Shu Yang

Excess Cholesterol Biosynthesis by Up-Regulated HMGCS1 in Colorectal Cancer Cells Induced M2-Like Tumor-Associated Macrophage Polarization Via Extracellular Vesicles

Background and Purpose: Acute graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (HSCT). Gastrointestinal damage is a priority target of acute GVHD, presented as intestinal barrier dysfunction. The first-line treatment for acute GVHD is glucocorticoid, which, however, brings inevitable side effects. A search for alternative or supplementary therapy has been ongoing. Experimental Approach: Acute GVHD mice were treated with artesunate (ART), dexamethasone (DXM) or ART combined with DXM. The progression and severity of acute GVHD was evaluated by weight loss, clinical scores and survival. Intestinal permeability was assessed by FD-4 leakage. Colonic tissues were analyzed by qPCR, western blotting and staining techniques. Th17 and Treg cells were analyzed by flow cytometry. Key Results: ART alleviated acute GVHD and prolonged survival. Damages in intestinal tract, liver and skin of acute GVHD mice were evidently mitigated by ART. Tight junction proteins Claudin-1/3 were increased while Claudin-2 decreased in intestinal epithelium, intestinal epithelial cell renewal was accelerated, mucus lining the epithelium was thickened, and intestinal permeability was decreased in the ART-treated acute GVHD mice. ART induced allo-T cells to differentiate to Treg instead of Th17 through inhibiting mTOR pathway. Significantly, half-dose DXM combined with ART showed potent protective effect on intestinal barrier comparable to full-dose DXM did. Conclusion and Implications: ART ameliorated intestinal damages in acute GVHD mice through restoring tight junctions and Th17/Treg balance. Moreover, ART synergized DXM against immune attack on intestinal barrier, indicating ART could be a supplementary strategy that reduced DXM-related side effects.

Background: Attenuated Oxaliplatin efficacy is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin de-efficacy during CRC treatment has not been completely elucidated. Methods: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC₅₀. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Results: CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and decreased Oxaliplatin response. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. Conclusion: Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α enhanced Oxaliplatin efficacy through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Background Oxaliplatin resistance is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy and the risks in relapse and metastasis. However, the mechanism of Oxaliplatin resistance has not been completely elucidated. Methods Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10 transporter. The Cancer Genome Atlas data was analyzed to figure out the correlation between the clinical manifestation and ABCC10 expression. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 assay was conducted to identify the CRC cell viability and Oxaliplatin IC₅₀. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Results CRC patients with higher ABCC10 were prone to relapse and metastasis. Differential ABCC10 expression in multiple CRC cell lines revealed a strong positive correlation between ABCC10 expression level and Oxaliplatin resistance. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was evidently elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. To explore a strategy to block ABCC10 in CRC cells, we paid a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that neither the inhibition of ERS nor the induction of mild ERS had anti-CRC effect. However, the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the induction of intense ERS. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS down-regulated ABCC10 expression via regulated IRE1-dependent decay activity. Conclusion Oxaliplatin was a substrate of ABCC10 efflux transporter. The intense ERS/IRE1α alleviated Oxaliplatin resistance through down-regulating ABCC10 in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Purpose Colorectal cancer (CRC) ranks 2nd in morbidity and 3rd in mortality among all cancers worldwide. 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is ubiquitously expressed in humans. Remarkably, the mevalonate pathway is often dysregulated in many cancers, suggesting tumor dependency on this classic metabolic pathway. In addition to altered substrates in the mevalonate pathway, it was recently found that HMGCS1 was highly expressed in prostate, melanoma and breast cancers. The transmembrane glycoprotein KIT is critical for the survival and development of various cells through activating downstream pathways and targets. Our previous research has proved that KIT is hyper-expressed in CRC and promotes CRC progression. However, whether HMGCS1 mediates the tumor-promoting effect of KIT signaling has not been unclosed. Material and Methods We investigated the regulatory mechanism of KIT signaling on HMGCS1 expression and the role of increased HMGCS1 in CRC development by the use of gene over-expression and knock-down techniques in CRC cells, biological function tests, protein-DNA binding detection, bioinformatics, database analysis and c-kit loss-of-function mutant mice (Wads^m/m). Results HMGCS1 was up-regulated by KIT-JNK-c-Jun signaling in CRC cells and could be a biomarker for CRC. Highly expressed HMCGS1 promoted CRC cell proliferation and invasion while inhibiting apoptosis. Our results provided evidence for the role of HMGCS1 in CRC progression and suggested that blocking KIT-JNK-c-Jun-HMGCS1 might be a new strategy for treating CRC patients. Conclusion Our results indicated that KIT signaling could up-regulate HMGCS1 in CRC mediated by Ap-1/c-Jun. Hyper-expressed HMGCS1 promotes CRC cell proliferation and invasion while inhibiting apoptosis.

