Wild-type HCT116 Cells Research Articles

Abstract C005: TP53 wildtype status can predict sensitivity to XPO1 inhibitors in Patient-Derived Cancer Models

Abstract Introduction: Selinexor (SEL) and eltanexor (ELT) are selective inhibitors of exportin 1 (XPO1), that lead to the nuclear accumulation and activation of key tumor suppressors (e.g. p53, p21, RB, and FOXO), resulting in selective cancer cell death. In the SIENDO trial of SEL maintenance for patients with advanced stage or relapsed endometrial cancer (EC), SEL reduced disease progression compared to placebo, with a more pronounced reduction seen in patients with TP53 wild-type (WT) EC (Makker et al, ASCO 2022). As TP53 is the most commonly mutated gene in human cancers, and p53 is a cargo of XPO1, we investigated whether TP53 wild type (WT) status could serve as a mechanistic predictor for cancer cell sensitivity to XPO1 inhibitors. Methods: PDO and PDX models representing 17 cancer types, each with duplicate TP53 WT and MUT models were used (4 models per cancer type), with an emphasis on models with common hotspot MUTs. Tumor types were included if biological replicate models with TP53 wild-type or TP53 mutations were available for comparison. SEL, ELT and a control agent (CDDP or staurosporine) IC50 values after 5 days of exposure were calculated using CellTiter Glo assay. Sensitivity to SEL and ELT was considered TP53 WT dependent for each cancer type if the IC50 values for the TP53 MUT models were at least 1.5x higher than the average IC50 for the TP53 WT models. Similarly, the IC50 of SEL at 72h was determined for HCT116 WT and R175H cells to directly evaluate the association between TP53 MUT and resistance to XPO1 inhibitors in a syngeneic background. Results: In PDX/PDO models, sensitivity to SEL and/or ELT was determined to be TP53 WT dependent in 9 cancer types (endometrial, ovarian, kidney, liver, esophageal, lung adeno and squamous, pancreatic, and bladder). For example, the 2 TP53 WT EC PDX tested were ~20 fold more sensitive to SEL and ELT than 2 EC PDX with mutations within the TP53 DNA binding domain (TP53 WT SEL IC50 = 31nM and 61nM; p.Y220C SEL IC50= 333nM; p.R213* SEL IC50 = 1542nM). Interestingly, the IC50 of SEL and ELT were lower than CDDP for the TP53 WT EC models, as well as in the kidney cancer models. For the remaining 8 indications (head and neck, gastric​, gallbladder​, cholangiocarcinoma, cervical​, breast​, mixed mullerian​, prostate​​), XPO1 inhibitor sensitivity was TP53 WT independent. Reduced sensitivity to SEL was also observed in syngeneic HCT116 cells with the TP53 R175H (IC50=343 nM) MUT compared to WT (IC50=174 nM). Notably, TP53 MUT status conferred resistance to the control agents in only models of two cancer types (esophageal and lung adenocarcinoma), demonstrating specificity between TP53 MUT status and XPO1 inhibitors. Conclusions: Using TP53 WT status as a biomarker of sensitivity to XPO1 inhibitors SEL and ELT is cancer-type specific. Further experiments are ongoing to validate these findings in CRISPR-engineered SNG-M EC cells with clinically relevant R175H and R248Q TP53 hotspot mutations, including experiments related to cancer-type specific SEL/ELT mechanism of action. Citation Format: Marie Maloof, Andrea Ellero, Pietro Taverna, Christopher J Walker. TP53 wildtype status can predict sensitivity to XPO1 inhibitors in Patient-Derived Cancer Models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C005.

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1370.

Abstract PR003: Overexpression of the small GTPase Rab27B promotes tumor growth by regulating autophagy flux in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite instable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. To analyze the therapeutic potential of Rab27B inhibition in CRC, we performed a high throughput drug screen to identify small-molecule inhibitors of Rab27B GTPase activity in vitro from a panel of ∼2,100 compounds contained within the KU-HTSL chemical library. This screening resulted in the identification of three compounds (BTB00809, BTB03584, and KM01532) that demonstrated Rab27B GTPase activity inhibition. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B promotes tumor growth by regulating autophagy flux in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR003.

