Rosuvastatin Ameliorates the Initiation and Progression of Lung Adenocarcinoma via Facilitating PLK1 Inhibition.

Lung cancer is still the most common cancer in the world, especially lung adenocarcinoma (LUAD). Despite years of effort, including the application of immunotherapy and targeted therapy, the survival rate of LUAD has not improved significantly. Exploring effective targets and combination drugs is crucial for the treatment of LUAD. We characterized differentially expressed genes between LUAD and normal lung tissue based on The Cancer Genome Atlas (TCGA) database and identified polo-like kinase 1 (PLK1) as the hub gene. Through an analysis using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), we obtained a combination of Chinese medicine with PLK1 inhibitor, whose biological function we confirmed by western blot and TdT-UTP nick-end labelling (TUNEL) assays. After combined analysis of protein expression with clinical characteristics, GNPNAT1, CCT6A, SMOX, UCK2, PLK1, HMMR and ANLN expression were significantly correlated with age, sex and stage. Among them, the survival rate was lower in patients with high PLK1 expression than in those with low PLK1 expression, making PLK1 a promising therapeutic target for LUAD. Stage and PLK1 expression could be used as independent prognostic factors for LUAD. By TCMSP analysis, tectoridin had the strongest correlation with PLK1. Tectoridin synergized with PLK1 inhibitor to suppress autophagy and ferroptosis but promoted caspase-3-mediated apoptosis in A549 cells. Our findings highlight a potential drug target and the combination therapy strategy of PLK1 inhibitor and tectoridin for LUAD patients.

Acute lymphoblastic leukemia is the most common cancer in children, and prognosis among adult patients is still poor. Recently, Cytokine Receptor-Like Factor 2 (CRLF2) was identified as a prognostic factor in ~6% of pediatric and adult patients with precursor B cell-acute lymphoblastic leukemia (B-ALL).1, 2 In addition, several independent studies also demonstrated that CRLF2 acts as an oncoprotein.3–6 The Children’s Oncology Group found that high expression of CRLF2 confers an adverse prognosis in high-risk B-ALL patients.7 The mechanism of CRLF2-associated leukemogenesis, however, is still unclear. In CRLF2-rearranged B-ALL cases, CRLF2 is overexpressed because of genetic rearrangements. Interestingly, about one-half of cases co-harbor an activating mutation in exon 16 of JAK2, most commonly involving R683 or I682.3, 6 In a murine progenitor B cell line, Ba/F3, it has been shown that the combination of CRLF2 overexpression and JAK2 R683G confers IL-3-independent growth, implicating their role in triggering leukemic proliferation.5, 6 As reflected in phenotypes, the signaling downstream of CRLF2 overexpression and JAK2 R683G is different from normal CRLF2 signaling.8 In this study, we aimed to take advantage of this discrepancy to identify potential therapy targeting this aberrant signaling seen in B-ALL. To identify kinase inhibitors against leukemic CRLF2 signaling, we subjected Ba/F3 cells with CRLF2 overexpression and JAK2 R683G to a kinase inhibitor screening system that consisted of 73 kinase inhibitors and other small molecules. An in vitro cell proliferation assay was coupled to observe growth inhibition (Figure 1a).9 Among all kinase inhibitors, we found Polo-like kinase 1 (PLK1) inhibitor, BI 2536, had the lowest IC50 (11 nM) while the IC50 of p38 mitogen-activated protein kinase inhibitor (VX-745), c-Jun N-terminal kinase inhibitor (JNK II) and Akt kinase inhibitor (GSK-690693) were all 1,000 times higher (Figure 1b). We verified this result by examining if PLK1 is preferentially inhibiting the growth of Ba/F3 cells with CRLF2 overexpression and JAK2 R683G but not Ba/F3 parental cells nor Ba/F3 cells with BCR/ABL. Another PLK1 inhibitor, volasertib, was used to treat these three cell lines. We found the IC50 of Ba/F3 cells with CRLF2 overexpression and JAK2 R683G was 9 and 7 times lower than the other two lines, respectively (Figure 1c, 1d and Figure S1). These findings suggest that the growth of Ba/F3 cells conferred by the combination of CRLF2 overexpression and JAK2 mutant is preferentially inhibited by the PLK1 inhibitor in vitro. Figure 1 Inhibition of Polo-like kinase 1 counteracts in vitro proliferation conferred by the aberrant CRLF2 signaling in leukemia To check if Plk1 is downstream of the aberrant signaling by CRLF2 overexpression and JAK2 mutation, we measured the abundance of Plk1 with immunoblotting. It showed that the expression of Plk1 was higher in Ba/F3 cells with CRLF2 overexpression and JAK2 R683G than in Ba/F3 parental cells (Figure 1e). Because phospho-Plk1 (Thr210) is the major phosphosite in activated Plk1,10 we immunoblotted phospho-Plk1 (Thr210) and found it was increased in Ba/F3 cells with CRLF2 overexpression and JAK2 R683G while Cdk1 was dephosphorylated for cell cycle entry as the downstream effect (Figure 1e). We immunoprecipitated endogenous Plk1 and carried out a non-radioactive kinase assay, which quantified the amount of ATP converted to ADP as result of Plk1 catalytic activity in vitro. As shown in Figure 1f, the in vitro Plk1 kinase activity from Ba/F3 cells with CRLF2 overexpression and JAK2 R683G is higher than in Ba/F3 parental cells. Taken together, CRLF2 overexpression and JAK2 activating mutation can lead to increased expression and activation of PLK1, a finding corroborating the high-throughput kinase inhibition assay as mentioned earlier. Because PLK1 is a major regulator of centrosomes in mitosis, we chose to study how dysregulation of PLK1 affects cell division. Centrosomes consist of centrioles and pericentriolar material. Within pericentriolar material are PLK1 substrates including γ-tubulin among others.11 At prophase and metaphase, PLK1 can recruit these proteins for centrosomal nucleation of microtubules at their minus ends. To examine PLK1 function for our Ba/F3 system in this regard, we carried out immunofluorescence staining of γ-tubulin, a specific marker for centrosomes. In normal mitosis, centrioles duplicate and summon pericentriolar material to form centrosomes that move toward two poles of the cell. That was our observation in Ba/F3 parental cells (Figure 1g and 1h). Nevertheless, in Ba/F3 cells with CRLF2 overexpression and JAK2 R683G, we noticed the abnormal separation of centrosomes presented and was surrounded by condensed chromosomes. This would undermine the normal chromosome partition in mitosis. This abnormal appearance of centrosomes and chromosomes was also phenocopied at the other extreme of the imbalanced PLK1 function where PLK1 was knocked down in SW962 cells.12 When both PLK1 and its negative regulator, myosin phosphatase-targeting subunit 1 (MYPT1), were knocked down, this mitotic abnormality was rescued. This implicates that either overexpression or knockdown of PLK1 causes its imbalanced function and hence abnormal mitosis. Interestingly, we also found MYPT1 Ser695 was hyperphosphorylated in Ba/F3 cells with CRLF2 overexpression and JAK2 R683G (Figure S2). This would inactivate the function of MYPT1.13 Taken together, this aberrant pattern of γ-tubulin in centrosomes could be indicative of PLK1 dysfunction in Ba/F3 cells with CRLF2 overexpression and JAK2 R683G. Next, we sought to validate the efficacy of PLK1 inhibition ex vivo. BI 2536 or vehicle control (DMSO) was used to treat Ba/F3 cells with CRLF2 overexpression and JAK2 R683G, which were then injected into tail veins of sublethally irradiated NOD-scid IL2Rγnull (NSG) mice that lacked functional T and B lymphocytes and natural killer cells. Fifteen days after cell injection, necropsy was performed to evaluate cell infiltration in mouse spleens and livers (Figure 2a). While the sizes of mouse spleens and livers from the no injection control were consistent with those of regular NSG mice, all of the mice receiving cell injection had hepatosplenomegaly that could be reduced with the BI 2536 treatment (Figure 2b to 2d). We found Ba/F3 cells with CRLF2 overexpression and JAK2 R683G treated with BI 2536 resulted in significantly smaller spleens (0.5% vs. 1.5% of total body weight, p<0.01) and livers (5.5% vs. 7.6% of total body weight, p<0.01) compared with what untreated cells would do (Figure 2e and 2f). This subsiding of hepatosplenomegaly suggests that the proliferation of Ba/F3 cells with CRLF2 overexpression and JAK2 R683G in mice was thwarted by the PLK1 inhibition, consistent with the result of high-throughput kinase inhibitor assay. Figure 2 Inhibition of Polo-like kinase 1 counteracts the growth mediated by aberrant CRLF2 signaling ex vivo and the proliferation of CRLF2-rearranged precursor B acute lymphoblastic leukemia in vitro The efficacy of the PLK1 inhibitor in our Ba/F3 cell system led us to examine its effect on human samples. We checked if any different effect of BI 2536 on B-ALL patient samples with CRLF2 overexpression and JAK2 activating mutation compared with wild-type CRLF2 and JAK2. We used two human B-ALL xenografts (CRLF2 overexpression/JAK2 I682F vs. CRLF2 normal expression/JAK2 wild type) that had been passaged in NSG mice to test (Figure 2g). Forty-eight hours prior to culturing the leukemia xenografts, hTERT-transformed human bone marrow stromal cells were seeded as the feeder layer.14, 15 Leukemia cells were seeded and treated either with BI 2536 or vehicle control (DMSO). Forty-eight hours later, the leukemia cells were harvested and assessed for viability. In comparison with vehicle control, the viability of CRLF2-rearranged B-ALL cells treated with BI 2536 was lower (48.8% of vehicle) than B-ALL cells without CRLF2 rearrangement or JAK2 mutations (105.3% of vehicle) (Figure 2h). In this particular case of B-ALL, CRLF2 rearrangement was associated with a higher sensitivity to the PLK1 inhibitor. To summarize, we identified PLK1 as a potential therapeutic target for human CRLF2-rearranged B-ALL with JAK2 activating mutations. From the high-throughput kinase inhibitor screening, ex vivo therapy in mouse transplantation experiments and in vitro viability assay for human xenografts, inhibition of PLK1 was shown to have a potent activity against Ba/F3 cells with CRLF2 overexpression with JAK2 R683G as well as human B-ALL cells with CRLF2 overexpression with JAK2 I682F. In addition, the immunofluorescence imaging implicate that PLK1 is dysregulated downstream of the aberrant CRLF2 signaling. More studies are warranted for understanding the particular mechanism behind these observations, and we suggest future therapy for this subset of B-ALL could be directed against PLK1 and its associated signaling pathways.

