) consisting of IL-13Rα2-binding (Pep-1) and cytolytic (Phor21) domains. This study demonstrates that particularly TNBC tissues and cells display the prominent expression of IL-13Rα2. Furthermore, Pep-1-Phor21 induced the rapid necrosis of tumor cells expressing cell-surface IL-13Rα2. Notably, IL-13Rα2 expression was found to be epigenetically regulated in breast cancer cells in that the inhibition of histone deacetylase (HDAC) or DNA methyltransferase (DNMT) upregulated IL-13Rα2 expression, thereby sensitizing them to Pep-1-Phor21. IL-13Rα2-negative non-malignant cells were refractory to these epigenetic effects. Consistent with its cytolytic activity, Pep-1-Phor21 readily destroyed IL-13Rα2-expressing breast cancer spheroids with HDAC or DNMT inhibition, further enhancing cytolytic activity. Therefore, the Pep-1-Phor21-mediated targeting of IL-13Rα2 is a potentially novel therapeutic strategy for TNBC. Given that tumor cells can be selectively sensitized to Pep-1-Phor21 via the epigenetic up-regulation of IL-13Rα2, a combined adjuvant approach involving Pep-1-Phor21 and epigenetic inhibitors may be an effective strategy.


Introduction
Breast cancer accounts for one of the commonest forms of malignancy in women and while advances in treatments have decreased mortality rates for many forms of breast cancer, highly metastatic types such as triple-negative breast cancer (TNBC) remain a considerable therapeutic challenge [1].In particular, as TNBC-type tumors lack the expression of the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor (EGF) receptor-2 (HER2), current treatments, therefore, consist of adjuvant-type approaches based on agents that include anthracyclines, taxanes, alkylating agents, and antimetabolites [2,3].However, these approaches lack tumor-specific targeting and produce considerable side effects and, consequently, TNBC is associated with faster relapse times and higher mortality rates [4,5].Moreover, the development of multi-drug resistance, where (for example) cancer cells over-express intracellular drug-efflux pumps, further reduces the effectiveness of conventional cytotoxic chemotherapies [6].Therefore, more selective and effective targeted therapies for TNBC-type breast cancer are urgently required.More selective approaches to tumor therapy include targeting receptors that are upregulated in an oncogenic setting with one putative candidate being the cell-surface protein, interleukin-13 receptor α2 (IL-13Rα2).Previous studies have shown that IL-13Rα2 is over-expressed in the latter stages of breast cancer, where increased expression levels correlate with higher rates of metastasis and a poorer prognosis [7,8].Further, IL-13Rα2, which is also called cancer/testis antigen (CT) 19, has insignificant expression in normal tissues [9,10].Initially, IL-13Rα2 was characterized as a decoy receptor, where it binds with high affinity to its natural cytokine ligand, IL-13 [11,12].However, recent studies have shown a more complex signaling function for IL-13Rα2 in brain tumor progression [12].Although the functional significance of IL-13Rα2 expression on tumor cells remains largely undefined, under certain circumstances, IL-13Rα2 signaling can mediate a variety of cellular and tissue responses that may impact tumorigenesis [13].Nevertheless, various IL-13Rα2targeting strategies involving cytotoxic therapeutics have been developed [14].For example, IL-13 conjugated with Pseudomonas exotoxin subunits (IL-13-PE) has been developed for glioblastoma therapy [15].However, treatment-induced neurotoxicity associated with the internalization of IL-13-PE [16,17] produced off-target tissue damage, attributed to the ability of IL-13-PE to bind to the alternative physiological receptor for IL-13, namely the IL-13Rα1/IL-4Rα heterodimer [18].Therefore, in pursuing a strategy of targeting IL-13Rα2expressing tumors, candidate drugs must display appropriate specificity and selectivity.Furthermore, IL-13Rα2 expression is regulated via epigenetic mechanisms [19,20] and, concerning breast cancer, epigenetic activity, including DNA methylation and histone modifications, has been linked to disease progression [21][22][23].Notably, ER expression can be re-established in breast cancer cells after treatment with histone deacetylase (HDAC) inhibitors [23], raising the possibility that similar mechanisms may also upregulate IL-13Rα2 expression, consequently sensitizing them to IL-13Rα2-targeting strategies.
To improve selectivity and specificity in targeting IL-13Rα2, we developed a hybrid cytolytic peptide (Pep-1-Phor21), which, unlike previous strategies, is not a cytotoxindrug conjugate.Instead, Pep-1-Phor21 consists of a unitary peptide structure bearing distinct receptor-binding (Pep-1) and cytolytic (Phor21) domains.The seven-amino-acid Pep-1 peptide was originally discovered by screening a phage display library for novel IL-13Rα2 ligands and it selectively binds IL-13Rα2 with high affinity at a site on the receptor distinct from that utilized for IL-13 binding [24].Pep-1 in conjugate form has been used as drug therapy and as a means of PET imaging IL-13Rα2-expressing gliomas [25,26].The Phor21 lytic domain corresponds to the previously described amphipathic membranedisrupting cytolytic peptide, which contains three sequence repeats of seven amino acids (KFAKFAK) [27].This cytolytic peptide kills cells through necrosis by acting on the plasma membrane and, therefore, bypasses multidrug resistance [28].Further, unlike the lytic peptides that kill cells by apoptosis, the Phor21 cytolytic peptide doesn't internalize since it elicits necrosis [29].Although the combination of Pep-1 with Phor21 (Pep-1-Phor21) has not previously been investigated, Phor21, when combined with a segment of the β-chain of chorionic gonadotropin (β-CG) (Phor21-βCG[ala]), was reportedly able to target cancer cells expressing the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) [30].Since Phor21-βCG [ala] has been shown to have a short in vivo half-life (~5 h), it is very unlikely to elicit an immune response and/or cause liver toxicity [28].We have demonstrated recently that the Pep-1-Phor21 cytolytic peptide shows anticancer properties against IL-13Rα2-expressing prostate cancer cells [31].
This study addresses the hypothesis that IL-13Rα2 is a druggable target for the treatment of breast cancer.To test this, we examined IL-13Rα2 expression in both non-TNBC and TNBC breast cancer using representative cell lines, and tissue cDNA-and micro-arrays.Further, we defined the specific activity of Pep-1-Phor21 against IL-13Rα2-expressing breast cancer cells and -transfected cells.To model more tumor-representative conditions, we determined in detail the activity of Pep-1-Phor21 against IL-13Rα2-positive TNBC spheroid cultures [32].To sensitize breast cancer cells to subsequent Pep-1-Phor1 treatment, we also examined the epigenetic regulation of IL-13Rα2 expression in breast cancer cells with a specific focus on HDAC and DNMT inhibitor treatments.Taken together, the results show that TNBC cells, in particular, express relatively high levels of IL-13Rα2 and the Pep-1-Phor21 peptide efficiently targets these tumor cells, rapidly destroying them through cytolysis.Furthermore, HDAC or DNMT inhibition selectively upregulated IL-13Rα2 expression and enhanced the effectiveness of Pep-1-Phor21 against tumor cells, raising the possibility that a combination drug therapy approach may be particularly effective.

