Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

Abstract

Human surfactant protein D (SP-D) belongs to the family of collectins that is composed of a characteristic amino-terminal collagenous region and a carboxy-terminal C-type lectin domain. Being present at the mucosal surfaces, SP-D acts as a potent innate immune molecule and offers protection against non-self and altered self, such as pathogens, allergens, and tumor. Here, we examined the effect of a recombinant fragment of human SP-D (rfhSP-D) on a range of breast cancer lines. Breast cancer has four molecular subtypes characterized by varied expressions of estrogen (ER), progesterone (PR), and epidermal growth factor (EGF) receptors (HER2). The cell viability of HER2-overexpressing (SKBR3) and triple-positive (BT474) breast cancer cell lines [but not of a triple-negative cell line (BT20)] was reduced following rfhSP-D treatment at 24 h. Upregulation of p21/p27 cell cycle inhibitors and p53 phosphorylation (Ser15) in rfhSP-D-treated BT474 and SKBR3 cell lines signified G2/M cell cycle arrest. Cleaved caspases 9 and 3 were detected in rfhSP-D-treated BT474 and SKBR3 cells, suggesting an involvement of the intrinsic apoptosis pathway. However, rfhSP-D-induced apoptosis was nullified in the presence of hyaluronic acid (HA) whose increased level in breast tumor microenvironment is associated with malignant tumor progression and invasion. rfhSP-D bound to solid-phase HA and promoted tumor cell proliferation. rfhSP-D-treated SKBR3 cells in the presence of HA showed decreased transcriptional levels of p53 when compared to cells treated with rfhSP-D only. Thus, HA appears to negate the anti-tumorigenic properties of rfhSP-D against HER2-overexpressing and triple-positive breast cancer cells.

Keywords: breast cancer; hyaluronic acid; immune surveillance; innate immunity; surfactant protein D.

Figure 1

SDS-PAGE (A) and immunoblot profile (B) of the purified recombinant form of human surfactant protein D (rfhSP-D). Expression of rfhSP-D under the bacteriophage T7 promoter, expressed as ~20 kDa insoluble protein. Eluted fractions were passed through a maltose–agarose column, which appeared as a single band at ~20 kDa. Immunoreactivity of affinity-purified rfhSP-D was examined via western blotting; lane 1: BSA as a negative control; lane 2: purified rfhSP-D (5 μg/ml). Binding of rfhSP-D (10 μg/ml) to breast cancer cell lines using fluorescence microscopy, following 1 h incubation at 4°C (C,D). The nucleus of the cells was stained with Hoechst (1:10,000), and both untreated (cells only) (C) and rfhSP-D-treated (D) cells were probed with polyclonal anti-human SP-D/FITC antibody (1:200). Membrane localization of the bound proteins was only detected in the treated cells, while no FITC was detected in the untreated control (cells only).

Figure 2

Flow cytometric analysis of apoptosis induction in breast cancer cells treated with 20 μg/ml of immobilized (A) and solution-phase (B) rfhSP-D for 24 h. For both Annexin V/FITC and DNA/PI staining, 12,000 cells were acquired and plotted. A significant difference was seen among treated and untreated (cells only) samples, as evident by the shift in the fluorescence intensity. The data were expressed as the mean of three independent experiments (n = 3) done in triplicates ± SEM. Statistical significance was established using the unpaired one-way ANOVA test (**p < 0.01 and ****p < 0.0001). The percentage of apoptosis was calculated by normalizing the treated cells with their untreated counterparts. Fluorescence microscopy analysis of apoptosis induction in breast cancer cells treated with rfhSP-D (20 μg/ml) for 24 h, using an Annexin V with a propidium iodide (PI) staining kit; untreated (cells only) controls (C) and rfhSP-D-treated cells (D). The nucleus was stained with Hoechst (1:10,000), and the cell membrane was stained positively with Annexin V and PI (1:200) in treated cell lines, suggesting that cells treated with rfhSP-D induced apoptosis at 24 h, where translocation of PS to the outer plasma membrane was able to bind Annexin V due to loss of membrane integrity and PI stain was taken, which stained the DNA of apoptotic cells. No Annexin V/PI staining was detected in untreated cells.

Figure 3

Caspase activation in breast cancer cell lines following rfhSP-D treatment at 24 h. Western blot analysis showed presence of cleaved caspase 3 at 17kDa in the cells treated with rfhSP-D for 24 h. (A) Western blot analysis showed presence of cleaved caspase 9 at 37kDa (B), suggesting induction of the intrinsic pathway.

