Mitotic Catastrophe Research Articles

Administering Docetaxel for Metastatic Hormone-Sensitive Prostate Cancer 1-6 Days Compared to More Than 14 Days after the Start of LHRH Agonist Is Associated with Better Clinical Outcomes Due to Androgen Flare.

Simple SummaryHormonal therapy with long-acting luteinizing hormone-releasing hormone (LHRH) for metastatic hormone-sensitive prostate cancer (MHSPC) results initially in testosterone flare followed by testosterone deprivation. Docetaxel is a chemotherapy that is effective against prostate cancer and specifically targets cells during cell division by stabilizing the mitotic spindle, which results in “mitotic catastrophe” and death of the dividing cancer cells. Combining LHRH and docetaxel was proved to be superior to LHRH treatment alone. Here, we show that simply by providing the first dose of docetaxel during testosterone flare, which occurs 1–6 days after LHRH initiation, patients could have better clinical outcomes, as testosterone drives specifically prostate cells into mitosis, priming them to cell kill by docetaxel.Docetaxel, when given at the beginning of androgen deprivation therapy (ADT) for patients with metastatic hormone-sensitive prostate cancer (MHSPC), results in significantly longer overall survival than ADT alone. We aimed to investigate if the delivery of the first dose of docetaxel during the testosterone flare associated with LHRH initiation results in better clinical outcomes, as testosterone induces mitosis of prostate cancer cells, and docetaxel specifically targets cells in mitosis. We analyzed data from the CHAARTED trial which randomized MHSPC patients to ADT alone or ADT plus docetaxel. We included only patients treated with LHRH agonist and docetaxel (n = 379). The only cutoff that resulted in differences in treatment outcomes was between patients who started docetaxel 1–6 days (n = 18) compared to more than 14 days from LHRH initiation (n = 297). Actuarial median overall survival was 72 versus 57 months (p = 0.2); progression-free survival was 49 versus 17 months (p = 0.06), and freedom from castrate-resistant prostate cancer was 51 versus 18 months (p = 0.04) for patients who started docetaxel 1–6 days compared to more than 14 days from LHRH initiation, respectively. Administering docetaxel 1–6 days from the initiation of LHRH agonist for patients with MHSPC could be associated with improved clinical outcomes.

IMB5476, a novel microtubule inhibitor, induces mitotic catastrophe and overcomes multidrug resistance in tumors

IMB5046 is a nitrobenzoate microtubule inhibitor we reported previously. During screening of its structural analogues, we identified a novel compound IMB5476 with increased aqueous solubility. Here, its antitumor activity and the underlying mechanism were investigated. IMB5476 disrupted microtubule networks in cells and arrested cell cycle at G2/M phase. It inhibited purified tubulin polymerization in vitro. Competition assay indicated that it bound to tubulin at the colchicine pocket. Further experiments proved that it induced cell death by mitotic catastrophe and apoptosis. Notably, it was a poor substrate of P-glycoprotein and exhibited potent cytotoxicity against drug-resistant tumor cells. In addition, IMB5476 could inhibit angiogenesis in vitro. IMB5476 also inhibited the growth of drug-resistant KBV200 xenografts in mice. Conclusively, our data reveal a novel nitrobenzoate microtubule inhibitor with improved aqueous solubility and can overcome multidrug resistance.

Exclusive modifications of NuMA in malignant epithelial cells: A potential therapeutic mechanism

NuMA (nuclear mitotic apparatus) protein is indispensable in the mitosis of human proliferating cells, both malignant and benign. The progression of mitosis requires stable spindles, which depend on the bipolar clustering of NuMA within the spindles. The phenanthridine PJ34 kills malignant epithelial cells during mitosis and targets NuMA. PJ34 treated healthy cells are not impaired. PJ34 exclusively blocks the post-translational modification of NuMA in a variety of malignant epithelial cells, but not in benign cells. This blockage of the post-translational modification of NuMA affects its protein-binding capacity, causing construction faults in the mitotic spindle poles of PJ34-treated cancer cells, leading to Mitotic Catastrophe cell death. PJ34 is a potent PARP1 inhibitor. Therefore its PARP independent exclusive cytotoxicity in human malignant cells, challenges the currently accepted notion that inhibition of PARP1 halts cancer by preventing DNA repair. Certain molecules that act as PARP1 inhibitors kill cancer cells by targeting other proteins and vital mechanisms.