Immunohistochemistry
For the detection of IL-13Rα2 expression in primary tumor tissue, immunohistochemical staining was performed on a breast cancer tissue array (BR1009, US Biomax) using a Vectastain ® Elite ABC-HRP kit (Vector Laboratories (#PK-6200)) [38].Briefly, duplicate cores on the array (formaldehyde-fixed tissue, paraffin sections) were first pre-treated with 1.0% hydrogen peroxide to block endogenous peroxidase activity followed by antigen unmasking with Retrievagen A solution (BD Biosciences (#BD 550524)).Subsequent sequential blocking treatments included incubation with 1.5% normal horse serum and avidin/biotin solution (Vector Laboratories (#SP-2001)).IL-13Rα2 was detected using mouse anti-human IL-13Rα2 (2K8, sc-134363, Santa Cruz Biotechnology Inc., diluted 1:200 (0.5 µg/mL final concentration)) primary antibody, biotinylated secondary antibody (1:500 dilution), and horseradish peroxidase (HRP)-conjugated avidin (1:500 dilution).Diaminobenzidine (DAB) enzyme substrate was used to visualize anti-IL-13Rα2 staining.The manual scoring of tissue cores for positive staining was based on the Allred scoring system in which the percentage of positive cells (proportion score: 0 [none]-5 [100%]) and the intensity of the reaction product (intensity score: 0 [none]-3 [strong]) in the entire tissue core were evaluated [36].The two scores were added together for a final score with a maximum score (strongly positive) of 8 and negative staining = 0.

Cell Surface Enzyme-linked Immunosorbent Assay (ELISA)
The cell surface expression of IL-13Rα2 was assessed by ELISA using non-permeabilized cells, as previously described [34,41].Briefly, cells were plated at 60-80% confluence in poly-L-lysine-coated wells of a 48-well plate.After 24 h in culture, cells were serum-starved for 2 h and then fixed with 4% (w/v) paraformaldehyde for 5 min.Following incubation with blocking buffer (1% bovine serum albumin (BSA) in TBS), cells were incubated with an anti-IL-13Rα2 mouse monoclonal antibody (sc-134363, Santa Cruz Biotechnology; diluted 1:800) followed by incubation with an HRP-conjugated anti-mouse IgG antibody.Following washing, surface staining was developed by incubating with 1-step Ultra TMB-ELISA substrate solution (Thermo-Fisher Scientific (#34029)) and absorbance was determined at 450 nm.

