Mitotic Cells Research Articles

Abstract 6612: Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress

Abstract Therapy resistance is responsible for 90% of cancer-related deaths. We identified a novel mechanism of therapy resistance in which cancer cells enter an endocycling cell state, undergoing repeated S and G phases without dividing. As a result, resistant endocycling cells are 40 times larger than mitotic cancer cell controls. Interestingly, we observed that these non-proliferative endocycling cells can eventually give rise to proliferative progeny weeks after chemotherapy is removed, modeling cancer recurrence. We hypothesize that this endocycling cancer cell state is a critical driver of therapy resistance and cancer lethality. We evaluated oxidative stress in endocycling cells that survived the days and weeks following chemotherapy treatment using DCF-DA staining. We found increased levels of reactive oxygen species (ROS) in endocycling prostate cancer cells compared to untreated mitotic cells when normalized to cell size. Given that mitochondria are major producers and regulators of ROS, we hypothesize that cells surviving in this resistant state alter mitochondrial structure and function in response to increased oxidative stress. We performed immunofluorescence and confocal microscopy on mitochondria in untreated cells and cells following chemotherapy treatment to observe their morphology. Surviving endocycling cells had increased mitochondrial fragmentation when compared to the untreated group, a result that was further supported by reduced expression of phospho-DRP1 Ser637 via western blot. To assess changes in glucose metabolism, we then utilized isotope tracing-based metabolomics with [U-13C]glucose on untreated and surviving endocycling cells following chemotherapy. Endocycling cells had decreased labeling of 13C in TCA cycle metabolites, suggesting a decrease in mitochondrial function when compared to untreated cells. Ongoing work involves further studying metabolic reprogramming as cells enter this resistant state with parallel labeling experiments using [1,2-13C]glucose and [U-13C]glutamine to perform mathematical modeling-based 13C-metabolic flux analysis. In addition, we engineered a cell line to track mitochondria in real-time to quantify mitochondrial dynamics underlying the morphology observed in untreated and surviving cells. Our goal is to characterize how this therapy-resistant cell state alters its mitochondrial structure and function to survive therapeutic stress. Identifying vulnerabilities of this phenotype will enable new approaches for eliminating drug resistance in cancer. Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress [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 6612.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [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 3347.

Abstract 4478: Development and characterization of kinase sparing TPX2-tethering Aurora kinase A ligands for cancer therapy

Abstract A major limitation in the treatment of cancer is the limited therapeutic index of currently available chemotherapies or molecularly targeted therapies. Drug related toxicities often necessitate restricted drug dosing regimens, resulting in subtotal tumor remissions, which represent an important cause for tumor recurrences and the development of therapy resistance. Following up on previous work suggesting Aurora kinase A (AURKA) as a promising therapeutic target for the treatment of TP53 altered liver carcinomas (Dauch D. et al., Nat. Med., 2016) and TP53; RB1 altered small cell lung carcinomas (Gong X. et al., Cancer Discovery, 2019), we here report on the development and characterization of first in class kinase sparing AURKA ligands, which selectively kill TP53 deficient liver cancer cells and TP53; RB1 deficient small cell lung cancer cells by tethering AURKA to its binding partner TPX2 in mitotic cells. Small molecule mediated stabilization of AURKA/TPX2 complexes resulted in formation of multipolar spindles and subsequent cell death through mitotic catastrophe. Mechanistically, we were able to show that the hypersensitivity of TP53 deficient liver cancer cells and TP53; Rb1 deficient small cell lung cancer cells towards AURKA ligands is due to low expression levels of the antiapoptotic protein MCL1, which is necessary for survival of cancer cells in prolonged mitosis. Our novel AURKA ligands do not affect the kinase function of AURKA and thus, in contrast to conventional AURKA kinase inhibitors, can be administered continuously to mice without inducing toxicity related to inhibition of kinase related non-canonical (non-mitotic) functions of AURKA in normal cells or TP53 and RB1 wildtype cancer cells. Preclinical therapy studies in cell-line derived xenograft (CDX) tumor models of small cell lung cancer (SCLC) and orthotopic models of TP53 altered hepatocellular carcinoma and TP53;Rb1 altered SCLC demonstrated marked monotherapeutic efficacy of AURKA ligands. Conceptually, our herein provided data suggest that small molecules designed to stabilize protein-protein interactions represent powerful anti-cancer drugs with exceptional therapeutic window and thus open a venue towards the development of novel cancer therapies. Citation Format: Athina Anastasia Moschopoulou, Melanie Henning, Benedikt Wagner, Dirk Flötgen, Juliander Reiner, Yulia Skliarenko, Stefan Zwirner, Clemens Hinterleitner, Marco Seehawer, Luana dArtista, Elke Rist, Tatu Pantsar, Thales Kronenberger, Daniel Dauch, Corinna Gehring-Khav, Antti Poso, Liudmila Andreeva, Matthias Schwab, Michael Forster, Wolfgang Albrecht, Stefan Laufer, Lars Zender. Development and characterization of kinase sparing TPX2-tethering Aurora kinase A ligands for cancer therapy [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 4478.