Piperine Increases Pentagamavunon-1 Anti-cancer Activity on 4T1 Breast Cancer Through Mitotic Catastrophe Mechanism and Senescence with Sharing Targeting on Mitotic Regulatory Proteins.

Pentagamavunon-1 performs more potent anti-cancer effects than curcumin against various cancer cells, but it remains to be optimized. Piperine shows the activity as an enhancer of a therapeutic agent. This study expects to achieve higher effectiveness of PGV-1 on 4T1 breast cancer cells through co-treatment with piperine with exploring the effect of cytotoxicity, mitotic catastrophe, cellular senescence, and target proteins of PGV-1 and piperine on the regulation of mitosis in TNBC cells (4T1). The assays emphasize MTT assay, May Grünwald-Giemsa staining, SA-β-galactosidase assay, and bioinformatics analysis, respectively, to elicit the respected activities. The results revealed that PGV-1 performed a cytotoxic effect with an IC50 value of 9 µM while piperine showed a lower cytotoxic effect with an IC50 value of 800 µM on 4T1 cells 24 h treatment. However, the combination treatment of both showed a synergistic cytotoxic enhancement effect with an average CI value < 1. Furthermore, the combination of PGV-1 and piperine induced mitotic catastrophe and senescence better than the single treatment. Treatment of 1 µM of PGV-1 and 400 µM of piperine increased the percentage of senescent cells by 33%. Bioinformatics analysis revealed that PGV-1 and piperine target proteins play a role in mitotic regulation, namely CDK1, KIF11, AURKA, AURKB, and PLK1, to contribute to mitotic catastrophe. Therefore, piperine increases the effectiveness of PGV-1 to suppress 4T1 cells growth synergistically that may occur through mitotic catastrophe and senescence targeting on mitotic regulatory proteins.

Autophagy and cancer treatment: four functional forms of autophagy and their therapeutic applications

Cancer is the leading cause of death worldwide. Drugs play a pivotal role in cancer treatment, but the complex biological processes of cancer cells seriously limit the efficacy of various anticancer drugs. Autophagy, a self-degradative system that maintains cellular homeostasis, universally operates under normal and stress conditions in cancer cells. The roles of autophagy in cancer treatment are still controversial because both stimulation and inhibition of autophagy have been reported to enhance the effects of anticancer drugs. Thus, the important question arises as to whether we should try to strengthen or suppress autophagy during cancer therapy. Currently, autophagy can be divided into four main forms according to its different functions during cancer treatment: cytoprotective (cell survival), cytotoxic (cell death), cytostatic (growth arrest), and nonprotective (no contribution to cell death or survival). In addition, various cell death modes, such as apoptosis, necrosis, ferroptosis, senescence, and mitotic catastrophe, all contribute to the anticancer effects of drugs. The interaction between autophagy and these cell death modes is complex and can lead to anticancer drugs having different or even completely opposite effects on treatment. Therefore, it is important to understand the underlying contexts in which autophagy inhibition or activation will be beneficial or detrimental. That is, appropriate therapeutic strategies should be adopted in light of the different functions of autophagy. This review provides an overview of recent insights into the evolving relationship between autophagy and cancer treatment.

Characterization of radiation-induced micronuclei associated with premature senescence, and their selective removal by senolytic drug, ABT-263

