Cell Sublines Research Articles

P3.02-089 Establishment of Highly Metastatic Lung Cancer Cell Sublines in Long-term Three-dimensional Low Attachment Cultures

Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. We investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential and examined the mechanisms underlying metastasis.

A newly synthesized nickel chelate can selectively target and overcome multidrug resistance in cancer through redox imbalance both in vivo and in vitro.

Induction of undesired toxicity and emergence of multidrug resistance (MDR) are the major obstacles for cancer treatment. Moreover, aggressive cancers are less sensitive towards existing chemotherapeutics. Therefore, selective targeting of cancers without inducing undesired side effects and designing proper strategies to overcome MDR has utmost importance in modern chemotherapy. Previously we revealed the anticancer properties of some transition metal chelates of Schiff base, but the effectiveness of nickel complex is still unrevealed. Herein, we synthesized and characterized a Schiff base nickel chelate, nickel-(II) N-(2-hydroxyacetophenone) glycinate (NiNG), through different spectroscopic means. NiNG proves to be a broad spectrum anticancer agent with considerable efficacy to overcome MDR in cancer. Antiproliferative effects of NiNG was evaluated using drug-resistant (CEM/ADR5000; NIH-MDR-G185; EAC/Dox), drug-sensitive aggressive (Hct116; CCRF-CEM; EAC/S) and normal (NIH-3T3) cells that reveal the selective nature of NiNG towards drug resistant and sensitive cancer cells without inducing any significant toxicity in normal cells. Moreover, NiNG involves reactive oxygen species (ROS)-mediated redox imbalance for induction of caspase 3-dependent apoptosis in aggressive drug-sensitive Hct116 and drug-resistant NIH-MDR-G185 cells through disruption of mitochondrial membrane potential. Moreover, intraperitoneal (i.p.) application of NiNG at non-toxic doses caused significant increase in the life-span of Swiss albino mice bearing sensitive and doxorubicin-resistant subline of Ehrlich ascites carcinoma cells. It is noteworthy that, in vitro NiNG can only overcome P-glycoprotein-mediated MDR while in vivo NiNG can overcome MRP1-mediated MDR in cancer. Therefore, NiNG has therapeutic potential to target and overcome MDR in cancer.

The role of WT1 isoforms in vasculogenic mimicry and metastatic potential of human triple negative breast cancer cells

Triple negative breast cancer (TNBC) is highly aggressive and has a few therapeutic treatments, so new targeted therapy and biomarkers are required to provide alternative choices for treating TNBC patients. Recent studies showed that vasculogenic mimicry (VM), the formation of blood channels by aggressive cancer cells that mimic endothelial cells, is a factor contributing to poor prognosis in TNBC. Wilms' tumor 1 (WT1) gene has been found to be highly expressed in TNBC, and has 4 major distinct isoforms; isoform A (−17AA/–KTS; −/−), isoform B (+17AA/–KTS; +/−), isoform C (−17AA/+KTS; −/+) and isoform D (+17AA/+KTS; +/+). The involvement of each WT1 isoform in TNBC progression remains largely unclear. In this study, WT1 isoform-overexpressing cell sublines were established from a TNBC cell line, MDA-MB-231, by stable transfection, and the aggressive behavior of the cell sublines were evaluated. Only the WT1 isoform B- and isoform C-overexpressing cell sublines showed the significant increase in VM forming capability compared to the parental cell line and other isoform cell sublines. qRT-PCR was used to explore the change in expression level of two VM-related genes, EphA2 and VE-cadherin. All WT1 isoform cell sublines showed up-regulation of EphA2 but the levels detected in the isoform B- and isoform C-cell sublines were higher than those observed in other cell sublines. In contrast, significant up-regulation of VE-cadherin was found only in isoform A- and isoform D-cell sublines. Isoform B- and isoform C-cell sublines showed higher rates of cell migration compared to those of other cell sublines, as determined by both wound healing and Transwell assays. Gelatin zymography revealed increased MMP-9 enzyme production in isoform D-cell subline compared to the parental cell line, but this change was not observed in other cell sublines. Western blot analysis showed significantly increased expression of β-catenin in isoform B- and isoform C-cell sublines, compared to parental cell line and other isoform cell sublines. In conclusion, our findings demonstrate that WT1 isoforms play different roles in modulating the VM-forming capacity and metastatic potential of TNBC cells.

