SN-38 Treatment Research Articles

TNF Signaling through RIP1 Kinase Enhances SN38-Induced Death in Colon Adenocarcinoma.

Elucidation of TNF-directed mechanisms for cell death induction and maintenance of tumor growth has revealed a role for receptor-interacting protein kinases 1 and 3 (RIPK1/RIP1 and RIPK3/RIP3), components of the necrosome complex, as determinants of cell fate. Here, the participation of TNF signaling was analyzed with regard to the cytotoxic action of different DNA-damaging agents in a panel of colon cancer cells. While most of these cell lines were insensitive to TNF, combination with these drugs increased sensitivity by inducing cell death and DNA damage, especially in the case of the topoisomerase inhibitor SN38. Changes in levels of RIP1 and RIP3 occurred following monotherapy with SN38 or in combination with TNF. Downregulation of RIP1 resulted in increased resistance to SN38, implying a requirement for RIP1 in mediating cytotoxicity through the TNF/TNFR signaling pathway. Downregulation of RIP1 in a xenograft model impaired tumor growth inhibition from SN38 treatment, suggesting the potential of RIP1 to determine the clinical outcome of irinotecan treatment. These results indicate that TNF plays a key role in determining the cytotoxic effectiveness of SN38 in colorectal cancer and suggests a re-evaluation of TNF-based interventions to enhance therapeutic efficacy.Implications: The capacity of RIP1 to influence drug sensitivity suggests RIP1 may have biomarker potential. Mol Cancer Res; 15(4); 395-404. ©2017 AACR.

Abstract 3693: Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor

Abstract It is well established that cell cycle arrest in response to treatment with DNA damaging agents can be abrogated in p53-defective cells by treatment with the Chk1 inhibitors, but our more recent studies have shown that some p53 wild-type tumors are also sensitive to checkpoint abrogation. Two possible explanations are cytoplasmic sequestration of p53 or a defect in p53 oligomerization. In this study, we investigated the DNA damage response and UCN-01 sensitivity of two p53 wildtype neuroblastoma cell lines: SK-N-SH, and SH-SY5Y. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. Both lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 and serine 20 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25 nM UCN-01 for 6 and 24 hours. The SK-N-SH showed now sensitivity to UCN-01 treatment whereas the SH-SY5Y abrogated S arrest within 6 hours and abrogated G2 arrest within 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 and repression of cyclin B in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation in p53 wildtype neuroblastomas. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Pawan Puli, Robert Lipski, Aime A. Levesque. Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3693.

Abstract 5045: Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC

Abstract Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents. Citation Format: Yoshitaka Yagi, Hiroaki Ozasa, Takahiro Tsuji, Yuichi Sakamori, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5045.

Nuclear translocation and activation of YAP by hypoxia contributes to the chemoresistance of SN38 in hepatocellular carcinoma cells.

Although hypoxia is a prominent feature contributing to the therapeutic resistance of hepatocellular carcinoma cells (HCC) against chemotherapeutic agents, including the Topoisomerase I inhibitor SN38, the underlying mechanism is not fully understood and its understanding remains a major clinical challenge. In the present study, we found that hypoxia-induced nuclear translocation and accumulation of YAP acted as a survival input to promote resistance to SN38 in HCC. The induction of YAP by hypoxia was not mediated by HIF-1α because manipulating the abundance of HIF-1α with CoCl2, exogenous expression, and RNA interference had no effect on the phosphorylation or total levels of YAP. The mevalonate-HMG-CoA reductase (HMGCR) pathway may modulate the YAP activation under hypoxia. Combined YAP inhibition using either siRNA or the HMGCR inhibitor statins together with SN38 treatment produced improved anti-cancer effects in HCC cells. The increased anti-cancer effect of the combined treatment with statins and irinotecan (the prodrug of SN-38) was further validated in a human HepG2 xenograft model of HCC in nude mice. Taken together, our findings identify YAP as a novel mediator of hypoxic-resistance to SN38. These results suggest that the administration of SN28 together with the suppression of YAP using statins is a promising strategy for enhancing the treatment response in HCC patients, particularly in advanced stage HCC cases presenting hypoxic resistance.