Radiotherapy is well-recognized as an efficient non-invasive remedy for cancer treatment. Since 10 Gy, a weekly total dose for conventional radiotherapy, was proven to create unreparable and residual DNA double-strand breaks (DSBs), they were found to give rise to mitotic failure, such as mitotic catastrophe, which resulted in multiple micronuclei associated with premature senescence. We demonstrated that pulverization of micronuclear DNA was caspase-dependent and triggered not ATM-dependent but DNA-PK-dependent DNA damage response, including phosphorylation of histone H2AX. Pulverization of micronuclear DNA and senescence-associated secretory phenotype (SASP) worsen tumor microenvironment after radiotherapy, so that senolytic drug was applied to eliminate senescent cancer cells. Prematurely senescent cancer cells with micronuclei caused by 10 Gy of γ-irradiation were subjected to 5 μM of ABT-263, a Bcl-2 family inhibitor, and selective cancer cell death by apoptosis was observed, while ABT-263 had little effect on growing cancer cells. Western blot analysis showed augmented expression of both apoptotic and anti-apoptotic proteins in senescent cells, indicating that increased apoptotic factors are essential for selective apoptotic cell death in combination with ABT-263. Our results suggested that selective elimination of senescent cells alleviates SASP and micronuclei-mediated the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) activation, both of which lead to unfavorable adverse effects caused by radiotherapy.

Abstract PO-131: RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer

Abstract Breast cancer (BC) is the second most diagnosed malignant disease in women, and one of the leading causes of cancer-related deaths. Triple-negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype of BC because it is highly metastatic and lacks targeted therapies. African American (AA) women have a higher death rate from BC than women of other races and ethnicities. The higher incidences of TNBCs and their aggressive growth in young AA women contributing to higher death rates indicate a biological basis for this difference. Thus, it is imperative to understand the molecular mechanisms that contribute to aggressive tumor growth in AA women, identify biomarkers to select patients who will respond to existing therapies, and develop effective therapeutics to reduce this disparity. Our studies identified that multiple TNBC cells derived from AA women are inherently chemoresistant and exhibit aggressive growth behavior compared to TNBC cells derived from European American (EA) patients. Our preliminary screenings showed that the DNA repair protein, RAD51, is overexpressed in AA TNBC patients and correlates a poor prognosis relative to EA TNBC patients. Analysis of AA and EA TNBC tumor specimens indicated the epigenetic regulation of RAD51 by promoter methylations and microRNAs. Furthermore, AA women diagnosed with TNBC, have a considerably lower incidence of germline BRCA1 mutations than women of other racial or ethnic groups. This indicates most TNBC tumors in AA patients are DNA repair proficient and have intact cell cycle checkpoint mechanisms that protect them from chemotherapy-induced DNA damage and promote therapeutic resistance. Our drug screenings identified CHK1 inhibitor, Prexasertib caused DNA repair deficiency in BRCA wild-type TNBC cells by promoting proteasome-mediated degradation of BRCA1 and RAD51 proteins. Therefore, we designed a synthetic lethality-based drug combination of Prexasertib with PARP inhibitors (PARPi) in DNA repair proficient TNBC cells. Data from our preclinical evaluations show Prexasertib and Olaparib cause increased DNA strand breaks, mitotic catastrophe, and synergistic TNBC cell lethality compared to individual drug treatments. Additionally, computational analysis of TCGA data revealed a RAD51 upregulation in TNBC tumors compared to normal breast tissues and other subtypes of BC which renders as a poor prognostic marker for these patients. Remarkably, there was an interesting discrepancy in RAD51 expression levels between different racial groupings, with AA and Asian BC patients having higher levels of RAD51 expression than Caucasian BC patients. Consistent with these observations, AA and Asian TNBC patients showed decreased survival probability. Together, our data indicate that RAD51 and its epigenetic regulators could be biomarkers for aggressive TNBC and racial disparity in BC therapeutic outcomes and suggests a novel combination therapy involving Prexasertib and Olaparib may improve prognosis and reduce racial disparity in TNBC. Citation Format: Ganesh N. Acharya, Chinnadurai Mani, Upender Manne, Komaraiah Palle. RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-131.

The histone deacetylase inhibitor OBP-801 has invitro/invivo anti-neuroblastoma activity.