Androgen-independent LNCaP cells are a subline of LNCaP cells with a more aggressive phenotype and androgen suppresses their growth by inducing cell cycle arrest at the G1phase.

Androgen deprivation therapy (ADT, surgical or chemical castration) is the mainstay treatment for metastatic prostate cancer (PCa); however, patients ineluctably relapse despite extremely low androgen levels. This evolution of PCa indicates its lethal progression. In this study, to mimic the traits of clinical PCa progression in vitro, we investigated the alterations in the cell biological characteristics in androgen-independent LNCaP cells (LNCaP-AI cells) compared with LNCaP cells. We also examined the effects of androgen on LNCaP and LNCaP-AI cell proliferation, androgen receptor (AR) expression and prostate-specific antigen (PSA) secretion. Furthermore, AR was silenced in the LNCaP and LNCaP-AI cells to detect the roles that AR plays in cell growth, apoptosis and PSA secretion. We found that prolonged androgen ablation increased the LNCaP-AI cell growth rate and cell invasiveness, and induced epithelial-mesenchymal transition in the LNCaP-AI cells. Moreover, despite the fact that the LNCaP and LNCaP-AI cells expressed equal amounts of AR protein, androgen induced a greater secretion of PSA in the LNCaP-AI cells than in the LNCaP cells. The proliferation of the LNCaP-AI cells was not dependent on, but was suppressed by androgen, which led to arrest at the G1 phase. Conversely, androgen significantly increased LNCaP cell proliferation by promoting the G1-S transition. Moreover, the silencing of AR suppressed LNCaP and LNCaP-AI cell growth by inducing cell cycle arrest at the G1 phase rather than promoting apoptosis, and reduced PSA secretion. On the whole, our data suggest that LNCaP-AI cells have a more more aggressive phenotype compared with the LNCaP cells; AR remains a critical factor in the LNCaP-AI cells, and androgen suppresses LNCaP-AI cell growth by blocking the cell cycle at the G1 phase.

Establishment and characterization of a triple negative basal-like breast cancer cell line with multi-drug resistance.

Triple-negative breast carcinoma (TNBC) is one of the most aggressive subtypes of breast cancer and is associated with an unfavorable prognosis. The management of TNBC is currently based on the use of classical cytotoxic drugs, i.e., anthracyclines and/or microtubule-binding agents (TBAs). However, conventional chemotherapy is not always effective in these tumors and a systemic relapse is often observed, potentially due to the development of multi-drug resistance (MDR). Therefore, an improved understanding of MDR mechanisms may improve the therapeutic strategies for TNBC. In the present study, a paclitaxel-resistant (TxR) breast cancer cell subline of HCC1806 TNBC cells was established and characterized. The resistance index of this subline was calculated according to the IC50 of HCC1806-TxR relative to the parental HCC1806 cells (16.86-fold). TxR-cells also exhibited cross-resistance to vinblastin, doxorubicin and etoposide (~14-, ~4- and ~3-fold, respectively). As assessed with reverse transcription-quantitative polymerase chain reaction, TxR-resistant cells exhibited the upregulated expression of a number of multidrug resistance-associated genes, including MDR-1, MRP-1, -5, -6 and YB-1. The TxR cells also exhibited an increased expression of MDR-related proteins including MDR1 and MRP-1, which led to a substantial increase (5.4-fold) of the paclitaxel efflux from TxR-cells. In addition, the pro-apoptotic protein Fas was downregulated, whereas the anti-apoptotic Bcl-2 was upregulated, in TxR-cells. This may explain why a reduced extent of apoptosis was observed when TxR cells were exposed to TBAs and topoisomerase type II inhibitors, relative to the parental HCC1806 cells. Thus, the HCC1806-TxR cell line may serve as an appropriate model for the analysis of chemoresistance mechanisms in TNBCs, and for the investigation of novel anticancer agents for overcoming MDR-mediated mechanisms in TNBC.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Plasma membrane expression of ZNF185 is a prognostic factor in pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the major causes of cancer-associated mortality globally due to its high potential for distant metastasis. To understand hematogenous metastasis, the molecular expression profiles of weak metastatic PDC cell subline BxPC-3 and highly liver-metastatic cell subline LM-BxPC-3 were compared, and zinc finger protein 185 (ZNF185) was identified as a molecule that is upregulated in LM-BxPC-3 cells. The aim of the present study was to evaluate the clinicopathological significance of ZNF185 in PDC. Using immunohistochemistry, ZNF185 expression was investigated in 182 patients with PDC, in association with numerous clinicopathological variables. The expression profile of ZNF185 was also characterized using xenograft models. In contrast to parent BxPC-3 cells in subcutaneous transplanted tumor foci, which only expressed ZNF185 on their plasma membrane (m)ZNF185, LM-BxPC-3 cells in liver-metastatic foci that were formed subsequent to transplantation all expressed cytoplasmic (c)ZNF185. Additionally, 51% of the cells at the periphery of the tumor foci expressed mZNF185. Expression of cZNF185, and of mZNF185 and cZNF185 combined was identified in 93 and 39% of clinical patients with PDC, respectively. Patients with mZNF185-negative and -positive PDC exhibited a median survival time of 30.2 months and 21.3 months, respectively. Multivariate analysis indicated that the expression of mZNF185 is closely associated with a shorter overall survival time. Increased marked venous invasion was more prevalent in patients who were mZNF185-positive, as compared with patients who were mZNF185-negative. These data suggest that the expression of mZNF185 is an independent and unfavorable prognosticator in patients with PDC. The results suggested that the amount and subcellular location of ZNF185 are correlated with the position of the cancer cells expressing it within the nests. Additionally, the subcellular location of ZNF185 may be important to its biological function.

