The natural anticancer compound rocaglamide selectively inhibits the G1‐S‐phase transition in cancer cells through the ATM/ATR‐mediated Chk1/2 cell cycle checkpoints

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over‐expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide‐A (Roc‐A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1‐S phase. Roc‐A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc‐A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR‐Chk1/Chk2 checkpoint pathway. However, Roc‐A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc‐A by a time‐resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc‐A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc‐A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.     

Acquired small cell lung cancer resistance to Chk1 inhibitors involves Wee1 up‐regulation

Platinum‐based chemotherapy has been the cornerstone treatment for small cell lung cancer (SCLC) for decades, but no major progress has been made in the past 20 years with regard to overcoming chemoresistance. As the cell cycle checkpoint kinase 1 (Chk1) plays a key role in DNA damage response to chemotherapeutic drugs, we explored the mechanisms of acquired drug resistance to the Chk1 inhibitor prexasertib in SCLC. We established prexasertib resistance in two SCLC cell lines and found that DNA copy number, messengerRNA (mRNA) and protein levels of the cell cycle regulator Wee1 significantly correlate with the level of acquired resistance. Wee1 small interfering RNA (siRNA) or Wee1 inhibitor reversed prexasertib resistance, whereas Wee1 transfection induced prexasertib resistance in parental cells. Reverse phase protein microarray identified up‐regulated proteins in the resistant cell lines that are involved in apoptosis, cell proliferation and cell cycle. Down‐regulation of CDK1 and CDC25C kinases promoted acquired resistance in parental cells, whereas down‐regulation of p38MAPK reversed the resistance. High Wee1 expression was significantly correlated with better prognosis of resected SCLC patients. Our results indicate that Wee1 overexpression plays an important role in acquired resistance to Chk1 inhibition. We also show that bypass activation of the p38MAPK signaling pathway may contribute to acquired resistance to Chk1 inhibition. The combination of Chk1 and Wee1 inhibitors may provide a new therapeutic strategy for the treatment of SCLC.     

Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase: a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy

5-fluorouracil (5-FU) is the first line component used in colorectal cancer (CRC) therapy however even in combination with other chemotherapeutic drugs recurrence is common. Mutations of the adenomatous polyposis coli (APC) gene are considered as the initiating step of transformation in familial and sporadic CRCs. We have previously shown that APC regulates the cellular response to DNA replication stress and recently hypothesized that APC mutations might therefore influence 5-FU resistance. To test this, we compared CRC cell lines and show that those expressing truncated APC exhibit a limited response to 5-FU and arrest in G1/S-phase without undergoing lethal damage, unlike cells expressing wild-type APC. In SW480 APC-mutant CRC cells, 5-FU-dependent apoptosis was restored after transient expression of full length APC, indicating a direct link between APC and drug response. Furthermore, we could increase sensitivity of APC truncated cells to 5-FU by inactivating the Chk1 kinase using drug treatment or siRNA-mediated knockdown. Our findings identify mutant APC as a potential tumor biomarker of resistance to 5-FU, and importantly we show that APC-mutant CRC cells can be made more sensitive to 5-FU by use of Chk1 inhibitors.     

Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics

Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.     

Chk1蛋白激酶及其抑制剂的研究进展

Chk1(checkpoint kinase 1,Chk1)属丝氨酸/苏氨酸蛋白激酶家族成员,在DNA损伤反应（DNA damage response,DDR）中是细胞周期检测点的核心蛋白。很多研究表明,Chk1蛋白激酶具有促进肿瘤细胞增生的功能,它的缺乏能使肿瘤细胞对放疗或化疗更加敏感,且其抑制剂与其它分子靶向药物联合应用具有“合成致死”的效应,因此Chk1可作为今后肿瘤治疗的新靶点。本文就Chk1蛋白激酶及其在肿瘤治疗中的作用作一综述。     

Mangrove dolabrane‐type of diterpenes tagalsins suppresses tumor growth via ROS‐mediated apoptosis and ATM/ATR–Chk1/Chk2‐regulated cell cycle arrest

Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti‐cancer drugs. In this study, we show that a group of dolabrane‐type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS‐mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S‐G2 phase via activation of the ATM/ATR—Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T‐cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs.     

