Promotes Cell Cycle Arrest Research Articles

ANTICANCER EFFECTS OF DIFFERENT FATTY ACID SYNTHASE INHIBITORS ON ORAL CANCER

<h3>Objectives</h3> Overexpression of fatty acid synthase (FASN) is associated with aggressiveness, poor prognosis, and risk of metastasis in several human cancers, including oral squamous cell carcinomas (OSCC). Here, we compared the in vitro effects of 3 FASN inhibitors on malignant phenotypes of the OSCC cell line SCC-9. <h3>Study Design</h3> SCC-9 cells were treated with C75, triclosan (TCS), and orlistat (ORL) in their respective IC12.5, IC25, and IC50. We analyzed proliferation using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, cell cycle, and apoptosis by flow cytometry. Cell migration and invasion were evaluated by transwell inserts and adhesion using type I collagen or fibronectin. <h3>Results</h3> C75 showed the lowest IC50, whereas ORL presented the highest IC50. Each drug promoted distinct morphologic changes in SCC-9 cells and was able to significantly reduce FASN activity. All drugs promoted cell cycle arrest in G0/G1 and induced apoptosis at IC50. C75 reduced cell migration and invasion and increased the adhesion of SCC-9 cells. TCS reduced adhesion, migration, and invasion of SCC-9 cells. ORL reduced cell migration but did not significantly modify the adhesion and invasion of SCC-9 cells. <h3>Conclusions</h3> FASN inhibitors affected significant steps of tumor progression, and TCS showed better anticancer effects on SCC-9 cells.

Ganoderic acid D prevents oxidative stress-induced senescence by targeting 14-3-3ε to activate CaM/CaMKII/NRF2 signaling pathway in mesenchymal stem cells.

Stem cell senescence is an important cause of aging. Delaying senescence may present a novel way to combat aging and age‐associated diseases. This study provided a mechanistic insight into the protective effect of ganoderic acid D (GA‐D) against human amniotic mesenchymal stem cell (hAMSCs) senescence. GA‐D, a Ganoderma lucidum‐derived triterpenoid, markedly prevented hAMSCs senescence via activating the Ca2+ calmodulin (CaM)/CaM‐dependent protein kinase II (CaMKII)/nuclear erythroid 2‐related factor 2 (Nrf2) axis, and 14‐3‐3ε was identified as a target of GA‐D. 14‐3‐3ε‐encoding gene (YWHAE) knockdown in hAMSCs reversed the activation of the CaM/CaMKII/Nrf2 signals to attenuate the GA‐D anti‐aging effect and increase senescence‐associated β‐galactosidase (SA‐β‐gal), p16 and p21 expression levels, including reactive oxygen species (ROS) production, thereby promoting cell cycle arrest and decreasing differentiation potential. YWHAE overexpression maintained or slightly enhanced the GA‐D anti‐aging effect. GA‐D prevented d‐galactose‐caused aging in mice by significantly increasing the total antioxidant capacity, as well as superoxide dismutase and glutathione peroxidase activity, and reducing the formation of malondialdehyde, advanced glycation end products, and receptor of advanced glycation end products. Consistent with the protective mechanism of GA‐D against hAMSCs senescence, GA‐D delayed the senescence of bone‐marrow mesenchymal stem cells in this aging model in vivo, reduced SA‐β‐gal and ROS production, alleviated cell cycle arrest, and enhanced cell viability and differentiation via regulating 14‐3‐3ε and CaM/CaMKII/Nrf2 axis. Therefore, GA‐D retards hAMSCs senescence by targeting 14‐3‐3ε to activate the CaM/CaMKII/Nrf2 signaling pathway. Furthermore, the in vivo GA‐D anti‐aging effect may involve the regulation of stem cell senescence via the same signal axis.

Phytochemical-rich Fractions from Foxtail Millet (Setaria italica (L.) P. Beauv) Seeds Exhibited Antioxidant Activity and Reduced the Viability of Breast Cancer Cells In Vitro by Inducing DNA Fragmentation and Promoting Cell Cycle Arrest.

