The IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis

Nutlin-3a is a small molecule MDM2 antagonist and potent activator of wild-type p53. Nutlin-3a disrupts MDM2 binding to p53, thus increasing p53 levels and allowing p53 to inhibit proliferation or induce cell death. Factors that control sensitivity to Nutlin-3a-induced apoptosis are incompletely understood. In this study we isolated cisplatin-resistant clones from MHM cells, an MDM2-amplified and p53 wild-type osteosarcoma cell line. Cisplatin resistance in these clones resulted in part from heightened activation of the IGF-1R/AKT pathway. Interestingly, these cisplatin resistant clones showed hyper-sensitivity to Nutlin-3a induced apoptosis. Increased Nutlin-3a sensitivity was associated with reduced authophagy flux and a greater increase in p53 levels in response to Nutlin-3a treatment. IGF-1R and AKT inhibitors further increased apoptosis by Nutlin-3a in parental MHM cells and the cisplatin-resistant clones, confirming IGF-1R/AKT signaling promotes apoptosis resistance. However, IGF-1R and AKT inhibitors also reduced p53 accumulation in Nutlin-3a treated cells and increased autophagy flux, which we showed can promote apoptosis resistance. We conclude the IGF-1R/AKT pathway has opposing effects on Nutlin-3a-induced apoptosis. First, it can inhibit apoptosis, consistent with its well-established role as a survival-signaling pathway. Second, it can enhance Nutlin-3a induced apoptosis through a combination of maintaining p53 levels and inhibiting pro-survival autophagy.     

Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

HPV-positive HNSCC cell lines but not primary human fibroblasts are radiosensitized by the inhibition of Chk1

Purpose

Despite the comparably high cure rates observed for HPV-positive HNSCC, there is still a great need for specific tumor radiosensitization due to the often severe side effects resulting from intense radiochemotherapy. We recently demonstrated that HPV-positive HNSCC cell lines are characterized by a defect in DNA double-strand break repair associated with a pronounced G2-arrest. Here we tested whether abrogation of this radiation-induced G2-arrest by the inhibition of Chk1 results in specific radiosensitization of HPV-positive HNSCC cells.

Materials and methods

Experiments were performed with five HPV and p16-positive (93-VU-147T, UM-SCC-47, UT-SCC-45, UD-SCC-2, UPCI-SCC-154) and two HPV and p16-negative HNSCC cell lines, as well as two normal human fibroblast strains. Chk1 was inhibited by the selective inhibitor PF-00477736. Cell cycle distribution was determined by flow cytometry, Chk1-activity via Western blot and cell survival by colony formation assay.

Results

With the exception of UPCI-SCC-154, the inhibition of Chk1 was found to abolish the pronounced radiation-induced G2-arrest in all HPV-positive cells utilized. All tumor cell lines that demonstrated the abrogation of G2-arrest also demonstrated radiosensitization. Notably, in G1-arrest-proficient normal human fibroblasts no radiosensitization was induced.

Conclusion

Abrogation of the G2 checkpoint through the inhibition of Chk1 may be used to selectively increase the cellular radiosensitivity of HPV-positive HNSCC without affecting the surrounding normal tissue.     

Chk1/2对肿瘤细胞凋亡的调节作用

目的:观察顺铂作用下K562细胞及白血病骨髓单个核细胞(BMMNC)的细胞周期变化和转染Chk1/2反义寡核苷酸对顺铂诱导下K562细胞及白血病BMMNC凋亡的影响.方法:用流式细胞仪检测顺铂作用下K562细胞及白血病BMMNC的细胞周期变化.转染Chk1和Chk2反义寡核苷酸于K562细胞及白血病BMMNC,检测顺铂作用下转染细胞的凋亡率.结果:10 μmol/L顺铂作用下K562细胞和白血病BMMNC均出现S期阻滞,转染Chk1/2反义寡核苷酸可明显增加顺铂诱导下K562细胞凋亡,转染Chk1反义寡核苷酸可明显增加顺铂诱导下白血病BMMNC的凋亡率,但转染Chk2反义寡核苷酸未增加白血病BMMNC的凋亡率.结论:Chk1在肿瘤细胞凋亡中有重要调节作用,可作为肿瘤增敏治疗的有效靶点,Chk2在肿瘤细胞凋亡中的作用需进一步研究.     