Patients with high-risk neuroblastoma have a poor prognosis; new therapeutic agents are therefore required. We investigated the antitumor effects of OBP-801, a novel histone deacetylase inhibitor, its underlying mechanism, and its potential as a therapeutic agent for patients with neuroblastoma. The study included five human neuroblastoma cell lines: IMR32, GOTO, KP-N-RTBM, SK-N-AS, and SH-SY5Y. We investigated cell proliferation, cell cycle status, protein expression patterns, and apoptosis in neuroblastoma cells after OBP-801 treatment invitro. Cell survival rate and cell cycle were analyzed using the WST-8 assay and flow cytometry, respectively. Apoptosis was detected using annexin V staining, and the expression of apoptosis-related proteins was investigated by western blotting. The antitumor activity of OBP-801 was examined in an invivo xenograft mouse model. Dose-effect curve analysis showed that the mean half-maximal inhibitory concentration value was 5.5 ± 5.9 nM for the MYCN-amplified cell lines (IMR32, GOTO, and KP-N-RTBM) and 3.1 ± 0.7 nM for the MYCN-nonamplified cell lines (SK-N-AS and SH-SY5Y). OBP-801 inhibited cell proliferation and growth in all the cell lines. It induced G2/M phase arrest through the p21 (CDKN1A) pathway, increasing histone H3 levels and, subsequently, apoptosis in human neuroblastoma cells. OBP-801 suppressed the growth of neuroblastoma cells in the mouse xenograft model. Overall, OBP-801 induces M-phase arrest and apoptosis in neuroblastoma cells via mitotic catastrophe. Our results indicate that OBP-801 is a promising therapeutic agent with fewer adverse effects for patients with neuroblastoma.

The deacetylation of Foxk2 by Sirt1 reduces chemosensitivity to cisplatin.

In multiple types of cancer, decreased tumour cell apoptosis during chemotherapy is indicative of decreased chemosensitivity. Forkhead box K2 (FOXK2), which is essential for cell fate, regulates cancer cell apoptosis through several post‐translational modifications. However, FOXK2 acetylation has not been extensively studied. Here, we evaluated the effects of sirtiun 1 (SIRT1) on FOXK2 deacetylation. Our findings demonstrated that SIRT1 inhibition increased FOXK2‐induced chemosensitivity to cisplatin and that K223 in FOXK2 was acetylated. Furthermore, FOXK2 K223 deacetylation reduced chemosensitivity to cisplatin in vitro and in vivo. Mechanistically, FOXK2 was acetylated by the acetyltransferase cAMP response element binding protein and deacetylated by SIRT1. Furthermore, cisplatin attenuated the interaction between FOXK2 and SIRT1. Cisplatin or SIRT1 inhibition enhanced FOXK2 acetylation, thereby reducing the nuclear distribution of FOXK2. Additionally, FOXK2 K223 acetylation significantly affected the expression of cell cycle–related and apoptosis‐related genes in cisplatin‐stimulated cancer cells, and FOXK2 K223 hyperacetylation promoted mitotic catastrophe, which enhanced chemosensitivity to cisplatin. Overall, our results provided insights into the mechanisms of SIRT1‐mediated FOXK2 deacetylation, which was involved in chemosensitivity to cisplatin.

The effect of paclitaxel on apoptosis, autophagy and mitotic catastrophe in AGS cells

Paclitaxel is an anti-microtubule agent that has been shown to induce cell death in gastric cancer. However, the detailed mechanism of action is unclear. In this study, we reveal that the paclitaxel-induced cell death mechanism involves mitotic catastrophe, autophagy and apoptosis in AGS cells. Paclitaxel induced intrinsic apoptosis by activating caspase-3, caspase-9 and PARP. In addition, the significant increase in autophagy marker LC3B-II, together with Atg5, class III PI3K and Beclin-1, and the down-regulation of p62 following paclitaxel treatment verified that paclitaxel induced autophagy. Further experiments showed that paclitaxel caused mitotic catastrophe, cell cycle arrest of the accumulated multinucleated giant cells at the G2/M phase and induction of cell death in 24 h. Within 48 h, the arrested multinucleated cells escaped mitosis by decreasing cell division regulatory proteins and triggered cell death. Cells treated with paclitaxel for 48 h were grown in fresh medium for 24 h and checked for CDC2, CDC25C and lamin B1 protein expressions. These proteins had decreased significantly, indicating that the remaining cells became senescent. In conclusion, it is suggested that paclitaxel-induced mitotic catastrophe is an integral part of the cell death mechanism, in addition to apoptosis and autophagy, in AGS cells.

