Parental Cancer Cell Lines Research Articles

Depolymerization of tubulin as the main molecular mechanism of the cytotoxic and antitumor activity of pyrrole-containing heterocyclic compounds

Introduction. Microtubules are highly dynamic polymers of α, β-tubulin dimers involves in a broad spectrum of the processes, such as intracellular transport and cell proliferation. This makes them an attractive molecular target for anti-cancer therapies. Substances that affect the dynamic state of tubulin microtubules are known as the mitotic poisons that are effectiveand widely used in the chemotherapy of various tumors. Mitotic poisons are able to interfere with polymerization (stabilization) or depolymerization of tubulin, which in turn leads to the arrest of cells in the M-phase (named as a mitotic catastrophe) and their subsequent death via activation of apoptotic mechanisms. However, the effectiveness of MP-based therapies is gradually decreasing over the time due to development of multiple drug resistance mechanisms in cancer cells. Thus, development of novel compounds selectively targeting tubulin and effectively overcoming multiple drugresistance phenotype in cancer is an urgent need in current oncology. Aim. To examine the cytotoxic and antitumor activities of several pyrrole-containing heterocyclic compounds (EPC-91, EPC-92 and PCA-93) against cancer cell lines with epithelial and mesenchymal origin, including those with multiple drug resistance phenotype. Materials and methods. Studies were performed on parental human cancer cell lines – triple-negative breast cancer HCC1806, gastrointestinal stromal tumor GIST T-1, osteosarcoma SaOS-2, – sensitive to chemotherapy (paclitaxel, doxorubicin) and their resistant sublines (HCC1806 Tx-R, GIST T-1 Tx-R, SaOS-2 Dox-R), as well as on murine colorectal adenocarcinoma cell line Colon-26, exhibiting primary resistance to the aforementioned chemotherapeutic agents. Results. The cytotoxic activities of EPC-91 and PCA-93 were due to their abilities to depolymerize tubulin. The results of immunofluorescence microscopy and Western blotting indicated that the compounds disrupt assembly of tubulin microtubules and prevent polymerization of α-tubulin in cancer cells. Inhibition of tubulin polymerizations led to significant increasein number of round-shaped and phospho-histone 3 (e. g. mitotic) cells, followed by their death through apoptosis. PCA-93 also exhibited potent anti-tumor effect against Colon-26 cells due to its anti-proliferative and proapoptotic activities. Conclusion. The data shown here illustrates potent cytotoxic activities of EPC-91 and PCA-93 against multiple cancer cell lines in vitro including those with multiple drug resistance phenotype. Similarly, PCA-93 was found to be highly effective against Colon-26 cell in vivo, thereby illustrating the attractive platform for the development of novel pyrrole-based agents exhibiting potent anti-tumor activities.

Abstract 4301: Increasing the therapeutic vulnerability of heterogenous cell phenotypes within prostate cancer

Abstract Phenotype heterogeneity is a hallmark of prostate adenocarcinoma and is a major source of recurrent disease. We tested pre-clinical therapies to eradicate phenotypically distinct cell populations derived from the parental DU145WT prostate cancer cell line. The derived cell lines are called DU145AA, DU145J7, and DU145G6. The DU145AA variant is a non-aggressive cell population while the DU145J7 and DU145G6 are aggressive phenotypes that invade through muscle and metastasize to bone. Flow cytometry analysis documented the phenotypic plasticity of the DU145 cell variants since DU145 aggressive lines expressed 1.3-fold increases in matrix metalloprotease 14 surface expression and a 2-fold increase in tri-methyl Histone H3 at lysine 27 as compared to the DU145WT parent line. The DU145AA non-aggressive line expressed an epithelial cell-cell lineage phenotype with up to 2-fold increases in E-cadherin, claudin 4, claudin 7, and a unique cytokeratin 6A expression and a 2-fold loss of Zinc finger E-box-binding homeobox 1, EVL, and Kindlin 2. A collaboration with the National Center for Advancing Translational Science (NCATS) resulted in the identification of pharmacological agents active in eradicating DU145J7 and DU145G6 cells as compared to the DU145WT population. The agents were selected from a quantitative high-throughput screen of 10,677 combined investigative and approved anti-cancer agents using the Cell Titer-Glo Luminescent Cell Viability assay. We independently validated 31 compounds from the assay that have 2-fold, or greater, differences in concentration to inhibit 50% cell growth (IC50) between the wild-type and aggressive variants of DU145 cells. The top 10 hits were tested for the efficacy of different combinations to eradicate defined admixtures of aggressive and non-aggressive phenotypes, including the DU145AA cells, to model tumor heterogeneity. Tested compounds with differential sensitivity between aggressive and non-aggressive cells were those inhibiting histone deacetylase (vorinostat, fimepinostat, and pracinostat), proteasomes (bortezomib, delanzomib, ixazomib), topoisomerases (gimatecan and daunorubicin), and other compounds identified independently by us targeting nicotinamide phosphoribosyl transferase (FK866) and DNA (bleomycin). Using admixtures of tumor cell phenotypes, we determined optimal combinations of compounds that would selectively eradicate the sub-populations. The results suggest that determination of phenotypes within tumor populations may eliminate the heterogenous tumor and prevent recurrent disease. Funding was provided by the University of Arizona Cancer Center (NCI-P30 CA23074 and NCI-R01 CA159406) and by the Partnership in Native American Cancer Prevention at the University of Arizona (U54CA143924) and Northern Arizona University (U54CA143925). Collaborators at NCATS were supported by the intramural research program. Citation Format: Allan I. Paxson, Loren H. Chang, Kendra D. Marr, Jaime M. Gard, Colin S. Nelson, Abhijeet Kapoor, William L. Harryman, Juan J. Marugan, Mark J. Henderson, Tino W. Sanchez, Anne E. Cress. Increasing the therapeutic vulnerability of heterogenous cell phenotypes within prostate 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 4301.