Radiation and SN38 treatments modulate the expression of microRNAs, cytokines and chemokines in colon cancer cells in a p53-directed manner.

Aberrant expression of miRNAs, cytokines and chemokines are involved in pathogenesis of colon cancer. However, the expression of p53 mediated miRNAs, cyto- and chemokines after radiation and SN38 treatment in colon cancer remains elusive. Here, human colon cancer cells, HCT116 with wild-type, heterozygous and a functionally null p53, were treated by radiation and SN38. The expression of 384 miRNAs was determined by using the TaqMan® miRNA array, and the expression of cyto- and chemokines was analyzed by Meso-Scale-Discovery instrument. Up- or down-regulations of miRNAs after radiation and SN38 treatments were largely dependent on p53 status of the cells. Cytokines, IL-6, TNF-α, IL-1β, Il-4, IL-10, VEGF, and chemokines, IL-8, MIP-1α were increased, and IFN-γ expression was decreased after radiation, whereas, IL-6, IFN-γ, TNF-α, IL-1β, Il-4, IL-10, IL-8 were decreased, and VEGF and MIP-1α were increased after SN38 treatment. Bioinformatic analysis pointed out that the highly up-regulated miRNAs, let-7f-5p, miR-455-3p, miR-98, miR-155-5p and the down-regulated miRNAs, miR-1, miR-127-5p, miR-142-5p, miR-202-5p were associated with colon cancer pathways and correlated with cyto- or chemokine expression. These miRNAs have the potential for use in colon cancer therapy as they are related to p53, pro- or anti-inflammatory cyto- or chemokines after the radiation and SN38 treatment.

Comet assay measures of DNA damage as biomarkers of irinotecan response in colorectal cancer in vitro and in vivo.

The use of irinotecan to treat metastatic colorectal cancer (CRC) is limited by unpredictable response and variable toxicity; however, no reliable clinical biomarkers are available. Here, we report a study to ascertain whether irinotecan-induced DNA damage measures are suitable/superior biomarkers of irinotecan effect. CRC-cell lines (HCT-116 and HT-29) were treated in vitro with irinotecan and peripheral blood lymphocytes (PBL) were isolated from patients before and after receiving irinotecan-based chemotherapy. Levels of in vitro-, in vivo-, and ex vivo-induced DNA damage were measured using the Comet assay; correlations between damage levels with in vitro cell survival and follow-up clinical data were investigated. Irinotecan-induced DNA damage was detectable in both CRC cell-lines in vitro, with higher levels of immediate and residual damage noted for the more sensitive HT-29 cells. DNA damage was not detected in vivo, but was measurable in PBLs upon mitogenic stimulation prior to ex vivo SN-38 treatment. Results showed that, following corrections for experimental error, those patients whose PBLs demonstrated higher levels of DNA damage following 10 h of SN-38 exposure ex vivo had significantly longer times to progression than those with lower damage levels (median 291 vs. 173 days, P = 0.014). To conclude, higher levels of irinotecan-induced initial and residual damage correlated with greater cell kill in vitro and a better clinical response. Consequently, DNA damage measures may represent superior biomarkers of irinotecan effect compared to the more often-studied genetic assays for differential drug metabolism.

UbcH10 expression can predict prognosis and sensitivity to the antineoplastic treatment for colorectal cancer patients.

Colorectal cancer (CRC) is one of the most frequent and deadly malignancies worldwide. Despite the progresses made in diagnosis and treatment, the identification of tumor markers is still a strong clinical need, because current treatments are efficacious only in a subgroup of patients. UbcH10 represents a potential candidate biomarker, whose expression levels could be employed to predict response or resistance to chemotherapy or targeted agents. UbcH10 mRNA and protein expression levels have been evaluated in a large group of CRC patients and correlated with clinico-pathological characteristics, including KRAS mutations. Moreover, the endogenous levels of UbcH10 and its role on cell growth have been evaluated in CRC cells. Finally, to investigate the impact of UbcH10 protein expression on the response to irinotecan, its active metabolite SN-38 and cetuximab treatment, UbcH10 silencing experiments were carried-out on two colon carcinoma cell lines, Caco-2, and DLD1. Overexpression of UbcH10 mRNA and protein was observed in the vast majority of patients analyzed. UbcH10 suppression decreased CRC cell growth rate (at least in part through deregulation of Cyclin B and ERK1) and sensitized them to pharmacological treatments with irinotecan, SN-38 and cetuximab (at least in part through a down-regulation of AKT). Taken together, these findings indicate that UbcH10 expression regulates CRC growth and could play an important role in the personalization of the therapy of CRC patients.

Schlafen-11 sensitizes colorectal carcinoma cells to irinotecan.

Schlafen-11 (SLFN11) showed a highly significant positive correlation with the response of topoisomerase inhibitors in cancer cell lines derived from prostate, lung, etc. However, this finding has not been validated in colorectal cancers (CRCs). Although irinotecan (CPT-11), a topoisomerase inhibitor, is one of the most important drugs in the treatment of advanced and/or metastatic CRC, resistance is a critical drawback to its clinical effectiveness. The present study aimed to investigate the mechanism of SLFN11 in the response of CRC cell lines to SN-38 (an active CPT-11 metabolite) treatment. Western blotting was used to measure protein expression levels of SLFN11 in human CRC cell lines. Then, SLFN11 expression was modulated by transfecting human CRC cell lines with vectors carrying the SLFN11 gene or specific SLFN11 small interfering RNAs. The effects of SN-38 treatment on CRC cells with different SLFN11 expression levels were detected, including inhibition of cell growth, induction of apoptosis, and cell cycle arrest. This study showed that SLFN11 expression varied between the CRC cell lines and high-level SLFN11 expression promoted SN-38-induced antiproliferative activity, apoptosis, and cell cycle arrest. Our results suggest that SLFN11 plays a key role in cell cycle arrest and/or induction of apoptosis in response to exogenous SN-38-induced DNA damage and might be used as a new predictive biomarker for CRC treatment.

Abstract 488: Inhibition of JNK signaling sensitizes hypoxic colon cancer cells to DNA-damaging agents

Abstract BACKGROUND: We have shown that hypoxia induces signaling through stress pathways in the HT29 colon adenocarcinoma cell line, and that modulation of signaling through MKK4/MKK7 exerts the opposite effects on oxaliplatin cytotoxicity under hypoxic conditions: higher sensitivity when MKK4 function is impaired and higher resistance when MKK7 is down- regulated. We also observed differential effects of JNK1 and JNK2 modulation on sensitivity of hypoxic HT29 cells to oxaliplatin treatment which implied a pro-survival function for JNK1. PURPOSE: Here we investigate how modulating signaling through JNK influences the sensitivity of hypoxic HT29 cells to oxaliplatin, SN-38 and 5-FU. We used pharmacological and molecular approaches to inhibit JNK signaling and expanded the cellular model in a panel of colon cancer cell lines. Sensitivity to drugs and induction of stress pathways were investigated in 12 colon cancer cell lines of different genetic backgrounds. We also assessed cell cycle changes and cell death characteristics upon treatment. In addition to HT29-derived cell lines stably expressing dominant negative constructs for MKK4 (HTS13), MKK7 (HTM9), JNK1 (HTJ1.3) and JNK2 (HTJ2.2), we also utilized several colon cancer cell lines (HCT116, RKO1, LoVo and SW620) in which a dominant negative construct for JNK1 or JNK2 was introduced by viral delivery. RESULTS: Our data demonstrate that stress signaling through JNK is induced by hypoxia to various extent and with differing temporal characteristics in all colon cancer cell lines assessed. In HT29 cells, treatment with oxaliplatin or SN-38 alone does not cause significant activation of signaling through JNK, whereas 5-FU treatment leads to induction of this pathway. Inhibition of JNK1 renders HT29 cells more sensitive to oxaliplatin and has no effect upon SN-38 treatment, while inhibition of either isoform sensitizes hypoxic HT29 cells to 5-FU. We also show that pharmacological inhibition of JNK by CC-401 augments cytotoxicity of oxaliplatin, SN-38 and of 5-FU in hypoxic HT29 cells, with CI50 of 0.42, 0.6 and 0.63, respectively. CC-401 and SP600125 have also demonstrated synergy with chemotherapy in other colon cancer cell lines. Finally, we show that colon cancer cell lines differ in their responses to oxaliplatin when JNK1 or JNK2 are down-regulated, from no effect (HCT116, LoVo) to increased sensitivity (HT29, SW620). CONCLUSIONS: Our data demonstrate that inhibition of the JNK pathway is a plausible approach to augment the cytotoxicity of oxaliplatin in colon cancer. However, inhibition of individual isoforms presents a complicated picture. Since enhancement of oxaliplatin cytotoxicity by this approach appears to be model-specific in colon cancer, it should be undertaken following the identification of molecular markers better able to predict the outcome. Citation Format: Irina A. Vasilevskaya, Muthu Selvakumaran, Lucia Cabal-Hierro, Peter J. O'Dwyer. Inhibition of JNK signaling sensitizes hypoxic colon cancer cells to DNA-damaging agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 488. doi:10.1158/1538-7445.AM2014-488

