Insensitive Cells Research Articles

Abstract 1067: Anti-cancer efficacy of imipridones in pancreatic cancer: single agent ONC212 or combination of ONC201 with IGF1-R inhibition

Abstract Pancreatic cancer is a highly chemo-resistant tumor type known to aggressively metastasize at an early stage with an overall five-year survival rate of ~6%. In this study we tested the efficacy of ONC201 and ONC212 in a panel of pancreatic cancer cell lines and patient-derived models in vitro and in vivo. ONC201 is the founding member of the imipridone class of small molecules with anti-proliferative and pro-apoptotic effects in various tumor types. Kline et al. and Allen et al. previously showed that ONC201 stimulates the integrated stress response by up-regulating ATF4, CHOP and DR5 followed by induction of apoptosis by up-regulating TRAIL. While the spectrum of ONC201 efficacy is broad among tumor types, pancreatic cancer cells are relatively insensitive. ONC201 exhibits low micromolar GI50 values in 3 out of 7 pancreatic cancer cell lines and 2 out of 9 patient-derived cells that were most sensitive among the pancreatic cell lines and samples tested. ONC201 induces the integrated stress response in both ONC201-sensitive and –insensitive pancreatic cancer cell lines. Interestingly, ONC201-treated resistant cells arrest in G1 and do not go through apoptosis. In order to address insensitivity to ONC201 in pancreatic cancer we explored two approaches. The first approach included treating with the ONC201 analogue ONC212, an imipridone that is currently under pre-clinical development. In vitro and in vivo studies consistently show a higher potency of ONC212 as compared to ONC201 in pancreatic cancer, especially in ONC201-insensitive models. We are currently investigating the mechanism of action of ONC212 in comparison to ONC201. The second approach to address ONC201-insensitive pancreatic cancer was combination treatment with the IGF1-R inhibitor AG1024. Western blot analysis of receptor tyrosine kinase expression levels in the panel of pancreatic cancer cell lines revealed a strong correlation between resistance to ONC201 and high expression of IGF1-R. Therefore we hypothesized that IGF1/IGF1-R might play a role in the resistance of pancreatic cancer cells to ONC201. Indeed, treatment of resistant pancreatic cancer cells with the IGF1-R inhibitor AG1024 sensitized the cells to ONC201. We are currently further exploring the involvement of IGF1-R in the ONC201 resistance mechanism. Furthermore, in-vivo studies are ongoing to validate the efficacy of the combination treatment. In summary, although pancreatic cancer is known to be refractory to many drugs, this study introduces two different approaches with imipridone small molecules ONC212 and ONC201 that show promising therapeutic potential for pancreatic cancer. Citation Format: Avital Lev, Amriti R. Lulla, Jessica Wagner, David T. Dicker, Wafik S. El-Deiry. Anti-cancer efficacy of imipridones in pancreatic cancer: single agent ONC212 or combination of ONC201 with IGF1-R inhibition [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 1067. doi:10.1158/1538-7445.AM2017-1067

Abstract 5850: Salicylate sensitizes prostate cancer (PrCa) to radiotherapy; activation of AMPK and suppression of lipogenesis

Abstract Radiotherapy (RT) is a key therapeutic modality for PrCa that achieves reasonable disease control rates in localized disease. However, this requires dose-escalated RT, which is associated with increased toxicity to surrounding tissues and leads to deterioration of patient quality of life. Improving the therapeutic ratio in PrCa RT is of high clinical importance. Aspirin has an established role in cancer prevention and studies suggested association with improved PrCa outcomes. It was shown that its metabolite salicylate (SAL) binds directly and activates the metabolic stress sensor AMP-activated kinase (AMPK). This is likely achieved through direct binding to the AMPK β1 subunit, leading to enhanced AMPK activity through suppression of AMPK α-subunit T172 de-phosphorylation. In this study we examined the ability of SAL to sensitize PrCa to RT. Hormone insensitive (PC3) and sensitive (LnCap) human PrCa cells, as well as PC3 xenografts grown in immunocompromised nude mice were subjected to SAL and RT treatments. SAL mediated a dose-dependent inhibition of proliferation and clonogenic survival in both PrCa cell lines and suppressed de novo lipogenesis (DNL). This was detected at μM concentrations of the drug, which can be safely achieved in human circulation. SAL (100-500 μM) radio-sensitized PC3 and LnCap cells, and enhanced the ability of SAL to inhibit DNL. In animals treated with conformal RT (10Gy) to the tumor xenografts, Salsalate feeding (chow diet containing 2.5gr/kg) enhanced the cytotoxicity of RT detected as reduced tumor growth kinetics and weight and volume at sacrifice. Salicylate treatment of cells or salsalate feeding of mice led to increased AMPK activity in cells and tumors, inhibitory phosphorylation of Acetyl-CoA Carboxylase and reduction of AktThr308 phosphorylation. These results indicate that SAL could improve radiation response in PrCa through a mechanism involving the suppression of DNL. This is achieved at clinically attainable doses of SAL. This work supports prospective investigation of SAL as a radio-sensitizer in early phase clinical trials of localized PrCa. Note: This abstract was not presented at the meeting. Citation Format: Theodoros Tsakiridis, Katarina Marcinko, Linsday Broadfield, Linda Villani, Carrie Gerdes, Danitra Maharaj, Tom Farell, James Wright, Gregory Steinberg. Salicylate sensitizes prostate cancer (PrCa) to radiotherapy; activation of AMPK and suppression of lipogenesis [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 5850. doi:10.1158/1538-7445.AM2017-5850

Abstract 2787: Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing

Abstract Epithelial ovarian cancer is a complex disease and patients vary considerably in response to treatment. A subset of patients responds well to endocrine therapy, however hormone receptor positivity does not predict response and there are no biomarkers in clinical use to help select patients that would benefit from endocrine agents. We aimed to identify markers of endocrine response in high-grade serous ovarian cancer (HGSOC) by analysis of genes that are differentially expressed in tumors from patients that are endocrine responders compared with non-responders, combined with analysis of estrogen (E2) regulated genes that differ between E2 sensitive and E2 insensitive HGSOC cell lines. Methods: Among women recruited to the Australian Ovarian Cancer Study, we identified 10 HGSOC patients treated with endocrine therapy (5 responders and 5 non-responders, based on GCIG CA125 response criteria) and performed RNAseq on cryopreserved tumor tissue. We also utilized paired E2 receptor (ER) positive HGSOC cell lines, derived from the same patient, with differential sensitivity to the growth effect of E2, PEO1 (E2 insensitive) and PEO4 (E2 sensitive). RNA was extracted following treatment with 0.1 nM E2 (or vehicle) for 24 hrs and gene expression changes in response to E2 were determined using RNAseq. Expression of selected ER responsive genes was validated using PCR array. Differential gene expression was determined using EdgeR. Pathway and Gene Ontology enrichment analysis of differentially expressed genes were performed using Metacore. Results: Between endocrine responders and non-responders, 27 genes were significantly differentially expressed (False Discovery Rate (FDR)<0.05, absolute log2 Fold-Change (FC)≥1). In the paired HGSOC cell lines, significantly more genes were regulated by E2 in E2-sensitive PEO4, compared with E2-insensitive PEO1 (896 genes compared to 56 genes, respectively, FDR<0.05, absolute log2FC≥1). In response to E2, up-regulation of NRP1 in PEO1, up-regulation of LTBP1, MYC, PDZK1 and down-regulation of S100A6 and CYP1A1 in PEO4 were validated by PCR array profiling of ER-related genes. Genes regulated by E2 in cell lines and also differentially expressed between endocrine responders and non-responders included anoctamin 1 (ANO1) and NOTCH-Regulated Ankyrin Repeat Protein (NRARP). Both were up-regulated in endocrine responders and also up-regulated by E2 in PEO4. Conclusion: The use of RNAseq in both tumors and cell line models was able to identify plausible sets of genes with prior evidence of association with endocrine sensitivity. In breast cancer, ANO1 has been associated with good prognosis following tamoxifen treatment, while NOTCH signaling, implicated in endocrine resistance, is negatively regulated by NRARP. Further validation is required to test the utility of these genes as predictive biomarkers that may aid in identifying HGSOC patients likely to respond to endocrine treatment. Citation Format: Cristina Mapagu, Sian Fereday, Australian Ovarian Cancer Study Group, David D. Bowtell, Paul R. Harnett, Anna deFazio. Identifying predictive markers of endocrine response in high-grade serous ovarian cancer using RNA sequencing [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 2787. doi:10.1158/1538-7445.AM2017-2787

Abstract 141: CD44 positive and sorafenib resistant hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128

Abstract The PI3K/AKT/mTOR pathway is activated in about 50% of patients with hepatocellular carcinoma (HCC). Allosteric mTOR inhibitors, also known as rapalogs, fail to show any significant clinical utility. In an effort to identify new pathways and compounds to treat advanced HCC, we considered the ATP-competitive mTOR inhibitor INK128. ATP-competitive mTOR inhibitors attenuate both mTORC1 and mTORC2. We evaluated INK128 in sorafenib sensitive and insensitive HCC cell lines, CD44low and CD44high HCC and those cell lines with acquired sorafenib resistance. CD44 was significantly increased in Huh7 cells made resistant to sorafenib. Forced expression of CD44 enhanced cellular proliferation and migration, and rendered the cells more sensitive to the anti-proliferative effects of INK128. INK128 suppressed CD44 expression by blocking phosphorylation of eukaryotic translation initiation factor 4EBP1 in HCC cells while allosteric mTOR inhibitors do not. Moreover, INK128 exhibited potent anti-proliferative and anti-migration effects on the mesenchymal-like HCC cells, CD44high and sorafenib resistant HCC cells compared to the allosteric mTOR inhibitor everolimus. Combination studies of INK128 and sorafenib showed an additive to synergistic anti-proliferative effect in CD44high HCC cells. Our findings suggest that ATP-competitive mTOR inhibitors are effective in treating advanced HCC patients who express CD44, and are insensitive or resistant to sorafenib. It also highlights the potential use of CD44 as a biomarker of response in these patients. Citation Format: Mohamed Badawi, Jihye Kim, Dhruvitkumar Sutaria, Tasneem Motiwala, Ryan Reyes, Nissar Wani, Samson Jacob, Mitch Phelps, Thomas Schmittgen. CD44 positive and sorafenib resistant hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128 [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 141. doi:10.1158/1538-7445.AM2017-141

Abstract 4194: Antitumor activity of multikinase inhibitors in HER2-positive gastric cancer cells

Abstract Background: The developments of novel anti-HER2 drugs including multikinase inhibitors have achieved advancing evolution for past several years, and the options in the treatment of HER2-positive malignant tumors have been increasing, especially in breast and lung cancers. On the other hand, regarding HER2-positive gastric cancer, trastuzumab is still the only anti-HER2 drug with the established clinical evidences. Afatinib, an irreversible human epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, binds to the kinase domain of HER2, and is one of the hopeful candidates as novel molecularly targeted drugs for HER2-positive gastric cancer. In this study, we investigated the antitumor effect of multikinase inhibitors including afatinib in gastric cancer cell lines. Method: We examined the relation between molecular profiles and multikinase inhibitors sensitivities in 12 gastric cancer cell lines: ECC10, GCIY, KATO-III, MKN7, MKN74, N87, NUGC3, NUGC4, OCUM-1, SH-10-TC, SNU-16, and SNU-216. In addition to multikinase inhibitors including afatinib, we also examined trastuzumab and gefitinib sensitivity in 12 cell lines. HER2 copy number alteration, HER2 mutation, and protein expression status of HER2 and its downstream pathway molecules of these cell lines were analyzed by qPCR, direct sequencing, and Western blotting, respectively. Cell proliferation was determined by a MTS assay. Result: Afatinib was effective in GCIY, N87, SNU-216, NUGC3, and NUGC4 cells. The range of IC50 for these cell lines was 2.1 to 39.4 nM. All these cell lines except for NUGC3 were HER2 amplified. NUGC4, the most afatinib sensitive cells, had also EGFR amplification. Interestingly, among the HER2 amplified cell lines, MKN7 cells were not sensitive to afatinib, nevertheless this cell line showed the activation of HER2 pathway with the expression of phospho-HER2 by Western blotting. In addition, MKN7 cells were not sensitive to other HER2-targeted drugs trastuzumab, and reported to be insensitive to lapatinib either. On the other hand, ECC10 with HER2-L755S mutation was not sensitive to afatinib. The range of IC50 in 7 insensitive cell lines was 0.80 to more than 10 µM. Three cell lines showed moderate sensitivity to gefitinib of which IC50 ranged from 100 to 1000uM. Inhibition rate of trastuzumab was under 25% in all 12 cell lines. Conclusion: Afatinib, a multikinase inhibitor, is highly effective to HER2 amplified gastric cancer cell lines, and may become a novel treatment option in HER2-positive gastric cancer treatment. On the other hand, afatinib is not always effective even if cancer cells have HER2 mutation or amplification. In this study, we report the detailed predictive biomarkers of sensitivity to afatinib and other several multikinase inhibitors in HER2-positive gastric cancer cells. Citation Format: Takahiro Yoshioka, Kazuhiko Shien, Kei Namba, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Toshiyoshi Fujiwara, Shinichi Toyooka. Antitumor activity of multikinase inhibitors in HER2-positive gastric cancer cells [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 4194. doi:10.1158/1538-7445.AM2017-4194