The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factor that may be exploited to develop therapeutic approaches.

Abstract 6244: Design, synthesis, and structure-activity relationships of substituted pyrido[3,2-d]pyrimidines as microtubule targeting agents and anti-cancer agents

Abstract Microtubules (MTs) are composed of αβ-tubulin heterodimers and are essential components of the cellular cytoskeleton involved in critical cellular functions, including mitosis, cell migration and signaling, intracellular trafficking, and angiogenesis. Microtubule targeting agents (MTAs) are highly successful chemotherapeutic agents because they target MT dynamics and interfere with MT-dependent cellular processes. The clinical efficacy of MTAs is limited by multidrug resistance due to expression of the P-glycoprotein (Pgp) drug efflux pump or increased expression of βIII-tubulin. This necessitates the development of novel MTAs effective against cell lines expressing these forms of drug resistance, which could offer potential advantages in patients who fail to respond to current MTAs, such as paclitaxel. Previously, we reported colchicine site (CS) binding compound 1 with a pyrido[3,2-d]pyrimidine scaffold and a 2-H substitution and N4-4’-methoxyaniline substitution inhibited colchicine binding and tubulin assembly (87% inhibition at 5 µM and IC50 = 1.3 µM, respectively). Compounds 2-9 were designed with a 2-CH3 substitution to evaluate the structure-activity relationships at the 4-position by using conformational restriction and isosteric replacements. Compounds 2-9 were synthesized, and the biological evaluation showed that compounds 2-9 inhibited tubulin assembly (7, IC50 = 0.72 μM) and binding of colchicine to tubulin (7, 97% inhibition at 5 µM, 81% at 0.5 µM). Compound 7 was found to be highly potent in the MCF-7 breast cancer cell line with an IC50 of 2.5 nM which was ~3.5-fold better than combretastatin-A4 (CA4). Compound 7 inhibited the growth of the human ovarian cancer cell line OVCAR-8 with an IC50 = 3.0 nM. For the Pgp overexpressing cell line NCI/ADR-RES, 7 was equally potent (IC50 = 3.0 nM), whereas paclitaxel was almost inactive (IC50 = 1,500 nM). Thus, we have identified 2-CH3 substituted pyrido[3,2-d]pyrimidines as a novel structural scaffold for inhibition of tubulin assembly and colchicine binding that have potent cytotoxic activity against the breast cancer cell line MCF-7 and against both the parental ovarian cancer cell line OVCAR-8 and its derivative Pgp overexpressing cell line NCI/ADR-RES. These compounds thus merit further study as potential cancer chemotherapeutic agents. Citation Format: Yesha Vijaykumar Shah, Ruoli Bai, Ernest Hamel, Aleem Gangjee. Design, synthesis, and structure-activity relationships of substituted pyrido[3,2-d]pyrimidines as microtubule targeting agents and anti-cancer agents. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6244.

Serum IL-6 level is associated with clinical outcome of intravesical gemcitabine therapy in T1 non-muscle-invasive bladder cancer.