Figure 4

(i) SP-D and Hyaluronic acid (HA) presence in different histotypes of neoplastic breast (A–L), and normal ductal mammary epithelium (M–O). (A,D,G,J). A high variability of SP-D expression between different histotypes is shown, although the positivity of the signal is always attributable to neoplastic cells, as can be observed from the higher- resolution images in the right part of the figure (B,E,H,K). AEC (red) chromogen was used to visualize the binding of anti-human SP-D antibodies. (C,F,I,L,O) Histochemical staining with Alcian Blue highlighted HA distribution in breast cancer and normal tissue sections; in particular, the staining was visible in tumor-associated stroma. (ii) Immunohistochemical and histochemical analysis in Luminal-B and Her2+/ER-/PR- breast carcinoma sections of SP-D (A–F) and HA (G–L) expression, respectively (B). A high variability of SP-D and HA expression within the same isotypes is shown. It is possible to notice a slight inverse correlation between SP-D and HA expression. AEC (red) chromogen was used to visualize the binding of anti-human SP-D antibodies, whereas histochemical staining with Alcian Blue highlighted HA distribution.

Figure 5

Interaction of rfhSP-D with HA. Binding of varied concentrations of rfhSP-D to HA (20 μg/ml) by ELISA (A). The effects of rfhSP-D on Breast cancer cell lines adhesion (B). Breast cancer cells were labeled with the FAST DiI fluorescent dye and allowed to adhere to 96 microtiter wells pre-coated with HA, rfhSP-D, and BSA. The data is expressed as the mean of three independent experiments. The adhesion of cells was measured after 35 minutes of incubation at 37°C under 5% CO₂. Results are expressed as adhesion percentage with reference to a standard curve established using an increasing number of FAST DiI -labeled cells. The data is expressed as the mean of three independent experiments (n = 3) done in triplicates ± SEM. Statistical significance was established using the unpaired one-way ANOVA test (**p < 0.01 and ***p < 0.001). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D -treated breast cancer cells.

Figure 6

Apoptosis induction in BT20, BT474, and SKBR3 cell lines following HA challenge in the presence and absence of rfhSP-D (A). The data were expressed as the mean of three independent experiments (n = 3). A significant difference was seen among treated and untreated samples, as made evident by the shift in the fluorescence intensity. Staurosporine (1 μM/ml) was used as a positive control. Proliferative effects of rfhSP-D treatment on BT474 and SKBR3 breast cancer cell lines (B). BT474 and SKBR3 cells were seeded in wells pre-coated with HA, HA + rfhSP-D, and rfhSP-D alone. The percentage of proliferative cells was evaluated by staining with mouse anti-human KI-67 antibody, and KI-67-stained cells were measured via flow cytometry. The data were generated from at least three independent experiments (n = 3) and presented as mean ± SD (*p < 0.1, **p < 0.01, and ****p < 0.0001). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells. The secretion levels of FL-SP-D were confirmed and analyzed via western blotting (C). Culture medium collected from BT20, BT474, and SKBR3 cell lines was passed through a maltose Agarose column, and the eluted fractions were validated via western blotting; FL-SP-D was detected at ~43 kDa only for BT474. Both BT20 and SKBR3 cell lines did not secrete any FL-SP-D. Secreted FL-SP-D by BT474 was tested for its ability to induce apoptosis (D). No effect of secreted FL-SP-D was seen in terms of cell viability and apoptosis induction.

Figure 7

Intracellular signaling to show phosphorylation of p53 in rfhSP-D-treated BT20 and SKBR3 cell lines (A). Breast cancer cell lines were allowed to adhere to HA or HA-bound rfhSP-D, and the phosphorylation status of p53 was evaluated using total cell lysates with a PathScan Antibody Array Kit (Cell Signaling). Data were generated from at least three independent experiments and presented as mean ± SD. rfhSP-D treatment resulted in upregulation of p21 and p27 cell cycle inhibitors (B). BT20, BT474, and SKBR3 (0.4 × 10⁶) cells were seeded in a six-well-plate pre-coated with rfhSP-D and HA + rfhSP-D. The treated cells were lysed and pelleted down. The pelleted cells were subjected to RNA isolation, followed by cDNA synthesis and qPCR. The comparative quantification method was performed to calculate the efficiencies of each gene for each individual PCR and is based on the second differential maximum method or takeoff analysis. The takeoff results obtained with p21/p27 primers were normalized with the housekeeping gene TBP. qPCR assay was conducted in triplicates, and error bars represent ± SEM. Unpaired one-way ANOVA test was used to determine the significance; *p < 0.05, **p < 0.01, and ***p < 0.001 (n = 3). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells.