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1.  Clinicopathological significance of LRP16 protein in 336 gastric carcinoma patients 
AIM: To investigate the expression of leukemia related protein 16 (LRP16), and the possible relationship between LRP16 expression and clinicopathological indices in 336 gastric carcinoma patients.
METHODS: Immunohistochemistry was used to detect LRP16 expression in 336 cases of paraffin-embedded gastric carcinoma tissues and 60 cases of distal normal mucosa. The relationships between LRP16 expression and patients’ age, tumor size, histological grade, clinical stage, metastatic status and prognosis were analysed.
RESULTS: The expression of LRP16 was 58.6% (197/336) in gastric carcinoma and 31.7% (19/60) in distal normal gastric mucosa. The expression of LRP16 in carcinoma was significantly higher than that in normal mucosa tissues (χ2 = 14.929, P = 0.001). LRP16 protein expression was found in 44.1% (63/143) carcinomas at stage I and II, and 69.4% (134/193) carcinomas at stage III and IV (χ2 = 21.804, P = 0.001), and in 56.9% (182/320) of cancers without metastasis but 93.8% (15/16) of those with metastasis (χ2 = 8.543, P = 0.003). The expression of LRP16 was correlated with tumor size, infiltrative depth, clinical stage, lymphatic invasion and distant metastasis (all P < 0.05). Follow-up data showed that there was a significant difference in median survival time between cancer patients with expression of LRP16 (27.0 mo) and those without (48.0 mo, Log rank =31.644, P = 0.001).
CONCLUSION: The expression of LRP16 may be associated with invasion, metastasis and prognosis of gastric cancer.
PMCID: PMC2761564  PMID: 19824120
Gastric neoplasms; Immunohistochemistry; Leukemia related protein 16; Prognosis
2.  Low Level of Low-Density Lipoprotein Receptor-Related Protein 1 Predicts an Unfavorable Prognosis of Hepatocellular Carcinoma after Curative Resection 
PLoS ONE  2012;7(3):e32775.
Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).
LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.
Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.
LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.
PMCID: PMC3299691  PMID: 22427881
3.  Expression of LRP and MDR1 in locally advanced breast cancer predicts axillary node invasion at the time of rescue mastectomy after induction chemotherapy 
Breast Cancer Research  2001;3(3):183-191.
Axillary node status after induction chemotherapy for locally advanced breast cancer has been shown on multivariate analysis to be an independent predictor of relapse. However, it has been postulated that responders to induction chemotherapy with a clinically negative axilla could be spared the burden of lymphadenectomy, because most of them will not show histological nodal invasion. P-glycoprotein expression in the rescue mastectomy specimen has finally been identified as a significant predictor of patient survival.
We studied the expression of the genes encoding multidrug resistance associated protein (MDR1) and lung cancer associated resistance protein (LRP) in formalin-fixed, paraffin-embedded tumor samples from 52 patients treated for locally advanced breast cancer by means of induction chemotherapy followed by rescue mastectomy. P-glycoprotein expression was assessed by means of immunohistochemistry before treatment in 23 cases, and by means of reverse-transcriptase-mediated polymerase chain reaction (RT-PCR) after treatment in 46 (6 failed). LRP expression was detected by means of immunohistochemistry, with the LRP-56 monoclonal antibody, in 31 cases before treatment. Immunohistochemistry for detecting the expression of c-erb-B2, p53, Ki67, estrogen receptor and progesterone receptor are routinely performed in our laboratory in every case, and the results obtained were included in the study. All patients had received between two and six cycles of standard 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) chemotherapy, with two exceptions [one patient received four cycles of a docetaxel-adriamycin combination, and the other four cycles of standard cyclophosphamide-methotrexate-5-fluorouracil (CMF) polychemotherapy]. Response was assessed in accordance with the Response Evaluation Criteria In Solid Tumors (RECIST). By these, 2 patients achieved a complete clinical response, 37 a partial response, and the remaining 13 showed stable disease. This makes a total clinical response rate of 75.0%. None achieved a complete pathological response.
MDR1 mRNA expression detected by RT-PCR was associated with the presence of invaded axillary nodes at surgery in 18/22 cases (81.8%), compared with 13/24 (54.2%) in the group with undetectable MDR1 expression. This difference was statistically significant (P < 0.05). LRP expression in more than 20% of tumor cells before any treatment was associated with axillary nodal metastasis after chemotherapy and rescue mastectomy in 17/23 cases, compared with 3/8 in nonexpressors. Again, this difference was highly significant (P < 0.01). LRP expression before treatment and MDR1 mRNA expression after treatment were significantly interrelated (P < 0.001), which might reflect the presence of chemoresistant clones liable to metastasize to the regional nodes. Persistence of previously detected MDR1-positivity after treatment (7/9 compared with 0/2 cases) was significantly associated with axillary node metastasis (P < 0.05). Finally, in a logistic regression multivariate model, histology other than ductal, a Ki67 labeling index of at least 20% and the combination of LRP and MDR1 positivity emerged as independent predictors of axillary node invasion at the time of rescue mastectomy.
The expression of different genes involved in resistance to chemotherapy, both before and after treatment with neoadjuvant, is associated with the presence of axillary node invasion at rescue surgery in locally advanced breast cancer. This might reflect the presence of intrinsically resistant clones before any form of therapy, which persist after it, and could be helpful both for prognosis and for the choice of individual treatment.
PMCID: PMC30705  PMID: 11305953
axillary nodes; chemotherapy; LRP; multidrug resistant; neoadjuvant
4.  Expression of Bmi-1, P16, and CD44v6 in Uterine Cervical Carcinoma and Its Clinical Significance 
Cancer Biology & Medicine  2012;9(1):48-53.
Bmi-1, a putative proto-oncogene, is a core member of the polycomb gene family, which is expressed in many human tumors. The p16 protein negatively regulated cell proliferation, whereas CD44v6 is associated with proliferation as an important protein. Additionally, CD44v6 is an important nuclear antigen closely correlated to tumor metastasis. The present study aims to investigate the expression and significance of Bmi-1, p16, and CD44v6 in uterine cervical carcinoma (UCC).
A total of 62 UCC, 30 cervical neoplasic, and 20 normal cervical mucosal tissues were used in the current study. The expression of Bmi-1, p16, and CD44v6 in these tissues was determined using immunohistochemical assay. The relationships among the expression of these indices, the clinicopathologic features of UCC, and the survival rate of UCC patients were also discussed. The correlation between Bmi-1 protein expression and p16 or CD44v6 protein in UCC was analyzed.
The expression of Bmi-1, p16, and CD44v6 was significantly high in cervical carcinoma compared with that in the cervical neoplasia and normal colorectal mucosa (P<0.05). The over-expression of Bmi-1 protein in UCC was apparently related to the distant metastasis (P<0.01) and the tumor, nodes and metastasis-classification, i.e. the TNM staging, World Health Organization (P<0.05). Nevertheless, the positive expression of p16 protein in UCC was not significantly associated with the clinicopathologic features (P>0.05). The Kaplan–Meier survival analysis showed that the over-expression of Bmi-1 significantly decreased the survival rate of UCC patients (P<0.05). A strong correlation indicated that there was statistical significance between the expression of Bmi-1 and CD44V6 proteins in UCC (r=0.419, P=0.001).
The over-expression of Bmi-1 and CD44v6 protein closely correlate to the tumorigenesis, metastasis, and prognosis of UCC. Bmi-1 and CD44v6 may be used to predict the prognosis of cervical carcinoma. Bmi-1 may indirectly regulate the expression of CD44v6 in UCC patients. The positive expression of p16 protein is possibly associated with the tumorigenesis, but not with the metastasis or prognosis of UCC.
PMCID: PMC3643647  PMID: 23691455
Bmi-1 protein; P16 protein; CD44v6; uterine cervical carcinoma; immunohistochemistry
5.  Chemokine CXCL14 is associated with prognosis in patients with colorectal carcinoma after curative resection 
The chemokine CXCL14 has been reported to play an important role in the progression of many malignancies such as breast cancer and papillary thyroid carcinoma, but the role of CXCL14 in colorectal carcinoma (CRC) remains to be established. The purpose of this study was to investigate the expression pattern and significance of CXCL14 in CRC progression.
265 colorectal carcinoma specimens and 129 matched adjacent normal colorectal mucosa specimens were collected. Expression of CXCL14 in clinical samples was examined by immunostaining. The effect of CXCL14 on colorectal carcinoma cell proliferation was measured by MTT assay, BrdU incorporation assay and colony formation assay. The impact of CXCL14 on migration and invasion of colorectal carcinoma cells was determined by transwell assay and Matrigel invasion assay, respectively.
CXCL14 expression was significantly up-regulated in tumor tissues compared with adjacent nontumorous mucosa tissues (P < 0.001). Tumoral CXCL14 expression levels were significantly correlated with TNM (Tumor-node-metastasis) stage, histodifferentiation, and tumor size. In multivariate Cox regression analysis, high CXCL14 expression in tumor specimens (n = 91) from stage I/II patients was associated with increased risk for disease recurrence (risk ratio, 2.92; 95% CI, 1.15-7.40; P = 0.024). Elevated CXCL14 expression in tumor specimens (n = 135) from stage III/IV patients correlated with worse overall survival (risk ratio, 3.087; 95% CI, 1.866-5.107; P < 0.001). Functional studies demonstrated that enforced expression of CXCL14 in SW620 colorectal carcinoma cells resulted in more aggressive phenotypes. In contrast, knockdown of CXCL14 expression could mitigate the proliferative, migratory and invasive potential of HCT116 colorectal carcinoma cells.
Taken together, CXCL14 might be a potential novel prognostic factor to predict the disease recurrence and overall survival and could be a potential target of postoperative adjuvant therapy in CRC patients.
PMCID: PMC3551837  PMID: 23294544
Colorectal carcinoma; CXCL14; Disease-free survival; Overall survival; Prognosis
6.  Stromal LRP1 in lung adenocarcinoma predicts clinical outcome 
LRP1 is a broadly-expressed receptor that binds multiple extracellular ligands and participates in protein clearance. LRP1 is expressed numerous cancers, but its role in lung cancer has not been characterized. Here, we investigate the relationship between LRP1 and lung cancer.
Experimental Design
LRP1 mRNA levels were determined in lung tumors from several large, multicenter studies. LRP1 protein localization was determined by immunohistochemical analysis of lung tumor microarrays. Normal fibroblasts, fibroblasts treated with the LRP1 inhibitor RAP, and LRP1 null fibroblasts were co-cultured with three independent lung cancer cell lines to investigate the role of LRP1 on tumor cell proliferation.
LRP1 mRNA levels are significantly decreased in lung tumors relative to non-tumorous lung tissue. Lower expression of LRP1 in lung adenocarcinomas correlates with less favorable clinical outcome in a cohort of 439 patients. Immunohistochemical analysis demonstrates that LRP1 is primarily expressed in stromal cells in 94/111 lung cancers, with very little protein found in cancer cells. A growth suppressive function of mouse embryonic fibroblast cells (MEF) was observed in three lung cancer cell lines tested (H460, H2347, and HCC4006 cells); growth suppression was blocked by the LRP1 inhibitor, RAP. LRP1 deletion in fibroblasts reduced the ability of MEF cells to suppress tumor cell mitosis. In a validation set of adenocarcinomas, we confirmed a significant positive correlation between both LRP1 mRNA and protein levels and favorable clinical outcomes.
LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non-cell autonomously suppress lung tumor cell proliferation.
PMCID: PMC3079007  PMID: 21325077
7.  Multidrug resistant proteins: P-glycoprotein and lung resistance protein expression in retinoblastoma 
The British Journal of Ophthalmology  2004;88(12):1521-1526.
Background/aim: Retinoblastoma is the commonest primary intraocular tumour in children. Chemotherapy now plays a big part in the treatment of these tumours. There is not much information about the role of the multidrug resistance proteins (MDR)—P-glycoprotein (P-gp) and vault protein lung resistance protein (LRP)—in retinoblastoma. The authors investigated the expression of P-gp and LRP in retinoblastoma and correlated them clinicopathologically.
Methods: Among 60 retinoblastomas, 40 tumours were not subjected to preoperative or postoperative chemotherapy and 20 tumours were subjected to postoperative chemotherapy. In this cohort 27 tumours had no invasion and 33 tumours had invasion of choroid, optic nerve, and orbit. P-gp and LRP expression were studied by immunohistochemistry. Immunoanalysis was done semiquantitatively.
Results: Among the 60 tumours P-gp was expressed in 23 (38%) tumours and LRP was expressed in 35 (58%). P-gp was expressed in 11/27 (40%) tumours with no invasion and in 12/33 (36%) tumours with invasion. LRP was expressed in 15/27 (55%) tumours with no invasion and in 20/33 (60%) tumours with invasion. Both P-gp and LRP were negative in three tumours with invasion, which had later developed bone marrow metastasis. There was no correlation between P-gp and LRP expression with invasion, differentiation and laterality of the tumours and response to treatment.
Conclusion: Retinoblastoma expresses P-gp and LRP intrinsically before chemotherapy and none of these proteins predicted the response to chemotherapy. Thus, further studies are needed to understand the significance of the expression of the P-gp and LRP proteins in retinoblastoma.
PMCID: PMC1772413  PMID: 15548804
lung resistance protein; P-glycoprtein; multidrug resistance; retinoblastoma; immunohistochemistry; chemotherapy
8.  Verification of predicted alternatively spliced Wnt genes reveals two new splice variants (CTNNB1 and LRP5) and altered Axin-1 expression during tumour progression 
BMC Genomics  2006;7:148.
Splicing processes might play a major role in carcinogenesis and tumour progression. The Wnt pathway is of crucial relevance for cancer progression. Therefore we focussed on the Wnt/β-catenin signalling pathway in order to validate the expression of sequences predicted as alternatively spliced by bioinformatic methods. Splice variants of its key molecules were selected, which may be critical components for the understanding of colorectal tumour progression and may have the potential to act as biological markers. For some of the Wnt pathway genes the existence of splice variants was either proposed (e.g. β-Catenin and CTNNB1) or described only in non-colon tissues (e.g. GSK3β) or hitherto not published (e.g. LRP5).
Both splice variants – normal and alternative form – of all selected Wnt pathway components were found to be expressed in cell lines as well as in samples derived from tumour, normal and healthy tissues. All splice positions corresponded totally with the bioinformatical prediction as shown by sequencing. Two hitherto not described alternative splice forms (CTNNB1 and LRP5) were detected. Although the underlying EST data used for the bioinformatic analysis suggested a tumour-specific expression neither a qualitative nor a significant quantitative difference between the expression in tumour and healthy tissues was detected. Axin-1 expression was reduced in later stages and in samples from carcinomas forming distant metastases.
We were first to describe that splice forms of crucial genes of the Wnt-pathway are expressed in human colorectal tissue. Newly described splicefoms were found for β-Catenin, LRP5, GSK3β, Axin-1 and CtBP1. However, the predicted cancer specificity suggested by the origin of the underlying ESTs was neither qualitatively nor significant quantitatively confirmed. That let us to conclude that EST sequence data can give adequate hints for the existence of alternative splicing in tumour tissues. That no difference in the expression of these splice forms between cancerous tissues and normal mucosa was found, may indicate that the existence of different splice forms is of less significance for cancer formation as suggested by the available EST data. The currently available EST source is still insufficient to clearly deduce colon cancer specificity. More EST data from colon (tumour and healthy) is required to make reliable predictions.
PMCID: PMC1523213  PMID: 16772034
9.  Clinicopathologic significance of GLUT1 expression and its correlation with Apaf-1 in colorectal adenocarcinomas 
AIM: To investigate the role of glucose transporter 1 (GLUT1) expression in colorectal carcinogenesis and evaluate the correlation with clinicopathological parameters and apoptosis-activating factor-1 (Apaf-1) expression in colorectal adenocarcinomas.
METHODS: We used tissue microarrays consisting of 26 normal mucosa, 50 adenomas, 515 adenocarcinomas, and 127 metastatic lesions. Medical records were reviewed and clinicopathological analysis was performed.
RESULTS: GLUT1 expression was absent in normal mucosa and low or moderately apparent in 19 cases (38.0%) of 50 adenomas. However, GLUT1 expression was detected in 423 (82.1%) of 515 adenocarcinomas and in 96 (75.6%) of 127 metastatic lesions. GLUT1 expression was significantly correlated with female gender (P = 0.009), non-mucinous tumor type (P = 0.045), poorer differentiation (P = 0.001), lymph node metastasis (P < 0.001), higher AJCC and Dukes stage (P < 0.001 and P < 0.001, respectively). There was a significant inverse correlation between GLUT1 expression and Apaf-1 expression (P = 0.001). In univariate survival analysis, patients with GLUT1 expression demonstrated poor overall survival and disease-free survival (P = 0.047 and P = 0.021, respectively, log-rank test).
CONCLUSION: GLUT1 expression was frequently increased in adenocarcinomas and metastatic lesions. GLUT1 expression was significantly correlated with poorer clinicopathologic phenotypes and survival of patients with colorectal adenocarcinomas.
PMCID: PMC3080722  PMID: 21528061
Adenocarcinoma; Colorectum; Glucose transporter 1; Apoptosis-activating factor-1; Prognosis; Survival
10.  Expression of the RON receptor tyrosine kinase and its association with gastric carcinoma versus normal gastric tissues 
BMC Cancer  2008;8:353.
Recepteur d'origine nantais (RON) is a receptor tyrosine kinase that is activated by a serum-derived, macrophage stimulating protein (MSP) growth factor and is expressed in many malignant tumors. The aim of the present study was to reveal the protein expression profile of RON and its relationship with clinicopathological characteristics of gastric carcinoma and prognosis.
Gastric carcinoma tissue from 98 patients, along with 29 specimens of paraneoplastic tissue and 10 specimens of normal gastric mucosa, were examined by immunohistochemistry (IHC). Western blot analysis of 19 samples of gastric carcinoma tissue and corresponding paraneoplastic tissue, 8 specimens of normal gastric mucosa, and 2 specimens of normal lymph node samples also detected expression of a splice variant of RON, RONΔ165. All samples obtained were accompanied by patient follow-up data that ranged from 3 to 89 months (median time: 22 months).
The rate of positive RON expression differed significantly between gastric carcinoma tissues [56.1%, (55/98)] and paraneoplastic tissues [25.6%, (8/29)] (p = 0.007). In contrast, RON expression was absent in normal gastric mucosa samples. RON expression positively correlated with the invasive depth of the tumor (p = 0.019), perigastric lymph nodes metastasis (p = 0.019), and TNM stage (p = 0.001). However, RON expression was independent of tumor growth pattern according to Bormann criteria (p = 0.209), histopathological grade (p = 0.196), and incidence of distant metastasis (p = 0.400). RON expression was not related to a patient's survival rate (p = 0.195). RONΔ165 was strongly expressed in fresh gastric carcinoma tissue, corresponding paraneoplastic tissue, and perigastric lymph nodes with metastatic carcinoma. In contrast, expression of RONΔ165 was not observed in normal gastric mucosa and normal lymph node tissue samples.
RON expression is significant in gastric carcinoma tissue and corresponding paraneoplastic tissue, but is not expressed in normal gastric mucosa. Expression of RONΔ165 was similarly observed in gastric carcinoma tissue and in metastases present in lymph node tissues. We hypothesize that RON and its splice variant play an important role in the occurrence, progression, and metastasis of gastric carcinoma, and therefore may represent a useful marker to evaluate the biological activity of gastric carcinoma.
PMCID: PMC2629483  PMID: 19040718
11.  The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer 
To detect the expression of multidrug resistance molecules P-glycoprotein (P-gp), Lung resistnce protein (LRP) and Multidrug resistance-associated protein (MRP) and analyze the relationship between them and the clinico-pathological features.
The expressions of P-gp, LRP and MRP in formalin-fixed paraffin-embedded tissue sections from 59 gastric cancer patients were determined by a labbelled Streptavidin-Peroxidase (SP) immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery.
The positive rates of P-gp, LRP, MRP were 86.4%, 84.7% and 27.1%, respectively. The difference between the positive rate of P-gp and MRP was significant statistically, as well as the difference between the expression of MRP and LRP. No significant difference was observed between P-gp and LRP, but the positively correlation between the expression of P-gp and LRP had been found. No significant correlation between the expression of P-gp, LRP, MRP and the grade of differentiation were observed. The expression of P-gp was correlated with clinical stages positively (r = 0.742), but the difference with the expression of P-gp in different stages was not significant.
The expressions of P-gp, LRP and MRP in patients with gastric cancer without prior chemotherapy are high, indicating that innate drug resistance may exist in gastric cancer.
PMCID: PMC2788536  PMID: 19930704
12.  Ectodomains of the LDL Receptor-Related Proteins LRP1b and LRP4 Have Anchorage Independent Functions In Vivo 
PLoS ONE  2010;5(4):e9960.
The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype.
Methods and Principal Findings
Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and γ-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain.
Conclusions and Significance
Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b) or perinatally (Lrp4) lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby preserving signaling thresholds that are critical for embryonic development, as well as for the clear, but poorly understood role of LRP1b in cancer.
PMCID: PMC2850915  PMID: 20383322
13.  NGAL Expression Is Elevated in Both Colorectal Adenoma–Carcinoma Sequence and Cancer Progression and Enhances Tumorigenesis in Xenograft Mouse Models 
There is growing evidence implicating that neutrophil gelatinase–associated lipocalin (NGAL) plays a role in the development and progression of cancers. However, the effect of NGAL in colorectal carcinoma (CRC) has not been clearly elucidated. In this study, we investigated the role of NGAL in the tumorigenesis and progression of CRC and evaluated the clinical value of NGAL expression.
Experimental Design
We examined NGAL expression in 526 colorectal tissue samples, including 53 sets of matched specimens (histologically normal mucosa, adenomas, and carcinomas) using immunohistochemical analysis. In CRCs, correlations between NGAL expression and clinicopathologic parameters were analyzed, and survival analysis was conducted. The role of NGAL was further tested using mouse xenograft models.
NGAL expression was elevated during the colorectal adenoma–carcinoma sequence both among the 526 cases (rs = 0.66, P < 0.001) and in the 53 sets of matched specimens (rs = 0.60, P < 0.001). In CRCs, NGAL expression was associated with cancer stage (P = 0.041) and tumor recurrence in stage II patients (P = 0.037). Survival analysis revealed that NGAL expression was an independent prognostic factor for overall survival (HR = 1.84, P = 0.004) and for disease-free survival of stage II patients (HR = 5.88, P = 0.021). In mouse models, the xenografts in cecum and spleen were heavier and more numerous in the group injected with NGAL-overexpressing CRC cells (P < 0.05).
NGAL overexpression may promote the tumorigenesis and progression of CRC. Detecting NGAL expression in tumor tissues may be useful for evaluating prognosis of patients with CRC.
PMCID: PMC3575684  PMID: 21622717
14.  Expression of the phosphorylated MEK5 protein is associated with TNM staging of colorectal cancer 
BMC Cancer  2012;12:127.
Activation of MEK5 in many cancers is associated with carcinogenesis through aberrant cell proliferation. In this study, we determined the level of phosphorylated MEK5 (pMEK5) expression in human colorectal cancer (CRC) tissues and correlated it with clinicopathologic data.
pMEK5 expression was examined by immunohistochemistry in a tissue microarray (TMA) containing 335 clinicopathologic characterized CRC cases and 80 cases of nontumor colorectal tissues. pMEK5 expression of 19 cases of primary CRC lesions and paired with normal mucosa was examined by Western blotting. The relationship between pMEK5 expression in CRC and clinicopathologic parameters, and the association of pMEK5 expression with CRC survival were analyzed respectively.
pMEK5 expression was significantly higher in CRC tissues (185 out of 335, 55.2%) than in normal tissues (6 out of 80, 7.5%; P < 0.001). Western blotting demonstrated that pMEK5 expression was upregulated in 12 of 19 CRC tissues (62.1%) compared to the corresponding adjacent nontumor colorectal tissues. Overexpression of pMEK5 in CRC tissues was significantly correlated to the depth of invasion (P = 0.001), lymph node metastasis (P < 0.001), distant metastasis (P < 0.001) and high preoperative CEA level (P < 0.001). Consistently, the pMEK5 level in CRC tissues was increased following stage progression of the disease (P < 0.001). Analysis of the survival curves showed a significantly worse 5-year disease-free (P = 0.002) and 5-year overall survival rate (P < 0.001) for patients whose tumors overexpressed pMEK5. However, in multivariate analysis, pMEK5 was not an independent prognostic factor for CRC (DFS: P = 0.139; OS: P = 0.071).
pMEK5 expression is correlated with the staging of CRC and its expression might be helpful to the TNM staging system of CRC.