Serum biomarkers are valuable tools to predict the prognosis of anticancer therapies. This study aimed to evaluate the impact of serum interleukin-6 (IL-6) level on the clinical outcome of intravesical gemcitabine (GEM) therapy in non-muscle-invasive bladder cancer (NMIBC). This retrospective study enrolled 71 patients initially diagnosed with T1 NMIBC who underwent intravesical GEM therapy between 2017 and 2019. The expression of IL-6 gene was examined by real-time PCR. Serum IL-6 level was determined by enzyme-linked immunosorbent assay (ELISA). The cell viability after gemcitabine treatment was measured by CCK-8 assay. The optimal serum IL-6 cutoff values for recurrence prediction were calculated using receiver-operating characteristic curve analysis with reference to cancer recurrence. Recurrence-free survival was compared by the log-rank test. Univariate and multivariate Cox regression analysis was conducted to identify the prognostic factors influencing recurrence-free survival after treatment with intravesical GEM. Increased expression and secretion of IL-6 were observed in GEM-resistant sublines compared with parental bladder cancer cell lines. Serum IL-6 level rendered a sensitivity of 82.6% and a specificity of 77.1% to correlate with the cancer recurrence after intravesical GEM. Patients with a high serum IL-6 level exhibited shorter recurrence-free survival after intravesical GEM. Moreover, serum IL-6 level in our NMIBC cohort was significantly associated with clinicopathological characteristics, such as tumor diameter, multifocality, concomitant CIS, and grade. Serum IL-6 level was an independent prognostic factor for recurrence-free survival in NMIBC patients treated with intravesical GEM. Given that it was significantly associated with clinical outcome of intravesical GEM therapy, serum IL-6 level might be used as a potential prognostic biomarker for intravesical GEM in T1 NMIBC.

Increased glucose influx and glycogenesis in lung cancer cells surviving after irradiation

Purpose Lung cancer is considered as one of the most frequent malignancies worldwide. Radiotherapy is the main treatment modality applied for locally advanced disease, but remnant surviving cancer tissue results in disease progression in the majority of irradiated lung carcinomas. Metabolic reprogramming is regarded as a cancer hallmark and is associated with resistance to radiation therapy. Here, we explored metabolic alterations possibly related to cancer cell radioresistance. Materials and methods We compared the expression of metabolism-related enzymes in the parental A549 lung cancer cell line along with two new cell lines derived from A549 cells after recovery from three (A549-IR3) and six (A549-IR6) irradiation doses with 4 Gy. Differential GLUT1 and GYS1 expression on proliferation and radioresistance were also comparatively investigated. Results A549-IR cells displayed increased extracellular glucose absorption, and enhanced mRNA and protein levels of the GLUT1 glucose transporter. GLUT1 inhibition with BAY-876, suppressed cell proliferation and the effect was significantly more profound on A549-IR3 cells. Protein levels of molecules associated with aerobic or anaerobic glycolysis, or the phosphate pentose pathway were similar in all three cell lines. However, glycogen synthase 1 (GYS1) was upregulated, especially in the A549-IR3 cell line, suggestive of glycogen accumulation in cells surviving post irradiation. GYS1-gene silencing repressed the proliferation capacity of A549, but this increased their radioresistance. The radio-protective effect of the suppression of proliferative activity induced by GYS1 silencing did not protect A549-IR3 cells against further irradiation. Conclusions These findings indicate that GYS1 activity is a critical component of the metabolism of lung cancer cells surviving after fractionated radiotherapy. Targeting the glycogen metabolic reprogramming after irradiation may be a valuable approach to pursue eradication of the post-radiotherapy remnant of disease.

Deletion of arylamine N-acetyltransferase 1 in MDA-MB-231 human breast cancer cells reduces primary and secondary tumor growth in vivo with no significant effects on metastasis.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. Previous studies showed that inhibition or depletion of NAT1 in breast cancer cells diminishes anchorage-independent growth in culture, suggesting that NAT1 contributes to breast cancer growth and metastasis. To further investigate the contribution of NAT1 to growth and cell invasive/migratory behavior, we subjected parental and NAT1 knockout (KO) breast cancer cell lines (MDA-MB-231, MCF-7, and ZR-75-1) to multiple assays. The rate of cell growth in suspension was not consistently decreased in NAT1 KO cells across the cell lines tested. Similarly, cell migration and invasion assays failed to produce reproducible differences between the parental and NAT1 KO cells. To overcome the limitations of in vitro assays, we tested parental and NAT1 KO cells in vivo in a xenograft model by injecting cells into the flank of immunocompromised mice. NAT1 KO MDA-MB-231 cells produced primary tumors smaller than those formed by parental cells, which was contributed by an increased rate of apoptosis in KO cells. The frequency of lung metastasis, however, was not altered in NAT1 KO cells. When the primary tumors of the parental and NAT1 KO cells were allowed to grow to a pre-determined size or delivered directly via tail vein, the number and size of metastatic foci in the lung did not differ between the parental and NAT1 KO cells. In conclusion, NAT1 contributes to primary and secondary tumor growth in vivo in MDA-MB-231 breast cancer cells but does not appear to affect its metastatic potential.

