Cell Testicular Cancer Research Articles

Estrogen receptor activates SRC and ERK1/2 and promotes tumorigenesis in human testicular embryonic carcinoma cells NT2/D1

Testicular germ cell tumors have the highest incidence in young men (between 15 and 44 years of age) and its etiology is still unclear, but its emergence on puberty suggests a hormone-dependent mechanism for the development of these tumors and their progression. We previously identified the estrogen receptor ESR1, ESR2, GPER and an isoform of ESR1, the ESR1-36 in human testicular embryonic carcinoma NT2/D1 cells, and the activation of SRC induced by ESR1 and ESR2 in these cells. Therefore, this study aimed to analyze the role of ER in the activation of ERK1/2, and the involvement of SRC and ERK1/2 on proliferation, migration, and invasion of the NT2/D1 cells. Our results showed that the activation of ESR1 (using ESR1-selective agonist PPT) or ESR2 (using ESR2-selective agonist DPN) increased phosphorylation of ERK1/2 in NT2/D1 cells. In the presence of the selective inhibitor for SRC-family kinases PP2, or the MEK specific inhibitor U0126, the effects of 17β-estradiol (E2) or PPT were blocked on proliferation and invasion of NT2/D1 cells. Finally, the proliferation, migration, and invasion of NT2/D1 cells simulated by E2 or ESR2 were also blocked by PP2 and U0126. This study provides novel insights into molecular mechanisms of ER in NT2/D1 cells by demonstrating that ER activates rapid responses molecules, including SRC and ERK1/2, which enhance the tumorigenic potential of testicular cancer cells.

The CDK Inhibitor Dinaciclib Improves Cisplatin Response in Nonseminomatous Testicular Cancer: A Preclinical Study.

Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP.

EDITING CDKN1A AND RASD1 IN A CELL CULTURE SYSTEM

Abstract As an individual ages, there is a higher incidence of cancer and infertility. Testicular germ cell cancer rates have been rising year after year. According to the American Cancer Society 1 of every 250 males will develop testicular cancer during their lifetime. In this research project, we aim to develop a molecular toolbox to analyze gene function in male germinal cells using a CRISPR/Cas9 cell culture system. This biotechnology allows for the targeted alteration and cutting of specific DNA strands. To achieve this, we will utilize cancer cell lines (SUSA and NT2/D1 testicular cancer cell lines) and knock out two genes of interest: cyclin-dependent kinase inhibitor 1 (CDKN1A) and ras-related dexamethasone-induced 1 (RASD1). Our objective is to investigate the effects of gene knockout on cell proliferation. For this purpose, we designed primers and single-guide RNAs using bioinformatic databases: Benchling and Chop Chop. The tools we used were employed to amplify human genomic DNA and assess the effectiveness of the guide RNAs through in vitro cutting of our genes of interest, followed by gel electrophoresis observations. Based on the results obtained from these experiments, our next step is to electroporate the guide RNA and Cas9 complex to alter gene function in our cell culture system. Through a better understanding of gene function in testicular cancer cell lines, we hope to inform future therapeutic targets for both testicular cancer and age-related infertility.

