Alternative mRNA splicing in colon cancer causes loss of expression of neural cell adhesion molecule

doi:10.1067/msy.2001.116415

Surgery

Volume 130, Issue 5, November 2001, Pages 834-843

https://doi.org/10.1067/msy.2001.116415 Get rights and content

Abstract

Background. The neural cell adhesion molecule (NCAM) has numerous isoforms resulting from alternative splicing of mRNA. The 3 major isoforms found in adult tissue are (1) a 120-kDa protein that is linked to the plasma membrane by glycosylphosphatidylinositol; (2) a 140-kDa form that has a transmembrane component and a cytoplasmic tail with unknown function; and (3) a 180-kDa isoform that has an intracellular protein that binds the cytoskeleton. NCAM is capable of homotypic binding and therefore plays a role in cell-cell adhesion for cells expressing the 180-kDa isoform by anchoring groups of cells into epithelial sheets. NCAM-180 is the isoform found in colonocytes, and loss of expression is associated with clinically aggressive colon cancers. Methods. Western blotting and reverse transcriptase-polymerase chain reaction were used to screen commercially available cell lines for NCAM-180 expression. For cell-line pairs with differential NCAM-180 expression, exon analysis was performed with reverse transcriptase-polymerase chain reaction to determine where the molecule was spliced, culminating in failed expression. These results were confirmed with exon analysis in colon cancers harvested at the time of laparotomy. Results. Analysis of a SW480 cell line (derived from a patient's primary colon cancer lesion) revealed NCAM-180 expression, whereas no expression was found in the SW620 cell line (derived from a metastatic lesion from the same patient). Exon analysis of NCAM mRNA transcripts from SW620 revealed that the transcripts were truncated after exon 12. This region correlates to an area between 2 fibronectin-III domains on the NCAM protein. Conclusions. The most common site for NCAM alternative splicing is between the 2 fibronectin-III domains corresponding to the border between exons 12 and 13 of the NCAM gene. Loss of NCAM-180 expression in aggressive colon carcinoma results from a splice defect in the same area, which may result in defective intracellular adhesion between colonocytes. (Surgery 2001;130:834-43.)

Section snippets

Colon cancer cell lines

The following 5 human colon cancer cell lines were obtained from the American Tissue Culture Collection (ATCC) (Rockville, Md):

1.
LS 174T (colon, adenocarcinoma CCL-188; passage number: 104)
2.
WiDr (colon, adenocarcinoma CCL-218; passage number: 19)
3.
COLO 205 (colon, adenocarcinoma CCL-222; passage number: unknown [6 at ATCC])
4.
SW620 (colon, adenocarcinoma, lymph node metastasis CCL-227; passage number: 83)
5.
SW480 (colon, adenocarcinoma CCL-228; passage number: 96).

The cell lines were grown according to

Cell-line screening

RT-PCR screening for NCAM exons 17 to 18 is presented in Fig 3.

. Expression of NCAM exons 17 to 18 in colon cancer cell lines. Six cell lines were screened for the presence of NCAM sequences from exons 17 to 18 in their mRNA. Glyceraldehyde-3-phosphate dehydrogenase is found in all cells and served as a positive control to ensure adequate RNA was extracted from cells and that the PCR reaction was complete. Human brain is known to contain large amounts of NCAM-L 180 and was used as a control for

Discussion

Previous studies from our laboratory demonstrated that NCAM-L 180 is present in normal colonic epithelium and benign colonic tumors. NCAM is an adhesion molecule capable of both heterotypic and homotypic binding. Adjacent cells expressing NCAM bind together via homotypic NCAM adhesion. NCAM-L 180 is found in mature neural tissues,²⁰ and because the intracelluar protein expressed by exon 18 links NCAM to the cytoskeleton,¹⁶ the 180-kDa isoform is believed to mediate cellular adhesion. Colonocyte

References (38)

WT. Chen
Membrane proteases: roles in tissue remodeling and tumour invasion
Curr Opin Cell Biol
(1992)
S Benchimol et al.
Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule
Cell
(1989)
C Goridis et al.
NCAM: structural diversity, function and regulation of expression
Semin Cell Biol
(1992)
JB Rothbard et al.
Differences in the carbohydrate structures of neural cell-adhesion molecules from adult and embryonic chicken brains
J Biol Chem
(1982)
J Roesler et al.
Tumor suppressor activity of neural cell adhesion molecule in colon carcinoma
Am J Surg
(1997)
U. Rutishauser
Neural cell-to-cell adhesion and recognition
Curr Opin Cell Biol
(1989)
S Goodison et al.
Multiple intron retention occurs in tumor cell CD44 mRNA processing
Am J Pathol
(1998)
G Dickson et al.
Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain
Cell
(1987)
HJ Gower et al.
Alternative splicing generates a secreted form of N-CAM in muscle and brain
Cell
(1988)
RP Lane et al.
Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31
Genomics
(1996)

T Brummendorf et al.

Neural cell recognition molecule F11: homology with fibronectin type III and immunoglobulin type C domains

Neuron

(1989)

AH Huber et al.

Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A

Neuron

(1994)

EC Woodhouse et al.

General mechanisms of metastasis

Cancer

(1997)

L Eisenbach et al.

Tumor cell surface determinants in host immunity against metastases

Semin Cancer Biol

(1991)

BR. Zetter

Cell motility in angiogenesis and tumor metastasis

Cancer Invest

(1990)

BR. Zetter

Cellular basis of site-specific tumor metastasis

N Engl J Med

(1990)

H Turley et al.