Abstract P077: SCO-101 mediates re-sensitization of irinotecan (SN38) resistant colorectal cancer cells

Abstract Introduction: Annually, more than 500.000 die from Colorectal Cancer (CRC). Three types of chemotherapy are currently being used worldwide to treat CRC patients: 5FU, oxaliplatin and irinotecan. Unfortunately, chemotherapy resistance is present or develops in the majority of metastatic CRC patients. Resistance to anti-cancer drugs represents the main cause of cancer-related deaths. Thus, re-sensitization of chemotherapy resistant cancer cells constitutes a highly unmet medical need. The small molecule drug, SCO-101, is an oral drug and has passed 4 Phase I clinical trials where it demonstrated excellent PK and favourable safety profile. Materials and Methods: We used pairs of parental (sensitive) and SN38-resistant (SN38 is the active metabolite of irinotecan) human colon cancer cell lines (HT29 and LoVo). Inhibition of ABCG2 was known re-sensitize the SN38 resistant cells and SCO-101 was investigated for effects on ABCG2 by dye-flux assays, bi-directinal fluz assays and vesicular uptake assays. The impact on the levels of ABCG2 was investigated in the presence or absence of SCO-101. Furthermore, a kinase screen revealed that the kinase SRPK1 was inhibited by SCO-101 and the importance of SRPK1 activity in re-sensitization of SN38 resistant colon cancer cells was investigated by two different synthetic SRPK1 inhbitors. The effect of SCO-101 was investigated by cell viability assays (MTT/PresoBlue) in vitro during 72h exposure to SCO-101 and/or SN38. Results: Updated results from the various flux assays clearly demonstrated that SCO-101 inhibited the activity of ABCG2 (BCRP). Protein analysis further demonstrated that SCO-101 causes degradation of ABCG2/BCRP and in silco docking predicted SCO-101 to bind in the part of ABCG2 whwere also SN38 binds. Cell viability assays with two different SRPK1 inhibitors demonstrated that this kinase is involved in re-sensitization to SN38. Exposing SN38-resistant cells to the combination of SCO-101 and SN38, had a significant inhibitory effect on cell viability compared to either drug alone, suggesting an additive or synergistic effect between these two drugs with SCO-101 re-sensitizing the resistant cells to SN38. Conclusion and future perspectives: These preclinical studies demonstrate that SCO-101 sensitizes SN38-resistant colon cancer cells to SN38 through inhibition of the SRPK1 kinase and inhibition/degradation of the ABCG2/BCRP drug efflux pump. In conclusion, SCO-101 represents a unique drug with an innovative dual mechanism of action. Scandion Oncology has reported the outcome of the first part of the ongoing Phase II clinical CORIST trial with metastatic CRC patients, who have developed acquired resistance to irinotecan containing treatment. These patients were be re-exposed to the combination of irinotecan containing treatment and SCO-101 (www.clinicaltrials.gov/ct2/show/NCT04247256?term=scandion&draw=2&rank=2). Citation Format: Jan Stenvang, Nicklas Lindland Roest, Peter Michael Vestlev, Bo Rode Hansen. SCO-101 mediates re-sensitization of irinotecan (SN38) resistant colorectal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P077.

Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells.

Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.