B-069 Performance Evaluation of a Ma2 Autoantibodies ELISA

Abstract Background Ma-2 is a neuronal nucleolar protein with purported involvement in RNA transcription and apoptosis regulation. Patients with Ma2 antibodies present with autoimmune encephalitis (limbic or brainstem). Ma-2 antibodies are a high-risk marker for paraneoplastic neurologic cause for autoimmune encephalitis (>75% of patients having either testicular cancer or non-small cell lung cancer). In older patients, there is a higher frequency of small cell lung cancer or other cancers especially when Ma 1 antibodies are also detected. Identification of Ma2 antibodies is important to help diagnose autoimmune encephalitis, guide the cancer search and aid treatment selection. The objective of this study was to develop and document the performance of a qualitative enzymatically amplified sandwich-type Ma2 Antibody ELISA. Methods The Ma2 ELISA was produced in-house utilizing commercially available protein and was performed on a Hamilton Star liquid handling instrument equipped with a plate washer and plate reader. Each assay plate included patient samples along with 3 independent negative serum calibrators and 4 quality controls (Both positive and negative controls per matrix [CSF or serum]) each tested in duplicate. Mean optical densities (OD) for patient specimens were compared to the average OD of the negative serum calibrators to generate a ratio. A ratio cut-off of 8 was selected for reporting positivity based on preliminary studies and was validated during the method performance assessment. To assess assay performance we measured precision (3 positive pools at or above the cut-off ratio for each specimen type were tested 20 times in one assay and in duplicate across 10 independent assays), accuracy comparing to the current reference method at Athena® Diagnostics (N = 10 positives / 40 negatives), analytical specificity (20 samples with high titer autoantibodies directed against other proteins), and the impact of common interferences (lipids, bilirubin, hemolysate were spiked into 2 positive and 1 negative sample per interference). Clinical performance of the assay for differentiating healthy donors without disease (N = 140) and those with non-Ma2 neurological autoimmunity (N = 23) from those with clinical phenotypes consistent with Ma-2 associated encephalitis (N = 8) was measured. Results Serum intra-assay imprecision (coefficient of variation; %CV) ranged from 3.0–6.5%. Inter-assay imprecision ranged from 21–29%. CSF intra-assay imprecision ranged from 3.5–10%. Inter-assay imprecision ranged from 13–15.5%. Accuracy was assessed via qualitative comparison to a reference method [western blot or nanoliter scale immunoassay]. There was perfect agreement between methods. To assess analytical specificity, samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N = 10] or Systemic Lupus Erythematosus [N = 10]) were tested in serum (and spiked into CSF); all were negative. Interference studies were conducted on 2 positive Ma2 serum and CSF samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. All samples remained positive under given interferents. A negative sample was tested for each matrix, both remained negative. Clinical specificity was 100% with all samples from patients without Ma-2 encephalitis testing negative (N = 183). All ELISA positives identified in this study had phenotypes consistent with Ma-2 autoimmune encephalitis (N = 8). Conclusion The performance of the Ma2 ELISA was acceptable for clinical laboratory implementation.

PiR-36249 and DHX36 together inhibit testicular cancer cells progression by upregulating OAS2.

PIWI-interacting RNAs (piRNAs) are a class of noncoding RNAs originally reported in the reproductive system of mammals and later found to be aberrantly expressed in tumors. However, the function and mechanism of piRNAs in testicular cancer are not very clear. The expression level and distribution of piR-36249 were detected by RT-qPCR and immunofluorescence staining assay. Testicular cancer cell (NT2) progression was measured by CCK8 assay, colony formation assay and wound healing assay. Cell apoptosis was assessed by flow cytometry and western blot. RNA sequencing and dual-luciferase reporter assay were conducted to identify the potential targets of piR-36249. The relationship between piR-36249 and OAS2 or DHX36 was confirmed using overexpression assay, knockdown assay, pull-down assay and RIP assay. piR-36249 is significantly downregulated in testicular cancer tissues compared to tumor-adjacent tissues. Functional studies demonstrate that piR-36249 inhibits testicular cancer cell proliferation, migration and activates the cell apoptosis pathway. Mechanically, we identify that piR-36249 binds to the 3'UTR of 2'-5'-oligoadenylate synthetase 2 (OAS2) mRNA. OAS2 has been shown in the literature to be a tumor suppressor modulating the occurrence and development of some tumors. Here, we show that OAS2 knockdown also promotes testicular cancer cell proliferation and migration. Furthermore, piR-36249 interacts with DHX36, which has been reported to promote translation. DHX36 can also bind to OAS2 mRNA, and knockdown of DHX36 increases OAS2 mRNA but downregulates its protein, indicating the enhancing effect of DHX36 on OAS2 protein expression. All these data suggest that piR-36249, together with DHX36, functions in inhibiting the malignant phenotype of testicular cancer cells by upregulating OAS2 protein and that piR-36249 may be used as a suppressor factor to regulate the development of testicular cancer.