P52-ZER6: a determinant of tumor cell sensitivity to MDM2-p53 binding inhibitors.

Targeting MDM2-p53 interaction has emerged as a promising antitumor therapeutic strategy. Several MDM2-p53 inhibitors have advanced into clinical trials, but results are not favorable. The lack of appropriate biomarkers for selecting patients has been assumed as the critical reason for this failure. We previously identified ZER6 isoform p52-ZER6 as an oncogene upregulated in tumor tissues. In this study we investigated whether p52-ZER6 acted as a blocker of MDM2-p53 binding inhibitors, and whether p52-ZER6 could be used as a biomarker of MDM2-p53 binding inhibitors. In p53 wild-type colorectal carcinoma HCT116, hepatocarcinoma HepG2 and breast cancer MCF-7 cells, overexpression of p52-ZER6 enhanced MDM2-p53 binding and promoted p53 ubiquitination/proteasomal degradation. Furthermore, overexpression of p52-ZER6 in the tumor cells dose-dependently reduced their sensitivity to both nutlin and non-nutlin class MDM2-p53 binding inhibitors. We showed that p52-ZER6 restored tumor cell viability, which was suppressed by nutlin-3, through restoring their proliferation potential while suppressing their apoptotic rate, suggesting that MDM2-p53 binding inhibitors might not be effective for patients with high p52-ZER6 levels. We found that nutlin-3 treatment or p52-ZER6 knockdown alone promoted the accumulation of p53 protein in the tumor cells, and their combinatorial treatment significantly increased the accumulation of p53 protein. In HCT116 cell xenograft nude mouse model, administration of shp52-ZER6 combined with an MDM2-p53 binding inhibitor nutlin-3 exerted synergistic antitumor response. In conclusion, this study reveals that p52-ZER6 might be a potential biomarker for determining patients appropriate for MDM2-p53 binding inhibition-based antitumor therapy, and demonstrates the potential of combinatorial therapy using MDM2-p53 binding inhibitors and p52-ZER6 inhibition.

Abstract 1816: Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers

Abstract Synthetic lethality occurs when a single gene alteration is compatible with cell viability, but an additional co-occurring genetic alteration leads to cell death. In the context of cancer therapy, synthetic lethality can occur through the inhibition of a target that is selectively essential to tumors harboring a specific genetic alteration. Gene paralog pairs represent one promising class of synthetic lethal cancer targets, wherein the function of one paralog is lost in tumor cells, rendering them dependent on the remaining paralog to carry out an essential cellular process. To identify essential gene paralog pairs as starting points for drug discovery programs, we mined publicly available CRISPR genetic loss-of-function data and associated molecular datasets collected across a diverse panel of cancer cell lines. We first identified pairs of gene paralogs where one paralog was essential in a subset of cell lines, and then filtered these genes based on function, known literature, enrichment in specific lineages and integration of external datasets. These efforts identified VPS4A as a synthetic lethal target in cancers harboring copy number loss of VPS4B. VPS4A and VPS4B are highly homologous AAA ATPases that carry out multiple essential cellular processes including nuclear membrane remodeling and endosomal membrane biogenesis. VPS4B loss occurs as a passenger deletion during loss of the tumor suppressors SMAD2 and SMAD4. Loss of VPS4B creates a genetic dependency on VPS4A to drive essential VPS4-dependent processes. VPS4B deletion occurs at a frequency of up to 3% in multiple solid tumor types including esophageal, head and neck, pancreatic and colorectal cancers. To further explore the potential of VPS4A as a therapeutic target in VPS4B-deleted tumors, we first validated the synthetic lethal relationship between VPS4A/B using isogenic cell line pairs. HCT116 cells with an engineered homozygous loss of VPS4B, but not wild-type HCT116 cells, showed profound cell kill in response to genetic silencing of VPS4A. Moreover, simultaneous siRNA-mediated knockdown of VPS4A and VPS4B resulted in cell death across a panel of cancer cell lines (e.g. H1975, Panc0403), while knockdown of either gene alone was compatible with cell viability. Encouraged by these results, we profiled several previously reported small-molecule inhibitors of VPS4A (e.g. DBeQ and MSC1094308) in a suite of biochemical assays. Notably, these molecules were inactive against VPS4A. We have discovered a novel series of VPS4A inhibitors and are advancing this inhibitor series through lead optimization. Potent, selective, and pharmacologically active VPS4A inhibitors are expected to be well tolerated and have strong single-agent activity in tumors bearing VPS4B homozygous deletions. Citation Format: Meredith Kuo, Jason Chen, Sacha Holland, Eugene Lurie, An-Angela Ngoc Van, Francesco Parlati, Tayna Santos, Eric Sjogren, Natalija Sotirovska, Susanne Steggerda, Andrew MacKinnon. Identification of novel VPS4A inhibitors for the treatment of VPS4B-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1816.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Upregulation of GCLC is Responsible for SFN‐induced Tumor Cell Proliferation