Abstract 5820: Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

Abstract While intensive chemotherapy regimens result in remission in approximately 80% of pediatric and 45% of adult ALL patients, new approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Early T-cell precursor (ETP) ALL, a subset of T-ALL associated with a higher rate of relapse and induction failure, with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. PIM protein kinases are overexpressed in a number of hematopoietic malignancies, including ALL, with its overexpression being associated with poor prognosis. Pan-PIM inhibitors (PIMi) are being tested in clinical trials in patients with relapsed/refractory multiple myeloma and acute myeloid leukemia. Analysis of the effects of PIMi (AZD1208/LGB321) on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Immunophenotype of all the PIMi insensitive cells lines was consistent with a more mature T-ALL phenotype. In contrast, sensitive cells, had an immature or ETP-ALL phenotype. Bioinformatics analysis of three independent T-ALL patient datasets (GSE28703, E-MEXP-313, and GSE62156) demonstrated overexpression of PIM1 in the majority of ETP-ALLs and in a small subset of non-ETP-ALL. We also identified 58 genes changing significantly between PIMi sensitive versus insensitive cell lines that were common to the differential genes between ETP versus non-ETP ALL cases from GSE28703, suggesting that high PIM1 expression along with 58 gene signature could be a driver of the ETP phenotype. Importantly, we have made the novel observation that, combination of Ponatinib, a tyrosine kinase inhibitor (TKI) with the PIMi is synergistically lethal to T-ALL cells with ETP phenotype. These agents appear to work together by inducing PARP and Caspase cleavage, sufficient to drive apoptosis. Following engraftment of NSG mice with H-SB2-Luc cells, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly abrogated leukemia as evidenced by optical scanning for luciferase cells, and reduced number of hCD45+ leukemic cells in the peripheral blood and bone marrow. Also, the dual therapy significantly prolonged the survival of the treated mice. In summary, our work demonstrates a strong preclinical rationale for a novel treatment strategy of combining PIMi and TKI to treat T-ALL patients with high PIM expression. Citation Format: Sathish Kumar Reddy Padi, Libia Luevano, Neha Singh, Jiin Song, Ritu Pandey, Jon C. Aster, Xue-Zhong Yu, Shikhar Mehrotra, Andrew Kraft. Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset [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 5820. doi:10.1158/1538-7445.AM2017-5820

P 234 - Glut 1 overexpression prevents glucose deprivation-induced prostate cancer cell death by increasing pentose phosphate pathway and glutathione

Although glucose metabolism was override in prostate cancer, it is recognized that there are metabolic differences between androgen-sensitive and castration-resistant phenotypes that might be responsible of progression or treatment success. Among the targets of glucose metabolism, glucose uptake is on the top of the cascade. The increase of glucose uptake by GLUTs overexpression in oncogenesis or the androgenic regulation of some GLUT transporters in other tissues is demonstrated. The prostate, at the beginning of carcinogenesis, depends on OXPHOS but then, at aggressive stages, tumors become again glycolytic like other cancers. Therefore, their resistance to glucose deprivation is different at the beginning or at later stages. In this work, we demonstrated by using androgen sensitive and insensitive cells that facilitative GLUT1 transporter overexpression protect prostate cancer cells from apoptosis caused by glucose removal. Glucose removal caused an increment in oxidative stress that stimulated androgen receptor activity and GLUT1 overexpression. This protection is mediated by a derivation towards pentose phosphate metabolic pathway and an increment of glutathione in androgen dependent prostate cancer cells.

MiR-124 contributes to glucocorticoid resistance in acute lymphoblastic leukemia by promoting proliferation, inhibiting apoptosis and targeting the glucocorticoid receptor

Acute lymphoblastic leukemia (ALL) is characterized by the accumulation of abnormal lymphoblasts in the bone marrow and blood. Though great progress has been made for improvement in clinical treatment during the past decades, some children with ALL still relapsed. Glucocorticoid (GC) resistance is an important clinical problem for ALL treatment failure. Therefore, further understanding of the mechanism of GC resistance and exploring novel therapeutic strategies are crucial for improving treatment outcome. The reported involvement of microRNAs (miRNAs) in drug resistance implied that deregulated miRNA expression might contribute to GC treatment response of ALL. However, individual miRNAs and their functional mechanisms potentially involved in the GC response are still largely unknown. In the present study, we found that miR-124 was up-regulated in prednisone insensitive human ALL cell line and prednisone-poor response ALL patients. Furthermore, it was found that miR-124 might contribute to GC resistance by promoting proliferation and inhibiting apoptosis of ALL cells. Importantly, we validated that miR-124, targeted and decreased the expression of glucocorticoid receptor (NR3C1), prevented the inhibitory effect of GC in ALL. These findings strongly suggest that miR-124 is critical in poor GC response and may serve as a potential therapeutic target in ALL with poor GC resistance.

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways

BackgroundMesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma.MethodsWe examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53.ResultsMetformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells.ConclusionThese data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

Epithelial-to-mesenchymal transition correlates with gefitinib resistance in NSCLC cells and the liver X receptor ligand GW3965 reverses gefitinib resistance through inhibition of vimentin.

