BRCA1 Insufficiency Induces a Hypersialylated Acidic Tumor Microenvironment That Promotes Metastasis and Immunotherapy Resistance

Abstract

Cancer metastasis is an extremely complex process affected by many factors. An acidic microenvironment can drive cancer cell migration toward blood vessels while also hampering immune cell activity. Here, we identified a mechanism mediated by sialyltransferases that induces an acidic tumor-permissive microenvironment (ATPME) in BRCA1-mutant and most BRCA1-low breast cancers. Hypersialylation mediated by ST8SIA4 perturbed the mammary epithelial bilayer structure and generated an ATPME and immunosuppressive microenvironment with increased PD-L1 and PD1 expressions. Mechanistically, BRCA1 deficiency increased expression of VEGFA and IL6 to activate TGFβ-ST8SIA4 signaling. High levels of ST8SIA4 led to accumulation of polysialic acid (PSA) on mammary epithelial membranes that facilitated escape of cancer cells from immunosurveillance, promoting metastasis and resistance to αPD1 treatment. The sialyltransferase inhibitor 3Fax-Peracetyl Neu5Ac neutralized the ATPME, sensitized cancers to immune checkpoint blockade by activating CD8 T cells, and inhibited tumor growth and metastasis. Together, these findings identify a potential therapeutic option for cancers with a high level of PSA.

Significance: BRCA1 deficiency generates an acidic microenvironment to promote cancer metastasis and immunotherapy resistance that can be reversed using a sialyltransferase inhibitor.

Graphical abstract

Figure 1.

