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Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.

Caspase-8 is crucial for cell death induction, especially via the death receptor pathway. The dysregulated expression or function of caspase-8 can promote tumor formation, progression and treatment resistance in different human cancers. Here, we show procaspase-8 is regulated during the cell cycle through the concerted inhibitory action of Cdk1/cyclin B1 and polo-like kinase 1 (Plk1). By phosphorylating S387 in procaspase-8 Cdk1/cyclin B1 generates a phospho-epitope for the binding of the PBD of Plk1. Subsequently, S305 in procaspase-8 is phosphorylated by Plk1 during mitosis. Using an RNAi-based strategy we could demonstrate that the extrinsic cell death is increased upon Fas-stimulation when endogenous caspase-8 is replaced by a mutant (S305A) mimicking the non-phosphorylated form. Together, our data show that sequential phosphorylation by Cdk1/cyclin B1 and Plk1 decreases the sensitivity of cells toward stimuli of the extrinsic pathway during mitosis. Thus, the clinical Plk1 inhibitor BI 2536 decreases the threshold of different cancer cell types toward Fas-induced cell death., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

ERK 1/2 are found to be hyperactive in many cancers. Active ERK 1/2 (pERK 1/2) are known to protect cancer cells from undergoing death receptor-mediated apoptosis, although the mechanism(s) behind this is poorly understood. Through in vitro kinase assays and mass-spectrometry we demonstrate that pERK 1/2 can phosphorylate pro-Caspase-8 at S387. Also, in EGFR-overexpressing Type I and II ovarian and breast cancer cell lines respectively, ERK 1/2 remain active only during the interphase. During this period, pERK 1/2 could inhibit Trail-induced apoptosis, most effectively during the G1/S phase. By knocking-down the endogenous pro-Caspase-8 using RNAi and replacing it with its non-phosphorylatable counterpart (S387A), a significant increase in Caspase-8 activity upon Trail stimulation was observed, even in the presence of pERK 1/2. Taken together, we propose that a combination of Trail and an inhibitor of ERK 1/2 activities could potentially enhance of Trail's effectiveness as an anti-cancer agent in ERK 1/2 hyperactive cancer cells., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi). We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA) targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SK-BR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neu-overexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as antiproliferative agents that display activity against neoplastic cells at very low doses.

Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50-80% 48 h after transfection and apoptosis was increased from 5 to 40-50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.

Background: Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference., Methods: Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis., Results: All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%-5% to 13%-50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation., Conclusions: PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses.

Simple Summary: Despite the recent advancement in the treatment of ovarian cancer, it continues to be a largely incurable disease, plagued by de novo and acquired resistance to standard chemotherapeutics and novel second-line therapeutics. Therefore, identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have demonstrated that low Caspase-8 expression correlates with poor prognosis in ovarian cancer patients. Moreover, the lack of Caspase-8 alters transcriptional regulation in ovarian cancer cells. In Caspase-8 knockout cells, increased BRD4 expression, increased CDK9 activity, and resistance to Carboplatin and Paclitaxel were observed. We have also shown that combining BRD4 inhibitors with Carboplatin, Paclitaxel, and CDK9 inhibitors synergistically sensitized these cells to undergo cell death. Ovarian cancer is one of the most lethal gynecological cancers worldwide, with approximately 70% of cases diagnosed in advanced stages. This late diagnosis results from the absence of early warning symptoms and is associated with an unfavorable prognosis. A standard treatment entails a combination of primary chemotherapy with platinum and taxane agents. Tumor recurrence following first-line chemotherapy with Carboplatin and Paclitaxel is detected in 80% of advanced ovarian cancer patients, with disease relapse occurring within 2 years of initial treatment. Platinum-resistant ovarian cancer is one of the biggest challenges in treating patients. Second-line treatments involve PARP or VEGF inhibitors. Identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have determined that low Caspase-8 expression in ovarian cancer patients leads to poor prognosis. High-Grade Serous Ovarian Cancer (HGSOC) cells lacking Caspase-8 expression showed an altered composition of the RNA Polymerase II-containing transcriptional elongation complex leading to increased transcriptional activity. Caspase-8 knockout cells display increased BRD4 expression and CDK9 activity and reduced sensitivities to Carboplatin and Paclitaxel. Based on our work, we are proposing three potential therapeutic approaches to treat advanced ovarian cancer patients who exhibit low Caspase-8 expression and resistance to Carboplatin and/or Paclitaxel—combinations of (1) Carboplatin with small-molecule BRD4 inhibitors; (2) Paclitaxel with small-molecule BRD4 inhibitors, and (3) small-molecule BRD4 and CDK9 inhibitors. In addition, we are also proposing two predictive markers of chemoresistance—BRD4 and pCDK9. [ABSTRACT FROM AUTHOR]

