Glycosphingolipid expression at breast cancer stem cells after novel thieno[2,3-b]pyridine anticancer compound treatment

Glycosphingolipid expression differs between human breast cancer stem cells (CSC) and cancer non-stem cells (non-CSC). We performed studies of viability, type of cell death, cancer stem cell percent and glycosphingolipid expression on CSC and non-CSC after treatment of MDA-MB-231 and MDA-MB-453 triple-negative breast cancer cells with a newly developed thienopyridine anticancer compound (3-amino-N-(3-chloro-2-methylphenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide, 1). Compound 1 was cytotoxic for both breast cancer cell lines and the majority of cells died by treatment-induced apoptosis. The percent of cancer stem cells and number of formed mammospheres was significantly lower. Glycosphingolipids IV6Neu5Ac-nLc4Cer and GalNAc-GM1b (IV3Neu5Ac-Gg5Cer) not reported previously, were identified in both CSCs and non-CSCs. IV6Neu5Ac-nLc4Cer had increased expression in both CSCs and non-CSCs of both cell lines after the treatment with 1, while GM3 (II3Neu5Ac-LacCer) had increased expression only on both cell subpopulations in MDA-MB-231 cell line. GalNAc-GM1b, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) and GM2 (II3Neu5Ac-GalNAcβ1-4Galβ1-4Glcβ1-1Cer) were increased only in CSCs of both cell lines while GD3 was decreased in CSC of MDA-MB-231 cell line. Due to its effect in reducing the percentage of cancer stem cells and number of mammospheres, and its influence upon several glycosphingolipid expressions, it can be concluded that compound 1 deserves attention as a potential new drug for triple-negative breast cancer therapy.

Flow cytometric analyses. Equal numbers of cells were seeded in 6-well plates and treated with 2 µM 1 and then analysed for apoptosis. After treatment with 1, the cells were trypsinized, washed with phosphate buffered saline (PBS) and resuspended in 100 µl of the binding buffer containing 5 µl Annexin-V-FITC and/or 5 µl of PI (Annexin-V-FITC Apoptosis Detection Kit I, BD Biosciences). The cells were incubated for 15 min at room temperature in the dark and thereafter analysed by flow cytometry (BD Accuri C6, BD Biosciences). The percentages of apoptotic cells (Annexin-V positive cells) were analysed using the FlowLogic Software (Inivai) and presented as mean ± standard deviation (SD).
Data acquisition of triple and fourfold stained samples was performed on a BD Accuri 6 cytometer and analysed using the FlowLogic Software. CD44 + cells and CD133 + were gated and CSC were determined. Glycosphingolipids Gb 4 Cer, nLc 4 Cer, IV 3 Neu5Ac-nLc 4 Cer, IV 6 Neu5Ac-nLc 4 Cer, GM3, GD3, GM2, Gg 3 Cer and   Fig. 2A). In the MDA-MB-453 cell line, concentration of 250 nM of 1 showed cytotoxicity after 48 h and the maximum of cytotoxic effect was after 72 h after treatment with 5 µM of 1 (Fig. 2B).
Compound 1-mechanism of cell death. To determine whether the MTT findings are due to cell death or cell cycle arrest, we subsequently determined the type of cell death induced by 48 h treatment with 2 µM of compound 1. The majority of cells died by treatment-induced apoptosis in both cell line as shown in Fig. 3.

Cancer stem cells.
In breast cancer cell lines, such as MDA-MB-231, a subset of markers, including CD44 + /CD24 − has been shown to enrich CSC 27 . Treatment with 1 resulted in a statistically significant decrease of the CD44 + /CD24 − subpopulation from 89.9% (untreated control) to 55.5% (Fig. 5A). In the MDA-MB-453 breast cancer cell line, expression of CD44 is very low and CD44 + /CD24 − subpopulation is not considered CSC subpopulation 6 , and this subpopulation significantly increases after treatment with 1 (Fig. 5C). Much more reliable marker of CSCs in the MDA-MB-453 cell line is CD133. After treatment with 1, a significant decrease of CD133 + subpopulation from 48.3% in untreated control to 19.4% was obtained (Fig. 5B).  In MDA-MB-453 cell line, the percentages of GM3, IV 3 Neu5Ac-nLc 4 Cer and IV 6 Neu5Ac-nLc 4 Cer positive cells were significantly increased only in CSC subpopulation, whilst IV 6 Neu5Ac-nLc 4 Cer positive cells decreased within non-CSC ( Fig. 6A, B, lower row). Percentage of GD3 positive cells was not affected by treatment with compound 1 (Fig. 6A, B, lower row). Compound 1 increases expression of IV 3 Neu5Ac-nLc 4 Cer in both CSC and non-CSC cells, while expression of GD3 was increased only in CSC subpopulation, and expression of IV 6 Neu5Ac-nLc 4 Cer in non-CSC cells (Fig. 6C, D, lower row). In MDA-MB-453 cell line, percentages of GM2, GalNAc-GM1b, Gg 3 Cer, and Gb 4 Cer positive cells were significantly increased only in CSC subpopulation, whilst percentage of GM2 was slightly decreased in non-CSC subpopulation (Fig. 7A, B, lower row). Compound 1 significantly increases the expression of GM2, GalNAc-GM1b, Gg 3 Cer, and Gb 4 Cer in CSC subpopulation (Fig. 7D, lower row), while expression of these GSLs was not affected by treatment with compound 1 in CSC of MDA-MB-453 cell line (Fig. 7C, lower row).