PMCID: PMC3337320  PMID: 22458985
15.  Myofibrillogenesis regulator-1 overexpression is associated with poor prognosis of gastric cancer patients 
AIM: To investigate the expression of myofibrillogenesis regulator-1 (MR-1) in relation to clinicopathological parameters and postoperative survival in a group of Chinese patients with gastric cancer.
METHODS: In our previous study of human whole-genome gene expression profiling, the differentially expressed genes were detected in the gastric cancer and its adjacent noncancerous mucosa. We found that MR-1 was associated with the location and differentiation of tumors. In this study, MR-1 protein expression was determined by immunohistochemistry in specimens of primary cancer and the adjacent noncancerous tissues from gastric cancer patients. A set of real-time quantitative polymerase chain reaction assays based on the Universal ProbeLibrary-a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid hydrolysis probes-was designed specifically to detect the expression of MR-1 mRNA. The correlation was analyzed between the expression of MR-1 and other tumor characteristics which may influence the prognosis of gastric cancer patients. A retrospective cohort study on the prognosis was carried out and clinical data were collected from medical records.
RESULTS: MR-1 mRNA and protein could be detected in gastric cancer tissues as well as in matched noncancerous tissues. MR-1 was up-regulated at both mRNA (5.459 ± 0.639 vs 1.233 ± 0.238, P < 0.001) and protein levels (34.2% vs 13.2%, P = 0.003) in gastric cancer tissues. Correlation analysis demonstrated that high expression of MR-1 in gastric cancer was significantly correlated with clinical stage (P = 0.034). Kaplan-Meier analysis showed that the postoperative survival of the MR-1 positive group tended to be poorer than that of the MR-1 negative group, and the difference was statistically significant (P = 0.002). Among all the patients with stage I-IV carcinoma, the 5-year survival rates of MR-1 positive and negative groups were 50.40% and 12.70%, respectively, with respective median survival times of 64.27 mo (95%CI: 13.41-115.13) and 16.77 mo (95%CI: 8.80-24.74). Univariate and multivariate analyses were performed to compare the impact of MR-1 expression and other clinicopathological parameters on prognosis. In a univariate analysis on all 70 specimens, 6 factors were found to be significantly associated with the overall survival statistically: including MR-1 expression, depth of invasion, distant metastasis, lymph node metastasis, vascular invasion and the tumor node metastasis (TNM) stage based on the 7th edition of the International Union against Cancer TNM classification. To avoid the influence caused by univariate analysis, the expressions of MR-1 as well as other parameters were examined in multivariate Cox analysis. Clinicopathological variables that might affect the prognosis of gastric cancer patients were analyzed by Cox regression analysis, which showed that MR-1 expression and TNM stage were independent predictors of postoperative survival. The best mathematical multivariate Cox regression model consisted of two factors: MR-1 expression and TNM stage. Our results indicated that MR-1 protein could act as an independent marker for patient overall survival [Hazard ratio (HR): 2.215, P = 0.043].
CONCLUSION: MR-1 is an important variable that can be used to evaluate the outcome, prognosis and targeted therapy of gastric cancer patients.
PMCID: PMC3471113  PMID: 23082061
Myofibrillogenesis regulator-1; Gastric cancer; Real-time quantitative reverse transcriptase-polymerase chain reaction; Immunohistochemistry; Poor prognosis
16.  Lgr5 is a potential marker of colorectal carcinoma stem cells that correlates with patient survival 
Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) has recently been identified as an intestinal stem cell marker. In order to determine whether Lgr5 is a potential marker of cancer stem cells, we investigated whether Lgr5 expression correlated with Ki-67 expression and prognosis in colorectal carcinoma.
Lgr5 and Ki-67 expression were evaluated by immunohistochemistry in 192 colorectal carcinoma specimens. Selection of side population (SP) cells was performed by staining with Hoechest 33342, and Lgr5 expression in Colo205 SP cells was then detected by immunofluorescence.
Lgr5 expression was significantly higher in carcinoma than in normal mucosa (P=0.001). Lgr5 was positively correlated with histological grade (P=0.001), depth of invasion (P=0.001), lymph node metastasis (P=0.001), distant metastasis (P=0.004), pTNM stage (P=0.001), and Ki-67 (r=0.446, P=0.001). Multivariate analysis showed that the effect of Lgr5 on survival was independent of Ki-67 (P=0.037). In the in vitro study, Hoechst low-staining cells were counted in 7% of the Colo205 colon cancer cell line population, and Lgr5 expression was strikingly stronger in Hoechst low-staining cells than in high-staining cells (P=0.001).
These findings suggest that Lgr5 may play an important role in the progression and prognosis of colorectal carcinoma, and may be a potential new therapeutic target for the treatment of colorectal cancer patients. It may also be considered as a potential marker for colorectal cancer stem cells (CSCs).
PMCID: PMC3506563  PMID: 23153436
Colorectal cancer; Lgr5; Prognosis; Cancer stem cells
17.  Death decoy receptor overexpression and increased malignancy risk in colorectal cancer 
AIM: To evaluate human epidermal growth factor receptor 2 (HER2) and death decoy receptor (DcR3) as colorectal cancer prognostic indicators.
METHODS: Colorectal carcinoma specimens from 300 patients were analyzed by immunohistochemistry to detect the staining patterns of HER2 and DcR3. Classification of HER2 staining was carried out using the United States Food and Drug Administration semi-quantitative scoring system, with scores of 0 or 1+ indicating a tumor-negative (normal expression) status and scores of 2+ and 3+ indicating a tumor-positive (overexpression) status. Classification of DcR3 was carried out by quantitating the percentage of positive cells within the stained section, with < 10% indicating a tumor-negative status and ≥ 10% indicating a tumor-positive status. Correlation of the HER2 and DcR3 staining status with clinicopathological parameters [age, sex, tumor size, differentiation, and the tumor, node, metastasis (pTNM) classification] and survival was statistically assessed.