The demand for millet-based diets has increased significantly in recent years due to their beneficial effects on human health. Foxtail Millet (Setaria italica (L.) P. Beauv, previously known as Panicum italicum L., referred as FTM in this manuscript) seeds have been not only used as astringent and diuretic agents, but they are also used to treat dyspepsia and rheumatism. Recent studies have shown that solvent extracts from FTM seeds exhibited antioxidant and antiinflammatory activities. However, the nature and antiproliferative potential of phytochemical constituents of solvent extracts are not much explored. Major objectives of this study are to generate and characterize the phytochemical-rich fractions from Foxtail millet seeds, test the antioxidant activity, and antiproliferative potential against cell lines representing carcinomas of the breast, and determine the mechanisms of cell growth inhibition. Phytochemical-rich fractions were generated by extracting the seeds using 70% ethanol (FTM-FP) and 10% alkali (FTM-BP). Antioxidant potential was determined by ferric reducing antioxidant power (FRAP) assay and DPPH radical scavenging activity assays. The antiproliferative potential was determined using sulforhodamine-B assay. The impact on cell cycle and DNA fragmentation was analyzed by staining the cells with DAPI followed by analyzing the stained cells using NC-3000. Analysis of the results showed the presence of phenolics and flavonoids in the FTM-FP and FTM-BP fractions. Both fractions exhibited antiproliferative potential against breast cancer cell lines. Mechanistically, both fractions induced G2/M cell cycle arrest and increased the fragmentation of DNA, which lead to the accumulation of cells in the Sub-G1 phase. In summary, results of this study demonstrated the potential of foxtail millet phytochemical fractions for retarding the proliferative potential of breast cancer cells.

Cucumber-Derived Nanovesicles Containing Cucurbitacin B for Non-Small Cell Lung Cancer Therapy.

PurposeIn recent years, a variety of nanoparticles with excellent anticancer and delivery properties have emerged for cancer therapy. However, potential toxicity, high production cost and complex preparation procedures have been obstacles to their use in biomedicine. Here, we obtained cucumber-derived nanovesicles (CDNVs) at high yield and low cost by simple juicing and ultracentrifugation. The anticancer effects of CDNVs were evaluated in vitro and in vivo.MethodsTransmission electron microscope, nanoparticle tracking analysis and laser particle size analysis were used to characterize the morphology, diameter and zeta potential of CDNVs, respectively. The anticancer effects of CDNVs in vitro were evaluated by MTT and apoptosis assays. The mechanism was further explored by measuring the protein levels of signal transducer and activator of transcription 3 pathway, reactive oxygen species, cell cycle distribution and caspase activity. In-vivo anticancer efficacy was evaluated by measuring tumor volume and weight of mice in three different treatment groups (CDNVs, cucurbitacin B and PBS).ResultsCDNVs inhibited proliferation of human non-small cell lung cancer cells by suppressing signal transducer and activator of transcription 3 activation, generating reactive oxygen species, promoting cell cycle arrest, and activating the caspase pathway. These CDNVs exhibited strong anticancer effects both in vitro and in vivo, and reduced the rate of tumor growth without obvious toxicity to mouse visceral organs. Compared with an equivalent dose of cucurbitacin B, CDNVs exerted stronger anticancer effects in vitro and in vivo.ConclusionThese results demonstrate that CDNVs suppress tumor growth. This study addresses the development of cancer therapeutic drugs using plant-derived nanovesicles that are cost-efficient, simple to produce in high yields, and provide an alternative approach to drug isolation that may help advance sustainability of medicinal plants.

Effects of Pennogenin 3-O-β-Chacotrioside, a Steroidal Saponin from Rhizoma paridis, on Proliferation Inhibition and Apoptosis Induction in Non-Small Cell Lung Cancer Cells

Rhizoma paridis is a traditional Chinese medicine that has been reported to have anti-cancer activity. However, the antitumor effect of pennogenin 3-O-β-chacotrioside (Formular: C45H72O17), a steroidal saponin isolated from its extract, in lung cancer have not been well studied, its potential mechanisms remain unclear. In this paper, its effect on the cell viability of non-small cell lung cancer (NSCLC) was examined using the Cell Counting Kit-8 assay, the potential target proteins were screened by antibody microarrays, the potential pharmacological mechanisms were predicted using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein–protein interaction (PPI) analyses. Moreover, to further validate the findings, cell apoptosis and cell cycle arrest were measured by flow cytometry, cell invasion and migration were measured by Transwell assays, and apoptosis-related protein expression levels were measured using western blotting. The results showed that, after treatment by pennogenin 3-O-β-chacotrioside, the cell viability was significantly reduced. In addition, thirteen target proteins related to apoptosis and platinum drug resistance were screened out, and their PPIs were constructed. Moreover, it was confirmed to have effects in inducing apoptosis and promoting cell cycle arrest in NSCLC cells. The expression levels of apoptosis-related proteins, such as caspase 8, caspase 9, phospho-ERK1/2, phospho-p38, phospho-Akt, were decreased following the treatment, whereas the expression levels of cleaved caspase 3 and cytochrome C were increased. The findings of the present study highlight the potential of pennogenin 3-O-β-chacotrioside as a novel therapeutic agent.