Abstract 2274: RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines

Abstract BACKGROUND: TNF is a dual role cytokine involved both in cell death and survival that is expressed in the tumor microenvironment. However, little is known about the molecular events that mark the biological outcomes of such opposite biological events. Recently, the protein kinase RIP1 has been identified as a new intermediate involved in the responses activated after DNA damage induction. PURPOSE: In this study we investigated how TNF modulates the sensitivity to SN38 in colon cancer cell lines. We focused further on HT29- and HCT116-derived cell lines in which RIP1 or RIP3 proteins expression were stably downregulated. RESULTS: We found that TNF increases cellular sensitivity to different chemotherapeutic drugs under both normoxic and hypoxic conditions over a panel of different colon adenocarcinoma cell lines. Combinations with SN38 were highly synergistic. In HT29 and HCT116 cell lines (TNF resistant and TNF sensitive respectively), we analyzed this interaction by studying P-H2-AX induction, cell proliferation (clonogenic assays and BrdU incorporation) and cell cycle distribution. To study the role of RIP1 and RIP3 on the biological responses triggered by SN38, HT29- and HCT116-derived cell lines expressing low levels of RIP1 and RIP3 were analyzed. While knockdown of RIP3 had no effect on SN38 cytotoxicity, RIP1 down regulation produced a 2.5-fold increased resistance to SN38 under hypoxic conditions. This change in SN38 sensitivity also correlated with a reduction in the levels of P-H2-AX in the presence of TNF, suggesting dependence of DNA damage induction by SN38 on RIP1. CONCLUSIONS: These data suggest that RIP1 may be a key intermediate in determining cell death following SN38 treatment. Additional studies in tumor samples are in progress. Citation Format: Lucia Cabal-Hierro, Peter J. O'Dwyer. RIP1-mediated sensitivity to SN38 induced by TNF in colon adenocarcinoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2274. doi:10.1158/1538-7445.AM2014-2274