The role of epithelial-to-mesenchymal transition in cancer drug resistance is increasingly acknowledged. We examined whether epithelial-to-mesenchymal transition affects gefitinib resistance in non-small cell lung cancer (NSCLC) cells. Cell viability was detected by CCK-8 assay, VIM expression levels were determined by quantitative real-time polymerase chain reaction. Western blot and immunocytochemistry were performed to determine the protein expression level of vimentin. We observed morphologic differences between gefitinib-sensitive and -insensitive cells. Compared with the sensitive parental cell line, HCC827, vimentin expression levels were increased in HCC827 cells with acquired gefitinib resistance. Vimentin expression was also markedly upregulated in cells with intrinsic gefitinib resistance, and upregulated vimentin expression was correlated with gefitinib sensitivity. Our previous study demonstrated that coadministration of gefitinib and GW3965 resulted in decreased cell proliferation and induced apoptosis. Therefore, we investigated the relationship among GW3965, vimentin, and gefitinib resistance in NSCLC cells by analysis of the expression of vimentin in cells treated with a combination of gefitinib and GW3965. Gefitinib treatment led to increased levels of intracellular vimentin, while combined treatment with gefitinib and GW3965 resulted in decreased vimentin expression levels through reduction of gefitinib drug resistance in NSCLC cells. Overall, these findings suggest that vimentin expression is associated with sensitivity to gefitinib, and our study highlights the potential usefulness of the drug, GW3965, for reversal of gefitinib resistance through inhibition of vimentin expression.

PD33-07 CCRL2 REGULATES PROSTATE CANCER ORGAN SPECIFIC METASTASIS THROUGH THE INTERACTION WITH CCR5

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2017PD33-07 CCRL2 REGULATES PROSTATE CANCER ORGAN SPECIFIC METASTASIS THROUGH THE INTERACTION WITH CCR5 Chia-Ling Hsieh, Kuan-Chou Chen, and Shian-Ying Sung Chia-Ling HsiehChia-Ling Hsieh More articles by this author , Kuan-Chou ChenKuan-Chou Chen More articles by this author , and Shian-Ying SungShian-Ying Sung More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3321AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To evaluate the expression level of chemokine receptors in response to chemokines released by reactive stroma, quantitative PCR to evaluate the mRNA level of chemokine receptors was performed. METHODS To check the expression level of CCRL2, we performed qRT-PCR and western blot analysis. Cell tracking assays were performed to analyze its role in chemoattraction. Proximity ligation assays were conducted to analyze the co-localization of CCRL2 and CCR5. To analyze the role of CCRL2 in regulation of cancer cell metastasis, we conducted in vivo analysis. Stromal cell overexpressed CCL5 were inoculated in renal capsule, followed by intra-peritoneal injection of prostate cancer cell overexpressed CCRL2 to determine the organ specific metastasis. To analyze the population of cells expression CCRL2, cell sorting assays was conducted. RESULTS We noticed the significant increasing of CCRL2 (86-fold) in androgen insensitive prostate cancer cells. Knockdown of CCRL2 declined 75% of migration activities induced by CCL5, suggests CCRL2 involve in CCL5 induce cancer migratory activities. IHC analysis of CCRL2 in paired prostate cancer patients was performed and revealed increasing of CCRL2 expression in malignant prostate cancer locus, whereas no CCRL2 can be detected in the benign region of same patient. Proximity ligation assays (PLAs) of benign, high grade tumor and tissues collected from patients less than 5-year survive demonstrated the PLA signals only in the lethal progression on patients. In vivo xenograft mice model studies demonstrated the organ specific cancer metastasis can be enhanced after overexpressed CCRL2 in cancer cells. Cell sorting assays indicated that CCRL2 only expressed in less than 10% population of prostate cancer cells. Molecular analysis of CCRL2 demonstrated that CCRL2 regulated EZH2 expression in prostate cancer cells. CONCLUSIONS Our studies indicated the increased expression of CCRL2 play a central role in cancer cell migration induced by chemokine CCL5. CCL5, secreted by bone stromal cells after interaction with dormancy cancer cell, may induce directional migration and invasion of circulating cancer cells. Clinical analyses demonstrated increased of CCRL2 in malignant prostate cancer. Inhibition of these homing mechanisms might significantly decreased cancer cell metastatic activities of AIPC patients. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e596 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Chia-Ling Hsieh More articles by this author Kuan-Chou Chen More articles by this author Shian-Ying Sung More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract A33: Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo

Abstract CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and lung cancer. We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is loss of the S phase cell cycle checkpoint response. This is through a number of mechanisms including the uncoupling of CHK1 activation with the destabilization of CDC25A. Loss of checkpoint by over-expressing components of the checkpoint or inhibition of Wee1, covert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. Loss of the S phase checkpoint provides cells with an adaptive advantage through introduction of moderate levels of genomic instability. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, but also involves a mechanism that is dependent in part on DNA-PK activity. Loss of S phase checkpoint function is predicted for <25% of melanomas and non-small cell lung squamous cell cancers. This is independent of other known risk factors, suggesting a significant proportion of melanoma and lung cancer patients could benefit from treatment with these drugs. Note: This abstract was not presented at the conference. Citation Format: Zay Yar Oo, Alexander Stevenson, Catherine Lanagan, Loredana Spoerri, Jill Larsen, Brian Gabrielli. Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A33.

Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells

Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin) and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer.

Effect of enzalutamide on sensitivity in prostate cancer cells to radiation by inhibition of DNA double strand break repair.