Cell Viability, Cytotoxicity, and Apoptosis Assays
Unless specified otherwise, 96-well black, µClear, half area, and flat-bottom plates (Greiner Bio-One (#675090)) were used for these assays.Cell viability was assessed using Alamar Blue according to the manufacturer's instructions (Thermo Fisher Scientific (#DAL1100)).Before this, the optimal cell density of MCF-10A, MCF7, and MDA-MB-231 required for cell viability was established as 40,000 cells per well (Figure S2).This was carried out by plating 10,000 to 80,000 cells per well and analyzing cell growth daily for up to 4 days by using Alamar Blue assay.Cell cytotoxicity was assessed using CellTox™ Green Cytotoxicity Assay (Promega (#G8743), Southampton, UK).Briefly, 40,000 cells per well were plated.After 24 h, the medium was replaced with medium containing 0.1% (v/v) CellTox Green Dye and the appropriate test compound.Fluorescence was measured using the 490 nm (excitation) and 525 nm (emission) settings on a POLARstar Omega microplate reader (BMG LABTECH).Fluorescence after the initial 30 min was considered zero, with subsequent fluorescence measurements obtained at 3 h intervals.Cells treated with the lysis buffer provided in the assay kit were used as the positive control (100% cytotoxicity).The combined measurement of cell viability, cytotoxicity, and apoptosis was determined by ApoTox-Glo TM Triplex Assay (Promega (#G6320)): Briefly, 40,000 cells per well were plated into the 96-well µClear ® half area black (for the cell viability and cytotoxic assays) or white (for the apoptosis assay) plate with a flat bottom (Greiner Bio-One (#655083)).After 24 h, the medium was replaced with fresh medium containing the test compound.After 6 h, 10 µL of cell viability/cytotoxicity reagent was added to each well and incubated for a further 1 h at 37 • C. Cell viability (fluorescence) was measured at 400 nm (excitation) and 505 nm (emission) settings.Cytotoxicity was measured at 485 nm (excitation) and 520 nm (emission).Apoptosis (luminescence) was determined by adding Caspase-Glo ® 3/7 reagent and incubating for 30 min at RT.

Statistical Analysis
Data were analyzed using GraphPad Prism version 6.0 software.All data are presented as the mean ± standard error of the mean (SEM) from three independent experiments [42].Statistical analysis was conducted using an unpaired Student's t-test or one-way analysis of variance with post-Tukey's multiple comparisons test with a significance of * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns = not significant.

Expression of IL-13Rα2 in Breast Cancer
For the determination of IL-13Rα2 mRNA expression, real-time RT-PCR was performed on breast cancer cDNA arrays containing multiple samples representative of the diverse breast cancer phenotypes.IL-13Rα2 mRNA expression was found to be significantly higher in breast cancer tissues compared with that in non-malignant breast tissues (Figure 1a).Further, IL-13Rα2 mRNA expression was significantly greater in TNBC-type tumors, compared to that in non-TNBC tumors (positive for at least one of ER, PR, HER2) or non-malignant breast tissue (Figure 1b).Immunohistochemical staining of tissue micro-array samples confirmed this pattern of expression at the protein level in that IL-13Rα2 expression was more predominantly detectable in TNBC-type tumors (Figure 1c).expression of IL-13Rα2 mRNA (Figure 2a) and this was confirmed at the protein level by immunoblotting (Figure 2b).In contrast, non-malignant MCF-10A and non-TNBC MCF-7 cancer cells did not exhibit detectable IL-13Rα2 protein expression.A cell-based ELISA was utilized to determine whether the IL-13Rα2 protein was appropriately localized at the cell surface and this analysis confirmed that IL-13Rα2 cell-surface expression was undetectable in MFC-10A and MCF-7 cells (Figure 2c).In comparison, TNBC-type cell lines exhibited prominent cell-surface IL-13Rα2 expression (MDA-MB-231: 613.3% ± 55.3, LM2: 863.5% ± 33.0; relative to MCF-10A).Therefore, IL-13Rα2 expression is similar in breast cancer tissue samples and representative cell lines with an expression more prominent in TNBC-type tumor cells.To validate cells for in vitro modeling studies, IL-13Rα2 expression was determined in the non-tumorigenic breast epithelial cell line (MCF-10A), a non-TNBC cell line (MCF-7), and two TNBC cell lines (MDA-MB-231 and its highly-metastatic sub-line, LM2 [7]).RT-PCR analysis revealed that only the TNBC-type cell lines had significantly elevated expression of IL-13Rα2 mRNA (Figure 2a) and this was confirmed at the protein level by immunoblotting (Figure 2b).In contrast, non-malignant MCF-10A and non-TNBC MCF-7 cancer cells did not exhibit detectable IL-13Rα2 protein expression.A cell-based ELISA was utilized to determine whether the IL-13Rα2 protein was appropriately localized at the cell surface and this analysis confirmed that IL-13Rα2 cell-surface expression was undetectable in MFC-10A and MCF-7 cells (Figure 2c).In comparison, TNBC-type cell lines exhibited prominent cell-surface IL-13Rα2 expression (MDA-MB-231: 613.3% ± 55.3, LM2: 863.5% ± 33.0; relative to MCF-10A).Therefore, IL-13Rα2 expression is similar in breast cancer tissue samples and representative cell lines with an expression more prominent in TNBC-type tumor cells.