Abstract P078: Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer

Abstract Oncogenic KRAS mutation is common in non-small cell lung cancer (NSCLC) and portends poor outcome. Direct KRASG12C inhibitors have clinical activity; however, intrinsic and acquired resistance to these drugs limits their utility. Aurora A Kinase (AURKA), a mitotic cell cycle regulator, has been considered as a key druggable KRAS effector and mediates adaptive resistance to direct KRASG12C inhibitors. We found that AURKA expression correlated with poor outcome of lung cancer patients in caBIG, GEO and TCGA, and its expression was greater in KRAS-mutated NSCLC cells resistant to KRASG12C inhibitor sotorasib (AMG510) compared to normal human tracheobronchial epithelial (NHTBE), lung fibroblast, and KRAS wild-type cells. We have previously demonstrated synergistic antitumor effects for combination AURKA and WEE1 inhibition. Here, we explored a novel combination of AURKA and KRASG12C inhibition in KRAS(G12C)-mutated NSCLC cells intrinsically resistant to the KRASG12C inhibitor, along with a combination of AURKA and WEE1 inhibition in mutant KRAS(G12C) lung cancer cells with acquired resistance to the inhibitor. To overcome resistance to the KRASG12C inhibitor sotorasib, we tested combination treatment with VIC-1911, a newly synthesized selective AURKA inhibitor, with sotorasib in sotorasib-resistant human lung cancer cells. Cooperative screening, Loewe plotting, and clonogenic survival assays were performed to determine synergistic antitumor effects. In addition, we established KRAS(G12C)-mutated human lung cancer cell lines with acquired resistance to sotorasib through the means of escalated incremental dosing or sorting non-quiescent cells after drug exposure. Synergy was further confirmed with cell cycle distribution, phenotypic mitotic catastrophe on confocal microscopy, and induction of apoptosis. Interestingly, cells intrinsically resistant to sotorasib showed profound synergistic suppressive effect on cancer cell survival with addition of VIC-1911 compared to sotorasib-sensitive cells (Loewe synergy scores: NCI-H1792=16.03; HCC44=14.37; NCI-H358=1.48). Further, the combination of AURKA and WEE1 inhibition synergistically induced greater cell death in NCI-H358 lung carcinoma cells harboring acquired resistance to sotorasib, with dramatic induction of Bim. Moreover, the combination of AURKA and WEE1 inhibition resulted in a synergistic tumor control in KRAS/TP53-mutated lung cancer xenograft models. Our findings suggest AURKA activation leads to both intrinsic and acquired resistance to sotorasib in KRAS(G12C)-mutated NSCLC; therefore, addition of AURKA inhibition to sotorasib may be a promising therapeutic approach in NSCLC with intrinsic resistance to direct KRASG12C inhibitors, while the combination of AURKA and WEE1 inhibition merits exploration in acquired resistance to these agents. Citation Format: Jong Woo Lee, Sundong Kim, Cindy Yang, Barbara Burtness. Aurora A kinase inhibition with VIC-1911 overcomes intrinsic and acquired resistance to KRASG12C inhibition in KRAS(G12C)-mutated lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P078.

STMN1 is highly expressed and contributes to clonogenicity in acute promyelocytic leukemia cells.

