Plasma Protein Profiling Reveal Osteoprotegerin as a Marker of Prognostic Impact for Colorectal Cancer

BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments.     

Activated cMET and IGF1R-Driven PI3K Signaling Predicts Poor Survival in Colorectal Cancers Independent of KRAS Mutational Status

Background

Oncogenic mutational analysis provides predictive guidance for therapeutics such as anti-EGFR antibodies, but it is successful only for a subset of colorectal cancer (CRC) patients.

Method

A comprehensive molecular profiling of 120 CRC patients, including 116 primary, 15 liver metastasis, and 1 peritoneal seeding tissue samples was performed to identify the relationship between v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) WT and mutant CRC tumors and clinical outcomes. This included determination of the protein activation patterns of human epidermal receptor 1 (HER1), HER2, HER3, c-MET, insulin-like growth factor 1 receptor (IGF1R), phosphatidylinositide 3-kinase (PI3K), Src homology 2 domain containing (Shc), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) kinases using multiplexed collaborative enzyme enhanced reactive (CEER) immunoassay.

Results

KRAS WT and mutated CRCs were not different with respect to the expression of the various signaling molecules. Poor prognosis in terms of early relapse (<2 years) and shorter disease-free survival (DFS) correlated with enhanced activation of PI3K signaling relative to the HER kinase pathway signaling, but not with the KRAS mutational status. KRAS WT CRCs were identified as a mixed prognosis population depending on their level of PI3K signaling. KRAS WT CRCs with high HER1/c-MET index ratio demonstrated a better DFS post-surgery. c-MET and IGF1R activities relative to HER axis activity were considerably higher in early relapse CRCs, suggesting a role for these alternative receptor tyrosine kinases (RTKs) in driving high PI3K signaling.

Conclusions

The presented data subclassified CRCs based on their activated signaling pathways and identify a role for c-MET and IGF1R-driven PI3K signaling in CRCs, which is superior to KRAS mutational tests alone. The results from this study can be utilized to identify aggressive CRCs, explain failure of currently approved therapeutics in specific CRC subsets, and, most importantly, generate hypotheses for pathway-guided therapeutic strategies that can be tested clinically.     

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.     

Serum levels of FAK and some of its effectors in adult AML: correlation with prognostic factors and survival

Focal adhesion kinase (FAK), human myofibrillogenesis regulator-1 (MR-1), ephrin receptor type A4 (EphA4), proto-oncogene tyrosine kinase Src (Src), and protein kinase C (PKC) are important markers in proliferation, survival, and migration in some cancers. However, the significance of each is still unclear in different malignancies, including acute myeloid leukemia (AML). Therefore, this study was conducted to investigate their serum levels in Egyptian adult de novo AML patients (n?=?70) against healthy volunteers (n?=?20). We managed to study the correlation between each pair and to investigate their association with diagnosis, prognosis, and survival. Serum levels were analyzed using enzyme-linked immunosorbent assay (ELISA). We found that FAK, MR-1, Src, and PKC serum levels were significantly higher in AML patients compared to control (p?<?0.0001), and this was associated with significantly lower EphA4 level (p?<?0.0001). Interestingly, we also observed a significant negative correlation of FAK (p?=?0.027), MR-1 (p?=?0.003), Src (p?=?0.038), and PKC (p?=?0.03) with patients’ overall survival (OS) while there was a positive significant correlation between EphA4 and OS (p?=?0.007). In conclusion, this study suggests that FAK, MR-1, EphA4, Src, and PKC may be used as early diagnostic and prognostic markers with high sensitivity and specificity in AML patients and thus may be incorporated into the patients’ early diagnostic and prognostic panels.