208 Background: Prostate cancer is the second leading cause of cancer-related deaths amongst men in North America. Data suggests that, following radiation therapy (XRT), androgen receptor (AR) enhances DNA damage repair and contributes to resistance of prostate cancer (PCa) cells to XRT. At present AR-pathway inhibition is the mainstay treatment of metastatic castration resistance prostate cancer (mCRPC). Enzalutamide (ENZA), a potent AR inhibitor is one of the approved drugs in this setting. The purpose of this study was to assess the potential radiosensitization of ENZA and its mechanism of action in hormone resistant PCa cells. Methods: The effect of ENZA alone or in combination with XRT was assessed on hormone-sensitive, (HS: LNCaP, PC3-T877A) and insensitive PCa cells (HI: PC3, PC3-AR V7, C4-2) using viability and clonogenic assays, cell cycle arrest and DNA damage analysis. Results: MTT assay demonstrates that ENZA significantly inhibits the proliferation of HS PCa cells in a dose dependent manner whereas CRPC required ENZA in combination with ADT (androgen deprivation therapy). Additionally, clonogenic assay proves that concurrent administration of ENZA or ADT+ENZA and XRT led to a supra-additive antitumor effect with the dose enhancement factor of 1.76±0.008 in LNCaP, 1.65±0.01 in PC3-T877A and 1.35±0.003 in C4-2 respectively at surviving fraction of 0.1. This effect was not observed in PC3 and PC3-AR V7 cells pre-treated with ENZA (in all cases DEF = 1 at SF = 0.1). Additionally, the level of γH2AX increased in HS cells and CRPC cells treated with ENZA/ADT+ENZA and XRT when compared to XRT alone. The enhanced H2AX activity remained unchanged up to 24 hours after combination treatment. Furthermore, there is an initial inhibition of DNA-PKcs in HS and CRPC cells treated with ENZA/ADT+ENZA administered before XRT. Conclusions: Our data suggest that the higher efficacy of ENZA/ENZA+ADT and XRT could be partially due to inhibition of DNA damage repair. Our results demonstrated a significant enhancement of XRT efficacy and confirms the rational for the ongoing combination clinical trials with XRT.

Histone demethylase KDM7a to control androgen receptor activity in hormone-sensitive prostate cancer.

262 Background: Prostate cancer can be controlled by androgen-hormone treatment until the cancer becomes refractory. It is believed that hormone sensitivity is largely dependent on androgen receptor (AR) activity. Here, we investigated the molecular mechanism whereby androgen receptor activity is regulated by histone demethylase KDM7a, which demethylates histone H3K9 and H3K27. Methods: The LNCaP, PC3, and DU145 cell lines were cultured at 37 °C in 5% CO2 in Minimum Essential Medium alpha, which was supplemented with 10% fetal bovine serum. We engineered AR-positive LNCaP cells to stably express a short-hairpin RNA against KDM7a mRNA from a lentiviral vector. To examine the mechanisms of AR regulation by KDM7a, we used various techniques of molecular biology, such as real-time PCR, western blot, and chromatin immunoprecipitation assay. Also, we performed immunohistochemical staining by using prostate cancer tissue microarray. Results: By measuring AR downstream gene expression following androgen stimulation, we found that a KDM7a-deficient cell line showed lower AR downstream gene expression compared to a control cells. KDM7a knock-down in LNCaP cell line caused decreased cell proliferation, compared to control cell line. Silencing KMD7a in AR insensitive cell lines did not cause differences in cell proliferation. Western blot analysis with modified-histone antibody revealed that the KDM7a-knock-down LNCaP cell line had increased H3K27 dimethylation. We confirmed KDM7a binding on AR target-gene promoters after hormone stimulation in chromatin-immunoprecipitation experiments. And increased H3K27 di-methylation was observed in KDM7a knock-down LNCaP stable cell. Finally, we observed that the KDM7a protein was significantly upregulated in prostate cancer tissue (compared to control tissue), and that this difference correlated with the Gleason score. Conclusions: These data suggested that KDM7a is potentially a good therapeutic target for prostate cancer drugs and can be used as potentially a good prognostic indicator for prostate cancer and related treatment strategies.

