Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3β/snail signalling pathway

Background:

Epithelial mesenchymal transition (EMT) is known to be associated with chemoresistance as well as increased invasion/metastasis. However, the relationship between EMT and resistance to an epidermal growth factor receptor (EGFR) -targeting drug in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we investigated the acquisition of EMT by gefitinib in HNSCC cell line (UMSCC81B).

Methods:

We isolated fibroblastoid variant (81B-Fb) from gefitinib-resistant UMSCC81B-GR3 cells obtained after increasing the doses of gefitinib treatment in vitro and examined EMT and its underlying mechanism.

Result:

81B-Fb cells exhibited fibroblast-like morphology, increased motility, loss of E-cadherin, acquisition of vimentin and snail expression. In 81B-Fb cells, downregulation of EGFR, which is mediated by increased ubiquitination, and activation of downstream protein kinase B (Akt), glycogen synthase kinase-beta (GSK-3β) signalling and upregulation of snail expression were observed compared with UMSCC81B cells. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, but not U0126, suppressed foetal bovine serum or heregulin-β1-induced phosphorylation of Akt/GSK-3β and snail expression together with the inhibition of 81B-Fb cell motility. Furthermore, forced expression of EGFR resulted in partial restoration of gefitinib sensitivity and reversal of EMT.

Conclusion:

These results suggest that EMT in the gefitinib-resistant cells is mediated by the downregulation of EGFR and compensatory activation of Akt/GSK-3β/snail pathway.     

Modulating effect of the PI3-kinase inhibitor LY294002 on cisplatin in human pancreatic cancer cells

Background

Chemoresistance is a serious problem in pancreatic cancer, but the mechanism of resistance and strategies against the resistance have not been elucidated. We examined the potential of the phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 to enhance the anti-tumor effect of cisplatin and investigated the mechanism of chemoresistance in pancreatic cancer cells using a combination therapy of cisplatin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, both in vitro and in vivo.

Methods

Cisplatin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, individually or in combination, were given to AsPC-1 and PANC-1 cell lines. Tumor growth, DNA fragments, and Akt phosphorylation were examined in vitro. To examine the therapeutic effect of cisplatin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, individually or combination an AsPC-1 tumor xenograft model was prepared for in vivo study.

Results

Cisplatin induced growth inhibition and Akt phosphorylation in pancreatic cancer cells. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 also inhibited cell proliferation but without showing Akt phosphorylation. However, the combination therapy markedly increased cleavage of caspase-3 and cytoplasmic histone-associated DNA fragments compared to the results with cisplatin alone. In the in vivo study, blocking the PI3K/Akt cascade with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 increased the efficacy of cisplatin-induced inhibition of tumor growth in nude mice, suppressing half the tumor growth with cisplatin alone. There were no detectable side effects in mice treated with combination therapy.

Conclusion

Our studies suggest that the PI3K/Akt pathway plays an important role in cisplatin resistance of pancreatic cancer cells. The augmentation of cisplatin with PI3K/Akt inhibitor may resolve the chemoresistance problem of cisplatin, and this might be a plausible strategy for achieving tolerance for chemotherapeutic agents in pancreatic cancer therapy.     

Phosphatidylinositol 3-kinase inhibitor(LY294002) induces apoptosis of human nasopharyngeal carcinoma in vitro and in vivo

Background

To evaluate whether PI3K/Akt pathway could effect on apoptosis and its mechanism in nasopharyngeal carcinoma cells.

Methods

The activation of the PI3K/Akt and its effect on CNE-2Z cells in vivo and in vitro was investigated by MTT assay, flow cytometry, western blot, ELISA, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays (TUNEL), and immunohistochemical analyses, using PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Results

The results showed that {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited the phosphorylating of Akt (S473), cell proliferation, and induced apoptosis in CNE-2Z cells. However, our experiment results also demonstrated that apoptosis-induced {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was directly regulated by caspase-9 activation pathway.

Conclusion

These data suggested that PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, induced apoptosis by caspase-9 activation pathway and might be as a potentially useful target for therapeutic intervention in nasopharyngeal carcinoma patients.     

Characterisation of fibronectin-mediated FAK signalling pathways in lung cancer cell migration and invasion

Background:

Focal adhesion kinase (FAK) is overexpressed in a variety of cancers, such as breast, colon, prostate, ovary, and lung cancers. However, the mechanism by which extracellular matrix fibronectin stimulates lung cancer cell migration and invasion through FAK remains to be investigated.