Abstract 1642: NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication

Abstract The post-translational modification of proteins implicated in mitosis in healthy and in cancer epithelial cells was measured by 2-D gels analysis. The post-translational modifications of NuMA in a variety of human epithelial cancer cells was prevented by treatment with a small phenanthridine, PJ34, while not impaired in similarly treated healthy epithelial cells. NuMA in the cancer cells was phosphorylated by pim1 kinase, and was polyADP-ribosylated by Tankyrase1. These post-translational modifications are inhibited by PJ34. Tankyrase1 is not expressed in healthy epithelial cells, and pim1 kinase was hardly expressed in these cells. Preventing the post-translational modification of NuMA in the epithelial cancer cells also suppressed the protein binding capacity of NuMA. NuMA clustering in the mitotic spindle poles, which stabilizes the mitotic spindle, and is required for the alignment of chromosomes in the spindle mid-zone was impaired. In epithelial cancer cells treated with PJ34, the mitotic spindle poles were distorted, extra-centrosomes were not clustered in multi-centrosomal cancer cells, and chromosomes were dispersed. Chromosomes dispersion violated mitosis termination. Mitosis arrested in the anaphase led to mitotic catastrophe cell death. PJ34 treated epithelial cancer cells were eradicated during 72-96 hours, regardless of their mutations, while treated healthy epithelial cells were not affected, and continued to proliferate. Citation Format: Malka Cohen-Armon. NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication [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 1642.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [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 6487.