Disrupted mammary bilayer structure and undetermined epithelial cell fate associated with elevated St8sia4. A–D, Representative images of the tumor-adjacent mammary gland (A) and mammary tumor tissues (C) co-stained with CK18/CK14 and the quantification of the percentage of disrupted basal layer (the length of either CK14 or collagen IV–positive cells versus perimeter of the mammary duct) in the length of the mammary gland (B) and tumor tissues (n = 50 ducts/per patient; D) from non-BRCA1 mutation carriers (n = 21) and BRCA1 mutation carriers (n = 9) by ImageJ (Fiji). Scale bar, 10 μm. E–H, Representative images of tumor-adjacent mammary glands (E) and mammary tumor tissues (G) co-stained with CK18/collagen IV, and the quantification of the percentage of disrupted basal layer in length in mammary glands (F) and tumor tissues (n = 50 ducts/per patient; H) from non-BRCA1 mutation carriers (n = 21 samples) and BRCA1 mutation carriers (n = 9 samples). Scale bar, 10 μm. I–K, Representative images of WT and MT tumor-adjacent mammary gland (I) and breast tumor tissues (J) co-stained with CK18 and CK14 and quantification of the percentage of the disrupted basal layer in length (K) from 8 and 10-month-old Brca1^MKO mice and control mice (n = 6 mice/group and 50 ducts/mouse were counted). Arrowheads, basal layer cells. Scale bar, 20 μm. L and M, Representative images of 10-month mammary gland tissues (L) co-stained with CK18 and CK14 and quantification of the percentage of the disrupted basal layer (M) in length (n = 8–10 mice/group and 150 ducts/mouse were counted; L). Arrowheads, basal layer cells. Scale bar, 20 μm. N and O, Representative images of tumor tissues from control mice and Brca1^MKO mice (N) and quantification of the percentage of the disrupted basal layer in length (n = 8–10 mice/group and 150 ducts/mouse were counted; O). Arrowheads, basal layer cells. Scale bar, 20 μm. P, The top 20 cytokines from 97 cytokine arrays probed with the serum of 6-month-old WT and Brca1^MKO mice presented by heat map (n = 3 mice/group). Q, The comparison of mRNA expressions of 22 sialyltransferases in B477 and G600 mammary epithelial cell lines as determined by qPCR and presented by volcano plot. R and S, Protein levels of E-Selectin, L-Selectin, and St8sia4 in age-matched WT mammary tissues and tumor tissues from Brca1^MKO mice (R), and the B477 control and G600 Brca1-MT cell lines by Western blots (n = 3–6 mice/group; S). T and U, Protein levels of ST8SIA4 in MCF10A185 (T), HCC1937 cell lines (U), and their controls as determined by Western blots. V, TNM box plots of ST8SIA4 gene expression in human normal (n = 403 patients) and breast tumor tissues (n = 1,097 patients); P = 2.63e−12. W, Violin plots of ST8SIA4 expressions in patients with breast cancer with WT BRCA1 (n = 431) and patients with low expression of BRCA1 (n = 361); P = 0.0002. X, Correlation expression between ST8SIA4 and SELE in general patients with BRCA (n = 1,100) from TCGA database. Y, Correlation expression between ST8SIA4 and SELE in patients with BRCA-basal cancer (n = 191) from TCGA database. Data are presented as means ± SD and P values determined by an unpaired Student t test (B, D, F, H, K, M, and O) and one-way ANOVA (O). ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Increased polysialic acid in mouse BRCA1-deficient cells and human BRCA1ness breast cancers. A, The mRNA expression levels of St8sia4 in B477 cells with the expression of shBrca1 DNA vectors at different concentrations. B and C, Distribution patterns (B) and quantification (C) of PSA in/on epithelial cells of normal MG by ImageJ with 7.46% as the signal threshold for all pictures (a), tumor-adjacent MG (b), and tumors (c) from 10-month-old Brca1^MKO mice (n = 5 mice/group) by IF staining with PSA. D and E, Distribution patterns (D) and quantification (E) of PSA in/on epithelial cells of breast cancer patients by ImageJ, with 42.3% as the signal threshold for all pictures (n = 9 BRCA1 mutation carriers and n = 9 non-BRCA1 mutation carriers) by staining with PSA. F and G, Representative images of fresh slices from the mammary gland (A) and tumor tissues (B) with a ratiometric fluorescent probe (n = 3 mice/group) and quantification of pH (C) in A and B, with the ratio of red and green intensities using the formula: (y = 31.8403–4.4898x, R = 0.9928). H and I, Representative images of breast cancer tissues with the antibody of BRCA1 by regular IHC or PSA by IF (H) and the quantification of PSA in patients with breast cancer (I) with the expression of BRCA1^high (n = 24 patients), BRCA1^inter (n = 34 patients), and BRCA1^low (n = 23 patients). J–L, Tumor images from B477-WT cells without or with OE-St8sia4 (J), tumor slice images with a ratiometric fluorescent probe (K), and quantification of tissue interstitial pH (L) in K. M–O, Tumor images from G600-Brca1 mutant cells without or with sgSt8sia4 (M), tumor slice images with the ratiometric fluorescent probe (N), and quantification of tissue interstitial pH (O) in N. Data are presented as means ± SD and P values determined by one-way ANOVA (A and C) and an unpaired Student t test (E, G, L, and O). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 3.

