TK1 Expression Research Articles

Multi-Omics Integration Analysis of TK1 in Glioma: A Potential Biomarker for Predictive, Preventive, and Personalized Medical Approaches

Multi-omics expression datasets obtained from multiple public databases were used to elucidate the biological function of TK1 and its effects on clinical outcomes. The Kaplan-Meier curve, a predictive nomogram mode, and the time-dependent receiver operating characteristic (ROC) curve were established to assess the role of TK1 expression in glioma prognosis. TK1 was overexpressed in glioma compared with normal samples, and patients with elevated expression of TK1 had poor overall survival. The ROC curves indicated a high diagnostic value of TK1 expression in patients of glioma; the areas under the ROC curve (AUC) were 0.682, 0.735, and 0.758 for 1 year, 3 years, and 5 years of glioma survival, respectively. For a model based on TK1 expression and other clinical characteristics, the values of AUC were 0.864, 0.896, and 0.898 for 1 year, 3 years, and 5 years, respectively. Additionally, the calibration curve indicated that the predicted and observed areas at 1 year, 3 years, and 5 years of survival were in excellent agreement. Three types of TK1 alterations-missense mutations, splice mutations, and amplifications-were identified in 25 of 2706 glioma samples. The TK1-altered group had better overall survival than the unaltered group. Single-cell function analysis showed that TK1 was positively associated with proliferation, the cell cycle, DNA repair, DNA damage, and epithelial-mesenchymal transition in glioma. Immunoinfiltration analysis indicated that TK1 expression might play different roles in low-grade glioma and glioblastoma multiforme tumor microenvironments, but TK1 expression was positively associated with activated CD4 and Th2, regardless of tumor grade. In summary, our findings identified TK1 as a novel marker for predicting clinical outcomes and a potential target for glioma.

TK-1, TP, Ang-2, and Tie-2 mRNA expression in plasma-derived microvesicles of chemo-refractory metastatic colorectal cancer patients.

Trifluridine/tipiracil and regorafenib are indicated for metastatic colorectal cancer (mCRC) patients' refractory to standard chemotherapy. No prognostic or predictive biomarkers are available for these agents. We assessed messenger ribonucleic acid (mRNA) expression of four biomarkers implicated in the mechanism of action of trifluridine/tipiracil (TK-1 and TP) and regorafenib (Ang-2 and Tie-2) in baseline plasma-derived microvesicles of chemo-refractory mCRC patients treated with these agents (trifluridine/tipiracil cohort and regorafenib cohort), to explore their prognostic and predictive role. Baseline characteristics of the two cohorts were not different. Ang-2 mRNA was not detectable. Only TK-1 expression measured as a continuous variable was associated with progression-free survival (HR=1.09, 95%CI: 0.99-1.21; p=0.07) and overall survival (HR=1.11, 95%CI: 1.00-1.22; p=0.04), confirmed at multivariate analysis for progression-free survival (p=0.02) with a positive trend for overall survival (p=0.08). Baseline mRNA levels of TK-1, TP and Tie-2 were not predictive of trifluridine/tipiracil and regorafenib benefit. Baseline mRNA levels of TK-1, TP and Tie-2 on plasma-derived microvesicles were not predictive of trifluridine/tipiracil and regorafenib benefit. Future studies should analyze the early modulation of these biomarkers to assess their potential predictive role.

Combined Inhibition of CDK2 and BCL2 Overrides Resistance to Targeting BTK and CDK4/6 in Mantle Cell Lymphoma Therapy

Mantle cell lymphoma (MCL) remains incurable due to the development of drug resistance, despite the plethora of therapies available. Each successive treatment failure is associated with a more rapidly proliferating disease and fewer treatment options. Understanding the drug-resistance mechanism and developing effective, well-tolerated, new therapies for MCL are urgently needed. Unrestrained proliferation of MCL cells is mainly driven by aberrant cyclin D1 expression and dysregulated CDK4 activity that promotes cell cycle progression from G1 to S. In our phase 1 clinical trial inhibiting CDK4/6 with palbociclib and BTK with ibrutinib (PALIBR) in recurrent MCL, the complete response rate (CR) was 42% compared to 21% in response to ibrutinib alone, despite a comparable 67% overall response rate. Moreover, 5 patients remain on therapy (2 CR and 3 PR) for ~ 8 years, suggesting that CDK4/6 inhibition deepens and prolongs the clinical response to BTK inhibition in MCL. However, resistance emerges in some patients. To elucidate the underpinnings, we undertook integrative longitudinal genomic analysis of sequential tissue and blood specimens (n=53) of 27 MCL patients before, during therapy and on progression, and 4 treatment-naïve MCL patients and 4 normal subjects as controls. Whole exome and whole transcriptome sequencing of freshly isolated MCL cells and B cells revealed that resistance to PALIBR is rooted in homozygous deletion of RB1 (direct target of CDK4/6) or compound copy number variation (cCNV) - concurrent hemizygous loss of RB1 and CDKN2A (p16, CDK4/6 inhibitor) and gain of CDK4. In other resistant patients, the RB protein is silenced by translational termination mutation. Single-cell RNA-sequencing (scRNA-seq, 210,000 cells) of corresponding PBMCs using a unique in-house MCL RNA reference library reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling; C3 represents non-proliferating, long-lived MCL cells; and C4 is highly proliferative, expanding with disease progression. RNA velocity analysis further uncovered that C2 fuels the transition to C4 with disease progression, whereas C3 is a terminal node (Fig 1). In C2 and C3 MCL cells, CDK2 is highly elevated, indicating evasion of CDK4/6 inhibition, and PIK3IP1 (negative PI3K regulator) is silenced, suggesting that compensatory PI3K activation in BTKi resistance (Chiron et al, Cancer Discovery 2014) is secondary to loss of cell cycle control. BCL2 is markedly elevated in C2, greater in C3 MCL cells along with increases in anti-apoptotic BCL2L1(BCL-XL) and BCL-2A1 and decreases in pro-apoptotic PMAIP1 (NOXA) expression. This suggests combined CDK2 and BCL2 inhibition as a rational approach to override CDK4/6i and BTKi resistance. Indeed, combined inhibition of CDK2 with PF-07104091 and BCL2 with venetoclax (PF-VEN) cooperatively impaired growth and killed ibrutinib-resistant MAVER-1 cells, more effectively in the isogenic derivative MAVER-1R cells which we generated to recapitulate cCNV in PALIBR resistance - depletion of RB protein through translational termination mutation (W99, AAF 75%) in RB1, loss of CDKN2A and gain of CDK4. Mechanistically, MAVER-1R cells are accelerated in G1 to S progression, based on elevation of CDK2 and TK1 expression and cell cycle profiling. They are intrinsically apoptotic and vulnerable to BCL2 inhibition due to a marked reduction in cells expressing anti-apoptotic BCL2L1 and IRF4, and universal expression of pro-apoptotic PMAIP1 (NOXA). Importantly, combined CDK2 and BCL2 inhibition synergistically kills MCL cells of a patient resistant to PALIBR ex vivo. In summary, by longitudinal genomic analysis, we have provided the first evidence that 1) resistance to CDK4/6i and BTKi stems from MCL intrinsic cCNV; 2) MCL cells comprise 4 major transcriptomically distinct clusters; cluster 2 expressing copious CDK2 and BCL2 is pivotal in fueling proliferation of MCL cells in resistance; 3) cluster 3 cells also accumulate with resistance due to longevity; 4) combined inhibition of CDK2 and BCL2 overrides resistance to CDK4i and BTKi, in MCL cell lines and primary MCL cells from PALIBR resistant patient. Combined inhibition of CDK2 and BCL2, therefore, represents a mechanism-based strategy to overcome CDK4/6i and BTKi resistance in MCL therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

External Validation of the Colon Life Nomogram for Predicting 12-Week Mortality in Dutch Metastatic Colorectal Cancer Patients Treated with Trifluridine/Tipiracil in Daily Practice.

