Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models

Abstract

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Keywords: breast cancer; carbonic anhydrase IX; hypoxia; invasion; tumour microenvironment.

Figure 1. The anti-proliferative effects of novel ureido-sulfamate CAIX inhibitors on the growth of breast cancer cell lines in vitro

A. The structures of the 5 novel ureido-sulfamate CAIX inhibitors, FC9396A, FC9403A, FC9398A, FC11409B, and S4 used in this study. B. The effect of the CAIX inhibitors, FC9396A, FC9403A, FC9398A, FC11409B, and S4 on the proliferation of MDA-MD-231 breast cancer cells in normoxia. Compounds were used at concentrations between 3 – 100 μM. Data represents mean of n = 5 assays; 6 replicates per assay, at day 5. C. The response to the CAIX inhibitors, FC9396A, FC9403A, FC9398A, FC11409B, and S4 on the proliferation of MDA-MD-231 breast cancer cells in hypoxia (0.5% O₂) Compounds were used at concentrations between 3 – 100 μM. n = 2 assays, 6 replicates per assay at day 5. D. The concentration response of a panel of various breast cancer cell lines to the CAIX inhibitor FC9396A in normoxic conditions. Compounds were used at concentrations between 3 – 100 μM. n = 3 assays, 6 replicates per assay at day 5. E. The anti-proliferative response of a panel of breast cancer cell lines to the CAIX inhibitor FC9398A used at concentrations between 3 – 100 μM, in hypoxia (0.5% O₂). n = 2 assays, 6 replicates per assay at day 5.

Figure 2. The effect of S4, FC9396A, FC9403A, and FC11409B on the ability of MDA-MB-231 spheroids to invade collagen

A. 3D collagen invasion assay. Shown are the results of treating MDA-MB-231 spheroids with the CAIX inhibitor FC11409B at time 0 and at 48 h. A, C, E and G are time 0; B, D, F, H, are time 48 hours. A and B are controls; C and D, FC11409B 10 μM; E and F, FC11409B 30 μM; G and H, FC11409B 100 μM. Original magnification of images = × 25. Scale bar = 0.5 mm. B. The effects of S4, FC9396A, FC9403A, and FC11409B on the ability of MDA-MB-231 spheroids to invade collagen. Inhibitors were used at 0, 3, 10, 30 and 100 μM. The invasive areas of each of the spheroids treated with the various concentrations of drug were normalized against the invasion area of the control spheroids. Results shown are mean ± SEM, *P < 0.01. n = 8 for all drug concentrations.

Figure 3. The effects of S4, FC9398A and FC9403A on breast cancer invasion within an explant invasion assay

A. Breast tumor explant invasion assays. Representative experiment showing the ability of FC9403A to inhibit the invasion of an ER+ breast tumor explant into collagen after 5 days. Magnification × 25. Scale bar = 0.5 mm. B. Collagen embedded explants showing control invasion over 15 days when measured by increased area. Data pooled from 26 separate biopsy samples (n= 214 – 177). Results shown = mean ± SEM, ***P < 0.001. C–E. Inhibition of invasion of collagen-embedded explants. C) S4 treatment; D) FC9398A treatment; E) FC9403A treatment. Explants were cultured for 15 days with measurements taken every 5 days. Mean ± SEM (n = 8) 21-25 explants per condition *P < 0.05, **P < 0.01, ***P < 0.001 of relevant control

Figure 4. The effects of S4, FC9398A and FC9403A, and FC11409B on breast cancer invasion

A. An example of decrease in tumor explant area in an invasive explant. Explant treated with 1 μM S4 over 10 days. Magnification × 25. B. Percentage of control explants showing invasive morphology over 15 days. Data shown are mean ± SEM (n = 230 explants). ***P < 0.001. C. Percentage of FC9398A treated explants showing invasive morphology. Explants cultured for 15 days with measurements taken every 5 days. Inhibitor was used at concentrations between 3 – 100 μM. Data shown are mean ± SEM n = 4. ***P < 0.001. D. Percentage of S4 treated explants showing invasive morphology. Explants cultured for 15 days with measurements taken every 5 days. Inhibitor was used at concentrations between 3 – 100 μM. Data shown are mean ± SEM n = 4. ***P < 0.001. E. Percentage of FC9403A treated explants showing invasive morphology. Explants cultured for 15 days with measurements taken every 5 days. Inhibitor was used at concentrations between 3 – 100 μM. Data shown are mean ± SEM n = 4. ***P < 0.001

Figure 5. Reversal of invasion in human breast cancer explants after treatment using ureido-sulfamate CAIX inhibitors

A. Representative images showing the reversal of invasion in a human breast cancer explant after 5 days of treatment using 30 μM FC9403A. Explants were monitored for 5 or 10 days and changes quantified using Image J. Original magnification × 25. B. The effect of FC9398A on explant invasion. Explants were allowed to invade into collagen plugs for 15 days, before treatment with 30 μM FC9398A. Data shown are mean ± SEM. N = 10 *P < 0.05; ***P < 0.001 compared to Day 0 control; ‡‡‡P < 0.001 compared to Day 10 control. C. The effect of S4 on explant invasion. Explants were allowed to invade into collagen plugs for 15 days, before treatment with 30 μM S4. Data shown are mean ± SEM. N = 10 − 13 *P < 0.05; **P < 0.05 compared to Day 0 control; + P < 0.05 compared to Day 10 control. D. The effect of FC9403A on explant invasion. Explants were allowed to invade into collagen plugs for 15 days, before treatment with 30 μM FC9403A. Data shown are mean ± SEM. N = 10 − 13 **P < 0.05; ***P < 0.001 compared to Day 0 control. ###P < 0.001 compared to Day 5 control; +++P < 0.001 compared with Day 10 control. E. Comparison of the effects of S4, FC9398A and FC9403A on invasive growth using the same biopsy tissue. Explants were allowed to invade into collagen plugs for 15 days, before treatment with 30 μM inhibitor. Data shown are mean ± SD. Samples in triplicate *P < 0.05; ***P < 0.001, compared to Day 0 control; ++P < 0.01; +++P < 0.001 compared with Day 5 control; ‡‡‡P < 0.001 compared with Day 10 control.

Figure 6. The effects of DTP348 and FC9398A on the MDA-MB-231 breast cancer xenograft model

A. The effect of DTP348 and FC9398A on relative mean tumor volume of MDA-MB-231 breast cancer xenografts. 10 mice were used per treatment and tumors measured over a 14 day period. Data shown are mean ± SD. *p < 0.05 compared with day matched control. B. Ki67 expression in xenografts collected on final day (Day 14) of study. Mean values of approximately 2000 cells counted from multiple fields. C. Percentage viability in treated xenografts was assessed by use of Image J software on hematoxylin stained sections. D. CAIX staining in an untreated MDA-MB-231 xenograft. Magnification X25. Brown cellular areas indicate CAIX expression. Light brown acellular areas are areas of necrosis. Cells were stained with hematoxylin (blue).