Simple Summary: Primary concurrent platinum-based chemoradiation (CRT) is the standard-of-care treatment for locally advanced cervical cancer. However, persistent, recurrent or metastatic disease remains a substantial cause of mortality in women worldwide. Biomarker research can help identify the potential mechanisms of chemo- and radioresistance, improve risk stratification and ultimately translate into novel treatment strategies. We report here that elevated pretreatment levels of caspase-8 and cyclin-dependent kinase 9 (CDK9) are associated with significantly improved relapse-free, distant metastasis-free and cancer-specific survival, while, in contrast, higher levels of phosphorylated CDK9 predict an increased risk of recurrence and distant metastases, and inferior cancer-specific survival. Introduction: After primary platinum-based chemoradiation of locally advanced uterine cervical cancer, a substantial proportion of women present with persistent, recurrent or metastatic disease, indicating an unmet need for biomarker development. Methods: We evaluated the clinical records of 69 cervical cancer patients (Federation of Gynecology and Obstetrics, FIGO Stage > IB3) who were subjected to definitive CRT. Immunohistochemical scoring of caspase-8, cyclin dependent kinase 9 (CDK9) and phosphorylated (phospho-)CDK9 (threonine (Thr) 186) was performed on pretreatment samples and correlated with the histopathological and clinical endpoints, including relapse-free survival (RFS), distant metastasis-free survival (DMFS), cancer-specific survival (CSS) and overall survival (OS). Results: Lower levels of caspase-8 were more prevalent in patients with a higher T-stage (p = 0.002) and a higher FIGO stage (p = 0.003), and were significantly correlated with CDK9 expression (p = 0.018) and inversely with pCDK9 detection (p = 0.014). Increased caspase-8 levels corresponded to improved RFS (p = 0.005), DMFS (p = 0.038) and CSS (p = 0.017) in the univariate analyses. Low CDK9 expression was associated with worse RFS (p = 0.008), CSS (p = 0.015) and OS (p = 0.007), but not DMFS (p = 0.083), and remained a significant prognosticator for RFS (p = 0.003) and CSS (p = 0.009) in the multivariate analyses. Furthermore, low pCDK9 staining was significantly associated with superior RFS (p = 0.004) and DMFS (p = 0.001), and increased CSS (p = 0.022), and remained significant for these endpoints in the multivariate analyses. Conclusion: Increased caspase-8 and CDK9 levels correlate with improved disease-related outcomes in cervical cancer patients treated with CRT, whereas elevated pCDK9 levels predict worse survival in this patient population. [ABSTRACT FROM AUTHOR]

Ovarian cancer exhibits the highest mortality rate among gynecological malignancies. Antimitotic agents, such as paclitaxel, are frontline drugs for the treatment of ovarian cancer. They inhibit microtubule dynamics and their efficiency relies on a prolonged mitotic arrest and the strong activation of the spindle assembly checkpoint (SAC). Although ovarian cancers respond well to paclitaxel, the clinical efficacy is limited due to an early onset of drug resistance, which may rely on a compromised mitosis exit associated with weakend intrinsic apoptosis. Accordingly, we aimed at overcoming SAC silencing that occurs rapidly during paclitaxel‐induced mitotic arrest. To do this, we used a specific anaphase‐promoting complex/cyclosome (APC/C) inhibitor to prevent a premature mitotic exit upon paclitaxel treatment. Furthermore, we investigated the role of the antiapoptotic BCL‐2 family member MCL‐1 in determining the fate of ovarian cancer cells lines with CCNE1 amplification that are challenged with clinically relevant dose of paclitaxel. Using time‐laps microscopy, we demonstrated that APC/C and MCL‐1 inhibition under paclitaxel prevents mitotic slippage in ovarian cancer cell lines and restores death in mitosis. Consistent with this, the combinatorial treatment reduced the survival of ovarian cancer cells in 2D and 3D cell models. Since a therapeutic ceiling has been reached with taxanes, it is of utmost importance to develop alternative strategies to improve the patient's survival. Thus, our study provides not only elements to understand the causes of taxane resistance in CCNE1‐amplified ovarian cancers but also suggests a new combinatorial strategy that may improve paclitaxel‐based efficacy in this highly lethal gynecological disease. What's new? About one‐fifth of patients with high‐grade serous ovarian cancer (HGSOC) exhibit CCNE1‐amplification, which is associated with resistance to paclitaxel and other antimitotic agents used as frontline therapies against HGSOC. Here, to reinforce mitotic arrest and overcome spindle assembly checkpoint silencing, a major resistance mechanism in ovarian cancer, paclitaxel was combined with inhibition of both anaphase‐promoting complex/cyclosome (APC/C) and the pro‐survival protein MCL‐1. This combination successfully reduced cell proliferation and reactivated apoptosis in ovarian cancer cell lines and patient‐derived tumor cells. The findings warrant further investigation of paclitaxel and inhibition of APC/C and MCL‐1 as a therapeutic strategy in HGSOC. [ABSTRACT FROM AUTHOR]

