: Ubiquitin-specific peptidase 10 (USP10) stabilizes both tumor suppressors and oncogenes in a context-dependent manner. However, the nature of USP10’s role in non-small cell lung cancer (NSCLC) remains unclear. By analyzing The Cancer Genome Atlas (TCGA) database, we have shown that high levels of USP10 are associated with poor overall survival in NSCLC with mutant p53, but not with wild-type p53. Consistently, genetic depletion or pharmacological inhibition of USP10 dramatically reduces the growth of lung cancer xenografts lacking wild-type p53 and sensitizes them to cisplatin. Mechanistically, USP10 interacts with, deubiquitinates, and stabilizes oncogenic protein histone deacetylase 6 (HDAC6). Furthermore, reintroducing either USP10 or HDAC6 into a USP10-knockdown NSCLC H1299 cell line with null-p53 renders cisplatin resistance. This result suggests the existence of a “USP10-HDAC6-cisplatin resistance” axis. Clinically, we have found a positive correlation between USP10 and HDAC6 expression in a cohort of NSCLC patient samples. Moreover, we have shown that high levels of USP10 mRNA correlate with poor overall survival in a cohort of advanced NSCLC patients who received platinum-based chemotherapy. Overall, our studies suggest that USP10 could be a potential biomarker for predicting patient response to platinum, and that targeting USP10 could sensitize lung cancer patients lacking wild-type p53 to platinum-based therapy.

(2016). Nucleus or cytoplasm? The mysterious case of SIRT1's subcellular localization. Cell Cycle: Vol. 15, No. 24, pp. 3337-3338.

MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating enzymes, which regulate MSH2 remain unknown. Here we report that ubiquitin-specific peptidase 10 (USP10) interacts with and stabilizes MSH2. USP10 deubiquitinates MSH2 in vitro and in vivo. Moreover, the protein level of MSH2 is positively correlated with the USP10 protein level in a panel of lung cancer cell lines. Knockdown of USP10 in lung cancer cells exhibits increased cell survival and decreased apoptosis upon the treatment of DNA-methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and antimetabolite 6-thioguanine (6-TG). The above phenotypes can be rescued by ectopic expression of MSH2. In addition, knockdown of MSH2 decreases the cellular mismatch repair activity. Overall, our results suggest a novel USP10-MSH2 pathway regulating DNA damage response and DNA mismatch repair.

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the MAPK signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and 2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and 2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CBP and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N-terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

MLH1 is a key DNA mismatch repair (MMR) protein, which plays an important role in maintenance of genomic stability and the DNA damage response (DDR). Here, we report that MLH1 is a novel substrate of HDAC6. HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo. Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLa complex. Moreover, we have identified four novel acetylation sites in MLH1 by mass spectrometry analysis. The deacetylation mimetic mutant, but not the wild-type and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSa-MutLa complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSa-MutLa complex by deacetylation of MLH1, leading to the tolerance of DNA damage.

We have previously discovered that HDAC6 regulates the DNA damage response (DDR) via modulating the homeostasis of a DNA mismatch repair protein, MSH2, through HDAC6’s ubiquitin E3 ligase activity. Here, we have reported HDAC6’s second potential E3 ligase substrate, a critical cell cycle checkpoint protein, Chk1. We have found that HDAC6 and Chk1 directly interact, and that HDAC6 ubiquitinates Chk1 in vivo and in vitro. Specifically, HDAC6 interacts with Chk1 via the DAC1 domain, which contains its ubiquitin E3 ligase activity. During the cell cycle, Chk1 protein levels fluctuate, peaking at the G2 phase, subsequently resolving via the ubiquitin-proteasome pathway, and thereby allowing cells to progress to the M phase. However, in HDAC6 knockdown non-small cell lung cancer (NSCLC) cells, Chk1 is constitutively active and fails to resolve post-ionizing radiation (IR), and this enhanced Chk1 activity leads to preferential G2 arrest in HDAC6 knockdown cells accompanied by a reduction in colony formation capacity and viability. Depletion or pharmacological inhibition of Chk1 in HDAC6 knockdown cells reverses this radiosensitive phenotype, suggesting that the radiosensitivity of HDAC6 knockdown cells is dependent on increased Chk1 kinase activity. Overall, our results highlight a novel mechanism of Chk1 regulation at the post-translational level, and a possible strategy for sensitizing NSCLC to radiation via inhibiting HDAC6’s E3 ligase activity.