Rectal Cancer Cell Lines Research Articles

Targeting Tri-Methyl-Histone H3 (Lys27) in Rectal Cancer: A Novel Strategy to Enhance Radiosensitivity and Predict Response to Neoadjuvant Therapy

Tri-Methyl-Histone H3 (Lys27) is a modification of histone H3 involved in gene regulation and chromatin organization. It plays a crucial role in cell response to ionizing radiation and affects DNA damage repair, with altered levels being linked to decreased radiation sensitivity. Our hypothesis is that high levels of Tri-Methyl-Histone H3 (Lys27) in rectal tumors can predict response to neoadjuvant chemoradiation, and targeting SETD2, the histone methyltransferase responsible for adding the tri-methyl group to Lys27 of histone H3, may enhance radiosensitivity in rectal cancer cells. Biopsy samples from 25 patients with locally advanced rectal adenocarcinoma (Stage II-III) were obtained for analysis. Immunohistochemistry (IHC) was used to evaluate Tri-Methyl-Histone H3 (Lys27) expression levels in the tumor biopsies. The staining intensity was scored semi-quantitatively. Samples with 100% positive staining were grouped as "Tri-Methyl-High," and all others were grouped as "Tri-Methyl-Low." Fisher's exact test was used to analyze the data to determine the correlation between Tri-Methyl-Histone H3 (Lys27) levels and response to neoadjuvant chemoradiation. The primary endpoint was either a sustained clinical complete response (cCR) at one year or a pathological complete response (pCR). To further understand the role of Tri-Methyl-Histone H3 (Lys27) in response to ionizing radiation (IR), clonogenic assays, γH2aX foci staining, and western blot analysis of DNA damage response proteins and histones were performed on two established human rectal cancer cell lines following either RNA inhibition (RNAi) or drug targeting of SETD2. Twenty-five patients with rectal cancer had pre-treatment biopsies analyzed for Tri-Methyl-Histone H3 (Lys27) levels, 11 were classified as Tri-Methyl-High and 14 as Tri-Methyl-Low. The groups were well balanced in terms of age, sex, clinical T/N stage, and neoadjuvant treatment approach. The overall combined complete response (cCR/pCR) rate was 36%, while the rate for Tri-Methyl-High patients was 9% (1/11) compared to 57% (8/14) in Tri-Methyl-Low patients (p = 0.03). After treating two colorectal cancer cell lines (SW837 and HCT116) with IR (5 Gy) in vitro, Tri-Methyl-Histone H3 (Lys27) levels increased 3-fold. SETD2 RNAi reduced Tri-Methyl-Histone H3 (Lys27) and increased cell death when combined with IR. In vitro treatment with a novel SETD2 inhibitor, EZM0414, led to a 2-fold increase in DNA damage following IR (5 Gy) as measured by γH2aX foci staining. EZM0414 treatment showed a 37% improvement in the dose enhancement ratio. Our study uncovers a new biomarker, Tri-Methyl-Histone H3 (Lys27), that could be used to predict response to neoadjuvant chemoradiation in rectal cancer patients. Our preclinical data indicates that targeting SETD2 to reduce Tri-Methyl-Histone H3 (Lys27) mediated DNA repair could improve the efficacy of radiation therapy for rectal cancer patients.