Methods:

The signalling pathways in fibronectin-mediated lung cancer cell migration and invasion were examined using western blotting. The metastasis function was detected by wound healing, migration and invasion assays. Further, RNA interference and kinase inhibitors were also used to study the downstream signals.

Results:

In this study, we examined the FAK signalling pathways in relation to calpain-2 and RhoA in fibronectin-mediated lung cancer cell migration and invasion. We found that A549 lung epithelial cells stimulated by fibronectin showed increased phosphorylation of FAK and its downstream targets, Src, ERK1/2, phosphatidylinositol 3′-kinase (PI3K), and Akt. Consistent with this observation, depletion of FAK by siRNA resulted in the inhibition of Src, ERK1/2, PI3K, and Akt activity. In addition, the Src inhibitor, PP2, blocked the phosphorylation of FAK, ERK1/2, PI3K, and Akt. Conversely, inhibition of MEK1/2 using PD98059 reduced the expression of matrix metalloproteinase-9 (MMP9) and calpain-2. The PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, further blocked the expression of MMP9 and RhoA. Inhibition of both MEK1/2 and PI3K caused reduced cell migration and invasion.

Conclusion:

Our data suggest that fibronectin-mediated activation of FAK that leads to lung cancer metastasis could occur through ERK or PI3K/Akt regulation of MMP9/calpain-2 or MMP9/RhoA activity, respectively.     

Snail regulates Nanog status during the epithelial–mesenchymal transition via the Smad1/Akt/GSK3β signaling pathway in non-small-cell lung cancer

The epithelial–mesenchymal transition (EMT), a crucial step in cancer metastasis, is important in transformed cancer cells with stem cell-like properties. In this study, we established a Snail-overexpressing cell model for non-small-cell lung cancer (NSCLC) and investigated its underlying mechanism. We also identified the downstream molecular signaling pathway that contributes to the role of Snail in regulating Nanog expression. Our data shows that high levels of Snail expression correlate with metastasis and high levels of Nanog expression in NSCLC. NSCLC cells expressing Snail are characterized by active EMT characteristics and exhibit an increased ability to migrate, chemoresistance, sphere formation, and stem cell-like properties. We also investigated the signals required for Snail-mediated Nanog expression. Our data demonstrate that {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, SB431542, LDN193189, and Noggin pretreatment inhibit Snail-induced Nanog expression during EMT. This study shows a significant correlation between Snail expression and phosphorylation of Smad1, Akt, and GSK3β. In addition, pretreatment with SB431542, LDN193189, or Noggin prevented Snail-induced Smad1 and Akt hyperactivation and reactivated GSK3β. Moreover, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 pretreatment prevented Akt hyperactivation and reactivated GSK3β without altering Smad1 activation. These findings provide a novel mechanistic insight into the important role of Snail in NSCLC during EMT and indicate potentially useful therapeutic targets for NSCLC.     

Bone marrow mesenchymal stem cells from leukemia patients inhibit growth and apoptosis in serum-deprived K562 cells

Background

The regulation of growth and apoptosis in K562 cells by human bone marrow mesenchymal stem cells (MSCs) from leukemia patients was investigated.

Methods

K562 cells were cocultured with leukemic MSCs under serum deprivation. Cell Counting Kit-8 (CCK-8), PI staining, Annexin V/PI binding and FACS assays were used to investigate cell proliferation, cell cycle status, and apoptosis of K562 cells cultures in the presence or absence of 10% serum. Western blotting was used to determine the levels of Akt, phosphorylated Akt (p-Akt), the BCL-2 family member Bad, and phosphorylated Bad (p-Bad) proteins in K562 cells after coculturing with MSCs. The effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific inhibitor of PI3K) on protein expression were also determined.

Results

K562 cell proliferation was inhibited by coculture with MSCs and the dominant cell cycle was the G₀-G₁ phase. The proportion of apoptotic K562 cells was decreased and the levels of p-Akt and p-Bad were upregulated after exposing K562 cells to MSCs. However, when {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was used, p-Akt and p-Bad proteins inK562 cells showed a significant reduction, while no distinct variation was seen in the nonphosphorylated Akt and Bad protein levels.

Conclusion

Leukemic MSCs can inhibit K562 cell expansion and modulate the cell cycle to a state of relative quiescence. This allows the K562 cells to endure adverse conditions such as serum starvation. The PI3K-Akt-Bad signaling pathway may be involved in this antiapoptotic process via phosphorylation of the Akt and Bad proteins. Blocking MSC-induced transduction of the PI3K-Akt-Bad pathway may be a potential strategy for a targeted therapy to combat leukemia.     