Contribution of transcription factor, SP1, to the promotion of HB-EGF expression in defense mechanism against the treatment of irinotecan in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a worst histological subtype than other ovarian malignant tumor. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for ovarian cancer therapy. The aims of this study were to validate the efficacy of HB-EGF–targeted therapy for OCCC and to identify the transcription factor that contributed to the induction of HB-EGF by SN38 treatment in OCCC cells. HB-EGF was highly expressed in OCCC cells, and an increase of HB-EGF was induced by SN38 which had only antitumor effect among conventional anticancer agents on OCCC. A specific inhibitor of HB-EGF, a cross-reacting material 197 (CRM197), led to a synergistic increase in the number of apoptotic OCCC cells with the treatment of SN38. The luciferase assay with 5′-deletion promoter constructs identified a GC-rich element between −125 and −178 (the distal transcription start site was denoted +1) as a cis-regulatory region, and the treatment of SN38 induced luciferase activity in this region. An in silico and chromatin immunoprecipitation analysis estimated that SP1 bound to the cis-regulatory region of HB-EGF in OCCC cells. Real-time PCR and cell viability assays showed that the transfection of a small interfering RNA targeting SP1 suppressed the expression of HB-EGF induced by SN38, resulting in the enhanced sensitivity of SN38. Taken together, these results indicate that induction of HB-EGF expression contributed to defense mechanism against treatment of SN38 through the transcriptional activity of SP1 in OCCC cells.

The Use of Olaparib (AZD2281) Potentiates SN-38 Cytotoxicity in Colon Cancer Cells by Indirect Inhibition of Rad51-Mediated Repair of DNA Double-Strand Breaks

Potent application of topoisomerase I inhibitor plus PARP inhibitor has been suggested to be an effective strategy for cancer therapy. Reportedly, mismatch repair (MMR)-deficient colon cancer cells are sensitive to topoisomerase I inhibitor, presumably due to microsatellite instability (MSI) of the MRE11 locus. We examined the synergy of SN-38, an active metabolite of irinotecan, in combination with the PARP inhibitor olaparib in colon cancer cells showing different MMR status, such as MSI or microsatellite stable (MSS) phenotype. Treatment with SN-38 and olaparib in combination almost halved the IC50 of SN-38 for a broad spectrum of colon cancer cells independent of the MMR status. Furthermore, olaparib potentiated S-phase-specific double-strand DNA breaks (DSB) induced by SN-38, which is followed by Rad51 recruitment. siRNA-mediated knockdown of Rad51, but not Mre11 or Rad50, increased the sensitivity to olaparib and/or SN-38 treatment in colon cancer cells. In vivo study using mouse xenograft demonstrated that olaparib was effective to potentiate the antitumor effect of irinotecan. In conclusion, olaparib shows a synergistic effect in colon cancer cells in combination with SN-38 or irinotecan, potentiated by the Rad51-mediated HR pathway, irrespective of the Mre11-mediated failure of the MRN complex. These results may contribute to future clinical trials using PARP inhibitor plus topoisomerase I inhibitor in combination. Furthermore, the synergistic effect comprising topoisomerase I-mediated DNA breakage-reunion reaction, PARP and Rad51-mediated HR pathway suggests the triple synthetic lethal pathways contribute to this event and are applicable as a potential target for future chemotherapy.

Abstract 568: Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma.

Abstract Cell cycle checkpoints prevent cells from dividing with damaged DNA by causing cell cycle arrest or apoptosis. DNA damage-induced cell cycle arrest resulting from treatment with the topoisomerase I inhibitor SN38 can be abrogated specifically in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). However, some p53 wild-type tumor cell lines have also been shown to be sensitive to checkpoint abrogation, suggesting that these cells have other defects in the p53 pathway that can be exploited for therapeutic purposes. Our recent studies revealed that cells that are sensitive to checkpoint abrogation lack p53 dimers and tetramers. Thus, one possible explanation for the sensitivity of p53 wildtype tumors to checkpoint abrogation is a defect in oligomerization. In the current study, we investigate the DNA damage response and UCN-01 sensitivity of three p53 wildtype neuroblastoma cell lines: SK-N-SH, SH-SY5Y, and IMR32. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. All three cell lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25-100 nM UCN-01 for 6 and 24 hours. The SK-N-SH and IMR32 showed only slight sensitivity to the highest concentration of UCN-01 treatment as evidenced by partial abrogation of S and G2 arrest. The SH-SY5Y, however, abrogated S arrest after 6 hours at the lowest concentration of UCN-01 and abrogated G2 arrest after 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possess levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The IMR32 and SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation p53 wildtype neuroblastomas, although more recent studies refute these reports. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53, while in IMR32, p53 was evenly distributed between the nucleus and cytoplasm. The results of this study suggest that oligomerization status alone is insufficient as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor. Citation Format: Robert Lipski, Aime A. Levesque. Analysis of p53 oligomerization and sensitivity to checkpoint abrogation of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2013-568

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancer cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the 3H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.