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and overexpressed in multiple human cancers, including leukemia and also many solid tumors. PLK1 knockdown has been shown to block proliferation of leukemic cell lines and the clonogenic potential of tumor cells grown from patients with cancer. PLK1 inhibition is a promising strategy for the treatment of some tumors. We aim to analyze expression of PLK1 in metastatic colorectal carcinoma. Retrospective analysis of colorectal carcinomas with hepatic metastasis during follow-up receiving neoadjuvant chemotherapy (NAC), based on oxaliplatin. Immunohistochemistry for PLK-1 in paraffin-embedded tissue from the primary and also from the metastasis. 50 patients. 32% showed good histopathological response. 43% of the primaries were positive for PLK1, as opposed to 23.5% of the metastasis. Expression of PLK1 was significantly reduced in metastasis compared with the primaries (p = 0.05), what could be due to therapy or to a phenotypic change of the metastatic nodule. Analysis of the prognostic influence of PLK1 expression showed significant association between PLK1 expression in metastasis and lower overall survival (p = 0.000). We have also found a significant association between PLK1 expression and histopathological response (p = 0.02). All the tumors with high expression of PLK1 showed minor response (11/11). This study shows the association between survival and poor histopathological response to therapy and high expression of PLK1 in metastasis. Our results could open a new therapeutic approach through the inhibition of PLK1.