Circular RNA ZFR promotes cell cycle arrest and apoptosis of colorectal cancer cells via the miR-147a/CACUL1 axis.

BackgroundColorectal cancer (CC) is one of the most prevalent malignancies worldwide. Nonetheless, its pathogenicity and molecular mechanisms have not been completely elucidated yet. The potential clinical value of circular RNAs (circRNAs) in tumor diagnosis, treatment, and prognosis has received considerable attention.MethodsHere, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) examined the levels of circular ZFR (circZFR) in CC cells. The expression of circZFR was knocked down in CC cells and cell viability was detected using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cell cycle progression was assessed by flow cytometry and the expression levels of cyclin-associated proteins were detected by western blot analysis. The transferase dUTP nick end labeling (TUNEL) assay was used to detect apoptosis and western blot analysis was used to evaluate the expression levels of apoptosis-associated proteins. Subsequently, the interactions between circZFR and microRNA (miR)-147a and between miR-147a and CDK2 associated cullin domain 1 (CACUL1) were predicted by the Encyclopedia of RNA Interactomes database and verified by luciferase reporter assays. Finally, plasmid transfection, CCK-8, and flow cytometry assays were used to explore the associated mechanism of action.ResultsCircZFR was highly expressed in CC cell lines. Interference with its expression inhibited proliferation and induced G1/S cell cycle arrest and apoptosis in CC cells. The expression levels of miR-147a and CACUL1 were decreased and increased, respectively, in CC cells. These data demonstrated that circZFR could target miR-147a and CACUL1 to regulate the cell cycle and apoptosis of CC cells and, ultimately, promote the progression of CC.ConclusionsKnockdown of the expression of circZFR upregulated miR-147a expression and reduced CACUL1 expression levels, thereby inhibiting the proliferation of CC cells and inducing cell cycle arrest and apoptosis.

Yohimbine, an α2-Adrenoceptor Antagonist, Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway.

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.

CUDC907, a dual phosphoinositide-3 kinase/histone deacetylase inhibitor, promotes apoptosis of NF2 Schwannoma cells.

Neurofibromatosis Type 2 (NF2) is a rare tumor disorder caused by pathogenic variants of the merlin tumor suppressor encoded by NF2. Patients develop vestibular schwannomas (VS), peripheral schwannomas, meningiomas, and ependymomas. There are no approved drug therapies for NF2. Previous work identified phosphoinositide-3 kinase (PI3K) as a druggable target. Here we screened PI3K pathway inhibitors for efficacy in reducing viability of human schwannoma cells. The lead compound, CUDC907, a dual histone deacetylase (HDAC)/PI3K inhibitor, was further evaluated for its effects on isolated and nerve-grafted schwannoma model cells, and primary VS cells. CUDC907 (3 nM IG50) reduced human merlin deficient Schwann cell (MD-SC) viability and was 5–100 fold selective for MD over WT-SCs. CUDC907 (10 nM) promoted cell cycle arrest and caspase-3/7 activation within 24 h in human MD-SCs. Western blots confirmed a dose-dependent increase in acetylated lysine and decreases in pAKT and YAP. CUDC907 decreased tumor growth rate by 44% in a 14-day treatment regimen, modulated phospho-target levels, and decreased YAP levels. In five primary VS, CUDC907 decreased viability, induced caspase-3/7 cleavage, and reduced YAP levels. Its efficacy correlated with basal phospho-HDAC2 levels. CUDC907 has cytotoxic activity in NF2 schwannoma models and primary VS cells and is a candidate for clinical trials.