Stathmin 1 (STMN1) is a microtubule-destabilizing protein highly expressed in hematological malignancies and involved in proliferation and differentiation. Although a previous study found that the PML-RARα fusion protein, which contributes to the pathophysiology of acute promyelocytic leukemia (APL), positively regulates STMN1 at the transcription and protein activity levels, little is known about the role of STMN1 in APL. In this study, we aimed to investigate the STMN1 expression levels and their associations with laboratory, clinical, and genomic data in APL patients. We also assessed the dynamics of STMN1 expression during myeloid cell differentiation and cell cycle progression, and the cellular effects of STMN1 silencing and pharmacological effects of microtubule-stabilizing drugs on APL cells. We found that STMN1 transcripts were significantly increased in samples from APL patients compared with those of healthy donors (all p < 0.05). However, this had no effect on clinical outcomes. STMN1 expression was associated with proliferation- and metabolism-related gene signatures in APL. Our data confirmed that STMN1 was highly expressed in early hematopoietic progenitors and reduced during cell differentiation, including the ATRA-induced granulocytic differentiation model. STMN1 phosphorylation was predominant in a pool of mitosis-enriched APL cells. In NB4 and NB4-R2 cells, STMN1 knockdown decreased autonomous cell growth (all p < 0.05) but did not impact ATRA-induced apoptosis and differentiation. Finally, treatment with paclitaxel (as a single agent or combined with ATRA) induced microtubule stabilization, resulting in mitotic catastrophe with repercussions for cell viability, even in ATRA-resistant APL cells. This study provides new insights into the STMN1 functions and microtubule dynamics in APL.

Survivin suppression heightens BZML-induced mitotic catastrophe to overcome multidrug resistance by removing therapy-induced senescent A549/Taxol cells

Mitotic catastrophe (MC) is a newly identified type of anticancer mechanism for multidrug resistance (MDR) prevention. However, the long cellular death process resulting from MC is not beneficial for anticancer treatment. BZML is a novel colchicine-binding site inhibitor which can overcome MDR by inducing MC; however, BZML-induced MC cells underwent a long cellular death process. Thus, to improve anticancer therapies based on drug-induced MC, BZML-induced MC was served as a model to further study the underlying molecular mechanisms in the process of MC. Here, BZML could induce p53-dependent senescence in A549/Taxol cells, a MDR cell line. This senescence was a secondary effect of MC in overcoming MDR. During MC, BZML-induced destruction of protein-degradation system contributed not only to an increase of p53 protein but also to the accumulation of survivin in nucleus of A549/Taxol cells. Importantly, the nuclear accumulation of survivin was not the inducer but the result of BZML-induced MC, and it promoted the survival of senescent cells. Moreover, it provided additional vulnerability and critical opportunities for sequentially applied therapies. Further, targeting survivin with YM155 accelerated the death of MC cells by timely eliminating therapy-induced senescent cells and strengthening the efficiency of BZML in overcoming MDR in A549/Taxol cells. Collectively, nuclear accumulation of survivin delayed cellular death during MC by promoting the survival of BZML-induced senescent A549/Taxol cells. Moreover, “one-two punch” approach to cancer treatment based on combination therapy with YM155 for survivin suppression might be a new strategy for potentiating MC to overcome MDR.

Dynamically Accumulating Homologous Recombination Deficiency Score Served as an Important Prognosis Factor in High-Grade Serous Ovarian Cancer.

Background: The homologous recombination (HR) pathway defects in cancers induced abrogation of cell cycle checkpoints, resulting in the accumulation of DNA damage, mitotic catastrophe, and cell death. Cancers with BRCA1/2 loss and other accumulation of similar genomic scars resulting in HRD displayed increased sensitivity to chemotherapy. Our study aimed to explore HRD score genetic mechanisms and subsequent clinical outcomes in human cancers, especially ovarian cancer. Methods: We analyzed TCGA data of HRD score in 33 cancer types and evaluated HRD score distribution and difference among tumor stages and between primary and recurrent tumor tissues. A weighted gene co-expression network analysis (WGCNA) was performed to identify highly correlated genes representing essential modules contributing to the HRD score and distinguish the hub genes and significant pathways. We verified HRD status predicting roles in patients’ overall survival (OS) with univariate and multivariate Cox regression analyses and built the predicting model for patient survival. Results: We found that the HRD score increased with the rise in tumor stage, except for stage IV. The HRD score tended to grow up higher in recurrent tumor tissue than in their primary counterparts (p = 0.083). We constructed 15 co-expression modules with WGCNA, identified co-expressed genes and pathways impacting the HRD score, and concluded that the HRD score was tightly associated with tumor cells replication and proliferation. A combined HRD score ≥42 was associated with shorter OS in 33 cancer types (HR = 1.010, 95% CI: 1.008–1.011, p < 0.001). However, in ovarian cancer, which ranked the highest HRD score among other cancers, HRD ≥42 cohort was significantly associated with longer OS (HR = 0.99, 95% CI: 0.98–0.99, p < 0.0001). We also built a predicting model for 3 and 5 years survival in HGSC patients. Conclusion: A quantitative HRD score representing the accumulated genomic scars was dynamically increasing in proliferating tumor cells since the HRD score was tightly correlated to tumor cell division and replication. We highlighted HRD score biomarker role in prognosis prediction of ovarian cancer.