RESULTS: Tumor-positive status for HER2 and DcR3 was found in 18.33% and 58.33% of the 300 colorectal carcinoma specimens, respectively. HER2 tumor-positive status showed a significant correlation with tumor size (P = 0.003) but not with other clinicopathological parameters. DcR3 tumor-positive status showed a significant correlation with tumor differentiation (P < 0.001), pTNM stage (P < 0.001), and lymph node metastasis (P < 0.001). However, correlation coefficient analysis did not indicate that a statistically significant correlation exists between tumor-positive status for the HER2 and DcR3 overexpression (P = 0.236). Patients with specimens classified as DcR3-overexpressing had a significantly worse overall survival (OS) rate than those without DcR3 overexpression (median OS: 42.11 vs 61.21 mo; HR = 50.27, 95%CI: 44.90-55.64, P < 0.001). HER2 overexpression had no significant impact on median OS (35.10 mo vs 45.25 mo; HR = 44.40, 95%CI: 39.32-49.48, P = 0.344). However, patients with specimens classified as both HER2- and DcR3-overexpressing had a significantly poorer median OS than those with only HER2 overexpression (31.80 mo vs 52.20 mo; HR = 35.10, 95%CI: 22.04-48.16, P = 0.006).
CONCLUSION: HER2 overexpression is not an independent prognostic marker of colorectal cancer, but DcR3 overexpression is highly correlated with lymph node metastasis and poor OS.
PMCID: PMC3989983  PMID: 24764685
Colorectal carcinoma; Human epidermal growth factor receptor 2; Death decoy receptor; Immunohistochemistry; Prognosis
18.  Expressions of the γ2 chain of laminin-5 and secreted protein acidic and rich in cysteine in esophageal squamous cell carcinoma and their relation to prognosis 
Chinese Journal of Cancer  2011;30(1):69-78.
Previous studies have shown that the expressions of the γ2 chain of laminin-5 and secreted protein acidic and rich in cysteine (SPARC) play important roles in oncogenesis and the development of carcinoma. To assess the expressions of laminin-5 γ2 chain and SPARC in esophageal squamous cell carcinoma (SCC), and to clarify the prognostic significance of the expressions of laminin-5 γ2 chain and SPARC in esophageal SCC, we detected the expressions of laminin-5 γ2 chain and SPARC in cancer tissue and corresponding normal mucosa from 116 patients with advanced (stages II–IV) esophageal SCC using the tissue microarray-based immunohistochemistry and analyzed the correlation of the expressions with clinicopathologic characteristics and survival. We found that in normal esophageal tissues, laminin-5 γ2 chain was expressed in the basement membrane, whereas in esophageal SCC tissues, laminin-5 γ2 chain was expressed in the cytoplasm of carcinoma cells, with a positive rate of 72.4%. SPARC was not detected in normal esophageal mucosa, but was expressed in stromal fibroblasts in 84.6% of esophageal SCC cases and in cancer cells in 7.8% of esophageal SCC cases. There was a significant correlation between laminin-5 γ2 chain and stromal SPARC expression in esophageal SCC (Spearman' s rho = 0.423, P < 0.001). The expressions of both laminin-5 γ2 chain and stromal SPARC were correlated with survival (P = 0.032 and P = 0.034, respectively). In stage-II esophageal SCC, the expression of laminin-5 γ2 chain was significantly correlated with survival (P = 0.023), while the expression of SPARC was not significantly correlated with survival (P = 0.154). Patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis than did those lacking elevated levels of laminin-5 γ2 chain expression and/or elevated levels of SPARC expression (P = 0.001). In stage-II esophageal SCC, patients with elevated levels of laminin-5 γ2 chain and SPARC expressions had a poorer prognosis (P < 0.001). These results suggest that laminin-5 γ2 chain and SPARC may play roles in the progression of esophageal SCC and their simultaneous expression is correlated with poorer prognosis, especially in patients with stage-II SCC.
PMCID: PMC4012265  PMID: 21192846
Laminin-5 γ2; SPARC; esophageal cancer; squamous cell carcinoma; pathology
19.  Clinical significance of Ankyrin repeat domain 12 expression in colorectal cancer 
Ankyrin repeat domain 12 (ANKRD12), is encoding a 224 kDa nuclear protein and most conserved at its N-terminal ankyrin repeats region and the C-terminal co-activator interacting domain. The aim of this study was to investigate the ANKRD12 mRNA expression in colorectal cancer (CRC) tumor tissues and the normal adjacent mucosa and its potential relevance to clinicopathological characteristics and prognosis.
Surgical specimens of tumor tissues (n = 68) and adjacent normal mucosa (n = 51) were obtained from CRC patients. The ANKRD12 mRNA expression was measured by quantitative real time reverse transcriptase polymerase chain reaction. The relationship between ANKRD12 mRNA expression and clinicopathological features was analyzed by appropriate statistics. Kaplan–Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between ANKRD12 expression and prognosis of CRC patients.
The relative mRNA expression of ANKRD12 were significantly lower in CRC tumor tissues than in the normal adjacent mucosa (P < 0.001), and the cases with low ANKRD12 expression showed a higher frequency of liver metastasis (P = 0.015). Kaplan–Meier analysis indicated that patients (CRC without liver metastasis) with low ANKRD12 expression had poor overall survival (P = 0.041). Multivariate analysis showed that low ANKRD12 expression was an independent predictor of overall survival.
This study revealed that ANKRD12 mRNA were down regulated in CRC tumor tissues and low ANKRD12 expression was correlated with liver metastasis and poor survival of CRC patients.
PMCID: PMC3689078  PMID: 23718802
Ankyrin repeat domain 12 (ANKRD12); Colorectal cancer; Metastasis; Prognostic factor
20.  Immunohistochemical Expression of Estrogen and Progesterone Receptors in Human Colorectal Adenoma and Carcinoma Using Specified Automated Cellular Image Analysis System: A Clinicopathological Study 
Oman Medical Journal  2011;26(5):307-314.
To evaluate the immunohistochemical expression of estrogen receptors (ER) and progesterone receptors (PR) in colorectal adenoma and adenocarcinoma and to correlate this immunohistochemical expression with different clinicopathological parameters.
The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas and a control group of 20 samples of non-tumorous colonic tissue, were included in the study.
The frequency of expression of ER and PR showed a gradual increase from control through adenoma to carcinoma. The frequencies of expression of ER in the control, adenoma and carcinoma were (10%, 15.15% and 42.42% respectively, p<0.001), while the frequency of expression for PR were (10%, 24.24% and 36.36% respectively, p<0.001). Strong ER and PR staining was mainly seen in carcinoma cases (42.42%, 36.36%, respectively) in comparison with adenoma (9.09%, 15.15%, respectively) and control (0%, 0%, respectively). The three digital parameters of ER and PR immunohistochemical expression (Area [A], Number of objects [N], and intensity [I]) were significantly increased in a sequence of normal mucosa-adenoma-carcinoma. There was a significant positive correlation between ER and PR in adenoma (r=0.312, p=0.034) and carcinoma (r=0.321, p=0.0398).
