Post-Operative Plasma Osteopontin Predicts Distant Metastasis in Human Colorectal Cancer

BackgroundThe overall prognosis of colorectal cancer (CRC) patients is unsatisfactory due to cancer metastasis after operation. This study aims to investigate the clinical significance of plasma osteopontin (OPN) levels as minimally invasive, predictive, and surrogate biomarkers for prognosis of CRC patients.MethodsThis randomized study design consists of pre-operative and post-operative plasma samples from a total of 79 patients. We determined plasma levels of OPN by ELISA and examined their correlation with the clinicopathological parameters of CRC patients. The effects of endogenous and exogenous OPN on CRC metastasis were investigated by examination of the effect on regulators of epithelial to messenchymal transition and migration assay.ResultsOur findings demonstrated for the first time the clinical correlation of plasma OPN with metastasis of CRC patients. High post-operative plasma OPN level (>153.02 ng/ml) associated with development of metastasis after curative resection (p<0.001). Moreover, post-operative plasma OPN level correlated with disease-free survival of CRC patients (p=0.009) and was an independent factor for predicting development of metastasis in CRC patients after curative resection (p=0.036). Our in vitro model showed that OPN ectopic expression induced DLD1 cell migration through Snail and Twist1 overexpression and E-cadherin repression, and secretory OPN level enhanced cell migration.ConclusionsThe results of the current study suggest that post-operative plasma OPN correlated with post-operative metastasis, suggesting that it is a potential non-invasive biomarker for the development of future metastasis in CRC patients. In addition, OPN was shown to be involved in the metastatic process and thus inhibition of OPN is a potential therapeutic approach to treat CRC patients.     

Prognostic Significance of Cyclin D1 Expression in Colorectal Cancer: A Meta-Analysis of Observational Studies

Objective

Cyclin D1 plays a vital role in cancer cell cycle progression and is overexpressed in many human cancers, including colorectal cancer (CRC). However, the prognostic value of cyclin D1 overexpression in colorectal cancer is conflicting and heterogeneous. We conducted a meta-analysis to more precisely evaluate its prognostic significance.

Methods

A comprehensive literature search for relevant studies published up to January 2014 was performed using PubMed, EMBASE, and ISI Web of Science. The pooled hazard ratio (HR) with 95% confidence intervals (CI) was used to estimate the effects.

Results

22 studies with 4150 CRC patients were selected to evaluate the association between cyclin D1 and overall survival (OS), disease-free survival (DFS) and clinicopathological parameters. In a random-effects model, the results showed that cyclin D1 overexpression in CRC was significantly associated with both poor OS (HR = 0.73, 95% CI: 0.63–0.85, P<0.001) and DFS (HR = 0.60, 95% CI: 0.44–0.82, P = 0.001). Additionally, cyclin D1 overexpression was significantly associated with more relative older patients (≥60 years) (OR 0.62, 95% CI 0.44–0.89, P = 0.009), T3,4 tumor invasion (OR 0.70, 95% CI 0.57–0.85, P<0.001), N positive (OR 0.75, 95% CI 0.60–0.95, P = 0.016) and distant metastasis (OR 0.60, 95% CI 0.36–0.99, P = 0.047) of CRC.

Conclusion

The meta-analysis results indicated that cyclin D1 is an unfavorable prognostic factor for CRC. Cyclin D1 overexpression might be associated with poor clinical outcome and some clinicopathological factors such as age, T category, N category and distant metastasis in CRC patients.     

Characterization of Macrophage Galactose-type Lectin (MGL) ligands in colorectal cancer cell lines

BackgroundThe Ca²⁺-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GalNAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor disease-free survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive.MethodsUsing a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics.ResultsHCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope.ConclusionsWe have identified cell surface MGL-ligands on CRC cell lines.General significanceAdvances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC.     

Comparative profiling of primary colorectal carcinomas and liver metastases identifies LEF1 as a prognostic biomarker

Purpose

We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).

Patients and Methods

We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.

Results

Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1.

Conclusion

Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.     

Receptor tyrosine kinases in cancer escape from BRAF inhibitors

The BRAF inhibitors (BRAFi) induce anti-tumor responses in nearly 60% of patients with advanced ^V600BRAF-mutant melanomas but only 5% of patients with ^V600BRAF-mutant colorectal carcinomas. Earlier studies of how a subset of melanoma that initially responds to BRAFi but later acquires drug resistance pointed to the importance of receptor tyrosine kinases (RTKs) in drug escape. In a pair of recent reports, this RTK-mediated mechanism of acquired BRAFi resistance in melanoma is re-surfacing in the context of innate or primary BRAFi resistance in ^V600BRAF-mutant colorectal carcinomas, suggesting potential upfront therapeutic strategies to prevent BRAFi resistance.^V600BRAF mutations are found in >50% of melanomas, nearly 100% of hairy cell leukemias but smaller subsets of more common human malignancies (e.g., colorectal, thyroid)¹. The in-human “druggability” of mutant BRAF has been best demonstrated in metastatic BRAF mutant melanomas using the novel small-molecule BRAF inhibitor (BRAFi) PLX4032/vemurafenib, producing survival benefits². Early clinical results of BRAFi in colorectal carcinoma, however, were disappointing, with only 5% of patients (1 of 21 patients) experiencing a partial response and 19% of patients (4 of 21 patients) experiencing minor responses³. This difference in the clinical results (melanoma vs. colorectal carcinoma) may relate less to their ontological origins but more to alternative states of a dynamic and plastic survival signaling network.The majority of BRAF mutant melanomas responds to BRAFi rapidly but acquires drug resistance within a median time of 6-7 months. The specific mechanisms of acquired BRAFi resistance are variegated but fall under two core pathways: 1) reactivation of RAF-MEK-ERK MAPK signaling, and 2) activation of MAPK-redundant signaling via the receptor tyrosine kinase (RTK)-PI3K-AKT pathway, which is parallel but interconnected to the MAPK pathway. MAPK reactivation can occur via NRAS activating mutations⁴, COT overexpression⁵, ^V600BRAF alternative splicing⁶, ^V600BRAF amplification⁷, and MEK1 activating mutation^8,9. MAPK-redundant signaling via RTK overexpression has been shown to result in AKT activation and RAS-CRAF-MEK signaling, bypassing mutant BRAF^4,10,11. The repertoire of RTK overexpressed appears restricted but shares a common pattern of PDGFRβ and EGFR overexpression, at least in melanoma cell lines with acquired resistance to vemurafenib⁴. It is unclear at present how this overexpression of a select number of wild-type RTKs contributes to the molecular details of survival pathway redundancy and cooperativity. Nevertheless, understanding how melanomas acquire BRAFi resistance via core pathways may shed key insights into mechanisms of innate BRAFi resistance in multiple malignancies. Hence, it came as not a complete surprise that a pair of papers published recently implicated RTKs in innate BRAFi resistance in colorectal cancer cell lines^12,13. Both studies pointed to EGFR activation and downstream signaling as a key component to innate BRAFi resistance, at least in a majority of colorectal carcinoma (CRC) cell lines examined.Corcoran et al.¹² showed that BRAF mutant CRC cell lines, in contrast to BRAF mutant melanoma cell lines, displayed innate resistance to growth inhibition by vemurafenib. An important clue implicating RTK involvement in innate vemurafenib resistance of BRAF mutant CRC cell lines came from the observation that p-ERK recovery occurred soon (hours to days) after vemurafenib treatment, unlike the kinetics of p-ERK recovery in BRAF mutant melanoma cell lines. This relatively rapid recovery of p-ERK post vemurafenib treatment in CRC cell lines is akin to that in melanoma cell lines with acquired BRAFi resistance driven by RTK overexpresion¹⁰. Corcoran et al. then traced this propensity for early p-ERK recovery to vemurafenib treatment (24 h)-dependent enhancement of (activated) RAS-GTP levels and MEK activity, parallel to elevated RAS-GTP levels in melanoma cell lines with RTK-driven, acquired BRAFi resistance⁴. In phospho-RTK arrays, they determined that the p-EGFR level (among others such as p-c-MET and p-IGF1R levels) was elevated in CRC cell lines relative to those in melanoma cells. Vemurafenib treatment (24 h) did not significantly enhance the p-EGFR level (but did elevate the p-IGFR1 level). Elevated p-EGFR levels in BRAF mutant CRC cell lines were correlated with elevated total EGFR levels (i.e., overexpressed compared with BRAF mutant melanoma cell lines). Thus, several observations correlated with innate BRAFi resistance in CRC cell lines: RTK (mostly consistently EGFR) overexpression (at baseline); upregulation of activation-associated phosphorylation of RTKs (at baseline); and upregulation of RAS-GTP levels (in response to BRAFi treatment). Curiously, although EGFR is highly phosphorylated at baseline, the RAS-GTP levels only rose in response