Sulforaphane (SFN), a sulfur‐containing compound that belongs to the isothiocyanate class of dietary phytochemicals found in cruciferous vegetables, has been known to cause a hormetic effect on cancer cell growth. We previously reported that SFN at low doses stimulated proliferation of HCT116 human colorectal carcinoma cells through Nrf2 activation in vitro and in vivo. Recently, we discovered that brusatol, a nuclear factor erythroid 2‐related factor 2 (Nrf2) inhibitor, significantly suppressed the SFN‐induced proliferation of HCT116 cells. Therefore, we hypothesized that SFN‐induced proliferation of HCT116 cells is mediated by an Nrf2 downstream gene(s). Transcriptomic profiling revealed a set of differentially expressed genes (DEGs) involved in glutathione (GSH) metabolism as compared between wildtype (WT) and Nrf2 knockout (KO) HCT116 cells treated with or without SFN. In particular, we found that glutamate‐cysteine ligase catalytic subunit (GCLC) was highly elevated in SFN‐treated WT HCT116 cells among the DEGs, but not in SFN‐treated Nrf2 KO HCT116 cells. To confirm if upregulation of γ‐glutamate‐cysteine ligase (γ‐GCL) is associated with SFN‐induced proliferation of HCT116 cells, inhibiting γ‐GCL by buthionine sulfoximine (BSO, a selective γ‐GCL inhibitor) and silencing GCLC by short hairpin RNA (shRNA) were carried out. The results showed that SFN‐induced proliferation of HCT116 cells was significantly reduced by γ‐GCL inhibition and abolished by GCLC knockdown. Furthermore, we confirmed that BSO suppressed SFN‐induced tumor growth of WT HCT116 xenograft mice and the tumor growth was not stimulated in both Nrf2 KO and GCLC shRNA HCT16 xenograft mice by SFN at a low dose. Collectively, these findings indicate that upregulation of GCLC‐dependent GSH biosynthesis by SFN promotes proliferation of HCT116 cells and thus GCL could be a potential target for chemotherapy.

Abstract 999: The transcriptional response in colorectal cancer cells varies across four clinically used chemotherapeutic drugs in terms of gene identity, magnitude of change, and p53 dependence