Expression of neutral GSL with terminal Gal residue at CSCs and non-CSCs.
There was no difference in the percentage of nLc 4 Cer positive cells in both CSC and non-CSC after treatment of MDA-MB-231 cells with compound 1 (Fig. 8A, B, upper row). Also, the expression of nLc 4 Cer was not affected by compound 1, in both cell subpopulations of this cell line (Fig. 8C, D, upper row).
The percentage of nLc 4 Cer positive cells was significantly increased in CSC subpopulation in MDA-MB-453 cell line treated with compound 1 (Fig. 8A, lower row), and not affected in non-CSC − subpopulation (Fig. 8B,

Discussion
We found that newly developed anticancer compound, 3-amino-N- Glycosphingolipids that were increased in both CSCs and non-CSCs after compound 1 treatment of MDA-MB-231 cells are acidic GSLs: gangliosides GM3 and IV 6 Neu5Ac-nLc 4 Cer. Sialic or N-acetyl-neuraminic acid (Neu5Ac) is added in the last step of their synthesis (Fig. 9A). The last step of GalNAc-GM1b, Gb 4 Cer and GM2 synthesis, that were increased only at CSCs after compound 1 treatment, includes GalNAc addition. GalNAc residue must be activated by binding to UDP-GalNAc. That is achieved mostly by conversion of UDP-GlcNAc to UDP-GalNAc (Fig. 9B). Fructose-6-P is common metabolite of glycolysis and UDP-GalNAc synthesis. Neu5Ac, www.nature.com/scientificreports/ needed for acidic GSL synthesis, and UDP-GalNAc share UDP-GlcNAc as common precursor (Fig. 9B) 30 . In non-treated MDA-MB-231 cells, we found a 23-fold higher GalNAc-GM1b expression in non-CSCs compared to CSCs, together with increased Gb 4 Cer and IV 6 Neu5Ac-nLc 4 Cer, all containing GalNAc as last sugar residue (Fig. 9A, C). In addition, gangliosides GM2 and GM3, were increased in non-treated non-CSCs, but not so dramatically as GalNAc-GM1b. These results indicate that glycolysis could be slower in non-treated non-CSCs in comparison to CSC, giving more precursors for UDP-GalNAc and Neu5Ac synthesis. Therefore, the findings at CSCs after compound 1 treatment of MDA-MB-231 cells, increased GM3, IV 6 Neu5Ac-nLc 4 Cer, GalNAc-GM1b, Gb 4 Cer, and GM2, could indicate CSC glycolysis slowdown. Cancer stem cells of glioma are more glycolytic than non-CSCs due to a mitochondrial voltage-dependent anion channel that controls the phenotype transition between glioma stem cells and non-stem cells 31 . The channel is highly expressed in non-CSC relative to CSC and coupled to a glycolytic rate-limiting enzyme platelet-type of phosphofructokinase on mitochondrion to inhibit kinase-mediated glycolysis required for CSC maintenance. During tumorigensis, distinct GalNAc transferases (GALNTs) can be differently expressed. Glycosylation of E-cadherin with GalNAc starts in the Golgi apparatus by glycosyltransferases called GALNTs 32 . E-cadherin combines mechanotransduction and EGFR signaling to regulate junctional tissue polarization and tight junction positioning 33 , GALNT3 preserves the epithelial state in trophoblast stem cells. The loss of GALNT3 expression diminishes O-GalNAc glycosylation and causes epithelial-mesenchymal transition 32 . Due to replacement of E-cadherin by N-cadherin in the mammary gland, fibrocystic changes and tumor formation occur 34 . N-cadherin causes FGFR upmodulation which results in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties 35 . We can speculate that GALNT responsible for GalNAc-GM1b synthesis is sensitive to similar effectors as GALNT3. There is no data in literature concerning GalNAc-GM1b expression in cancer stem cells. We found lower GalNAc-GM1b in CSCs compared to non-CSC MDA-MB231. This finding is in accordance with the results of Guan et al. During induced epithelial-mesenchymal transition of breast cells, there is significantly reduced Gg4 and its synthase B3GALT4 19 . Neutral glycosphingolipid Gg4 is a direct precursor of GM1b. GM1b is further a direct precursor of GalNAc-GM1b, as it is presented in Fig. 9A. On the other hand, GALNT14 shows the opposite effects. It catalyzes O-glycosylation of EGF-containing fibulin-like extracellular matrix protein 2. This significantly increases the invasion ability of breast cancer cell lines (MCF-7 and MBA-MD-231) 36,37 . GALNT14 www.nature.com/scientificreports/ is related to the chemosensitivity of breast cancer. Osterix, a zinc finger-containing transcription factor, decreases chemosensitivity and enhances anti-apoptosis by upregulating GALNT14 38 . Osterix has also important roles in facilitating breast cancer invasion 39 . In our study, treated CSCs acquired a phenotype closer to non-treated non-CSCs. Malignancy is not only defined by tumour-specific molecules, or their genes, but it can be caused by disorganization of cell membrane components 40 . Glycosphingolipids are important cell membrane components being able to influence final cell behaviour. Gb3Cer plays an essential role in the maintenance of epithelial cancer cell properties. Depletion of Gb3Cer by deletion of the key enzyme lactosylceramide 4-alpha-galactosyltransferase (A4GALT) induces epithelial-to-mesenchymal transition, enhances chemoresistance, and increases CD44 + /CD24 − cells 41 . The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cholera toxin is able to induce transition after binding to its receptor, Gb3Cer 42 . Liang et al. described greatly reduced levels of Gb3Cer in breast CSCs in comparison to cancer non-stem cells (non-CSCs) 15 . Whilst we did not determine Gb3Cer, the enzyme A4GALT was obviously active in our study because Gb3Cer is direct precursor of Gb 4 Cer and Gb 4 Cer was found elevated in CSCs after treatment with compound 1 42 . We have not found elevated GM2 and GD3 in CSCs as was earlier reported 15 15 . They proved increased GM2 and GD3 in organic solvent extract of GSLs using methods of Orbitrap-Fourier transform (FT) mass spectrometry (MS) and high-performance liquid thin layer chromatography (HPTLC)-immunostaining. That means, GSLs from the plasma membrane and from Golgi are included in final findings. Their flow-cytometry results have not proved elevated GM2 and CSCs. The percentage of GD3 was reported as elevated, but due to their gating strategy, they had excluded the most GD3 positive cells from analyses of their CSC markers (CD44 + /CD24 − ) 15 . Therefore, our final results are not comparable.

