Cytokinetic effects of Wee1 disruption in pancreatic cancer

Abstract

The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker gH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genomescale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

<p>miR-526b-3p serves as a prognostic factor and regulates the proliferation, invasion, and migration of glioma through targeting WEE1</p>

“…WEE1, a member of the tyrosine kinase family, is a G2/M checkpoint regulatory protein involved in the regulation of cell cycle progression 27. Massive studies reported that WEE1 expression was upregulated in various tumors, and its oncogenic roles were also widely reported, which suggested that WEE1 may be a good therapeutic candidate target in cancer treatment 28–30. In glioma, several studies reported that WEE1 was overexpressed in glioma and promoted glioma cells proliferation, migration, and invasion 31,32.…”

Wee1 Inhibitor AZD1775 Combined with Cisplatin Potentiates Anticancer Activity against Gastric Cancer by Increasing DNA Damage and Cell Apoptosis

“…Likewise, AZD1775 inactivated G 2 /M checkpoint to abrogate G 2 /M arrest induced by cisplatin (Figures 2(c), 2(d), and S2 ) [ 28 ], indicating DNA damage enhancement of AZD1775-cisplatin combination partially due to G 2 /M checkpoint abrogation in our work. Beyond replication stress initiated by G 2 /M checkpoint abrogation, AZD1775-induced DNA damage has also been attributed to AZD1775's effect on dephosphorylation and activation of CDK2 during S phase which regulates overall timing of DNA replication [ 11 , 30 ]. AZD1775 can cause deficiency in homologous recombination repair [ 31 ], which serves as another approach to AZD1775-induced DNA damage.…”

“…Nevertheless, disputable opinions exist demonstrating that p53 status is indispensable for AZD1775's anticancer activity [ 10 , 19 , 24 ]. DNA damage rather than premature mitosis (a typical phenotype of G 2 /M checkpoint defects) has recently been proven to be the primary cytotoxic consequence of AZD1775 in some cases [ 11 , 30 ]. As mentioned above, apart from through a p53-reliant G 2 /M checkpoint defect, AZD1775 can also generate DNA damage-associated cytotoxicity through a p53-independent manner, such as homologous recombination defect and DNA replication disruption by inactivating CDK2 [ 11 , 30 , 31 ].…”

“…DNA damage rather than premature mitosis (a typical phenotype of G 2 /M checkpoint defects) has recently been proven to be the primary cytotoxic consequence of AZD1775 in some cases [ 11 , 30 ]. As mentioned above, apart from through a p53-reliant G 2 /M checkpoint defect, AZD1775 can also generate DNA damage-associated cytotoxicity through a p53-independent manner, such as homologous recombination defect and DNA replication disruption by inactivating CDK2 [ 11 , 30 , 31 ]. Consistent with these studies, GC cells with p53 mutation and wild type have a similar response to Wee1 blockade [ 8 ] and our findings also reveal that the response to AZD1775 and enhanced efficacy of AZD1775 plus cisplatin were independent of p53 status ( Figure 1 ).…”

Profiling of tRNA Halves and YRNA Fragments in Serum and Tissue From Oral Squamous Cell Carcinoma Patients Identify Key Role of 5′ tRNA-Val-CAC-2-1 Half

“…Supporting our findings, Green module-overtargeted FBXO3 has been indeed reported to be downregulated in multiple cancers and to suppress cell proliferation partly through the degradation of cyclin D1 (65). On the other hand, the nuclear protein Wee1 (the product of the Green module-overtargeted WEE1 gene) was recently recognized as a tumor suppressor that regulates the progression of pancreatic cancer cells via delaying G2, which allows for longer time to repair post-replication errors in genomically unstable cancers (66). Similarly, the RB1 gene is mutated in a variety of human cancers and represents a classical prototype of tumor suppressor, inhibiting cell proliferation primarily through targeting of the E2F transcription factor and, additionally, through transcription-independent mechanisms (67).…”