The role of St8sia4 in lung metastasis. A, The protein levels of St8sia4 in EMT6 and 545 parental cells and cells expressing sgSt8sia4 cells by Western blot. B, Representative images of mammary ducts co-stained by the antibodies of CK18/CK14 from Balb/c control (Ctr) mice, adjacent mammary glands from Balb/c mice implanted with EMT6 parental cells, OE-St8sia4-EMT6 cells at days 14 and 26, as well as OE-St8sia4 mice treated with pan inhibitor 3Fax-P-Neu5Ac (STi) at the concentration of 20 mg/kg for 7 consecutive days. The quantification is shown on the right (n = 3 mice/group). Scale bar, 10 μm. C–E, Representative images of tumor-adjacent mammary gland structures from nude mice with fat pad implantation of 545 and OE-St8sia4–545 cells by IF staining of CK18 and CK14 (C), tumor tissues with OE-St8sia4–545 cells by PSA/E-cadherin and CK18/collagen IV (D), and CK18/vimentin (n = 3 mice/group; E). Scale bar, 10 μm. F and G, The migration assay of EMT6 cells and EMT6-OE-St8sia4 (F) and the quantification of migrated cells (G). Scale bar, 1 mm. H and I, The migration assay of 545-Ctr cells and 545-OE-St8sia4 cells (H) and the quantification of migrated cells (I). Scale bar, 1 mm. J, Tumor volumes from nude mice with fat pad implantation of 545 and 545-OE-St8sia4 cells for 32 days (n = 8 mice/group). K and L, Representative images of the lung from nude mice with fat pad implantation of 545, 545-OE-St8sia4 (K) and quantified GFP intensities in the lungs (n = 8 mice/group; L). Scale bar, 2 mm. M, Protein levels of St8sia4 in 545 and 628W cells as determined by Western blot. N, Protein levels of St8sia4 with the expression of sgSt8sia4 as determined by Western blot. O, Tumor volumes from nude mice with fat pad implantation of 628 cells and 628 cells expressing sgSt8sia4 cells for 32 days (n = 8 mice/group). P and Q, Representative images of the lung from nude mice with fat pad implantation of 628W and 628W-sgSt8sia4 cells (P) and quantified GFP intensity in the lungs (Q) in P, respectively (n = 7 mice/group). Scale bar, 2 mm. Data are presented as means ± SD and P values were determined by one-way ANOVA (B) and an unpaired Student t test (G, I, J, L, O, and Q). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 4.

St8sia4 is upregulated by elevated Vegfa/Il6-TGFβ signal in mammary epithelial cells. A, Relative expressions of Vegfa and Il6 in B477-shBrca1 cells at different concentrations or G600-OE-mBrca1 cells by qPCR. B, Expressions of Vegfa and Il6 in G600 control and G600 with mBrca1 cells determined by qPCR. C and D, Promoter activities of Vegfa (C) and Il6 (D) in both B477 and G600 cells without or with the expression of mBrca1 by luciferase activity assay. E and F, Protein level of St8sia4 in B477 cells expressing shBrca1, shBrca1/sgVegfa, and shBrca1/sgIl6 (E) and G600 cells expressing OE-Brca1, OE-Brca1/OE-Vegfa, and OE-Brca1/OE-Il6 (F) by Western blots. G and H, Protein levels of Vegfa, Il6, pSmad3, and pStat3 in the mammary gland (G) and breast tumor tissues (H) in age-matched WT and Brca1^MKO mice were determined by Western blot (n = 3 mice/group). I, Heat map showing expression profiles of sialyltransferase genes induced by TGFβ determined by qPCR. J, Protein levels of St8sia4, pStat3, and pSmad3 in 628W and G600 cells without or with TGFβ inhibitor (LY2107961) treatment as determined by Western blots. K, Protein levels of Vegfa, Il6, TGFβ, and St8sia4 in B477 cells expressing OE-Vegfa, OE-Il6, and in G600 cells expressing sgVegfa and sgIl6 by Western blots. L, Protein levels of St8sia4, pStat3, and pSmad3 in EMT6 cells expressing either Vegfa or Il6 treated with LY2109761 inhibitor as determined by Western blots. M, Protein levels of St8sia4, pStat3, and pSmad3 in W628 cells expressing either sgVegfa or sgIl6 treated with TGFb as determined by Western blots. N and O, Representative images of PSA (N) on the cell membrane of G600 cells either treated with 5 ng/mL TGFβ for 48 hours, or 10 μmol/L LY2109761 for 48 hours, or transfection of Brca1 cDNA for 72 hours and quantified cell membrane PSA positive dots (O). n = 18 pictures/group from three independent experiments. Scale bar, 10 μm. P, The gene expression correlation between ST8SIA4 and TGFβ in patients with breast cancer from the TCGA database by Linkedomics. Q, Summary of factors involved in the upregulation of St8sia4 that induces the acidic environment in mammary tissues. Data are presented as means ± SD and P values were determined by one-way ANOVA (C and J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

