MYO10 regulates genome stability and cancer inflammation through mediating mitosis.

Genomic instability can promote inflammation and tumor development. Previous research revealed an unexpected layer of regulation of genomic instability by a cytoplasmic protein MYO10; however, the underlying mechanism remained unclear. Here, we report a protein stability-mediated mitotic regulation of MYO10 in controlling genome stability. We characterized a degron motif and phosphorylation residues in the degron that mediate ß-TrCP1-dependent MYO10 degradation. The level of phosphorylated MYO10 protein transiently increases during mitosis, which is accompanied by a spatiotemporal cellular localization change first accumulating at the centrosome then at the midbody. Depletion of MYO10 or expression of MYO10 degron mutants, including those found in cancer patients, disrupts mitosis, increases genomic instability and inflammation, and promotes tumor growth; however, they also increase the sensitivity of cancer cells to Taxol. Our studies demonstrate a critical role of MYO10 in mitosis progression, through which it regulates genome stability, cancer growth, and cellular response to mitotic toxins.

Chemical screen identifies shikonin as a broad DNA damage response inhibitor that enhances chemotherapy through inhibiting ATM and ATR.

DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.

MYO10 drives genomic instability and inflammation in cancer.

Genomic instability is a hallmark of human cancer; yet the underlying mechanisms remain poorly understood. Here, we report that the cytoplasmic unconventional Myosin X (MYO10) regulates genome stability, through which it mediates inflammation in cancer. MYO10 is an unstable protein that undergoes ubiquitin-conjugating enzyme H7 (UbcH7)/ß-transducin repeat containing protein 1 (ß-TrCP1)­dependent degradation. MYO10 is upregulated in both human and mouse tumors and its expression level predisposes tumor progression and response to immune therapy. Overexpressing MYO10 increased genomic instability, elevated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)­dependent inflammatory response, and accelerated tumor growth in mice. Conversely, depletion of MYO10 ameliorated genomic instability and reduced the inflammation signaling. Further, inhibiting inflammation or disrupting Myo10 significantly suppressed the growth of both human and mouse breast tumors in mice. Our data suggest that MYO10 promotes tumor progression through inducing genomic instability, which, in turn, creates an immunogenic environment for immune checkpoint blockades.

Corrigendum: Shikonin Inhibits Cancer Through P21 Upregulation and Apoptosis Induction.

[This corrects the article DOI: 10.3389/fphar.2020.00861.].

Shikonin Inhibits Cancer Through P21 Upregulation and Apoptosis Induction.

Shikonin is a natural naphthoquinone compound and has demonstrated potent anti-cancer activities; however, the underlying molecular mechanisms remained elusive. Here we report that Shikonin inhibited the growth of a wide range of human cancer cell lines, illustrating a broad anticancer effect. Mechanistically, we show that Shikonin arrested the cell cycle at the G2/M phase, inhibited the ERK-dependent cell growth signal, and induced cell death in both P53 wild type and mutant cancer cells, which collectively contributed to the growth inhibitory effect of Shikonin. A pan-apoptosis inhibitor largely suppressed Shikonin-induced cell death, suggesting an important role of apoptosis in this process. Intriguingly, Shikonin also activated autophagy and inhibition of autophagy by depleting critical autophagic genes further increased Shikonin-induced cell death, indicating a protective role of autophagy. In uncovering the molecular mechanisms underlying these effects of Shikonin, we found that Shikonin induced a robust upregulation of P21 independent of the P53 status, upregulated autophagy genes, as well as inhibited expression of genes required for cell growth. Using mouse tumor models, we confirmed the strong anticancer effect of Shikonin in vivo. Together, our data reveal a broad range of pharmacological functions of Shikonin, involving simultaneous growth inhibition, cell cycle arrest, autophagy activation and apoptosis induction through regulating expression of critical genes involved in these pathways. Our study may facilitate the development of Shikonin in cancer therapy as a single agent or in combination with other anticancer therapies.

Protein phosphatase 2A controls ongoing DNA replication by binding to and regulating cell division cycle 45 (CDC45).