Radiation alters PD-L1/NKG2D ligand levels in lung cancer cells and leads to immune escape from NK cell cytotoxicity via IL-6-MEK/Erk signaling pathway.

We investigated whether radiation influences the susceptibility of non-small cell lung cancer (NSCLC) cells to NK cell mediated cytotoxicity. We found radiation treatment increased expression of programmed cell death ligand 1 (PD-L1), but decreased NK group 2, member D (NKG2D) ligand expressions in A549 and H157 NSCLC cells. Both types of changes would have protected tumor cells from the cytotoxic action of NK cells. Consistently, we detected similar alteration in these molecules in radioresistant A549R26-1 and H157R24-1 subline cells. Higher PD-L1 level was also observed in tumors of A549R26-1 cell-derived xenografts than tumors of parental A549 (A549P) cell-derived xenografts. Accordingly, we found radioresistant cells were more resistant to the cytotoxic action of NK cells than parental cells, and such resistance was decreased when neutralizing antibody (Ab) of PD-L1 was added to the radioresistant cell/NK cell co-cultures. In mechanism studies, we found that IL-6-MEK/Erk signaling contributed most significantly to the up-regulation of PD-L1/down-regulation of NKG2D ligands in radioresistant cells. The addition of the MEK/Erk inhibitor increased the susceptibility of A549R26-1 and H157R24-1 cells to NK-cell cytotoxicity while no significant effect was observed in parental cells. Moreover, we detected enhanced NK-cell cytotoxicity to radioresistant cells when PD-L1 Ab and MEK/Erk inhibitor were added together to co-cultures of tumor/NK cells compared to when PD-L1 Ab was used alone. We suggest that combined use of PD-L1 Ab and MEK/Erk inhibitor may offer better therapeutic benefits than PD-L1 Ab alone to treat NSCLC patients who are receiving radiotherapy or who are at the radioresistant stage.