Cytotoxic Activity of Pep-1-Phor21 against IL-13Rα2-Positive Breast Cancer Cells
Having validated its selectivity, Pep-1-Phor21 activity was further tested against breast cancer cell lines representative of TNBC and non-TNBC phenotypes and of defined IL-13Rα2 expression.When grown as monolayers, IL-13Rα2-negative non-malignant MCF-10A cells or malignant non-TNBC MCF-7 cells did not exhibit any appreciable loss of viability (Alamar Blue assay) after treatment with Pep-1-Phor21 (dose-response range = 0-120 µM) or its constitutive peptide subunits (Pep-1, Phor21) for 24 h (Figure 4a).In contrast, IL-13Rα2-positive TNBC-type cell lines, MDA-MB-231 and LM2, were highly sensitive to Pep-1-Phor21 treatment and their sensitivity increased as the time of incubation with Pep-1-Phor21 was increased.Notably, LM2 cells, which exhibited the most prominent surface IL-13Rα2 expression, were the most susceptible to Pep-1-Phor21 (Figure 4b).Furthermore, the measurement of cell death associated with a loss of cell membrane integrity (CellTox Green assay, detects the dead cell DNA) showed that only Pep-1-Phor21 had a demonstrable cytotoxic effect on IL-13Rα2-positive cell lines (Figure 4c) with LM2 cells again being the most susceptible (24 µM Pep-1-Phor21, 3 h: relative cytotoxicity = 96.9%± 0.4 of maximal activity).In contrast, Pep-1-Phor21 (≤120 µM) did not exhibit appreciable cytotoxic activity against IL-13Rα2-negative cells (MCF-10Aand MCF-7).Since the 3 h treatment seems to be optimal for analyzing the dose-dependent effect of the peptide on the tumor cell lines used in the study, we used the treatment of peptides for 3 h in further experiments unless otherwise indicated.