The SPATA20 Gene Modifies Rectal Cancer Radiosensitivity

INTRODUCTION: Rectal cancer response to neoadjuvant chemoradiotherapy correlates with outcomes, and is highly variable between patients, ranging from a complete response to minimal or no response. We have previously identified an association between SPATA20 gene expression and response in patients. The goal of this study was to establish and test a rectal cancer cell model to study SPATA20. METHODS: HRT18 rectal cancer cell lines were transfected with a lentiviral construct containing small interfering RNAs (siRNA) targeting SPATA20 mRNA to create knockdown cell lines. Western blot was subsequently used for knockdown validation. Wildtype HRT18 and knockdown HRT18 cell lines were plated in 96 well plates and subsequently irradiated with 10 Gy. Cell viability was assessed at 24 hours interval using CellTiter-Glo Luminescent Cell Viability Assay for both irradiated and non-irradiated controls (Figure 1). RESULTS: Decreased expression of SPATA20 was confirmed in knockdown HRT18 cell lines compared with wildtype HRT18 using Western blot. SPATA20 knockdown by itself did not significantly affect cell viability (p = 0.18). Knockdown SPATA20 cells were significantly more sensitive to radiation when compared with wildtype cells, resulting in a 17% decrease in cell viability (p < 0.0001). CONCLUSION: We established a SPATA20 knockdown rectal cancer model to study the effects of radiation. Manipulation of SPATA20 correlates with radiation response, signifying it as a possible modulator of radiosensitivity in rectal cancer.

Inhibition of DNAJC12 Inhibited Tumorigenesis of Rectal Cancer via Downregulating HSPA4 Expression.

Background Dysregulation of DnaJ heat shock protein family (HSP40) member C12 (DNAJC12) is implicated in the malignancy progression of multiple cancers. The current study aimed to determine the biology function and mechanism of DNAJC12 in rectal cancer (RC). Methods RC tissues, adjacent tissues, RC cell lines, and normal colorectal epithelial cell lines were collected to analyze DNAJC12 expression. The abilities of DNAJC12 on proliferation, migration, and apoptosis of RC cells were detected by CCK-8, wound healing, and flow cytometry assays. Co-IP assays were carried out to confirm the association between DNAJC12 and HSPA4. The effect of DNAJC12 on tumor growth was detected by using the xenograft model of nude mice. Results Elevation of DNAJC12 was uncovered in RC tissues and cell lines. DNAJC12 upregulation facilitated RC cell proliferation and migration and induced apoptosis, while DNAJC12 interference showed the opposite results. Besides, HSAP4 served as a potential binding protein for DNAJC12. Rescue experiments revealed that elevated of HSAP4 restored the impact of DNAJC12 silencing on the cell functions. Finally, DNAJC12 silencing hampered tumor growth of RC in vivo. Conclusion In summary, this study highlighted a key player of DNAJC12 in modulating the malignant biological progression of RC via DNAJC12/HSPA4 axis, displaying a potential therapeutic target for RC.

Cytotoxic effect of paraprobiotic yogurts on cancer cell lines

Cancer is a cellular disorder caused by changes in the mechanisms that control cell growth and differentiation. Breast cancer is the second most common cancer in women following lung cancer. Today, due to the side effects of treatment methods, many efforts have been made to discover natural compounds with a selective power in limiting cancers. This study was performed to investigate the cytotoxicity effects of paraprobiotic yogurt on breast and rectal cancer cell lines (MDA-Md, SKBR3, and SW 480). After culturing and amplifying the cancer cell line to determine the effect of supernatant toxicity, these cells were exposed to different doses of paraprobiotic supernatant, which was performed after 72 hours of MTT assay. According to the results, after adding different dilutions of paraprobiotic yogurt supernatant, YB-B could significantly inhibit the cell viability in three categories of cancer cells (MDA-Md, SKBR3, and SW 480) and had the greatest impact on SKBR3 (p≤0.05). The effects of cytotoxicity increased with elevating the concentration of the extract with the highest percentage of growth inhibition being related to the concentrations of 1/4, 1/3, and 1/2 (p = 0.05 and p = 0.01), respectively. Thus, the use of paraprobiotic yogurt supernatant as an effective substance in the treatment of cancer is recommended. Accordingly, future research could explore its substances to be potentially used in the treatment of cancer.

Inhibition of DNA-PK may improve response to neoadjuvant chemoradiotherapy in rectal cancer.

Treatment of locally advanced rectal cancer includes chemoradiation and surgery, but patient response to treatment is variable. Patients who have a complete response have improved outcomes; therefore, there is a critical need to identify mechanisms of resistance to circumvent them. DNA-PK is involved in the repair of DNA double-strand breaks caused by radiation, which we found to be increased in rectal cancer after treatment. We hypothesized that inhibiting this complex with a DNA-PK inhibitor, Peposertib (M3814), would improve treatment response.We assessed pDNA-PK in a rectal cancer cell line and mouse model utilizing western blotting, viability assays, γH2AX staining, and treatment response. The three treatment groups were: standard of care (SOC) (5-fluorouracil (5FU) with radiation), M3814 with radiation, and M3814 with SOC.SOC treatment of rectal cancer cells increased pDNA-PK protein and increased γH2AX foci, but this was abrogated by the addition of M3814. Mice with CT26 tumors treated with M3814 with SOC did not differ in average tumor size but individual tumor response varied. The clinical complete response rate improved significantly with the addition of M3814 but pathological complete response did not. We investigated alterations in DNA repair and found that Kap1 and pATM are increased after M3814 addition suggesting this may mediate resistance.When the DNA-PK inhibitor, M3814, is combined with SOC treatment, response improved in some rectal cancer models but an increase in other repair mechanisms likely diminishes the effect. A clinical trial is ongoing to further explore the role of DNA-PK inhibition in rectal cancer treatment.

Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer

Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment. ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.

Studies on Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p on Irradiation Resistance in Rectal Cancer Cells

<h3>Purpose/Objective(s)</h3> To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism. <h3>Materials/Methods</h3> Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected. <h3>Results</h3> The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway. <h3>Conclusion</h3> The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.

CircTUBGCP3 Contributes to the Malignant Progression of Rectal Cancer.

Circular RNA (circRNA) tubulin gamma complex associated protein 3 (circTUBGCP3) has been reported to play an oncogenic role in colorectal cancer and osteosarcoma. We further assessed the role and working mechanism of circTUBGCP3 in rectal cancer progression. Colony formation assay and transwell assays were performed to analyze cell colony formation ability and motility. Flow cytometry was utilized to assess cell cycle progression and cell apoptosis. The production of lactate and the consumption of glucose were evaluated by fluorescence-based glucose/lactate assay kit to analyze cell glycolysis. The intermolecular interaction was verified by dual-luciferase reporter assay. In vivo experiments were carried out to analyze the role of circTUBGCP3 in tumor growth using xenograft tumor model. CircTUBGCP3 was significantly up-regulated in rectal cancer tissues and cell lines. CircTUBGCP3 interference inhibited the colony formation ability, migration, invasion, cell cycle progression, and glycolysis and promoted the apoptosis in rectal cancer cells. CircTUBGCP3 negative regulated microRNA-375 (miR-375) expression through interacting with it and circTUBGCP3 silencing-mediated effects in rectal cancer cells were largely based on the up-regulation of miR-375. Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) was a target of miR-375, and ROCK1 was regulated by circTUBGCP3/miR-375 axis in rectal cancer cells. MiR-375 overexpression suppressed the malignant behaviors of rectal cancer cells partly through down-regulating ROCK1. CircTUBGCP3 interference restrained rectal cancer progression in vivo. CircTUBGCP3 acted as an oncogene to promote the malignant phenotypes of rectal cancer cells by modulating miR-375/ROCK1 axis.

Analysis of MicroRNA Expression Changes During the Course of Therapy In Rectal Cancer Patients.

MicroRNAs (miRNAs) regulate gene expression in a tissue-specific manner. However, little is known about the miRNA expression changes induced by the therapy in rectal cancer (RC) patients. We evaluated miRNA expression levels before and after therapy and identified specific miRNA signatures reflecting disease course and treatment responses of RC patients. First, miRNA expression levels were assessed by next-generation sequencing in two plasma samplings (at the time of diagnosis and a year after) from 20 RC patients. MiR-122-5p and miR-142-5p were classified for subsequent validation in plasma and plasma extracellular vesicles (EVs) on an independent group of RC patients (n=107). Due to the intrinsic high differences in miRNA expression levels between samplings, cancer-free individuals (n=51) were included in the validation phase to determine the baseline expression levels of the selected miRNAs. Expression levels of these miRNAs were significantly different between RC patients and controls (for all p <0.001). A year after diagnosis, miRNA expression profiles were significantly modified in patients responding to treatment and were no longer different from those measured in cancer-free individuals. On the other hand, patients not responding to therapy maintained low expression levels in their second sampling (miR-122-5p: plasma: p=0.05, EVs: p=0.007; miR-142-5p: plasma: p=0.008). Besides, overexpression of miR-122-5p and miR-142-5p in RC cell lines inhibited cell growth and survival. This study provides novel evidence that circulating miR-122-5p and miR-142-5p have a high potential for RC screening and early detection as well as for the assessment of patients’ outcomes and the effectiveness of treatment schedule.

Effect of mistletoe extract on tumor response in neoadjuvant chemoradiotherapy for rectal cancer: a cohort study

BackgroundMistletoe extract, used as a complementary chemotherapeutic agent for cancer patients, has anticancer effects against various malignancies. The aim of the present study was to evaluate the effect of mistletoe extract (Abnoba Viscum Q®) on tumor responses to neoadjuvant chemoradiotherapy (NCRT) for locally advanced rectal cancer.MethodsThis study included patients with rectal cancer who underwent NCRT between January 2018 and July 2020. In the mistletoe group (MG), the patients were administered Abnoba Viscum Q® subcutaneously during chemoradiotherapy—maintained just before surgery. Patient demographics, clinical outcomes, histopathological outcomes, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay results were compared between the MG and non-mistletoe group (NMG). Two rectal cancer cell lines (SNU-503 and SNU-503R80Gy) were treated with Abnoba Viscum Q® to assess its mechanistic effects in vivo.ResultsOverall, the study included 52 patients (MG: n = 15; NMG: n = 37). Baseline demographics between the two groups were similar, except carbohydrate antigen 19-9 levels and tumor location from the anal verge. There was no difference in the clinical stage between the two groups. A better tumor response in the MG, relative to the NMG, was observed with respect to tumor regression grade (TRG), T stage, and overall tumor–node–metastasis stage. Tumor response was significantly better in the MG than in the NMG in terms of pathologic complete response rate (53.3% vs. 21.6%, P = 0.044), good TRG response (66.7% vs. 32.4%, P = 0.024), T downstaging (86.7% vs. 43.2%, P = 0.004), and overall downstaging (86.7% vs. 56.8%, P = 0.040). The toxicities during NCRT were minimal in both groups. More apoptotic cells were noted in MG samples than in the NMG samples on TUNEL staining. Cleaved caspase-3 level following treatment with Abnoba Viscum Q® was higher in SNU-503R80Gy cells than in SNU-503 cells.ConclusionPatients treated with chemoradiation combined with mistletoe extract showed better outcomes than patients not treated with mistletoe extract in terms of tumor responses. This diversity in treatment may improve the efficacy of NCRT, leading to better oncologic outcomes. Prospective and randomized studies with long-term follow-up are warranted to confirm and extend these results.

Effect of the ACY‑1 gene on HER2 and TRAIL expression in rectal carcinoma

The incidence of rectal carcinoma (RC) is increasing and the age at onset of the disease is reducing. Therefore, elucidating the pathogenesis of RC is beneficial for early diagnosis and improving the prognosis. Aminoacylase-1 (ACY-1) is abnormally expressed in various malignant tumor tissues. Furthermore, the human epidermal growth factor receptor-2 (HER2) gene is involved in tumor metastasis and invasion, while tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumor cell apoptosis. The aim of the present study was to investigate the effect of the ACY-1 gene on the expression of HER2 and TRAIL in RC. Cancerous and adjacent tissues from RC patients were collected. ACY-1 expression was analyzed by immunohistochemistry. The rectal cancer cell lines HT29 and SW620, and normal colorectal mucosal epithelial fetal human cells were cultured in vitro. ACY-1 gene and protein expression levels were tested by reverse transcription-quantitative PCR and western blotting. ACY-1 small interfering RNA (siRNA) was transfected into HT29 and SW620 cells. Cell proliferation was detected by thiazolyl blue MTT assay. Caspase-3 activity was assessed using a commercial kit. HER2 and TRAIL expression levels were determined by western blotting. ACY-1 expression was significantly increased in cancer tissue compared with adjacent tissue (P<0.05). ACY-1 expression was elevated in HT29 and SW620 cells compared with normal colorectal mucosal epithelial cells (P<0.05). ACY-1 siRNA transfected into HT29 cells downregulated its expression, inhibited cell proliferation, enhanced caspase-3 activity, reduced HER2 expression and upregulated TRAIL expression (P<0.05). ACY-1 expression was found to be increased in rectal cancer tissue. Therefore, targeting the ACY-1 gene may regulate HER2 and TRAIL expression levels, and may reduce the occurrence and inhibit the development of rectal cancer.

High-Dose Vitamin C Exerts Its Anti-cancer Effects in a Xenograft Model of Colon Cancer by Suppressing Angiogenesis.

Several studies have been conducted to investigate the anti-cancer effects of vitamin C (VC). However, the effect of high-dose VC administration on tumor angiogenesis remains unclear. Focusing on our high-dose VC, our study investigated the effect of high-dose VC (4 g/kg) on vascular endothelial growth in mice with xenografts of a rectal cancer cell line referred to as Colon 26. Male mice harboring Colon 26 tumors were established, and high-dose VC solution was orally administered once daily for 14 d. On the final day of the study, the lower limb tumor tissues and serum samples were collected and analyzed for the expression of tumor angiogenesis related proteins as well as the levels of reactive oxygen species (ROS). Oral VC administration decreased tumor volumes and increased p53 and endostatin levels. In addition, plasma and in tumor part ROS levels and tissue hypoxia inducible factor-1α (HIF-1α) were reduced by VC administration. In addition, the levels of vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor D (VEGFD) were decreased by VC administration. These results suggest that VC exerts its anti-cancer effects by suppressing angiogenesis.

Abstract 6178: Loss of DAB2IP promotes resistance to radiation in rectal cancer by activating HIF-1α

Abstract Rectal cancer is one of the most common cancers in the United State. It shows that approximately 72.20% of the diagnosed rectal cancers are T3-4N0 or TxN+ from the SEER database. Preoperative radiotherapy has been performed as a prevalent therapy to decrease the local recurrence after total mesorectal excision. However, some patients could not get benefits from the local radiotherapy because of the resistance to radiation. While different expressions of numerous proteins have been found in radiation resistant cancer, the mechanism has been still veiled. Previously, we reported that DAB2IP, a novel member of the Ras GTPase-activating protein family, was down-regulated in various human cancers. DAB2IP functions as a scaffold protein to balance proliferation and apoptosis, and also inhibits metastasis in prostate cancer cells. In CRC, loss of DAB2IP increases the properties of cancer stem cells by regulating the NF-kB signal pathway, which also indicates the poor survival outcome. However, the role and mechanism of DAB2IP involving the resistance to radiation has not been elucidated. In this study, firstly, we compared the expression of DAB2IP in rectal cancers sensitive (42 patients) and non-sensitive (47 patients) to radiation by immunohistochemistry. Our data showed that non- or low-expression of DAB2IP was found in patients resistant to radiation (P&amp;lt;0.001). And these patients had worse disease free survival than high DAB2IP expression patients (p&amp;lt;0.01). Multivariate analyses indicated that DAB2IP expression level was an independently prognostic marker for survival of patients with rectal cancer (p&amp;lt;0.05). Then, we explored the biological function of DAB2IP by knocking down endogenous DAB2IP in cultured rectal cancer cell line SW837. Our results showed that knocking down DAB2IP in SW837 increased the survival rate when exposed to 2Gy dose radiation demonstrated by MTT assay and Anexin V/PI assay in flow cytometery. In xenograft model, SW837 with DAB2IP knocking down grew faster by recording the growth curve even if given total 10Gy dose radiation. Moreover, we found the amount of cleaved PARP and phosphorylated γ-H2AX decreased in DAB2IP knocking down cells exposed to 2Gy dose radiation for 6h. In addition, loss of DAB2IP could promote HIF-1α expression in nucleus and activate the signal pathway by HIF-1α luciferase analysis. In SW837 with DAB2IP knocking down, knocking down the HIF-1a by siRNA could almost rescue DAB2IP loss inducing the resistance to radiation. Taken together, our data provides strong evidence that loss of DAB2IP may contribute to the resistance to radiation in rectal cancer by activating the HIF-1α pathway. Citation Format: Shenglan Yang, Jiang Min, Liang liu, Daxing Xie. Loss of DAB2IP promotes resistance to radiation in rectal cancer by activating HIF-1α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6178.

MiR-1224-5p acts as a tumor suppressor via inhibiting the malignancy of rectal cancer through targeting SLC29A3.

The emphasis of our study was to determine the physiological function of miR-1224-5p in rectal cancer (RC) and its in-depth mechanism. First, the expression of miR-1224-5p in RC tissues was analyzed using public data from the TCGA database. Then, miR-1224-5p expression in RC cell lines SW480 and SW837 was measured using the qRT-PCR assay. The subsequent CCK-8 assay was executed to assess the function of miR-1224-5p in the viability of the RC cell. Bioinformatics prediction prompted that SLC29A3 may be a potential target gene for miR-1224-5p. Western blotting and dual-luciferase reporter assays were performed to affirm the above forecasting. Kaplan-Meier analysis and Cox multivariate analysis were carried out to assess the relationship between SLC29A3 and prognosis. Finally, CCK-8, colony formation assay, and transwell assay were used for functional analysis of miR-1224-5p/ SLC29A3 axis in vitro. MiR-1224-5p was expressed at low levels in RC tissues and cell lines. Up-regulation of miR-1224-5p inhibited SW480 cell viability, while inhibition of miR-1224-5p enhanced the viability of SW837 cells. What is more, we affirmed that miR-1224-5p could direct target SLC29A3, which was expressed at high levels in RC tissues. In addition, SLC29A3 could be used as an independent predictive factor of prognosis in patients with RC, and the higher SLC29A3 expression, the lower survival rate. Finally, cellular functional experiments evidenced that miR-1224-5p mimic can reduce the cell viability, invasion, and migration, while overexpression of SLC29A3 presented an opposite effect. Importantly, co-transfection experiments indicated that SLC29A3 can reverse miR-1224-5p-mediated inhibition in the malignant progression of RC cells. Our work raised the possibility that miR-1224-5p functioned as a tumor suppressor in RC, which achieved its function via targeting SLC29A3.

Abstract A56: Identification of circulating miRNA signatures in rectal cancer

Abstract Colorectal cancer is the third most common cancer and the second leading cause of cancer death in Europe. Therefore, the search for new biomarkers to enable facilitate early diagnosis, prognosis, and individualized treatment is particularly warranted. From a clinical point of view, malignancies in the colon and the rectum (RC) present two distinct entities that require different treatment strategies, so CRC might not be considered as a single enteropathogenic entity. MicroRNA (miRNAs) are small noncoding RNA molecules involved in the post-transcriptional and translational regulation of gene expression. The discovery that miRNAs may act either as oncogenes or tumor suppressors has initiated extensive research. It was recently discovered that extracellular miRNAs circulate in the bloodstream and that such circulating miRNAs are remarkably stable. This has raised the possibility that miRNAs might serve as novel markers for disease diagnosis and prognosis. In the present study, we aimed to identify circulating miRNAs enabling early diagnosis and prognosis of RC. MiRNA expression levels (from plasma and plasma exosomes) were characterized by next-generation sequencing (NGS) in 24 patients with RC collected at 2-time intervals. The first sampling was collected at the time of diagnosis and the second sampling was conducted around 1 year after the diagnosis. The expression levels of miR-122 and miR-142 were identified to be associated with overall survival in RC patients. Their functional characteristics were tested in vitro on rectal cancer cell lines HRA16 and SW1463. Increased levels of miRNAs were associated with decreased long-term survival of cells. Identified miRNAs are currently being verified on a larger population of RC patients. This has raised the question whether miRNA can serve as biomarkers for rectal cancer, but prior to its clinical applications, clinical trials are warranted. Acknowledgments: This work was supported by AZV 17-30920A, GA17-16857S, and GA UK 302119. Citation Format: Klara Cervena, Alena Opattova, Barbara Pardini, Alessio Naccarati, Pavel Vodicka, Veronika Vymetalkova. Identification of circulating miRNA signatures in rectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A56.

PITPNC1 fuels radioresistance of rectal cancer by inhibiting reactive oxygen species production.

Neoadjuvant radiotherapy is a commonly used method for the current standard-of-care for most patients with rectal cancer, when the effects of radioresistance are limited. The phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1), a lipid-metabolism-related gene, has previously been proved to manifest pro-cancer effects in multiple types of cancer. However, whether PITPNC1 plays a role for developing radioresistance in rectal cancer patients is still unknown. Therefore, this study aims to investigate the role of PITPNC1 in rectal cancer radioresistance. Patient-derived tissue were used to detect the difference in the expression level of PITPNC1 between radioresistant and radiosensitive patients. Bioinformatic analyses of high-throughput gene expression data were applied to uncover the correlations between PITPNC1 level and oxidative stress. Two rectal cancer cell lines, SW620, and HCT116, were selected in vitro to investigate the effect of PITPNC1 on radioresistance, reactive oxygen species (ROS) generation, apoptosis, and proliferation in rectal cancer. PITPNC1 is highly expressed in radioresistant patient-derived rectal cancer tissues compared to radiosensitive tissue; therefore, PITPNC1 inhibits the generation of ROS and improves the extent of radioresistance of rectal cancer cell lines and then inhibits apoptosis. Knocking down PITPNC1 facilitates the production of ROS while application of the ROS scavenger, N-acetyl-L-cysteine (NAC), could reverse this effect. PITPNC1 fuels radioresistance of rectal cancer via the inhibition of ROS generation.

Retraction.

Retraction: 'Circular RNA circMTO1 acts as an antitumor factor in rectal cancer cell lines by downregulation of miR-19b-3p',by Mingfeng Fan, Yueqing Wang, Shubo Gao, J Cell Biochem. The above article, published online on 30 December 2019 on Wiley Online Library, has been retracted by agreement between the authors, the journal's Editor in Chief, Prof. Dr. Christian Behl, and Wiley Periodicals LLC. The authors asked to retract this manuscript and stated that not all co-authors agreed on the publication, initially, but were not available for further comments during the investigation. The retraction has been agreed following an investigation based on allegations raised by a third party. The authors stated that not all co-authors agree on the publication. Raw data could not be provided and thus the overall validity of the results could not be confirmed.

MicroRNA-130a modulates a radiosensitivity of rectal cancer by targeting SOX4

Radioresistance poses a major challenge in the treatment of advanced rectal cancer. Therefore, understanding the detailed mechanisms of radioresistance may improve patient response to irradiation and the survival rate. To identify the novel targets that modulate the radiosensitivity of rectal cancer, we performed small RNA sequencing with human rectal cancer cell lines. Through bioinformatics analysis, we selected microRNA-310a (miR-130a) as a promising candidate to elucidate radioresistance. miR-130a was dramatically upregulated in radiosensitive rectal cancer cells and overexpression of miR-130a promotes rectal cancer cell radiosensitivity. Mechanically, miR-130a reversed the epithelial-mesenchymal transition phenotype of rectal cancer cells following inhibition of cell invasion upon irradiation. Moreover, miR-130a also inhibited the repair of irradiation-induced DNA damage followed by cell death. We identified that SOX4 was a direct target of miR-130a. Overexpression of SOX4 reversed the promotion activity of miR-130a on radiosensitivity. Together, our findings suggest that miR-130a functions as a radiosensitizer in rectal cancer and reveals a potential therapeutic target and preoperative prognostic marker for radiotherapy.

Effects of metformin and phenformin on apoptosis and epithelial-mesenchymal transition in chemoresistant rectal cancer.

Recurrence and chemoresistance in colorectal cancer remain important issues for patients treated with conventional therapeutics. Metformin and phenformin, previously used in the treatment of diabetes, have been shown to have anticancer effects in various cancers, including breast, lung and prostate cancers. However, their molecular mechanisms are still unclear. In this study, we examined the effects of these drugs in chemoresistant rectal cancer cell lines. We found that SW837 and SW1463 rectal cancer cells were more resistant to ionizing radiation and 5‐fluorouracil than HCT116 and LS513 colon cancer cells. In addition, metformin and phenformin increased the sensitivity of these cell lines by inhibiting cell proliferation, suppressing clonogenic ability and increasing apoptotic cell death in rectal cancer cells. Signal transducer and activator of transcription 3 and transforming growth factor‐β/Smad signaling pathways were more activated in rectal cancer cells, and inhibition of signal transducer and activator of transcription 3 expression using an inhibitor or siRNA sensitized rectal cancer cells to chemoresistant by inhibition of the expression of antiapoptotic proteins, such as X‐linked inhibitor of apoptosis, survivin and cellular inhibitor of apoptosis protein 1. Moreover, metformin and phenformin inhibited cell migration and invasion by suppression of transforming growth factor β receptor 2‐mediated Snail and Twist expression in rectal cancer cells. Therefore, metformin and phenformin may represent a novel strategy for the treatment of chemoresistant rectal cancer by targeting signal transducer and activator of transcription 3 and transforming growth factor‐β/Smad signaling.

Establishment, functional and genetic characterization of three novel patient-derived rectal cancer cell lines.

AIMTo establish patient-individual tumor models of rectal cancer for analyses of novel biomarkers, individual response prediction and individual therapy regimens.METHODSEstablishment of cell lines was conducted by direct in vitro culturing and in vivo xenografting with subsequent in vitro culturing. Cell lines were in-depth characterized concerning morphological features, invasive and migratory behavior, phenotype, molecular profile including mutational analysis, protein expression, and confirmation of origin by DNA fingerprint. Assessment of chemosensitivity towards an extensive range of current chemotherapeutic drugs and of radiosensitivity was performed including analysis of a combined radio- and chemotherapeutic treatment. In addition, glucose metabolism was assessed with 18F-fluorodeoxyglucose (FDG) and proliferation with 18F-fluorothymidine.RESULTSWe describe the establishment of ultra-low passage rectal cancer cell lines of three patients suffering from rectal cancer. Two cell lines (HROC126, HROC284Met) were established directly from tumor specimens while HROC239 T0 M1 was established subsequent to xenografting of the tumor. Molecular analysis classified all three cell lines as CIMP-0/ non-MSI-H (sporadic standard) type. Mutational analysis revealed following mutational profiles: HROC126: APCwt, TP53wt, KRASwt, BRAFwt, PTENwt; HROC239 T0 M1: APCmut, P53wt, KRASmut, BRAFwt, PTENmut and HROC284Met: APCwt, P53mut, KRASmut, BRAFwt, PTENmut. All cell lines could be characterized as epithelial (EpCAM+) tumor cells with equivalent morphologic features and comparable growth kinetics. The cell lines displayed a heterogeneous response toward chemotherapy, radiotherapy and their combined application. HROC126 showed a highly radio-resistant phenotype and HROC284Met was more susceptible to a combined radiochemotherapy than HROC126 and HROC239 T0 M1. Analysis of 18F-FDG uptake displayed a markedly reduced FDG uptake of all three cell lines after combined radiochemotherapy.CONCLUSIONThese newly established and in-depth characterized ultra-low passage rectal cancer cell lines provide a useful instrument for analysis of biological characteristics of rectal cancer.