Lewis y antigen promotes the proliferation of ovarian carcinoma-derived RMG-I cells through the PI3K/Akt signaling pathway

Background

Lewis y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cell surface. Elevated expression of Lewis y has been found in 75% of ovarian tumor, and the high expression level is correlated to the tumor''s pathological staging and prognosis. This study was to investigate the effect and the possible mechanism of Lewis y on the proliferation of human ovarian cancer cells.

Methods

We constructed a plasmid encoding α1,2-fucosyltransferase (α1,2-FT) gene and then transfected it into ovarian carcinoma-derived RMG-I cells with lowest Lewis y antigen expression level. Effect of Lewis y on cell proliferation was assessed after transfection. Changes in cell survival and signal transduction were evaluated after α-L-fucosidase, anti-Lewis y antibody and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.

Results

Our results showed that the levels of α1,2-FT gene and Lewis y increased significantly after transfection. The cell proliferation of ovarian carcinoma-derived RMG-I cells sped up as the Lewis y antigen was increased. Both of α-L-fucosidase and anti-Lewis y antibody inhibited the cell proliferation. The phosphorylation level of Akt was apparently elevated in Lewis y-overexpressing cells and the inhibitor of PI3K, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, dramatically inhibited the growth of Lewis y-overexpressing cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of α1,2-FT were attenuated significantly by the monoantibody to Lewis y and by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions

Increased expression of Lewis y antigen plays an important role in promoting cell proliferation through activating PI3K/Akt signaling pathway in ovarian carcinoma-derived RMG-I cells. Inhibition of Lewis y expression may provide a new therapeutic approach for Lewis y positive ovarian cancer.     

BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt

Background:

Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS.

Methods:

The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment.

Results:

BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity.

Conclusions:

BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC.     

Cellular senescence predicts treatment outcome in metastasised colorectal cancer

Background:

Cellular senescence is a terminal cell-cycle arrest that occurs in response to activated oncogenes and DNA-damaging chemotherapy. Whether cancer cell senescence at diagnosis might be predictive for treatment outcome is unknown.

Methods:

A senescence index (SI) was developed and used to retrospectively correlate the treatment outcome of 30 UICC stage IV colorectal cancer (CRC) patients with their SI at diagnosis.

Results:

5-Fluorouracil/leucovorin-treated CRC patients achieved a significantly longer progression-free survival when presenting with SI-positive tumours before therapy (median 12.0 vs 6.0 months; P=0.044).

Conclusion:

Cancer cell senescence predicts treatment outcome in metastasised CRC. Prospective analyses of larger patient cohorts are needed.     

Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells

Background:

Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown.

Methods:

In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1.

Results:

Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)–Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens.

Conclusion:

Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K–Akt signalling pathway and resultant upregulation of cyclin D1 levels.     

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:

Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.

Methods:

Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.

Results:

Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.

Conclusion:

Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.     

A leukotriene B4 receptor-2 is associated with paclitaxel resistance in MCF-7/DOX breast cancer cells

Background:

Breast cancer is the most common malignancy in women. Although chemotherapeutic agents, such as paclitaxel, are effective treatments for the majority of breast cancer patients, recurrence is frequent and often leads to death. Thus, there is an urgent need to identify novel therapeutic targets that sensitise tumour cells to existing chemotherapy agents.

Methods:

The levels of leukotriene B₄ receptor-2 (BLT2) in multidrug-resistant MCF-7/DOX cells were determined using quantitative PCR and FACS analysis. The potential role of BLT2 in the paclitaxel resistance of MCF-7/DOX cells was assessed using a pharmacological inhibitor and small interfering RNA knockdown, and the BLT2-associated resistance mechanism was assessed.

Results:

The expression levels of BLT2 were markedly upregulated in MCF-7/DOX cells. The inhibition of BLT2 by pre-treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283 or siBLT2 knockdown significantly sensitised MCF-7/DOX cells to paclitaxel and induced significant levels of apoptotic death, suggesting that BLT2 mediates paclitaxel resistance. We also demonstrated that BLT2-induced paclitaxel resistance was associated with the upregulation of P-glycoprotein. Finally, co-treatment with a BLT2 inhibitor and paclitaxel markedly reduced tumour growth in an MCF-7/DOX in vivo model.

Conclusion:

Together, our results demonstrate that BLT2 is a novel therapeutic target that sensitises drug-resistant breast cancer cells to paclitaxel.     