Simple SummaryPredicting prognosis in cancer patients is needed to guide decision making. In order to predict survival, nomograms can be used to estimate chances of survival based on clinical characteristics. In order to identify metastatic colorectal cancer (mCRC) patients with a very short life expectancy (less than 12 weeks) after receiving multiple standard treatments, the Colon Life nomogram was previously developed. Before a nomogram can be used in daily practice, it is essential to show that it accurately predicts survival in different real-life populations and can be used to guide clinical decision making. This is called external validation. We externally validated the Colon Life nomogram in a cohort of patients with refractory mCRC who were treated with a last treatment option, trifluridine/tipiracil, in daily practice. We demonstrated that the nomogram severely overestimated 12-week mortality and therefore should not be used in clinical practice in its present form. We also showed that quality of life reported by patients themselves can improve the prediction of survival, stressing the importance of patient-reported outcomes. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model and include quality of life.Background: Predicting prognosis in refractory metastatic colorectal cancer (mCRC) patients is needed to guide decision making. The Colon Life nomogram was developed to predict 12-week mortality in refractory mCRC patients. The aim of this study is to validate the Colon Life nomogram in last line/refractory patients receiving trifluridine/tipiracil (FTD/TPI) in daily practice. Methods: The validation cohort consists of 150 QUALITAS study patients, an observational substudy of the Prospective Dutch CRC cohort, who were treated with FTD/TPI between 2016 and 2019. Model performance was assessed on discrimination, calibration, and clinical usefulness. The additional prognostic value of baseline quality of life (QoL) and thymidine kinase (TK1) expression in tissue was explored. Results: Of the 150 patients, 25 (16.7%) died within 12 weeks of starting FTD/TPI treatment. The C-statistic was 0.63 (95% C.I. 0.56–0.70). The observed/expected ratio was 0.52 (0.37–0.73). The calibration intercept and slope were −1.06 (−1.53 to −0.58) and 0.41 (0.01–0.81), respectively, which indicated overestimation of 12-week mortality by the nomogram. Decision curve analysis showed the nomogram did not yield a positive net benefit at clinically meaningful thresholds for predicted 12-week mortality. Addition of QoL to the nomogram improved the C-statistic to 0.85 (0.81–0.89). TK1 expression was associated with progression-free survival but not with overall survival. Conclusion: We demonstrated evident miscalibration of the Colon Life nomogram upon external validation, which hampers its use in clinical practice. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model including QoL.

TFAP4 Activates IGF2BP1 and Promotes Progression of Non-Small Cell Lung Cancer by Stabilizing TK1 Expression through m6A Modification.

Our findings revealed that TFAP4 activated IGF2BP1 and facilitated NSCLC progression by stabilizing TK1 expression via m6A modification, which offered new insights into the diagnosis and treatment of NSCLC.

Methylation status of TK1 correlated with immune infiltrates in prostate cancer.

TK1 is overexpressed in numerous cancers and is associated with to a poor prognosis. However, the relationship between methylation status of TK1 and Immune Infiltrates in Prostate Cancer (PCa) is unknown. The goal of this study was to use comprehensive bioinformatic analyses to elucidate the involvement relationship between methylation status of TK1 and Immune Infiltrates in PCa. TK1 mRNA expression and methylation data in PCa were investigated via GEPIA, TIMER, and UALCAN coupled with MEXPRESS data resources. We employed the LinkedOmics data resource to determine the signaling cascades linked to TK1 expression. Single-cell analysis was performed using the CancerSEA data resource. GeneMANIA and CancerSEA were used to analyze the correlation between TK1 and TK1 coexpressed genes. In addition, TIMER and TISIDB were adopted to assess tumor-invading immune cells and immunomodulators. CTD was utilized to detect the drugs acting on TK1. This study found that TK1 was overexpressed in PCa, and its contents were linked to tumor stage and prognosis. Genes co-expressed with TK1 were enriched in cascades involved in the ribosome, cell cycle, oxidative phosphorylation, DNA replication, oocyte meiosis, and the proteasome. The expression of TK1 along with its methylation status was found to be linked to tumor-invading immune cells, as well as PCa immunomodulators. We also examined the prospect of employing TK1 as a possible target for PCa therapy. This work provides the clinical value of TK1 hypermethylation in PCa and highlights new insights into its novel immunomodulatory functions.

NF-κB-Activated lncRNACASC9 Promotes Bladder Cancer Progression by Regulating the TK1 Expression.