Cytotoxic effects of 15d-PGJ2 against osteosarcoma through ROS-mediated AKT and cell cycle inhibition

Polo-like kinase 1 (PLK1), a critical cell cycle regulator, has been identified as a potential target in osteosarcoma (OS). 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a prostaglandin derivative, has shown its anti-tumor activity by inducing apoptosis through reactive oxygen species (ROS)-mediated inactivation of v-akt, a murine thymoma viral oncogene homolog, (AKT) in cancer cells. In the study analyzing its effects on arthritis, 15d-PGJ2 mediated shear-induced chondrocyte apoptosis via protein kinase A (PKA)-dependent regulation of PLK1. In this study, the cytotoxic effect and mechanism underlying 15d-PGJ2 effects against OS were explored using OS cell lines. 15d-PGJ2 induced significant G2/M arrest, and exerted time- and dose-dependent cytotoxic effects against all OS cell lines. Western blot analysis showed that both AKT and PKA-PLK1 were down-regulated in OS cell lines after treatment with 15d-PGJ2. In addition, transfection of constitutively active AKT or PLK1 partially rescued cells from 15d-PGJ2-induced apoptosis, suggesting crucial roles for both pathways in the anti-cancer effects of 15d-PGJ2. Moreover, ROS generation was found treatment with 15d-PGJ2, and its cytotoxic effect could be reversed with N-acetyl-l-cysteine. Furthermore, inhibition of JNK partially rescued 15d-PGJ2 cytotoxicity. Thus, ROS-mediated JNK activation may contribute to apoptosis through down-regulation of the p-Akt and PKA-PLK1 pathways. 15d-PGJ2 is a potential therapeutic agent for OS, exerting cytotoxicity mediated through both AKT and PKA-PLK1 inhibition, and these results form the basis for further analysis of its role in animal studies and clinical applications.     

Distinct DNA damage determines differential phosphorylation of Chk2

Checkpoint kinase 2 (Chk2) has been implicated in DNA damage signaling. By using BJ human fibroblasts, HCT116 colorectal cancer cells and HeLa cervical cancer cells, we further detailed phosphorylation kinetics of Chk2 under treatment with neocarcinostatin (NCS) or doxorubicin (Dox). After NCS treatment, phosphorylation of Chk2 Thr68 occurs in 3 min, followed by phosphorylation of Ser19 and Ser33/35. In ATM deficient fibroblasts, NCS does not induce phosphorylation of NBS1 Ser343 and Chk2 Ser19 and Ser33/35, however Chk2 Thr68 is still phosphorylated, indicating that ATM is essential for phosphorylation of these residues when treated with NCS. By using Chk2-deficient HCT116 cells re-expressing phospho-mutant Chk2 (T68A), we found that inhibition of Thr68 phosphorylation enhances Ser19 phosphorylation in NCS treated cells. Interestingly, in contrast to NCS, Dox does not induce Ser33/35 phosphorylation in HeLa and HCT116 cells. Phosphorylation of Thr68 is sustained until 3 to 4 hours, and phosphorylation of Ser19 occurs 70 to 80 min after Dox treatment. These results demonstrate that Chk2 s involved in the early stages of DNA damage response. Differential phosphorylation kinetics of these residues suggests that DNA damage determines intermolecular and intramolecular interaction of Chk2, which may regulate phosphorylation.     

Chk1/2和Plk1蛋白在子宫内膜癌中的表达

目的 Chk1/2（checkpoint kinase 1、2）和Plk1（polo like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶,本研究检测3种激酶在子宫内膜癌及正常子宫内膜组织中的表达,探讨3种蛋白在两者之间的表达差异、与子宫内膜癌临床病理特征的关系及3种蛋白表达的相关性。方法 应用免疫组化SP法检测44例子宫内膜癌组织和21例正常子宫内膜组织中Chk1、Chk2和Plk1蛋白的表达情况。结果 Chk1、Chk2和Plk1蛋白在子宫内膜癌患者中的阳性率分别为47.7％、75.0％和31.8％,在正常子宫内膜中的阳性率分别为61.9％、61.9％和4.8％;Plk1蛋白在子宫内膜癌组织中的表达显著高于正常子宫内膜组织（P＜0.01）,而Chk1、Chk2的表达差异无统计学意义（P>0.05）。Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的子宫内膜癌患者中差异无统计学意义（P>0.05）;但Chk1的表达在不同分化程度的子宫内膜癌患者中差异有统计学意义（P<0.01）。Spearman等级相关分析,在44例子宫内膜癌患者中,Chk2与Plk1间的表达呈正相关（r＝0.482,P＝0.001）。结论 Plk1可能成为子宫内膜癌比较理想的治疗靶点,而CHK1/2在子宫内膜癌中表达及意义还有待进一步研究。     

miR-203a-3p靶向AKT2增强骨肉瘤细胞的放射敏感性

目的:研究miR-203a-3p对骨肉瘤MG-63细胞凋亡和放射敏感性的影响及潜在作用机制.方法:qRT-PCR检测AKT2 mRNA的表达水平及不同放射剂量(0、2、4、6、8 Gy)照射后MG-63细胞中miR-203a-3p的表达水平,克隆形成实验检测不同放射剂量处理后细胞存活分数,流式细胞术测定MG-63细胞凋...     

