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A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10/245 (4%) matched normal tissue (P < 0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11–2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07–2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil-based chemotherapy. (Cancer Sci 2011; 102: 1855–1861)
Colorectal carcinoma (CRC) is one of the most common human cancers in the world,(1) and is frequently diagnosed at advanced stages that require a multimodality approach for treatment. In addition to surgery and radiotherapy, contemporary treatment options for advanced CRC include multidrug fluoropyrimidine-based chemotherapeutic regimens as well as novel targeted therapies.(2) However, despite substantial progress in the understanding of CRC pathobiology in recent years, it remains a challenge in translating this knowledge to allow the identification of patients at risk for treatment failure and poor prognosis. As such, much effort has been instilled in the field of biomarker discovery to advance towards effective personalized therapy and improved clinical outcome.(3)
Recently, cell adhesion molecules (CAMs) have caught the attention of cancer researchers, due to their counter-intuitive roles in cancer invasion and metastasis. Cell adhesion molecules are known to aid intercellular adherence, yet they have also been shown to promote cell motility, invasion, and migration.(4) Activation of the canonical Wnt signaling pathway has been strongly suggested to hold a direct causal relationship to CRC initiation and progression.(5) Interestingly, several CAMs associated with the Wnt pathway have been implicated in human cancer development and progression, and their overexpression has been linked with features of aggressive disease and worse prognosis.(6–8) In particular, two members belonging to the L1 family of CAMs within the immunoglobulin-like superfamily, L1-CAM and neurone glial-related (Nr)-CAM, have been identified as targets of the β-catenin–Tcf complex. L1-CAM has been shown to be exclusively detected at the invasive front of colon cancer tissue, and its expression promoted tumorigenesis and conferred metastasis in colon cancer cells.(9) L1-CAM expression has also been identified as a marker for poor prognosis in patients with CRC.(10) Although the roles of L1-CAM in CRC biology have been well-characterized, that of Nr-CAM remain relatively obscure.
Neurone glial-related cell adhesion molecule is a 200–220 kDa transmembrane protein composed of six Ig-like domains and five FnIII repeats in the extracellular region plus a highly conserved cytoplasmic tail, and is known to be involved in the development and function of the mammalian nervous system.(11) First identified in neural tissue, Nr-CAM functions as an adhesion molecule and mediates axonal outgrowth and guidance along with other aspects of neuronal tissue development.(12–16) Nr-CAM was initially believed to be present exclusively in the nervous system,(11) but subsequent studies revealed that it is expressed in a variety of normal non-neural tissues and cells including those of the pancreas, adrenal gland, placenta, testis, thyroid, endothelium, and lens fibre.(17–21) More significantly, Nr-CAM overexpression has been confirmed in various neural(22–24) as well as non-neural cancers such as pancreatic adenocarcinoma,(19) renal cell carcinoma, papillary thyroid carcinoma,(25) malignant melanoma, and colonic carcinoma.(6) Furthermore, Nr-CAM overexpression has been shown to enhance cancer cell motility and invasiveness in vitro,(6) and is associated with aggressive clinical phenotypes.(23,24) Given the potential significance of Nr-CAM in cancer pathobiology, its clinical relevance in human CRC deserves to be investigated. Therefore, the aim of this study was to examine the protein expression of Nr-CAM in a well-characterized retrospective series of primary human CRC and matched normal tissues, as well as to evaluate Nr-CAM expression as a possible biomarker.
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In this study, we examined the protein expression of Nr-CAM in a cohort of primary human CRC samples and evaluated its utility as a possible biomarker. We showed that Nr-CAM expression occurred in all CRC cases but was rarely observed in normal tissue. This is in keeping with a previous study that detected significant Nr-CAM RNA expression in a small number of CRC cell lines and patient samples, but not in normal colonic tissue.(6) More significantly, we noted that a subset of CRC samples displayed a greater degree of Nr-CAM expression, which correlated with adverse clinicopathological features, including the presence of vascular invasion as well as nodal and distant metastasis. Our results suggest that Nr-CAM overexpression is a strong prognostic biomarker in advanced CRC, independent of several clinicopathological and molecular variables.
It is known that Nr-CAM is a downstream target of canonical Wnt signaling.(6) However, in our study, we observed that 72% of Nr-CAM overexpressed cases did not show cytoplasmic/nuclear β-catenin expression, suggesting that other upstream factors, apart from oncogenic β-catenin activation, might be responsible for regulating Nr-CAM expression. In line with this thinking, we found a novel relationship between Nr-CAM and p53 expression, suggesting that normal functional p53 signaling might directly or indirectly suppress Nr-CAM expression. In colorectal cancers, early oncogenic activation of β-catenin is followed by a sharp increase in p53 mutation frequency.(36) p53 downregulates β-catenin signaling by enhancing β-catenin degradation through the ubiquitin–proteasome system.(37) Of note, inactivating mutations of p53 have been associated with activation of β-catenin.(35) It may be hypothesized that p53 mutation leads to oncogenic activation of β-catenin signaling, and consequently results in upregulation of Nr-CAM gene expression. Interestingly, we also observed that loss of membranous β-catenin staining was positively correlated with Nr-CAM overexpression. It has been shown previously that L1-CAM disrupts the E-cadherin/β-catenin complex at adherens junctions, resulting in β-catenin activation and a positive feedback loop.(38) It remains to be investigated if such a signaling loop exists for Nr-CAM as well.