Genomic replication is a highly regulated process and represents both a potential benefit and liability to rapidly dividing cells; however, the precise post-translational mechanisms regulating genomic replication are incompletely understood. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that regulates a diverse array of cellular processes. Here, utilizing both a gain-of-function chemical biology approach and loss-of-function genetic approaches to modulate PP2A activity, we found that PP2A regulates DNA replication. We demonstrate that increased PP2A activity can interrupt ongoing DNA replication, resulting in a prolonged S phase. The impaired replication resulted in a collapse of replication forks, inducing dsDNA breaks, homologous recombination, and a PP2A-dependent replication stress response. Additionally, we show that during replication, PP2A exists in complex with cell division cycle 45 (CDC45) and that increased PP2A activity caused dissociation of CDC45 and polymerase α from the replisome. Furthermore, we found that individuals harboring mutations in the PP2A Aα gene have a higher fraction of genomic alterations, suggesting that PP2A regulates ongoing replication as a mechanism for maintaining genomic integrity. These results reveal a new function for PP2A in regulating ongoing DNA replication and a potential role for PP2A in the intra-S-phase checkpoint.

Regulatory cross-talk determines the cellular levels of 53BP1 protein, a critical factor in DNA repair.

DNA double strand breaks (DSBs) severely disrupt DNA integrity. 53BP1 plays critical roles in determining DSB repair. Whereas the recruitment of 53BP1 to the DSB site is key for its function, recent evidence suggests that 53BP1's abundance also plays an important role in DSB repair because recruitment to damage sites will be influenced by protein availability. Initial evidence has pointed to three proteins, the ubiquitin-conjugating enzyme UbcH7, the cysteine protease cathepsin L (CTSL), and the nuclear structure protein lamin A/C, that may impact 53BP1 levels, but the roles of each protein and any interplay between them were unclear. Here we report that UbcH7-dependent degradation plays a major role in controlling 53BP1 levels both under normal growth conditions and during DNA damage. CTSL influenced 53BP1 degradation during DNA damage while having little effect under normal growth conditions. Interestingly, both the protein and the mRNA levels of CTSL were reduced in UbcH7-depleted cells. Lamin A/C interacted with 53BP1 under normal conditions. DNA damage disrupted the lamin A/C-53BP1 interaction, which preceded the degradation of 53BP1 in soluble, but not chromatin-enriched, cellular fractions. Inhibition of 53BP1 degradation by a proteasome inhibitor or by UbcH7 depletion restored the 53BP1-lamin A/C interaction. Depletion of lamin A/C, but not CTSL, caused a similar enhancement in cell sensitivity to DNA damage as UbcH7 depletion. These data suggest that multiple pathways collectively fine-tune the cellular levels of 53BP1 protein to ensure proper DSB repair and cell survival.

A 10-year follow-up of a child with mild case of xeroderma pigmentosum complementation group D diagnosed by whole-genome sequencing.

BACKGROUND: Most patients with xeroderma pigmentosum complementation group D (XP-D) from Western countries suffer from neurological symptoms, whereas Japanese patients display only skin manifestations without neurological symptoms. We have previously suggested that these differences in clinical manifestations in XP-D patients are attributed partly to a predominant mutation in ERCC2, and the allele frequency of S541R is highest in Japan. METHODS: We diagnosed a child with mild case of XP-D by the evaluation of DNA repair activity and whole-genome sequencing, and followed her ten years. RESULTS: Skin cancer, mental retardation, and neurological symptoms were not observed. Her minimal erythema dose was 41 mJ/cm(2) , which was slightly lower than that of healthy Japanese volunteers. The patient's cells showed sixfold hypersensitivity to UV in comparison with normal cells. Post-UV unscheduled DNA synthesis was 20.4%, and post-UV recovery of RNA synthesis was 58% of non-irradiated samples, which was lower than that of normal fibroblasts. Genome sequence analysis indicated that the patient harbored a compound heterozygous mutation of c.1621A>C and c.591_594del, resulting in p.S541R and p.Y197* in ERCC2: then, patient was diagnosed with XP-D. Y197* has not been described before. CONCLUSION: Her mild skin manifestations might be attributed to the mutational site on her genome and daily strict sun protection. c.1621A>C might be a founder mutation of ERCC2 among Japanese XP-D patients, as it was identified most frequently in Japanese XP-D patients and it has not been found elsewhere outside Japan.

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

UbcH7 regulates 53BP1 stability and DSB repair.

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.