Abstract Heterogeneity in the p53 response to traditional chemotherapy is recognized but its significance has yet to be exploited in the clinic. Further investigation could provide insight into the relative importance of individual p53 target genes and identify unique characteristics of specific drugs, guiding therapy selection. Wild-type or p53 null HCT-116 cells were treated with oxaliplatin, cisplatin, CPT-11 (irinotecan), or 5-Fluorouracil (5-FU) at an IC50 dose for 8 hours, in triplicate. Total cellular RNA was isolated and Clariom™ D microarrays were used to measure changes in RNA expression relative to untreated cells. TAC software was used to calculate fold-changes and statistical significance. Western blot was used to confirm treatment-induced increase in p53 and to further investigate p53 target heterogeneity at the protein level. The top 100 p53-dependent genes upregulated (FC>1.5) by each drug were identified, which revealed BTG2 as the top upregulated gene after oxaliplatin, cisplatin, and 5-FU treatment. Eight genes appeared in all four lists (GPR87, FAS, TP53INP1, DCP1B, POLH, PDGFA, MDM2, and PHLDA3) but variations in magnitude of change across drugs were noted for several including GPR87, FAS, TP53INP1, and MDM2. Moreover, heterogeneity in the magnitude of change of p53 targets was observed by western blot, which also showed differential upregulation of p21 and DR5 across drug treatment conditions. The lists contained numerous genes that were unique to each treatment, with oxaliplatin regulating the highest number of unique genes (31/100). Several transcripts outside of the top 100 were also identified as unique in each drug treatment including BAX, which showed slight preferential upregulation by 5-FU. Evaluation of the p53-independent transcriptional regulation revealed a strikingly significant and unique regulation of histone modifications by 5-FU compared to the other treatments. Beyond the specific genes mentioned, variation in the magnitude of change and identification of the unique targets after each drug treatment highlights potentially important mechanistic differences between the chemotherapeutic drugs. The appearance of eight transcripts in the top 100 upregulated genes for all four drug treatments indicates their importance as well as the contribution of the p53 response to the molecular effects of chemotherapy. We are currently further evaluating the relevance of these genes and their unique contributions to the action of the chemotherapeutic drugs. Citation Format: Lindsey Carlsen, Lanlan Zhou, Liz J. Hernández Borrero, Arunasalam Navaraj, Shengliang Zhang, Christoph Schorl, Wafik S. El-Deiry. The transcriptional response in colorectal cancer cells varies across four clinically used chemotherapeutic drugs in terms of gene identity, magnitude of change, and p53 dependence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 999.

Ring finger 20/ring finger 40/WW domain-containing adaptor with coiled-coil complex interacts with p53 to regulate gene transcription in DNA damage response.

p53 is one of the most important tumor suppressor genes, and its primary function is to act as a transcriptional activator to control cell cycle arrest, DNA repair and cellular metabolism by recognizing and binding to specific DNA sequences. Defects in the ring finger (RNF)20/RNF40/WW domain-containing adaptor with coiled-coil (WAC) complex, one of the histone H2B ubiquitination E3 ligases, have been reported to be a key factor in oncogenesis, cancer cell migration and invasion. Histone H2B mono-ubiquitination has been demonstrated to be essential for maintaining the functionality of the p53 tumor suppressor protein. The aim of the present study was to identify any sites in the p53 DNA-binding domain (DBD) specific to the RNF20/RNF40/WAC complex that may be involved in the gene regulation in DNA damage response. The results demonstrated that p53 and the RNF20/RNF40/WAC complex interacted with each other, and the coiled-coil regions in RNF20, RNF40 and WAC were identified to directly interact with p53. The R282 site in the p53 DBD, one of the frequent missense mutations associated with p53 mutation-dependent cancer, was demonstrated to be the key binding site for the RNF20/RNF40/WAC complex. Furthermore, knockout of RNF20/RNF40 suppressed the expression levels of p53 and its target genes in HCT116 cells compared with those in wild-type HCT116 cells. Consistent with these results, the R282W mutation in p53 inhibited the expression levels of p53 and its downstream genes by inactivating the interaction between p53 and RNF20/RNF40 compared with those in wild-type HCT116 cells. In conclusion, the results of the present study revealed the molecular mechanism of the interaction between the RNF20/RNF40/WAC complex and p53, and demonstrated that these proteins regulated gene transcription in the DNA damage response.

Artemisia annua L. Polyphenol-Induced Cell Death Is ROS-Independently Enhanced by Inhibition of JNK in HCT116 Colorectal Cancer Cells.