Background: CDK 4/6 inhibitors (CDK 4/6i) with endocrine therapy (ET) combination therapy have improved outcomes in patients (pts) with hormonal receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-) advanced breast cancer (ABC). However, most pts eventually develop resistance to these drugs, and one third never respond. Aside from HR positivity, predictive markers of clinical benefit from CDK 4/6i remains elusive. We aimed to identify biomarkers of response to palbociclib (PAL) and analyze potential therapeutic targets to reverse resistance. Methods: PEARL trial is a multicenter phase 3 study that assigned 601 postmenopausal HR+/HER2- ABC pts, whose disease progressed on aromatase inhibitors (AIs), to receive PAL + ET vs capecitabine (CAPE). We performed a differential gene expression analysis in pre-treatment tumors in extreme responders to PAL using the HTG EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Inc.), containing 2534 cancer related genes. Samples were subset in 2 categories: refractory (progressive disease as best response) vs sensitive (progression-free survival (PFS) within the upper quartile). Cox regression and Significance Analysis of Microarrays (SAM) analysis adjusting for multiple comparisons were performed. Results: We analyzed 455 (75.7%) pts with pre-treatment tumors available [from them, PAL + ET arm: 229 (50.3%) pts; CAPE arm: 226 (49.7%) pts]. Fifty genes (false discovery rate (FDR)&amp;lt;0.05) were differentially expressed in pts sensitive vs refractory to PAL (E2F target genes, epithelial-to-mesenchymal transition (EMT) and cell cycle genes, mainly). Unsupervised hierarchical clustering of pts based on the expression of these genes revealed two clusters. Cluster 1 is composed mostly of resistant tumors, highly proliferative (Ki67≥20%: 70%) with a great proportion of luminal B (59%) and non-luminal tumors (19%). Cluster 2 is composed of sensitive, low proliferative (Ki67&amp;lt;20%: 58%), mostly luminal A tumors (75%). There was no difference in ESR1 mutations distribution between the two clusters (Table 1). Forty genes were up-regulated and associated with resistance, including CCNE1 and PLK1 (Polo Like Kinase 1). In the whole cohort, pts with high levels (&amp;gt; median) of PLK1 (PLK1-high) treated with PAL, had a worse PFS in a multivariate model (5.7 months (m) vs 9.3 m of median PFS in PLK1-High vs -Low; HR=1.64, 95% CI (1.25-2.34), p=0.0008; adjusted model for confounders: age, site of disease, sites of metastasis, prior chemotherapy and Ki67). There were no differences in population treated with CAPE (9.9 m vs 9.4 m, PLK1-High vs -Low; HR=0.82, 95% CI (0.56-1.21), p=0.3189). In the METABRIC cohort, PLK1-High was associated with worse overall survival in HR+/HER2- BC but not in triple negative nor in HER2+ tumors. Among HR+/HER2- tumors, PLK1 expression was higher in luminal B and HER2-enriched intrinsic subtypes. We interrogated DepMap database and found that in BC cells lines there was an inverse correlation between PLK1 expression and effect on cell viability of CDK4 CRISPR knock-out (Pearson correlation r:0.54, p=0.009), but not of CDK6 knock-out. Also, HR+/HER2-/High Ki67 BC cell lines (HCC1428, EFM19 and MCF7) showed resistance to PAL on cell proliferation assays but sensitivity to the PLK1 inhibitor BI-2536. Conclusion: High expression of PLK1 is associated with intrinsic resistance to PAL and ET, this might be overcome with PLK1 inhibition. Table 1PATIENT CHARACTERISTICSCluster 1Cluster 2ALLn=57n=47n=104RespondersSensitive42 (73.68%)14 (29.79%)56 (53.85%)Refractory15 (26.32%)33 (70.21%)48 (46.15%)ESR1Mutated9 (15.79%)13 (27.66%)22 (21.15%)Wild type45 (78.95%)34 (72.34%)79 (75.96%)Unknown3 (5.26%)0 (0%)3 (2.88%)PriorQTN42 (73.68%)31 (65.96%)73 (70.19%)Y15 (26.32%)16 (34.04%)31 (29.81%)SubtypeLumA43 (75.44%)10 (21.28%)53 (50.96%)LumB14 (24.56%)28 (59.57%)42 (40.38%)Non Luminal0 (0%)9 (19.15%)9 (8.65%)MetastasisOne21 (36.84%)15 (31.91%)36 (34.62%)Multiple36 (63.16%)32 (68.09%)68 (65.38%)KI67 20%KI67&amp;lt;2033 (57.89%)7 (14.89%)40 (38.46%)KI67≥2016 (28.07%)33 (70.21%)49 (47.12%)Unknown8 (14.04%)7 (14.89%)15 (14.42%)Objective ResponseComplete1 (1.75%)0 (0%)1 (0.96%)Partial16 (28.07%)6 (12.77%)22 (21.15%)Progressive15 (26.32%)33 (70.21%)48 (46.15%)Stable25 (43.86%)8 (17.02%)33 (31.73%) Citation Format: Angel Guerrero- Zotano, Christoph Zielinski, Miguel Gil-Gil, Manuel Ruiz-Borrego, Eva M. Ciruelos, Montserrat Munoz, Begoña Bermejo, Mireia Margeli, Antonio Antón, Tibor Csöszi, Andrés García-Palomo, Ana Santaballa, Jose Luis Alonso, Antonio Fernández, Massimo Corsaro, Jesús Herranz, Paula López, Rosalia Caballero, Christiane Thallinger, Miguel Martin. Plk1 expression &amp; efficacy of palbociclib in advanced hormonal receptor-positive breast cancer patients from PEARL study (GEICAM 2012-03) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-01.