ER and PR expression increased in a sequence of; normal colonic mucosa-adenoma-carcinoma, and a positive correlation was observed between ER and PR expression in colonic adenoma and carcinoma specimen indicating that ER and PR may play a role in colorectal carcinogenesis.
PMCID: PMC3215447  PMID: 22125723
Estrogen receptors; Progesterone receptors; Colorectal adenoma; Colorectal carcinoma
21.  An LRP5 Receptor with Internal Deletion in Hyperparathyroid Tumors with Implications for Deregulated WNT/β-Catenin Signaling 
PLoS Medicine  2007;4(11):e328.
Hyperparathyroidism (HPT) is a common endocrine disorder with incompletely understood etiology, characterized by enlarged hyperactive parathyroid glands and increased serum concentrations of parathyroid hormone and ionized calcium. We have recently reported activation of the Wnt signaling pathway by accumulation of β-catenin in all analyzed parathyroid tumors from patients with primary HPT (pHPT) and in hyperplastic parathyroid glands from patients with uremia secondary to HPT (sHPT). Mechanisms that may account for this activation have not been identified, except for a few cases of β-catenin (CTNNB1) stabilizing mutation in pHPT tumors.
Methods and Findings
Reverse transcription PCR and Western blot analysis showed expression of an aberrantly spliced internally truncated WNT coreceptor low-density lipoprotein receptor–related protein 5 (LRP5) in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%). Stabilizing mutation of CTNNB1 and expression of the internally truncated LRP5 receptor was mutually exclusive. Expression of the truncated LRP5 receptor was required to maintain the nonphosphorylated active β-catenin level, transcription activity of β-catenin, MYC expression, parathyroid cell growth in vitro, and parathyroid tumor growth in a xenograft severe combined immunodeficiency (SCID) mouse model. WNT3 ligand and the internally truncated LRP5 receptor strongly activated transcription, and the internally truncated LRP5 receptor was insensitive to inhibition by DKK1.
The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/β-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention.
Gunnar Westin and colleagues report the expression of an aberrantly spliced LRP5 receptor in primary and spontaneous parathyroid tumors and implicate it in the deregulated activation of the Wnt/β-catenin signaling pathway.
Editors' Summary
The parathyroid glands—four rice-sized glands in the neck—maintain a normal calcium balance in the body, to maintain strong bones and essential cellular functions. The glands release parathyroid hormone as a response to a decrease in blood calcium level. By stimulating calcium release from bone and its absorption in the gut, parathyroid hormone restores the blood calcium level. However, 100,000 new individuals in the US develop hyperparathyroidism (HPT) annually, characterized by enlarged, overactive parathyroid glands and high blood levels of calcium. Primary HPT (pHPT) is usually caused by a benign tumor (a non-life-threatening growth) in one of the parathyroid glands. Secondary HPT (sHPT) occurs in response to calcium regulatory disturbances, linked to vitamin D deficiency, and more or less invariably develops in patients with uremic kidney disease.
Why Was This Study Done?
HPT is usually treated by surgical removal of the enlarged parathyroid glands, which is done with great efficiency. However, ideally, doctors would like to know what drives the overgrowth of the parathyroid glands to be able to develop drugs for treatment or disease prophylaxis. Researchers recently reported that the cells in enlarged parathyroid glands from patients with HPT contain high amounts of β-catenin. This protein is part of the Wnt signaling pathway, which has been found to be disrupted in many tumor entities in other organs. In the absence of Wnt proteins, a group of proteins called the β-catenin destruction complex marks β-catenin so that it is rapidly destroyed. When Wnt proteins bind to a cell-surface receptor called Frizzled and a coreceptor called LRP5, the destruction complex is inhibited and β-catenin accumulates. This accumulation induces the production of other proteins (in particular, c-Myc) that stimulate cell growth and division. The accumulation of β-catenin in the enlarged parathyroid glands of patients with HPT could, therefore, significantly contribute to the overgrowth of their glands—but what causes β-catenin accumulation? In this study, the researchers have investigated this question to try to identify a target for drugs to treat HPT.
What Did the Researchers Do and Find?
The researchers looked for genetic changes (mutations) in β-catenin that stabilize the protein and measured the expression of LRP5 in abnormal parathyroid gland tissue from 37 patients with pHPT and 20 with uremia and sHPT. All the samples contained high levels of β-catenin, but only four contained a β-catenin–stabilizing mutation. All the sHPT samples and 32 pHPT samples (but none of the samples containing the β-catenin stabilizing mutation) expressed a mutated LRP5, with the central region deleted. To investigate the functional consequences of this internally deleted LRP5 protein, the researchers used a technique called RNA interference to block its expression in a human parathyroid tumor cell line. They found that expression of the mutated, short LRP5 is required for accumulation of β-catenin, expression of c-Myc, and continued growth of the cell line in test tubes and in animals.
What Do These Findings Mean?
The accumulation of β-catenin in all the enlarged parathyroid glands examined so far strongly implicates abnormal Wnt/β-catenin signaling in the development of pHPT and sHPT. These new findings identify which part of the signaling pathway is altered. The expression data and functional data together suggest that an internally deleted LRP5 coreceptor is often responsible for the accumulation of β-catenin. The functional data also show that expression of shortened LRP5 is necessary for the abnormal growth of parathyroid tumor cells. Exactly how the internally deleted coreceptor activates β-catenin signaling in parathyroid gland cells, or why a shorter-than-normal LRP5 is made, are not yet known. However, because these findings indicate that internally deleted LRP5 has a fundamental role in activating Wnt signaling in HPT, drugs that inactivate this aberrant protein but leave the normal protein unscathed might provide a nonsurgical treatment for this common hormone disorder.
Additional Information.
Please access these Web sites via the online version of this summary at
edlinePlus has encyclopedia pages on hyperparathyroidism, primary hyperparathyroidism, and secondary hyperparathyroidim (in English and Spanish)
Information is available for patients from the US National Institute of Diabetes and Digestive and Kidney Diseases on hyperparathyroidism, which includes links to organizations that help people with hyperparathyroidism
Wikipedia maintains pages on Wnt signaling pathway and on β-catenin (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2082644  PMID: 18044981
22.  HMG-CoA reductase expression in primary colorectal cancer correlates with favourable clinicopathological characteristics and an improved clinical outcome 
Diagnostic Pathology  2014;9:78.