c-Jun N-terminal kinase (JNK) is activated by chemotherapeutic reagents including natural plant polyphenols, and cell fate is determined by activated phospho-JNK as survival or death depending on stimuli and cell types. The purpose of this study was to elucidate the role of JNK on the anticancer effects of the Korean plant Artemisia annua L. (pKAL) polyphenols in p53 wild-type HCT116 human colorectal cancer cells. Cell morphology, protein expression levels, apoptosis/necrosis, reactive oxygen species (ROS), acidic vesicles, and granularity/DNA content were analyzed by phase-contrast microscopy; Western blot; and flow cytometry of annexin V/propidium iodide (PI)-, dichlorofluorescein (DCF)-, acridine orange (AO)-, and side scatter pulse height (SSC-H)/DNA content (PI)-stained cells. The results showed that pKAL induced morphological changes and necrosis or late apoptosis, which were associated with loss of plasma membrane/Golgi integrity, increased acidic vesicles and intracellular granularity, and decreased DNA content through downregulation of protein kinase B (Akt)/β-catenin/cyclophilin A/Golgi matrix protein 130 (GM130) and upregulation of phosphorylation of H2AX at Ser-139 (γ-H2AX)/p53/p21/Bak cleavage/phospho-JNK/p62/microtubule-associated protein 1 light chain 3B (LC3B)-I. Moreover, JNK inhibition by SP600125 enhanced ROS-independently pKAL-induced cell death through downregulation of p62 and upregulation of p53/p21/Bak cleavage despite a reduced state of DNA damage marker γ-H2AX. These findings indicate that phospho-JNK activated by pKAL inhibits p53-dependent cell death signaling and enhances DNA damage signaling, but cell fate is determined by phospho-JNK as survival rather than death in p53 wild-type HCT116 cells.

Overexpression of RNF126 Promotes the Development of Colorectal Cancer via Enhancing p53 Ubiquitination and Degradation.

BackgroundRING finger protein 126 (RNF126), as a novel E3 ubiquitin ligase, plays an oncogenic role in several solid cancers. But its potential role in colorectal cancer (CRC) that harbored 50% mutant p53, to our knowledge, is rarely reported.Materials and MethodsWe investigated the clinical significance and relationship of RNF126 and p53 in CRC tissues and cells. Meanwhile, WB, qRT-PCR, co-IP, MTT, and transwell were used to investigate the function and molecular mechanism of RNF126 in regulating malignant biology in vitro.ResultsRNF126 was overexpressed in human CRC specimens, which was tightly associated with tumor size (P=0.021), T stage (P=0.030), lymph node metastasis (P=0.006), TNM stage (P=0.001), and the poor survival (P=0.003) of CRC patients. RNF126 had no association with p53 mutation in CRC specimens, and in p53 mutant Colo-205 and SW620 cells. However, in p53 wildtype HCT116 and HCT-8 cells, RNF126 silencing upregulated p53 and p21 but inhibited Rb phosphorylation at Serine 780 (pRb), which was inhibited by p53siRNA. Conversely, RNF126 overexpression downregulated p53 and p21 but promoted pRb expression, which was reversed by a classic proteasome inhibitor, MG132. However, the mRNA levels of above target genes were unchanged, implying a ubiquitination dependent post-translational modification involving in above regulation. Meanwhile, RNF126 was co-immunoprecipitated with p53 and p21 to form a triple complex. RNF126 silencing and overexpression inhibited and promoted p53 ubiquitination and degradation in vitro, respectively. In addition, p53siRNA reversed RNF126 silencing-inhibited cell proliferation, drug resistance, and cell mobility in HCT116 cells. Conversely, MG132 inhibited RNF126 overexpression-promoted above cell biology in HCT-8 cells.ConclusionOverexpression of RNF126 was remarkably associated with multiple advanced clinical characters of CRC patients independent of mutant p53. RNF126 promotes cell proliferation, mobility, and drug resistance in CRC via enhancing p53 ubiquitination and degradation.

Versicolorin A, a precursor in aflatoxins biosynthesis, is a food contaminant toxic for human intestinal cells

Aflatoxin B1 (AFB1) is the most potent carcinogen among mycotoxins. Its biosynthesis involves the formation of versicolorin A (VerA), whose chemical structure shares many features with AFB1. Our data revealed significant levels of VerA in foodstuff from Central Asia and Africa. Given this emerging food risk, it was of prime interest to compare the toxic effects of the two mycotoxins against cells originating from the intestinal tract. We used human colon cell lines (Caco-2, HCT116) to investigate the cytotoxic process induced by the two mycotoxins. Contrary to AFB1, a low dose of VerA (1 µM) disturbed the expression level of thousands of genes (18 002 genes). We show that the cytotoxic effects of low doses of VerA (1–20 µM) were stronger than the same low doses of AFB1 in both Caco-2 and HCT116 cell lines. In Caco-2 cells, VerA induced DNA strand breaks that led to apoptosis and reduced DNA replication of dividing cells, consequently inhibiting cell proliferation. Although VerA was able to induce the p53 signaling pathway in p53 wild-type HCT116 cells, its toxicity process did not mainly rely on p53 expression since similar cytotoxic effects were also observed in HCT116 cells that do not express p53. In conclusion, this study provides evidence of the risk of food contamination by VerA and shed light on its toxicological effect on human colon cells.

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

ObjectivesRING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair.Materials and methodsIn this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair.ResultsWe found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60.ConclusionsTaken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

Synthetic Lethal Interaction between Apto-253 and IDH1/2 Mutations

APTO-253 is a small molecule with a novel mechanism of action and potent antiproliferative activity against cell lines derived from a wide range of human malignancies. A Phase 1 study of APTO-253 was completed in patients with advanced solid tumors. It was well tolerated and produced evidence of antitumor activity in patients but did not cause myelosuppression even at the maximum tested dose. A phase 1a/b trial of APTO-253 in patients with relapsed/refractory acute leukemias (including AML) and high-risk MDS (NCT02267863) is currently underway. We previously reported that APTO-253 was converted intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. Both APTO-253 and Fe(253)3 stabilized DNA quadruplex (G4) structures in the Myc promotor region to reduce the expression of Myc (Figure 1). Stabilization of G4 structures has been reported to stall replication forks and produce DNA strand breaks. Treatment of cancer cells with APTO-253 produced time and concentration-dependent γH2AX foci formation and DNA double strand breaks that have to be repaired by homologous recombination. Loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 whose magnitude was similar to the effects of PARP inhibitors, olaparib. Thus, APTO-253 is a member of the limited repertoire of drugs which can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. The goal of this project was to identify synthetic lethal interactions in addition to BRCA1/2 deficiency that can guide combination drug studies. The normal function of the isocitrate dehydrogenase (IDH) enzymes is to catalyze the conversion of isocitrate to α-ketoglutarate (αKG) in the citric acid cycle. IDH1 mutations occur in more than 70% of low-grade gliomas, 20% of high-grade glioblastomas and with a frequency of about 10% in AML, cholangiocarcinomas, melanomas and chondrosarcomas. Additionally, mutations are also identified in IDH2 in about 4% of gliomas and 10% of AMLs. IDH1/2 alterations are heterozygous missense mutations that confer a neomorphic activity on the encoded enzyme such that they covert αKG to (R)-2-hydroxyglutarate [(R)-2HG]. (R)-2HG is an oncometabolite with pleiotropic effects on cell biology including chromatin methylation and cellular differentiation. It was reported that IDH1/2 mutations induce a homologous recombination defect that renders tumor cells exquisitely sensitive to PARP inhibitors. We used 2 pairs of isogenic IDH1 wild type and mutant cell lines to test the hypothesis that cells carrying IDH1/2 mutations are hypersensitive to APTO-253. Induction of the expression of IDH1 R132H in THP-1 AML cells increased the 2-HG concentration ~150-fold whereas induction of wild-type IDH1 expression increased it only 2-fold (doi: 10.1038/nm.3788). THP-1 cells expressing the mutant IDH1 R132H were hypersensitive the treatment of APTO-253 compared to cells expressing the wild-type IDH1 (Figure 2). Comparison of wild-type and isogenic IDH1 R132H HCT116 cells disclosed a ~100-fold higher level of (R)-2-HG in the mutant cells (doi: 10.1126/scitranslmed.aal2463), and an increase in sensitivity to the cytotoxic effect of APTO-253 (Figure 3). These results are consistent with the observation that both APTO-253 and increased levels of 2-HG cause DNA damage that depends on homologous recombination for repair and that this favors a synthetic lethal interaction. We surmise that the ability of APTO-253 to markedly reduce the expression of Myc further contributes to the favorable interaction. This data further supports the conclusion that defects in homologous DNA repair can enhance the potency of APTO-253 and suggest the use of this non-myelosuppressive drug in both IDH1/2 mutant AML and solid tumors. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Howell:Aptose Biosciences, Inc: Consultancy, Research Funding.