Cytotoxic Activity of Pep-1-Phor21 against IL-13Rα2-Positive Breast Cancer Cells
Having validated its selectivity, Pep-1-Phor21 activity was further tested against breast cancer cell lines representative of TNBC and non-TNBC phenotypes and of defined IL-13Rα2 expression.When grown as monolayers, IL-13Rα2-negative non-malignant MCF-10A cells or malignant non-TNBC MCF-7 cells did not exhibit any appreciable loss of viability (Alamar Blue assay) after treatment with Pep-1-Phor21 (dose-response range = 0-120 µM) or its constitutive peptide subunits (Pep-1, Phor21) for 24 h (Figure 4a).In contrast, IL-13Rα2-positive TNBC-type cell lines, MDA-MB-231 and LM2, were highly sensitive to Pep-1-Phor21 treatment and their sensitivity increased as the time of incubation with Pep-1-Phor21 was increased.Notably, LM2 cells, which exhibited the most prominent surface IL-13Rα2 expression, were the most susceptible to Pep-1-Phor21 (Figure 4b).Furthermore, the measurement of cell death associated with a loss of cell membrane integrity (CellTox Green assay, detects the dead cell DNA) showed that only Pep-1-Phor21 had a demonstrable cytotoxic effect on IL-13Rα2-positive cell lines (Figure 4c) with LM2 cells again being the most susceptible (24 µM Pep-1-Phor21, 3 h: relative cytotoxicity = 96.9%± 0.4 of maximal activity).In contrast, Pep-1-Phor21 (≤120 µM) did not exhibit appreciable cytotoxic activity against IL-13Rα2-negative cells (MCF-10Aand MCF-7).Since the 3 h treatment seems to be optimal for analyzing the dose-dependent effect of the peptide on the tumor cell lines used in the study, we used the treatment of peptides for 3 h in further experiments unless otherwise indicated.mary necrosis (Figure 5b).In contrast, only staurosporine preferentially activated cellular apoptosis, as demonstrated by increased caspase 3/7 activation above baseline activity (Figure 5c).The Pep-1-Phor21 or ionomycin-treated cells showed lower caspase activity than untreated or Pep-1-or Pho21-treated cells, which is probably due to the loss of cellular components by necrosis.Therefore, Pep-1-Phor21 may be particularly effective against TNBC-type tumors that express high levels of IL-13Rα2.Furthermore, Pep-1-Phor21 has a cytolytic mode of action, rapidly inducing necrotic cell death.4a).MDA-MB 231 and LM2 cells were treated with Pep-1-Phor21 at 24 µM (maximal effective concentration against LM2, as determined in dose-response analysis, Alamar Blue, Figure 4a).Pep-1-Phor21 had a significant cytotoxic effect only against IL- To more precisely delineate the mode of cell killing, we concomitantly measured the cell viability, cytotoxicity, and apoptosis (ApoTox-Glo Triplex Assay) of IL-13Rα2positive TNBC MDA-MB-231 cells after treating them with Pep-1-Phor21 (20 µM).As comparative controls, cells were treated with the cytotoxic compound ionomycin (150 µM), or the apoptosis inducer staurosporine (10 µM).The exposure of MDA-MB-231 cells to either Pep-1-Phor21 or control compounds produced a significant loss of viability (loss of internal live-cell protease activity, Figure 5a).However, only Pep-1-Phor21 or ionomycin (but not staurosporine) induced a significant increase in cytotoxicity (increased dead-cell protease activity, released from cells with impaired membrane integrity), consistent with primary necrosis (Figure 5b).In contrast, only staurosporine preferentially activated cellular apoptosis, as demonstrated by increased caspase 3/7 activation above baseline activity (Figure 5c).The Pep-1-Phor21 or ionomycin-treated cells showed lower caspase activity than untreated or Pep-1-or Pho21-treated cells, which is probably due to the loss of cellular components by necrosis.Therefore, Pep-1-Phor21 may be particularly effective against TNBC-type tumors that express high levels of IL-13Rα2.Furthermore, Pep-1-Phor21 has a cytolytic mode of action, rapidly inducing necrotic cell death.

Epigenetic Modulation of IL-13Rα2 Expression in Breast Cancer Cells
Given previous reports that IL-13Rα2 expression can be upregulated in certain cancers via epigenetic mechanisms [19], we sought to clarify whether IL-13Rα2 expression could be similarly enhanced in breast cancer cells, sensitizing them to Pep-1-Phor21 in the process.In particular, we determined the effects of treatment with an HDAC inhibitor (TSA), or with a DNMT (5-aza-dC) on IL-13Rα2 expression.The treatment of IL-13Rα2negative non-TNBC MCF-7 cells with either TSA or 5-aza-dC for 24 h induced the detectable expression of IL-13Rα2 mRNA (Figure 6a), total IL-13Rα2 protein (Figure 6b), and cell-surface IL-13Rα2 expression (Figure 6c) in a dose-dependent manner.In contrast, this induction was not demonstrable in non-malignant MCF-10A cells.Although the TNBCtype cell lines already expressed relatively high levels of IL-13Rα2, significantly increased expression was detectable in 10µM TSA-treated MDA-MB-231 cells, with the LM2 subline exhibiting further marginal increases in expression.

Epigenetic Modulation of IL-13Rα2 Expression in Breast Cancer Cells
Given previous reports that IL-13Rα2 expression can be upregulated in certain cancers via epigenetic mechanisms [19], we sought to clarify whether IL-13Rα2 expression could be similarly enhanced in breast cancer cells, sensitizing them to Pep-1-Phor21 in the process.In particular, we determined the effects of treatment with an HDAC inhibitor (TSA), or with a DNMT (5-aza-dC) on IL-13Rα2 expression.The treatment of IL-13Rα2-negative non-TNBC MCF-7 cells with either TSA or 5-aza-dC for 24 h induced the detectable expression of IL-13Rα2 mRNA (Figure 6a), total IL-13Rα2 protein (Figure 6b), and cell-surface IL-13Rα2 expression (Figure 6c) in a dose-dependent manner.In contrast, this induction was not demonstrable in non-malignant MCF-10A cells.Although the TNBC-type cell lines already expressed relatively high levels of IL-13Rα2, significantly increased expression was detectable in 10µM TSA-treated MDA-MB-231 cells, with the LM2 sub-line exhibiting further marginal increases in expression.

