Proteomic Profiling of Chemotherapy Responses in FOLFOX-Resistant Colorectal Cancer Cells.

Chemoresistance mechanisms of colorectal cancer remain largely elusive. We aim to compare the difference of chemotherapy responses between FOLFOX-resistant and wild-type colorectal cancer cells by proteomic profiling to suggest novel treatment targets. FOLFOX-resistant colorectal cancer cells DLD1-R and HCT116-R were developed by chronic exposure to progressive FOLFOX doses. Proteomic profiling of FOLFOX-resistant and wild-type cells under FOLFOX exposure were conducted by mass-spectrometry-based protein-analysis technology. Verification of selected KEGG pathways was conducted by Western blot. DLD1-R had significantly higher FOLFOX-chemoresistance (10.81 times) than its wild-type counterpart. A total of 309 and 90 differentially expressed proteins were identified in DLD1-R and HCT116-R, respectively. In terms of gene ontology molecular function, RNA binding and cadherin binding ranked first for DLD1 and HCT116 groups, respectively. For gene set enrichment analysis, ribosome pathway and DNA replication were significantly up-regulated and down-regulated in DLD1-R, respectively. The most significantly up-regulated pathway in HCT116-R was regulation of the actin cytoskeleton. Up-regulations in the ribosome pathway (DLD1-R) and actin cytoskeleton (HCT116-R) were verified by Western blot. There were several significantly altered signaling pathways in FOLFOX-resistant colorectal cancer cells under FOLFOX with notable up-regulations in the ribosomal process and actin cytoskeleton.

Suppression of small nucleolar RNA host gene 8 (SNHG8) inhibits the progression of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies around the world with high mortality. Accumulating evidences demonstrate that long non-coding RNAs (lncRNAs) play critical roles in CRC tumorigenesis by regulating different pathways of carcinogenesis. SNHG8 (small nucleolar RNA host gene 8), a lncRNA, is highly expressed in several cancers and acts as an oncogene that promotes cancer progression. However, the oncogenic role of SNHG8 in CRC carcinogenesis and the underlying molecular mechanisms remain unknown. In this study, we explored the role of SNHG8 in CRC cell lines by performing a series of functional experiments. Similar to the data reported in the Encyclopedia of RNA Interactome, our RT-qPCR results showed that SNHG8 expression was significantly upregulated in CRC cell lines (DLD-1, HT-29, HCT-116, and SW480) compared to the normal colon cell line (CCD-112CoN). We performed dicer-substrate siRNA transfection to knockdown the expression of SNHG8 in HCT-116 and SW480 cell lines which were expressing high levels of SNHG8. SNHG8 knockdown significantly reduced CRC cell growth and proliferation by inducing autophagy and apoptosis pathways through the AKT/AMPK/mTOR axis. We performed wound healing migration assay and demonstrated that SNHG8 knockdown significantly increased migration index in both cell lines, indicating reduced migration abilities of cells. Further investigation showed that SNHG8 knockdown suppresses epithelial to mesenchymal transition and reduces cellular migratory properties of CRC cells. Taken together, our study suggests that SNHG8 acts as an oncogene in CRC through the mTOR-dependent autophagy, apoptosis, and EMT pathways. Our study provides a better understanding the role of SNHG8 in CRC at molecular level and SNHG8 might be used as novel therapeutic target for CRC management.

Proteomic profiling of metabolic proteins as potential biomarkers of radioresponsiveness for colorectal cancer.