Anticancer effects of natural phytochemicals in anaplastic thyroid cancer (Review).

Anaplastic thyroid cancer (ATC) is an aggressive and lethal malignancy having a dismal prognosis. Phytochemicals are bioactive components obtained from plants that have been proven useful to treat numerous diseases. Phytochemicals are also an important source of novel anticancer drugs and an important area of research due to the numerous available candidates that can potentially treat cancers. This review discusses naturally occurring phytochemicals and their derivatives that show promising anticancer effects in anaplastic thyroid cancer. Anticancer effects include cell growth inhibition, induction of apoptosis, promoting cell cycle arrest, suppressing angiogenesis, modulating autophagy, and increasing the production of reactive oxygen species. Phytochemicals are not only prospective candidates in the therapy of anaplastic thyroid cancer but also exhibit potential as adjuvants to improve the anticancer effects of other drugs. Although some phytochemicals have excellent anticancer properties, drug resistance observed during the use of resveratrol and artemisinin in different anaplastic thyroid cancer cell lines is still a problem. Anaplastic thyroid cancer cells have several biological, clinical, and drug-resistance features that differ from differentiated thyroid cancer cells. Phytochemicals such as resveratrol and quercetin exhibit different biological effects in anaplastic thyroid cancer and differentiated thyroid cancer. Tumor cells depend on increased aerobic glycolysis by mitochondrial oxidative phosphorylation to provide energy for their rapid growth, invasiveness, and drug resistance. Phytochemicals can alter signaling cascades, modulate the metabolic properties of cancer cells, and influence the mitochondrial membrane potential of anaplastic thyroid cancer cells. These findings enrich our knowledge of the anticancer effects of phytochemicals and highlight alternative therapies to prevent drug resistance in anaplastic thyroid cancer.

Co-targeting of specific epigenetic regulators in combination with CDC7 potently inhibit melanoma growth

SummaryMelanoma is a highly aggressive skin cancer that frequently metastasizes, but current therapies only benefit some patients. Here, we demonstrate that the serine/threonine kinase cell division cycle 7 (CDC7) is overexpressed in melanoma, and patients with higher expression have shorter survival. Transcription factor ELK1 regulates CDC7 expression, and CDC7 inhibition promotes cell cycle arrest, senescence, and apoptosis, leading to inhibition of melanoma tumor growth and metastasis. Our chemical genetics screen with epigenetic inhibitors revealed stronger melanoma tumor growth inhibition when XL413 is combined with the EZH2 inhibitor GSK343 or BRPF1/2/3 inhibitor OF1. Mechanistically, XL413 with GSK343 or OF1 synergistically altered the expression of tumor-suppressive genes, leading to higher apoptosis than the single agent alone. Collectively, these results identify CDC7 as a driver of melanoma tumor growth and metastasis that can be targeted alone or in combination with EZH2 or BRPF1/2/3 inhibitors.

Statins inhibit proliferation and induce apoptosis in triple-negative breast cancer cells

TP53 (p53) is mutated in 80–90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines (atorvastatin, p < 0.01; simvastatin p < 0.05) indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker.

Cannabidiol Antiproliferative Effect in Triple-Negative Breast Cancer MDA-MB-231 Cells Is Modulated by Its Physical State and by IGF-1.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.

MiRNA-651-3p regulates EMT in ovarian cancer cells by targeting ZNF703 and via the MEK/ERK pathway

miRNAs are non-coding single-stranded RNA molecules with many functions. Several miRNAs have been found to be dysregulated in ovarian cancer; however, the role of miR-651-3p in ovarian cancer remains unknown. Here, the expression level of miR-651-3p in ovarian tissue samples was determined via qRT-PCR, and then miR-651-3p was overexpressed and downregulated to study the functional changes in ovarian cancer cells. Based on previous research and database predictions, we analyzed the binding and regulatory effects of miR-651-3p on zinc finger protein 703 (ZNF703). We additionally evaluated the effect of miR-651-3p on epithelial–mesenchymal transition (EMT) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways in ovarian cancer cells. We found that miR-651-3p was downregulated in ovarian cancer tissues. miR-651-3p expression was associated with inhibited proliferation, invasion, and migration of ovarian cancer cells and promoted cell cycle arrest. Additionally, miR-651-3p was found to target ZNF703 and affect EMT in ovarian cancer by activating the MEK/ERK signaling pathway. MiR-651-3p was downregulated in ovarian cancer, and suppressed the malignant biological behavior of ovarian cancer by inhibiting ZNF703 and the MEK/ERK pathway. Our findings on miR-651-3p provided new insights for the diagnosis and treatment of ovarian cancer.