to vemurafenib treatment.Corcoran et al. further showed that small-molecule EGFR inhibitors (EGFRi) could downregulate, partially or completely, the RAS-GTP level induced by vemurafenib treatment. The combination of vemurafenib (BRAFi) and gefitnib (EGFRi) could synergistically reduce p-ERK levels and the net growth inhibition of most but not all CRC cell lines studied, suggesting that survival in some CRC cell lines may also depend on other RTKs and downstream signaling (e.g., AKT). Consistently, the combination of vemurafenib and erlotinib (EGFRi) stabilized the growth of, but did not cause significant regression of, CRC xenografts. Simultaneous inhibition or genetic knockdown of multiple RTKs was not explored, leaving unresolved the issue of how multiple RTKs may potentially play cooperative survival roles at baseline or in response to kinase inhibitor therapy.Prahallad et al.¹³ also compared CRC and melanoma cell lines and showed that EGFR expression is generally higher in CRC cell lines. Vemurafenib treatment (6 h) of the WiDr CRC cell line led to an induction in p-EGFR and p-AKT levels, concomitant with the expected suppression of p-MEK and p-ERK. MEK inhibition, by AZD6244 treatment, similarly led to the rebound phosphorylation of EGFR. Based on earlier literature showing that the ERK kinase phosphorylates Cdc25c, activating its phosphatase activity, and that Cdc25c can dephosphorylate EGFR, Prahallad et al. went on to show that Cdc25c knockdown mimicked vemurafenib treatment in inducing p-EGFR levels. As predicted, vemurafenib treatment of CRC cell line inhibited Cdc25c phosphorylation at a key threonine (Thr 48), which was previously demonstrated to be a key event for its phosphatase activity. Addition of an EGFRi (cetuximab or gefitnib) to the BRAFi vemurafenib treatment downregulated the baseline level of p-ERK and the BRAFi-induced p-AKT level (but not the baseline p-AKT level). Moreover, addition of an EGFRi sensitized CRC cell lines to growth inhibition by vemurafenib in vitro but did not induce tumor regression in vivo, again suggesting incomplete survival signaling blockade. Accordingly, it has been shown that the effect of vemurafenib in shrinking CRC tumor xenografts was enhanced by combining with an AKT inhibitor (MK-2206)¹⁴. Moreover, in this study, the addition of vemurafenib to erlotinib treatment also resulted in increased anti-tumor activity and improved survival in xenograft models. It should be pointed out that Prahallad et al. did not formally assess BRAFi and EGFRi synergy, nor did they examine the diversity of RTK overexpression/activity and its contribution to downstream survival signaling (e.g., AKT).These works, along with prior studies^4,10, highlight the importance of expression and activity level of RTKs as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines (Figure 1). An important question remains as to whether the diversity of RTK overexpression and/or upregulation participates in and contributes to the full BRAFi resistance phenotype. A recent study afforded us a systems-wide view of the RTKinome reprogramming in response to MEK inhibition in the so-called triple-negative breast cancer cell lines¹⁵. The balance of the MAPK vs. RTK network signaling may be dynamically influenced by kinase inhibitors targeting RAF or MEK. This daunting diversity of RTK expression/activity may corner us into abandoning a combination of RTK inhibitors (already approved for clinical usage) with a BRAF inhibitor. Instead, we might need to resort to downstream pathway inhibitors not yet approved for clinical usage (e.g., an inhibitor of MEK with an inhibitor of the PI3K-AKT-mTORC1/2 axis) before we have a chance to corner BRAF mutant cancers into death.Open in a separate windowFigure 1Upregulation of receptor tyrosine kinase(s) (RTKs) as a key sensitivity determinant of BRAFi resistance in BRAF mutant cancer cell lines. (A) In BRAF mutant melanoma cell lines, RTKs are generally expressed at very low levels and contribute minimally to survival signaling, resulting in a strong addiction to mutant BRAF signaling and sensitivity to BRAFi. When BRAF mutant melanoma cell lines acquire BRAFi resistance, they upregulate the expression and activity of PDGFRb and other RTKs, resulting in reactivation of MEK-ERK as well as MAPK-redundant PI3K-AKT survival signaling. (B) In BRAF mutant colorectal carcinoma (CRC) cell lines, EGFR and other RTKs are upregulated by overexpression and some level of activation, resulting in MAPK-redundant survival signaling and conferring innate or primary BRAFi resistance. Treatment of CRC cell lines wth a BRAF or a MEK inhibitor can further activate EGFR and potentially other RTKs and stimulate GTP-RAS levels, consolidating innate BRAFi resistance. Red denotes mutated protein (e.g., BRAF); gray symbols denote weak signaling or interactions; multiplicity of protein symbols denotes overexpression; P in blue denotes activation-associated phosphorylation.     