Abstract 2874: Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1

Abstract The efficacy of image-guided radiotherapy (RT) has been linked to directing increased chromosomal double strand breaks (DSBs) to tumor cells while sparing surrounding normal tissue. DSBs can block cell cycle progression and promote mitotic catastrophe, cell death and senescence. End processing of persistent DSBs releases single strand DNA (ssDNA), driving cGAS/STING signaling and anti-tumor immune response. DSB repair, whether by non-homologous end-joining (NHEJ) or homologous recombination (HR), is a resistance mechanism, supporting cell survival, repopulation and immune evasion. Decades of effort to block DSB repair to enhance benefits of RT has yet to produce approved agents. Toxicity due to lack of cancer specificity may prevent the current generation of targeted agents from reaching the clinic. An answer may come from examining cancer metabolic reprogramming as a determinant of intrinsic radiation resistance. Along with other glucose metabolites, the Warburg effect increases N-acetyl glucosamine (GlcNAc) biosynthesis, which not only supports N-glycosylation but also modification of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). OGT-dependent O-GlcNAcylation confers radiation resistance, in part by accelerating DSB repair. OGT regulates γH2AX, 53BP1 and BRCA1 foci kinetics and suppresses ssDNA formation at DSB ends, limiting HR but favoring NHEJ for S/G2 DSB repair. Blocking OGT induces radiation sensitization in vitro and in vivo. In cells, OGT inhibition results in hyperresection, persistent RPA and RAD51 foci and accumulation of cytosolic DNA. One mediator may be the histone methyltransferase EZH2, which is rapidly recruited to deposit H3K27me3 at DSBs, promoting NHEJ repair. Irradiation of EZH2 KO cells results in hyperresection that cannot be suppressed by O-GlcNAcylation. Cancer cells may also accumulate NAD+, a substrate for PARP1 incorporated into poly-ADP-ribose to initiate DSB repair. Much like OGT inhibition, blocking PARP1 induces hyperresection and shifts repair toward HR and away from NHEJ. Importantly, increasing O-GlcNAcylation can suppress ssDNA formation even in PARP1 KO cells. In turn, inhibiting PARP1 enhances hyperesection in EZH2 KO cells, leading to a marked increase in cytosolic ssDNA. Our data assign complementary roles for EZH2 and PARP1 in DSB repair pathway choice by independently limiting 5' end resection. This directs DSBs to rapid, imprecise repair by NHEJ over slow and precise repair by HR. An implication is that PARP inhibitor resistance due to the Warburg effect may be mediated by EZH2. Driving hyperresection by targeting O-GlcNAcylation or H3K27 trimethylation along with PARP inhibition may saturate the capacity of HR and produce irreversible damage in cancer cells. Targeting impacts of metabolic reprogramming on DSB repair may not only enhance the therapeutic index of radiation but also sharpen synthetic lethality with HR deficiency. Citation Format: Elena E. Efimova, Sera Averbek, Donald J. Wolfgeher, Ding Wu, Yue Liu, Stephen J. Kron. Cancer metabolism in radiation resistance: Complementary roles of O-GlcNAc transferase and PARP1 [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 2874.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P<0.05) and widespread γH2AX positivity in >95% tumor cells in tumor core and >85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [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 5113.

Abstract 5112: Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor, comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbor focal amplifications of the MYC oncogene and are particularly prone to tumor recurrence and leptomeningeal spread which currently remains incurable. Therefore, there is an urgent need for the development of therapeutic modalities that can spare the vulnerable, developing brain while having potent antitumor efficacy against recurrent MB. Through proteomic profiling of cyclophosphamide-resistant patient-derived MYC-driven Group 3 MB cell line SU_MB002, we identified nuclear envelope (NE) proteins to be highly abundant and enriched in Group 3 MB compared to normal tissue. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the NE, is aberrantly presented to the cell surface in MYC-driven Group 3 MB cell lines. Immunohistochemistry analysis of LBR on MB patient tumor samples identified that positive LBR staining correlated with a significantly worse prognosis and was also identified to be enriched in recurrent tissue compared to their matched primary samples. Cell surface expression flow cytometry analysis identified highly abundant cell surface expression in all MYC-driven Group 3 MB cell lines with low to no expression in human neural stem cells and other MB subgroups. Mechanistically, high resolution microscopy of endogenously HALO-tagged LBR reveals LBR cell surface presentation to be linked to ER or ER-like vesicles that are directly trafficked to the cell surface. Transcriptomic analysis of LBR cell surface positive and negative cells identify that cell surface positivity is significantly linked to cell division and mitotic processes (cell cycle process, mitotic cell cycle process, chromosome segregation, cell division, nuclear division). LBR mislocalization stems from mitosis at the time of nuclear envelope dissolution and is enriched in the hyperproliferative, therapy-resistant subpopulation of MB cells following exposure to chemotherapy and radiation in vitro and in vivo and may act as a marker for the brain tumor initiating cells that seed recurrence. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing relapsed or treatment-refractory MB and have generated single domain antibodies in an effort to engineer LBR-specific CAR T cells. The development of therapies against NE proteins like LBR may provide patients a novel therapeutic opportunity that may be able to treat therapy-resistant, hyperproliferative MYC-driven Group 3 MB cells. Citation Format: Yujin Suk, Jorge Ibañez-Vega, Iqra Chaudhry, Martin Rossotti, Minomi Subapanditha, Petar Miletic, Stefan Custers, Laura Escudero, Alberto Delaidelli, Takuma Nakashima, Yuxi Xiao, Jason Moffat, Hiromichi Suzuki, Poul Sorensen, Kevin Henry, Chitra Venugopal, Giedre Krenciute, Sheila Singh. Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma [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 5112.