Targeting multiple genes containing long mononucleotide A-T repeats in lung cancer stem cells

BackgroundIntratumour heterogeneous gene expression among cancer and cancer stem cells (CSCs) can cause failure of current targeted therapies because each drug aims to target the function of a single gene. Long mononucleotide A-T repeats are cis-regulatory transcriptional elements that control many genes, increasing the expression of numerous genes in various cancers, including lung cancer. Therefore, targeting A-T repeats may dysregulate many genes driving cancer development. Here, we tested a peptide nucleic acid (PNA) oligo containing a long A-repeat sequence [A(15)] to disrupt the transcriptional control of the A-T repeat in lung cancer and CSCs.MethodsFirst, we separated CSCs from parental lung cancer cell lines. Then, we evaluated the role of A-T repeat gene regulation by counting the number of repeats in differentially regulated genes between CSCs and the parental cells of the CSCs. After testing the dosage and effect of PNA-A15 on normal and cancer cell toxicity and CSC phenotypes, we analysed genome-wide expression to identify dysregulated genes in CSCs.ResultsThe number of A-T repeats in genes differentially regulated between CSCs and parental cells differed. PNA-A15 was toxic to lung cancer cells and CSCs but not to noncancer cells. Finally, PNA-A15 dysregulated a number of genes in lung CSCs.ConclusionPNA-A15 is a promising novel targeted therapy agent that targets the transcriptional control activity of multiple genes in lung CSCs.

GLUD1 Promotes Cetuximab Resistance in Metastasis Colorectal Cancer

Background: Colorectal cancer (CRC) is one of the most common causes of cancer mortality worldwide, among which metastatic colorectal cancer (mCRC) is the deadliest one. Cetuximab as a monoclonal antibody targeting epidermal growth factor receptor (EGFR) has improved the survival of mCRC patients. However, primary and acquired resistance to cetuximab are often found during therapy. Therefore, it is important to study the mechanism of cetuximab resistance and find more potential biomarkers. Methods: We previously established the acquired cetuximab resistant cell lines Caco2-CR and NCI-H508-CR by treating their parental cetuximab sensitive colorectal cancer cell lines, Caco2 and NCI-H508, with continuously increasing concentrations of cetuximab. Microarray analysis was utilized to detect the expression profiling of resistant cells and sensitive cells. Gene expression data of mCRC treated with cetuximab (GSE5851) was obtained from GEO. Patient-derived xenografts (PDX) were established using the tumor tissues from either primary lesions or liver metastases of mCRC patients without previous treatment. The metabolic flux of 13 C labelled glutamine in NCI-H508-CR and its parental cells was analyzed. Findings: Microarray analysis indicated that GLUD1, a key enzyme in glutamine metabolism, was one of the differentially expressed genes between Caco2-CR and Caco2. The expression of GLUD1 in the cetuximab resistant patients from GSE5851 dataset were higher than that in the sensitive ones. The same results were seen in animal PDX models. The glutamine metabolism was stronger in NCI-H508-CR compared with NCI-H508 and inhibition of glutamine metabolism could reduce the cetuximab resistance. GLUD1 could promote cetuximab resistance by generating more α-KG to support tricarboxylic acid (TCA) cycle. Furthermore, the expression of GLUD1 in resistant cells could be promoted by the nucleus translocated FOXO3. Interpretation: In this study，we found that GLUD1 can promote cetuximab resistance in mCRC, and it was transcriptional regulated by FOXO3. Funding: This work was supported by the National Natural Science Foundation of China (81772511). Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Ethics Committee of Zhongshan Hospital, Fudan University

Cdc20 induces the radioresistance of bladder cancer cells by targeting FoxO1 degradation.

Ionizing radiation is a conventional therapy for cancer patients, but patients often experience distant metastasis and recurrence, which lead to a poor prognosis after the implementation of this treatment. Moreover, the underlying mechanisms by which radioresistance contributes to metastatic potential is still elusive. Here, we explored the molecular mechanisms that contribute to radioresistance in bladder cancer. To achieve this, we established two irradiation-resistant (IR) cell lines, T24R and 5637R, which were derived from parental bladder cancer cell lines. Cell viability was detected by CCK-8 assay, while migration and invasion abilities were examined by wound healing and Transwell chamber assays, respectively. Furthermore, the role of Cdc20 in the regulation of epithelial to mesenchymal transition (EMT) in IR cells was explored by Western blotting, immunoprecipitation and immunofluorescence staining. The IR cells exhibited EMT properties, and our data showed that Cdc20 expression was significantly elevated in IR cells. Remarkably, Cdc20 silencing reversed the EMT phenotype in IR cells. Mechanistically, Cdc20 governed IR-mediated EMT in part by governing forkhead box O1 (FoxO1) degradation. Taken together, our findings showed that the inactivation of Cdc20 or the activation of FoxO1 might be a potential strategy to overcome radioresistance in bladder cancer.