TIME induced by elevated St8sia4 in mice and patients with breast cancer. A and B, The immune cell population analysis by CyTOF technology with antibodies of CD45⁺/CD11b⁺/Ly6G⁺/Ly6C⁻ for PMN-MDSC, CD45⁺/CD11b⁺/Ly6G⁻/Ly6C⁺ for M-MDSC, CD45⁺/CD3⁺/CD4⁺/CD8⁻ for CD4 T cells, CD45⁺/CD3⁺/CD4⁻/CD8⁺ for CD8 T cells (A), and quantification of the immune cell populations (B) in mammary tissues from control mammary gland (MG), mammary tumor tissues from 14 days after implantation of EMT6 cells, EMT6-OE-St8sia4 cells, EMT6-OE-St8sia4/sgSt8sia4 cells, 26 days after implantation of EMT6 cells, EMT6-OE-St8sia4 cells, EMT6-OE-St8sia4/sgSt8sia4 cells in Balb/c mice (n = 3–5 mice/group). C–E, Representative images of macrophages in breast tumor tissues from Balb/c mice with fat pad implantation of EMT6 and EMT6-OE-St8sia4 cells by staining with F4/80/CD86 for M1-like (C), F4/80/CD206 for M2-like (D), and the quantified macrophage number (n = 30 pictures from 5 mice/group; E). Scale bar, 10 μm. F and G, Representative images of MDSC in breast tumor (BT) from nude mice with fat pad implantation of B477, G600, G600-sgVegfa, and G600-sgIl6 cells by staining with S100a9 (F) and quantified S100A9-positive MDSC (n = 18 pictures from 6 mice/group; G). Scale bar, 50 μm. H and I, Representative images of MDSC in the spleen (H) from nude mice with fat pad implantation of 545, 545-OE-St8sia4, 628W, and 628W-sgSt8sia4 cells (C) by staining with S100a9 and quantified S100A9-positive MDSC (n = 18 pictures from 6 mice/group; I). Scale bar, 50 μm. J, Western blot showing protein level of TGFβ signaling in spleen and bone marrow from Brca1-WT and Brca1^MKO mice. K–M, Correlations between the expression of St8sia4 immune cell populations of MDSC (K), macrophage (L), and Treg (M) in breast cancer patients from TISIDB. N, Breast cancer patients' survival outcomes with different levels of St8sia4 expression and CD8⁺ T-cell infiltration. Data are presented as means ± SD and P values were determined by one-way ANOVA (B, G, and I) and an unpaired Student t test (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 6.

Delivery of STi and Stattic by nanoparticles restores the mammary gland structure and inhibits metastasis. A, Drug delivery plan with an intraperitoneal injection (IP). The mice were divided into four groups: vehicle-treated control, Stattic only (10 mg/kg), 3Fax-P-Neu5Ac only (20 mg/kg), and combination treatment of Stattic (10 mg/kg) and 3Fax-P-Neu5Ac (20 mg/kg). Cells for the 3Fax-P-Neu5Ac treatment group were pretreated with 200 μmol/L 3Fax-P-Neu5Ac for 5 days and a total of 1×10⁶ pretreated or untreated cells were implanted into the fat pad of 5-weeks-old nude mice. 3Fax-P-Neu5Ac was injected every day for 7 consecutive days. Once the tumor size reached approximately 50 mm³, Stattic was injected every two days until day 25. B and C, Tumor images (B) and growth curve (C) of four groups of mice from days 7 to 25. D and E, Representative images of breast tumor co-stained with CK18 and collagen IV (D) and the quantification of disrupted BM length (E) from the same cohort of mice in B (n = 5 mice/group). Scale bar, 10 μm. F and G, Representative images of tumor-adjacent mammary glands co-stained with CK18 and CK14 (F) and the quantification disrupted basal layer length (G) from the same cohort of mice in B (n = 5 mice/group). Scale bar, 10 μm. H, Drug delivery plan with nanoparticles (NP). A total of 1×10⁶ 628W cells were implanted into the fat pad of 5 weeks old nude mice. The mice were divided into four groups: PBS (n = 5 mice), PEGs (nanoparticle without drug; n = 4 mice), Stattic inhibitor only (n = 6 mice), and 3Fax-P-Neu5AC only groups (n = 6 mice). Seven days after implantation when the average tumor size reached about approximately 50 mm³, the first nanoparticles (Stattic 10 mg/kg, 3Fax-P-Neu5Ac 20 mg/kg) were delivered through tail vein injection, the second delivery was on day 11, and the third delivery was on day 15. Samples were harvested on day 26. I, Representative images of enrichment nanoparticles at the tumor site at 6, 24, and 48 hours after tail vein injection. J and K, Representative tumor images (J) and tumor volume (K) from four groups of mice on day 26 (D). L and M, Representative lung images in brightfield and lung images with metastatic GFP signals (L) and quantified GFP intensity (M). Scale bar, 2 mm. N and O, Representative images of breast tumor tissues using the ratiometric fluorescent probe (N) from the same cohort of mice in C and quantified tissue pH (O). Scale bar, 0.2 mm. Data are presented as means ± SD and P values were determined by one-way ANOVA (D, F, and H). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Figure 7.