Sensitization of cancer cells to paclitaxel-induced apoptosis by canagliflozin

Cancer cells consume more glucose and usually overexpress glucose transporters which have become potential targets for the development of anticancer drugs. It has been demonstrated that selective SGLT2 inhibitors, such as canagliflozin and dapagliflozin, display anticancer activity. Here we demonstrated that canagliflozin and dapagliflozin synergistically enhanced the growth inhibitory effect of paclitaxel in cancer cells including ovarian cancer and oral squamous cell carcinoma cells. Canagliflozin also inhibited glucose uptake via GLUTs. The combination of paclitaxel and WZB117, a GLUT inhibitor, exhibited a strong synergy, supporting the notion that inhibition of GLUTs by canagliflozin may also account for the synergy between canagliflozin and paclitaxel. Mechanistic studies in ES-2 ovarian cancer cells revealed that canagliflozin potentiated paclitaxel-induced apoptosis and DNA damaging effect. Paclitaxel in the nanomolar range elevated abnormal mitotic cells as well as aneuploid cells, and canagliflozin further enhanced this effect. Furthermore, canagliflozin downregulated cyclin B1 and phospho-BUBR1 upon spindle assembly checkpoint (SAC) activation by paclitaxel, and may consequently impair SAC. Thus, paclitaxel disturbed microtubule dynamics and canagliflozin compromised SAC activity, together they may induce premature mitotic exit, accumulation of aneuploid cells with DNA damage, and ultimately apoptosis.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.

The Arabidopsis BUB1/MAD3 family protein BMF3 requires BUB3.3 to recruit CDC20 to kinetochores in spindle assembly checkpoint signaling

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes.

We present novel workflows for Q-FISH nanoscopy with the potential for prognostic applications and resolving novel chromatin compaction changes. DNA-fluorescence in situ hybridization (DNA-FISH) is a routine application to visualize telomeres, repetitive terminal DNA sequences, in cells and tissues. Telomere attrition is associated with inherited and acquired diseases, including cancer and cardiomyopathies, and is frequently analyzed by quantitative (Q)-FISH microscopy. Recently, nanoscopic imaging techniques have resolved individual telomere dimensions and their compaction as a prognostic marker, in part leading to conflicting conclusions still unresolved to date. Here, we developed a comprehensive Q-FISH nanoscopy workflow to assess telomeres with PNA telomere probes and 3D-Stimulated Emission Depletion (STED) microscopy combined with Dynamic Intensity Minimum (DyMIN) scanning. We achieved single-telomere resolution at high, unprecedented telomere coverage. Importantly, our approach revealed a decrease in telomere signal density during mitotic cell division compared to interphase. Innovatively expanding FISH-STED applications, we conducted double FISH targeting of both telomere- and chromosome-specific sub-telomeric regions and accomplished FISH-STED in human cardiac biopsies. In summary, this work further advanced Q-FISH nanoscopy, detected a new aspect of telomere compaction related to the cell cycle, and laid the groundwork for future applications in complex cell types such as post-mitotic neurons and muscle cells.

Induction of somatic cell haploidy by premature cell division.

Canonical mitotic and meiotic cell divisions commence with replicated chromosomes consisting of two sister chromatids. Here, we developed and explored a model of premature cell division, where nonreplicated, G0/G1-stage somatic cell nuclei are transplanted to the metaphase cytoplasm of mouse oocytes. Subsequent cell division generates daughter cells with reduced ploidy. Unexpectedly, genome sequencing analysis revealed proper segregation of homologous chromosomes, resulting in complete haploid genomes. We observed a high occurrence of somatic genome haploidization in nuclei from inbred genetic backgrounds but not in hybrids, emphasizing the importance of sequence homology between homologs. These findings suggest that premature cell division relies on mechanisms similar to meiosis I, where genome haploidization is facilitated by homologous chromosome interactions, recognition, and pairing. Unlike meiosis, no evidence of recombination between somatic cell homologs was detected. Our study offers an alternative in vitro gametogenesis approach by directly reprogramming diploid somatic cells into haploid oocytes.