Abstract P6-02-04: Atorvastatin insensitivity is associated with increased lipid droplets accumulation and fatty acid metabolism in breast cancer cells

Abstract Lipophilic statins, including atorvastatin, may exert significant antiproliferative and proapoptotic effects in breast cancer as demonstrated in both clinical trials and cell models. However, heterogeneity in treatment response still remains a noteworthy challenge to be addressed. In this regard, we previously proposed a multigene signature including genes involved in cholesterol biosynthesis, which were dysregulated upon atorvastatin treatment and was shown to predict statin responsiveness in breast cancer (BC) cell lines and primary tumors. To further delineate the molecular mechanisms underlying this variability, we sought to characterize the differential statin-induced effects on intracellular lipid regulation observed in BC cell lines based on their sensitivity to atorvastatin treatment. BC cells were classified as insensitive (MCF-7 and T47D) or sensitive (MDA-MB-231) to atorvastatin by virtue of growth inhibition rate consequent to treatment with doses ranging up to 10 μM. Under complete culture conditions, atorvastatin-induced decrease in cell proliferation was inversely correlated to a progressive accumulation of neutral lipids in lipid droplets (LDs) in the insensitive cells following 72 hours treatment. Interestingly, in the sensitive MDA-MB-231 cells no significant change in LDs formation was observed despite the very potent antiproliferative effects (60% impairment of growth). However, in correspondence of severe inhibition of growth rate of at least 80% by atorvastatin treatment, MDA-MB-231 cells displayed a consistent reduction in LDs accumulation, suggesting beneficial effects of LDs biosynthesis on proliferation of BC cells. Transcriptional profiling of genes involved in lipid metabolism using microarrays and validated by qRT-PCR ruled out the likelihood that atorvastatin treatment altered lipid uptake and export mechanisms in sensitive cells in the presence of exogenous lipid supply. Nevertheless, gene ontology analysis indicated that an induction of cell stress responses was likely associated to atorvastatin sensitivity. Significant deregulation of genes involved in the fatty acid metabolic process, including biosynthesis of monounsaturated fatty acids (MUFAs), and cholesterol biosynthesis were instead linked to atorvastatin insensitivity. Accordingly, we found that the magnitude of the induction of the mRNAs of stearoyl-CoA desaturase (SCD), the key effector in MUFAs metabolism, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the established target of statins, were consistently lower in sensitive cells compared to the insensitive counterpart in response to atorvastatin treatment. Therefore, we suggest that the ability to significantly increase the number of stored neutral lipids in response to statin treatment may likely confer a proliferative advantage to BC cells. Our results also identified MUFAs metabolism as an attractive pathway to be further investigated in view of finding promising biomarkers for unraveling the molecular basis of statin sensitivity in breast cancer. Citation Format: Lettiero B, Kimbung S, Borgquist S. Atorvastatin insensitivity is associated with increased lipid droplets accumulation and fatty acid metabolism in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-04.

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex classI-related chainAandB in cancer cells.

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low-dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage-dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage-dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Displacement of Coilin in Cajal Body Is Related to Drug Resistance of Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is one of the most prevalent childhood cancers worldwide. Although the 5-year event-free survival rate of ALL patients have been improved to about 80% in latest years, there are approximately 15% of ALL cases in China remained to be poorly responsive to chemotherapy treatments, and therefore manifested in poor prognosis. Daunorubicin (DNR) is a key agent (Fig.1), as a chemical can cause DNA damages, in the formula for the treatments of ALL patients, the drug resistance to DNR will directly contributed to the failing of treatments and poor prognosis. In clinic, there is an urgent need for the development of assays that can be used for the prediction of DNR resistance, especially the involvement of visual indicators. Coilin is a major scaffold and marker protein of the functional nuclear structure - Cajal body (CB). We found that DNR was able to induce vast nucleolar accumulation of coilin in ALL leukemia cell lines of RS4;11 and Reh. Such accumulation of coilin was dose-dependently correlated to the increase of drug concentrations (Fig.2). At the molecular level, we also observed that the expression levels of coilin increased based on treatment of DNR(Fig.3B), while the expression levels of p27 protein was much lower in DNR insensitive cells with less significant changes of Coilin-indicated CB morphology as compared to the sensitive cells (Fig.3A). The over-expression of Coilin inhibited p27 expression and coilin knock-down increased the expression of p27(Fig.3 C,D). Our study suggested that coilin could be considered as a visual maker for the detection of daunorubicin resistance in ALL cells, and the mechanism possibly involved the regulation of drug sensitivity -related protein p27 through Cajal body -mediated RNA processing, during which coilin is an important participator with crucial functions.Results1.Daunorubicin inhibits topoisomerase II activity2.Daunorubicin(DNR)-induced DNA damage triggers the nucleolar accumulation of coilin3.Coilin inhibit the expression of p27 and may play a role in the drug resistance of leukemia through p27 [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Targeting of PIM Protein Kinases in a Subset of T-Cell Acute Lymphoblastic Leukemia