Retaining MKP1 expression and attenuating JNK-mediated apoptosis by RIP1 for cisplatin resistance through miR-940 inhibition

The elucidation of chemoresistance mechanisms is important to improve cancer patient survival. In this report, we investigated the role and mechanism through which receptor-interacting protein 1 (RIP1), a mediator in cell survival and death signaling, participates in cancer''s response to chemotherapy. In lung cancer cells, knockdown of RIP1 substantially increased cisplatin-induced apoptotic cytotoxicity, which was associated with robust JNK activation. The expression of the JNK inactivating phosphatase, MKP1, was substantially reduced in RIP1 knockdown cells. Although MKP1 protein stability was not altered by RIP1 suppression, the synthesis rate of MKP1 was dramatically reduced in RIP1-suppressed cells. Furthermore, we found that the expression of miR-940 was substantially increased in RIP1 knockdown cells. Knockdown of miR-940 restored MKP1 expression and attenuated cisplatin-induced JNK activation and cytotoxicity. Importantly, ectopic expression of MKP1 effectively attenuated cisplatin-induced JNK activation and cytotoxicity. In addition, activation of the JNK upstream signaling kinase, MKK4, was also potentiated in RIP1 knockdown cells. Altogether, our results suggest that RIP1 contributes to cisplatin resistance by suppressing JNK activation that involves releasing miR-940-mediated inhibition of MKP1 and suppressing activation of MKK4. Intervention targeting the RIP1/miR-940/MKP1/JNK pathway may be used to sensitize platinum-based chemotherapy.     

Enhancement of cellular radiation sensitivity through degradation of Chk1 by the XIAP-XAF1 complex

X-linked inhibitor of apoptosis (XIAP) and Chk1 are potential molecular targets in radiotherapy. However, their molecular association in the regulation of radiation sensitivity has been rarely studied. Here, we show that XIAP modulates radiation sensitivity by regulating stability of Chk1 in lung cancer cells. Both Chk1 and XIAP are highly expressed in various lung cancer cells. Overexpression of XIAP increased cell survival following genotoxic treatments by preventing downregulation of Chk1. However, XIAP reversed Chk1-protective activity in the presence of XIAP-associated factor 1 (XAF1) by degrading Chk1 via ubiquitination-dependent proteasomal proteolysis. The XIAP-XAF1 complex-mediated Chk1 degradation also required CUL4A and DDB1. Chk1 or XIAP was associated with DDB1 and CUL4A. Depletion of CUL4A or DDB1 prevented the XIAP-XAF1-mediated Chk1 degradation suggesting involvement of a CUL4A/DDB1-based E3 ubiquitin ligase in the process or its collaboration with XIAP E3 ligase activity. Taken together, our findings show that XIAP plays a dual role in modulation of Chk1 stability and cell viability following IR. In the absence of XAF1, XIAP stabilizes Chk1 under IR with corresponding increase of cell viability. By contrast, when XAF1 is overexpressed, XIAP facilitates Chk1 degradation, which leads to enhancement of radiation sensitivity. This selective regulation of Chk1 stability by XIAP and XAF1 could be harnessed to devise a strategy to modulate radiation sensitivity in lung cancer cells.     