Abstract 5354: 3-phosphoinositide-dependent kinase-1 (PDK1, PDPK1) is a driver of osimertinib acquired resistance in EGFR mutant NSCLC

Abstract Osimertinib, the only third-generation EGFR-TKI, showed incomplete responses to T790M-mutant NSCLC due to acquired resistance caused by activation of bypass pathways. We developed osimertinib-acquired resistant H1975-OSIR (T790M/L858R mutant) isogenic cells and TC386-OSIR isogenic PDXs. Neither H1975-OSIR nor TC386-OSIR PDXs developed additional mutations in EGFR. The H1975-OSIR clone showed 100 fold higher resistance to osimertinib compared with H1975 cells. TC386-OSIR PDX was developed through continuous in-vivo treatment for 8 months and the residual PDXs were passaged for several generations under continuous osimertinib treatment. TC386-OSIR fourth resistant generation (RG4) showed significantly higher resistance than initial generations (RG1). H1975-OSIR xenografts were developed in non-humanized and humanized NSG mice under osimertinib pressure. H1975-OsiR tumors were significantly less sensitive to osimertinib than their parental counterparts in both mouse models. Dose dependent antitumor activity of osimertinib (5mg/kg and 10mg/kg) was observed in H1975-parental tumors, whereas no treatment effect was observed for H1975-OsiR tumors with increasing doses. The tumor microenvironment was enriched with higher infiltration of tumor associated macrophages (TAM) and lower numbers of tumor infiltrating lymphocytes (TIL) in H1975-OSIR vs H1975 tumors. RPPA analysis of residual tumor tissues showed a distinct set of proteins upregulated in H1975-OsiR vs H1975-parental, among which PDK1 was the most upregulated. PDK1 was also significantly upregulated in H1975-OsiR tumors treated with osimertinib vs controls. PDK1 was not altered in any treatment groups in H1975-parental tumors. PDK1 and pPDK1 expression was many-fold higher in both H1975-OSIR cells and TC386-OSIR PDXs as compared to their parental counterparts by western blot and mass spec proteomics. Selective inhibition by the PDK inhibitor, BX 795, and CRISPR knock-out (KO) restored osimertinib sensitivity in resistant cells. Colony forming assays showed that the PDK1 KO clone was as sensitive as H1975-parental cells whereas a PDK overexpressing clone (OE) restored resistance. In-vivo inhibition of PDK1 by treating mice with BX-795 in both H1975-OSIR xenografts and TC386-OSIR PDXs significantly enhanced the antitumor activity of osimertinib. PDK1 KO dysregulated PI3K/Akt/mTOR signaling by downregulating Akt and mTOR phosphorylation and promoted cell cycle arrest at the G1 phase. NCI-H1975-OSIR and PDK1 OE cells showed a high level of nuclear localization of the activated Yes-associated protein pYAP(Y357). PDK1 KO cells significantly reduced nuclear localization of pYAP(Y357). The level of YAP and pYAP was upregulated in osimertinib resistant xenograft tumors and residual tumor biopsies. Taken together, we identified PDK1 as a drug able target to treat osimertinib acquired resistance. Citation Format: Ismail M. Meraz, Mourad Majidi, Bingliang Fang, Feng Meng, Lihui Gao, RuPing Shao, Renduo Song, Feng Li, Min Jin Ha, Qi Wang, Jing Wang, Elizabeth Shpall, Sung Yun Jung, Franziska Haderk, Philippe Gui, Jonathan W. Riess, Victor Olivas, Trever G. Bivona, Jack A. Roth. 3-phosphoinositide-dependent kinase-1 (PDK1, PDPK1) is a driver of osimertinib acquired resistance in EGFR mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5354.