Roles of Chk1 in cell biology and cancer therapy

The evolutionally conserved DNA damage response (DDR) and cell cycle checkpoints preserve genome integrity. Central to these genome surveillance pathways is a protein kinase, Chk1. DNA damage induces activation of Chk1, which then transduces the checkpoint signal and facilitates cell cycle arrest and DNA damage repair. Significant progress has been made recently toward our understanding of Chk1 regulation and its implications in cancer etiology and therapy. Specifically, a model that involves both spatiotemporal and conformational changes of proteins has been proposed for Chk1 activation. Further, emerging evidence suggests that Chk1 does not appear to be a tumor suppressor; instead, it promotes tumor growth and may contribute to anticancer therapy resistance. Recent data from our laboratory suggest that activating, but not inhibiting, Chk1 in the absence of chemotherapy might represent an innovative approach to suppress tumor growth. These findings suggest unique regulation of Chk1 in cell biology and cancer etiology, pointing to novel strategies for targeting Chk1 in cancer therapy.     

下调Chk1和Chk2基因促进射线照射后HeLa细胞凋亡

目的 探讨下调Chk1和Chk2基因对HeLa细胞射线照射后细胞周期和凋亡的影响及其作用机制.方法 应用流式细胞仪检测不同剂量60Co照射引起的HeLa细胞周期和凋亡的动力学变化,以激光共聚焦显微镜和Western blot法检测转染Chk1和Chk2正义寡核苷酸(sODN)和反义寡核苷酸(AsODN)对Chk1和Chk2蛋白表达的影响,以AnnexinV-PI法、凋亡细胞的周期特异性检测法及透射电镜观察单独或联合转染Chk1和Chk2反义寡核苷酸对射线照射后HeLa细胞凋亡的影响.结果 不同剂量的60Co照射可引起Hela细胞小同程度的G2/M期阻滞,15 Gy的60Co照射48 h后,G2/M期细胞可达75.53%±3.72%.当细胞周期阻滞解除后,细胞凋亡明显增加.转染Chk1 AsODN组的早期凋亡、晚期凋亡和死亡细胞共有94.42%±4.78%,明显高于转染Chk1 sODN组(44.35%±2.08%,P<0.0001);转染Chk2 AsODN组的凋亡细胞共有93.08%±5.24%,明显高于转染Chk2 sODN组(48.98%±3.35%,P<0.0001);而共转染Chk1和Chk2 AsODN组的凋亡细胞共有94.26%±4.92%,明显高于共转染Chk1和Chk2 sODN组(42.46%±2.56%,P<0.0001);共转染Chk1和Chk2AsODN组与仅转染Chk1 AsODN或Chk2 AsODN组比较,差异无统计学意义(P>0.05).凋亡细胞的周期特异性检测结果显示,转染Chk1和Chk2 sODN引起的凋亡主要来自于G1期细胞,而转染Chk1 AsODN或Chk2 AsODN后,G1期、S期、G2/M期的凋亡细胞均较对应sODN转染组明显增加,尤其共转染Chk1和Chk2 AsODN组更为显著.结论 射线照射激活细胞周期检测点信号传导通路引起细胞自我保护,是临床上产生放疗抵抗的主要原因,而阻断该信号通路的关键激酶Chk1或Chk2,可显著增强细胞的放射敏感性,其机制在于改变了凋亡细胞的周期特异性,凋亡细胞可以来自G1、S、G2/M各周期的细胞.     

G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer

Rigorous quality control steps, termed checkpoints, tightly regulate progression through the cell cycle. DNA-damaging chemotherapy and radiation activate functional cellular checkpoints. These checkpoints can facilitate DNA repair and promote cell death in unrepaired cells. There are at least three DNA damage checkpoints - at G1/S, S, and G2/M - as well as a mitotic spindle checkpoint. Most cancer cells harbour mutations in tumour suppressors and/or oncogenes, which impair certain cell checkpoints. Inhibiting the remaining cell checkpoints - particularly after exposure of cancer cells to chemotherapy and/or radiation - allows cell death, a strategy now being employed in cancer therapeutics. With our increasing knowledge of cell cycle regulation, many compounds have been developed to inhibit specific checkpoint components, particularly at the G2/M transition. One such target is checkpoint kinase-1 (Chk1). We review here the molecular framework of the cell cycle, the rationale for targeting Chk1, the preclinical concepts related to the development of Chk1 inhibitors, and the efficacy and safety results from Chk1 inhibitors now in phase I/II trials.     

