Dictyostelium Differentiation-inducing Factor Derivatives Reduce the Glycosylation of PD-L1 in MDA-MB-231 Human Breast Cancer Cells

Objectives Triple-negative breast cancer (TNBC) is a metastatic and intractable cancer with limited treatment options. Refractory cancer cells often express the immune checkpoint molecules programmed death-ligand 1 (PD-L1) and PD-L2, which inhibit the anticancer effects of T cells. Differentiation-inducing factors, originally found in Dictyostelium discoideum, and their derivatives possess strong antiproliferative activity, at least in part by reducing cyclin D1 expression in various cancer cells, but their effects on PD-L1/PD-L2 have not been examined. In this study, we investigate the effects of six DIF compounds (DIFs) on the expression of PD-L1/PD-L2 and cyclin D1/D3 in MDA-MB-231 cells, a model TNBC cell line. Methods MDA-MB-231 cells were incubated for 5 or 15 h with or without DIFs, and the mRNA expression of cyclin D1, PD-L1, and PD-L2 were assessed by quantitative polymerase chain reaction (qPCR). Whereas, MDA-MD-231 cells were incubated for 12 or 24 h with or without DIFs, and the protein expression of cyclins D1 and D3, PD-L1, and PD-L2 were assessed by Western blotting. Results As expected, some DIFs strongly reduced cyclin D1/D3 protein expression in MDA-MB-231 cells. Contrary to our expectation, DIFs had little effect on PD-L1 mRNA expression or increased it transiently. However, some DIFs partially reduced glycosylated PD-L1 and increased non-glycosylated PD-L1 in MDA-MB-231 cells. The level of PD-L2 was very low in these cells. Conclusions Since PD-L1 glycosylation plays an important role in preventing T cells from attacking cancer cells, such DIFs may promote T cell attack on cancer cells in vivo.

Breast cancer is the most common cancer among women, accounting for about 25% of all new female cancers each year 16,17) .Triple-negative breast cancer (TNBC) is a subtype of breast cancer that does not express the estrogen receptor, progesterone receptor, or human epidermal receptor 2, with clinical features that include being highly proliferative, metastatic, heterogenous, and refractory.Due to the lack of targetable receptors, targeted hormone therapies for TNBC are not an option [18][19][20][21] . Terefore, innovative breast cancer treatments, including novel anticancer and antimetastatic drugs, are needed.PD-L1 is expressed in 20% of TNBCs, suggesting that it may serve as a therapeutic target in this disease [22][23][24] .
In this study, we assessed the effects of six representative DIF compounds (Figure 1) on the expression of PD-L1 and PD-L2 in MDA-MB-231 cells.The results showed that although DIF compounds had little effect on PD-L1/PD-L2 protein expression or slightly decreased it, some of the DIF compounds significantly reduced glycosylated PD-L1 and increased non-glycosylated PD-L1, which may reduce PD-L1 activity.Thus, such compounds might inhibit PD-1/PD-L1 signaling, thereby facilitating activation of T cells that then attack cancer cells.

Cells and reagents
Human MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown and maintained at 37°C with 5% CO 2 and 95% air in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 4.5 g/L glucose, and antibiotics (75 μg/mL penicillin and 50 μg/mL streptomycin).DIF compounds were synthesized as previously described 34) and stored as 10 mM stock solutions in dimethyl sulfoxide (DMSO) at -20°C.

Cell proliferation assay
MDA-MB-231 cells were incubated in 12-well plates (Corning, New York, NY, USA) with each well containing 1 ml of DMEM-FBS (2 × 10 4 cells/1 mL/well) until cells attached to the bottom of the wells.After removal of the media, cells were incubated for 3 days with 1 mL DMEM-FBS containing 0.2% (v/v) DMSO or 10 or 20 μM DIF compounds.Then, the cells were observed by phase-contrast microscopy and re-incubated with 1 mL of fresh DMEM-FBS containing 5% (v/v) of the cell number indicator Alamar blue (Wako Pure Chemical Industries, Osaka, Japan) until color of the media had changed.Relative cell number was determined by measuring absorbance at 570 nm (reference at 595 nm), as described previously 31,34) .

Quantitative polymerase chain reaction (PCR)
MDA-MB-231 cells were grown in 10 cm tissue culture dishes (Corning) to 60-80% confluence in DMEM-FBS.Then the media were removed, and cells were incubated for 5 and 15 h with 10 mL DMEM-FBS containing 0.2% (v/v) DMSO or 10 and 20 μM DIF compounds.After washing the cells with 10 mL phosphate-buffered saline (PBS), cells were lysed in 1 mL Isogen (Nippon Gene Co., Ltd., Tokyo, Japan).Total RNAs were prepared from the lysates, followed by reverse transcription of 1 μg RNA into cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.We performed quantitative PCR (qPCR) to assess the relative mRNA levels of cyclin D1, PD-L1, and PD-L2 by using the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) and LightCycler Nano Instrument (Roche, Basel, Switzerland), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal control.The following primers were used: cyclin D1, Hs00765553_ m1; PD-L1, Hs00204257_m1; PD-L2, Hs00228839_ m1; and GAPDH, Hs99999905_m1 (Thermo Fisher Scientific).