Surgery, radiation therapy (RT), and chemotherapy are commonly used treatment modalities for CRC management. The locally advanced CRC is managed with preoperative RT or in combination of chemoradiotherapy whereas palliative RT is recommended for metastatic CRC patients to enhance overall survival and reduce distressing symptoms. There are many biomarkers established based on tumour staging, grading and molecular characteristics of patients (e.g., mutation, DNA methylation, and gene expression profiling). Interestingly, none of these markers are adequately validated for RT scheme. In order to establish the radioresponsive biomarker in CRC, we established a mouse xenograft tumour model and applied radiation to the tumours. We identified 9 metabolic proteins, namely PGK1, PGAM1, ENO1, PKM, TKT, GLUD1, LDHA, GAPDH, and MDH2, which are differentially expressed in tumours with different radioresponsiveness. Furthermore, we validated their expression in tumours from the unirradiated, poorly responded and highly responded tumour groups. In addition, we analysed their expressions in clinical samples from the public database. Extensive literature studies shown that these metabolic proteins are associated with key biochemical pathways including, glycolysis, ammonia detoxification, carcinogenesis, and drug responses. Further studies are needed to translate our findings into clinical use. SIGNIFICANCE: With the increasing incidence of colorectal cancer (CRC) globally, it is crucial to establish strategic treatment protocol by personalizing cancer treatment. Despite the well-established treatment protocols for CRC in the past decades, the mortality remains high. There is a trend of applying personalized treatment to improve patient survival. It has been reported that biomarkers may be used to predict treatment outcomes or to adjust individual treatment protocols. This project aims to identify specific metabolic proteins as biomarkers for CRC radioresponsiveness. Using bioinformatical analysis, we have identified 9 metabolic proteins which could be used as potential biomarkers for radiation therapy in CRC tumours.

Advances in High Throughput Proteomics Profiling in Establishing Potential Biomarkers for Gastrointestinal Cancer.

Gastrointestinal cancers (GICs) remain the most diagnosed cancers and accounted for the highest cancer-related death globally. The prognosis and treatment outcomes of many GICs are poor because most of the cases are diagnosed in advanced metastatic stages. This is primarily attributed to the deficiency of effective and reliable early diagnostic biomarkers. The existing biomarkers for GICs diagnosis exhibited inadequate specificity and sensitivity. To improve the early diagnosis of GICs, biomarkers with higher specificity and sensitivity are warranted. Proteomics study and its functional analysis focus on elucidating physiological and biological functions of unknown or annotated proteins and deciphering cellular mechanisms at molecular levels. In addition, quantitative analysis of translational proteomics is a promising approach in enhancing the early identification and proper management of GICs. In this review, we focus on the advances in mass spectrometry along with the quantitative and functional analysis of proteomics data that contributes to the establishment of biomarkers for GICs including, colorectal, gastric, hepatocellular, pancreatic, and esophageal cancer. We also discuss the future challenges in the validation of proteomics-based biomarkers for their translation into clinics.

RAMS11 promotes CRC through mTOR-dependent inhibition of autophagy, suppression of apoptosis, and promotion of epithelial-mesenchymal transition.

BACKGROUND: Long non-coding RNAs (lncRNAs), a class of non-coding RNAs (ncRNAs) associated with diverse biological processes of cells. Over the past decades, cumulating research evidences revealed that abnormal expressions of lncRNAs are associated with colorectal cancer (CRC) initiation, progression, metastasis, and resistance to therapies. Moreover, their usefulness as candidate biomarkers for CRC diagnosis and prognosis are well evident throughout previous literature. In the current study, we examined the role and molecular mechanisms of newly identified lncRNA named RNA associated with metastasis-11 (RAMS11) in CRC development. METHODS: The expression of RAMS11 in CRC cell lines DLD-1, HT-29, HCT-116, and SW480 and colon normal cells CCD-112-CoN were evaluated by quantitative RT-qPCR. The results showed that the RAMS11 is significantly upregulated in CRC cell lines compared to the normal cells. The CCK-8 proliferation assay, colony formation assay, and migration assay were performed to evaluate the biological and physiological functions of RAMS11 in vitro. To decipher the molecular mechanisms of RAMS11 medicated CRC progression, we further performed western blot analysis of the key pathway proteins (e.g., AMPK, AKT, and mTOR). RESULTS: Our results revealed that higher expression of RAMS11 is associated with increased CRC proliferation, migration, and development of metastasis. Knockdown of RAMS11 induced autophagy, apoptosis along with reduction of epithelial-mesenchymal transition (EMT) suggesting that RAMS11 is involved in CRC progression. The molecular mechanisms of RAMS11 indicated that knockdown of RAMS11 significantly inhibited CRC carcinogenesis through mTOR-dependent autophagy induction. CONCLUSIONS: In sum, our results suggested that RAMS11 is an important oncogene in CRC pathogenesis. Targeting RAMS11 could be a potential therapeutic strategy for CRC management.