A meta-analysis of local-regional therapy (LRT) in liver metastases from genitourinary cancers.

e16606 Background: Liver metastases (Mets) from genitourinary cancers are difficult to treat and many of them are resistant to systemic therapy. Local-Regional Therapies (LRT) are becoming popular in treating Liver Mets. In this Meta-analysis, we analyze the outcomes of LRT for Liver Mets from Genitourinary Cancers. Methods: We searched PubMed, Google scholar for case reports, case studies, retrospective studies with patients who underwent LRT like Radiofrequency Ablation (RF), Chemoembolization (CE), Microwave Ablation (MA), Cryoablation for liver metastases for from Genitourinary Cancers including Prostate Cancer (PC), Testicular Cancer (TC) and Renal Cell Cancer (RCC). We also included 3 patients from Dignity Health Cancer Center, Phoenix, AZ. Results: Total number of patients in the study is 54, 3 of them from our Center. 39 are men and 15 are women. Mean and median age of the patients was 62.3, 65 years respectively. Number of patients with RCC, PC, TC are 43, 6, 5 respectively. 28 patients had CE, 23 had RFA and 3 had MA with the total number of procedures being 144, 41 and 5 respectively. Mean survival for all the patients is 62.7 months, mean survival for RCC, TC and PC are 68.9, 45.8 and 24.4 months respectively. Median number of systemic therapies received by the patients prior to getting LRT was 2 (range 1-4). We used RECIST criteria to quantify the benefit from LRT, 23 patients had Complete Response (CR), 6 had Partial Response (PR), 14 had Stable Disease (SD) and 8 had Progressive Disease (PD), there was no data for 2 patients. 43 patients had Clinical Benefit. We present a case of castration resistant metastatic PC from our facility. A 78-year man diagnosed metastatic PC in 2007 who had Prostatectomy, salvage radiation, intermittent androgen deprivation and Combined deprivation therapy. But his PSA continued to rise, flutamide was added. In 2017, he had liver metastasis, had RFA twice for 2 liver lesions and had CR. He continues to be on relugolix with good quality of life. Patients in the study are people with advanced metastatic cancer who underwent various systemic therapies like chemotherapy, radiation, immunotherapy and hormonal therapy prior to getting the LRT. LRT was done as the patients failed or did not tolerate systemic therapies. The mean survival for RCC patients in the study group is greater than the average survival of stage IV RCC, the lower mean survival for PC and TC can be explained by the limited number of this patient population in the study. Conclusions: Combination of LRT with systemic therapy for previously heavily treated Liver Mets from genitourinary cancers is a promising strategy for patients with otherwise poor prognosis. [Table: see text]

Ursolic Acid against Prostate and Urogenital Cancers: A Review of In Vitro and In Vivo Studies.

Prostate cancer is the second most diagnosed form of cancer in men worldwide and accounted for roughly 1.3 million cases and 359,000 deaths globally in 2018, despite all the available treatment strategies including surgery, radiotherapy, and chemotherapy. Finding novel approaches to prevent and treat prostate and other urogenital cancers effectively is of major importance. Chemicals derived from plants, such as docetaxel and paclitaxel, have been used in cancer treatment, and in recent years, research interest has focused on finding other plant-derived chemicals that can be used in the fight against cancer. Ursolic acid, found in high concentrations in cranberries, is a pentacyclic triterpenoid compound demonstrated to have anti-inflammatory, antioxidant, and anticancer properties. In the present review, we summarize the research studies examining the effects of ursolic acid and its derivatives against prostate and other urogenital cancers. Collectively, the existing data indicate that ursolic acid inhibits human prostate, renal, bladder, and testicular cancer cell proliferation and induces apoptosis. A limited number of studies have shown significant reduction in tumor volume in animals xenografted with human prostate cancer cells and treated with ursolic acid. More animal studies and human clinical studies are required to examine the potential of ursolic acid to inhibit prostate and other urogenital cancers in vivo.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-β signalling pathway outcomes in TGCTs.

