Prognostic impact of Schlafen 11 in bladder cancer patients treated with platinum-based chemotherapy.

The utility of Schlafen 11 (SLFN11) expression as a predictive biomarker for platinum-based chemotherapy has been established for cancers from different histologies. However, the therapeutic relevance of SLFN11 in bladder cancer (BC) is unknown. Here, we examined the clinicopathologic significance of SLFN11 expression across 120 BC cases by immunohistochemistry. We divided the cases into two cohorts, one including 50 patients who received adjuvant or neoadjuvant platinum-based chemotherapy, and the other including 70 BC patients treated by surgical resection without chemotherapy. In the cohort of 50 BC cases treated with platinum-based chemotherapy, the SLFN11-positive group (n = 25) showed significantly better overall survival than the SLFN11-negative group (n = 25, P = .012). Schlafen 11 expression correlated significantly with the expression of luminal subtype marker GATA3. Multivariate analyses identified SLFN11 expression as an independent prognostic predictor (odds ratio, 0.32; 95% confidence interval, 0.11-0.91; P = .033). Conversely, in the cohort of 70 BC cases not receiving platinum-based chemotherapy, the SLFN11-positive group (n = 29) showed significantly worse overall survival than the SLFN11-negative group (n = 41, P = .034). In vitro analyses using multiple BC cell lines confirmed that SLFN11 KO rendered cells resistant to cisplatin. The epigenetic modifying drugs 5-azacytidine and entinostat restored SLFN11 expression and resensitized cells to cisplatin and carboplatin in SLFN11-negative BC cell lines. We conclude that SLFN11 is a predictive biomarker for BC patients who undergo platinum-based chemotherapy and that the combination of epigenetic modifiers could rescue refractory BC patients to platinum derivatives by reactivating SLFN11 expression.

Establishment of oxaliplatin-resistant gastric cancer organoids: importance of myoferlin in the acquisition of oxaliplatin resistance.

BACKGROUND: The attainment of drug resistance in gastric cancer (GC) is a problematic issue. Although many studies have shown that cancer stem cells (CSCs) play an important role in the acquisition of drug resistance, there is no clinically available biomarker for predicting oxaliplatin (L-OHP) resistance in relation to CSCs. Organoid technology, a novel 3D cell culture system, allows harboring of patient-derived cancer cells containing abundant CSCs using niche factors in a dish. METHODS: In this study, we established L-OHP-resistant gastric cancer organoids (GCOs) and evaluated their gene expression profile using microarray analysis. We validated the upregulated genes in the L-OHP-resistant GCOs compared to their parental GCOs to find a gene responsible for L-OHP resistance by qRT-PCR, immunohistochemistry, in vitro, and in vivo experiments. RESULTS: We found myoferlin (MYOF) to be a candidate gene through microarray analysis. The results from cell viability assays and qRT-PCR showed that high expression of MYOF correlated significantly with the IC50 of L-OHP in GCOs. Immunohistochemistry of MYOF in GC tissue samples revealed that high expression of MYOF was significantly associated with poor prognosis, T grade, N grade, and lymphatic invasion, and showed MYOF to be an independent prognostic indicator, especially in the GC patients treated with platinum-based chemotherapy. The knockdown of MYOF repressed L-OHP resistance, cell growth, stem cell features, migration, invasion, and in vivo tumor growth. CONCLUSIONS: Our results suggest that MYOF is highly involved in L-OHP resistance and tumor progression in GC. MYOF could be a promising biomarker and therapeutic target for L-OHP-resistant GC cases.

Schlafen 11 predicts response to platinum-based chemotherapy in gastric cancers.

BACKGROUND: Although unresectable or recurrent gastric cancers (GC) are frequently treated with platinum-based chemotherapy, response to treatment remains unpredictable. Because Schlafen 11 (SLFN11) is recently identified as a critical determinant of platinum sensitivity, we investigated the potential clinical utility of SLFN11 in the treatment of GC. METHODS: We analysed the correlation between SLFN11 expression and overall survival in 169 GC patients by our established immunohistochemical approach. The impact of SLFN11 expression on the response to platinum and transition of SLFN11 expression upon long-term treatment with platinum were examined using GC cell lines and organoids. RESULTS: GC patients with high-SLFN11 expression exhibited significantly better survival than those with low-SLFN11 expression, and the significance increased when we selected patients treated with platinum-based chemotherapy. Knockout of SLFN11 and reactivation of SLFN11 in GC cells conferred resistance and sensitivity to platinum, respectively. In GC cells and organoids, long-term treatment with oxaliplatin suppressed SLFN11 expression while imparting drug resistance. The acquired resistance to oxaliplatin was reversed by reactivation of SLFN11 with epigenetic modifying drugs. CONCLUSIONS: This is the first report revealing definitive clinical implications of SLFN11 in the treatment of GC patients and providing novel strategies for the drug selection based on SLFN11 expression.

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer.

5-Fluorouracil (5-FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5-FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS-mutant organoids and cell lines in comparison with KRAS wild-type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3-positive patients was significantly worse compared with that of KHDRBS3-negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3-positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage-independent growth by maintaining stem cell-like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

Immunohistochemical analysis of SLFN11 expression uncovers potential non-responders to DNA-damaging agents overlooked by tissue RNA-seq.

DNA-damaging agents include first-line drugs such as platinum (cisplatin, carboplatin), topoisomerase inhibitors (etoposide, doxorubicin), and replication inhibitors (cytarabine, gemcitabine). Despite their wide and long usage, there is no clinically available biomarker to predict responses to these drugs. Schlafen 11 (SLFN11), a putative DNA/RNA helicase, recently emerged as a dominant determinant of sensitivity to these drugs by enforcing the replication block in response to DNA damage. Since the clinical importance of SLFN11 is implicated, a comprehensive analysis of SLFN11 expression across human organs will provide a practical resource to develop the utility of SLFN11 in the clinic. In this study, we established a scoring system of SLFN11 expression by immunohistochemistry (IHC) and assessed SLFN11 expression in ~ 700 malignant as well as the adjacent non-tumor tissues across 16 major human adult organs. We found that the SLFN11 expression is tissue specific and varies during tumorigenesis. Although The Cancer Genome Atlas (TCGA) is a prevailing tool to assess gene expression in various malignant and normal tissues, our IHC data exhibited obvious discrepancy from the TCGA data in several organs. Importantly, SLFN11-negative tumors, potentially non-responders to DNA-damaging agents, were largely overrated in TCGA because TCGA samples are a mixture of infiltrating immune cells, including T cells, B cells, and macrophages, which have strong SLFN11 expression. Thus, our study reveals the significance of immunohistochemical procedures for evaluating expression of SLFN11 in patient samples and provides a robust resource of SLFN11 expression across adult human organs.

Molecular biological analysis of 5-FU-resistant gastric cancer organoids; KHDRBS3 contributes to the attainment of features of cancer stem cell.

5-FU is one of the key drugs in the treatment of gastric cancer (GC). Much evidence has shown that cancer stem cells (CSCs) play a key role in the acquisition of drug resistance. The organoid is a novel 3D cell culture system technology that sustains stem-cell-driven formation of near-physiological, self-renewing tissues using specific niche factors in a dish. In this study, we established GC organoids (GCOs) and gradually treated them with higher concentrations of 5-FU. We successfully harvested four 5-FU-resistant GCOs, which were supported by significant changes in the expression of molecules related to 5-FU metabolism. We then performed microarray analysis using three normal gastric organoids and three pairs of 5-FU-resistant and parental GCOs. Through the comparison of expression profiles and further validation, we chose KHDRBS3 as a target gene. We found KHDRBS3 to be an independent prognostic factor in GC patients, especially in GC patients treated with 5-FU chemotherapy. We also determined that KHDRBS3 might play an important role in the acquisition of stem cell-like features, such as multi-drug resistance and organoid formation, by regulating CD44 variant expression. We found KHDRBS3, which is thought to play an important role in the acquisition of characteristics of CSCs in GC, to be a promising candidate marker for predicting therapeutic effect and prognosis in GC patients.

Uc.63+ contributes to gastric cancer progression through regulation of NF-kB signaling.

BACKGROUND: The transcribed ultraconserved regions (T-UCRs) are a novel class of long non-coding RNAs and are involved in the development of several types of cancer. Although several different papers have described the oncogenic role of Uc.63+, there are no reports mentioning its importance in gastric cancer (GC) biology. METHODS: In this study, we evaluated Uc.63+ expression using clinical samples of GC by qRT-PCR, and also assessed the correlation between Uc.63+ expression and clinico-pathological factors. RESULTS: The upregulation of Uc.63+ was significantly correlated with advanced clinico-pathological features. Knockdown of Uc.63+ significantly repressed GC cell growth and migration, whereas overexpression of Uc.63+ conversely promoted those of GC cells. In situ hybridization of Uc.63+ revealed its preferential expression in poorly differentiated adenocarcinoma. We further conducted a microarray analysis using MKN-1 cells overexpressing Uc.63- and found that NF-κB signaling was significantly upregulated in accordance with Uc.63+ expression. CONCLUSION: Our results suggest that Uc.63+ could be involved in GC progression by regulating GC cell growth and migration via NF-κB signaling.