Long noncoding RNAs (lncRNA) are involved in cancer development, but the roles of most lncRNAs are undocumented. In this study, we identified lncRNAs that were abnormally expressed in bladder cancer. We found that lncRNACASC9 plays an important role in the progression of bladder cancer. CASC9 was highly expressed in bladder cancer cells and tissues, and the prognosis of bladder cancer patients with high expression of CASC9 was poor. The results of colony formation assays, CCK-8 assays, EdU assays, transwell assays, mouse xenograft models, and tail vein injection lung metastasis model showed that CASC9 could promote bladder cancer cells growth and metastasis both in vitro and in vivo. Mechanistically, through FISH experiments, luciferase reporter experiments, and RIP experiments, we proved that CASC9 regulated the expression of TK1 by adsorbing miR-195-5p, thereby exerting an oncogenic effect in bladder cancer. Taken together, our findings support that the CASC9/miR-195-5p/TK1 axis is a critical pathway in the tumorigenesis and progression of bladder cancer, implicating a new therapeutic direction for the treatment of bladder cancer.

Identification of Novel Immune Cell-Relevant Therapeutic Targets and Validation of Roles of TK1 in BMSCs of Systemic Lupus Erythematosus.

Objective: Systemic lupus erythematosus (SLE) displays the characteristics of abnormal activity of the immune system, contributing to diverse clinical symptoms. Herein, this study was conducted for discovering novel immune cell-relevant therapeutic targets. Methods: The abundance of diverse immune cells was estimated in PBMCs of SLE and healthy controls from the GSE50772 dataset with CIBERSORT approach. Immune cell-relevant co-expression modules were screened with WGCNA and relevant characteristic genes were determined with LASSO algorithm. Inflammatory chemokines were measured in serum of twenty SLE patients and twenty controls through ELISA. Bone marrow mesenchymal stem cells (BMSCs) were isolated and TK1 expression was measured in BMSCs through RT-qPCR and western blotting. TK1-overexpressed and TK-1-silenced BMSCs of SLE were conducted and apoptosis and cell cycle were measured with flow cytometry. Apoptosis-, cell cycle- and senescence-relevant proteins were tested with western blotting. Results: We determined three co-expression modules strongly linked to immune cells. Five characteristic genes (CXCL1, CXCL2, CXCL8, CXCR1 and TK1) were screened and ROC curves proved the excellent diagnostic performance of this LASSO model. Inflammatory chemokines presented widespread up-regulations in serum of Systemic lupus erythematosus patients, demonstrating the activation of inflammatory response. TK1 expression was remarkably elevated in SLE BMSCs than controls. TK1 overexpression enhanced IL-1β expression, apoptosis, cell cycle arrest, and senescent phenotypes of SLE BMSCs and the opposite results were proved in TK1-silenced SLE BMSCs. Conclusion: Collectively, our findings demonstrate that silencing TK1 alleviates inflammation, growth arrest and senescence in BMSCs of SLE, which highlights TK1 as a promising therapeutic target against SLE.

Comprehensive Analysis of Immune-Related Prognosis of TK1 in Hepatocellular Carcinoma.

Increased expression of TK1 is associated with the progression of a variety of tumors. However, the relationship of TK1 expression with immune cell infiltration and its prognostic value in hepatocellular carcinoma (HCC) are still unknown. In this study the TCGA database was used to evaluate TK1 expression and its impact on survival in patients with HCC. Compared with normal tissue, TK1 in the liver tissue of patients with HCC was significantly up-regulated at both the mRNA and protein levels. Furthermore, TK1 expression was significantly related to pathological stage, tumor stage and lymph node metastasis, with high TK1 expression being an unfavorable prognostic factor for HCC. TK1 expression was also significantly associated with the infiltration of B cells, T cells, and dendritic cells in HCC. Single-cell sequencing analysis revealed that TK1 was associated with relatively large changes in T cells, especially gamma-delta T cells. A prognostic risk score based on TK1-related immune genes (CD40LG and TNFRSF4) was established using COX regression analysis. By integrating the immune-related risk score model with clinical features, a nomogram was constructed to predict the survival rate of HCC patients (1 year, 3-year and 5-year AUC of 0.782, 0.783 and 0.771, respectively). Knockdown of the target gene for TK1 was found to have significant anti-apoptosis and pro-proliferation effects on HepG2 cells. The level of TK1 in the serum and liver tissue of patients with HCC was significantly increased relative to healthy controls. These findings highlight the role of TK1 in the tumor immune response of HCC patients and in the proliferation and apoptosis of HepG2 cells. TK1 could therefore be a potential immunotherapy target for HCC patients, while the two immune genes related to TK1 (CD40LG And TNFRSF4) may be promising prognostic biomarkers in HCC.

