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Blockade of integrin β3 signals to reverse the stem-like phenotype and drug resistance in melanoma

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Abstract

Purpose

Cancer cells with stem-like phenotype are frequently proliferative and show high resistance to chemotherapeutic agents. Specific cell markers to identify the cancer stem cells and reverse the drugs resistance are urgent needs in clinic cancer treatment.

Methods

To identify the potential role of integrin β3 in melanoma stem cells. Flow cytometry and immunofluorescence were performed to detect the expression levels of integrin β3 and integrin β3 related signal molecules. qRT-PCR and western blotting were used to detect the signaling pathways induced by integrin β3. Colony formation analysis and melanoma-bearing mice treatment by chemotherapeutic agents and integrin β3 inhibitors were used to detect the curative effects.

Results

We proved that integrin β3 could serve as a marker of stem-like cancer cells in melanoma, along with the acquired chemotherapeutic drugs resistance. Furthermore, we observed that the membrane–proximal complex of integrin β3 with KRAS and Galectin-3 on the surface of melanoma cancer cells could recruit the RalB, resulting in the activation of TBK1. The phosphorylated TBK1 facilitates the activation of NF-κB signaling pathway, leading to the stem-like phenotype and drug resistance development in melanoma. Herein, the combination of cilengitide, an integrin β3 inhibitor, and chemotherapeutic agents were capable of suppressing the tumor growth and reversing the drug resistance induced by integrin β3.

Conclusion

These findings identified integrin β3 as a driver of melanoma stem-like cells with drug resistance and revealed an innovative strategy in clinic melanoma treatment.

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Acknowledgements

This study was supported by the fund of Health and Family Planning Commission of Zhejiang Province (2017189412).

Funding

This study was supported by the fund of Health and Family Planning Commission of Zhejiang Province (2017189412).

Author information

Authors and Affiliations

  1. Department of Dermatology, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, 158 Shangtang Road, Hangzhou, 310014, People’s Republic of China

    Xiaohua Tao, Wei Lu, Yang Ding & Yi Tang

  2. Department of Dermatology, Ningbo First Hospital, 59 Liuting Road, Ningbo, Zhejiang, People’s Republic of China

    Xiaoxia Zhu

Authors
  1. Xiaoxia Zhu
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  3. Wei Lu
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Corresponding author

Correspondence to Yi Tang.

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Conflict of interest

Author Yi Tang has received research grants from Health and Family Planning Commission of Zhejiang Province in China. Author Xiaoxia Zhu declares that he has no conflict of interest. Author Xiaohua Tao declares that he has no conflict of interest. Author Wei Lu declares that she has no conflict of interest. Author Yang Ding declares that he has no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Electronic supplementary material

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280_2018_3760_MOESM1_ESM.tif

Supplementary material 1 Supplementary figure 1. Activation of integrin β3/Galectin-3/KRAS results in the stem-like phenotype and drug resistance. (a) Relative KRAS expression in integrin β3 positive A375 cells treated with NRAS siRNA silence or not. (b) Relative activated GTP-KRAS level in integrin β3 positive or negative A375 cells. (c) Immunofluorescence of integrin β3 and KRAS in sorted β3- and the β3+ populations from A375 cells. Scale bar is 10 μm. (d) Immunofluorescence of integrin β3 and Galectin-3 in sorted β3- and the β3+ populations from A375 cells. Scale bar is 10 μm. The data was presented as the means ± SEM from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant (TIF 2474 KB)

280_2018_3760_MOESM2_ESM.tif

Supplementary material 2 Supplementary figure 2. Integrin β3/KRAS complex formation is independent of integrin β3 RGD binding domain. (a) Quantification of colonies formed by the β3+ population from A375 cells (D119A mutant or not) treated with PBS and cilengitide (10nM). (b-c) Cell viability of sorted β3+ population from A375 cells (D119A mutant or not) treated with DOX (1μg/ml), DOX(1μg/ml) combined with cilengitide (10nM), MTX (0.5 μg/ml) and MTX (0.5 μg/ml) combined with cilengitide (10nM) for 24h. The data was presented as the means ± SEM from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant (TIF 2161 KB)

280_2018_3760_MOESM3_ESM.tif

Supplementary material 3 Supplementary figure 3. Integrin β3/Galectin-3/KRAS regulates the macropinocytosis and intracellular ROS. (a) Relative micropinocytosis in integrin β3 positive or negative A375 cells. (b) Relative ROS level in integrin β3 positive or negative A375 cells. (c) Quantification of colonies formed by the β3+ population from A375 cells treated with PBS, MK-2206 (50nM) and SCH772984 (10nM). (d) Cell viability of sorted β3+ population from A375 cells treated with DOX (1μg/ml), DOX(1μg/ml) combined with MK-2206 (50nM) and SCH772984 (10nM), MTX (0.5 μg/ml) and MTX (0.5 μg/ml) combined with MK-2206 (50nM) and SCH772984 (10nM) for 24h. The data was presented as the means ± SEM from three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant (TIF 2344 KB)

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Zhu, X., Tao, X., Lu, W. et al. Blockade of integrin β3 signals to reverse the stem-like phenotype and drug resistance in melanoma. Cancer Chemother Pharmacol 83, 615–624 (2019). https://doi.org/10.1007/s00280-018-3760-z

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  • DOI: https://doi.org/10.1007/s00280-018-3760-z

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