An association between tumor-specific HMG-CoA reductase (HMGCR) expression and good prognosis has previously been demonstrated in breast and ovarian cancer. In this study, the expression, clinicopathological correlates and prognostic value of HMGCR expression in colorectal cancer was examined.
Immunohistochemical expression of HMGCR was assessed in tissue microarrays with primary tumours from 557 incident cases of colorectal cancer in the Malmö Diet and Cancer Study. Pearson’s Chi Square test was applied to explore the associations between HMGCR expression and clinicopathological factors and other investigative biomarkers. Kaplan Meier analysis and Cox proportional hazards modeling were used to assess the relationship between HMGCR expression and cancer-specific survival (CSS) according to negative vs positive HMGCR expression.
A total number of 535 (96.0%) tumours were suitable for analysis, of which 61 (11.4%) were HMGCR negative. Positive cytoplasmic HMGCR expression was associated with distant metastasis-free disease at diagnosis (p = 0.002), lack of vascular invasion (p = 0.043), microsatellite-instability (p = 0.033), expression of cyclin D1 (p = <0.001) and p21 (p = <0.001). Positive HMGCR expression was significantly associated with a prolonged CSS in unadjusted Cox regression analysis in the entire cohort (HR = 1.79; 95% CI 1.20-2.66) and in Stage III-IV disease (HR = 1.71; 95% CI 1.09-2.68), but not after adjustment for established clinicopathological parameters.
Findings from this prospective cohort study demonstrate that HMGCR is differentially expressed in colorectal cancer and that positive expression is associated with favourable tumour characteristics and a prolonged survival in unadjusted analysis. The utility of HMGCR as a predictor of response to neoadjuvant or adjuvant statin treatment in colorectal cancer merits further study.
Virtual slides
The virtual slides for this article can be found here:
PMCID: PMC4000148  PMID: 24708688
HMG-CoA reductase; Immunohistochemistry; Colorectal cancer; Prognosis
23.  The Internally Truncated LRP5 Receptor Presents a Therapeutic Target in Breast Cancer 
PLoS ONE  2009;4(1):e4243.
Breast cancer is a common malignant disease, which may be caused by a number of genes deregulated by genomic or epigenomic events. Deregulated WNT/β-catenin signaling with accumulation of β-catenin is common in breast tumors, but mutations in WNT signaling pathway components have been rare. An aberrantly spliced internally truncated LRP5 receptor (LRP5Δ666–809, LRP5Δ) was shown recently to be resistant to DKK1 inhibition, and was required for β-catenin accumulation in hyperparathyroid tumors and parathyroid tumor growth.
Methodology/Principal Findings
Here we show, by reverse transcription PCR and Western blot analysis, that LRP5Δ is frequently expressed in breast tumors of different cancer stage (58–100%), including carcinoma in situ and metastatic carcinoma. LRP5Δ was required in MCF7 breast cancer cells for the non-phosphorylated active β-catenin level, transcription activity of β-catenin, cell growth in vitro, and breast tumor growth in a xenograft SCID mouse model. WNT3 ligand, but not WNT1 and WNT3A augmented the endogenous β-catenin activity of MCF7 cells in a DKK1-insensitive manner. Furthermore, an anti-LRP5 antibody attenuated β-catenin activity, inhibited cell growth, and induced apoptosis in LRP5Δ-positive MCF7 and T-47D breast cancer cells, but not in control cells.
Our results suggest that the LRP5Δ receptor is strongly implicated in mammary gland tumorigenesis and that its aberrant expression present an early event during disease progression. LRP5 antibody therapy may have a significant role in the treatment of breast cancer.
PMCID: PMC2627768  PMID: 19158955
24.  Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis 
BMC Cancer  2014;14:2.
Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC.
Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients.
MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients.
Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC.
PMCID: PMC3890584  PMID: 24383423
MEKK3; ESCC; Diagnosis; Dysplasia; Immunohistochemistry; Prognosis
25.  Low-Density Lipoprotein Receptor Related protein-1 (LRP1)-Dependent Cell Signaling Promotes Neurotrophic Activity in Embryonic Sensory Neurons 
PLoS ONE  2013;8(9):e75497.
Developing sensory neurons require neurotrophic support for survival, neurite outgrowth and myelination. The low-density lipoprotein receptor-related protein-1 (LRP1) transactivates Trk receptors and thereby functions as a putative neurotrophin. Herein, we show that LRP1 is abundantly expressed in developing dorsal root ganglia (DRG) and that LRP1-dependent cell signaling supports survival, neurite extension and receptivity to Schwann cells even in the absence of neurotrophins. Cultured embryonic DRG neurons (E15) were treated with previously characterized LRP1 ligands, LRP1-receptor binding domain of α2-macroglobulin (RBD), hemopexin domain of MMP-9 (PEX) or controls (GST) for two weeks. These structurally diverse LRP1 ligands significantly activated and sustained extracellular signal-regulated kinases (ERK1/2) 5-fold (p<0.05), increased expression of growth-associated protein-43(GAP43) 15-fold (P<0.01), and increased neurite outgrowth 20-fold (P<0.01). Primary sensory neurons treated with LRP1 ligands survived > 2 weeks in vitro, to an extent equaling NGF, a finding associated with canonical signaling mechanisms and blockade of caspase-3 cleavage. LRP1 ligand-induced survival and sprouting were blocked by co-incubation with the LRP1 antagonist, receptor associated protein (RAP), whereas RAP had no effect on NGF-induced activity. Site directed mutagenesis of the LRP1 ligand, RBD, in which Lys1370 and Lys1374 are converted to alanine to preclude LRP1 binding, were ineffective in promoting cell signaling, survival or inducing neurite extension in primary sensory neurons, confirming LRP1 specificity. Furthermore, LRP1-induced neurite sprouting was mediated by Src-family kinase (SFK) activation, suggesting transactivation of Trk receptors. Co-cultures of primary embryonic neurons and Schwann cells showed that LRP1 agonists promoted axonal receptivity to myelination to Schwann cells. Collectively, these findings identify LRP1 as a novel and perhaps essential trophic molecule for sensory neuronal survival and development.
PMCID: PMC3781060  PMID: 24086544

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