INVESTIGATING MALE INFERTILITY: EDITING GENES IMPORTANT FOR SPERMATOGENESIS IN A CELL CULTURE SYSTEM

Abstract The four levels of aging include the specific disease, the systemic complications that lead to the disease, the loss of cellular integrity that affects the system, and macromolecule malfunction that alters cellular integrity. Similarly, this project investigates infertility and testicular cancer, misregulation of the endocrine system, problems with germ and Sertoli cell maintenance, and ultimately gene expression changes that alter cellular proliferation and death. Infertility affects 4.5–6% of North American males and up to 15% of couples worldwide. Infertility may be caused by unknown genetic factors, as up to 2,300 genes are pertinent to male fertility. This research project aims to create the molecular toolbox needed to evaluate gene function in cultured male germ cells. We will use mouse primary spermatogonial stem cells and a human testicular cancer cell line to knock out the functions of genes Phosphoprotein 1 (Spp1) and Inhibitor of DNA Binding 4 (Id4) in the testis to determine the effect of the knockouts on male germ cell proliferation and cell death. We designed single guide RNAs (gRNAs) using online bioinformatic tools and amplified the genes from human and mouse genomic DNA to demonstrate the effectiveness of our gRNAs in vitro. These gRNAs and Cas9 compose the beginnings of the molecular toolbox used to electroporate cultured cells. This research will contribute to the greater scientific community by providing insight into the function of Spp1 and Id4 relating to male fertility and providing a methodology for future research in aging-related fertility diseases and testicular cancer.

First Evidence of the Expression and Localization of Prothymosin α in Human Testis and Its Involvement in Testicular Cancers.

Prothymosin α (PTMA) is a phylogenetically conserved polypeptide in male gonads of Vertebrates. In Mammals, it is a ubiquitous protein, and, possessing a random-coil structure, it interacts with many other partners, in both cytoplasmic and nuclear compartments. PTMA has been widely studied during cell progression in different types of cancer because of its anti-apoptotic and proliferative properties. Here, we provided the first evidence of PTMA expression and localization in human testis and in two testicular cancers (TC): classic seminoma (CS) and Leydig cell tumor (LCT). Data showed that its protein level, together with that of proliferating cell nuclear antigen (PCNA), a cell cycle progression marker, increased in both CS and LCT samples, as compared to non-pathological (NP) tissue. Moreover, in the two-cancer tissue, a decreased apoptotic rate and an increased autophagic flux was also evidenced. Results confirmed the anti-apoptotic action of PTMA, also suggesting that it can act as a switcher from apoptosis to autophagy, to favor the survival of testicular cancer cells when they develop in adverse environments. Finally, the combined data, even if they need to be further validated, add new insight into the role of PTMA in human normal and pathological testicular tissue.

Pannexin1 inhibits autophagy of cisplatin-resistant testicular cancer cells by mediating ATP release

ABSTRACT Pannexin1 (Panx-1) is a gap junction channel protein that mediates the release of intracellular ATP during autophagy, and thus plays an important role in tumor cell apoptosis and chemo-resistance. However, the role of Panx-1 in cisplatin-resistance of testicular cancer cells remains unclear. We found that cisplatin-resistant I-10 testicular cancer cell lines (I-10/CDDP) autophagy-associated proteins (p62, p-mTOR, mTOR and LC3) exhibited high levels of autophagy in their expression, while LC3-II expression was more significantly in the presence of lysosomal degradation blocked by chloroquine (CQ). Xenograft models using I-10/CDDP cells with knockdown ATG5 and ATG7 were established in mouse models and showed blockade of autophagic flux and inhibition of tumor growth. In addition, inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN), as well as shRNA-mediated knockdown promoted autophagy in the I-10/CDDP cells, which was accompanied by a decrease in the levels of extracellular ATP. In contrast, overexpression of Panx-1 decreased autophagy of I-10/CDDP cells and increased extracellular ATP levels. To further determine the effect of panx-1-mediated ATP release on the autophagy of I-10/CDDP cells, apyrase was used to hydrolyze the extracellular ATP. Apyrase promoted autophagy in I-10/CDDP cells city by decreasing extracellular ATP, regardless of Panx-1 expression. This study demonstrated for the first time that Panx-1-mediated ATP release inhibits autophagy of I-10/CDDP cells, which provides a potential therapeutic strategy for cisplatin-resistant testicular cancer.