PLK1 Inhibition Sensitizes Breast Cancer Cells to Radiation via Suppressing Autophagy.

A Novel Role for PLK1 in Leukemia Stem Cell Function in Acute Myeloid Leukemia

The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-kappaB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-kappaB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity.

Polo-like kinase-1 (PLK1) plays a crucial role in cell cycle progression, and it is considered a potential therapeutic target in many cancers. Although the role of PLK1 is well established in triple-negative breast cancer (TNBC) as an oncogene, its role in luminal BC is still controversial. In this study, we aimed to evaluate the prognostic and predictive role of PLK1 in BC and its molecular subtypes. A large BC cohort (n = 1208) were immunohistochemically stained for PLK1. The association with clinicopathological, molecular subtypes, and survival data was analysed. PLK1 mRNA was evaluated in the publicly available datasets (n = 6774), including The Cancer Genome Atlas and the Kaplan-Meier Plotter tool. 20% of the study cohort showed high cytoplasmic PLK1 expression. High PLK1 expression was significantly associated with a better outcome in the whole cohort, luminal BC. In contrast, high PLK1 expression was associated with a poor outcome in TNBC. Multivariate analyses indicated that high PLK1 expression is independently associated with longer survival in luminal BC, and in poorer prognosis in TNBC. At the mRNA levels, PLK1 expression was associated with short survival in TNBC consistent with the protein expression. However, in luminal BC, its prognostic value significantly varies between cohorts. The prognostic role of PLK1 in BC is molecular subtype-dependent. As PLK1 inhibitors are introduced to clinical trials for several cancer types, our study supports evaluation of the pharmacological inhibition of PLK1 as an attractive therapeutic target in TNBC. However, in luminal BC, PLK1 prognostic role remains controversial.

HDAC Inhibitor Synergizes with GL-V9, a Derivative of Wogonin, to Suppress Acute Myeloid Leukemia By Downregulating ESPL1 and PLK4

Polo-like kinase 1 (PLK1) is an evolutionarily conserved serine/threonine kinase essential for cell mitosis. As a master cell cycle regulator, p21/Waf1 plays a critical role in cell cycle progression. Nitric oxide (NO.) has been shown to down-regulate PLK1 and up-regulate p21/Waf1 independent of cGMP. Here, the respective roles of p38 MAPK and p21/Waf1 in NO.-mediated PLK1 repression were investigated using differentiated U937 cells that lack soluble guanylate cyclase. NO. was shown to down-regulate both PLK1 mRNA and protein. Nuclear run-on assays and mRNA stability studies demonstrated that the effect of NO. on PLK1 expression was associated with decreased transcription without changes in transcript stability. SB202190, a p38 MAPK inhibitor, prevented transcriptional repression of PLK1 by NO.. Transfection with dominant-negative p38 MAPK mutant eliminated the NO. effect on both p21/Waf1 and PLK1 gene expression. Knockdown of p21/Waf1 with siRNA also substantially reduced the regulatory effect of NO. on PLK1. Reporter gene experiments showed that NO. decreased activity of the PLK1 proximal promoter, an effect that was blocked by p38 MAPK inhibitor. Deletion or mutation of the CDE/CHR promoter site, an element regulated by p21/Waf1, increased base-line promoter activity and abolished NO. repression of the PLK1 promoter. Likewise, electrophoretic mobility shift assays with CDE/CHR probe revealed a NO.-mediated change in protein-probe complex formation. Competition with various unlabeled CDE/CHR mutant sequences showed that NO. increased nuclear protein binding to intact CHR. These results demonstrate that a NO.-p38 MAPK-p21/Waf1 signal transduction pathway represses PLK1 through a canonical CDE/CHR promoter element.