Towards image-based cancer cell lines authentication using deep neural networks

Although short tandem repeat (STR) analysis is available as a reliable method for the determination of the genetic origin of cell lines, the occurrence of misauthenticated cell lines remains an important issue. Reasons include the cost, effort and time associated with STR analysis. Moreover, there are currently no methods for the discrimination between isogenic cell lines (cell lines of the same genetic origin, e.g. different cell lines derived from the same organism, clonal sublines, sublines adapted to grow under certain conditions). Hence, additional complementary, ideally low-cost and low-effort methods are required that enable (1) the monitoring of cell line identity as part of the daily laboratory routine and 2) the authentication of isogenic cell lines. In this research, we automate the process of cell line identification by image-based analysis using deep convolutional neural networks. Two different convolutional neural networks models (MobileNet and InceptionResNet V2) were trained to automatically identify four parental cancer cell line (COLO 704, EFO-21, EFO-27 and UKF-NB-3) and their sublines adapted to the anti-cancer drugs cisplatin (COLO-704rCDDP1000, EFO-21rCDDP2000, EFO-27rCDDP2000) or oxaliplatin (UKF-NB-3rOXALI2000), hence resulting in an eight-class problem. Our best performing model, InceptionResNet V2, achieved an average of 0.91 F1-score on tenfold cross validation with an average area under the curve (AUC) of 0.95, for the 8-class problem. Our best model also achieved an average F1-score of 0.94 and 0.96 on the authentication through a classification process of the four parental cell lines and the respective drug-adapted cells, respectively, on a four-class problem separately. These findings provide the basis for further development of the application of deep learning for the automation of cell line authentication into a readily available easy-to-use methodology that enables routine monitoring of the identity of cell lines including isogenic cell lines. It should be noted that, this is just a proof of principal that, images can also be used as a method for authentication of cancer cell lines and not a replacement for the STR method.

Abstract 6340: Re-sensitization of irinotecan/SN38 resistant colorectal cancer cells by SCO-101

Abstract Introduction: Annually, more than 1 million people are diagnosed with Colorectal Cancer (CRC) and more than 500.000 die from CRC. Only three types of chemotherapy are currently being used worldwide to treat CRC patients: 5FU, oxaliplatin and irinotecan. However, chemotherapy resistance is present or develops in the majority of metastatic CRC patients. Resistance to anti-cancer drugs represents the main cause of cancer-related deaths. Thus, re-sensitization of chemotherapy resistant cancer cells constitutes a highly unmet medical need. Materials and Methods: The drug, SCO-101, is an oral drug and has previously passed 4 Phase I clinical trials where it demonstrated excellent PK (T1/2=15 hours) and favourable safety profile. We used two pairs of parental (sensitive) and SN38-resistant (SN38 is the active metabilote of irinotecan) human colon cancer cell lines (HT29 and LoVo). Cell viability in vitro during 72h exposure to SCO-101 and/or SN38 was adressed by MTT assays. The level of the SN38 efflux transpoter, ABCG2/BCRP, was investigated in the presence or absence of SCO-101. Additionally, various flux assays were applied to investigate the effect of SCO-101 on ABCG2/BCRP activity. A kinase screen revealed that the kinase SRPK1 was inhibited by SCO-101 and the importance of SRPK1 activity in re-sensitization of SN38 resistant colon cancer cells was investigated by two different synthetic SRPK1 inhbitors. Results: In parental HT29 and LoVo cells, SCO-101 and SN38 each had a dose-dependent effect on cell viability and no combinational effects were observed between the drugs. When exposing SN38-resistant cells to the combination of SCO-101 and SN38, a significant inhibitory effect on cell viability compared to either drug alone was observed, suggesting an additive or synergistic effect between these two drugs with SCO-101 re-sensitizing the resistant cells to SN38. Flux assays with ABCG2 /BCRP substrates clearly demonstrated that SCO-101 inhibited the activity of ABCG2 and protein analysis further demonstrated that SCO-101 causes degradation of ABCG2/BCRP. Cell viability assays with two different SRPK1 inhibitors demonstrated that this kinase is involved in re-sensitization to SN38. Conclusion and future perspectives: These preclinical studies demonstrate that SCO-101 sensitizes SN38-resistant colon cancer cells to SN38 through two different mechanisms of actions e.g. inhibition of the SRPK1 kinase and degradation of the ABCG2/BCRP drug efflux pump. We are now preparing a clinical phase II study enrolling metastatic CRC patients, who have developed acquired resistance to irinotecan containing treatment. These patients will be re-exposed to the combination of irinotecan containing treatment and SCO-101. Primary end-points will be safety and objective response rate according to RECIST 1.1. Secondary end-points will be clinical benefit rate. The study has been approved by the Danish Medicines Agency. In conclusion, SCO-101 represents a unique drug with an innovative dual mechanism of action. Citation Format: Jan Stenvang, Sophie Ambjørner, Khwajanezrabodin Sedighi, Peter Michael Vestlev, Lasse Saaby, Birger Brodin, Nils Brunner. Re-sensitization of irinotecan/SN38 resistant colorectal cancer cells by SCO-101 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6340.