A comprehensive analysis of ncRNA-mediated interactions reveals potential prognostic biomarkers in prostate adenocarcinoma

As one of common malignancies, prostate adenocarcinoma (PRAD) has been a growing health problem and a leading cause of cancer-related death. To obtain expression and functional relevant RNAs, we firstly screened candidate hub mRNAs and characterized their associations with cancer. Eight deregulated genes were identified and used to build a risk model (AUC was 0.972 at 10 years) that may be a specific biomarker for cancer prognosis. Then, relevant miRNAs and lncRNAs were screened, and the constructed primarily interaction networks showed the potential cross-talks among diverse RNAs. IsomiR landscapes were surveyed to understand the detailed isomiRs in relevant homologous miRNA loci, which largely enriched RNA interaction network due to diversities of sequence and expression. We finally characterized TK1, miR-222-3p and SNHG3 as crucial RNAs, and the abnormal expression patterns of them were correlated with poor survival outcomes. TK1 was found synthetic lethal interactions with other genes, implicating potential therapeutic target in precision medicine. LncRNA SNHG3 can sponge miR-222-3p to perturb RNA regulatory network and TK1 expression. These results demonstrate that TK1:miR-222-3p:SNHG3 axis may be a potential prognostic biomarker, which will contribute to further understanding cancer pathophysiology and providing potential therapeutic targets in precision medicine.

Comprehensive Nursing can Relieve the Negative Emotions of Patients undergoing Breast Cancer Resection and Reduce Postoperative

To explore the effect of comprehensive nursing on adverse emotions and postoperative complications of breast cancer patients undergoing mastectomy. Altogether 180 patients who received treatment in our hospital from May 2017 to May 2019 were selected as the research participants and divided into group A and group B. Among them, 100 cases in group A received comprehensive nursing, 80 cases in group B received routine nursing. The surgical indications, upper limb function, serum NGF, TK1 and CA15-3 expression level, VAS score, SAS, SDS score, quality of life SF-36 score were detected, and the incidence rate of postoperative complications and nursing quality score were compared. Compared with group B, group A had less postoperative bed time, intraoperative blood loss, length of hospital stay, better recovery of upper limb function, lower expression levels of serum NGF, TK1 and CA15-3, lower VAS score, SAS and SDS score, higher quality of life SF-36 score, lower incidence of postoperative complications and higher nursing quality score. Comprehensive nursing can relieve the negative emotions of patients undergoing breast cancer resection and reduce the incidence of postoperative complications.

Long Non-Coding RNA CCAT2 Promotes the Development of Esophageal Squamous Cell Carcinoma by Inhibiting miR-200b to Upregulate the IGF2BP2/TK1 Axis.

Long non-coding RNAs (lncRNAs) have been shown to play important roles in human cancers, including esophageal squamous cell carcinoma (ESCC). In the current study, we identified CCAT2 as a relevant lncRNA and investigated its role in the progression of ESCC. RT-qPCR was adopted to detect CCAT2 expression in collected clinical samples, ESCC cell lines, and a normal cell line. We tested the correlation between CCAT2 expression and the prognosis of ESCC. RT-qPCR or immunoblotting was adopted to detect the expression of relevant factors in ESCC tissues or cells. Cell proliferation, apoptosis, migration, and invasion were examined by colony formation assay, flow cytometry, scratch assay, and Transwell assay, respectively, while subcutaneous tumorigenesis in nude mice was adopted to examine the role of CCAT2 in tumorigenesis of ESCC cells in vivo. Bioinformatics analysis, dual luciferase reporter assay, and RIP were conducted for the target relationship profiling. Me-RIP was adopted to detect m6A modification level of TK1 in ESCC tissues or cells. Upregulated CCAT2, IGF2BP2, and TK1 expression and inhibited miR-200b expression were observed in ESCC cells and tissues. CCAT2 bound to miR-200b and reduced its expression, leading to upregulated IGF2BP2 expression. IGF2BP2 improved TK1 mRNA stability to enhance its expression by recognizing its m6A modification. CCAT2 promoted the migration and invasion of ESCC cells in vitro, and tumorigenesis in vivo by upregulating TK1 expression, while overexpression of miR-200b reversed these effects of CCAT2. Overall, this study suggests that CCAT2 competitively binds to miR-200b to alleviate its inhibitory effects on IGF2BP2 expression, resulting in elevated TK1 expression, and an ensuing promotion of the development of ESCC.