Augmented cancer resistance and DNA damage response phenotypes in PPM1D null mice

The p53-induced serine/threonine phosphatase, protein phosphatase 1D magnesium-dependent, delta isoform (PPM1D) (or wild-type p53-induced phosphatase 1 (Wip1)), exhibits oncogenic activity in vitro and in vivo. It behaves as an oncogene in rodent fibroblast transformation assays and is amplified and overexpressed in several human tumor types. It may contribute to oncogenesis through functional inactivation of p53. Here, we show that the oncogenic function of PPM1D is associated with its phosphatase activity. While overexpressed PPM1D may be oncogenic, PPM1D null mice are resistant to spontaneous tumors over their entire lifespan. This cancer resistance may be based in part on an augmented stress response following DNA damage. PPM1D null mice treated with ionizing radiation display increased p53 protein levels and increased phosphorylation of p38 MAP kinase, p53, checkpoint kinase 1 (Chk1), and checkpoint kinase 2 (Chk2) in their tissues compared to their wild-type (WT) counterparts. Male PPM1D null mice show a modest reduction in longevity, reduced serum insulin-like growth factor 1 (IGF-1) levels, and reduced body weight compared to WT mice. The PPM1D null mouse phenotypes indicate that PPM1D has a homeostatic role in abrogating the DNA damage response and may regulate aspects of male longevity.     

Chk1/2和Plk1蛋白在宫颈良恶性病变组织中的表达及其意义

背景与目的：Chk1/2（checkpoint kinase 1/2）和Plk1（polo-like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶.本研究主要探讨3种激酶蛋白在宫颈良恶性病变中的表达差异、与宫颈癌临床病理特征的关系及3种激酶在宫颈癌组织中表达之间的相互关系.方法：应用免疫组化SP法检测43例宫颈癌组织和20例慢性宫颈炎性组织中Chk1、Chk2和Plk1蛋白的表达情况.结果：Chk1、Chk2和Plk1蛋白在宫颈癌患者中的阳性率分别为76.7%、60.5%和32.6%,在慢性宫颈炎患者中的阳性率分别为30.0%、35.0%和0;Chkl和Plk1蛋白在宫颈癌组织中的表达显著高于慢性宫颈炎组织（P＜0.01）,而Chk2的表达差异无显著性（P＞0.05）.Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的宫颈癌患者中差异无显著性（P＞0.05）;但Chk1和Plkl的表达在不同分化程度的宫颈癌患者中差异有显著性（P＜0.05）.Spearman等级相关分析,在43例宫颈癌患者中,Chk1与Chk2的表达呈正相关（r=0.492,P=0.001）.结论：Chk1和Plk1可能成为比较理想的宫颈癌治疗靶点.     

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。     

Stim1在骨肉瘤组织和细胞中的表达水平及与顺铂耐药性的关系

目的:探讨Stim1在骨肉瘤组织和细胞中的表达水平及与顺铂耐药性的关系.方法:采用免疫组织化学染色检测了50例骨肉瘤患者的肿瘤组织中Stim1的表达.通过用高浓度的顺铂处理人骨肉瘤细胞系U-2 OS来建立耐药性骨肉瘤细胞(U-2 OS/DDP细胞).通过RT-PCR和蛋白质印迹检测细胞中Stim1、ATF4、CHOP和...     

Immunostaining of thymidylate synthase and p53 for predicting chemoresistance to S-1/cisplatin in gastric cancer

High expression of thymidylate synthase (TS) and inactivation of p53 are allegedly associated with chemoresistance. The authors evaluated TS and p53 expression in gastric cancer treated with neoadjuvant S-1/cisplatin chemotherapy. Paraffin sections of pretreatment biopsy and surgical specimens from 41 gastric cancers were immunostained for TS and p53 protein after appropriate antigen retrieval. Fifty-one cases without neoadjuvant chemotherapy were also studied. In the pretreatment biopsies, high expression of TS was seen in 8% of the histologic responders, in 28% of the nonresponders and in 31% of the controls. High expression of p53 was observed in 56% of the nonresponders, but in 8% of the responders and in 29% of the controls (P<0.01 and P<0.05, respectively). The TS- and/or p53-high phenotype was seen in 76% of the nonresponders and in 54% of the controls, but in 8% of the responders (P<0.0001 and P<0.005, respectively). The data of the surgical specimens were consistent with those of the pretreatment biopsies. These results suggest that immunostaining for TS and p53 protein is useful for pretreatment selection of gastric cancer patients unresponsive to S-1/cisplatin chemotherapy.     