Synthesis and Antiproliferative Evaluation of 3-Chloroazetidin-2-ones with Antimitotic Activity: Heterocyclic Bridged Analogues of Combretastatin A-4.

Antimitotic drugs that target tubulin are among the most widely used chemotherapeutic agents; however, the development of multidrug resistance has limited their clinical activity. We report the synthesis and biological properties of a series of novel 3-chloro-β-lactams and 3,3-dichloro-β-lactams (2-azetidinones) that are structurally related to the tubulin polymerisation inhibitor and vascular targeting agent, Combretastatin A-4. These compounds were evaluated as potential tubulin polymerisation inhibitors and for their antiproliferative effects in breast cancer cells. A number of the compounds showed potent activity in MCF-7 breast cancer cells, e.g., compound 10n (3-chloro-4-(3-hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) and compound 11n (3,3-dichloro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one), with IC50 values of 17 and 31 nM, respectively, and displayed comparable cellular effects to those of Combretastatin A-4. Compound 10n demonstrated minimal cytotoxicity against non-tumorigenic HEK-293T cells and inhibited the in vitro polymerisation of tubulin with significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that β-lactam 10n caused a mitotic catastrophe by targeting tubulin. In addition, compound 10n promoted apoptosis by regulating the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2 and Mcl-1. Molecular docking was used to explore the potential molecular interactions between novel 3-chloro-β-lactams and the amino acid residues of the colchicine binding active site cavity of β-tubulin. Collectively, these results suggest that 3-chloro-2-azetidinones, such as compound 10n, could be promising lead compounds for further clinical anti-cancer drug development.

The Molecular Effects of a PARP-1/-2 Inhibitor on Proton and Photon Radiotherapy in Head and Neck Cancer Cells

<h3>Purpose/Objective(s)</h3> The responses of human head and neck cancer (HNSCC) cells to photon (XRT) and proton (PRT) radiotherapy can be enhanced by a poly-(ADP-ribose) polymerase (PARP)-1 and PARP-2 inhibitor (PARPi) with undefined underlying mechanisms. We hypothesize that elucidating these mechanisms will facilitate a personalized selection of PARPi in combination with XRT or PRT. Herein, the effects of PARPi on XRT and PRT induced cell death, cell cycle distribution, and related protein expression in HNSCC cells were defined. <h3>Materials/Methods</h3> Human papillomavirus (HPV)-negative HN5, SqCC/Y1, and HPV-positive UPCI-SCC-154, UM-SCC-47 cell lines were tested. Cells were exposed to a 4 Gy PRT or XRT with or without PARPi (1 µM, 1 hour [h] before radiation). Cell cycle distribution, cell necrosis and apoptosis (staining with Annexin-VFITC) were determined using terminal deoxy-nucleotidyltransferase dUTP nickend labeling assay and were analyzed by flow cytometry. Cell mitotic catastrophe (co-staining for cytoplasm with γ- tubulin for nucleus with Dapi), and senescence (assessed using SA-β-gal) were examined. Protein expressions were determined by western blot or by flow cytometry. <h3>Results</h3> PARPi significantly increased XRT and PRT induced cellular senescence at 6 days in the two HPV+ cell lines, with higher percentages of senescence in PRT than XRT cells (<i>P</i> all < 0.05); in the two HPV- cell lines, the PARPi significantly increased the ratio of XRT-induced senescence (<i>P</i> all < 0.01). PARPi caused significantly more cells to undergo mitotic catastrophe after PRT (in 3 cell lines) or XRT (in the two HPV- cell lines), with higher rates of mitotic catastrophe induced by PARPi in PRT vs. XRT group (<i>P</i> all < 0.05). The PARPi led to a trend (non-significant) of increased cell necrosis at 48 h after PRT (in 3 cell lines) or XRT (in 2 cell lines). No effect of PARPi on XRT or PRT induced apoptosis was noted. At 24 h, PARPi significantly prolonged XRT (in two HPV- cell lines) and PRT (in HN5 cells) induced cell cycle arrest at G2/M phase; trends of this prolongation were noted after PRT in SqCC/Y1 and UPCI-SCC-154 cells, and after XRT in UPCI-SCC-154 cells. PARPi caused trends of increasing ratios of specific protein (53BP1 and RAD51 [DNA repair related]; Ki-67 [cell cycle related]) abundant cells at 24 h after PRT (in two HPV- cell lines); and increased cleaved caspase-3 expression after XRT or PRT in four cell lines tested. <h3>Conclusion</h3> In conclusion, PARPi induced higher levels of mitotic catastrophe in combination with PRT versus XRT, promoted XRT and PRT induced senescence, while it led to a trend of increased cell necrosis after PRT and XRT. These upregulating effects of PARPi were cell line dependent. Cell line based delayed DNA damage repair and increase in radiation-induced cell cycle arrest at G2/M phase may have served as parts of the underlying mechanisms. Further studies on personalized therapy of XRT or PRT in combination with PARPi to improve the efficacy of radiotherapy are warranted.

