Fumagillin regulates stemness and malignancies in cancer stem-like cells derived from liver cancer via targeting to MetAP-2

Abstract

Background: Cancer relapse is associated with the presence of cancer stem-like cells (CSCs), which lead to multidirectional differentiation and unrestricted proliferative replication. Fumagillin, a myocotoxin produced by the saprophytic filamentous fungus Aspergillus fumigatus, has been reported to affect malignant characteristics in hepatocellular cancer cells. However, its exact role in CSCs is still unknown.

Methods: CSCs were enriched by culturing cancer cells in serum-free medium. The effects of fumagillin on malignant cell characteristics and mitochondrial function were measured. The regulatory role of fumagillin on methionine aminopeptidase-2 (MetAP-2) was assessed.

Results: When it was supplemented in medium, fumagillin treatment inhibited sphere formation and the maintenance of stemness of CSCs without disturbing cell growth. Fumagillin also decreased stemness-related markers and the aldehyde dehydrogenase 1 (ALDH1)-positive proportion, which demonstrated that fumagillin decreases stemness in CSCs. It was also found to inhibit malignant traits in CSCs, including cell proliferation, invasion, and tumor formation, and sensitize CSCs to chemoagents, including sorafenib and doxorubicin, by promoting chemoagent-induced apoptosis. Moreover, fumagillin treatment was found to disturb mitochondrial membrane homeostasis, ATP synthesis and mitochondrial transcriptional activity. In addition, we found that fumagillin decreased MetAP-2 protein levels and exerted anti-CSC effects potentially by regulating MetAP-2. We also found that fumagillin treatment activated p53 and its transcriptional activity and thus caused cell cycle blockade. Moreover, fumagillin treatment significantly decreased tumor formation in nude mice.

Conclusion: This work offers evidence for fumagillin as a specific inhibitor of liver cancer CSCs and proposes a novel strategy for cancer therapy.

Fig 1. Fumagillin affects stemness of CSCs derived from hepatocellular carcinoma cells.

A. By being cultured in serum-free medium, sphere formation was observed from day 1 to 10 derived from Huh-7 and SNU-449 cells. B. western blot was performed to detect stemness related factors in Huh-7 spheres, including Oct4, CD44, Sox2. *P<0.05, **P<0,01, vs. Parental cell group. C. CCK-8 assay was performed to detect inhibition rate of Fumagillin to cell viability of Huh-7 or SNU-449 CSCs. D. Huh-7 or SNU-449 cells were seeded and allowed to attach overnight. With the presence of Fumagillin for 24–96 h, cell morphology was observed and after 96-hour incubation, stemness related factors in Huh-7 were detected by western blot. *P<0.05, **P<0.01, vs. Mock group.

Fig 2. Fumagillin affects ALDH1+ positive proportion.

A. Spheres were cultured with different concentration of Fumagillin and mophology was observed. B. western blot was performed to detect stemness related factors. *P<0.05, **P<0.01, vs. Mock group. After Fumagillin treatment, ALDH1 staining was performed and analyzed followed by flow cytometry (C&D). *P<0.05, vs. ALDH1+/Fumagillin- group.

Fig 3. Fumagillin affects malignant behaviors in CSCs.

A. After Fumagillin treatment, cell viability of Huh-7 or SNU-449 CSCs from day 1–5 was measured by performing CCK-8 assay. *P<0.05, vs. Mock group. B. after being treated with Fumagillin for 48 and 72 h, cell cycle distribution of Huh-7 or SNU-449 CSCs was measured by performing PI staining followed by flow cytometry assay. *P<0.05, vs. Mock group. C. After Fumagillin treatment for 48 h, invasion was measured by performing Transwell assay in Huh-7 or SNU-449 CSCs. *P<0.05, vs. Mock group. E. After being co-cultured with Fumagillin for 12 days in soft agar, tumor formation was measured in Huh-7 or SNU-449 CSCs. *P<0.05, vs. Mock group.

Fig 4. Fumagillin sensitized CSCs to chemoagents.

A. Fumagillin was co-cultured with chemoagents, including Sorafenib or Doxorubicin for 24 h, cell viability was measured by performing CCK-8 assay. *P<0.05, vs. Fumagillin group. B. The effects of Fumagillin on sorafenib or Doxorubicin-induced apoptosis were measured by performing Annexin V-FITC/PI double staining followed by flow cytometry. *P<0.05, vs. Doxorubicin group; #P<0.05, vs. Sorafenib group. C. After treatment, apoptosis-specific protein, including cleaved PARP1 and cleaved caspase-3, were detected by performing western blot. *P<0.05, vs. Doxorubicin group; #P<0.05, vs. Sorafenib group.

Fig 5. Fumagillin regulates mitochondrial function and homeostasis.

After being treated with Fumagillin for 6–24 h, ATP synthesis (A) and mitochondrial transcriptional activity (B) were measured in Huh-7 or SNU-449 cells. *P<0.05, vs. Mock group. C. After being treated with Fumagillin for 24 h, mitochondrial homeostasis was measured by performing JC-1 staining followed by flow cytometry assay.

Fig 6. Fumagillin treatment decreased MetAP-2.

A. After Fumagillin treatment, MetAP-2 mRNA (left panel) and protein (right panel) were measured in Huh-7 or SNU-449 CSCs. *P<0.05, vs. Mock group. B. GEPIA database presents the transcriptional level of MetAP-2 in LIHC tissues (left panel) and its relation with overall survival rate (right panel). By transfecting lentivirus, MetAP-2 was efficiently overexpressed (C, *P<0.05, vs vector group) or downregulated (D, *P<0.05, vs shScrambled group). E. Overexpressed MetAP-2 is not affected by Fumagillin treatment. F. After being treated with Fumagillin for 24 h or MetAP-2 knockdown, mitochondrial homeostasis was measured by performing JC-1 staining followed by flow cytometry assay.

Fig 7. Fumagillin affects stemness, proliferation and invasion partially via downregulating MetAP-2.

With the presence of Fumagillin, the effects of MetAP-2 knockdown on sphere formation (A), expression of stemness related factors (B), cell cycle distribution (C, *P<0.05, vs. shScrambled group), and cell invasion (D). After PFT-α treatment, the effects of Fumagillin on p53 and p21 was measured by performing western blot.

Fig 8. Fumagillin pre-treatment inhibited tumor growth in nude mice.

Huh-7 CSCs were pretreated with Fumagillin for 48h, then been injected into nude mice. Every five days from day 10 after injection, volume of tumors was measured (A). In tumor sections, MetAP-2 (B) and Ki67 positive staining (C) were measured by performing immunohistochemistry measured. D. MetAP-2, P53 and P21 were measured by performing western blot in tumors (n = 4 for each group). *p<0.05, vs. Mock group.