Carbenoxolone enhances inhibitory effect of RSL3 against cisplatin-resistant testicular cancer cells by promoting ferroptosis

To investigate the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatinresistant testicular cancer cells (I-10/DDP) and the effect of carbenoxolone on the activity of RSL3 against testicular cancer. MTT assay was used to evaluate the survival rate of I-10/DDP cells following treatment with RSL3 (1, 2, 4, 8, 16 or 32 μmol/L) alone or in combination with carbenoxolone (100 μmol/L) or after treatment with Fer-1 (2 μmol/L), RSL3 (4 μmol/L), RSL3+Fer-1, RSL3+carbenoxolone (100 μmol/L), or RSL3+Fer-1+carbenoxolone. Colony formation assay was used to assess the proliferation ability of the treated cells; wounding-healing assay and Transwell assay were used to assess the invasion and migration ability of the cells. The expression of GPX4 was detected using Western blotting, the levels of lipid ROS were detected using C11 BODIPY 581/591 fluorescent probe, and the levels of Fe2+ were determined with FerroOrange fluorescent probe. RSL3 dose-dependently decreased the survival rate of I-10/DDP cells, and the combined treatment with 2, 4, or 8 μmol/L RSL3 with carbenoxolone, as compared with RSL3 treatment alone, resulted in significant reduction of the cell survival rate. The combination with carbenoxolone significantly enhanced the inhibitory effect of RSL3 on colony formation, wound healing rate (P=0.005), invasion and migration of the cells (P < 0.001). Fer-1 obviously attenuated the inhibitory effects of RSL3 alone and its combination with carbenoxolone on I-10/DDP cells (P < 0.01). RSL3 treatment significantly decreased GPX4 expression (P=0.001) and increased lipid ROS level (P=0.001) and Fe2+ level in the cells, and these effects were further enhanced by the combined treatment with carbenoxolone (P < 0.01). Carbenoxolone enhances the inhibitory effect of RSL3 on the proliferation, invasion and migration of cisplatin-resistant testicular cancer cells by promoting RSL3-induced ferroptosis.

NPS-1034 Induce Cell Death with Suppression of TNFR1/NF-κB Signaling in Testicular Cancer.

Background and objectives: NPS-1034 with a dual inhibitory effect on Met and Axl kinase receptors has exhibited therapeutic potential in previous models. However, no study on treating testicular cancer (TC) cell lines with NPS-1034 has been established. Materials and Methods: In this study, a series of in vitro examinations of the apoptotic effect induced by NPS-1034 in TC cell lines was conducted to clarify the molecular interactions involved. Results: A decrease in cell viability rate was observed following NPS-1034 treatment, as shown in the MTT assay. Induction of the apoptotic effect was observed in TC cells as the sub-G1 and Annexin-PI populations increased in a dose-dependent manner. The involvement of the tumor receptor necrosis factor receptor 1 (TNFR1) pathway was later determined by the proteome array and western blotting. A reduction in TNFR1 and NF-κB downstream protein expressions, an upregulation of cleaved caspase-3 and -7, and a downregulation of survivin and claspin all reassured the underlying mechanism of the TNFR1 involved in the apoptotic pathway induced by NPS-1034. Conclusions: Our findings provide evidence for a potential underlying TNFR1 pathway involved in NPS-1034 treatment. This study should offer new insights into targeted therapy for TC.