Western blot analysis
MDA-MB-231 cells were incubated in 12-well plates (Corning) with each well containing 1 ml of DMEM-FBS (5-10 × 10 4 cells/mL/well) until cells attached to the bottom of the wells.After removal of the media, cells were incubated for 12 and 24 h with 1 mL DMEM-FBS containing 0.2% (v/v) DMSO or 10 or 20 μM DIF compounds.Then cells were washed with 1 mL PBS, harvested by adding 0.1-0.2ml of SDS-sample buffer solution (in proportion to cell density: relative cell number), crushed and heated by sonication, and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Western blot analysis was performed as previously described 35) .Briefly, proteins were resolved by SDS-PAGE and electrotransferred to membranes.After blocking with 0.3% non-fat dry-milk powder in a Tris-buffered saline (10 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween 20; designated TBS-T hereafter) for 1 h at room temperature, the membranes were incubated for 1 h at room temperature with primary antibodies against cyclin D1, cyclin D3, PD-L1, PD-L2, or GAPDH in TBS-T.After washing with TBS-T, membranes were incubated for 1 h at room temperature with anti-mouse alkaline phosphatase or anti-rabbit IgG secondary antibody in TBS-T.Visualization of the protein bands was performed in an alkaline buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) containing 5-bromo-4chloro-3-indolyl-phosphate (62.5 μg/ml) and nitro blue tetrazolium (125 μg/ml); the labeled protein bands were quantified with Adobe Photoshop Element 2020 (Adobe, San Jose, CA, USA) and ImageJ software (version 1.53a).

Statistical analyses
Statistical analyses were performed using the Welch's t-test (two-tailed) with p<0.05 considered statistically significant.

Selection of six DIF compounds for evaluation
We first compared and evaluated the antitumor activity of six DIF compounds in MDA-MB-231 cells and LM8 murine osteosarcoma cells for comparison (Table 1).Because the antiproliferative and antimigratory activities of the natural compounds, DIF-1 and DIF-3, are comparatively weak in MDA-MB-231 cells and LM8 cells, we selected three compounds, one derivative of DIF-1 and two derivatives of DIF-3.Of the three compounds, Br-DIF-1 (1-(3,5-dibromo-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) is charac-teristic in the following points.The antiproliferative activity of Br-DIF-1 is comparatively weak in both cell lines but it strongly suppresses the serum-induced migration of MDA-MB-231 cells 43) and lysophosphatidic acid-induced migration of LM8 cells 36) (Table 1); therefore, Br-DIF-1 may be a good lead compound for the development of antimetastatic agents.Whereas, DIF-3(+1) hexan-1-one) are characteristic in the following points.The antiproliferative and antimigratory activities of DIF-3(+1) and Bu-DIF-3 are comparatively strong in both cell lines (Table 1); therefore, DIF-3(+1) and Bu-DIF-3 may be good lead compounds for the development of antiproliferative and antimetastatic agents.On the other hand, the antiproliferative and antimigratory activities of DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one) are weak in both cell lines (Table 1), but as this derivative has strong antimalarial activity 45) , we used it for subsequent comparative analyses.In this study, we reconfirmed the effects of these DIF compounds on cell proliferation and morphology in MDA-MB-231 cells (Figure 2), where we used DIF-1, Br-DIF-1, DIF-1(+2), DIF-3, and DIF-3(+1) at 20 μM and Bu-DIF-3 at 10 μM since Bu-DIF-3 at more than 10 μM is highly toxic to the cell.The concentrations of these compounds are omitted hereafter unless otherwise needed.

Effects of DIFs on cyclin D1 expression in MDA-MB-231 cells
DIF-1 and DIF-3 suppress cell proliferation, at least in part, by reducing cyclin D1 expression in many cancer cell lines 33,35,42,[46][47][48] . T confirm the general actions of DIFs, we first evaluated the effects of the six DIF compounds on cyclin D1 mRNA expression in MDA-MB-231 cells (Figure 3A).Cyclin D1 mRNA expression was not significantly suppressed after 5 and 15 h of incubation with DIF-1 and its derivatives except for Br-DIF-1, which slightly suppressed expression at 5 h.In contrast, DIF-3, DIF-3(+1), and Bu-DIF-3 significantly suppressed cyclin D1 mRNA expression after 5 h; after 15 h of incubation, only DIF-3(+1) significantly suppressed expression.
Next, we examined the effects of DIFs on the protein expression of D-type cyclins D1 and D3 in MDA-MB-231 cells; note that the cells were alive and healthy up to 24 h of incubation with the DIFs (Figure 4) until the cells were collected for Western blot analysis.Expression of cyclins D1 and D3 was not significantly changed after 12 and 24 h of incubation with DIF-1 and its derivatives (Figure 5A); however, DIF-3, DIF-3(+1), and Bu-DIF-3 significantly suppressed expression of both cyclins D1 and D3 protein after 12 and 24 h of incubation except for cyclin D1 with DIF-3 at 24 h (Figure 5B).These results showed that at the indicated concentrations, DIF-1 compounds had little effect on cyclins D1 and D3 protein expression but DIF-3 compounds reduced their expression in MDA-MB-231 cells.