Development of novel DNAJB6-KIAA1522-p-mTOR three-protein prognostic prediction models for CRC

BackgroundTo evaluate the prognostic value of DNAJB6, KIAA1522, and p-mTOR expression for colorectal cancer (CRC) and to develop effective prognostic models for CRC patients.MethodsThe expression of DNAJB6, KIAA1522, and p-mTOR (Ser2448) was detected using immunohistochemistry in 329 CRC specimens. The prognostic values of the three proteins in the training cohort were assessed using Kaplan-Meier curves and univariate and multivariate Cox proportional hazards models. Prediction nomogram models integrating the three proteins and TNM stage were constructed. Subsequently, calibration curves, receiver operating characteristic (ROC) curves, the concordance index (C-index), and decision curve analysis (DCA) were used to evaluate the performance of the nomograms in the training and validation cohorts.ResultsThe three proteins DNAJB6, KIAA1522, and p-mTOR were significantly overexpressed in CRC tissues (each P < 0.01), and their expression was an independent prognostic factor for overall survival (OS) and disease-free survival (DFS) (each P < 0.05). The area under the ROC curves (AUC) and C-index values were approximately 0.7. Additionally, the calibration curves showed that the predicted values and the actual values fit well. Furthermore, DCA curves indicated that the clinical value of the nomogram models was higher than that of TNM stage. Overall, the novel prediction models have good discriminability, sensitivity, specificity and clinical utility.ConclusionThe nomograms containing DNAJB6, KIAA1522, and p-mTOR may be promising models for predicting postoperative survival in CRC.     

P21-activated kinase 4 overexpression in metastatic gastric cancer patients

INTRODUCTION: P21-activated kinase 4 (PAK), a subfamily of serine/threonine kinases originally known as a regulator of cytoskeletal dynamics and cell motility, has recently been revealed to play a key role in oncogenic signaling pathways. We studied the frequency and clinical features of PAK4-overexpressed metastatic gastric cancer. PATIENTS AND METHODS: PAK4 overexpression was screened by Western blot in 18 human gastric cancer cell lines. Immunohistochemical staining of PAK4 protein was performed in tumor specimens of 49 metastatic gastric cancer patients who received palliative capecitabine/cisplatin as first-line treatment. RESULTS: PAK4 protein overexpression was detected strongly in five gastric cell lines (AGS, MGK-28, MKN-74, SNU-216, SNU-601) and weakly in four cell lines (KATOIII, MKN-1, SNU-620, and SNU-719). PAK4 knockdown by small interfering RNA induced apoptosis in PAK4-overexpressed AGS gastric cancer cells. Immunohistochemical staining revealed PAK4 overexpressions in 4 (8.1%) of 49 metastatic gastric cancer specimens. None of the four patients with PAK4(+) responded to capecitabine/cisplatin chemotherapy, and PAK4(+) gastric cancer patients had a trend of poorer survival compared with PAK(-)(P = .876). CONCLUSIONS: We demonstrated PAK4 overexpression in a subset of gastric cancer patients, implicating a role in gastric cancer tumorigenesis. Its prognostic significance and efficacy as a drug target should be further studied.     