Cancer Susceptibility Candidate 9 (CASC9) Promotes Colorectal Cancer Carcinogenesis via mTOR-Dependent Autophagy and Epithelial-Mesenchymal Transition Pathways.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Many recent studies have demonstrated that different long non-coding RNAs (lncRNAs) are involved in the initiation, advancement, and metastasis of many cancers including CRC. Cancer susceptibility candidate 9 (CASC9) is an lncRNA that has been reported in many cancers, but its role in CRC is poorly understood. In this study, we aimed to examine the expression of CASC9 in CRC cell lines and to determine the mechanism of action of CASC9 in CRC carcinogenesis. METHODS: The expression of CASC9 in CRC tissues was compared with normal samples from publicly available datasets in The Cancer Genome Atlas (TCGA) and The Encyclopedia of RNA Interactomes (ENCORI). CASC9 expression was further verified in four CRC cell lines (DLD1, HT-29, SW480, and HCT-116) and normal colorectal cell line (CCD-112CoN) by real-time quantitative polymerase chain reaction (RT-qPCR). After gene silencing in HCT-116 and SW480, Cell Counting Kit-8 assay, clonogenic assay, and wound healing assay were performed to evaluate cell proliferation, viability, and migration index of cells. Western blotting was used to explore the key pathways involved. RESULTS: CASC9 was significantly upregulated as analyzed from both public datasets TCGA and ENCORI where its overexpression was associated with poor survival of CRC patients. Similarly, CASC9 was significantly overexpressed in the CRC cell lines compared with normal cells studied. The silencing of CASC9 in HCT-116 and SW480 attenuated cell proliferation and migration significantly. Furthermore, pathways investigations showed that silencing of CASC9 significantly induced autophagy, promoted AMP-activated protein kinase (AMPK) phosphorylation, inhibited mTOR and AKT signaling pathways, and altered epithelial-mesenchymal transition (EMT) marker protein expression. CONCLUSION: We demonstrated that silencing of CASC9 contributes to the reduced CRC cell proliferation and migration by regulating autophagy and AKT/mTOR/EMT signaling. Therefore, CASC9 plays an important role in carcinogenesis, and its expression may act as a prognostic biomarker and a potential therapeutic target of CRC management.

Autophagy-Modulating Long Non-coding RNAs (LncRNAs) and Their Molecular Events in Cancer.

Cancer is a global threat of health. Cancer incidence and death is also increasing continuously because of poor understanding of diseases. Although, traditional treatments (surgery, radiotherapy, and chemotherapy) are effective against primary tumors, death rate is increasing because of metastasis development where traditional treatments have failed. Autophagy is a conserved regulatory process of eliminating proteins and damaged organelles. Numerous research revealed that autophagy has dual sword mechanisms including cancer progressions and suppressions. In most of the cases, it maintains homeostasis of cancer microenvironment by providing nutritional supplement under starvation and hypoxic conditions. Over the past few decades, stunning research evidence disclosed significant roles of long non-coding RNAs (lncRNAs) in the regulation of autophagy. LncRNAs are RNA containing more than 200 nucleotides, which have no protein-coding ability but they are found to be expressed in most of the cancers. It is also proved that, autophagy-modulating lncRNAs have significant impacts on pro-survival or pro-death roles in cancers. In this review, we highlighted the recently identified autophagy-modulating lncRNAs, their signaling transduction in cancer and mechanism in cancer. This review will explore newly emerging knowledge of cancer genetics and it may provide novel targets for cancer therapy.