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.     

p53反义RNA对人肺癌细胞表型和顺铂敏感性的影响

目的 研究外源p53反义RNA对有p53基因248密码点突变的人肺癌细胞系恶性表型和顺铂敏感性的影响。方法 构建p53反义RNA真核细胞表达载体PEGFP－p53（AS),经酶切图证明反向联结质粒。Lipofectin介导转染有p53基因248密码点突变的人肺癌细胞系801D，经G418筛选获耐受克隆。稀释法建立单细胞克隆系，应用PCR检测外源基因，荧光显微镜检测细胞绿色荧光蛋白表达，p53单抗免疫组化染色检测p53突变蛋白表达，体外集落形成实验检测细胞生长，流式细胞仪检测细胞周期，MTT法检测细胞对顺铂药物敏感性。结果 酶切图证明了p53-cDNA反向联结于质粒，构建了PEGFP－p53(AS),并建立了转染单细胞克隆系PEGFP－p53(AS)-801D及空载细胞系PEGFP－801D。PCR检测外源p53基因和neo基因存在于转染细胞系，而荧光显微镜下发现其胞浆有绿荧光蛋白表达。免疫组化染色p53突变蛋白在801D细胞系为阳性，而PEGFP－p53(AS)-801D为阴性，证明外源反义p53在转染细胞稳定表达并封闭内源突变蛋白表达。与母系相比，PEGFP－p53(AS)-801D集落形成抑制率为61％（P＜0．01），流式细胞检测该细胞系G1期细胞数明显增中，出现G1期阻滞的表现。MTT检测PEGFP－p53(AS）－801D对顺铂比母系更为敏感。结论 有p53基因248密码点突变的801D细胞恶性增殖明显，对顺铂耐药，外源p53反义RNA可封闭突变蛋白表达，抑制细胞体外恶性增殖，增加对顺铂的药物敏感性。转染p53反义RNA可抑制p53突变的恶性表型或恢复野生型p53基因功能。     

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.     

Antitumor effect of WEE1 blockade as monotherapy or in combination with cisplatin in urothelial cancer

Overcoming cisplatin (CDDP) resistance is a major issue in urothelial cancer (UC), in which CDDP-based chemotherapy is the first-line treatment. WEE1, a G₂/M checkpoint kinase, confers chemoresistance in response to genotoxic agents. However, the efficacy of WEE1 blockade in UC has not been reported. MK-1775, a WEE1 inhibitor also known as AZD-1775, blocked proliferation of UC cell lines in a dose-dependent manner irrespective of TP53 status. MK-1775 synergized with CDDP to block proliferation, inducing apoptosis and mitotic catastrophe in TP53-mutant UC cells but not in TP53-WT cells. Knocking down TP53 in TP53-WT cells induced synergism of MK-1775 and CDDP. In UMUC3 cell xenografts and two patient-derived xenograft lines with MDM2 overexpression, in which the p53/cell cycle pathway was inactivated, AZD-1775 combined with CDDP suppressed tumor growth inducing both M-phase entry and apoptosis, whereas AZD-1775 alone was as effective as the combination in RT4 cell xenografts. Drug susceptibility assay using an ex vivo cancer tissue-originated spheroid system showed correlations with the in vivo efficacy of AZD-1775 alone or combined with CDDP. We determined the feasibility of the drug susceptibility assay using spheroids established from UC surgical specimens obtained by transurethral resection. In conclusion, WEE1 is a promising therapeutic target in the treatment of UC, and a highly specific small molecule inhibitor is currently in early phase clinical trials for cancer. Differential antitumor efficacy of WEE1 blockade alone or combined with CDDP could exist according to p53/cell cycle pathway activity, which might be predictable using an ex vivo 3D primary culture system.     

Combined inhibition of Chk1 and Wee1 as a new therapeutic strategy for mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive, incurable disease, characterized by a deregulated cell cycle. Chk1 and Wee1 are main regulators of cell cycle progression and recent data on solid tumors suggest that simultaneous inhibition of these proteins has a strong synergistic cytotoxic effect. The effects of a Chk1 inhibitor (PF-00477736) and a Wee1 inhibitor (MK-1775) have been herein investigated in a large panel of mature B-cell lymphoma cell lines. We found that MCL cells were the most sensitive to the Chk1 inhibitor PF-00477736 and Wee1 inhibitor MK-1775 as single agents. Possible involvement of the translocation t(11;14) in Chk1 inhibitor sensitivity was hypothesized. The combined inhibition of Chk1 and Wee1 was strongly synergistic in MCL cells, leading to deregulation of the cell cycle, with increased activity of CDK2 and CDK1, and activation of apoptosis. In vivo treatment with the drug combination of mice bearing JeKo-1 xenografts (MCL) had a marked antitumor effect with tumor regressions observed at non-toxic doses (best T/C%=0.54%). Gene expression profiling suggested effect on genes involved in apoptosis. The strong synergism observed by combining Chk1 and Wee1 inhibitors in preclinical models of MCL provides the rationale for testing this combination in the clinical setting.