Background: Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited medical treatment options. We previously identified polo-like kinase 1 (PLK1) as one of most overexpressed genes in ACC; thus PLK1 represents a potential treatment target for this cancer type. Some PLK1 inhibitors are under evaluation in clinical trials for other solid organ malignancies, and seem to be more effective in TP53 mutated tumours. The aim of this study was to evaluate PLK1 protein levels in a large series of ACC and assess the in vitroefficacy of PLK1 inhibitors in two different ACC cell lines. Methods: 104 formalin-fixed paraffin-embedded ACC tissue samples with available genetic data were investigated. Nuclear PLK1 protein expression was evaluated by immunohistochemistry and a semi-quantitative H-score was calculated. PLK1 expression levels were correlated to clinical and histological parameters. Efficacy of PLK1-specific inhibitor Volasertib (0-200 nM) was tested in the standard NCI-H295R ACCcell line, which presents PLK-1 overexpression and a large TP53 deletion, and in the newly established MUC1 cell line, which bears a frameshift mutation in TP53. Cell proliferation was analysed using DNA fluorescence and cell apoptosis by Caspase Glo 3/7 assay. Results: Nuclear PLK1 expression was classified as high in 59% of ACC samples, with a significant difference noted between TP53-mutated (n=24) and wild-type (n=80) cases (87.5 vs 51%, p<0.01). PLK1 levels did not correlate with either progression-free or overall survival. H295R cells showed a significant time- and dose-dependent reduction of cell proliferation compared to vehicle control after 72h of Volasertib treatment (p<0.005 per trend, p=0.01 by 200nM by non-parametric two-way ANOVA). A less pronounced and non-significant trend towards inhibited proliferation was observed in MUC1 cells. Cell apoptosis was significantly higher in the H295R cells treated with 175nM and 200nM Volasertib when compared to control (p<0.05), while there was no significant difference in MUC1 cells. Conclusion:In this pilot study, we propose PLK1 inhibitors as promising candidates for treatment of a subset of ACC patients that may be pre-selected according to the tumour molecular pattern. We plan to extend functional experiments to further PLK1 inhibitors, including additional ACC cell lines with a different molecular profile.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. LIN28 homolog A (LIN28A) is a RNA-binding protein, which serves a fundamental role in cell development and pluripotency. Polo-like kinase 4 (PLK4) is a member of the polo-like kinase family, which primarily takes part in the mitotic regulation. Overexpression of LIN28A has been demonstrated in ovarian cancer; however, the expression of PLK4 and the correlation between the expression of LIN28A and PLK4 in EOC has not been discussed. In the present study, the mRNA and protein levels of LIN28A and PLK4 were evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry in ovarian tissues of patients. Results demonstrated significantly increased expression in EOC compared with benign epithelial ovarian tumors. High expression of LIN28A and PLK4 was detected at the advanced pathological stage. Furthermore, PLK4 expression was positively correlated with LIN28A (r=0.555; P=0.039). The median survival analysis of patients with EOC with LIN28A and PLK4 double positive expression was 14 months, compared with 30 months in single positive and 60 months in double negative patients by Kaplan-Meier analysis (P<0.05). The expressions of LIN28A and PLK4 was elevated in different EOC cell lines compared to with a normal ovarian cell line. The 293T cells transfected with LIN28A plus a PLK4 plasmid were the fastest-growing group. These results suggest that co-expression of LIN28A and PLK4 may be associated with poor prognosis of EOC and could serve as promising prognostic biomarkers and therapeutic targets in EOC. LIN28A and PLK4 may be used along with traditional morphological and clinical characteristics for predicting prognosis.