A Retrospective Observational Study of the Relationship between Single Nucleotide Polymorphisms Associated with the Risk of Developing Colorectal Cancer and Survival

BackgroundThere is variability in clinical outcome for patients with apparently the same stage colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) mapping to chromosomes 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and Xp22 have robustly been shown to be associated with the risk of developing CRC. Since germline variation can also influence patient outcome the relationship between these SNPs and patient survivorship from CRC was examined.MethodsAll enrolled into the National Study of Colorectal Cancer Genetics (NSCCG) were genotyped for 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and xp22 SNPs. Linking this information to the National Cancer Data Repository allowed patient genotype to be related to survival.ResultsThe linked dataset consisted of 4,327 individuals. 14q22.22 genotype defined by the SNP rs4444235 showed a significant association with overall survival. Specifically, the C allele was associated with poorer observed survival (per allele hazard ratio 1.13, 95% confidence interval 1.05–1.22, P = 0.0015).ConclusionThe CRC susceptibility SNP rs4444235 also appears to exert an influence in modulating patient survival and warrants further evaluation as a potential prognostic marker.     

Expression of Cyclin D1 Is Associated with ��-Catenin Expression and Correlates with Good Prognosis in Colorectal Adenocarcinoma

Background

The aim of this study is to investigate the prevalence and prognostic impact of β-catenin and cyclin D1 expression in colorectal carcinoma (CRC) patients.

Method

We evaluated immunohistochemial expression of β-catenin and cyclin D1 using 2-mm cores from 220 CRC patients for tissue microarray, and its significance was statistically evaluated.

Result

Positive expression of β-catenin and cyclin D1 was found in 72.5% (158 of 218 cases) and 59.4% (129 of 217 cases) of CRC patients, respectively. Expression of β-catenin was significantly correlated with tumor location (P = .017), differentiation (P = .010), lymph node metastasis (P = .032), preoperative carcinoembryonic antigen level (P = .032), and cyclin D1 expression (P = .005). Expression of cyclin D1 was significantly correlated with recurrence and/or metastasis (P = .004). In univariate analysis, β-catenin expression predicted more favorable overall survival (P = .022) and cyclin D1 expression predicted both more favorable overall survival and relapse-free survival (P = .004 and P = .006, respectively). Multivariate analysis showed that tumor stage and expression of cyclin D1 were independent prognostic factors significantly associated with overall survival and relapse-free survival.

Conclusion

This study shows that expression of β-catenin and cyclin D1 is associated with favorable clinicopathologic variables and it is a clinically significant prognostic indicator for CRC patients.     

Genetic polymorphism in a VEGF-independent angiogenesis gene ANGPT1 and overall survival of colorectal cancer patients after surgical resection

Background

The VEGF-independent angiogenic signaling plays an important role in the development of colorectal cancer (CRC). However, its implication in the clinical outcome of CRC has not been reported. This study aimed to investigate the association between genetic variations in several major VEGF-independent signaling pathway genes and the overall survival of CRC patients.

Methods

Seven single nucleotide polymorphisms (SNPs) in four important VEGF-independent angiogenic genes (ANGPT1, AMOT, DLL4 and ENG) were genotyped in a Chinese population with 408 CRC patients.

Results

One SNP, rs1954727 in ANGPT1, was significantly associated with CRC overall survival. Compared to patients with the homozygous wild-type genotype of rs1954727, those with heterozygous and homozygous variant genotypes exhibited a favorable overall survival with a hazard ratio (HR) of 0.89 (95% confidence interval [CI] 0.55–1.43, P = 0.623), and 0.32 (95% CI 0.15–0.71, P = 0.005), respectively (P trend = 0.008). In stratified analysis, this association remained significant in patients receiving chemotherapy (P trend = 0.012), but not in those without chemotherapy. We further evaluated the effects of chemotherapy on CRC survival that was stratified by rs1954727 genotypes. We found that chemotherapy resulted in a significantly better overall survival in the CRC patients (HR = 0.44, 95% CI 0.26–0.75, P = 0.002), which was especially prominent in those patients with the heterozygous genotype of rs1954727 (HR = 0.45, 95%CI 0.22–0.92, P = 0.028).

Conclusion

Our data suggest that rs1954727 in ANGPT1 gene might be a prognostic biomarker for the overall survival of CRC patients, especially in those receiving chemotherapy, a finding that warrants validation in larger independent populations.     

Effects of the m6Am methyltransferase PCIF1 on cell proliferation and survival in gliomas

BackgroundPrevious studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2′-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells.MethodsWe constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry.FindingsDownregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival.InterpretationThese results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.     