CHIR99021, trough GSK-3β Targeting, Reduces Epithelioid Sarcoma Cell Proliferation by Activating Mitotic Catastrophe and Autophagy.

Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor versus normal tissue samples and associated to poor prognosis in sarcoma patients. Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.

Recent Advances of WEE1 Inhibitors and Statins in Cancers With p53 Mutations.

p53 is among the most frequently mutated tumor suppressor genes given its prevalence in >50% of all human cancers. One critical tumor suppression function of p53 is to regulate transcription of downstream genes and maintain genomic stability by inducing the G1/S checkpoint in response to DNA damage. Tumor cells lacking functional p53 are defective in the G1/S checkpoint and become highly dependent on the G2/M checkpoint to maintain genomic stability and are consequently vulnerable to Wee1 inhibitors, which override the cell cycle G2/M checkpoint and induce cell death through mitotic catastrophe. In addition to the lost tumor suppression function, many mutated p53 (Mutp53) proteins acquire gain-of-function (GOF) activities as oncogenes to promote cancer progression, which manifest through aberrant expression of p53. In cancer cells with GOF Mutp53, statins can induce CHIP-mediated degradation of Mutp53 within the mevalonate pathway by blocking the interaction between mutp53 and DNAJA1. Therefore, targeting critical downstream pathways of Mutp53 provides an alternative strategy for treating cancers expressing Mutp53. In this review, we summarize recent advances with Wee1 inhibitors, statins, and mevalonate pathway inhibitors in cancers with p53 mutations.

Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.

Wee1 Kinase: A Potential Target to Overcome Tumor Resistance to Therapy.

During the cell cycle, DNA suffers several lesions that need to be repaired prior to entry into mitosis to preserve genome integrity in daughter cells. Toward this aim, cells have developed complex enzymatic machinery, the so-called DNA damage response (DDR), which is able to repair DNA, temporarily stopping the cell cycle to provide more time to repair, or if the damage is too severe, inducing apoptosis. This DDR mechanism is considered the main source of resistance to DNA-damaging therapeutic treatments in oncology. Recently, cancer stem cells (CSCs), which are a small subset of tumor cells, were identified as tumor-initiating cells. CSCs possess self-renewal potential and persistent tumorigenic capacity, allowing for tumor re-growth and relapse. Compared with cancer cells, CSCs are more resistant to therapeutic treatments. Wee1 is the principal gatekeeper for both G2/M and S-phase checkpoints, where it plays a key role in cell cycle regulation and DNA damage repair. From this perspective, Wee1 inhibition might increase the effectiveness of DNA-damaging treatments, such as radiotherapy, forcing tumor cells and CSCs to enter into mitosis, even with damaged DNA, leading to mitotic catastrophe and subsequent cell death.