     

Focal adhesion kinase: predictor of tumour response and risk factor for recurrence after neoadjuvant chemoradiation in rectal cancer

Rectal cancer represents about 30% of colorectal cancers, being around 50% locally advanced at presentation. Chemoradiation (CRT) followed by total mesorectal excision is the standard of care for these locally advanced stages. However, it is not free of adverse effects and toxicity and the complete pathologic response rate is between 10% and 30%. This makes it extremely important to define factors that can predict response to this therapy. Focal adhesion kinase (FAK) expression has been correlated with worse prognosis in several tumours and its possible involvement in cancer radio‐ and chemosensitivity has been suggested; however, its role in rectal cancer has not been analysed yet. To analyse the association of FAK expression with tumour response to CRT in locally advanced rectal cancer. This study includes 73 patients with locally advanced rectal cancer receiving standard neoadjuvant CRT followed by total mesorectal excision. Focal adhesion kinase protein levels were immunohistochemically analysed in the pre‐treatment biopsies of these patients and correlated with tumour response to CRT and patients survival. Low FAK expression was significantly correlated with local and distant recurrence (P = 0.013). Low FAK expression was found to be a predictive marker of tumour response to neoadjuvant therapy (P = 0.007) and patients whose tumours did not express FAK showed a strong association with lower disease‐free survival (P = 0.01). Focal adhesion kinase expression predicts neoadjuvant CRT response in rectal cancer patients and it is a clinically relevant risk factor for local and distant recurrence.     

局部黏着斑激酶作为肿瘤治疗靶点的研究进展

局部黏着斑激酶（focal adhesion kinase,FAK）是一种非受体型酪氨酸蛋白激酶,是细胞内重要的骨架蛋白与多种信号通路的关键分子。FAK在肿瘤发生、发展、迁移和侵袭的各个阶段都具有重要作用,FAK已经被当作潜在的肿瘤治疗靶点来研究。该综述将对FAK与肿瘤的关系以及FAK作为肿瘤治疗靶点的研究进展进行探讨。     

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.     

Prognostic and clinical significance of focal adhesion kinase expression in breast cancer: A systematic review and meta-analysis

BackgroundThe prognostic significance of focal adhesion kinase (FAK) in breast cancer remains controversial. Here, we conducted a meta-analysis to explore the prognostic value of FAK expression in breast cancer.Materials and methodsPossible prognostic significance of protein or mRNA expression of FAK in breast cancer was investigated with searches of electronic databases for relevant publications. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were extracted from eligible studies.ResultsA total of eight eligible studies which included 2604 participants were analyzed in this meta-analysis. Increased expression of FAK protein was found to significantly correlate with shorter overall survival (OS) (HR = 1.43, 95% CI: 1.12–1.83; P = 0.004), and not with disease-free survival (HR = 1.31, 95% CI: 0.92–1.85; P = 0.14). Elevated FAK protein expression was also associated with negative estrogen receptor (ER) expression (OR, 1.34; 95% CI, 1.06–1.68; P = 0.01), negative progesterone receptor (PR) expression (OR, 1.54; 95% CI, 1.22–1.93; P < 0.001), positive human epidermal growth factor receptor 2 (HER2) expression (OR, 1.64; 95% CI, 1.28–2.09; P < 0.001), triple-negative breast cancer (TNBC) (OR, 1.57; 95% CI, 1.14–2.17; P = 0.006), high nuclear grade (OR, 1.70; 95% CI, 1.05–2.78; P = 0.03), high Ki-67 expression level (OR, 2.87; 95% CI, 1.94–4.24; P < 0.001), and positive p53 status (OR, 2.28; 95% CI, 1.58–3.29; P < 0.001).ConclusionOur meta-analysis identifies an association between increased FAK protein expression and worse OS among breast cancer patients. Moreover, enhanced FAK expression is associated with negative ER expression, negative PR expression, positive HER2 expression, TNBC, high nuclear grade, high Ki-67 expression level, and positive p53 status in breast carcinoma.     