Combinatory treatment with αPD1 and STi to the mammary tumor with elevated St8sia4. A and B, Representative images of mammary tumors co-stained with PD-L1 and CK18 (A) from the WT mammary gland tissues (n = 3 mice), EMT6-Ctrl (n = 5 mice), the mice with OE-St8sia4-EMT6 (n = 6 mice), and the mice with OE-St8sia4/sgSt8sia4 (n = 6 mice). The quantification of PD-L1–positive cells (B) in A by ImageJ. Arrowheads, cell membrane. Scale bar, 10 μm. C–E, Representative images of spleen co-stained with PD1 and CD3 antibodies (C) from the same cohort of mice in A. Quantifications of PD1-positive cells (D) and CD3/PD1 double–positive cells (E) in C by ImageJ. Arrowheads, cell membrane. Scale bar, 10 μm. F, Drug delivery plan of the combination treatment. The mice were divided into 5 groups: vehicle-treated control (n = 5 mice), EMT6 OE-St8sia4, OE-St8sia4+αPD1, OE-St8sia4+STi NPs, and OE-St8sia4+αPD1+STi NPs (n = 6 mice). A total of 5×10⁵ cells were implanted into the fat pad of Balb/c mice. αPD1 antibody (0.2 mg/mouse) was injected intraperitoneally on days 7 and 15, and STi nanoparticles (NP; 20 mg/kg) were delivered through the tail vein on days 9, 13, and 17. Samples were harvested on day 26. G and H, Tumor images (G) and growth curve (H) of 5 groups of mice from days 7 to 26 (n = 5 mice/EMT6-Ctrl/group, n = 6 mice/ in OE-EMT6) with αPD1 treatment or STi treatment only and combination treatment with αPD1 and STi. I, tSNE analysis of different immune cells from the treatment of the same cohort of mice in G and H. J, The percentage of different immune cells from the CD45⁺ cell population, including PMN-MDSC (CD11b⁺/Ly6G⁺/Ly6C⁻), M-MDSC (CD11b⁺/Ly6G⁻/Ly6C⁺), CD4⁺ T-cell (CD3⁺/CD4⁺/CD8⁻), and CD8⁺ T-cell (CD3⁺/CD4⁻/CD8⁺; n = 3 mice/group). K–N, Representative images of IHC staining of the tumor with the antibody of caspase-3 (K) and PSA (M) and the quantifications (L) in K and N in M of the same cohort of mice in G and H. Scale bar, 20 μm. O, Representative tumor images from Balb/c mice with implantation of EMT6 cell (n = 4 mice), EMT6 cells with OE-St8sia4 (n = 5 mice), EMT6 cells with OE-St8sia4 and treated with STi+αPD1 (n = 5 mice), EMT6 cells with OE-St8sia4 and treated with STi⁺αPD1⁺αCD8 (0.3 mg/mouse; n = 5 mice). P, Tumor growth curves of the same cohort of mice in O. Q, CD8 cell population by CyTOF without or with αCD8 antibody treatment in the same cohort of mice in O analyzed by FlowJo 10.0. Data are presented as means ± SD and P values were determined by one-way ANOVA (D, E, and F). *, P <0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.