Abstract 80: Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance

Abstract Background: Sorafenib is the only approved systemic agent for hepatocellular carcinoma (HCC). However, the efficacy of sorafenib is modest for HCC with low objective response rate and short time to tumor progression, indicating that the inherent and acquired resistance underlies the limited success of sorafenib in HCC. We conducted a pooled shRNA screening in mouse xenografts of human HCC cells treated with sorafenib, and intended to identify genes that are involved in the therapeutic efficacy of sorafenib. Materials and Methods: We introduced a pool of 3920 shRNAs targeting 1960 genes, including various kinases, transcriptional factors, phosphatase and cytokine, into Huh7 HCC cells by retrovirus infection. The Huh7 cells after infection with pooled shRNAs were implanted subcutaneously in immunocompromised mice, and were treated with vehicle or sorafenib at the dose of 30mg/kg/day for 4 weeks. Genomic DNAs were extracted from HCC xenografts, PCR-amplified of the shRNA regions, and deep-sequenced of the PCR products to quantify the abundance of each shRNA. Candidate genes were identified from those shRNAs with statistically different frequencies between vehicle-treated and sorafenib-treated xenografts, and were further validated by their impacts on sorafenib-sensitivity in HCC cells. Results: By calculating the abundance frequencies of each shRNA of sorafenib- treated xenografts versus that of vehicle-treated xenografts, we found that 3 candidate sorafenib- resistance genes because their shRNA abundance was significantly decreased in sorafenib-treated xenografts. Among them, only the cyclin dependent kinase 5 (CDK5) was further investigated because its expression was confirmed to be increased in sorafenib- treated xenografts. In PLC5 and a sorafenib-resistant subline of Huh7 cells (Huh7-SR), downregulation of CDK5 by RNA interference or roscovitine, a CDK5 inhibitor, enhanced the sensitivity to sorafenib- induced growth-suppression. On the other hand, overexpression of CDK5 increased the resistance to sorafenib in Huh7 cells. Mechanistically, sorafenib combined with shRNA-mediated CDK5 downregulation augmented the induction of apoptosis, and resulted in an increased downregulation of multiple anti-apoptosis molecules such as XIAP and survivin in PLC5 and Huh7-SR cells. In 8 paired human HCC tissues, we found 3 of them have unequivocally increased expression of CDK5 in HCC cells of post-sorafenib HCC tissues compared with those of pre-treatment tissues. Conclusions: Our results suggest that increased CDK5 expression may mediate resistance to sorafenib in HCC in vivo, and targeting CDK5 is warranted further studies in the treatment of HCC. (This study was supported by grants MOST 103-2314-B-002 -181 -MY2, MOST 105-2314-B-002-194-, MOST 103-2314-B-002 -090 - & MOST 104-2314-B-002 -073). Citation Format: Tzu-Hsuan Lin, Wen-Chi Feng, Yu-Yun Shao, Li-Chun Lu, Ya-Jhen Chen, Ann-Lii Cheng, Chih-Hung Hsu. Pooled shRNA screening using mouse xenografts of human hepatocellular carcinoma cells identifies CDK5 as a potential mechanism mediating sorafenib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 80. doi:10.1158/1538-7445.AM2017-80

Abstract 4325: The role of tumor cell heterogeneity in the targeted therapy of ALK-positive neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. One of its hallmarks is high histological heterogeneity. The primary tumors are composed of neuroblastic cancer cells, Schwannian stroma and some infiltrating immune cells. The origin and functions of Schwannian stroma still remain controversial. It is suggested that they are tumor-infiltrating normal cells. Studies using Bone Marrow Stromal Cells (BMSCs) or Schwann cells to mimic Schwannian stroma have shown both tumor promoting or suppression effects. An alternative hypothesis is that Schwannian cells are derived from cancer stem-like cells since they shared the same genetic lesions with neuroblastic tumor cells. Our laboratory had reported that in the SK-N-SH cell line, both N-type cells (neuroblastic cells) and S-type cells (Schwann-like cells) shared almost identical genetic background. These cell lines may serve as relevant models to study NB tumor heterogeneity. Genome-wide sequencing for driver oncogenes in familial NB uncovered mutations in Anaplastic Lymphoma Kinase (ALK) gene. ALK point mutations have been found in 8-12% of all NB patients, and are restricted to the kinase domain. Aberrant activation of ALK signaling, and its downstream target, PI3K/AKT, STAT3 and ERK1/2, can contribute to NB tumorigenesis. Thus, ALK is an intensely studied therapeutic target. Sublines of N-type and S-type cells were isolated from an early passage of SK-N-SH. Surprisingly, over 90% have cell morphology that resembled S-type cells. Sequencing analysis revealed that all harbored the same ALK F1174L mutation, indicating they were tumor-derived. Western blotting analysis showed that N-type cells, but not S-type cells, expressed ALK protein. This may explain the insensitivity of S-type cells towards ALK inhibitor, TAE684. Next, co-culture experiments showed that S-type cells protected N-type cells from apoptosis induced by TAE684. Western blotting analysis showed that ALK, AKT and STAT3 signaling were stimulated in the co-culture. Furthermore, conditioned medium (CM) from Schwann-like cells activated these downstream signaling molecules in N-type cells, indicating secreted factors from S-type cells contributed to the pathway stimulations. As AKT and STAT3 were ALK downstream targets, ALK ligand could be a potential factor. We further confirmed these results in two additional ALK WT N-type cell lines, IMR 32 and BE (2)-M17. Co-cultivation and CM experiments had demonstrated robust STAT3 activation even without ALK stimulation. Overall, tumor-derived S-type cells could prevent N-type cells from apoptosis via secreted factors. The signaling involved could be separated into an ALK-independent STAT3 activation and a ligand-activated ALK-AKT pathway. Future work will focus on identifying STAT3 stimulators and ALK ligands in CM of S-type cells. This work was supported by a General Research Fund grant (#14100514) from the Hong Kong University Grants Committee to A.C. Citation Format: Jing LI, Penelope M. OR, Andrew M. CHAN. The role of tumor cell heterogeneity in the targeted therapy of ALK-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2017-4325