Abstract 344: Blockade of retrograde transport in triple negative breast cancer excludes ErbB-2 isoforms from the nucleus and abrogates tumor growth

Abstract Triple negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of the receptor tyrosine kinase ErbB-2/HER2. Due to its heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive, and chemotherapy has been the standard of care for early and metastatic TNBC. ErbB-2 is classically located at the membrane of BC cells, where it triggers signalling cascades and promotes oncogenesis. However, we have demonstrated that ErbB-2 is also localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). ErbB-2 migrates to the nucleus via retrograde transport. The small molecule Retro-2 is a non-toxic inhibitor of the retrograde transport route that protects cells from the deleterious effects of toxins and viruses. Here, we revealed that Retro-2 evicts both WTErbB-2 and ErbB-2c from the nuclei. Using BC models from several molecular subtypes, we demonstrated that Retro-2 specifically halts the proliferation of cells expressing NErbB-2 in a dose-dependent manner, whilst did not inhibit cell proliferation in the ErbB-2-negative MCF10A normal breast cell line. Additionally, Retro-2 decreased the expression of genes induced by NErbB-2 (cyclin D1 and Erk5) and promoted cell cycle arrest at G0/G1 phase and apoptosis. Even more, in preclinical models (including xenografts and tumor explants), Retro-2 treatment resulted in the eviction of NErbB-2 and abrogation of tumor growth. Our mechanistic studies demonstrated that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and plasma membrane, and of ErbB-2c at the Golgi, further preventing its sorting to the endoplasmic reticulum. These findings shed light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport and on the biology of ErbB-2 splicing variants. Together, our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC. Citation Format: Santiago Madera, Franco Izzo, Maria F. Chervo, Agustina Dupont, Violeta A. Chiauzzi, Sofia Bruni, Ezequiel Petrillo, Diego Montero, Sharon Merin, Maria F. Mercogliano, Cecilia J. Proietti, Roxana Schillaci, Rosalia I. Cordo Russo, Patricia V. Elizalde. Blockade of retrograde transport in triple negative breast cancer excludes ErbB-2 isoforms from the nucleus and abrogates tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 344.

PCDHB14 promotes ferroptosis and is a novel tumor suppressor in hepatocellular carcinoma.

Liver cancer, a result of multifactorial interplay between heredity and the environment, is one of the leading causes of cancer-related death worldwide. Hepatocellular carcinoma (HCC) is the most common histologic type of primary liver cancer. Here, we reported that deficiency in PCDHB14, a member of the cadherin superfamily, participates in the progression of HCC. We found that PCDHB14 is inactivated by aberrant methylation of its promoter in HCC patients and that PCDHB14 functions as a tumor suppressor to promote cell cycle arrest, inhibit cell proliferation, and induce ferroptosis. Furthermore, PCDHB14 ablation dramatically enhanced diethylenenitrite-induced HCC development. Mechanistically, PCDHB14 is induced by p53, and increased PCDHB14 downregulates the expression of SLC7A11, which is critical for ferroptosis. This effect is mediated by accelerated p65 protein degradation resulting from PCDHB14 promoting E3 ubiquitin ligase RNF182-mediated ubiquitination of p65 to block p65 binding to the promoter of SLC7A11. This study reports the new discovery that PCDHB14 serves as a potential prognostic marker for HCC.

ZNF582 promoter methylation predicts cervical cancer radiosensitivity and ZNF582 protein overexpression reduces radiosensitivity by cell cycle arrest in S phase

ABSTRACT This study aimed to investigate the relationship between ZNF582 promoter methylation (ZNF582m ) level and radiosensitivity of cervical cancer and its biological basis. This was a prospective multicenter clinical study, comprising two independent cohorts of locally advanced cervical cancer patients. Exfoliated cervical cells were collected at 0, 24, 30, 36, 48, and 64 Gy to test ZNF582m levels. Radiotherapy response was evaluated according to RECIST Version. RT-PCR and WT were used to detect the mRNA and protein expression levels; MTT and flow cytometry were used to detect the cell viability and cell cycle, respectively. While clone formation and subcutaneous tumorigenesis in nude mice were used to detect the growth of HeLa cells with/without ZNF582 overexpression. In the first cohort, 22 cases achieved complete remission (CR) or partial response (PR), and the other 28 cases exhibited stable disease (SD). Radiotherapy reduced ZNF582m levels among all patients. Initial lever of ZNF582m was significantly higher in the Responder (CR + PR) group than in the SD group. Also, patients with higher initial lever ZNF582m were more sensitive towards radiotherapy than ZNF582m- low patients. The second cohort confirmed the above results. The amplitude of ZNF582m levels were related to the radiotherapeutic response; some patients of ZNF582m- low showed a transient increase in ZNF582m , and present greater radiosensitivity than other ZNF582m- low patients. In vitro, ZNF582 protein overexpression promoted cell cycle arrest in S phase. These results suggested that higher ZNF582m levels predicted greater radiosensitivity in clinical cervical cancer cases. Overexpressed ZNF582 conferred radioresistance by cell cycle arrest in vitro.

