From the perspective of a protein-based treatment tool, C. perfringens iota toxin is relatively unexplored yet possesses promising potential for targeting breast cancer, as recent evidence now implicates LSR and CD44 as functional facilitators of iota cytotoxicity 35 36 . Thus, identifying the precise mechanisms of interaction between iota toxin with LSR and CD44 is a necessary step towards developing targeted therapies for breast cancer. Previous cancer studies have shown that LSR is positively correlated with ERα expression, tumor initiating cells, and chemoresistance 14 21 . Others have shown that high CD44 expression correlates to a basal-like phenotype and unfavorable prognosis in breast cancer patients 17 18 19 . Given this, we first assessed LSR and CD44 expression in a panel of well-characterized breast cancer cell lines. LSR was detectable in the majority of luminal and basal-like subtypes, while the claudin-low lines had little to no expression of LSR (Figure 1A,B). Conversely, CD44 expression was readily detectable in claudin-low lines while basal-like and luminal lines had little to below detectable expression levels via western blot analysis.

To further understand the role LSR and CD44 play in breast cancer susceptibility to iota toxin, we evaluated the effects of toxin exposure over time. Cells were treated with control (Ia only) or with varying concentrations of Ia and Ib as indicated (Figure 1C), under normal growth conditions. Toxin sensitivity, easily observed by robust cell rounding and altered morphology, was documented at 0, 1, 2, 4, 6, and 8 h post treatment. Results show that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were moderately sensitive, and LSR-/CD44+ lines were interestingly resistant to the cytotoxic effects of iota toxin in both a time and dose-dependent manner (Figure 1C; Table 1; cell rounding quantified in Additional file 1: Figure S1A). Furthermore, in fibroblasts and hepatocytes, binding of LSR to free fatty acids induces a conformational change that mediates binding of apoproteins B- and E-containing lipoproteins, leading to their subsequent internalization and degradation 37 38 39 . Thus, to further verify that iota toxin sensitivity is conveyed through LSR, we treated two LSR + cell lines moderately sensitive to iota toxin with oleic acid. Iota cytotoxicity was enhanced by oleic acid (Figure 1D; Table 2; cell rounding quantified in Additional file 1: Figure S1A).

(−) Resistant, 0 to 10% cell rounding; (+) Sensitive, 11 to 50% cell rounding; (++) Highly Sensitive, >50% cell rounding. Toxin Sensitivity recorded at 8 h post treatment.

(−) Resistant, 0 to 10% cell rounding; (+) Sensitive, 11 to 50% cell rounding; (++) Highly Sensitive, >50% cell rounding. Toxin Sensitivity at 8 h post treatment.

To confirm the observed cell death, cytotoxicity assays were performed on highly sensitive, LSR+/CD44- MCF-7 and resistant LSR-/CD44+ Hs578t cells when treated with low and high concentrations of iota toxin (Figure 1E). Results indicated that highly sensitive MCF-7 cells had a significant increase in cytotoxicity in a time- and dose- dependent manner when challenged with iota toxin (P < 0.01). However, resistant Hs578t cells did not have significant changes in cytotoxicity compared to controls treated with Ia only (Figure 1E). No significant alteration in expression or activation (via cleavage) of cytochrome c or caspase-3 was detected via western blot analysis, suggesting that cell death was not mediated through these apoptotic pathways (data not shown). Collectively, these data suggest that expression of LSR and not CD44 is required for sensitivity of breast cancer cells to iota toxin.