Treatment with the PARP inhibitor,niraparib, sensitizes colorectal cancer cell lines to irinotecan regardless of MSI/MSS status

Background

Cells with homologous recombination (HR) deficiency, most notably caused by mutations in the BRCA1 or BRCA2 genes, are sensitive to PARP inhibition. Microsatellite instability (MSI) accounts for 10-15% of colorectal cancer (CRC) and is hypothesized to lead to HR defects due to altered expression of Mre11, a protein required for double strand break (DSB) repair. Indeed, others have reported that PARP inhibition is efficacious in MSI CRC.

Methods

Here we examine the response to niraparib, a potent PARP-1/PARP-2 inhibitor currently under clinical evaluation, in MSI versus microsatellite stable (MSS) CRC cell lines in vitro and in vivo. We compiled a large panel of MSI and MSS CRC cell lines and evaluated the anti-proliferative activity of niraparib. In addition to testing single agent cytotoxic activity of niraparib, we also tested irinotecan (or SN-38, the active metabolite of irinotecan) activity alone and in combination with niraparib in vitro and in vivo.

Results

In contrast to earlier reports, MSI CRC cell lines were not more sensitive to niraparib than MSS CRC cell lines¸ suggesting that the MSI phenotype does not sensitize CRC cell lines to PARP inhibition. Moreover, even the most sensitive MSI cell lines had niraparib EC50s greater than 10 fold higher than BRCA-deficient cell lines. However, MSI lines were more sensitive to SN-38 than MSS lines, consistent with previous findings. We have also demonstrated that combination of niraparib and irinotecan was more efficacious than either agent alone in both MSI and MSS cell lines both in vitro and in vivo, and that niraparib potentiates the effect of irinotecan regardless of MSI status.

Conclusions

Our results support the clinical evaluation of this combination in all CRC patients, regardless of MSI status.     

Regulation of RAP1B by miR-139 suppresses human colorectal carcinoma cell proliferation

BackgroundMicroRNAs (miRNAs) are strongly implicated in carcinogenesis, but their specific roles in the major cancers have yet to be fully elucidated.MethodsThe expression levels of miR-139 in colorectal carcinoma and paired normal tissues were examined using real-time PCR assays. Potential functions of miR-139 were evaluated in colorectal carcinoma cell lines (SW480, SW620, LS174 T, and HCT116) using miR-139 mimics, anti-miR-139, and siRNA RAP1B.ResultsIn this study, we determined that miR-139 is down-regulated in colorectal carcinoma (CRC) tissues. Lower miR-139 expression correlates with more advanced CRC and lower overall survival of patients with CRC. The ectopic expression of miR-139 in human CRC cells decreased cell growth and tumorigenicity, whereas the silencing of miR-139 promoted cell growth. Mechanistic studies revealed that miR-139 repressed the activity of a reporter gene fused to the 3′-untranslated region of RAP1B, whereas miR-139 silencing up-regulated the expression of the reporter gene. RNAi-mediated knockdown of RAP1B phenocopied the antiproliferative effect of miR-139, whereas the overexpression of RAP1B blocked miR-139-mediated antiproliferative effects in CRC cells.ConclusionsTaken together, these results demonstrated that miR-139 decreases proliferation by directly targeting RAP1B, defining miR-139 as a new putative tumour suppressor miRNA in CRC.     

High Expression of Testes-Specific Protease 50 Is Associated with Poor Prognosis in Colorectal Carcinoma

Background

Testes-specific protease 50 (TSP50) is normally expressed in testes and abnormally expressed in breast cancer, but whether TSP50 is expressed in colorectal carcinoma (CRC) and its clinical significance is unclear. We aimed to detect TSP50 expression in CRC, correlate it with clinicopathological factors, and assess its potential diagnostic and prognostic value.

Methodology/Principal Findings

TSP50 mRNAs and proteins were detected in 7 CRC cell lines and 8 CRC specimens via RT-PCR and Western blot analysis. Immunohistochemical analysis of TSP50, p53 and carcinoembryonic antigen (CEA) with tissue microarrays composed of 95 CRCs, 20 colorectal adenomas and 20 normal colorectal tissues were carried out and correlated with clinicopathological characteristics and disease-specific survival for CRC patients. There was no significant correlation between the expression levels of TSP50 and p53 (P = 0.751) or CEA (P = 0.663). Abundant expression of TSP50 protein was found in CRCs (68.4%) while it was poorly expressed in colorectal adenomas and normal tissues (P<0.0001). Thus, CRCs can be distinguished from them with high specificity (92.5%) and positive predictive value (PPV, 95.6%). The survival of CRC patients with high TSP50 expression was significantly shorter than that of the patients with low TSP50 expression (P = 0.010), specifically in patients who had early-stage tumors (stage I and II; P = 0.004). Multivariate Cox regression analysis indicated that high TSP50 expression was a statistically significant independent risk factor (hazard ratio  = 2.205, 95% CI = 1.214–4.004, P = 0.009).