Deferasirox shows inhibition activity against cervical cancer in vitro and in vivo

ObjectiveIron depletion may be a novel therapeutic strategy for cancer. This study aimed to assess the inhibition effects of deferasirox (DFX), an oral iron chelator, on cervical cancer. MethodsIn this study, we performed immunohistochemical analysis, enzyme-linked immunoassay, cell viability and invasive ability assay, cell cycle and apoptosis analysis, protein expression investigation, molecular mechanism investigation, and in vivo murine xenograft model to evaluate the impact of DFX on cervical cancer. ResultsThe cervical cancer cell lines viability decreased and cell apoptosis was induced after DFX incubation. Additionally, DFX promoted cell cycle arrest by regulating the expression of cell cycle regulators cyclin D1, cyclin E and proliferating cell nuclear antigen (PCNA) in cervical cancer cell lines. DFX also decreased cell invasion by upregulating the expression of NDRG1 and downregulating c-Myc. The activation of Akt and the MEK/ERK signaling pathway was inhibited by DFX. DFX also significantly suppressed xenograft tumor growth, decreased the levels of ferritin in serum and tumor tissue, reduced iron deposits and reactive oxygen species (ROS) levels in xenografts of DFX-treated group compared with the control group, with no serious side effects. ConclusionPresent study demonstrated the inhibitory effect of DFX against cervical cancer, and provided a potential therapeutic agent for cervical cancer.

MicroRNA-30c-2-3p represses malignant progression of gastric adenocarcinoma cells via targeting ARHGAP11A

ABSTRACT MicroRNAs are crucial tumor regulators to tumor development and progression. MiR-30c-2-3p can suppress malignant progression of tumor cells, but no study has reported the modulatory process of miR-30c-2-3p in gastric adenocarcinoma (GA). We herein investigated role of miR-30c-2-3p in GA cells. Here, we evaluated gene level in cancer cells by qRT-PCR. CCK-8, colony formation, flow cytometry, and transwell assays revealed biological functions of miR-30c-2-3p and ARHGAP11A. Genes downstream of miR-30c-2-3p were acquired through bioinformatics analysis. Our results suggested a low level of miR-30c-2-3p in GA tissue and cells, while its high expression could repress the malignant progression and promote cell cycle arrest and apoptosis of GA cells. Besides, ARHGAP11A was downstream of miR-30c-2-3p, with up-regulated ARHGAP11A facilitating malignant progression and repressing cell cycle arrest and apoptosis of GA cells. In addition, changes in GA cell functions caused by high ARHGAP11A expression could be partially offset by enhancing miR-30c-2-3p. Thus, our observations indicated that miR-30c-2-3p was a tumor repressor that could inhibit GA progression via modulating ARHGAP11A.

Identifying Deubiquitinase Regulating the DNA Damage Response Pathway

In response to DNA damage, cells often activate Mec1‐dependent DNA damage checkpoint, resulting in the activation of the effector kinase Rad53 that promotes cell cycle arrest and DNA repair. The checkpoint proteins and the DNA damage response pathway are often regulated by ubiquitination, a reversible post‐translational modification, raising the possibility that checkpoint activation may be regulated by a deubiquitinating enzyme. To test this, we screened a collection of yeast deubiquitinating enzyme deletion mutants and monitored their responses to DNA damage agents. Interestingly, we find that one deubiquitinating enzyme deletion mutant displayed consistently decreased levels of checkpoint activation. Further analysis indicated that this mutant does not affect either the abundance or the stability of checkpoint activators, but it rather has a profound effect on Mec1 itself. This analysis suggests deubiquitinating enzyme as a novel regulator for Mecl1 and DNA damage checkpoint pathway.