Selective cyclooxygenase-2 silencing mediated by engineered E. coli and RNA interference induces anti-tumour effects in human colon cancer cells

Background:

Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required.

Methods:

We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi).

Results:

A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects.

Conclusion:

Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.     

A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation

Background:

Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool.

Methods:

Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT–PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies.

Results:

Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell–cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts.

Conclusion:

Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.     

Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability

Background:

Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood.

Methods:

In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity.

Results:

We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling.

Conclusions:

Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance.     

The TERT variant rs2736100 is associated with colorectal cancer risk

Background:

Polymorphic variation at the 5p15.33 (TERT–CLPTM1L) locus is associated with the risk of many cancers but a relationship with colorectal cancer (CRC) risk has yet to be defined.

Methods:

We used data from six genome-wide association studies (GWAS) of CRC, linkage disequilibrium mapping and imputation, to examine the relationship between 73 single-nucleotide polymorphisms at 5p15.33 and CRC risk in detail.

Results:

rs2736100, which localises to intron 2 of TERT, provided the strongest evidence of an association with CRC (P=2.28 × 10⁻⁴). The association was also shown in an independent series of 10 047 CRC cases and 6918 controls (P=0.02). A meta-analysis of all seven studies (totalling 16 039 cases, 16 430 controls) provided increased evidence of association (P=2.49 × 10⁻⁵; per allele odds ratio=1.07). The association of rs2736100 on CRC risk was shown to be independent of 15 low-penetrance variants previously identified.

Conclusion:

The rs2736100 association demonstrates an influence of variation at 5p15.33 on CRC risk and further evidence that the 5p15.33 (TERT–CLPTM1L) locus has pleiotropic effects (reflecting generic or lineage-specific effects) on cancer risk.     

Molecular alterations associated with liver metastases development in colorectal cancer patients

Background:

Understanding the molecular biology of colorectal cancer (CRC) provides opportunities for effective personalised patient management. We evaluated whether chromosomal aberrations, mutations in the PI(3)K signalling pathway and the CpG-island methylator phenotype (CIMP) in primary colorectal tumours can predict liver metastases.

Methods:

Formalin-fixed paraffin-embedded material from primary colorectal tumours of three different groups were investigated: patients with CRC without metastases (M0, n=39), patients who were treated with hyperthermal intraperitoneal chemotherapy for CRC metastases confined to the peritoneum (PM, n=46) and those who had isolated hepatic perfusion for CRC metastases confined to the liver (LM, n=48).

Results:

All samples were analysed for DNA copy number changes, PIK3CA, KRAS, BRAF mutations, CIMP and microsatellite instability. The primary CRCs of the LM group had significantly higher frequency of amplified chromosome 20q (P=0.003), significantly fewer mutations in the PI(3)K signalling pathway (P=0.003) and fewer CIMP high tumours (P=0.05). There was a strong inverse correlation between 20q and the PI(3)K pathway mutations.

Conclusion:

The development of CRC liver metastases is associated with amplification of chromosome 20q and not driven by mutations in the PI(3)K signalling pathway.     

C/EBP-β-activated microRNA-223 promotes tumour growth through targeting RASA1 in human colorectal cancer

Background:

Evidences have shown that the RAS signalling pathway plays an important role in colorectal cancer (CRC). Moreover, RAS-GTPase-activating proteins (RASGAPs) as RAS signalling terminators are associated with tumourigenicity and tumour progression. In this study, we used bioinformatics analysis to predict and study important miRNAs that could target RAS p21 GTPase-activating protein 1 (RASA1), an important member of RASGAPs.

Methods:

The levels of RASA1 and miR-223 were analysed by real-time PCR, western blotting or in situ immunofluorescence analyses. The functional effects of miR-223 and the effects of miR-223-targeted inhibitors were examined in vivo using established assays.

Results:

Upregulation of miR-223 was detected in CRC tissues (P<0.01) and was involved in downregulation of RASA1 in CRC tissues. Furthermore, the direct inhibition of RASA1 translation by miR-223 and the activation of miR-223 by CCAAT/enhancer binding protein-β (C/EBP-β) were evaluated in CRC cells. An in vivo xenograft model of CRC suggested that the upregulation of miR-223 could promote tumour growth and that the inhibition of miR-223 might prevent solid tumour growth.

Conclusions:

These results identify that C/EBP-β-activated miR-223 contributes to tumour growth by targeting RASA1 in CRC and miR-223-targeted inhibitors may have clinical promise for CRC treatment via suppression of miR-223.