Application of RNA-sequencing to identify transcriptome modification by DCLK1 in colorectal cancer cells.

Doublecortin like kinase 1 (DCLK1) is a cancer stem cell marker for the colorectal cancer (CRC). It plays critical roles in the oncogenesis, progression and metastasis of CRC. DCLK1 can be an intriguing therapeutic target for CRC treatment. However, the molecular mechanism of how DCLK1 functions is unclear currently. In our research, we aim to apply RNA-Sequencing (RNA Seq) technology, a high throughput massively Next Generation Sequencing approach, to monitor transcriptome changes due to DCLK1 over-expression in the CRC cells. In order to achieve our goal, RNA from quadruplicate samples from two clones of isogenic DCLK1 stable over-expression cells and the parental wild type HCT116 cells was sent for RNA Seq on the Illumina NextSeq500 platform. Differentially expressed (DE) genes were evaluated by t-test (P <0.05 and fold-change ±1.5 or greater) using two methods: (1) FWER; and (2) Benjamani and Hochberg FDR (false discovery rate) which corrects for multiple comparisons. Gene networks and functional analysis were evaluated using Ingenuity Pathways Analysis (IPA). We identified 1463 DE genes common for both DCLK1 overexpression clone A and clone B cells. IPA results indicated that 72 canonical pathways were significantly modified by DCLK1 over-expression (P<0.05), among which 9 out of the top 10 pathways are involved in the cell cycle regulation, indicating that DCLK1 might play its tumorigenesis role via activation of pathways facilitating cell proliferation, repression of pathways inhibiting cells proliferation and function against pathways facilitating cell apoptosis. Cell cycle analysis results confirmed the IPA findings, which demonstrated that DCLK1 over-expression cells had much less G0/G1 cells but much more S and G2/M cells (P<0.05). In conclusion, DCLK1 over-expression significantly modified transcriptome profile of CRC cancer cells. Control of the cell cycle regulation might be one of the critical mechanism for DCLK1 function. Our findings provide more direct evidence for the development of DCLK1 as a therapeutic target for CRC treatment, and will be of great benefit for the discovery of novel therapeutic target within the DCLK1 molecular network for the treatment of colorectal cancer patients.

Proteomic Analysis of miR-195 and miR-497 Replacement Reveals Potential Candidates that Increase Sensitivity to Oxaliplatin in MSI/P53wt Colorectal Cancer Cells

Most patients with advanced colorectal cancer (CRC) eventually develop resistance to systemic combination therapy. miR-195-5p and miR-497-5p are downregulated in CRC tissues and associated with drug resistance. Sensitization to 5-FU, oxaliplatin, and irinotecan by transfection with miR-195-5p and miR-497-5p mimics was studied using cell viability and clonogenic assays in cell lines HCT116, RKO, DLD-1, and SW480. In addition, proteomic analysis of transfected cells was implemented to identify potential targets. Significantly altered proteins were subjected to STRING (protein-protein interaction networks) database analysis to study the potential mechanisms of drug resistance. Cell viability analysis of transfected cells revealed increased sensitivity to oxaliplatin in microsatellite instable (MSI)/P53 wild-type HCT116 and RKO cells. HCT116 transfected cells formed significantly fewer colonies when treated with oxaliplatin. In sensitized cells, proteomic analysis showed 158 and 202 proteins with significantly altered expression after transfection with miR-195-5p and miR-497-5p mimics respectively, of which CHUK and LUZP1 proved to be coinciding downregulated proteins. Resistance mechanisms of these proteins may be associated with nuclear factor kappa-B signaling and G1 cell-cycle arrest. In conclusion, miR-195-5p and miR-497-5p replacement enhanced sensitivity to oxaliplatin in treatment naïve MSI/P53 wild-type CRC cells. Proteomic analysis revealed potential miRNA targets associated with the cell-cycle which possibly bare a relation with chemotherapy sensitivity.

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1−/− cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Trichostatin A induces p53-dependent endoplasmic reticulum stress in human colon cancer cells.

Trichostatin A (TSA) has been demonstrated to exhibit various anticancer effects that influence cell cycle arrest, cell proliferation and apoptosis of cancer cells. A potential association between TSA and endoplasmic reticulum (ER) function has been suggested but its anticancer mechanism involving the induction of ER stress is unknown. p53 has previously been demonstrated to regulate ER function in response to stress but its role involving TSA and ER stress in cancer cells is poorly understood. The current study identified that TSA induced ER stress in wild type (WT) HCT116 human colon cancer cells. Following TSA treatment, the ER stress markers GRP78 and GRP94 significantly increased without hyperacetylation of their promoter regions. The inositol-requiring enzyme 1 α (IRE1α)/X-box binding protein 1 (XBP1) pathway was implicated due to an association of phosphorylated IRE1α and spliced XBP1 with ER stress. However, luciferase reporter assay indicated that splicing events were attenuated in HCT116 TP53(-/-) cells. Furthermore, cell viability and apoptosis were revealed to depend on p53 during TSA treatment. Cell viability increased and the apoptosis rate decreased in HCT116 TP53(-/-) cells compared with WT HCT116 cells undergoing TSA treatment. In conclusion, the current study revealed that TSA may induce ER stress via a p53-dependent mechanism in colon cancer cells. This provides information that may assist the development of treatments that exploit the anticancer function of TSA.

Abstract 2463: Long noncoding RNAs in tumor responses toward lovastatin

Abstract Background: Lovastatin is one of FDA-approved drugs used to treat hypercholesterolemia to reduce cardiovascular diseases. It is a compound of the statin family derived from type of species of Aspergillus terruses. Lovastatin targets hepatocytes and blocks the cholesterol synthesis pathway by inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl glutaryl CoA reductase (HMG-CoA reductase). This leads to decreased production of cholesterol and many other biochemical end products, including ubiquinone and dolichol. These biochemical end products are important for cancer progression. When lovastatin blocks their production, it leads to increased apoptosis of cancer cells. Therefore, lovastatin has promising anticancer activities but the detailed mechanisms remain poorly understood. Long noncoding RNAs are originally thought as nonfunctional. Emerging evidence suggest that lncRNAs have crucial roles in many biologic functions like apoptosis and cell cycle, translation control, epigenetic regulation and splicing regulation. In this study, we set out to identify and determine Lnc-RNAs that can regulate tumor cell responses towards lovastatin treatment. Methods: PC3, Heya8, HCT116 cells were obtained from ATCC. HCT116 p53-/- cells were gifts from Dr. Yin-Yuan Mo. The cytotoxic effects of lovastatin on those cell lines were evaluated by several approaches like MTS assays and morphologic changes. The qPCR array was used to identify lncRNAs whose levels are changed by lovastatin treatment. The regulation of lncRNA by lovastatin was further confirmed in multiple cell lines. Results and Conclusion: Lovastatin treatment had cytotoxic effects in multiple cell lines we evaluated. Through profiling array, we have identified NEAT1, MALAT1, and RMST as a responder to lovastatin. The lncRNAs identified had a significant increase in the expression compared to the nontreated cells in a dose- and time-dependent manner. The RMST mRNA expression in the HCT116 p53-/- cell line was about 25-fold more than its level in the HCT116 wild-type cells after treatment of 20 μM lovastatin for 48 hours, suggesting a role of tumor suppressor p53 gene in regulating the RMST expression. We knocked out RMST using CRISPR-Cas9 approach and found that RMST is an important mediator for lovastatin to downregulate mTOR activities and reduce colony formation. Taken together, our data suggest that lncRNAs can be stimulated by lovastatin to elicit cellular responses. Citation Format: Entkhab M. Alanisi, Jiuhui Wang, Yande Guo, Daotai Nie. Long noncoding RNAs in tumor responses toward lovastatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2463.