New approaches are needed to treat and cure T-cell acute lymphoblastic leukemia (T-ALL) patients who are currently failing to achieve a remission with chemotherapy. While very intensive chemotherapy regimens result in remission in many T-ALL patients, early T-cell precursor (ETP)-ALL, defined by the absence of CD4, CD8, and CD1a and frequent expression of one or more myeloid markers, is associated with a higher rate of relapse and induction failure with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. The PIM serine/threonine protein kinase has been identified as a potential novel target for the development of new treatment regimens for the cure of hematopoietic malignancies.To explore the potential association of elevated PIM1 expression with ETP-ALL, we analyzed a publically available St. Judes gene expression dataset GSE28703. Results demonstrated that PIM1 is highly expressed in 75% of patients with ETP-ALL and in 13% of non-ETP ALL. PIM1 overexpression in immature or ETP-ALL is further validated using two other publically available independent cohorts: E-MEXP-313 and GSE62156.Based on this observation, we then tested the ability of pan-PIM inhibitors (AZD1208/LGB-321) on the growth of 6 human T-ALL cell lines. qRT-PCR and western blot analysis confirmed, PIM inhibitor sensitive (XTT assay: IC50 < 1 micromolar) cell lines (H-SB2, DU.528, and KOPT-K1) had elevated levels of PIM1 (>9 folds, p<0.01), activated JAK/STAT pathway and lower levels of MYC. Sensitive cells (H-SB2 and DU.528), had an immature or ETP-ALL phenotype (CD4-/CD8- and TdT-). Insensitive CUTLL1, SUP-T1, and HPB-ALL (XTT assay: IC50 > 10 micromolar) cell lines express significantly low PIM1, activated NOTCH mutations, activated AKT signaling, and higher levels of MYC. Immunophenotype of all the PIM inhibitor insensitive cells lines was consistent with a more mature T-ALL phenotype (CD4+/CD8+ and TdT+). When we selected NOTCH-mutant mature T-ALL cells for resistance to gamma secretase inhibitors (SUP-T1-Persister cells) they developed elevated PIM1 kinase levels and were sensitive to the growth inhibitory activities of PIM inhibition. Thus, PIM appears to be a potential target for chemotherapeutic treatment intervention in T-ALL and especially ETP-ALL tumor cells.While PIM kinase inhibitors block ETP-ALL growth they do not induce cell death. The observation that treatment with PIM inhibitors activated ERK and STAT phosphorylation, along with the knowledge that T-ALL cells have increased levels of SRC family tyrosine kinases suggested that adding a broadly active tyrosine kinase inhibitor could enhance the killing of these tumor cells. We have made the novel observation that the combination of Ponatinib, a tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia, with a PIM kinase inhibitor is synergistically lethal to ETP-ALL cells (CI < 1.0). These agents synergize to induce PARP and Caspase cleavage, sufficient to drive apoptosis. In murine models, following engraftment of NOD/scid/IL-2Rγ null (NSG) mice with H-SB2-luc cell xenografts, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly blocked leukemia growth as evidenced by optical scanning for luciferase producing cells (Figure 1, p<0.05), and reduced the number of leukemic cells (hCD45 staining) in the peripheral blood and bone marrow. This dual therapy significantly prolonged the survival (p<0.005) of the treated mice. This outcome was obtained at one-tenth of normal dose of Ponatinib used in animals, suggesting that in the humans the side effects induced by this agent could be avoided by using this combination therapy.Taken together, these findings highlight the important role for PIM protein kinase activity in T-ALL and especially in the ETP-ALL, phenotype. Tumor xenograft experiments provide strong preclinical rationale for a novel treatment strategy of combining PIM and tyrosine kinase inhibitors for treatment of patients with PIM overexpressing T-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of biomarker microRNAs for predicting the response of colorectal cancer to neoadjuvant chemoradiotherapy based on microRNA regulatory network.

Preoperative radiotherapy or chemoradiotherapy has become a standard procedure for treatment of patients with locally advanced colorectal cancer (CRC). However, patients’ responses to treatment are different and personalized. MicroRNAs (miRNAs) are promising biomarkers for predicting personalized responses. In this study, we collected 30 publicly reported miRNAs associated with chemoradiotherapy of CRC. We extracted 46 differentially expressed miRNAs from samples of responders and non-responders to preoperative radiotherapy from the Gene Expression Omnibus dataset (Student's t test, p-value < 0.05 and |fold-change| > 2). We performed a systematic and integrative bioinformatics analysis to identify biomarker miRNAs for prediction of CRC responses to chemoradiotherapy. Using the bioinformatics model, miR-198, miR-765, miR-671-5p, miR-630, miR-371-5p, miR-575, miR-202, miR-483-5p and miR-513a-5p were screened as putative biomarkers for treatment response. Literature validation and functional enrichment analysis were exploited to confirm the reliability of the predicted miRNAs. Quantitative polymerase chain reaction showed that seven of the candidates were significantly differentially expressed between radiosensitive and insensitive CRC cell lines. The unique target genes of miR-198 and miR-765 were altered significantly upon transfection of specific miRNA mimics in the radiosensitive cell line. These results demonstrated the predictive power of our model and suggested that miR-198, miR-765, miR-630, miR-371-5p, miR-575, miR-202 and miR-513a-5p could be used for predicting the response of CRC to preoperative chemoradiotherapy.