Conclusion

Our data demonstrate that TSP50 is a potential effective indicator of poor survival for CRC patients, especially for those with early-stage tumors.     

A missense variant in PTPN12 associated with the risk of colorectal cancer by modifying Ras/MEK/ERK signaling

BackgroundThe classical protein tyrosine phosphatases (PTPs) have been widely reported to be associated with various human malignancies including colorectal cancer (CRC). However, there are few comprehensive analyses of the association between the classical PTP genes and CRC risk.MethodsFirst, a bioinformatics analysis was performed to identify missense variants within the classical PTP gene family. Second, exome-wide association data and an independent population study were conducted to evaluate effects of candidate variants on CRC risk. Finally, functional assays based on signaling pathways were applied to uncover the potential pathogenic mechanism.ResultsWe identified that PTPN12 rs3750050 G allele presented a 19% increase the risk of CRC, with an OR of 1.19 (95% CI = 1.09–1.30, P = 1.015×10⁻⁴) under an additive model in the combined analysis. Furthermore, biochemical assays illustrated that rs3750050 could impair the inhibitory effect of PTPN12 on Ras/MEK/ERK signaling by impeding SHC dephosphorylation, increase the expression of cyclin D1 and ultimately lead to aberrant cell proliferation, thus contributing to CRC pathogenesis.ConclusionOur study highlights that PTPN12 rs3750050 could increase CRC risk by modifying Ras/MEK/ERK signaling. This work provides a novel insight into the roles of genetic variants within PTP genes in the pathogenesis of CRC.     

Effects of a selectively bred novelty-seeking phenotype on the motivation to take cocaine in male and female rats

Background

Although colorectal cancer (CRC) is generally not considered to be a hormone-dependent malignancy, several sex-related differences in incidence, molecular characteristics and survival have been reported. Epidemiological studies have consistently shown that increased exposure to female sex hormones is associated with a lower risk of CRC in women, and cyclin D1, an important downstream effector in estrogen-mediated signaling, is commonly activated in CRC. In this study, we analyzed the prognostic significance of cyclin D1 expression in CRC, with particular reference to sex-related differences, in tumors from a large, prospective, population-based cohort.

Methods

Using tissue microarrays and immunohistochemistry, the fraction and intensity of cyclin D1 expression was evaluated in 527 incident CRC cases from the Malmö Diet and Cancer Study. The χ² and Spearman's rho (ρ) tests were used for comparison of cyclin D1 expression and relevant clinicopathological characteristics. Kaplan-Meier analysis and Cox proportional hazards modeling were used to assess the effect of cyclin D1 expression on cancer-specific survival (CSS) in univariate and multivariate analysis, adjusted for established prognostic factors.

Results

Cyclin D1 intensity was significantly lower in male compared with female CRC (P = 0.018). In the full cohort, cyclin D1 expression was associated with a significantly prolonged CSS (hazard ratio (HR) = 0.69; 95% CI 0.49 to 0.96, P = 0.026) but subgroup analysis according to gender revealed a strongly accentuated prognostic effect of cyclin D1 in male CRC (HR = 0.48; 95% CI 0.31 to 0.74, P < 0.001), which was in contrast to female CRC, where cyclin D1 was not prognostic (HR = 1.05; 95% CI 0.62 to 1.78, P = 0.864) (P _interaction = 0.024). The prognostic value of cyclin D1 was not retained in multivariate analysis, either in the full cohort or in male CRC.

Conclusions

Cyclin D1 expression is strongly associated with prolonged survival in male CRC. These findings not only support an important role for cyclin D1 in colorectal carcinogenesis, but also add further weight to the accumulating evidence that CRC is indeed a hormone-dependent malignancy, for which prognostic and treatment-predictive molecular biomarkers should be evaluated differently in women and men.     

Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.