A Study of Hyaluronic Acid's Theoretical Reactivity and of Magnetic Nanoparticles Capped with Hyaluronic Acid.

Hyaluronic acid (HA) has attracted much attention in tumor-targeted drug delivery due to its ability to specifically bind to the CD44 cellular receptor, which is widely expressed on cancer cells. We present HA-capped magnetic nanoparticles (HA-MNPs) obtained via the co-precipitation method, followed by the electrostatic adsorption of HA onto the nanoparticles' surfaces. A theoretical study carried out with the PM3 method evidenced a dipole moment of 3.34 D and negatively charged atom groups able to participate in interactions with nanoparticle surface cations and surrounding water molecules. The ATR-FTIR spectrum evidenced the hyaluronic acid binding to the surface of the ferrophase, ensuring colloidal stability in the water dispersion. To verify the success of the synthesis and stabilization, HA-MNPs were also characterized using other investigation techniques: TEM, EDS, XRD, DSC, TG, NTA, and VSM. The results showed that the HA-MNPs had a mean physical size of 9.05 nm (TEM investigation), a crystallite dimension of about 8.35 nm (XRD investigation), and a magnetic core diameter of about 8.31 nm (VSM investigation). The HA-MNPs exhibited superparamagnetic behavior, with the magnetization curve showing saturation at a high magnetic field and a very small coercive field, corresponding to the net dominance of single-domain magnetic nanoparticles that were not aggregated with reversible magnetizability. These features satisfy the requirement for magnetic nanoparticles with a small size and good dispersibility for long-term stability. We performed some preliminary tests regarding the nanotoxicity in the environment, and some chromosomal aberrations were found to be induced in corn root meristems, especially in the anaphase and metaphase of mitotic cells. Due to their properties, HA-MNPs also seem to be suitable for use in the biomedical field.

The fission yeast NDR kinase Orb6 and its signalling pathway MOR regulate cytoplasmic microtubule organization during the cell cycle.

Microtubule organization and reorganization during the cell cycle are achieved by regulation of the number, distribution and activity of microtubule-organizing centres (MTOCs). In fission yeast, the Mto1/2 complex determines the activity and distribution of cytoplasmic MTOCs. Upon mitosis, cytoplasmic microtubule nucleation ceases; inactivation of the Mto1/2 complex is triggered by Mto2 hyperphosphorylation. However, the protein kinase(s) that phosphorylates Mto2 remains elusive. Here we show that a conserved signalling network, called MOR (morphogenesis Orb6 network) in fission yeast, negatively regulates cytoplasmic MTOCs through Mto2 phosphorylation to ensure proper microtubule organization. Inactivation of Orb6 kinase, the most downstream MOR component, by attenuation of MOR signalling leads to reduced Mto2 phosphorylation, coincident with increased number of both Mto2 puncta and cytoplasmic microtubules. These defects cause the emergence of uncoordinated mitotic cells with cytoplasmic microtubules, resulting in reduced spindle assembly. Thus, the regulation of Mto2 by the MOR is crucial for cytoplasmic microtubule organization and contributes to reorganization of the microtubule cytoskeletons during the cell cycle.

The molecular basis of cell memory in mammals: The epigenetic cycle.

Cell memory refers to the capacity of cells to maintain their gene expression program once the initiating environmental signal has ceased. This exceptional feature is key during the formation of mammalian organisms, and it is believed to be in part mediated by epigenetic factors that can endorse cells with the landmarks required to maintain transcriptional programs upon cell duplication. Here, we review current literature analyzing the molecular basis of epigenetic memory in mammals, with a focus on the mechanisms by which transcriptionally repressive chromatin modifications such as methylation of DNA and histone H3 are propagated through mitotic cell divisions. The emerging picture suggests that cellular memory is supported by an epigenetic cycle in which reversible activities carried out by epigenetic regulators in coordination with cell cycle transition create a multiphasic system that can accommodate both maintenance of cell identity and cell differentiation in proliferating stem cell populations.

