Written on the front
Today's recommendation is published by the Department of Biotechnology and Bioindustrial Science, National Cheng Kung University, Tainan, Taiwan, China on March 25, 2021 in Cell Death and Differentiation (2020if:15.).828, JCRQ1), corresponding author of Professor Jan-Jong Hung, showed that USP24 promotes drug resistance during cancer**.
Background:
Deubiquitinase (DUB) is a specific enzyme that regulates a variety of cellular functions by regulating the ubiquitin molecule. Ubiquitin-specific peptidases belong to the superfamily of the DUB superfamily, which has been implicated in many human disease processes. Drug resistance, on the other hand, is caused by drugs against various diseases**, such as bacterial infections and cancers, which reduces the effectiveness of the drug. Drug resistance remains an important issue for the prevention of various diseases, including cancer.
Summary section
Here, the authors found that USP24 not only inhibits DNA damage repair (DDR) activity by decreasing RAD51 expression, leading to tumor genome instability and cancer stemness, but also increases the levels of the ATP-binding cassette (ABC) transporter P-GP, ABCG2 and Ezrin enhance the pumping of paclitaxel from cancer cells, thereby creating resistance during cancer**. A novel USP24 inhibitor, NCI677397, was screened for specific inhibition of the catalytic activity of USP24. This inhibitor has been identified to inhibit drug resistance by reducing genomic instability, cancer stemness, and pumping drugs out of cancer cells. Understanding the role and molecular mechanism of USP24 in drug resistance will facilitate the development of novel USP24 inhibitors in the future. The authors' study provides new insights into the clinical significance of USP24 inhibitors in blocking drug resistance during chemotherapy.
What to study
1.USP24 is modulating drug resistance during cancer**
The absence of USP24 in cancer cells and macrophages significantly inhibits the malignant ability of cancer cells, suggesting that USP24 is a potential target for drug development. Since drug resistance is a major factor in the development of cancer** and malignancy, there is a need to elucidate the role of USP24 in drug resistance during **. The chemotherapy drugs paclitaxel, camptothecin (CPT), and cisplatin were used to induce resistance in cell lines, and increased levels of USP24 were found in these resistant cell lines. Knockdown of USP24 in these resistant cell lines partially inhibits drug resistance. In addition, paclitaxel-sensitive A549 cells with or without USP24 are treated with paclitaxel for 3 months to generate drug-resistant cell lines. Interestingly, paclitaxel** induced resistance in the A549 cell line three months later, but the absence of USP24 completely blocked resistance. The A549-resistant cell line T24 with and without USP24 knockdown is injected into SCID mice, and then the mice are treated with paclitaxel. Deletion of USP24 significantly enhances the cytotoxic effects of paclitaxel, reducing tumor size and tumor weight.
Figure 1USP24 positively modulates drug resistance.
Conclusions: USP24 positively regulates drug resistance during cancer**.
2.USP24 stabilizes the ABC transporter to draw the drug from cancer cells
The ABC transporter is critical for drug resistance, and paclitaxel-induced increased levels of P-GP, ABCG2, and MRP1 in T24 lung cancer cells compared to A549 cells or other resistant cell lines. The levels of P-GP, ABCG2, and EzRin decreased after USP24 knockdown, suggesting that USP24 acts as a deubiquitinating enzyme to stabilize P-GP, ABCG2, and Ezrin, enhancing the ability to pump drugs from cancer cells. In addition, knockdown of USP24 reduced the protein stability of P-GP and ABCG2. This effect of USP24 on P-GP and ABCG2 can be reversed by MG132 and chloroquine treatments, respectively, suggesting that USP24 stabilizes P-GP and ABCG2 in a proteasome- and lysosome-dependent manner, respectively. In addition, USP24 also stabilizes EzRin and insulates it from proteomic degradation. USP24 can interact with P-GP, ABCG2, and Ezrin, and knockdown of USP24 increases the ubiquitination of both P-GP and ABCG2. In vitro enzymatic analysis showed that purified recombinant USP24 could reduce the ubiquitination signal of ABCG2 and P-GP. These findings suggest that USP24 stabilizes these ABC transporters, which pump drugs. Based on previous studies, EzRin can assist with ABC transporters on cell membranes, and here, the authors found that USP24 not only stabilizes EzRin but also increases ABCG2.
Figure 2USP24 stabilizes the ABC transporter, which pumps out the drug.
Previous studies have also shown that cancer stemness is associated with drug resistance. After knockdown of USP24, the level of CD44 and the formation of spheroids decreased. In addition, the levels of USP24, CD133, CD44, ABCG2, and NanoG in spheroid cells were higher than in parental cells. Deletion of USP24 significantly reduced the levels of cancer stem cell markers such as CD44, ABCG2, NanoG, CD133, and SOX2. Subsequently, the authors directly measured the intracellular paclitaxel concentration by mass spectrometry. Deletion of USP24 reduced paclitaxel-treated levels of paclitaxel-resistant T24 cells from 1108 increased to 2727 nm。The relationship between USP24, ABCG2, and cancer stem cell markers was then investigated in a clinical lung cancer cohort. The data showed a high positive correlation between the levels of USP24 and ABCG2 in lung cancer patients. All the results suggest that USP24 stabilizes the ABC transporter and increases the stemness profile of cancer to induce drug resistance.
Figure 3USP24 promotes cancer stemness.
Conclusions: USP24 stabilizes ABC transporters and increases cancer stemness characteristics to induce drug resistance.
3.The entry of USP24 into the nucleus induces genomic instability and reduces DNA damage repair activity under DNA damage conditions
In order to **USP24 drug resistance effects between drug-sensitive and drug-resistant cancer cell lines. The authors' RNA sequencing data showed a significant increase in mRNA levels at RAD51 by USP24 knockdown. In fact, the protein and mRNA levels of RAD51 were increased in USP24 knockdown A549 cells. To study the transcriptional activity of RAD51, the authors found that the E2F binding site in the RAD51 promoter is essential for USP24-mediated RAD51 expression. Previously, the authors have shown that E2F4 is a substrate for USP24, and here the authors found that overexpression of E2F4 reversed the effects of USP24-KD on Rad51 mRNA levels and promoter activity, suggesting that USP24-stable E2F4 may inhibit Rad51 expression. Subsequently, the authors found that E2F4 can be recruited within the promoter of RAD51 and that knockdown of USP24 can eliminate this recruitment. Therefore, the authors investigated the role of USP24 in DDR activity during cancer progression. First, knockdown of USP24 inhibits CPT-induced apoptosis, cytotoxicity, and subG1 populations in A549 cells, but this effect is reversed by E2F4 overexpression or RAD51 knockdown. The authors also found that knockdown of USP24 increased the number of lesions and the recruitment of RAD51 into the chromosome.
Figure 4USP24-stable E2F4 inhibits RAD51 transcriptional activity.
Next, the authors further investigated the mechanism by which USP24 regulates DDR. Here, the authors found that USP24 can be transported into the nucleus of lung cancer cells under ultraviolet irradiation, CPT, and cisplatin treatment. All conserved ATM phosphorylated conserved sequences in USP24 are mutated individually and then overexpressed in cells, and then the distribution of USP24 mutant proteins after line irradiation is detected. Results 352, S1422, and S1620 are important for the nuclear localization of USP24. Knockdown of USP24 decreases the cytotoxicity of CPT, increases -H2ax and Mono-Ub--H2AX levels, increases the number of lesions, and increases HR-mediated GFP signaling, but E2F4 abolishes all effects. Whole genome sequencing of A549 and paclitaxel-induced resistant cell line T24 was performed by whole genome sequencing and analyzed using CircOS software. The results of the study showed that paclitaxel-induced resistance increased the proportion of structural variants and translocations, but there were no differences in deletions, duplications, or inversions, and that these effects were abrogated by knockdown of USP24, suggesting that USP24 enhances TMB not only by increasing drug pumping from cells, but also by inducing genomic instability.