Glycan signatures for the identification of cisplatin-resistant testicular cancer cell lines: Specific glycoprofiling of human chorionic gonadotropin (hCG).

BackgroundTesticular cancer (TC) is the most frequent type of cancer among young men aged between 15 and 34 years. TC is treated using cisplatin, but 3%–5% of TC patients fail to respond to cisplatin, with a very bad to fatal prognosis. Accordingly, it is most important to quickly and readily identify those TC patients who are resistant to cisplatin treatment.MethodsThis study seeks to investigate changes in the glycosylation associated with cisplatin resistance to TC cell lines.ResultsA specific glycoprofiling of human chorionic gonadotropin (hCG) was analysed in three TC cell lines and one cell line of female origin. A typical calibration curve for hCG glycoprofiling showed a dynamic range up to 50 ng/ml, with a limit of detection of 0.3 ng/ml and assay reproducibility represented by relative standard deviation of 3.0%. Changes in the glycan signatures on hCG were analysed in cisplatin‐sensitive cell lines and in their cisplatin‐resistant sub‐lines using an enzyme‐linked lectin assay (ELLA) protocol. An immobilised antibody was applied to a selective capture of hCG from a cytoplasmic fraction of cell lysates with final incubation using a lectin from a panel of 17 lectins.ConclusionThe results suggest that one particular lectin Dolichos biflorus agglutinin (DBA) can selectively discriminate sensitive TC cell lines from resistant TC cell lines. Moreover, there are additional lectins which can provide useful information about the strength of cisplatin resistance.

PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer.

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

Limbic Encephalitis with HU-Antibodies in T-cell Anaplastic Lymphoma. A Case Report

Limbic encephalitis is a rare paraneoplastic neurological syndrome usually associated with small cell lung cancers, testicular and breast cancers or B-cell lymphomas. We herein report the first patients with paraneoplastic limbic encephalitis associated with HU antibodies and anaplastic T-cell lymphoma.

Abstract 2127: Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents

Abstract Testicular germ cell tumors (TGCTs) are the most frequent solid cancers of males between the ages of 16 to 39 and have the highest mortality in this age group. Testicular cancer is highly curable with platinum-based chemotherapeutics. However, 15-20% of patients are resistant and succumb to their disease. The mechanisms for cisplatin sensitivity/resistance in testicular cancer is unclear. Previously we showed that cisplatin refractory testicular cancer is highly sensitive to the DNA methyltransferase inhibitors (DNMTi), 5-Aza-2'-deoxycytidine (5-AZA-DC) and guadecitabine in vivo and in vitro. High expression of DNMT3B correlates with sensitivity to DNMTi in TGCT cells. Based on these preclinical findings we recently completed a phase 1 clinical trial which demonstrated the combination of guadecitabine and cisplatin was tolerable and effective in treating patients with platinum refractory germ cell cancer. To better understand the mechanism of chemotherapy sensitivity/resistance in testicular cancer we generated a series of cisplatin resistant cell models. Genome wide methylome array and de novo transcriptional profiling of cisplatin resistant TGCT cells revealed decreased global H3K27 methylation due to suppression of the polycomb repressive complex 2 (PRC2 ). To better understand the relationship between cisplatin and 5-AZA-DC sensitivity, 5-AZA-DC resistant testicular cancer cells were also generated. Remarkably, there was a reciprocal relationship between cisplatin and 5-AZA-DC resistance, with cisplatin resistance associated with increased sensitivity to 5-AZA-DC and 5-AZA-DC resistance associated with increased sensitivity to cisplatin. Transcriptomics profiling and other studies in 5-AZA-DC resistant cells revealed increased H3K27me3, repression of polycomb target genes and a dramatic decreased in DNMT3B expression, the exact opposite of the situation in cisplatin resistant cells. Further knockdown of DNMT3B resulted in induction of H3K27me3, decreased sensitivity to 5-AZA-DC and increased sensitivity to cisplatin. These data suggest that DNA methylation and polycomb signaling are interconnected drivers of chemotherapy and epigenetic therapy responses in TGCTs. Ongoing H3K27 me3 ChIP-Seq, global methylation analysis, and validation studies will be described to further elucidate these mechanisms with the goal of devising novel epigenetic-based therapies for testicular and potentially other cancers. Citation Format: Ratnakar Singh, Emmanuel Bikorimana, Zeeshan Fazal, Cliff Yerby, Hannah Baldwin, Aleyah Destiny Hattab, Megan Tomlin, Andrea K. Corbet, Raya Iman Boyd, Khadeeja Shahid, Doha Naguib Ahmed Mohamed Shokry, Sarah Joy Spinella, Michael J. Spinella. Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2127.