Abstract 4757: Radioresistance is linked with stem-like properties via activation of aryl hydrocarbon receptor

Abstract Most cancer patients receive radiotherapy in the course of their disease and the occurrence of radioresistance is associated with a poor prognosis for cancer patients. The molecular pathways that drive the enhanced tumorigenic potential during the development of radioresistance are poorly understood. Here, we demonstrate that the aryl hydrocarbon receptor (AhR) plays a vital and previously unrecognized role in the maintenance of stem-like cells that are isolated from radioresistant epithelial lung cancer and nasopharyngeal carcinoma. AhR promotes the stem-like phenotype and drives metastasis by directly targeting the promoters of ‘stemness’ genes, such as the ATP-binding cassette sub-family G member 2 (ABCG2) gene. Cancer cell populations enriched for ABCG2 expression (CD338+) show an elevated ‘stemness’ signature and are more prone to cancer progression in vitro and in vivo. Ubiquitin C-terminal hydrolase-L3 (UCHL3), a deubiquitinating enzyme, may counteract AhR, while IKKα, a bone fide chromatin modifier, may directly activate stemness related genes. The radioresistant cancer cell sublines are associated with a hybrid state in epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), and elevated tumor associated macrophages and proinflammatory factors. Moreover, the radioresistant sublines display high levels of oncometabolites, including α-ketoglutarate, and treatment of cancer cells with α-ketoglutarate enhances their stem-like properties through activation of the AhR. Thus AhR is functionally linked with radioresistance in lung cancer and nasopharyngeal carcinoma, and it drives tumorigenesis by an epithelial and mesenchymal composition, tumor associated macrophages and oncometabolites. Note: This abstract was not presented at the meeting. Citation Format: SHuang Liu, Bin Yan, Yongguang Tao. Radioresistance is linked with stem-like properties via activation of aryl hydrocarbon receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2017-4757

Characterization of a novel p110β-specific inhibitor BL140 that overcomes MDV3100-resistance in castration-resistant prostate cancer cells.