Mitosis targeting in non-small lung cancer cells by inhibition of PAD4

PAD4 expression and activity were significantly up-regulated in lung cancer tissues suggesting that PAD4 could be a possible target for lung cancer treatment. In this study we had demonstrated that PAD4 expression was higher in lung cancer patients whom with lymphnode metastasis and pleural invasion. Inhibiting PAD4 with a small molecular inhibitor could induce apoptosis and suppress growth in lung cancer cells. We used RNA-sequencing to further investigate transcriptional changes that induced by PAD4 inhibition, and results suggested its affected mostly on the cell cycle, mitotic cell cycle process, p53 signaling pathway. By using image flow cytometry analysis, we found that PAD4 inhibited by YW3-56 could accumulate cells in the G1/G0 phases and reducing the fraction of G2/M and S phase cells. Quantification of different phase of mitosis in cells treated with YW3-56 revealed an increasing trend of telophase and prophase cells. Taken together, our data indicated that PAD4 inhibitor could affect cell cycle and mitosis of lung cancer cells, and targeting PAD4 could be a promising strategy for discovery novel anti-NSCLC treatments.

Glypican-1-targeted antibody–drug conjugate inhibits the growth of glypican-1-positive glioblastoma

Glioblastoma is the deadliest form of brain tumor. The presence of the blood–brain barrier (BBB) significantly hinders chemotherapy, necessitating the development of innovative treatment options for this tumor. This report presents the in vitro and in vivo efficacy of an antibody–drug conjugate (ADC) that targets glypican-1 (GPC1) in glioblastoma. The GPC1-ADC was created by conjugating a humanized anti-GPC1 antibody (clone T2) with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl linkers. Immunohistochemical staining analysis of a glioblastoma tissue microarray revealed that GPC1 expression was elevated in more than half of the cases. GPC1-ADC, when bound to GPC1, was efficiently and rapidly internalized in glioblastoma cell lines. It inhibited the growth of GPC1-positive glioma cell lines by inducing cell cycle arrest in the G2/M phase and triggering apoptosis in vitro. We established a heterotopic xenograft model by subcutaneously implanting KALS-1 and administered GPC1-ADC intravenously. GPC1-ADC significantly inhibited tumor growth and increased the number of mitotic cells. We also established an orthotopic xenograft model by intracranially implanting luciferase-transfected KS-1-Luc#19. After injecting Evans blue and resecting brain tissues, dye leakage was observed in the implantation area, confirming BBB disruption. We administered GPC1-ADC intravenously and measured the luciferase activity using an in vivo imaging system. GPC1-ADC significantly inhibited tumor growth and extended survival. In conclusion, GPC1-ADC demonstrated potent intracranial activity against GPC1-positive glioblastoma in an orthotopic xenograft model. These results indicate that GPC1-ADC could represent a groundbreaking new therapy for treating glioblastoma beyond the BBB.

Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

SAS-6 (SASS6) is essential for centriole formation in human cells and other organisms but its functions in the mouse are unclear. Here, we report that Sass6-mutant mouse embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In contrast, SAS-6 is not required for centriole formation in mouse embryonic stem cells (mESCs), but is essential to maintain centriole architecture. Of note, centrioles appeared after just one day of culture of Sass6-mutant blastocysts, from which mESCs are derived. Conversely, the number of cells with centrosomes is drastically decreased upon the exit from a mESC pluripotent state. At the mechanistic level, the activity of the master kinase in centriole formation, PLK4, associated with increased centriolar and centrosomal protein levels, endow mESCs with the robustness in using a SAS-6-independent centriole-biogenesis pathway. Collectively, our data suggest a differential requirement for mouse SAS-6 in centriole formation or integrity depending on PLK4 activity and centrosome composition.