Antiproliferative and antimigratory effects of DIF on MDA-MB-231 cells
DIF-1, DIF-3, and their derivatives have antiproliferative and/or antimigratory activities in a variety of tumor cells in vitro and in vivo [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] , and are expected to be lead compounds for the development of anticancer agents. W previously showed that several DIF derivatives, such as DIF-3(+1) and Bu-DIF-3, strongly suppressed both the proliferation and serum-induced migration of MDA-MB-231 43) (Table 1), while another group showed that 30 μM DIF-1 significantly suppressed the proliferation of MCF-7 human breast cancer cells in vitro, at least in part via the reduction of cyclin D1 expression 51) .More recently, the same group has shown that 30 μM DIF-1 inhibited the proliferation and migration of MDA-MB-231 cells in vitro, and 150 mg/kg DIF-1 (administered every 12 h for 14 days to mice) inhibited the growth and metastasis of TNBC cells in vivo 48) .Herein, we confirmed that DIF-3(+1) and Bu-DIF-3, the most promising DIF derivatives as anticancer agents evaluated in this study, may suppress cell proliferation in MDA-MB-231 cells, at least in part by reducing the expression of cyclins D1 and D3 (Figure 3A, 5B).

Suppression of PD-L1 glycosylation by DIF
As described in the Introduction section, because MDA-MB-231 cells express the immune checkpoint molecules PD-L1 and PD-L2 24,44) , these cells can be used for screening candidate agents targeting PD-L1/PD-L2.Here, we evaluated the effects of six DIFs (Figure 1) on the expression of PD-L1 and PD-L2 in MDA-MB-231cells (Figure 3B, C,  5C-F).Initially, we expected the DIFs to suppress expression of PD-L1 and PD-L2; contrary to our expectations, Br-DIF-1, DIF-3, DIF-3 (+1), and Bu-DIF-3 transiently increased the mRNA expression of PD-L1 at 5 h (Figure 3B), whereas the total protein expression of PD-L1 was not significantly increased after 12 and 24 h of incubation with the DIFs (Figure 5C, D).The reason for the transient increase in PD-L1 mRNA expression is unknown, but, for example, DIFs might induce a transient increase in PD-L1 mRNA expression as a secondary effect of arresting cell cycle.In any case, since protein expression is generally regulated by the balance between mRNA translation and protein degradation, any PD-L1 protein synthesized at 5 h may have been degraded to some extent by 12 h during incubation with DIFs.
The expression of PD-L2 protein was decreased by treatment with Br-DIF-1 at 24 h and DIF-3 and DIF-3(+1) at 12 h (Figure 5E, F).However, as the expression level of PD-L2 is considerably low in MDA-MB-231 cells (Figure 5E, F), it is unknown whether these levels of PD-L2 are involved in immunosuppression of T cells.

Conclusions
In this study, we assessed the effects of six DIF compounds on the expression of PD-L1 and PD-L2 in MDA-MB-231 cells and found that Br-DIF-1, DIF-3(+1), and Bu-DIF-3 reduced PD-L1 glycosylation.Our results suggest that such compounds might inhibit PD-L1/PD-1 signaling and thereby reduce immunosuppression of T cells, thus facilitating their attack of cancer cells (Figure 6), although it is currently unknown whether such a DIF-induced reduction in glycosylation actually affects T cell activity.Accordingly, DIFs may be good lead compounds for the development not only of antiproliferative and antimigratory (antimetastatic) drugs but also of immunotherapy drugs against TNBC and possibly some other metastatic cancers.It may be worth investigating the inhibitory effects of other DIF derivatives (~50 DIF derivatives in total) 34,40,43) on PD-L1 and PD-L2 protein expression in MDA-MB-231 cells as well as in other cancer cells.

Figure 1
Figure 1 Chemical structure of six differentiation-inducing factor (DIF) compounds

Figure 2
Figure 2 Effects of DIF compounds on cell growth and morphology in MDA-MB-231 cells.Cells were incubated for 3 days with 0.2% DMSO or 20 μM DIF-1 or its derivatives, or with 20 μM DIF-3 or DIF-3(+1) or 10 μM Bu-DIF-3, and relative cell number was determined; data are the mean ± standard deviation of a single experiment performed in triplicate (A).**p < 0.01 vs. DMSO control.Representative photos of the cells at Day 3 are shown in (B).

Figure 3
Figure 3 Effects of DIFs on the mRNA expression of cyclin D1 (A), PD-L1 (B), and PD-L2 (C) in MDA-MB-231 cells, as assessed by qPCR.Cells were incubated for 5 or 15 h in the presence of DMSO (control) or the indicated DIFs.The mean ± standard deviation of 3 or 4 independent experiments relative to control are shown.*p < 0.05, **p < 0.01 vs. DMSO control.GAPDH is glyceraldehyde-3-phosphate dehydrogenase.