The article discusses the Polo-like kinase (Plk) 1 as a target for novel anti-cancer agents as it induces apoptotic behavior of cancer cells. It examines the expression of death-associated protein kinase (DAPK) following the cells' treatment with Plk1-mismatch (MM) small interfering ribonucleic acid (siRNA). It reveals pro-apoptotic activation of DAPK upon mitotic arrest, induced by the Plk1 inhibitor BI6727 which is a design for optimal cancer therapy under proper dosing.

Background: Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference.Methods: Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis.Results: All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%-5% to 13%-50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation.Conclusions: PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses. [ABSTRACT FROM AUTHOR]

Simple Summary: The current standard therapy of ovarian cancers comprises a reductive surgery followed by a combination of taxane-platinum-based primary chemotherapy. However, despite an initial positive response, patients in the advanced stage showed relapse within months or even weeks. Thus, there is a need to find combinatorial therapies that permit overcoming the paclitaxel-associated resistance in patients. Here, we found that MRIA9, a newly developed small-molecule inhibitor of the salt-inducible-kinase 2, interferes with the cell division of cancer cells. More importantly, MRIA9 increases paclitaxel efficiency in eliminating ovarian cancer cells and patient derived cancer cells by inducing apoptosis or programmed cell death. Thus, our study indicates that MRIA9 might represent a novel therapeutical tool for translational studies to overcome paclitaxel resistance in ovarian cancer. The activity of the Salt inducible kinase 2 (SIK2), a member of the AMP-activated protein kinase (AMPK)-related kinase family, has been linked to several biological processes that maintain cellular and energetic homeostasis. SIK2 is overexpressed in several cancers, including ovarian cancer, where it promotes the proliferation of metastases. Furthermore, as a centrosome kinase, SIK2 has been shown to regulate the G2/M transition, and its depletion sensitizes ovarian cancer to paclitaxel-based chemotherapy. Here, we report the consequences of SIK2 inhibition on mitosis and synergies with paclitaxel in ovarian cancer using a novel and selective inhibitor, MRIA9. We show that MRIA9-induced inhibition of SIK2 blocks the centrosome disjunction, impairs the centrosome alignment, and causes spindle mispositioning during mitosis. Furthermore, the inhibition of SIK2 using MRIA9 increases chromosomal instability, revealing the role of SIK2 in maintaining genomic stability. Finally, MRIA9 treatment enhances the sensitivity to paclitaxel in 3D-spheroids derived from ovarian cancer cell lines and ovarian cancer patients. Our study suggests selective targeting of SIK2 in ovarian cancer as a therapeutic strategy for overcoming paclitaxel resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: CDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9's upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9's activity holds the potential to be a highly effective anti-cancer therapeutic. Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90. [ABSTRACT FROM AUTHOR]

Caspase-8 is a cysteine - aspartate specific protease that classically triggers the extrinsic apoptotic pathway, in response to the activation of cell surface Death Receptors (DRs) like FAS, TRAIL-R and TNF-R. Besides it's roles in triggering death receptor-mediated apoptosis, Caspase-8 has also been implicated in the onsets of anoikis, autophagy and pyroptosis. Furthermore, Caspase-8 also plays a crucial pro-survival function by inhibiting an alternative form of programmed cell death called necroptosis. Low expression levels of pro-Caspase-8 is therefore associated with the malignant transformation of cancers. However, the long-held notion that pro-Caspase-8 expression/activity is generally lost in most cancers, thereby contributing to apoptotic escape and enhanced resistance to anti-cancer therapeutics, has been found to be true for only a minority of cancers types. In the majority of cases, pro-Caspase-8 expression is maintained and sometimes elevated, while it's apoptotic activity is regulated through different mechanisms. This supports the notion that the non-apoptotic functions of Caspase-8 offer growth advantage in these cancer types and have, therefore, gained renewed interest in the recent years. In light of these reasons, a number of therapeutic approaches have been employed, with the intent of targeting pro-Caspase-8 in cancer cells. In this review, we would attempt to discuss - the classic roles of Caspase-8 in initiating apoptosis; it's non-apoptotic functions; it's the clinical significance in different cancer types; and the therapeutic applications exploiting the ability of pro-Caspase-8 to regulate various cellular functions. [ABSTRACT FROM AUTHOR]