Chromatin accessibility analysis reveals that TFAP2A promotes angiogenesis in acquired resistance to anlotinib in lung cancer cells.

Anlotinib, a multitarget tyrosine kinase inhibitor, is effective as a third-line treatment against non-small cell lung cancer (NSCLC). However, acquired resistance occurs during its administration. To understand the molecular mechanisms of anlotinib resistance, we characterized chromatin accessibility in both the parental and anlotinib-resistant lung cancer cell line NCI-H1975 through ATAC-seq. Compared with the parental cells, we identified 2666 genomic regions with greater accessibility in anlotinib-resistant cells, in which angiogenesis-related processes and the motifs of 21 transcription factors were enriched. Among these transcription factors, TFAP2A was upregulated. TFAP2A knockdown robustly diminished tumor-induced angiogenesis and partially rescued the anti-angiogenic activity of anlotinib. Furthermore, transcriptome analysis indicated that 2280 genes were downregulated in anlotinib-resistant cells with TFAP2A knocked down, among which the PDGFR, TGF-β, and VEGFR signaling pathways were enriched. Meanwhile, we demonstrated that TFAP2A binds to accessible sites within BMP4 and HSPG2. Collectively, this study suggests that TFAP2A accelerates anlotinib resistance by promoting tumor-induced angiogenesis.

Ceramide Kinase Is Upregulated in Metastatic Breast Cancer Cells and Contributes to Migration and Invasion by Activation of PI 3-Kinase and Akt.

Ceramide kinase (CerK) is a lipid kinase that converts the proapoptotic ceramide to ceramide 1-phosphate, which has been proposed to have pro-malignant properties and regulate cell responses such as proliferation, migration, and inflammation. We used the parental human breast cancer cell line MDA-MB-231 and two single cell progenies derived from lung and bone metastasis upon injection of the parental cells into immuno-deficient mice. The lung and the bone metastatic cell lines showed a marked upregulation of CerK mRNA and activity when compared to the parental cell line. The metastatic cells also had increased migratory and invasive activity, which was dose-dependently reduced by the selective CerK inhibitor NVP-231. A similar reduction of migration was seen when CerK was stably downregulated with small hairpin RNA (shRNA). Conversely, overexpression of CerK in parental MDA-MB-231 cells enhanced migration, and this effect was also observed in the non-metastatic cell line MCF7 upon CerK overexpression. On the molecular level, CerK overexpression increased the activation of protein kinase Akt. The increased migration of CerK overexpressing cells was mitigated by the CerK inhibitor NVP-231, by inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway and the Rho kinase, but not by inhibition of the classical extracellular signal-regulated kinase (ERK) pathway. Altogether, our data demonstrate for the first time that CerK promotes migration and invasion of metastatic breast cancer cells and that targeting of CerK has potential to counteract metastasis in breast cancer.

MiR-450a-5p inhibits autophagy and enhances radiosensitivity by targeting dual-specificity phosphatase 10 in esophageal squamous cell carcinoma.

Radioresistance reduces the success of therapy for patients with ESCC. Enhancing our understanding of the cardinal principles of radioresistance may improve the response of patients to irradiation. MicroRNAs perform a key role in posttranscriptional regulation, which is linked with the response of tumors to irradiation. Here, we successfully constructed a radioresistant cell line model, ECA109R, from parental esophageal cancer cell line ECA109. We used RNA-Seq analysis and qRT-PCR to compare the miRNA expression profiles of the ECA109 and ECA109R cell lines. The results revealed that miR-450a-5p was downregulated in the radioresistant cells. Functional analysis indicated that miR-450a-5p increases cellular radiosensitivity and suppresses autophagy in ESCC cells. We utilized a luciferase reporter assay to identify the target gene, DUSP10, as an indispensable regulator of the p38 and SAPK/JNK signaling pathways. Upregulation or downregulation of DUSP10 expression could reverse the effects of miR-450a-5p overexpression or inhibition. Tumor xenograft experiments verified that miR-450a-5p overexpression could increase sensitivity to radiation therapy in vivo. In general, our findings indicate that miR-450a-5p is a latent radiosensitizer and may represent a potential novel therapeutic target for radioresistance in ESCC.