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells

BackgroundThymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells.MethodsWe generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments.ResultsBinding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73–66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments.ConclusionsThe antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

The modulation study of multiple drug resistance in bladder cancer by curcumin and resveratrol.

Gemcitabine (GCB), which functions via the inhibition of DNA synthesis, is commonly used in the treatment of bladder cancer; however, its response rate is not satisfactory due to the development of drug resistance. The potential for phytochemicals to reverse drug resistance in bladder cancer tumor cells was evaluated. A human bladder cancer cell line, T24, was cultured, and GCB-resistant cells (T24-GCB) were also established. The acquired resistance of T24-GCB to GCB was measured using an MTT assay. The gene expression of ATP-binding cassette (ABC) transporter protein family members was analyzed using reverse transcription-quantitative PCR analysis, and western blotting was performed to verify ABC family protein, cytoplasmic thymidine kinase (TK) and poly (ADP-ribose) polymerase (PARP) expression on whole cell lysates. Subsequently, resveratrol and curcumin were used to evaluate their modulation potential in decreasing the drug resistance of T24-GCB cells to GCB using MTT and migration assays. T24-GCB cells have increased drug resistance ability, with an 18.75-fold higher ID50 value compared with native T24 cells (105 vs. 5.6 nM). T24-GCB cells also exhibit increased cross resistance to mitomycin C and paclitaxel. The mRNA expression of ABCC2 in T24-GCB cells increased compared with that in native T24 cells. Via western blot analysis, it was determined that the expression of ABCC2 protein was also increased in T24-GCB cells. Conversely, the expression of ABCB1, ABCG2, deoxycytidine kinase (DCK), TK1 and TK2 decreased. Following curcumin and resveratrol treatment alone or combined with GCB, additive cytotoxic enhancement was observed, and the migratory abilities of T24-GCB cells were significantly decreased. Western blot analysis revealed that ABCC2 protein expression increased, and DCK, TK1 and TK2 expression decreased following co-treatment of T24-GCB cells with GCB + curcumin or resveratrol compared with GCB alone. Of note, there was a marked increase in cleaved-PARP expression in T24-GCB cells treated with a combination of GCB + curcumin or resveratrol. Both curcumin and resveratrol could reverse the drug resistance of T24-GCB cells in an additive pattern though PARP enhancement without changes in ABCC2 and DCK, TK1 and TK2 expression.

Depicting Changes in Tumor Biology in Response to Cetuximab Monotherapy or Combination Therapy by Apoptosis and Proliferation Imaging Using 18F-ICMT-11 and 18F-FLT PET.

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7–specific isatin-5-sulfonamide 18F-ICMT-11 (18F-(S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluoro-phenoxymethyl)-pyrrolidine-1-sulfonyl)isatin) and 18F-FLT (3′-deoxy-3′-18F-fluorothymidine), were used to detect early treatment-induced changes in tumor biology and determine whether any of these changes indicate a response to cetuximab, administered as monotherapy or combination therapy with gemcitabine. Methods: In mice bearing cetuximab-sensitive H1975 tumors (non–small lung cancer), the effects of single or repeated doses of the antiepidermal growth factor receptor antibody cetuximab (10 mg/kg on day 1 only or on days 1 and 2) or a single dose of gemcitabine (125 mg/kg on day 2) were investigated by 18F-ICMT-11 or 18F-FLT on day 3. Imaging was also performed after 2 doses of cetuximab (days 1 and 2) in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer). For imaging–histology comparison, tumors were evaluated for proliferation (Ki-67 and thymidine kinase 1 [TK1]), cell death (cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and target engagement (epidermal growth factor receptor expression) by immunohistochemistry, immunofluorescence, and immunoblotting, respectively. Tumor and plasma were analyzed for thymidine and gemcitabine metabolites by liquid chromatography–mass spectrometry. Results: Retention of both tracers was sensitive to cetuximab in H1975 tumors. 18F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab (75%) and combination treatment (46%). Although a single dose of cetuximab was insufficient to induce apoptosis, it did affect proliferation. Significant reductions in tumor 18F-FLT uptake (44%–50%; P < 0.001) induced by cetuximab monotherapy and combination therapy were paralleled by a clear decrease in proliferation (Ki-67 decrease, 72%–95%; P < 0.0001), followed by a marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly reduced. Neither imaging tracer depicted the gemcitabine-induced tumor changes. However, cleaved caspase-3 and Ki-67 staining did not significantly differ after gemcitabine treatment whereas TK1 expression and thymidine concentrations increased. No cetuximab-induced modulation of the imaging tracers or other response markers was detected in the insensitive model of HCT116. Conclusion: 18F-ICMT-11 and 18F-FLT are valuable tools to assess cetuximab sensitivity depicting distinct and time-variant aspects of treatment response.