Chk1-induced CCNB1 overexpression promotes cell proliferation and tumor growth in human colorectal cancer

The high morbidity and mortality of colorectal cancer pose a significant public health problem worldwide. Here we assessed the pro-cancer efficacy and mechanism of action of CCNB1 in different colorectal cancer cells. We provided evidence that CCNB1 mRNA and protein level were upregulated in a subset of human colorectal tumors, and positively correlated with Chk1 expression. Repression of Chk1 caused a significant decrease in cell proliferation and CCNB1 protein expression in colorectal cancer cells. Furthermore, downregulation of CCNB1 impaired colorectal cancer proliferation in vitro and tumor growth in vivo. Specifically, suppression of CCNB1 caused a strong G₂/M phase arrest in both HCT116 and SW480 cells, interfering with the expression of cdc25c and CDK1. Additionally, CCNB1 inhibition induced apoptotic death in certain colorectal cancer cells. Together, these results suggest that CCNB1 is activated by Chk1, exerts its oncogenic role in colorectal cancer cells, and may play a key role in the development of a novel therapeutic approach against colorectal cancer.     

程序性死亡受体1及程序性死亡受体配体1抑制剂在小细胞肺癌治疗中的进展

小细胞肺癌(SCLC)约占肺癌的15%,是一种神经内分泌肿瘤,生长迅速、极具侵袭性,容易早期发生远处转移。尽管SCLC对一线化疗敏感,但容易短时间内复发。在过去的5年间,免疫治疗在SCLC中取得了一定的成果,特别是在程序性死亡受体1和程序性死亡受体配体1方面,SCLC治疗模式已经发生改变。文章对免疫检查点抑制剂在SCLC中的探索研究做一综述,以期为广大临床工作者提供参考。     

Targeting PTPs with small molecule inhibitors in cancer treatment

Protein tyrosine phosphorylation plays a major role in cellular signaling. The level of tyrosine phosphorylation is controlled by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Disturbance of the normal balance between PTK and PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. A number of PTPs have been implicated in oncogenesis and tumor progression and therefore are potential drug targets for cancer chemotherapy. These include PTP1B, which may augment signaling downstream of HER2/Neu; SHP2, which is the first oncogene in the PTP superfamily and is essential for growth factor-mediated signaling; the Cdc25 phosphatases, which are positive regulators of cell cycle progression; and the phosphatase of regenerating liver (PRL) phosphatases, which promote tumor metastases. As PTPs have emerged as drug targets for cancer, a number of strategies are currently been explored for the identification of various classes of PTP inhibitors. These efforts have resulted many potent, and in some cases selective, inhibitors for PTP1B, SHP2, Cdc25 and PRL phosphatases. Structural information derived from these compounds serves as a solid foundation upon which novel anti-cancer agents targeted to these PTPs can be developed.     

Id-1 promotes proliferation of p53-deficient esophageal cancer cells

The helix-loop-helix protein inhibitor of differentiation and DNA binding (Id-1) is known to promote cellular proliferation in several types of human cancer. Although it has been reported that Id-1 is over-expressed in esophageal squamous cell carcinoma (ESCC), its function and signaling pathways in esophageal cancer are unknown. In our study, we investigated the direct effects of Id-1 on esophageal cancer cell growth by transfecting an Id-1 expression vector into an ESCC cell line (HKESC-3), which showed serum-dependent Id-1 expression. Ectopic Id-1 expression resulted in increased serum-independent cell growth and G1-S phase transition, as well as up-regulation of mouse double minute 2 (MDM2) and down-regulation of p21Waf1/Cip1 protein expressions in the transfectant clones in a p53-independent manner. However, overexpression of Id-1 had no effect on the pRB, CDK4 and p16INK4A expressions. Stable transfection of Id-1 antisense expression vector to inhibit the expression of endogenous Id-1 in another ESCC cell line (HKESC-1) reversed the effects on MDM2 and p21Waf1/Cip1. In addition, Id-1 expression protected ESCC cells from Tumor Necrosis Factor (TNF)-alpha-induced apoptosis by up-regulating and activating Bcl-2. In conclusion, our study provides evidence for the first time that Id-1 plays a role in both proliferation and survival of esophageal cancer cells. Our findings also suggest that unlike prostate, hepatocellular and nasopharyngeal carcinomas in which Id-1 induces cell proliferation through inactivation of p16INK4A/RB pathway, the increased cell proliferation observed in ESCC cells may be mediated through a different mechanism.     

