Retracted: Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression

The aim of this study was to explore the relationship between the expression of HOXD antisense growth-associated long noncoding RNA (HAGLROS) and prognosis of patients with colorectal cancer (CRC), as well as the roles and regulatory mechanism of HAGLROS in CRC development. The HAGLROS expression in CRC tissues and cells was detected. The correlation between HAGLROS expression and survival time of CRC patients was investigated. Moreover, HAGLROS was overexpressed and suppressed in HCT-116 cells, followed by detection of cell viability, apoptosis, and the expression of apoptosis-related proteins and autophagy markers. Furthermore, the association between HAGLROS and miR-100 and the potential targets of miR-100 were investigated. Besides, the regulatory relationship between HAGLROS and PI3K/AKT/mTOR pathway was elucidated. The results showed that HAGLROS was highly expressed in CRC tissues and cells. Highly expression of HAGLROS correlated with a shorter survival time of CRC patients. Moreover, knockdown of HAGLROS in HCT-116 cells induced apoptosis by increasing the expression of Bax/Bcl-2 ratio, cleaved-caspase-3, and cleaved-caspase-9, and inhibited autophagy by decreasing the expression of LC3II/LC3I and Beclin-1 and increasing P62 expression. Furthermore, HAGLROS negatively regulated the expression of miR-100, and HAGLROS controlled HCT-116 cell apoptosis and autophagy through negatively regulation of miR-100. Autophagy related 5 (ATG5) was verified as a functional target of miR-100 and miR-100 regulated HCT-116 cell apoptosis and autophagy through targeting ATG5. Besides, HAGLROS overexpression activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. In conclusion, a highly expression of HAGLROS correlated with shorter survival time of CRC patients. Downregulation of HAGLROS may induce apoptosis and inhibit autophagy in CRC cells by regulation of miR-100/ATG5 axis and PI3K/AKT/mTOR pathway.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.     

PB-10, a thiazolo[4,5-d] pyrimidine derivative,targets p21-activated kinase 4 in human colorectal cancer cells

Targeting p21-activated kinase 4 (PAK4) is a potential therapeutic strategy against human colorectal cancer (CRC). In this study, we synthesized a series of novel thiazolo[4,5-d]pyrimidine derivatives (PB-1–12) and identified PB-10 (PAK4 IC₅₀ = 15.12 μM) as a potential and potent PAK4 inhibitor. Our results showed that PB-10 significantly suppressed the proliferation and colony formation of human CRC cells. PB-10 also arrested HCT-116 CRC cells at sub G0/G1 phase while promoting the expression of proapoptotic proteins. In addition, PB-10 inhibited migration, invasion, and adhesion as well as the PAK4 downstream signaling pathway in HCT-116 cells. Molecular docking analysis showed possible binding modes between PB-10 and PAK4. Our study provides a novel compound that may block the PAK4 signaling in CRC cells.     

5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells.     

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (?Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p     

Metformin: A Potential Therapeutic Agent for Recurrent Colon Cancer

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties. However, most of the studies to evaluate therapeutic efficacy of metformin have been on primary cancer. No information is available whether metformin could be effectively used for recurrent cancer, specifically colorectal cancer (CRC) that affects up to 50% of patients treated by conventional chemotherapies. Although the reasons for recurrence are not fully understood, it is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs/CSLCs). Therefore, development of non-toxic treatment strategies targeting CSCs would be of significant therapeutic benefit.In the current investigation, we have examined the effectiveness of metformin, in combination with 5-fluorouracil and oxaliplatin (FuOx), the mainstay of colon cancer therapeutics, on survival of chemo-resistant colon cancer cells that are highly enriched in CSCs/CSLCs. Our data show that metformin acts synergistically with FuOx to (a) induce cell death in chemo resistant (CR) HT-29 and HCT-116 colon cancer cells, (b) inhibit colonospheres formation and (c) enhance colonospheres disintegration. In vitro cell culture studies have further demonstrated that the combinatorial treatment inhibits migration of CR colon cancer cells. These changes were associated with increased miRNA 145 and reduction in miRNA 21. Wnt/β-catenin signaling pathway was also down-regulated indicating its pivotal role in regulating the growth of CR colon cancer cells. Data from SCID mice xenograft model of CR HCT-116 and CR HT-29 cells show that the combination of metformin and FuOX is highly effective in inhibiting the growth of colon tumors as evidenced by ∼50% inhibition in growth following 5 weeks of combination treatment, when compared with the vehicle treated controls. Our current data suggest that metformin together with conventional chemotherapy could be an effective treatment regimen for recurring colorectal cancer (CRC).     

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.     

lH-Pyrazolo[3,4-b]quinolin-3-amine derivatives inhibit growth of colon cancer cells via apoptosis and sub G1 cell cycle arrest

A series of lH-pyrazolo[3,4-b]quinolin-3-amine derivatives were synthesized and evaluated for anticancer efficacy in a panel of ten cancer cell lines, including breast (MDAMB-231 and MCF-7), colon (HCT-116, HCT-15, HT-29 and LOVO), prostate (DU-145 and PC3), brain (LN-229), ovarian (A2780), and human embryonic kidney (HEK293) cells, a non-cancerous cell line. Among the eight derivatives screened, compound QTZ05 had the most potent and selective antitumor efficacy in the four colon cancer cell lines, with IC₅₀ values ranging from 2.3 to 10.2?µM. Furthermore, QTZ05 inhibited colony formation in HCT-116 cells in a concentration-dependent manner. Cell cycle analysis data indicated that QTZ05 caused an arrest in the sub G1 cell cycle in HCT-116 cells. QTZ05 induced apoptosis in HCT-116 cells in a concentration-dependent manner that was characterized by chromatin condensation and increase in the fluorescence of fluorochrome-conjugated Annexin V. The findings from our study suggest that QTZ05 may be a valuable prototype for the development of chemotherapeutics targeting apoptotic pathways in colorectal cancer cells.     