Targeting Breast Cancer Spheroids with Pep-1-Phor21
To more rigorously test the activity of Pep-1-Phor21, we utilized breast cancer cells cultured as spheroids, which exhibit growth characteristics more consistent with solid tumors [43].All cell lines cultured in the spheroid format maintained their relative IL-13Rα2 expression status (Figure 8a) and, consistent with this, Pep-1-Phor21 had a dose-dependent cytotoxic effect only on MDA-MB-231 or LM2 spheroids (Figure 8b).However, calculated IC 50 values for Pep-1-Phor21 activity against spheroids were moderately higher than those observed under monolayer conditions-MDA-MB-231, IC 50 = 22.98 µM ± 1.5; LM2, IC 50 = 12.22 µM ± 2.5-consistent with the moderately lower IL-13Rα2 mRNA expression levels measured in spheroid cultures (Figure S1).As observed with monolayer cells, the Phor21 lytic peptide subunit displayed no significant cytotoxic activity against spheroid cultures.
To visualize cell killing, confocal fluorescence microscopy was performed on breast cancer spheroids treated with Pep-1-Phor21.This analysis revealed that only IL-13Rα2-positive (TNBC) spheroids exhibited significant dead-cell staining (EthD-1) with the concomitant disruption of spheroid integrity after Pep-1-Phor21 treatment (Figure 9).After 3 h of treatment, dead-cell staining was present throughout the disrupted spheroid with only a few remaining live cells (Vybrant DiO staining) detectable within the inner core of the spheroid.Furthermore, given the potentially altered growth characteristics of cancer cells in spheroids, we further tested the ability of HDAC or DNMT inhibitors to modulate IL-13Rα2 expression.Indeed, as with monolayer cells, TSA or 5-aza-dC treatments significantly increased IL-13Rα2 mRNA expression in non-TNBC MCF-7 spheroids, whereas MCF-10A spheroids maintained their resistance to the modulating effects of these compounds (Figure 10a).Only treatment with 10µM TSA or 5-aza-dC significantly increased IL-13Rα2 expression in TNBC MDA-MB-231 spheroids.Notably, this up-regulation of IL-13Rα2 expression concomitantly enhanced the sensitivity of spheroids to subsequent Pep-1-Phor21 treatment in that non-TNBC MCF-7 spheroids developed sensitivity to Pep-1-Phor21-mediated cytotoxicity (Figure 10b).MDA-MB-231 (TSA-or 5-aza-dC-treated) and LM2 (TSA-treated) spheroids also exhibited a further significant increase in relative sensitivity to Pep-1-Phor21.Therefore, IL-13Rα2 expression in breast cancer spheroids is selectively amenable to epigenetic upregulation and Pep-1-Phor21 rapidly disrupts spheroid integrity, efficiently killing multiple layers of IL-13Rα2-positive tumor cells.
cultured as spheroids, which exhibit growth characteristics more consistent with solid tumors [43].All cell lines cultured in the spheroid format maintained their relative IL-13Rα2 expression status (Figure 8a) and, consistent with this, Pep-1-Phor21 had a dose-dependent cytotoxic effect only on MDA-MB-231 or LM2 spheroids (Figure 8b).However, calculated IC50 values for Pep-1-Phor21 activity against spheroids were moderately higher than those observed under monolayer conditions-MDA-MB-231, IC50 = 22.98µM ± 1.5; LM2, IC50 = 12.22µM ± 2.5-consistent with the moderately lower IL-13Rα2 mRNA expression levels measured in spheroid cultures (Figure S1).As observed with monolayer cells, the Phor21 lytic peptide subunit displayed no significant cytotoxic activity against spheroid cultures.To visualize cell killing, confocal fluorescence microscopy was performed on breast cancer spheroids treated with Pep-1-Phor21.This analysis revealed that only IL-13Rα2positive (TNBC) spheroids exhibited significant dead-cell staining (EthD-1) with the concomitant disruption of spheroid integrity after Pep-1-Phor21 treatment (Figure 9).After 3 h of treatment, dead-cell staining was present throughout the disrupted spheroid with only a few remaining live cells (Vybrant DiO staining) detectable within the inner core of the spheroid.Furthermore, given the potentially altered growth characteristics of cancer