P53 Dimers Associate with a Head-to-Tail Response Element to Repress Cyclin B Transcription

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug.

Abstract 2220: p53 dimers associate with a head-to-head element to repress cyclin B transcription

Abstract DNA damage activates cell cycle checkpoints to promote cell cycle arrest. p53 is a major player in checkpoints by activating expression of the cdk inhibitor p21 and repressing expression of cyclin B. DNA damage-induced S and G2 arrest following treatment with the topoisomerase I inhibitor SN38 is abrogated in p53-defective cells by treatment with the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wildtype MCF10A cells with derivatives whose p53 function is inhibited by overexpression of the tetramerization domain (MCF10A/OD) or overexpression of shRNA against p53 (MCF10A/αp53) (Levesque et al, Oncogene 24, 3786, 2005). Treatment of MCF10A/OD with SN38 followed by UCN-01 results in abrogation of S, but not G2, arrest, while the MCF10A/αp53 abrogated both S and G2 arrest. The S abrogation correlated with reduced p21, consistent with the requirement of p53 tetramerization for activation of p21 expression. Failure of G2 abrogation correlated with reduced cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B expression. In the present study, we confirmed that the p53-dependent repression of cyclin B in response to DNA damage occurs at the transcriptional level by analyzing cyclin B mRNA levels. Treatment of MCF10A and MCF10A/OD with SN38 resulted in reduction of cyclin B mRNA levels whereas treatment of MCF10A/αp53 with SN38 did not. This reflects the change in protein levels following SN38 treatment. We next analyzed p53 oligomerization using glutaraldehyde crosslinking. Following SN38 treatment, MCF10A contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. MCF10A/OD, however, possessed monomers and dimers, but lacked trimers and tetramers. These results suggest that p53 monomers or dimers are involved in repression of cyclin B. One model to explain the p53-mediated repression of cyclin B in MCF10A/OD is that p53 dimers, rather than tetramers, bind to a p53 response element in the cyclin B promoter. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-tail orientation, the cyclin B promoter contains similar elements oriented head-to-head. We performed both chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA) to determine whether p53 binds to this head-to-head element. ChIP assays revealed that p53 associates with this element in MCF10A and MCF10A/OD but not in MCF10A/αp53. We performed EMSA using a biotin-labeled probe containing the head-to-head element and observed shifts in mobility consistent with the molecular weight of tetramers and dimers using MCF10A nuclear extract (NE), but observed only the dimeric shift with MCF10A/OD NE, and no shift with MCF10A/αp53 NE. Taken together, these results suggest p53 dimers associate with the head-to-head element to repress cyclin B transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2220. doi:1538-7445.AM2012-2220

A Systems Biology Approach Identifies SART1 as a Novel Determinant of Both 5-Fluorouracil and SN38 Drug Resistance in Colorectal Cancer