MicroRNA-211 expression promotes colorectal cancer cell growth in vitro and in vivo by targeting tumor suppressor CHD5

Background

Chromodomain-helicase-DNA-binding protein 5 (CHD5) is a newly identified tumor suppressor that is frequently downregulated in a variety of human cancers. Our previous work revealed that the low expression of CHD5 in colorectal cancer is correlated with CHD5 promoter CpG island hypermethylation. In this study, we investigated the effect of microRNA-211 (miR-211)-regulated CHD5 expression on colorectal tumorigenesis.

Methodology/Principal Findings

miR-211 was predicted to target CHD5 by TargetScan software analysis. A stably expressing exogenous miR-211 colorectal cancer cell line (HCT-116^miR-211) was generated using lentiviral transduction and used as a model for in vitro and in vivo studies. The expression level of miR-211 in HCT-116^miR-211 cells was upregulated by 16-fold compared to vector control cells (HCT-116^vector). Exogenous miR-211 directly binds to the 3′-untranslated region (3′-UTR) of CHD5 mRNA, resulting in a 50% decrease in CHD5 protein level in HCT-116^miR-211 cells. The levels of cell proliferation, tumor growth, and cell migration of HCT-116^miR-211 cells were significantly higher than HCT-116^vector cells under both in vitro and in vivo conditions, as determined using the methods of MTT, colony formation, flow cytometry, scratch assay, and tumor xenografts, respectively. In addition, we found that enforced expression of miR-211 in HCT-116 cells was able to alter p53 pathway-associated regulatory proteins, such as MDM2, Bcl-2, Bcl-xL, and Bax.

Conclusion/Significance

Our results demonstrate that CHD5 is a direct target of miR-211 regulation. Enforced expression of miR-211 promotes tumor cell growth at least in part by downregulating the expression level of the CHD5 tumor suppressor. Our results provide a better understanding of the association of between miR-211-regulated CHD5 expression and CHD5 function in colorectal tumorigenesis.     

Matrix stiffness mediates stemness characteristics via activating the Yes-associated protein in colorectal cancer cells

Matrix stiffness is an essential physical microenvironment in solid cancer. However, its influence on cancer stemness still remains elusive. Colorectal cancer (CRC) cell line HCT-116 was cultured in the matrix with various stiffness. The siYAP was applied to detect the changes of stemness markers. The cancer stemness markers, Yes-associated protein (YAP), Lamin A/C and downstream protein molecules, and their activation were measured after the treatment with anti-β1-integrin and FAK inhibitors. In CRC tissue samples, collagen deposition and the expression of α-SMA and CD133 were detected. The study found that the expression level of stemness markers and Lamin A/C increased as the matrix stiffness raised and was regulated by YAP activation in CRC stem cells. Inhibition of β1-integrin and FAK activation in a high stiffness cell culture medium significantly decreased the activation of YAP, PI3K, and AKT. Collagen was highly deposited in the CRC invasive tumor front (ITF), and the expression of CD133 was higher in ITF compared with normal tissue and the tumor cells. Moreover, the expression level of α-SMA was positively correlated with CD133 expression level. Together, our results suggest that activation of YAP in CRC plays an important role in the promotion of cancer stem cell properties by extracellular matrix stiffness in CRC.     

Global Phosphotyrosine Proteomics Identifies PKCδ as a Marker of Responsiveness to Src Inhibition in Colorectal Cancer

Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.     

Identification of hub genes in colorectal cancer based on weighted gene co-expression network analysis and clinical data from The Cancer Genome Atlas

Colorectal cancer (CRC) is one of the most common tumors worldwide and is associated with high mortality. Here we performed bioinformatics analysis, which we validated using immunohistochemistry in order to search for hub genes that might serve as biomarkers or therapeutic targets in CRC. Based on data from The Cancer Genome Atlas (TCGA), we identified 4832 genes differentially expressed between CRC and normal samples (1562 up-regulated and 3270 down-regulated in CRC). Gene ontology (GO) analysis showed that up-regulated genes were enriched mainly in organelle fission, cell cycle regulation, and DNA replication; down-regulated genes were enriched primarily in the regulation of ion transmembrane transport and ion homeostasis. Weighted gene co-expression network analysis (WGCNA) identified eight gene modules that were associated with clinical characteristics of CRC patients, including brown and blue modules that were associated with cancer onset. Analysis of the latter two hub modules revealed the following six hub genes: adhesion G protein-coupled receptor B3 (BAI3, also known as ADGRB3), cyclin F (CCNF), cytoskeleton-associated protein 2 like (CKAP2L), diaphanous-related formin 3 (DIAPH3), oxysterol binding protein-like 3 (OSBPL3), and RERG-like protein (RERGL). Expression levels of these hub genes were associated with prognosis, based on Kaplan–Meier survival analysis of data from the Gene Expression Profiling Interactive Analysis database. Immunohistochemistry of CRC tumor tissues confirmed that OSBPL3 is up-regulated in CRC. Our findings suggest that CCNF, DIAPH3, OSBPL3, and RERGL may be useful as therapeutic targets against CRC. BAI3 and CKAP2L may be novel biomarkers of the disease.     

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.