The roles of JK-1 (FAM134B) expressions in colorectal cancer

The aims of the present study are to investigate the clinicopathological correlations of JK-1(FAM134B) expression and its relationship to carcinogenesis in a colorectal adenoma–adenocarcinoma model. JK-1(FAM134B) protein expression was studied in a colon cancer cell line by Western blot and immunocytochemistry. JK-1(FAM134B) expression profiles at mRNA and protein levels were investigated in cancer tissues from 236 patients with colorectal adenocarcinoma and 32 patients with colorectal adenoma using real-time polymerase chain reaction and immunohistochemistry. The findings were then correlated with the clinicopathological features of these tumours. JK-1(FAM134B) protein was demonstrated in the colon cancer cells by Western blot. The protein was located in the nuclei of the tumour cells at both cellular and tissue levels. In colorectal adenocarcinomas, lower levels of JK-1(FAM134B) protein expression were associated with younger age (p=0.032), larger tumour size (p=0.004), advanced cancer stages (p=0.016) and higher rates of cancer recurrence (p=0.04). Also, lower levels of JK-1(FAM134B) mRNA expression were associated with advanced cancer stages (p=0.02) and presence of lymphovascular invasion (p=0.014). Higher JK-1(FAM134B) mRNA and protein expression levels were identified in adenomas and non-neoplastic mucosae, compared to carcinomas (p=0.005). To conclude, JK-1(FAM134B) mRNA expression and JK1 (FAM134B) protein levels varied with the different stages of progression of colorectal tumours. The expression levels of the gene were associated with clinicopathological features in patients with colorectal adenocarcinoma suggesting that JK-1(FAM134B) gene has roles in controlling some steps in the development of the invasive phenotypes from colorectal adenoma to early staged as well as advanced staged colorectal adenocarcinomas.     

Focal Adhesion Kinase Functions as an Akt Downstream Target in Migration of Colorectal Cancer Cells

Migration is a complex process that, besides its various physiological functions in embryogenesis and adult tissues, plays a crucial role in cancer cell invasion and metastasis. The focus of this study is the involvement and collaboration of Akt, focal adhesion kinase (FAK), and Src kinases in migration and invasiveness of colorectal cancer cells. We show that all three kinases can be found in one protein complex; nevertheless, the interaction between Akt and Src is indirect and mediated by FAK. Interestingly, induced Akt signaling causes an increase in tyrosine phosphorylation of FAK, but this increase is attenuated by the Src inhibitor SU6656. We also show that active Akt strongly stimulates cell migration, but this phenomenon is fully blocked by FAK knockdown or partly by inhibition of Src kinase. In addition, we found that all three kinases were indispensable for the successful invasion of colorectal cancer cells. Altogether, the presented data bring new insights into the mechanism how the phosphatidylinositol-3-kinase (PI3-K)/Akt pathway can influence migration of colorectal adenocarcinoma cells. Because FAK is indispensable for cell movements and functions downstream of Akt, our results imply FAK kinase as a potential key molecule during progression of tumors with active PI3-K/Akt signaling.     

Understanding the Roles of FAK in Cancer: Inhibitors,Genetic Models,and New Insights

Focal adhesion kinase (FAK) is a protein tyrosine kinase that regulates cellular adhesion, motility, proliferation and survival in various types of cells. Interestingly, FAK is activated and/or overexpressed in advanced cancers, and promotes cancer progression and metastasis. For this reason, FAK became a potential therapeutic target in cancer, and small molecule FAK inhibitors have been developed and are being tested in clinical phase trials. These inhibitors have demonstrated to be effective by inducing tumor cell apoptosis in addition to reducing metastasis and angiogenesis. Furthermore, several genetic FAK mouse models have made advancements in understanding the specific role of FAK both in tumors and in the tumor environment. In this review, we discuss FAK inhibitors as well as genetic mouse models to provide mechanistic insights into FAK signaling and its potential in cancer therapy.     

Discovery of a Novel Immune Gene Signature with Profound Prognostic Value in Colorectal Cancer: A Model of Cooperativity Disorientation Created in the Process from Development to Cancer