Scientific Reports
| (2020) 10:11876 | https://doi.org/10.1038/s41598-020-68516-y www.nature.com/scientificreports/ influence co-localized fibroblast growth factor (FGF) receptor action, that is involved in cancer pathogenesis 46 : low level of GM3 activates and high level inhibits FGF signal transduction 47 . Therefore, we could assume that increased GM3 in CSCs, after treatment with compound 1, contributes to inhibition of FGF signaling and thereby reduces cancer progression. Sialylation of the nLc 4 Cer to form IV 6 Neu5Ac-nLc 4 Cer is catalysed by enzyme sialyl transferase to nLc 4 Cer (ST6Gal). An increase in infiltrating lymphocytes is influenced by high expression of ST6Gal-II in triple negative breast cancers that correspond to our breast cancer model 48 . Triple negative breast cancer lymphocyte infiltration correlates with better overall survival and better chemotherapeutic responses 49 .
Concerning findings in MDA-MB-453 cells, only increased percent of both GalNAc-GM1b + CD44 + / CD24 − (data not shown) and GalNAc-GM1b + CD133 + cells after compound 1 treatment (Fig. 7A) corresponded to MDA-MB-231 GSL findings. Yang et al., detected 31 patients containing CSCs among 88 primary TNBCs, using CD44 + /CD24 − , aldehyde dehydrogenase family 1 member A1 (ALDH1A1) and CD133 markers. Eight cases were positive for both CD44 + /CD24 − and ALDH1A1, 10 cases were positive for both CD44 + /CD24 − and CD133, 9 cases were positive for both ALDH1A1 and CD133, while only 4 cases showed positivity of all the three CSC markers 50 . Correlation between CSC markers was weak, implying that most breast cancer cells do not express these markers concurrently. Knowing that CD133 phenotype is not significantly associated with worse progression-free survival, we can assume that CD133 is less reliable CSC marker compared to CD44 + /CD24 −50 . Expression of MDA-MB-231 glycosphingolipids found in this study could have higher impact for clinical implications. Dramatically reduction of GalNAc glycosylation observed in MDA-MB-231 CSCs after compound 1 treatment, characterized earlier to be enrolled in either epithelial-mesenchymal transition or in its reversal, could indicate possible biochemical pathway of CSC reduction by compound 1.

Conclusions
The novel thieno[2,3-b]pyridine anticancer compound 1 was cytotoxic for the breast cancer cells, cell death being mediated by apoptosis. The percent of cancer stem cells was significantly lower. Glycosphingolipids IV 6 Neu5Ac-nLc 4 Cer and GalNAc-GM1b, not reported previously, were identified in both breast cancer stem cells and cancer non-stem cells. IV 6 Neu5Ac-nLc 4