It is well established that glycosylation is necessary for proper functioning of CD44 in certain pathways 12 40 41 42 . While the glycosylation status of LSR is not characterized, sequence analysis indicates LSR contains residues as potential N-glycosylation sites. Thus, to determine whether glycosylation of LSR and/or CD44 plays a role in iota toxin sensitivity, we first assessed the glycosylation status of LSR. MCF-7 cells were treated with vehicle control or de-glycosylation agents Tunicamycin or Swainsonine (25 μg/ml, each). Western blot analysis indicated no shift in electrophoretic migration by SDS PAGE, suggesting that LSR was not heavily glycosylated (Additional file 2: Figure S2A). To verify that the de-glycosylating agents were functioning properly in the same cell line, expression of a known glycoprotein (E-cadherin) was assessed 43 44 . Treatment of MCF-7 cells with tunicamycin or swainsonine decreased the levels of glycosylated E-cadherin (top band) and increased the non-glycosylated (bottom) bands, thus confirming activity of the de-glycosylation agents (Additional file 2: Figure S2A). To confirm that the de-glycosylation agents did not affect the role of LSR cytotoxicity, LSR+/CD44- MCF-7 cells were cultured with or without Tunicamycin, followed by iota toxin. As shown in Additional file 2: Figure S2B and Additional file 3: Table S1, sensitivity of breast cancer cells to iota toxin was not altered by tunicamycin, suggesting that glycosylation does not play a role in toxin susceptibility.

Knowing that glycosylation is necessary for CD44 function in certain pathways 12 40 41 42 , we sought to determine whether glycosylation was required to convey toxin resistance. When LSR-/CD44+ Hs578t cells were treated as described above, sensitivity to iota toxin was not altered, suggesting that deglycosylation of CD44 does not affect sensitivity to iota cytotoxicity (Additional file 2: Figure S2B and Additional file 3: Table S1).

Previous studies have determined that LSR and CD44 have multiple variants/isoforms that occur in cancer 45 46 47 . We performed variant specific qRT-PCR analysis in order to determine whether there was a correlation between iota toxin sensitivity and expression of LSR and/or CD44 variants. Although expression of LSR and CD44 variants varied among cell lines, there was no correlation between expression of LSR and/or CD44 variants with sensitivity to iota toxin (Additional file 4: Figure S3 and Additional file 5: Figure S4).

To directly test the ability of LSR to convey iota toxin cytotoxicity, we stably knocked down LSR in MCF-7 and MDA-MB-231 cells resulting in decreased expression (64% and 46%, respectively) compared to control (Figure 2A). LSR knockdown lines were subjected to treatment with iota toxin, and as shown in Figure 2A and Table 3, reduced LSR expression in MCF-7 cells resulted in decreased sensitivity compared to scrambled control. This result could be due to the high expression of LSR in MCF-7 cells, suggesting there were still sufficient receptor numbers on the surface to facilitate intoxication. Conversely, knockdown of LSR in MDA-MB-231 cells, which have significantly lower basal levels of LSR expression (Figure 1A) resulted in a significant decrease in toxin sensitivity compared to scrambled, suggesting that reduction of LSR diminishes iota toxin sensitivity (Figure 2A; cell rounding quantified in Additional file 1: Figure S1B). To confirm our findings, LSR was overexpressed in two iota toxin resistant (LSR-/CD44+) cell lines, Hs578t and SUM159 (Figure 2B), and then treated with iota toxin. It is of note that overexpression of LSR in Hs578t and SUM159 cells did not increase sensitivity to iota toxin. While there were a few cells that succumbed to the toxin effects, the vast majority of the cell population remained resistant to the toxin (Figure 2B; Table 3; cell rounding quantified in Additional file 2: Figure S2B). These data suggested that introduction of LSR into LSR negative breast cancer cells does not increase sensitivity to iota toxin.

(−) Resistant, 0 to 10% cell rounding; (+) Sensitive, 11 to 50% cell rounding; (++) Highly Sensitive, >50% cell rounding. Toxin Sensitivity at 8 h post treatment.