Our previous studies demonstrated that the class IA PI3K/p110β is critical in castration-resistant progression of prostate cancer (CRPC) and that targeting prostate cancer with nanomicelle-loaded p110β-specific inhibitor TGX221 blocked xenograft tumor growth in nude mice, confirming the feasibility of p110β-targeted therapy for CRPCs. To improve TGX221's aqueous solubility, in this study, we characterized four recently synthesized TGX221 analogs. TGX221 analog efficacy were examined in multiple prostate cancer cell lines with the SRB cell growth assay, Western blot assay for AKT phosphorylation and cell cycle protein levels. Target engagement with PI3K isoforms was evaluated with cellular thermal shift assay. PI3K activity was determined with the Kinase-Glo Plus luminescent kinase assay. Cell cycle distribution was evaluated with flow cytometry after propidium iodide staining. As expected, replacing either one of two major functional groups in TGX221 by more hydrophilic groups dramatically improved the aqueous solubility (about 40-fold) compared to TGX221. In the CETSA assay, all the analogs dramatically shifted the melting curve of p110β protein while none of them largely affected the melting curves of p110α, p110γ, or Akt proteins, indicating target-specific engagement of these analogs with p110β protein. However, functional evaluation showed that only one of the analogs BL140 ubiquitously inhibited AKT phosphorylation in all CRPC cell lines tested with diverse genetic abnormalities including AR, PTEN, and p53 status. BL140 was superior than GSK2636771 (IC50 5.74 vs 20.49 nM), the only p110β-selective inhibitor currently in clinical trials, as revealed in an in vitro Kinase-Glo assay. Furthermore, BL140 exhibited a stronger inhibitory effect than GSK2636771 on multiple CRPC cell lines including a MDV3100-resistant C4-2B cell subline, indicating BL140 elimination of MDV3100 resistance. Mechanistic studies revealed that BL140 blocked G1 phase cell cycle entry by reducing cyclin D1 but increasing p27kip1 protein levels. These studies suggested that BL140 is a promising p110β-specific inhibitor with multiple superb properties than GSK2636771 worthy for further clinical development.

Genomic profiling of long non-coding RNA and mRNA expression associated with acquired temozolomide resistance in glioblastoma cells.

Temozolomide (TMZ) is an alkylating chemotherapeutic agent widely used in anti-glioma treatment. However, acquired TMZ resistance represents a major clinical challenge that leads to tumor relapse or progress. This study investigated the genomic profiles including long non-coding RNA (lncRNA) and mRNA expression associated with acquired TMZ resistance in glioblastoma (GBM) cells in vitro. The TMZ-resistant (TR) of GBM sub-cell lines were established through repetitive exposure to increasing TMZ concentrations in vitro. The differentially expressed lncRNAs and mRNAs between the parental U87 and U87TR cells were detected by human lncRNA microarray method. In this study, we identified 2,692 distinct lncRNAs demonstrating >2-fold differential expression with 1,383 lncRNAs upregulated and 1,309 lncRNAs downregulated. Moreover, 4,886 differential mRNAs displayed 2,933 mRNAs upregulated and 1,953 mRNAs downregulated. Further lncRNA classification and subgroup analysis revealed the potential functions of the lncRNA-mRNA relationship associated with the acquired TMZ resistance. Gene ontology and pathway analysis on mRNAs showed significant biological regulatory genes and pathways involved in acquired TMZ resistance. Moreover, we found the ECM-receptor interaction pathway was significantly downregulated and ECM related collagen I, fibronectin, laminin and CD44 were closely associated with the TR phenotype in vitro. Our findings indicate that the dysregulated lncRNAs and mRNAs identified in this work may provide novel targets for overcoming acquired TMZ resistance in GBM chemotherapy.

PI3K/AKT/mTOR pathway promotes progestin resistance in endometrial cancer cells by inhibition of autophagy

Endometrial cancer (EC) is now one of the most common malignant tumors in young women. In all, 90% of young patients with EC have a high expression of progesterone recep tor, can be treated with progestin, and have very good prognosis. However, some of the young EC patients are resistant to progestin, the mechanism of which is unclear. To illuminate the mechanism by which endometrial cells acquire progestin resistance, we treated Ishikawa cells by slowly increasing dosage of progestin and established a progestin-resistant cell subline. We show here that progesterone resistant cells acquire increased proliferation rate and interestingly decreased autophagy. To uncover the mechanism by which cells increase proliferation and bypass autophagy, we found higher activation of phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway was necessary to this malignant acquirement by RNAi technique. Further, we elucidated that activation of mTOR was sufficient and necessary for progestin resistance. RAD001, an inhibitor of mTOR, decreased phosphorylation of mTOR and inhibited proliferation of progestin-resistant cancer cells by promoting autophagy. Thus, our results indicated that mTOR can be a target to treat the progestin-resistant EC.

MicroRNA profiles involved in trifluridine resistance.

Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A microRNA analysis of three colorectal cell lines was conducted to investigate causes of FTD resistance. Drug resistant sublines of DLD-1, HCT-116, and RKO cells were developed by continuous administration of increasing doses of FTD for 5 months. The let-7d-5p gene, which maps to chromosome 9q22.32, was downregulated in the FTD-resistant DLD-1 sublines. DLD-1 cells became more resistant to FTD when let-7d-5p was knocked down and more sensitive when let-7d-5p was overexpressed. The FTD-resistant sublines were not cross-resistant to 5-fluorouracil (5-FU); 5-FU sensitivity was affected only slightly when let-7d-5p as overexpressed or knocked down. These data indicate that let-7d-5p increases sensitivity of FTD but not 5-FU and that let-7d-5p is a potential clinical marker of treatment sensitivity.

Calpain and AR-V7: Two potential therapeutic targets to overcome acquired docetaxel resistance in castration-resistant prostate cancer cells.

Docetaxel-based chemotherapy has been widely used as the first-line treatment for castration-resistant prostate cancer (CRPC) patients. However, the mechanisms of docetaxel-resistance remain unclear. In the present study with the establishment of 2 invitro models of docetaxel-resistant CRPC cell sublines, we firstly reported that activation of calpain may play a promotional role in the resistance of docetaxel in prostate cancer, meanwhile using the calpain inhibitor combined with docetaxel improved the efficiency of docetaxel in docetaxel-resistant cell sublines. Moreover, we also found that the expression of androgen-independent constitutively and transcriptionally active androgen receptor splice variant-7(AR-V7) remained high in the docetaxel-resistant CRPC cell subline Rv1-DR, and that it may be involved in acquired docetaxel-resistance of CRPC. However, a novel importin-β inhibitor (importazole) was only capable of slightly decreasing the transcriptional activity of the AR signaling pathway via blocking nuclear import of AR-FL and various non-specific AR-Vs, instead of AR-V7. These findings suggest that calpain and AR-V7 may serve as important biomarkers in the treatment of CRPC, and targeting calpain and AR-V7 may provide a new approach in overcoming docetaxel-resistance.

Membrane-enriched proteome changes and prion protein expression during neural differentiation and in neuroblastoma cells

BackgroundThe function of the prion protein, involved in the so-called prion diseases, remains a subject of intense debate and the possibility that it works as a pleiotropic protein through the interaction with multiple membrane proteins is somehow supported by recent reports. Therefore, the use of proteomic and bioinformatics combined to uncover cellular processes occurring together with changes in the expression of the prion protein may provide further insight into the putative pleiotropic role of the prion protein.ResultsThis study assessed the membrane-enriched proteome changes accompanying alterations in the expression of the prion protein. A 2D-DIGE approach was applied to two cell lines after prefractionation towards the membrane protein subset: an embryonic stem cell line and the PK1 subline of neuroblastoma cells which efficiently propagates prion infection. Several proteins were differentially abundant with the increased expression of the prion protein during neural differentiation of embryonic stem cells and with the knockdown of the prion protein in PK1 cells. The identity of around 20% of the differentially abundant proteins was obtained by tandem MS. The catalytic subunit A of succinate dehydrogenase, a key enzyme for the aerobic energy metabolism and redox homeostasis, showed a similar abundance trend as the prion protein in both proteomic experiments. A gene ontology analysis revealed “myelin sheath”, “organelle membrane” and “focal adhesion” associated proteins as the main cellular components, and “protein folding” and “ATPase activity” as the biological processes enriched in the first set of differentially abundant proteins. The known interactome of these differentially abundant proteins was customized to reveal four interactors with the prion protein, including two heat shock proteins and a protein disulfide isomerase.ConclusionsOverall, our study shows that expression of the prion protein occurs concomitantly with changes in chaperone activity and cell-redox homeostasis, emphasizing the functional link between these cellular processes and the prion protein.

Subcloning and characterization of highly metastatic cells derived from human esophageal squamous cell carcinoma KYSE150 cells by in vivo selection.

Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer-related deaths worldwide. Despite the research progress in understanding the disease, the mechanism underlying the metastasis is still unclear. Here, we successfully generated a highly metastatic cell subline, designated as KYSE150-LuM, derived from an esophageal squamous cell carcinoma cell line (KYSE150) by in vivo selection. To elucidate the mechanisms driving metastasis, we characterized the gene expression differences between LuM cells and parent KYSE150 cells. IL-6, IL-1β, and LCN2, previously associated with tumor growth and metastasis, were up-regulated in LuM cells. Recent studies on cancer have increasingly focused on the tumor microenvironment, from which these cytokines are released. The fact that these three cytokines (IL-6, IL-1β, LCN2) were up-regulated in LuM cells indicates that these highly metastatic cells obtained through in vivo selection will be a useful resource for further studies on elucidating the mechanisms underlying the tumor microenvironment which is associated with cytokine-related tumor growth and metastasis. Moreover, LuM cells could disseminate to the lung in shorter period of time in vivo, indicating their utility for in vivo experiments of metastasis and new therapeutic targets in a shorter period of time than currently possible.

Amigo2-upregulation in Tumour Cells Facilitates Their Attachment to Liver Endothelial Cells Resulting in Liver Metastases

Since liver metastasis is the main cause of death in cancer patients, we attempted to identify the driver gene involved. QRsP-11 fibrosarcoma cells were injected into the spleens of syngeneic mice to isolate tumour sub-populations that colonize the liver. Cells from liver metastatic nodules were established and subsequently injected intrasplenically for selection. After 12 cycles, the cell subline LV12 was obtained. Intravenous injection of LV12 cells produced more liver metastases than QRsP-11 cells, whereas the incidence of lung metastases was similar to that of QRsP-11 cells. LV12 cells adhered to liver-derived but not to lung-derived endothelial cells. DNA chip analysis showed that amphoterin-induced gene and open reading frame 2 (Amigo2) was overexpressed in LV12 cells. siRNA-mediated knockdown of Amigo2 expression in LV12 cells attenuated liver endothelial cell adhesion. Ex vivo imaging showed that suppression of Amigo2 in luciferase-expressing LV12 cells reduced attachment/metastasis to liver to the same level as that observed with QRsP-11 cells. Forced expression of Amigo2 in QRsP-11 cells increased liver endothelial cell adhesion and liver metastasis. Additionally, Amigo2 expression in human cancers was higher in liver metastatic lesions than in primary lesions. Thus, Amigo2 regulated tumour cell adhesion to liver endothelial cells and formation of liver metastases.

MiR-205 inhibits cell growth by targeting AKT-mTOR signaling in progesterone-resistant endometrial cancer Ishikawa cells.

PurposemiR-205 is significantly up-regulated in endometrioid adenocarcinoma. In this study, the significant anticancer effect of a miR-205 inhibitor was investigated in both endometrial carcinoma and progesterone-resistant endometrial carcinoma cells.ResultsCompared with Ishikawa endometrial cancer cells, miR-205 was expressed at higher levels in a progesterone-resistant (PR) sub-cell line. Inhibition of miR-205 suppressed the growth of cancer cells in a dose- and time-dependent manner. Moreover, the miR-205 inhibitor induced a marked increase in the percentage of Ishikawa-PR cells in G2/M phases and a decrease in the percentage of cells in G0/G1 and S phases. In addition, miR-205 inhibitor-treated tumor cells exhibited increased apoptosis. Moreover, miR-205 was found to negatively regulate PTEN expression and lead to autophagy and activation of the AKT/mTOR pathway in PR cells, and PTEN protein levels significantly decreased with development of progesterone resistance in endometrial cancer cells. Western blot assay showed up-regulated autophagy, as indicated by expression of LC3-II/LC3-I and beclin1, in Ishikawa cells; in particular, autophagy was markedly induced in PR cells treated with the miR-205 inhibitor.Materials and MethodsWe measured and analyzed cell growth curves with and without miR-205 inhibition with the MTT assay, miR-205 expression by qRT-PCR, cell cycle and apoptosis using annexin V/propidium iodide staining and flow cytometry, and autophagy, apoptosis, and AKT-mTOR signaling by western blotting.ConclusionsInhibition of miR-205, which targets the AKT-mTOR pathway, in endometrial cancer cells provides a potential, new treatment for PR endometrial carcinoma.