Discussion
Although the 5-year survival rate for breast cancer has significantly improved, in part due to the development of endocrine-based therapies [44], significant challenges to successful therapy remain, in that breast tumors exhibit heterogeneous phenotypes, including receptor-positive (ER + , PR + , HER2 + ), triple-negative (ER − , PR − , HER2 − ), and combinations of these variants.Therefore, selecting appropriate treatments can be problematic, and to compound this, the development of drug resistance poses a major hurdle to achieving a permanent cure [45].TNBC poses particularly significant challenges in that these aggressive tumors are refractory to hormone receptor-targeting strategies, with treatment predominantly confined to a combination of surgery, radiotherapy, and chemotherapy with cytotoxic drugs.Improved therapies for TNBC-type tumors are therefore required [46], and we hypothesized that our cytolytic peptide-based approach to targeting IL-13Rα2-expressing tumors would provide a selective and potentially more effective strategy.
IL-13Rα2 is reportedly over-expressed in a variety of malignancies [47], and targeting this receptor, therefore, has garnered considerable interest as a possible therapeutic strategy [16,24].With regard to TNBC, previous studies have shown an association between predominant IL-13Rα2 expression and metastasis [7,48] and our findings corroborate these findings in that elevated expression of IL-13Rα2 was readily detectable in TNBCtype tumors and cell lines.Furthermore, IL-13Rα2-mediated signaling has been shown to

Discussion
Although the 5-year survival rate for breast cancer has significantly improved, in part due to the development of endocrine-based therapies [44], significant challenges to successful therapy remain, in that breast tumors exhibit heterogeneous phenotypes, including receptor-positive (ER + , PR + , HER2 + ), triple-negative (ER − , PR − , HER2 − ), and combinations of these variants.Therefore, selecting appropriate treatments can be problematic, and to compound this, the development of drug resistance poses a major hurdle to achieving a permanent cure [45].TNBC poses particularly significant challenges in that these aggressive tumors are refractory to hormone receptor-targeting strategies, with treatment predominantly confined to a combination of surgery, radiotherapy, and chemotherapy with cytotoxic drugs.Improved therapies for TNBC-type tumors are therefore required [46], and we hypothesized that our cytolytic peptide-based approach to targeting IL-13Rα2expressing tumors would provide a selective and potentially more effective strategy.
IL-13Rα2 is reportedly over-expressed in a variety of malignancies [47], and targeting this receptor, therefore, has garnered considerable interest as a possible therapeutic strat-egy [16,24].With regard to TNBC, previous studies have shown an association between predominant IL-13Rα2 expression and metastasis [7,48] and our findings corroborate these findings in that elevated expression of IL-13Rα2 was readily detectable in TNBC-type tumors and cell lines.Furthermore, IL-13Rα2-mediated signaling has been shown to promote the metastasis of ER-negative breast cancer cells [8], and targeting IL-13Rα2 may be a particularly relevant strategy for these metastasis-prone tumors.In formulating an IL-13Rα2-targeting strategy, the Pep-1 sequence motif was selected, as it exhibits a high affinity for IL-13Rα2, binding the receptor at an extracellular site that is distinct from the IL-13-binding region [24] and theoretically avoiding off-target effects.Moreover, incorporating the cell-surface-active lytic Phor21 motif predicted that in contrast to exotoxin-drug conjugates, internalization would not be required for the cell-killing activity of Pep-1-Phor21.Indeed, we demonstrate that Pep-1-Phor21 rapidly mediates cytolytic-based necrosis, consistent with previous studies in which lytic peptides of this type caused rapid membrane disruption [27,49].Given these properties, Pep-1-Phor21 should, therefore, circumvent many intracellularly active drug-resistance mechanisms.We have shown recently that Pep-1-Phor21 kills pancreatic cancer cells by targeting IL-13Rα2 since the cells lost sensitivity to this hybrid peptide when the receptor expression was downregulated in these cells by using its siRNA [31].While this work was in progress, two more anti-cancer peptides that target IL-13Rα2 were identified [50,51].The first peptide, which is derived from the D1 domain of IL-13Rα2, has been shown to exhibit therapeutic activity against metastatic colorectal cancer by competitively inhibiting IL-13 binding to IL-13Rα2 and thereby the receptor signaling [50].The second peptide, which was isolated as an IL-13Rα2 binding peptide by screening a T7 phage display library, has shown effects in glioblastoma therapy when conjugated with a lytic peptide [51].
As a relatively small peptide (34 amino acids, molecular weight = 3.5 kDa), Pep-1-Phor-21 should be able to penetrate vascular endothelium and solid tumor structures.However, standard monolayer cultures poorly represent the physical barriers encountered within the in vivo tumor microenvironment and, consequently, can be poor predictors of drug efficacy [52].To address such limitations, multi-cellular spheroid models have been developed [53][54][55][56], which, in addition to presenting more relevant tumor cell gene expression patterns [54], also permit the analysis of the presence of extracellular matrix proteins and cell-cell interactions [57].The latter is particularly relevant in that tight junctions between different cell types within tumors can limit efficient drug penetration [58].This study reports data from such a 3-D spheroid model which utilized live cell imaging in conjunction with cytotoxicity assays to quantitatively analyze spheroid growth.Pep-1-Phor21-mediated cytotoxicity was less efficient against spheroids (moderately higher IC 50 values), and although the requirement for the penetrance of multiple cell layers could be partly responsible for this observation, IL-13Rα2 mRNA expression was also moderately reduced in spheroid-cultured cells (Figure S1).Indeed, given the rapid physical disruption of spheroid integrity, we speculate that the latter observation forms the predominant basis for the differences in cytotoxic effect observed between monolayer and spheroid cultures.This reduction in IL-13Rα2 expression is potentially explained by the findings of previous studies in that the central layers of multi-cellular spheroids were shown to be hypoxic [59,60] and, notably, IL-13Rα2 mRNA expression in glioblastoma cells was reportedly decreased under hypoxic conditions [61].Whether hypoxia within the breast cancer spheroids accounts for the moderate reduction in IL-13Rα2 expression requires further investigation.Nevertheless, despite such obstacles, diffuse dead cell staining and spheroid rupture were rapidly detectable post-treatment, indicating that Pep-1-Phor21 could efficiently penetrate breast cancer spheroids, selectively killing IL-13Rα2-expressing tumor cells in the process.
Epigenetic changes, including DNA methylation and histone modifications, have been linked to the progression of breast cancer with markedly different epigenetic profiles discernible between the early and late stages of breast cancer [21][22][23].Of note, it has been shown that ER expression can be re-established in breast cancer cells through treatment with HDAC inhibitors [23], and we demonstrate in this study that IL-13Rα2 expression is similarly regulated in that the inhibition of either histone deacetylation or DNA methylation produced an upregulation of IL-13Rα2 expression, enhancing consequent susceptibility to Pep-1-Phor21 treatment.Encouragingly, this modulation of IL-13Rα2 expression was only demonstrable in malignant cells, pointing to potentially specific tumor cell targeting when using Pep-1-Phor21 in combination with epigenetically active anti-cancer compounds.The reason for this selective effect of epigenetic modifiers on breast cancer cells remains unclear.However, studies on other cancers revealed that IL-13Rα2 expression requires AP-1/c-jun signaling and this pathway was shown to be quiescent in non-malignant cells, which, similar to the findings of this study, were refractory to HDAC inhibitor-induced IL-13Rα2 expression [19].Whether these specific signaling events also account for the variable expression of IL-13Rα2 in the various sub-types of breast cancer requires further study.It is also possible that combinations of epigenetically active drugs will more potently upregulate IL-13Rα2 in breast cancer cells and, in this regard, it is notable that combination therapy with 5-azacytidine and the HDAC inhibitor Entinostat is currently undergoing evaluation in clinical trials as a therapy for advanced breast cancer [62].