Altered biodistribution of deglycosylated extracellular vesicles through enhanced cellular uptake

ABSTRACT Extracellular vesicles (EVs) from cancer are delivered both proximal and distal organs. EVs are highly glycosylated at the surface where EVs interact with cells and therefore has an impact on their properties and biological functions. Aberrant glycosylation in cancer is associated with cancer progression and metastasis. However, the biological function of glycosylation on the surface of EV is uncovered. We first demonstrated differential glycosylation profiles of EVs and their originated cells, and distinct glycosylation profiles in a brain-metastatic subline BMD2a from its parental human breast cancer cell line, MDA-MB-231-luc-D3H2LN by lectin blot. We then investigated the roles of surface glycoconjugates on EV uptake. N- and/or O-glycosylation removal of fluorescent-labelled BMD2a EVs enhanced cellular uptake to endothelial cells, suggesting that surface glycosylation has inhibitory effects on cellular uptake. Biodistribution of glycosylation-deprived BMD2a EVs administrated intravenously into mice was further analysed ex vivo using near-infrared lipophilic dye. EVs treated with O-deglycosylation enzymes enhanced the accumulation of EVs to the lungs after 24 h from the injection, while N-deglycosylation did not markedly alter biodistribution. As the lungs are first organs in which intravenous blood flows, we suggest that surface glycosylation of cancer-derived EVs avoid promiscuous adhesion to proximal tissues to be delivered to distant organs.

Heme oxygenase-1 in osteosarcoma.

The present study was planned to analyze serum heme oxygenase-1 levels in osteosarcoma patients. Twenty five histopathologically confirmed cases of osteosarcoma localized without metastasis of all the ages attending the Orthopedic Clinics were included in the study group and twenty five patients having musculoskeletal pain (age and sex matched) served as control. Five ml of venous blood was collected aseptically from antecubital vein and serum was be separated by centrifugation and analyzed the same day. Routine biochemistry investigations were performed as per standard enzymatic methods by autoanalyzer. Serum Heme oxygenase-1 was analyzed by enzyme-linked immunosorbent assay. In osteosarcoma patients, serum HO-1 levels were increased as compared to patients having musculoskeletal pain (P < 0.05). Workers have found that HO-1 induction in prostate cancer cell lines (PC3) cells restored the proliferation of osteoblasts, which was inhibited during co-culture with parental prostate cancer cell line PC3 cells. However, no concrete data are available on blood levels of HO in osteosarcoma. Major role of HO-1 is the protection against oxidative injury, additionally, it regulates cell proliferation, modulates inflammatory response and facilitates angiogenesis. Findings of the present study suggests that pharmacological agents that regulate HO activity or HO-1 gene silencing may become powerful tools for preventing the onset or progression of various cancers and sensitize them to anticancer therapies.

Development of a Novel Quinoline Derivative as a P-Glycoprotein Inhibitor to Reverse Multidrug Resistance in Cancer Cells.

Multidrug resistance (MDR) is one of conventional cancer chemotherapy’s limitations. Our group previously synthesized a series of quinoline-based compounds in an attempt to identify novel anticancer agents. With a molecular docking analysis, the novel compound 160a was predicted to target p-glycoprotein, an MDR candidate. The purpose of this study is to evaluate 160a’s MDR reversal effect and investigate the underlying mechanism at the molecular level. To investigate 160a’s inhibitory effect, we used a series of parental cancer cell lines (A549, LCC6, KYSE150, and MCF-7), the corresponding doxorubicin-resistant cell lines, an MTS cytotoxicity assay, an intracellular doxorubicin accumulation test, and multidrug resistance assays. The Compusyn program confirmed, with a combination index (CI) value greater than 1, that 160a combined with doxorubicin exerts a synergistic effect. Intracellular doxorubicin accumulation and transported calcein acetoxymethyl (AM) (a substrate for p-glycoprotein) were both increased when cancer cells with MDR were treated with compound 160a. We also showed that compound 160a’s MDR reversal effect can persist for at least 1 h. Taken together, these results suggest that the quinoline compound 160a possesses high potential to reverse MDR by inhibiting p-glycoprotein-mediated drug efflux in cancer cells with MDR.