Repurposing homoharringtonine for thyroid cancer treatment through TIMP1/FAK/PI3K/AKT signaling pathway

Summary Homoharringtonine (HHT), an alkaloid isolated from Cephalotaxus, is an effective anti-leukemia agent and exhibits inhibitory effects in various solid tumors. However, the impacts of HHT treatment on thyroid cancer (TC) remain unclear. Our findings demonstrated that HHT exhibited remarkable anti-TC activity that involved inhibiting cell proliferation, invasion, and migration, as well as inducing apoptosis. Proteomics analysis revealed that the expression of the tissue inhibitor of metalloproteinase 1 (TIMP1) was downregulated in TC cells after HHT treatment. TIMP1 overexpression promoted TC progression and partially reversed the anti-TC effects of HHT, while TIMP1 downregulation inhibited TC progression and enhanced the anti-TC effects of HHT. Furthermore, TIMP1 re-expression attenuated the enhancement of anti-TC effects of HHT induced by TIMP1 knockdown. Mechanistically, HHT exerted anti-TC effects by downregulating TIMP1 expression and then inactivating the FAK/PI3K/AKT signaling pathway. Taken together, our study demonstrated that HHT could inhibit TC progression by inhibiting the TIMP1/FAK/PI3K/AKT signaling pathway.

Table S3.The sequence for siRNAs and shRNAs targeting TIMP1, related to STAR

Figure S2 .
Figure S2.Semi-quantitative analysis of protein expression in Figure 1F, related to Figure 1.Semi-quantification analysis of apoptosis-and EMT-related proteins in TPC-1 and 8505C cells with HHT treatment for 48h (Fig. 1F).GAPDH was used as the control.Data are presented as mean ± SD. **p < 0.01, ***p < 0.001.

Figure S3 .
Figure S3.HHT suppresses BCPAP and FTC-133 cells in vitro, related to Figure 1.(A) CCK8 assay was performed to detect cell viability of BCPAP and FTC-133 cells treated with increased gradients of HHT for 48 h.(B) CCK8 assay was perfprmed to detect cell viability of BCPAP and FTC-133 cells with HHT treatment for 24, 48, and 72h.(C) Colony formation assay was used to detect the clonogenic ability of BCPAP and FTC-133 cells with HHT treatment for 14 days.(D) The effect of HHT on the cell apoptosis was detected by flow cytometry.(E) Western blotting analysis of the expression of Bax, Bcl2, Cleaved-caspase 3, E-cadherin, and N-cadherin with HHT treatment for 48h.GAPDH was used as the control.(F) Transwell assay was used to detect the cell invasion ability.(G) The wound healing assay was performed to detect the cell migration rate.Data are presented as mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure S4 .
Figure S4.HHT downregulates the expression of NIS at both mRNA and protein levels, related to Figure 1.(A) RT-qPCR was performed to analyze the mRNA level of NIS in TC cells after HHT treatment for 48h.GAPDH was used as the control.(B) Western blotting analysis of the expression of NIS in TC cells with HHT treatment for 48h.GAPDH was used as the control.Data are presented as mean ± SD. ***p < 0.001.

Figure S7 .
Figure S7.The knockdown efficiency of TIMP1 in TC cells after transfected with TIMP1 siRNA, related to Figure 4. RT-qPCR detected the mRNA level of TIMP1 in TPC-1 and 8505C cells transfected with TIMP1 siRNA.GAPDH was used as the control.Data are presented as mean ± SD. ns, no significance, **p < 0.01, ***p < 0.001.

Figure S11 .
Figure S11.TIMP1 is overexpressed in TC tissues, related to Figure 8. IHC staining detected the TIMP1 expression of normal thyroid tissues and TC tissues.IHC staining score was based on the IRS system.**p < 0.01.

Figure S12 .
Figure S12.The correlation between high TIMP1 expression and invasive clinical features, related to Figure 8.The high mRNA expression of TIMP1 in TC tissues from the TCGA data was correlated to extrathyroidal invasion, multifocality, lymph node metastases, higher T stage, and higher TNM stage.*p < 0.05, **p < 0.01, ***p < 0.001.