Conclusions
We have shown that IL-13Rα2 exhibits potential as a therapeutic target, particularly for TNBC types of breast cancer.Furthermore, IL-13Rα2-expressing cells are efficiently targeted with Pep1-Phor21, even within solid spheroid cultures, consistent with the small molecular weight of this type of drug and its rapid cytolytic mode of action.Importantly, drug-induced cytolysis could be enhanced by treatment with epigenetically active anticancer compounds, suggesting that the combination adjuvant therapy of Pep1-Phor21 with such compounds may be a more effective treatment for non-TNBC-type tumors.

Figure 1 .
Figure 1.Expression of IL-13Rα2 in breast cancer tissue.(a) RT-PCR analysis of IL-13Rα2 mRNA expression in cDNA array samples (TissueScan™, Origene) derived from breast cancer (number of samples (n = 60)) and non-malignant (n = 7) tissue.Shown is the fold-change relative to mean

Figure 9 .
Figure 9. Confocal cell imaging of breast cancer spheroids treated with Pep-1-Phor21.Indicated cell lines established as spheroids (48 h) were treated with Pep-1-Phor21 or Phor21 (3 h, 30 µM) and assessed for the presence of live and dead cells using confocal fluorescent microscopy.IL-13Rα2-positive TNBC spheroids (MDA-MB-231, LM2) exhibited diffuse dead cell staining (red fluorescence, EthD-1) with the concomitant disruption of spheroid integrity after Pep-1-Phor21 treatment.Some foci of live cells (green fluorescence, Vybrant DiO) were detectable within the core of the disrupted spheroid after 3 h.In contrast, IL-13Rα2-negative MCF-10A breast epithelial and non-TNBC MCF-7 cells exhibited only viable cell staining with no observable loss of spheroid structure post-treatment.