Inhibiting autophagy by silencing ATG5 and ATG7 enhances inhibitory effect of DDP on DDP-resistant I-10 testicular cancer cells

To observe the changes in autophagy of cisplatin-resistant I-10 testicular cancer cells (I-10/DDP cells) in response to cisplatin treatment and the effect of silencing ATG5 and ATG7 on autophagy and proliferation of cisplatin-treated cells. I-10/DDP cells treated with 15 μmol/L cisplatin for 12 h were examined for expressions of LC3 and p62 by Western blotting and for autophagy level through transmission electron microscopy and mCherry-GFP-LC3B. I-10/DDP cells were transfected with short hairpin RNAs shRNA-ATG5 or shRNA-ATG7 via Lipfectamine2000, the empty vector (NC group), or Lipfectamine2000 alone (blank control group), and the cellular expressions of ATG5 and ATG7 were detected with Western blotting. The transfected cells were treated with 15 μmol/L cisplatin for 12 h, after that the expressions of LC3 and p62 were detected with Western blotting. Transmission electron microscopy and mCherry-GFP-LC3B were used to detect autophagy level in the cells. MTT assay and colony-forming assay were performed to assess the cell survival fraction and colony formation ability of the treated cells, respectively. After cisplatin treatmert, the expression level of LC3 II increased significantly (P < 0.001), the expression level of p62 decreased (P < 0.05), and the number of autophagosomes increased in I-10/DDP cells. The cells transfected with shRNA-ATG5 or shRNA-ATG7 showed significantly decreased expressions of ATG5 or ATG7 (P=0.005 or P < 0.001). Cisplatin treatment of the transfected cells obviously reduced the cellular expression of LC3 II (P < 0.001), increased the expression of p62 (P < 0.001), and decreased the number of autophagosomes, cell survival fraction and colony formation ability of the cells (P < 0.001). Silencing ATG5 and ATG7 inhibits cisplatin-mediated autophagy and enhances the inhibitory effect of cisplatin on inhibiting cell proliferation.

Cancer Testis Antigen, NOL4, Is an Immunogenic Antigen Specifically Expressed in Small-Cell Lung Cancer.

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.

Family history of any cancer for childhood leukemia patients in Sweden.

Acute lymphoblastic leukemia (ALL) is the most common childhood leukemia, while the other types, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML) are much rarer. While data on familial risks for childhood ALL have been emerging, such data for the other childhood leukemias are hardly available. We aim to fill in the gap of knowledge by assessing familial clustering of each childhood leukemia with childhood and adult leukemia and with any cancer. We identified 4461 childhood leukemias from the Swedish Cancer Registry and obtained their family members from the Multigeneration Register. Standardized incidence ratios (SIRs) were 3.34 for singleton siblings both diagnosed with ALL before age 20 years and 1.64 for those who had a family member diagnosed with ALL in adult age. Other childhood leukemias showed no familial risk, but childhood ALL risk was increased to 1.40 when adult family members were diagnosed with CLL. Childhood ALL was associated with endometrial cancer, and female ALL patients showed increased risk when family members were diagnosed with testicular cancer, melanoma, and skin squamous cell carcinoma. Childhood CLL was associated with rectal cancer, and childhood AML was associated with pancreatic and bladder cancers. As most of these associations are reported for the first time, there is a need to replicate the findings from independent sources.