Background: BAL0891 is a novel dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) which affects mitotic spindle assembly checkpoint integrity resulting in aberrant mitotic progression of cancer cells. Since unlike other organs, bladder urothelium is highly regenerative in certain circumstances, there are high chances of cell cycle inhibitors to have anti-cancer activity over bladder cancer. In this study, we investigated the therapeutic potential of BAL0891 in bladder cancer compared to single TTK or PLK1 inhibitor and discovered the best therapeutic combination partner for BAL0891. Methods: We performed meta-analysis of the mRNA expression levels of bladder cancer using Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Representative bladder cancer cell lines (253J, 253J-BV, J82, RT4, T24, J82, HT1197, and HT1376) were treated with the dual protein kinase inhibitors, BAL0891, in comparison with TTK inhibitor (BAY1217389) or PLK1 inhibitor (onvansertib). BAL0891 was combined with CDK4/6 inhibitor, another cell cycle inhibitor working at different point from BAL0891. Cell viability was assessed using CCK-8 assay during 72 hours after treatment. Expression levels of TTK and PLK1 were measured by immunoblotting. Cell cycle analysis and cell apoptosis detection were performed on T24 cell. Results: The mRNA expression levels of both TTK (16.7 times) and PLK1 (10.0 times) were significantly upregulated only in bladder cancers compared to normal bladder tissues (P&amp;lt;0.05). Compared to TTK or PLK1 inhibitor, BAL0891 was sensitive in 75% of bladder cancer cell lines (IC50 &amp;lt;0.1 uM) and significantly reduced proliferation of bladder cancer cell lines depending on the dosage. TTK and PLK1 expression levels of cancer cells were associated with the sensitivity to BAL0891. BAL0891 significantly induced necrosis and increased in polyploidy and G2/M arrest in T24 cell. The anti-cancer activity of BAL0891 was further enhanced in certain cell lines (RT4, and J82) by combination of CDK4/6 inhibitor. Conclusion: This is the first study to suggest the potent anti-cancer activity of BAL0891 in bladder cancer. BAL0891 effectively worked on bladder cancer by two tracks which not only induced G2/M arrest but also increased in polyploidy. Furthermore, BAL0891 in combination with CDK4/6 inhibitor promoted anti-cancer effects. Lastly, this study suggests the use of TTK and PLK1 as the predictive biomarker for BAL0891. These data support further investigation of BAL0891 for the treatment of bladder cancer patients. Citation Format: Jee Soo Park, Ji-eun Lee, Myung Eun Lee, Jongchan Kim, Won Sik Jang, Jae Jung Lee, Jun Seung Lee, Minkyung Kang, Won Sik Ham. The potential anti-cancer activity of dual TTK/PLK1 inhibitor, BAL0891, in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5726.

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase essential for regulating multiple stages of cell cycle progression in mammals. Aberrant regulation of PLK1 has been observed in numerous human cancers and is linked to poor prognoses. However, its role in the pathogenesis of colorectal cancer (CRC) in the Middle East remains unexplored. PLK1 overexpression was noted in 60.3% (693/1149) of CRC cases and was significantly associated with aggressive clinico-pathological parameters and p-ERK1/2 overexpression. Intriguingly, multivariate logistic regression analysis identified PLK1 as an independent predictor of lymph node metastasis. Our in vitro experiments demonstrated that CRC cells with high PLK1 levels were resistant to 5-Fu treatment, while those with low PLK1 expression were sensitive. To investigate PLK1's role in chemoresistance, we used the specific inhibitor volasertib, which effectively reversed 5-Fu resistance. Interestingly, forced PLK1 expression activated the CRAF-MEK-ERK signaling cascade, while its inhibition suppressed this cascade. PLK1 knockdown reduced epithelial-to-mesenchymal transition (EMT) progression and stem cell-like traits in 5-Fu-resistant cells, implicating PLK1 in EMT induction and stemness in CRC. Moreover, silencing ERK1/2 significantly mitigated chemoresistance, EMT, and stemness properties in CRC cell lines that express PLK1. Furthermore, the knockdown of Zeb1 attenuated EMT and stemness, suggesting a possible link between EMT activation and the maintenance of stemness in CRC. Our findings underscore the pivotal role of PLK1 in mediating chemoresistance and suggest that PLK1 inhibition may represent a potential therapeutic strategy for the management of aggressive colorectal cancer subtypes.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis