Colorectal Cancer Cell Lines HCT116 Research Articles

CRISPR/Cas9-mediated inactivation of miR-34a and miR-34b/c in HCT116 colorectal cancer cells: comprehensive characterization after exposure to 5-FU reveals EMT and autophagy as key processes regulated by miR-34.

The miR-34a and miR-34b/c encoding genes represent direct targets of the p53 transcription factor, and presumably mediate part of the tumor suppressive effects of p53. Here, we sought to determine their functional relevance by inactivating miR-34a and/or miR-34b/c using a CRISPR/Cas9 approach in the colorectal cancer (CRC) cell line HCT116. Concomitant deletion of miR-34a and miR-34b/c resulted in significantly reduced suppression of proliferation after p53 activation, enhanced migration, invasion and EMT, as well as reduced sensitivity to chemotherapeutics, increased stress-induced autophagic flux, decreased apoptosis and upregulation of autophagy-related genes after 5-FU treatment. However, inactivation of singular miR-34a or miR-34b/c had little effects on the aforementioned processes. RNA-Seq analysis revealed that concomitant deletion of miR-34a/b/c caused EMT signature enrichment, impaired gene repression by the p53-DREAM pathway and elevated autophagy after 5-FU treatment. A gene signature comprised of mRNAs significantly upregulated after combined inactivation of miR-34a and miR-34b/c showed a significant association with the invasive colon cancer subtype CMS4 and poor overall survival in two CRC patient cohorts, and with 5-FU resistance in CRC cell lines. In miR-34a/b/c-deficient cells the upregulated miR-34 target FOXM1 directly induced p62 and ATG9A, which increased autophagy and consequently attenuated apoptosis and rendered the miR-34a/b/c-KO cells more resistant to 5-FU. Inhibition of autophagy by depletion of ATG9A or chloroquine re-sensitized miR-34a/b/c-deficient HCT116 cells to 5-FU. In summary, our findings show a complementary role of miR-34a and miR-34b/c in the regulation of EMT and autophagy which may be relevant for CRC therapy in the future.

Effect of 3, 4-Dihydroxyphenylethanol on Antioxidant Enzymes Activity and Malondialdehyde Level in HCT-116 Cell Line

Background: There is much evidence-based research on the anti-cancerous effect of antioxidants. Dihydroxyphenylethanol is a potent antioxidant that has several activities, such as oxidant stress control, cell proliferation inhibitory activity, and apoptosis induction. The present study aimed to evaluate the effect of dihydroxyphenylethanol on antioxidant enzyme activity and malondialdehyde level in the HCT-116 cell line. Materials and Methods: In this study, a human colorectal cancer cell line HCT-116 was treated with different concentrations of 3, 4-dihydroxyphenylethanol (50, 100, 150, and 200 μM) for 24 h. Then, the level of malondialdehyde and activity of enzymes of catalase, superoxide dismutase, and glutathione peroxidase were measured by colorimetric methods. Results: The results showed that 3,4-dihydroxyphenylethanol administration resulted in significant reduction of the malondialdehyde concentration and also significant increase of the antioxidant enzymes (e.g., catalase, superoxide dismutase, and glutathione peroxidase) in the treatment groups compared to the control group (P<0.05). Conclusion: Overall, 3, 4-dihydroxyphenylethanol may result in reduction of oxidative stress on HCT-116 line through changes in concentration of the malondialdehyde and antioxidant enzymes activity.

Osthole inhibits malignant phenotypes and induces ferroptosis in KRAS-mutant colorectal cancer cells via suppressing AMPK/Akt signaling.

Ferroptosis is a form of cell death driven by iron-dependent lipid peroxidation. Intriguingly, KRAS-mutant cancers are particularly vulnerable to ferroptosis. Osthole is a natural coumarin extracted from Cnidium spp. and other Apiaceous plants. In the present study, we explored the antitumor potential of osthole in KRAS-mutant colorectal cancer (CRC) cells. Cell viability assay, EdU incorporation assay, flow cytometry, tumor xenograft model, western blot, immunochemistry staining, immunofluorescence, transcriptome RNA sequencing and quantitative reverse transcription-PCR were performed to evaluate the influence of osthole treatment on KRAS-mutant CRC cells. We found that osthole treatment suppressed proliferation and tumor growth of KRAS-mutant CRC cell lines HCT116 and SW480. Moreover, osthole treatment increased ROS production and induced ferroptosis. Osthole treatment also promoted autophagy, but inhibition of autophagy by ATG7 knockdown or 3-MA showed no influence on osthole-induced ferroptosis. In comparison, osthole increased lysosomal activation, and co-treatment with lysosome inhibitor Baf-A1 attenuated osthole-induced ferroptosis. Besides, osthole treatment reduced the phosphorylation of AMPK, Akt and mTOR in HCT116 and SW480 cells, while restored AMPK signaling by AMPK agonist AICAR partially abrogated ferroptosis induced by osthole treatment. Finally, co-treatment with osthole increased the cytotoxicity of cetuximab in KRAS-mutant CRC cells in vitro and in vivo. Our results suggested that the natural product osthole exerted its anticancer effects in KRAS-mutant CRC cells via inducing ferroptosis, and this was partially through inhibiting AMPK/Akt/mTOR signaling. Our results may expand our current knowledge for the use of osthole as an anticancer agent.

Chemo-radiotherapy with 177Lu-PLGA(RGF)-CXCR4L for the targeted treatment of colorectal cancer.

More than 1.9 million new cases of colorectal cancer and 935,000 deaths were estimated to have occurred worldwide in 2020. Therapies for metastatic colorectal cancer include cytotoxic chemotherapy and targeted therapies in multiple lines of treatment. Nevertheless, the optimal use of these agents has not yet been resolved. Regorafenib (RGF) is an Food and Drug Administration (FDA)-authorized multikinase inhibitor indicated for patients with metastatic colorectal cancer, non-responding to priority lines of chemotherapy and immunotherapy. Nanoparticles have been used in specific applications, such as site-specific drug delivery systems, cancer therapy, and clinical bioanalytical diagnostics. C-X-C Chemokine receptor type 4 (CXCR4) is the most widely-expressed chemokine receptor in more than 23 human cancer types, including colorectal cancer. This research aimed to synthesize and preclinically evaluate a targeted nanosystem for colorectal cancer chemo-radiotherapy using RGF encapsulated in Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles coated with a CXCR4 ligand (CXCR4L) and 177Lu as a therapeutic β-emitter. Empty PLGA and PLGA(RGF) nanoparticles were prepared using the microfluidic method, followed by the DOTA and CXCR4L functionalization and nanoparticle radiolabeling with 177Lu. The final nanosystem gave a particle size of 280 nm with a polydispersity index of 0.347. In vitro and in vivo toxicity effects were assessed using the HCT116 colorectal cancer cell line. 177Lu-PLGA(RGF)-CXCR4L nanoparticles decreased cell viability and proliferation by inhibiting Erk and Akt phosphorylation and promoting apoptosis. Moreover, in vivo administration of 177Lu-PLGA(RGF)-CXCR4L significantly reduced tumor growth in an HCT116 colorectal cancer xenograft model. The biokinetic profile showed hepatic and renal elimination. Data obtained in this research justify additional preclinical safety trials and the clinical evaluation of 177Lu-PLGA(RGF)-CXCR4L as a potential combined treatment of colorectal cancer.

Metformin Induces a Caspase 3-Unrelated Apoptosis in Human Colorectal Cancer Cell Lines HCT116 and SW620.

To study the effects of metformin on the proliferation and growth of human colorectal cancer cell lines HCT116 and SW620. The antiproliferative effect of metformin was assayed using an MTS reagent and its ability to inhibit colony formation was demonstrated using a clonogenic assay. Flow cytometry using YO-PRO-1/PI was performed to examine the effects of metformin on apoptosis and cell death of HCT116 and SW620. Caspase 3 activities were measured in caspase-3 activity tests using a caspase-3 activity kit. Furthermore, Western blots were performed with anti-PARP1, anti-caspase 3, and anti-cleaved caspase 3 to confirm whether caspase activation was present or not. Both MTS proliferation assays and clonogenic assays showed that metformin inhibited the proliferation and growth of HCT116 and SW620 cells in a concentration-dependent manner. Flow cytometric analysis identified early apoptosis and metformin-induced cell death in both cell lines. However, caspase 3 activity could not be detected. Cleavage of both PARP1 and pro-caspase 3 was not observed in the Western blot, confirming the absence of caspase 3 activations. This present study suggests a caspase 3-unrelated apoptosis mechanism of metformin-induced cell death in human colorectal cancer cell lines HCT116 and SW620.

Oxaliplatin: an effective treatment in colorectal cancer

Colorectal cancer is one of the most common malignancies around the world. Oxaliplatin is a new platinum compoundwith promising activity in colorectal cancer treatment. Based on the analysis of preclinical data. Because Oxaliplatincan lead to ICD (Immunogenic cell death), the study aims to figure out whether increasing concentrations and treatmentdurations with Oxaliplatin can kill colorectal cancer cells to test whether Oxaliplatin has a significant effect on colorectalcancer treatment. Using the human colorectal cancer cell line HCT116, the cancer cells will be treated with Oxaliplatinand PBS (negative control) at concentrations 1 mM, 10 mM, or 100 mM in a dose-dependent and duration-dependentmanner (1,3,6,12,24 hours ). Measure killing in vitro with MTT assay and measure tumor size in vivo. Also, measureHMGB-1 released by western blot and CRT by FACS. The positive control is cisplatin, and the negative is PBS/DMSOfor in vitro and saline solution for in vivo. The study’s findings will supply insight into the latent capacity of Oxaliplatinas a treatment for colorectal cancer in the future.

CREATION OF ISOGENIC CELL LINE MODELS OF 17P DELETION TO STUDY CLONAL EVOLUTION AND MECHANISMS OF THERAPY RESISTANCE IN CLL AND DLBCL

Introduction: A deletion of chromosome 17p (del17p), on which the tumour suppressor gene TP53 is located, is a prevalent genetic aberration in many cancer types. In chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), and other B cell malignancies this chromosomal mutation is associated with high fitness advantage in the setting of therapy, and the most adverse clinical outcome. Whilst resistance to the chemoimmunotherapies often used in the treatment of CLL and DLBCL can be explained by the functional loss of p53, such deactivation does not explain the increase in aggressive behaviour of the malignant cells. To study this, we aimed to develop in-vitro models of 17p deletion in CLL and DLBCL by creating TP53 (15.6 kbp) and 17p (20.5 Mbp) deletions by means of CRISPR-Cas9 genome editing. Methods: The efficiency of the gene editing machineries were firstly assessed in the adherent human colorectal cancer cell line HCT116 prior to their application on the B-cell lines, which are less amenable to genetic manipulation. TP53 gene removal (15.6 kbp) was performed by transient transfection of a vector encoding Cas9 as well as single guide (sg) RNAs targeting upstream and downstream of the TP53 coding region. For deletion of 17p (20.5 Mbp) sgRNAs targeting exon 2 of GEMIN4 (telomeric) and intron 5 of MAP2K3 (centromeric) were designed. PCR was used to confirm deletions. Results: Isogenic HCT116 cell lines with hemizygous and homozygous TP53 deletion, as well as hemizygous 17p deletion were obtained (Figure 1). HG-3 cells, a CLL cell line, were successfully transduced with inducible-Cas9, and subsequently with the sgRNAs for removal of TP53 and 17p. Keyword: Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Bradykinin and Neurotensin Analogues as Potential Compounds in Colon Cancer Therapy.

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential.

Proteomic Signature of Extracellular Vesicles Associated with Colorectal Cancer.

The proteins of extracellular vesicles (EVs) provide proteomic signatures that reflect molecular features of EV-producing cells, including cancer cells. Detection of cancer cell EV proteins is of great interest due to the development of novel predictive diagnostic approaches. Using targeted mass spectrometry with stable-isotope-labeled peptide standards (SIS), we measured in this study the levels of 34 EV-associated proteins in vesicles and whole lysate derived from the colorectal cancer (CRC) cell lines Caco-2, HT29 and HCT116. We also evaluated the abundance of 13 EV-associated proteins (FN1, TLN1, ITGB3, HSPA8, TUBA4A, CD9, CD63, HSPG2, ITGB1, GNAI2, TSG101, PACSIN2, and CDC42) in EVs isolated from blood plasma samples from 11 CRC patients and 20 healthy volunteers. Downregulation of TLN1, ITGB3, and TUBA4A with simultaneous upregulation of HSPG2 protein were observed in cancer samples compared to healthy controls. The proteomic cargo of the EVs associated with CRC represents a promising source of potential prognostic markers.

Potential effect of chloroquine and propranolol combination to treat colorectal and triple-negative breast cancers

Drug repositioning explores the reuse of non-cancer drugs to treat tumors. In this work, we evaluated the effect of the combination of chloroquine and propranolol on colorectal and triple-negative breast cancers. Using as in vitro models the colorectal cancer cell lines HCT116, HT29, and CT26, and as triple-negative breast cancer models the 4T1, M-406, and MDA-MB-231 cell lines, we evaluated the effect of the drugs combination on the viability, apoptosis, clonogenicity, and cellular migratory capacity. To explore the in vivo effects of the combination on tumor growth and metastasis development we employed graft models in BALB/c, nude, and CBi mice. In vitro studies showed that combined treatment decreased cell viability in a dose-dependent manner and increased apoptosis. Also, we demonstrated that these drugs act synergically and that it affects clonogenicity and migration. In vivo studies indicated that this drug combination was effective on colorectal models but only partially on breast cancer. These results contributed to the search for new and safe treatments for colorectal and triple-negative carcinomas.

Super Carbonate Apatite-miR-497a-5p Complex Is a Promising Therapeutic Option against Inflammatory Bowel Disease.

The incidence of inflammatory bowel disease (IBD) is increasing worldwide. It is reported that TGF-β/Smad signal pathway is inactivated in patients with Crohn's disease by overexpression of Smad 7. With expectation of multiple molecular targeting by microRNAs (miRNAs), we currently attempted to identify certain miRNAs that activate TGF-β/Smad signal pathway and aimed to prove in vivo therapeutic efficacy in mouse model. Through Smad binding element (SBE) reporter assays, we focused on miR-497a-5p. This miRNA is common between mouse and human species and enhanced the activity of TGF-β/Smad signal pathway, decreased Smad 7 and/or increased phosphorylated Smad 3 expression in non-tumor cell line HEK293, colorectal cancer cell line HCT116 and mouse macrophage J774a.1 cells. MiR-497a-5p also suppressed the production of inflammatory cytokines TNF-α, IL-12p40, a subunit of IL-23, and IL-6 when J774a.1 cells were stimulated by lipopolysaccharides (LPS). In a long-term therapeutic model for mouse dextran sodium sulfate (DSS)-induced colitis, systemic delivery of miR-497a-5p load on super carbonate apatite (sCA) nanoparticle as a vehicle restored epithelial structure of the colonic mucosa and suppressed bowel inflammation compared with negative control miRNA treatment. Our data suggest that sCA-miR-497a-5p may potentially have a therapeutic ability against IBD although further investigation is essential.

Prognostic value of SLC4A4 and its correlation with the microsatellite instability in colorectal cancer.

To explore new biomarkers related to microsatellite instability in order to better predict prognosis and guide medication. The "limma" R package was used to identify differentially expressed genes in GSE24514, and then weighted correlation network analysis was used to select key genes. Different cell types in the tumor microenvironment were identified and analyzed by single-cell sequencing, with a Lasso regression model used to screen prognostic variables. Furthermore, the correlation between microsatellite instability and potential prognostic variables was explored, as well as the expression characteristics and clinical characteristics of the prognostic variables in the TCGA, UALCAN, and HPA databases. PCR assay was used to investigate the expression of SLC4A4 in colorectal cancer cell lines. Finally, we further verified the expression of SLC4A4 by immunohistochemistry. First, 844 differentially expressed genes in GSE24514 were identified. Subsequently, weighted co-expression network analysis (WGCNA) of GSE24514 obtained all the genes significantly associated with microsatellite instability (MSI), a total of 1452. Analysis of GSE166555 single cell sequencing data set yielded 1564 differentially expressed genes. The gene sets obtained from the above three analysis processes were intersected, and 174 genes were finally obtained. The Lasso regression model revealed two potential prognostic genes, TIMP1 and SLC4A4, of which, there was a stronger correlation between microsatellite instability and SLC4A4. The mRNA and protein expression of SLC4A4 was significantly decreased in tumors, and patients with low SLC4A4 expression had a poor prognosis. In addition, SLC4A4 was specifically expressed in epithelial cells. In the microenvironment of colorectal cancer, malignant cells have a strong interaction with different stromal cells. PCR showed that SLC4A4 was significantly down-regulated in colorectal cancer cell lines Caco-2, HCT116 and HT29 compared with normal control NCM460 cell lines. Immunohistochemistry also showed low expression of SLC4A4 in colorectal cancer. SLC4A4, as a tumor suppressor gene, is significantly downregulated and positively correlated with microsatellite instability, thus it may be combined with microsatellite instability to guide colorectal cancer treatment.

Determination of the Antioxidant and Antiproliferative Properties of Pomegranate Peel Extract Obtained by Ultrasound on HCT-116 Colorectal Cancer Cell Line

Colorectal cancer (CRC) is one of the most common cancers in the world. When treating patients, therapeutic agents have side effects; hence, the use of natural compounds found in medicinal plants including pomegranate. Ultrasound assisted extraction (UAE) is a new technique evolving to the detriment of traditional methods such as maceration. In this study, we investigated the antioxidant and anticancer effect of pomegranate peel extracts obtained by maceration and UAE at three different ultrasonic power levels (P1 = 10 W; P2 = 50 W; P3 = 100 W) on HCT-116 colorectal cancer cells. Phytochemical screening highlighted the presence of primary and secondary metabolites in pomegranate peels. In addition, the ethanolic extract obtained by UAE at 50 W was shown to be the most concentrated in phenolic and flavonoid compounds and have the most powerful antioxidant activity, which reached a maximum activity of 92% as determined by DPPH test. Similarly, the MTT cell viability test showed that the extract obtained by UAE at 50 W had the most potent inhibitory effect compared to the other extracts. In conclusion, the UAE at 50 W was shown to be the most suitable and efficient extraction technique to obtain bioactive compounds from pomegranate peel extracts that can be used in the treatment of CRC.

Abstract 824: Statistical optimization of stable Quercetin nanoparticles for tumor targeting

Abstract Quercetin is a well-known flavonoid with a significant in vitro anticancer effect and low toxicity profile. Despite of that, its clinical use is still limited because of its low solubility, bioavailability, and instability in physiological conditions. The aim of this study was to develop and optimize stable quercetin nanoparticles targeting tumor cells within the body. Quercetin nanoparticles were synthesized using solvent emulsification technique using zein, beta-cyclodextrin, and pectin at different concentrations. The prepared formulations were optimized using factorial response surface analysis. The stability of the optimized quercetin nanoparticles in conditioned media, obtained from subculture of colorectal cancer cell line HCT-116, was compared to the stability of the free drug powder over 24 h. Moreover, the cytotoxic effect of Quercetin powder and the optimized Quercetin nanoparticles were evaluated against two colorectal cancer lines (HT-29, and HCT116), using MTT assay, under normoxic conditions. Annexin-V/FITC apoptotic assay was used to detect the number of cells that underwent apoptosis using flow cytometry. The optimized formulation was composed of 10 mg of zein and 31.35 mg beta-cyclodextrin, with no pectin. It had particle size of 221 nm, polydispersity index of 0.115, zetapotential of 7.65 mV. The stability of the optimized quercetin nanoparticles in the conditioned media of HCT-116 was significantly higher than the free drug powder over 24 h. In HT-29, the optimized nanoparticles and the free drug had IC50 of 29.33 µM and 60.78 µM, respectively. In HCT-116, optimized nanoparticle and the free drug had IC50’s 31.11 µM and 59.20 µM, respectively. The flow cytometric analysis after annexin-V/FITC staining showed that treatment with the optimized quercetin nanoparticles significantly induced total apoptosis in the two colorectal cancer lines (HT-29 and HCT 116) when compared to the free drug and plain nanoparticles. In conclusion, the optimized quercetin nanoparticles were found to be capable of increasing drug stability in physiological fluids as well as increasing its cytotoxic effect in two colorectal cancer cell lines by apoptosis induction when compared to the drug in powder form as well as plain nanoparticles. Citation Format: Nourhan Khaled Hassan, Eman El-labbad, Jayalakshmi jagal, Mohamed Haider, Ibrahim El-sayed. Statistical optimization of stable Quercetin nanoparticles for tumor targeting [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 824.

Abstract 3101: Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells

Abstract Introduction: Autophagy pathway activation and upregulation is an important mechanism for cancer cell survival upon exposure to chemotherapeutic agents. LC3, a ubiquitin-like protein, plays a key role in the regulation of cargo selection and autophagosome formation. Given small molecule inhibitors have not been well developed to target LC3, we aim to design and synthesize an LC3 binder and evaluate its activity in the inhibition of autophagy pathway in cancer cells. Methods: The crystal structure of LC3 and p62 complex (PDB: 2ZJD) was used to assist our design of helical sulfonyl-γ-AApeptides to the LC3 interacting region of p62. The peptidomimetics were synthesized on the solid phase and purified by HPLC. We used fluorescent polarization assay to assess the binding affinity of the peptidomimetics to LC3. We then utilized an LC3 HiBiT reporter cell line (U2OS) from Promega, which was engineered to have a fused human LC3-HiBiT with a HaloTag in between to study the effect of the lead LC3 binder on autophagic flux. In addition, confocal microscopy enabled direction visualization of the colocalization of FITC-labeled LC3 binder with LC3 and lysosome, respectively. Cytotoxicity of LC3 binder was evaluated in colorectal cancer cell line HCT116. Results: We designed and synthesized 6 helical sulfonyl-γ-AApeptides (JC1-6) to target the binding interface of LC3 and p62. These peptidomimetic compounds were designed to retain essential LC3 and p62 interactions at the W and L regions and yet have different functional groups on the helical scaffold. Compounds JC1 and JC6 have binding affinity of Kd 280 nM and 150 nM with LC3, respectively. FITC-labelled JC6 was able to penetrate U2OS and HCT116 cells. It had intracytoplasmic colocalization with not only lysosomes but also LC3 aggregates. Direct visualization of intracytoplasmic LC3 aggregates showed that JC6 at 50 µM significantly inhibited the autophagic degradation of LC3 aggregates. This effect was augmented by the presence of 2 µM autophagy activator PP242. Lastly, JC6 demonstrated cytotoxicity with IC50 approximately 50 µM in HCT116 cells bearing baseline autophagy activation compared to lack of cytotoxicity in U2OS cells with no baseline autophagy activity. Conclusion: We designed and synthesized novel helical sulfonyl-γ-AApeptide LC3 binders. With the ability of cellular penetration, they demonstrated inhibition of autophagic flux and preliminary cytotoxicity in cancer cells. Future studies may focus on structural activity relationship and further evaluation of its mechanisms of action in cancer cells. Citation Format: Mentalla Mahmoud, Jianyu Chen, Jianfeng Cai, Hao Xie. Discovery of helical sulfonyl-γ-AApeptides targeting LC3 in cancer cells [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 3101.

Abstract 1385: Oncogenic regulation of autophagy and DNA emission

Abstract Background: Cancer cells release higher levels of cell-free DNA (cfDNA) into the circulatory system than normal cells. However, the molecular mechanisms underlying the process of programmed DNA elimination remain poorly understood. Emerging evidence has indicated that oncogenes such as HRAS can be released through extracellular vesicles (EVs). Moreover, studies have shown an active mechanism of DNA emission through autophagy. Autophagy is an evolutionarily conserved process that degrades and recycles cytoplasmic materials such as organelles and proteins in the lysosomes. Interestingly, Microtubule-associated protein 1 light chain-3 (LC3B) is known to degrade nuclear lamina components in HRAS-induced cells, potentially producing a pool of cytoplasmic chromatin. Cytoplasmic chromatin is ultimately degraded in lysosomes or re-routed outside of the cells. The aim of our study was to evaluate the role of autophagy proteins in circulating tumor (ctDNA) and EV-associated DNA emission in cancer cell lines harboring oncogenes (RAS and BRAF) as this aspect of the process is relatively understudied. Methods: We have assessed protein and RNA expression of autophagy genes (LC3B, ATG5 and ATG7) in IEC-18 cells that have been transduced with the HRAS oncogene (RAS3 cells) and human colorectal cancer cell lines SW620 and HCT116 using RT-qPCR and western blot (WB). All conditions were compared to their respective control cells (IEC-18, CCD-841 CoN and CCD18-Co cells. To determine the role of LC3 in nuclear membrane degradation, we employed transmission electron microscopy (TEM). We have further validated the role of autophagy genes in EV-DNA emission by shRNA knockdown using shRNA against highly expressed autophagy genes, such as LC3B in RAS3 cells. EV-DNA was measured using droplet digital PCR (ddPCR). Results: To determine whether autophagy expels oncogenic DNA via EVs in KRAS-mutant CRC cells, we focused on the role of ATG5, LC3B and ATG7, as this aspect of the process is understudied. Our data shows higher LC3B and ATG7 expression in transformed RAS3 cells, compared to control cell lines (IEC18). Moreover, ATG5 is overexpressed in SW620 and HT29 colorectal cancer cells compared to control cell lines. LC3 is known to degrade the nuclear membrane in HRAS-activated cells. Indeed, we observed degradation of the nuclear membrane in RAS3 cells leading to the accumulation of cytosolic DNA shown by TEM. Furthermore, overexpression of LC3B in IEC18 cells using GFP-tagged LC3B resulted in increased EV emission. These findings suggest that the autophagy axis may contribute to oncogenic DNA emission via EVs. Lastly, shRNA knockdown of LC3 resulted in reduction of EV-DNA emission (ddPCR). Impact: EV biogenesis and autophagy pathways play pivotal and interconnected roles during DNA processing and extracellular emission. Therefore, understanding the detailed mechanisms is critical for biomarker discovery and potential therapeutic strategies targeting this process. Citation Format: Thupten Tsering, Kyle Dickinson, Laura Montermini, Janusz Rak, Julia Burnier. Oncogenic regulation of autophagy and DNA emission [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 1385.

Abstract 893: Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer

Abstract Metastatic (m) colorectal cancer (CRC) is an incurable, frequently lethal disease present in approximately 25% of newly diagnosed CRC patients, and almost 50% of the patients with CRC will develop metastatic disease. For mCRC, while systemic therapies (cytotoxic therapy, targeted therapy, immunotherapy or their combinations) have extended patient’s life expectancy, not all patients are candidates for these treatments. Immunotherapy has achieved significant curative effects in patients with solid tumors; however, a considerable proportion of mCRC patients (~90%) are not responsive to immune checkpoint blockade (ICB) leaving a gap in the CRC immunotherapy options. Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses against a wide variety of tumor cells; however, they are frequently dysfunctional in cancer patients. NK cells from CRC patients exhibit decreased expression of activating receptors and reduced cytokine production after stimulation with CRC cells with a more accentuated phenotype in advanced stages of the disease. Memory-like (ML) NK cells differentiated after IL-12, IL-15, and IL-18 activation have been shown to overcome limitations associated with deficient tumor recognition and poor anti-tumor activity. In clinical trials, ML NK cells were safe and active against acute myeloid leukemia (Romee R et al, Sci Transl Med, 2016). Preclinically, ML NK cells exhibited improved responses against melanoma (Marin ND et al, Clin Cancer Res, 2021) and ovarian cancers, compared to conventional NK cells. Here, we hypothesized that memory-like differentiation will enhance multiple aspects of the NK cell response against CRC cells. Allogeneic ML NK cells displayed enhanced IFN-γ production against four CRC cell lines DLD-1 (p=0.015), SW480 (p=0.0005), HT-29 (p=0.0005) and HCT116 (p=0.001), as well as primary patient derived CRC tumoroids (p=0.0156), compared to conventional (c) NK cells. ML NK cells also exhibited superior and sustained killing of CRC cell lines over time compared to cNK cells, as measured by Incucyte assays and using CRC tumoroids. Furthermore, IFN-γ production was significantly reduced after blockade of NKG2D, DNAM-1 and NKp46 (p<0.01) revealing mechanistic insights into how ML NK cells recognize CRC targets. Finally, using a xenograft model of CRC in NSG mice, we demonstrated that ML NK cells exhibited superior control of Luciferase expressing HCT116 cells compared to cNK cells (p=0.01) as measured by bioluminescent imaging (BLI). Collectively, these findings demonstrate that ML NK cells exhibit enhanced responses against CRC cells, and thus warrants further investigation in clinical trials for CRC patients, especially those who are not candidates for standard of care therapy or failed ICB. Citation Format: Nancy D. Marin, Michelle Becker-Hapak, Quazim A. Alayo, Melissa Berrien-Elliot, Lynne Marsala, Naomi Sonnek, Miriam T. Jacobs, Jennifer A. Foltz, Alice Zhou, Jennifer Tran, Pamela Wong, Celia Cubit, Kimberly Hwang, Timothy Schappe, Ryan C. Fields, Matthew A. Ciorba, Todd A. Fehniger. Memory like differentiation enhances in vitro and in vivo NK cell responses against 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 893.

The novel anti-cancer fluoropyrimidine NUC-3373 is a potent inhibitor of thymidylate synthase and an effective DNA-damaging agent

IntroductionFluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU.MethodsHuman colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage.ResultsWe demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU.ConclusionTaken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

H2O2 transit through the mitochondrial intermembrane space promotes tumor cell growth in vitro and in vivo

Cancer cells experience increased levels of oxidant stress as a consequence of oncogene activation, nucleotide biosynthesis, and growth factor receptor signaling. Mitochondria contribute to this redox stress by generating reactive oxygen species (ROS) along the electron transport chain, which are released to the matrix and the intermembrane space (IMS). Assessing the contribution of mitochondrial ROS in cancer cells is technically difficult, as electron transport chain inhibitors can increase or decrease ROS generation, while they also block oxidative phosphorylation and ATP synthesis. Mitochondria-targeted antioxidant compounds can scavenge ROS in the matrix compartment but do not act on ROS released to the IMS. We assessed the importance of mitochondrial ROS for tumor cell proliferation, survival, and for tumor xenograft growth by stably expressing a hydrogen peroxide (H2O2) scavenger, peroxiredoxin-5, in the mitochondrial IMS (IMS-Prdx5) in 143B osteosarcoma and HCT116 colorectal cancer cell lines. IMS-Prdx5 attenuates hypoxia-induced ROS signaling as assessed independently in cytosol and IMS, HIF-1α stabilization and activity, and cellular proliferation under normoxic and hypoxic culture conditions. It also suppressed tumor growth invivo. Stable expression of nondegradable HIF-1α only partially rescued proliferation in IMS-Prdx5-expressing cells, indicating that mitochondrial H2O2 signaling contributes to tumor cell proliferation and survival through HIF-dependent and HIF-independent mechanisms.

Synergistic effects of complex drug combinations in colorectal cancer cells predicted by logical modelling

Drug combinations have been proposed to combat drug resistance in cancer, but due to the large number of possible drug targets, in vitro testing of all possible combinations of drugs is challenging. Computational models of a disease hold great promise as tools for prediction of response to treatment, and here we constructed a logical model integrating signaling pathways frequently dysregulated in cancer, as well as pathways activated upon DNA damage, to study the effect of clinically relevant drug combinations. By fitting the model to a dataset of pairwise combinations of drugs targeting MEK, PI3K, and TAK1, as well as several clinically approved agents (palbociclib, olaparib, oxaliplatin, and 5FU), we were able to perform model simulations that allowed us to predict more complex drug combinations, encompassing sets of three and four drugs, with potentially stronger effects compared to pairwise drug combinations. All predicted third-order synergies, as well as a subset of non-synergies, were successfully confirmed by in vitro experiments in the colorectal cancer cell line HCT-116, highlighting the strength of using computational strategies to rationalize drug testing.