Figure 5The entry of SP24 into the nucleus induces genomic instability.
Conclusions: USP24 reduces DDR activity during cancer progression and favors chemotherapy-induced cell death, but may lead to genomic instability and ultimately drug resistance. USP24 induces genomic instability by inhibiting DDR entry into the nucleus, which may be the reason for the resistance of USP24 to drug-sensitive cell lines and drug-resistant cell lines.
4.Novel specific USP24 inhibitors block the enzymatic activity of USP24 to inhibit drug resistance in cancer** processes
The authors then screen for novel specific USP24 inhibitors to prevent resistance during cancer**. The authors used the Modeller module in Chimaera software to create a homologous model and screen inhibitors from the National Cancer Institute (NCI) compound library using a structure-based strategy. To further elucidate the interaction between NCI677397 and USP24, docking analysis revealed the presence of many hydrogen bonds between the binding sites in NCI677397 and USP24. The analysis showed that both compounds, NCI677397 and NCI158067, reduced the levels of BAX, BRD7 and P300. WP1130 (a compound that inhibits lymphoma progression) does not inhibit the activity of USP24 with these substrates in lung cancer. Treatment of A549 cells with NCI677397 also increases the ubiquitination of p300 by NCI677397. These two compounds also inhibit the catalytic activity of recombinantly purified USP24 protein. Interestingly, NCI677397 did not block the catalytic activity of USP7, USP9X, or USP10, but NCI158067 inhibited the catalytic activity of USP7 and USP10, suggesting that NCI677397 exhibited higher specificity in blocking USP24 enzyme activity.
Figure 6NCI677397 interconnects with USP24.
Conclusions: NCI677397 has higher specificity and efficacy for the inhibition of USP24.
5.NCI677397 inhibits chemotherapy-induced resistance in vitro and in vivo
The authors then investigated the inhibitory effect of NCI677397 on drug resistance in different cancer cell lines. NCI677397 significantly blocked the resistance induced by different stimuli in different cellsIn addition, NCI677397 treatment reduced the level of P-GP and inhibited the spheroid-forming ability of T24 cells. This finding suggests that the loss of catalytic activity of USP24 reduces the stemness profile of cancer, which may be critical for drug resistance. Subsequently, the authors found that NCI677397 increased the concentration of intracellular paclitaxel from 4 under paclitaxel treatment78 nm increased to 32136 nm。
Figure 7NCI677397 inhibits chemotherapy-induced resistance in vitro.
The role of NCI677397 in inhibiting drug resistance has also been validated in vivo. NCI677397 cotreatment enhances the cytotoxic effect of paclitaxel on tumor size and weight in SCID mice. In SCID mice injected with cancer cells, a high positive correlation between USP24 and cancer stem cell markers was also found.
Figure 8NCI677397 inhibits chemotherapy-induced resistance in vivo.
Conclusions: NCI677397 inhibits chemotherapy-induced resistance in vitro and in vivo.
Conclusions and discussions
The authors' study showed that USP24 increases TMB and induces the drug to be pumped out of cells, thereby inducing drug resistance. NCI677397, a novel, USP24-specific inhibitor, can stabilize the genome, reduce cancer stemness characteristics, and increase intracellular drug concentrations to prevent the emergence of drug resistance. In the future, NCI677397 will be modified to optimize the inhibition of resistance to chemotherapy, and the safety of the drug will also be evaluated in vitro and in vivo. In addition, since genomic instability and drug pumping are critical and pervasive issues in resistance induction, the authors will investigate the effects of USP24 inhibitors on targeted** and immune**-induced resistance in the future.
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