Background: Oxaliplatin resistance is a challenge in treating colorectal cancer (CRC) patients, contributory to the failure in chemotherapy. However, the mechanism of Oxaliplatin resistance has not been completely elucidated. In this study, we explored the key molecule involved in the Oxaliplatin resistance, which could be a candidate therapeutic target to attenuate chemo-resistance in CRC cells.Methods: Microarray screening, western blot and qPCR on clinic CRC samples were conducted to select the target gene ABCC10. The Oncomine Oncology Database and the Cancer Genome Atlas (TCGA) data were analyzed to figure out the correlation between the clinical manifestation and ABCC10. ABCC10 knock-down in CRC cells was conducted to identify its role in the Oxaliplatin resistance. Cell counting kit-8 (CCK-8) assay was conducted to identify the CRC cell viability and Oxaliplatin IC₅₀. Flow cytometry was conducted to detect the cell apoptosis exposed to Oxaliplatin. The intracellular Oxaliplatin accumulation was measured by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS).Results: ABCC10 transporter was correlated with CRC relapse and metastasis, indicating a CRC-promoting effect of ABCC10. In ABCC10 knock-down CRC cells the Oxaliplatin sensitivity was significantly elevated due to an increase of intracellular Oxaliplatin accumulation resulted from the diminished drug efflux. We next explored a strategy to inhibit ABCC10 in CRC cells, paying a special interest in the endoplasmic reticulum stress (ERS) / unfolded protein response (UPR) that plays a dual role in tumor development. We found that the CRC cell viability was profoundly decreased and the pro-apoptotic factor CHOP and apoptosis were increased by the intense ERS/UPR instead of the inhibited and mild ERS/UPR. Significantly, the Oxaliplatin sensitivity of CRC cells was enhanced in response to the intense ERS, which was blocked by inhibiting IRE1α branch of UPR. Finally, we figured out that the intense ERS/UPR down-regulated ABCC10 via regulated IRE1-dependent decay (RIDD) activity. Conclusion: Oxaliplatin was a substrate of ABCC10. The intense ERS/IRE1α elicited anti-CRC effects through down-regulating ABCC10 so as to increase Oxaliplatin sensitivity, in addition to inducing CHOP. We suggested that introduction of intense ERS/UPR could be a promising strategy to restore chemo-sensitivity when used in combination with Oxaliplatin or other chemotherapeutic drugs pumped out by ABCC10.

Carcinoembryonic antigen (CEA) is highly expressed in embryo and colorectal cancer (CRC) and has been widely used as a marker for CRC. Emerging evidence has demonstrated that elevated CEA levels promote CRC progression. However, the mechanism of the increased CEA expression in patients with primary and recurrent CRC is still an open question. In this study, we showed that c‐KIT, ELK1, and CEA were hyperexpressed in patients with CRC, especially patients with recurrent disease. From bioinformatics analysis, we picked ELK1 as a candidate transcription factor (TF) for CEA; the binding site of ELK1 within the CEA promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assays. Overexpression of ELK1 increased CEA expression in vitro, while knockdown of ELK1 decreased CEA. Upregulated ELK1 promoted the adhesion, migration, and invasion of CRC cells, however knockdown of CEA blocked the activities of ELK1‐overexpressed CRC cells. Furthermore, we explored the role of c‐KIT‐ERK1/2 signaling in activation of ELK1. Blocking c‐KIT signaling using Imatinib or ISCK03 reduced p‐ELK1 expression and consequently decreased CEA levels in CRC cells, as did blocking the ERK1/2 pathway by U0126. Compared with wild type littermates, the c‐kit loss‐of‐functional Wads^m/m mice showed lowered c‐KIT, ELK1, and CEA expression. In conclusion, our study revealed that ELK1, which was activated by c‐KIT‐ERK1/2 signaling, was a key TF for CEA expression. Blocking ELK1 or its upstream signaling could be an alternative way to decelerate CRC progression. Besides being a biomarker for CRC, CEA could be used for guiding targeted therapy.