There is considerable evidence implicating Nr-CAM expression in cancer initiation and progression. Forced expression of Nr-CAM in NIH3T3 fibroblasts through retroviral transduction stimulated cell growth, increased cell motility, proliferation, and induced rapid tumorigenesis in nude mice. Similarly, human melanoma cells of more advanced stages express high levels of Nr-CAM and form tumors in mice, whereas those lacking Nr-CAM do not.(6) Clinically, Nr-CAM gene overexpression was observed in highly proliferative ependymoma, which conferred a poor prognosis.(23) Additionally, high-risk neuroblastomas negative for MYCN amplification showed upregulation of the Nr-CAM gene in comparison to the high-risk MYCN-amplified or intermediate-risk counterparts.(24) Notably, an apparently contrasting role of Nr-CAM may surface depending on tumor type. This is illustrated in pancreatic carcinoma, whereby poorly differentiated, invasive, and metastatic tumors showed a loss of cell surface Nr-CAM expression, compared to their early stage counterparts showing Nr-CAM overexpression.(19) Nonetheless, such a counter-intuitive finding remains consistent with the role of Nr-CAM as an adhesion molecule, as its overexpression could inhibit tumor metastasis yet promote cell–cell interactions that facilitate carcinogenesis during early stages. Another interesting observation in our study was that Nr-CAM overexpression correlated with peritoneal metastasis at diagnosis, supporting a previous study suggesting that CAMs are involved in organ selectivity during metastasis.(39) Taken together, aberrant Nr-CAM expression occurs in several human cancers and is associated with features of aggressive disease, however, the exact function might be tumor-specific and remains to be explored.
In the present study, we provide data to suggest that poor outcome conferred by Nr-CAM overexpression could be attributed to abrogation of survival benefit from 5-FU-based chemotherapy. This is a major finding, which is supported with several lines of corroborative in vitro evidence for a plausible underlying mechanism. Previously, Nr-CAM was shown to promote tumorigenesis in mice through metalloprotease-mediated shedding of its ectodomain, resulting in apoptotic resistance through constitutive activation of MAPK and AKT survival pathways, accompanied by enhanced cell motility and proliferation.(40) In addition, our results show that Nr-CAM overexpression was associated with p53 expression, which is known to be associated with apoptotic resistance in colon cancer cells.(41) Indeed, it may be speculated that Nr-CAM overexpression confers resistance to 5-FU-evoked apoptosis in CRC cells. Nonetheless, as Nr-CAM overexpression is correlated with disease stage, it remains to be examined in larger studies if there exists a differential response to 5-FU-based chemotherapy at various stages of disease progression. Future work should investigate whether other CAMs, such as L1-CAM, have predictive value for chemoresistance in CRC and in other tumor types as well. It may be that combinations of such markers will show the strongest predictive value for response to chemotherapy.
Cell adhesion molecules have been identified as potential therapeutic targets for cancer. In particular, there is emerging evidence that targeted antibody-based treatment against CAMs may delay the process of tumor progression.(4) Inoculation of antisense Nr-CAM in nude mice resulted in slower growth of glioblastoma(42) and melanoma cells.(40) Likewise, injecting antibodies and/or antisense RNA to L1-CAM into nude mice carrying human ovarian tumors(43) as well as intrahepatic cholangiocarcinoma(44) resulted in a significant reduction of tumor growth. Given that Nr-CAM overexpression may predict poor response to chemotherapy, patient stratification by Nr-CAM status may provide a more personalized approach to colon cancer therapy. Nr-CAM is an attractive protein for the development of novel agents in CRC targeted therapy, especially in those tumors expressing high levels of Nr-CAM.
The limitations of our study were the relatively small sample size, and the incomplete inclusion of known biomarkers such as status of CpG island methylator phenotype. In addition, the selection of patients for chemotherapy was not randomized, introducing a possible selection bias to our results. The advantages of our study, however, include the adequacy of follow-up and extensive data on disease characteristics, details of treatment, as well as other commonly studied tumoral molecular events, including MSI, KRAS mutation, and BRAF mutation. Thus, this allowed us to establish an effect of Nr-CAM on patient survival independent of several clinicopathological and other molecular prognostic markers.
In conclusion, this study provides the first evidence to suggest that tumoral Nr-CAM overexpression is an independent poor prognostic marker in advanced CRC. In addition, survival benefit from 5-FU-based chemotherapy may be abrogated by Nr-CAM overexpression. Our findings have several clinical implications, given the emerging roles of CAMs and related pathways as potential biomarkers and targets for cancer therapy.