Rapid induction of p21WAF1 but delayed down-regulation of Cdc25A in the TGF-beta-induced cell cycle arrest of gastric carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15INK4B, p21WAF1/Cip1 and p27Kip1) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620 cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21, while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4 and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied.     

ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.     

Commensal microbiota contributes to predicting the response to immune checkpoint inhibitors in non-small-cell lung cancer patients

Immunotherapy against cancer, through immune checkpoint inhibitors targeting the programmed cell death-1/programmed cell death-ligand 1 axis, is particularly successful in tumors by relieving the immune escape. However, interindividual responses to immunotherapy are often heterogeneous. Therefore, it is essential to screen out predictive tumor biomarkers. In this study, we analyzed the commensal microbiota in stool samples and paired sputum samples from 75 metastatic non-small-cell lung cancer (NSCLC) patients at baseline and during treatment with immune checkpoint inhibitors. Results showed distinct microbes’ signatures between the gut microbiota and paired respiratory microbiota. The alpha diversity between the gut and respiratory microbiota was uncorrelated, and only the gut microbiota alpha diversity was associated with anti-programmed cell death-1 response. Higher gut microbiota alpha diversity indicated better response and more prolonged progression-free survival. Comparison of bacterial communities between responders and nonresponders showed some favorable/unfavorable microbes enriched in responders/nonresponders, indicating that commensal microbiota had potential predictive value for the response to immune checkpoint inhibitors. Generally, some rare low abundance gut microbes and high abundance respiratory microbes lead to discrepancies in microbial composition between responders and nonresponders. A significant positive correlation was observed between the abundance of Streptococcus and CD8⁺ T cells. These results highlighted the intimate relationship between commensal microbiota and the response to immunotherapy in NSCLC patients. Gut microbiota and respiratory microbiota are promising biomarkers to screen suitable candidates who are likely to benefit from immune checkpoint inhibitor-based immunotherapy.     

p28, A first in class peptide inhibitor of cop1 binding to p53

Background:

A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination.

Methods:

In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions.

Results:

The L1 loop (aa 112–124), a region within the S7–S8 loop (aa 214–236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53^wt and p53^mut cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53^wt and p53^mut cells were in response to sustained increases in p53.

Conclusion:

These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.     

Immune checkpoint inhibitors in advanced non–small cell lung cancer

The emergence of immune checkpoint inhibitors for the treatment of cancer has led to major changes to the therapeutic landscape of lung cancer. Improvements in overall survival relative to standard chemotherapy have been observed in the first‐line and second‐line therapy settings for patients with advanced non–small cell lung cancer (NSCLC) who are treated with immune checkpoint inhibitors. Consequently, every patient with advanced‐stage NSCLC is now a candidate for immune checkpoint inhibitor therapy. However, it is clear that the benefit from therapy is not universal, and identification of biomarkers to select therapy has assumed importance. In addition to programmed cell death receptor ligand 1 expression, both tissue‐based and blood‐based markers are under evaluation to select patients. In an era of increasing costs of care and potential for toxicities related to immune checkpoint inhibition, proper patient selection is critical to the optimal use of this new class of agents. In addition, development of novel combination approaches has also emerged as an important way to improve the efficacy of immune checkpoint inhibition. Studies in earlier stages of NSCLC are already underway with the hope of improving the cure rate. In this article, the authors review the current landscape of immune checkpoint inhibitors in the treatment of advanced NSCLC. Cancer 2018;124:248‐61 . © 2017 American Cancer Society.