TK1 overexpression is associated with the poor outcomes of lung cancer patients: a systematic review and meta-analysis.

The study aimed to unveil the effect of TK1 expression on the clinicopathological significance and prognosis in patients with lung cancer. Results & methodology: Studies for meta-analysis were selected according to a thorough literature search in databases (PubMed, EMBASE and Chinese databases). Ten studies containing 1393 lung cancer patients were investigated in our analysis. The TK1 overexpression was associated with poorer overall survival(OS) in lung cancer patients (hazard ratio=1.881; 95% CI:1.318-2.684, Z=3.48; p =0.001). Furthermore, The TK1 expression is associated with the clinicopathological features of lung cancer patients (tumor type, age, lymph node metastasis, distant metastasis and tumor, node, metastasis [TNM] stages). Discussion & conclusion: The TK1 expression might have a supportive implication in assessing biological behavior and prognosis of lung cancer patients.

Abstract OT3-05-09: A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer (Maintain trial)

Abstract Background Cyclin dependent kinase 4/6 inhibitors (CDK4/6i), including palbociclib and ribociclib (R), have demonstrated remarkable benefit in progression free survival (PFS) in patients (pts) with hormone receptor positive (HR+), HER2- metastatic breast cancer (MBC) when combined with anti-estrogen therapy. Switching between anti-estrogen therapies at disease progression is standard of care in the treatment of HR+ MBC. We evaluate the strategy of switching anti-estrogen therapy to fulvestrant (F) and maintaining CDK4/6 inhibition with R in pts with HR+, HER2- MBC who have progressed on an aromatase inhibitor (AI) + CDK4/6i. Trial Design Phase II, multi-center, randomized, double-blind, placebo-controlled trial to evaluate F +/- R in pts with HR+, HER2- MBC who have previously progressed on any AI + CDK4/6i. Pts can be screened and registered at two different time points: Scenario 1: Before receiving any CDK4/6i Scenario 2: At the time of progression of disease (POD) while being treated with an AI + CDK4/6i In scenario 1, the study will provide pts with letrozole + R, but pts will not be randomized until they demonstrate POD. At randomization, pts will be assigned 1:1 to either a) F + R or b) F + placebo, with treatment given in 4-week cycles. F will be given as a 500 mg dose intramuscularly every 2 weeks for 3 times and then every 4 weeks, as per standard of care. R or placebo will be given orally at 600 mg daily, 3 weeks on/1 week off. CT scans and bone scan are to be performed prior to every third cycle. Serum and whole blood samples and optional tissue biopsies for biomarker assessment will be performed prior to study treatment (scenario 1), prior to randomization to R +/- F, and when the patient goes off study. Main Eligibility Criteria 1. Age ≥ 18 years with unresectable or metastatic BC 2. Estrogen and/or progesterone receptor positive, HER2 negative, as per ASCO-CAP 3. Postmenopausal status or receiving ovarian suppression 4. Measurable or unmeasurable disease; stable CNS disease allowed Specific Aims Primary: Progression free survival (PFS), defined as the time from randomization to POD or death. Secondary: Objective response rate (ORR), clinical benefit rate (CBR = ORR + stable disease rate), overall survival (OS), and duration of response. Pts in scenario 1 will also be assessed for PFS, OS, CBR, and safety while receiving AI + R (pre-randomization). Biomarker assessment will include amplification of cyclin D1 and cyclin E, phosphoRb and TK1 expression, Rb1 and p16 loss, and ctDNA for ESR1 and PIK3CA mutations. Target Accrual 132 pts accrued from 11 academic medical centers in the U.S, with a goal of completing accrual in two years (˜60 to 72 pts in each scenario). Statistical Methods Assuming a median PFS of 3.8 months with F alone, we predict that F + R will lead to a median PFS of at least 6.5 months. A one-sided log-rank test with a sample size of N=120 and alpha=0.025, achieves 80% power to detect a difference in PFS of 2.7 months. N=132 pts allows for a 10% drop-out rate. Contact Kevin Kalinsky, MD, MS 212-305-1945 kk2693@cumc.columbia.edu. Citation Format: Kalinsky K, Mundi PS, Chiuzan C, Accordino MK, Trivedi MS, Sparano JA, Oh SY, Tiersten A, O'Regan R, Esteva FJ, Jain S, Mayer IA, Forero A, Vaklavas C, Baer L, Crew K, Hershman DL. A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer (Maintain trial) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-05-09.

Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses.

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P < 0.001; 397 up, 531 down). These clustered in pathways involved in signalling, sterol/lipid metabolism, cell proliferation/hypertrophy and apoptosis. Signalling genes included some implicated in adrenal adenomas but also upregulated genes associated with cyclic AMP and downregulated genes associated with aldosterone synthesis. Sterol metabolism genes were those promoting cholesterol supply (Scarb1, Sqle, Apoa1) and disposal (Cyp27a1, Cyp7b1). Oil red O staining showed lipid depletion consistent with reduced expression of genes involved in lipid synthesis. Genes involved in steroidogenesis (Star, Cyp11a1, Cyp11b1) were modestly affected (P < 0.05; <1.3-fold). Increased Ki67, Ccna2, Ccnb2 and Tk1 expression complemented immunohistochemical evidence of a 3-fold change in cell proliferation. Growth arrest genes, Cdkn1a and Cdkn1c, which are known to be active in hypertrophied cells, were increased >4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu,) were downregulated and apoptotic cells (Tunel staining) were fewer (P < 0.001) and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways.

A randomized phase II trial of fulvestrant with or without ribociclib after progression on aromatase inhibition plus cyclin-dependent kinase 4/6 inhibition in patients with unresectable or metastatic hormone receptor positive, HER2 negative breast cancer.

TPS1112 Background: CDK4/6i, including palbociclib and ribociclib (R), have demonstrated remarkable benefit in progression free survival (PFS) in patients (pts) with HR+, HER2- MBC with anti-estrogen therapy. Switching between anti-estrogen therapies at disease progression is standard of care in the treatment of HR+ MBC. We evaluate the strategy of switching anti-estrogen therapy to fulvestrant (F) and maintaining CDK4/6 inhibition with R in pts with HR+, HER2- MBC who have progressed on an AI + CDK4/6i. Methods: Trial Design Phase II, multi-center, randomized, double-blind, placebo-controlled trial to evaluate F +/- R in pts with HR+, HER2- MBC who have previously progressed on any AI + CDK4/6i: Screened at 2 different scenarios: Scenario 1: Before receiving any CDK4/6i Scenario 2: Time of progression of disease (POD) while being treated with an AI + CDK4/6i Intervention At randomization, pts assigned 1:1 to either a) F + R or b) F + placebo, with treatment given in 4-week cycles. Major Eligibility Criteria 1, Metastatic BC, 2. HR+ HER2-, 3. Measurable or unmeasurable disease Specific Aims Primary: PFS. Secondary: Objective response rate, clinical benefit rate, overall survival, and duration of response. Biomarker assessment: amplification of cyclin D1 and cyclin E, phosphoRb and TK1 expression, Rb1 and p16 loss, and ctDNA for ESR1 and PIK3CA mutations. Statistical Methods Assuming a median PFS of 3.8 months with F alone, we predict that F + R will lead to a median PFS of at least 6.5 months. A one-sided log-rank test with a sample size of N = 120 and alpha = 0.025, achieves 80% power to detect a difference in PFS of 2.7. With a 10% dropout, n = 132. Clinical trial registry number NCT02632045.