Chemotherapy response rates for advanced colorectal cancer remain disappointingly low, primarily because of drug resistance, so there is an urgent need to improve current treatment strategies. To identify novel determinants of resistance to the clinically relevant drugs 5-fluorouracil (5-FU) and SN38 (the active metabolite of irinotecan), transcriptional profiling experiments were carried out on pretreatment metastatic colorectal cancer biopsies and HCT116 parental and chemotherapy-resistant cell line models using a disease-specific DNA microarray. To enrich for potential chemoresistance-determining genes, an unsupervised bioinformatics approach was used, and 50 genes were selected and then functionally assessed using custom-designed short interfering RNA (siRNA) screens. In the primary siRNA screen, silencing of 21 genes sensitized HCT116 cells to either 5-FU or SN38 treatment. Three genes (RAPGEF2, PTRF, and SART1) were selected for further analysis in a panel of 5 colorectal cancer cell lines. Silencing SART1 sensitized all 5 cell lines to 5-FU treatment and 4/5 cell lines to SN38 treatment. However, silencing of RAPGEF2 or PTRF had no significant effect on 5-FU or SN38 sensitivity in the wider cell line panel. Further functional analysis of SART1 showed that its silencing induced apoptosis that was caspase-8 dependent. Furthermore, silencing of SART1 led to a downregulation of the caspase-8 inhibitor, c-FLIP, which we have previously shown is a key determinant of drug resistance in colorectal cancer. This study shows the power of systems biology approaches for identifying novel genes that regulate drug resistance and identifies SART1 as a previously unidentified regulator of c-FLIP and drug-induced activation of caspase-8.

Irinotecan induces steroid and xenobiotic receptor (SXR) signaling to detoxification pathway in colon cancer cells

BackgroundResistance to chemotherapy remains one of the principle obstacles to the treatment of colon cancer. In order to identify the molecular mechanism of this resistance, we investigated the role of the steroid and xenobiotic receptor (SXR) in the induction of drug resistance. Indeed, this nuclear receptor plays an important role in response to xenobiotics through the upregulation of detoxification genes. Following drug treatments, SXR is activated and interacts with the retinoid X receptor (RXR) to induce expression of some genes involved in drug metabolism such as phase I enzyme (like CYP), phase II enzymes (like UGT) and transporters (e.g. MDR1).ResultsIn this study, we have shown that endogenous SXR is activated in response to SN-38, the active metabolite of the anticancer drug irinotecan, in human colon cancer cell lines. We have found that endogenous SXR translocates into the nucleus and associates with RXR upon SN-38 treatment. Using ChIP, we have demonstrated that endogenous SXR, following its activation, binds to the native promoter of the CYP3A4 gene to induce its expression. RNA interference experiments confirmed SXR involvement in CYP3A4 overexpression and permitted us to identify CYP3A5 and MRP2 transporter as SXR target genes. As a consequence, cells overexpressing SXR were found to be less sensitive to irinotecan treatment.ConclusionsAltogether, these results suggest that the SXR pathway is involved in colon cancer irinotecan resistance in colon cancer cell line via the upregulation of select detoxification genes.

Regulation of the Aurora-A gene following topoisomerase I inhibition: implication of the Myc transcription Factor

During the G2 phase of the cell cycle, the Aurora-A kinase plays an important role in centrosome maturation and progression to mitosis. In this study, we show in colorectal cell lines that Aurora-A expression is downregulated in response to topoisomerase I inhibition. Using chromatin immunoprecipitation assays, we have observed that the Myc transcription factor and its Max binding partner are associated with the Aurora-A promoter during the G2 phase of the cell cycle. RNA interference experiments indicated that Myc is involved in the regulation of the Aurora-A gene. Following topoisomerase I inhibition, the expression of Myc decreased whereas Mad was upregulated, and the association of Myc and Max with the promoter of the kinase was inhibited. In parallel, an increased association of Mad and Miz-1 was detected on DNA, associated with an inhibition of the recruitment of transcriptional coactivators. Interestingly, a gain of H3K9 trimethylation and HP1γ recruitment was observed on the Aurora-A promoter following sn38 treatment, suggesting that this promoter is located within SAHF foci following genotoxic treatment. Since Aurora-A is involved in centrosome maturation, we observed as expected that topoisomerase I inhibition prevented centrosome separation but did not affect their duplication. As a consequence, this led to G2 arrest and senescence induction.These results suggest a model by which the Aurora-A gene is inactivated by the G2 checkpoint following topoisomerase I inhibition. We therefore propose the hypothesis that the coordinated overexpression of Myc and Aurora-A, together with a downregulation of Mad and Miz-1 should be tested as a prognosis signature of poor responses to topoisomerase I inhibitors.