Given that introduction of LSR into LSR^low/CD44^high expressing, claudin-low cell lines did not increase sensitivity to iota toxin and that CD44 can facilitate iota cytotoxicity in non-breast cancer cells 35 36 , we sought to determine if iota toxin binding to CD44 was resulting in resistance to cytotoxicity. The claudin-low breast cancer cell line SUM1315mo was chosen as a model because they are LSR^low/CD44^low and importantly, sensitive to iota toxin. Consistent with our prior results, stable knockdown of LSR in SUM1315mo cells (approximate 83% reduction in LSR; Figure 3A) significantly decreased toxin sensitivity in a time and dose dependent manner while overexpression significantly increased cytotoxicity compared to control cells (P < 0.05; Figures 3B,C and 4; cell rounding quantified in Additional file 1: Figure S1C,D). This was contradictory to our data with CD44^high expressing Hs578t and SUM159 cells. In the absence of CD44, overexpression of LSR in SUM1315mo cells increased sensitivity to iota toxin. This supports the hypothesis that CD44 expression in breast cancer cells may provide resistance to iota toxin. To directly test the functional role of CD44 in conveying toxin sensitivity, CD44 was overexpressed in our model (Figure 4B) and then treated with iota toxin. Reintroduction of CD44 into SUM1315mo cells indeed resulted in resistance to iota toxin (P < 0.05; Figure 4C, D; cell rounding quantified in Additional file 1: Figure S1E). Sensitivity of these cells was reduced to control levels even in the presence of high concentrations of iota toxin, directly demonstrating that CD44 expression conveys resistance to intoxication.

While CD44 promotes iota cytotoxicity in non-breast cancer cells 35 36 , our data is the first to illustrate that CD44 may actually confer resistance to iota toxin in breast cancer. To determine the mechanisms of resistance conveyed by CD44, we further investigated aspects of toxin endocytosis via lysosome formation. LSR knockout and knockin cells (SUM1315mo), as well as CD44 knockin cells were treated with Ia only (control) or a high concentration of iota toxin (Ia 100 ng/ml + Ib 200 ng/ml). When challenged with iota toxin, significantly lower levels of lysosomes were evident in LSR knockout cells compared to scramble control cells (P < 0.001; Figure 5A). Correspondingly, LSR-overexpression increased the number of lysosomes versus controls. Moreover, CD44-overexpression in the cells directly demonstrated a significant reduction in lysosome formation when challenged with the toxin compared to controls and LSR + cells (P < 0.001). These data correlate LSR expression with enhanced toxin-stimulated lysosome formation and establish a mechanism for CD44-based inhibition via lysosome formation.

To confirm our results of toxin endocytosis and incorporation into lysosomes, cells were treated with or without iota toxin, washed, and the lysates analyzed for internalized iota toxin (Ib) via western blotting. Similar to results observed with lysosome formation, knockdown of LSR significantly decreased levels of intracellular toxin compared to scrambled control, while overexpression increased toxin endocytosis (P < 0.05; Figure 5B). These data strongly confirm that LSR is not only a critical receptor for iota toxin cytotoxicity in breast cancer, but that it increases endocytosis of the toxin. Interestingly, reintroduction of CD44 resulted in a significant decrease in detectable Ib when compared to the control or LSR-overexpressing cells. This indicates that expression of CD44 in breast cancer cells confers iota toxin resistance by inhibiting endocytosis, a role not previously defined for CD44.

Cellular chemotherapeutic resistance is a major factor involved in poor response and reduced survival in breast cancer patients 48 . A common and successful targeted therapy for ERα-positive breast cancers includes anti-estrogen drugs, such as tamoxifen. However, an emerging problem has been that ~33% of patients given tamoxifen therapy for five years develop recurrent tumors, and of those, 26% subsequently die 49 50 51 . Previous studies in our laboratory show that LSR is positively correlated with ERα expression 21 . Tamoxifen-resistant breast cancers are derived from ERα-positive tumors, and thus are likely to have high expression of LSR making them potential candidates for successful treatment with iota toxin. Ultimately, such information opens up the possibility that iota toxin represents a novel, targeted therapeutic for breast cancer.

To directly evaluate iota toxin susceptibly of tamoxifen-resistant, MCF-7-derived breast cancer cell lines, ERα-positive TMX2-4 and TMX2-11, as well as ERα-negative TMX2-28, cells were assessed for LSR and CD44 expression. All three tamoxifen-resistant lines expressed LSR, but not CD44 (Figure 6A), via western blot analysis. When treated with iota toxin, all three tamoxifen-resistant lines were readily susceptible (Figure 6B,C; *P < 0.05; cell rounding quantified in Additional file 1: Figure S1F). These data suggest that iota toxin has the potential as a targeted therapy for tamoxifen-resistant breast cancers.