Immune response-related genes play a major role in colorectal carcinogenesis by mediating inflammation or immune-surveillance evasion. Although remarkable progress has been made to investigate the underlying mechanism, the understanding of the complicated carcinogenesis process was enormously hindered by large-scale tumor heterogeneity. Development and carcinogenesis share striking similarities in their cellular behavior and underlying molecular mechanisms. The association between embryonic development and carcinogenesis makes embryonic development a viable reference model for studying cancer thereby circumventing the potentially misleading complexity of tumor heterogeneity. Here we proposed that the immune genes, responsible for intra-immune cooperativity disorientation (defined in this study as disruption of developmental expression correlation patterns during carcinogenesis), probably contain untapped prognostic resource of colorectal cancer. In this study, we determined the mRNA expression profile of 137 human biopsy samples, including samples from different stages of human colonic development, colorectal precancerous progression and colorectal cancer samples, among which 60 were also used to generate miRNA expression profile. We originally established Spearman correlation transition model to quantify the cooperativity disorientation associated with the transition from normal to precancerous to cancer tissue, in conjunction with miRNA-mRNA regulatory network and machine learning algorithm to identify genes with prognostic value. Finally, a 12-gene signature was extracted, whose prognostic value was evaluated using Kaplan–Meier survival analysis in five independent datasets. Using the log-rank test, the 12-gene signature was closely related to overall survival in four datasets (GSE17536, n = 177, p = 0.0054; GSE17537, n = 55, p = 0.0039; GSE39582, n = 562, p = 0.13; GSE39084, n = 70, p = 0.11), and significantly associated with disease-free survival in four datasets (GSE17536, n = 177, p = 0.0018; GSE17537, n = 55, p = 0.016; GSE39582, n = 557, p = 4.4e-05; GSE14333, n = 226, p = 0.032). Cox regression analysis confirmed that the 12-gene signature was an independent factor in predicting colorectal cancer patient’s overall survival (hazard ratio: 1.759; 95% confidence interval: 1.126–2.746; p = 0.013], as well as disease-free survival (hazard ratio: 2.116; 95% confidence interval: 1.324–3.380; p = 0.002).     

Targeting of the protein interaction site between FAK and IGF-1R

The interaction of focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) plays an important role in cancer cell survival. Targeting this interaction with small molecule drugs could be a novel strategy in cancer therapy. By a series of pull-down assays using GST-tagged FAK fragments and His-tagged IGF-1R intracellular fragments, we showed that the FAK-NT2 (a.a. 127-243) domain directly interacts with the N-terminal part of the IGF-1R intracellular domain. Overexpressed FAK-NT2 domain was also shown to co-localize with IGF-1R in pancreatic cells. Computational modeling was used to predict the binding configuration of these two domains and to screen for small molecules binding to the interaction site. This strategy successfully identified a lead compound that disrupts FAK/IGF-1R interaction.     

Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization.

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n = 7) and TB-48 (n = 3) inhibited AP 125I-Ang II internalization (80+/-7% inhibition; p<0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n = 3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n = 5) or herbimycin A [150 ng/ml] (n = 4) also inhibited MSC 125I-Ang II internalization (82+/-11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.     

Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples

Aberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.     

Re-expression of AKAP12 inhibits progression and metastasis potential of colorectal carcinoma in vivo and in vitro

Background

AKAP12/Gravin (A kinase anchor protein 12) is one of the A-kinase scaffold proteins and a potential tumor suppressor gene in human primary cancers. Our recent study demonstrated the highly recurrent loss of AKAP12 in colorectal cancer and AKAP12 reexpression inhibited proliferation and anchorage-independent growth in colorectal cancer cells, implicating AKAP12 in colorectal cancer pathogenesis.

Methods

To evaluate the effect of this gene on the progression and metastasis of colorectal cancer, we examined the impact of overexpressing AKAP12 in the AKAP12-negative human colorectal cancer cell line LoVo, the single clone (LoVo-AKAP12) compared to mock-transfected cells (LoVo-CON).

Results

pCMV6-AKAP12-mediated AKAP12 re-expression induced apoptosis (3% to 12.7%, p<0.01), migration (89.6±7.5 cells to 31.0±4.1 cells, p<0.01) and invasion (82.7±5.2 cells to 24.7±3.3 cells, p<0.01) of LoVo cells in vitro compared to control cells. Nude mice injected with LoVo-AKAP12 cells had both significantly reduced tumor volume (p<0.01) and increased apoptosis compared to mice given AKAP12-CON. The quantitative human-specific Alu PCR analysis showed overexpression of AKAP12 suppressed the number of intravasated cells in vivo (p<0.01).

Conclusion

These results demonstrate that AKAP12 may play an important role in tumor growth suppression and the survival of human colorectal cancer.