MCF-7, T47D, ZR-75-1, HCC1937, MDA-MB-468, BT-20, HCC1143, MDA-MB-231, Hs578t, BT-549, AU565, SKBR3, M99005, MCF-10AI, MCF-10AIII, and MCF-10AIV cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). SUM159, SUM149, Sum190, and SUM1315mo cells were obtained from Asterand (Detroit, MI, USA). TMX2-4, TMX2-11 and TMX2-28 cells were a kind gift from Dr. Kathleen Arcaro (University of Massachusetts, Amherst). Cells were cultured according to manufacturer’s recommendations and passaged via trypsinization when approximately 80% confluent.

Iota toxin components Ia and Ib were purified as described previously 70 . For toxin sensitivity assays, cells were seeded at 1 - 3×10⁴ concentrations, enabling confluency 48 h later. Cells were then treated as either a control (10 ng Ia/ml) or with varying concentrations of iota toxin (Ia 10 ng/ml + Ib 20 ng/ml; Ia 25 ng/ml + Ib 50 ng/ml; Ia 50 ng/ml + Ib 100 ng/ml; Ia 100 ng/ml + Ib 200 ng/ml) and cultured under normal growth conditions. Observations to determine toxin sensitivity, indicated by a rounded morphology indicative of cell death, were made at 0, 1, 2, 4, 6, and 8 h post treatment. For oleic acid assays, cells were treated in the presence or absence of 0.8 mM oleic acid at 37°C, 30 min prior to added toxin. Images were obtained by Spot Advanced version 4.5 (Sterling Heights, Michigan).

RFP containing HuSH shRNA plasmids containing Homo sapiens LSR specific shRNA and Myc-DDK-tagged TrueORF clones of Homo sapiens LSR and CD44 were obtained from OriGene Technologies (cat# TF303412, RC223636, and RC221771; Rockville, MD). Cells were transfected using TurboFectin 8.0 (Thermo Scientific, Rockford, IL) according to manufacturer’s instructions. For stable transfection, cells were passaged at a 1:10 dilution into fresh growth medium containing 2.5 μg/ml Puromycin or 500–900 μg/ml of G418 (Life Technologies, Grand Island, NY). Control cells were simultaneously transfected with an empty plasmid vector and selected in antibiotic-containing medium as described above.

Cells were lysed in RIPA Buffer (50 mM Tris Base, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.25% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Halt™ Thermo Scientific, Rockford, IL). Equal protein concentrations of total cell lysates, as determined by the Coomassie Plus Protein Assay (Thermo Scientific, Rockford, IL), were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (BioExpress, Kaysville, UT). Membranes were blocked in 5% non-fat milk in TBST (1.0 M Tris–HCl, 5.0 M NaCl, 0.1% Tween) for 1 h at room temperature, then incubated with primary antibody against LSR (1:750; sc-133765), HCAM (CD44; 1:500; sc-7297), E-cadherin (1:500; sc-7870; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-Ib (1:1000) overnight at 4°C in TBST containing 5% BSA. Membranes were then washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (GE Healthcare, Piscataway, NJ) in TBST with 5% milk for 1 h at room temperature. Mouse monoclonal α-tubulin antibody was used to evaluate equal protein loading across all lanes at a 1:5000 dilution (T6199; Sigma Aldrich, St. Louis, MO). WesternBright ECL Kit (Bioexpress, Kaysville, UT) was used to detect peroxidase activity. NIH Image J64 software was used to quantify western blots.

Immunocytofluorescence was performed as previously described 71 . Briefly, cells were grown on 8-well chamber slides (Research Products International, Mt. Prospect, IL,) and fixed/permeabilized in ice-cold methanol:acetone. Following fixation, cells were blocked with 1% BSA and 5% normal horse serum in PBS, stained with the indicated primary antibody (1:100 dilution of anti-LSR, sc-133765 or anti-HCAM (CD44), sc-7297) for 1 h at 4°, washed, and then incubated for 30 min with an anti-rabbit or anti-mouse Alexa Fluor 488 secondary antibody (1:1000 dilution, Invitrogen). Coverslips were applied with ProLong® Gold Antifade Reagent and DAPI (Life Technologies). Imaging was performed on a Nikon DiaPhot microscope with digital camera and NIS-Elements 4.11.00 (Nikon Instruments Inc., Melville, NY). All cell lines and samples were obtained in compliance with the Helsinki Declaration and performed in accordance with the guidelines of the North Carolina Central University Institutional Review Board, approval 1201027.

Cells were grown under normal growth conditions until approximately 70% confluent, and then serum starved overnight. Cells were subsequently treated with 25 μg/ml of either Tunicamycin (MP Biomedical LLC, Solon, OH) or Swainsonine (Calbiochem, San Diego, CA), or vehicle control. Twenty-four hours post treatment, cell lysates were collected and analyzed via western blot analysis to determine glycosylation status of LSR.

Cells were grown under normal growth conditions until approximately 70% confluent. Cells were then treated as either a control (10 ng Ia/ml), or with iota toxin consisting of Ia (100 ng/ml) plus Ib (200 ng/ml), and cultured under normal growth conditions for 30 min. Following treatment, LysoTracker Green DND-26 (Cell Signaling Technology, Danvers, MA) was diluted 1:20,000 (50 nM) and added directly into growth medium, followed by imaging on a NIS-Elements 4.11.00. A minimum of three replicate wells was plated for each independent experiment, with a minimum of five fields imaged per well.

Cells were seeded at 1- 3×10⁴ concentrations, obtaining confluency 48 h later. Cells were then treated as controls (10 ng Ia/ml) or with iota toxin at low (Ia 10 ng/ml + Ib 20 ng/ml) or high (Ia 100 ng/ml + Ib 200 ng/ml) concentrations followed by culturing under normal growth conditions for 0, 2, 4, 6, and 8 h. Post treatment, cytotoxicity was determined using a CytoTox-Fluor™ Cytotoxicity Assay (Promega, Madison, WI) per manufacturer’s instructions.

RT-qPCR amplification reactions were conducted in duplicate using 1X Brilliant II SYBR® Green QPCR MasterMix (Agilent Technologies, Cary, NC) in the presence of variant specific primers (800 nM each; Additional file 3: Table S2) and 40 ng of cDNA (based on total RNA) in 20 μl. A non-template reaction was used as negative control. PCR conditions consisted of denaturation at 95°C for 10 min, activation of the DNA polymerase, followed by 40 cycles of 95°C for 15 seconds and specific annealing temperatures for each splicing variant for 1 min. Melting curves were generated after amplification at 95°C for 15 seconds, 60°C for 30 seconds and 95°C for 15 seconds. All reactions were conducted in a Stratagene Mx3005P detection system (Stratagene, La Jolla, CA). Amplification efficiency of each pair of primers was calculated using standard curve dilutions and incorporated into the calculation for relative expression differences as previously described 72 . The optimal normalization factor was calculated as the geometric mean of the reference targets B2M, SDHA, UBC and YWHAZ.

RT-qPCR amplification reactions were conducted in duplicate using 1X Brilliant II SYBR® Green QPCR MasterMix (Agilent Technologies, Cary, NC) in the presence of optimized variant specific primers (800 nM; Supplementary Table 1) and 100 ng of cDNA (based on total RNA) in 20 μl. A non-template reaction was used as negative control. PCR conditions were the same as those used for CD44 analysis. All reactions were conducted in an Applied Biosystems’ 7500 Real Time PCR system (Grand Island, NY). Raw data were normalized to GAPDH and analyzed via the comparative CT (^ΔΔCT) method.

To evaluate toxin sensitivity, relative percent of cell rounding was determined via visualization using light microscopy for each treatment group and statistical significances between treatment groups and across cell lines was determined by student t-test using GraphPad Prism version 3.02 software (GraphPad Software Inc., San Diego, CA), significance was set at P < 0.05. Differences in statistical significance between cell lines regarding CD44 and LSR expression levels (total and variants), was determined by ANOVA and post-hoc two-tailed comparisons. Significance was set at P < 0.05 and a Bonferonni correction was used to adjust the P-value of t-tests. Graphs were plotted in Microsoft Excel as mean ± S.D.