The distribution of the deleted in colon cancer (DCC) protein in human tissues

Cancer Res

(1995)

MA Reale et al.

Expression and alternative splicing of the deleted in colorectal cancer (DCC) gene in normal and malignant tissues

Cancer Res

(1994)

GJ Cole et al.

Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM 1986

J Cell Biol

(1986)

Cited by (53)

Tumor adhesion molecule targeting for breast cancer nanomedicine
2022, Targeted Nanomedicine for Breast Cancer Therapy
The majority of breast cancer-related deaths are caused by metastasis. The metastatic distribution of cancer is a complex process that involves the versatility of both tumor cells and interacting cells. Cell adhesion molecules are membrane receptors that mediate cellular and membrane interactions and transduce receptor signals and are important for three kinds of metastasis: cell adhesion, invasion, and diffusion of blood into the surrounding tissues and homing of blood cells. Adhesion molecules play a critical role in the initiation of carcinogenesis. The classification of breast carcinoma subtypes aids in the detection, prognosis, and therapeutic selection. The expansion of this research will also enable pathologists to learn more about how various molecules and molecular groups work together, and how this impacts their tumor differentiation and classification abilities, which will provide them with more resources to learn. Nanoparticles (NPs) provide various benefits over conventional molecules, such as high payloads, tunable shapes, customizable surface properties, controllable drug kinetics, and advanced pharmacokinetics. As a result, several NP-based systems capable of targeting plaques, the hallmark of atherosclerosis, have been established as an innovative means of enhancing diagnostic sensitivity and therapeutic efficacy. In this chapter, various forms of tumor adhesion molecules and nanotherapies in breast cancer management are discussed.
Discovery of novel transcripts of the human tissue kallikrein (KLK1) and kallikrein-related peptidase 2 (KLK2) in human cancer cells, exploiting Next-Generation Sequencing technology
2019, Genomics
Citation Excerpt :
Alternative splicing of cancer-related genes has previously been shown to play important roles in cell cycle control, proliferation, apoptosis, angiogenesis, as well as invasion and metastasis [30]. In spite of the fact that the relationship between particular splicing events and the etiology of cancer is not yet fully understood, novel splice variants may constitute new, attractive molecular biomarkers that could be exploited for diagnostic and/or prognostic purposes, potential biomarkers for response or resistance to treatment, as well as therapeutic targets [31–37]. The family of KLKs constitutes a prominent example, as all its members have been shown to be subjected to intense alternative splicing.
Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.
A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer
2018, EBioMedicine
Citation Excerpt :
Many of the genes with predicted AS events were involved in processes relevant to tumor progression, migration, and invasion, such as NCAM1 which encodes for a neural adhesion molecule capable of heterotypic and homotypic binding. Loss of expression of the NCAM-180 isoform due to truncated transcripts was previously reported for SW620 cells and is linked to CRC [80]. Other examples for differential AS events in CRC progression identified in our study are the EMT-associated splicing switches of the genes CD44 and FGFR2.
Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis.
Effect of NCAM on aged-related deterioration in vision
2016, Neurobiology of Aging
Citation Excerpt :
NCAM expression, including expression of its isoforms, is highly regulated in the central nervous system during development (Paz et al., 1995; Rafuse et al., 2000). NCAM dysregulation has been correlated with cancer and tumor progression in humans, suggesting that NCAM may contribute to age-associated changes in cellular architecture and function (Gattenlohner et al., 2009; Huerta et al., 2001; Sasaki et al., 1998). Different isoforms of NCAM play distinct roles in biological processes, such as mediating neurite outgrowth, myoblast fusion, and synaptic maturation (Berezin, 2010).
The neural cell adhesion molecule (NCAM) is involved in developmental processes and age-associated cognitive decline; however, little is known concerning the effects of NCAM in the visual system during aging. Using anatomical, electrophysiological, and behavioral assays, we analyzed age-related changes in visual function of NCAM deficient (−/−) and wild-type mice. Anatomical analyses indicated that aging NCAM −/− mice had fewer retinal ganglion cells, thinner retinas, and fewer photoreceptor cell layers than age-matched controls. Electroretinogram testing of retinal function in young adult NCAM −/− mice showed a 2-fold increase in a- and b-wave amplitude compared with wild-type mice, but the retinal activity dropped dramatically to control levels when the animals reached 10 months. In behavioral tasks, NCAM −/− mice had no visual pattern discrimination ability and showed premature loss of vision as they aged. Together, these findings demonstrate that NCAM plays significant roles in the adult visual system in establishing normal retinal anatomy, physiology and function, and in maintaining vision during aging.
Serum markers in small cell lung cancer: Opportunities for improvement
2013, Biochimica et Biophysica Acta - Reviews on Cancer
Citation Excerpt :
Since these variants are especially detected during prenatal life, they are sometimes referred to as embryonic variants. However, they are also detected in adults, albeit to a lesser extent and in limited tissues [80,82,83]. These variants may provide a target for a more accurate detection of cancerous cells.
Lung cancer is one of the leading causes of death from malignancy worldwide. In particular small cell lung cancers, which comprise about 15–20% of all lung cancers, are extremely aggressive and cure rates are extremely low. Therefore, new treatment modalities are needed and detection at an early stage of disease, as well as adequate monitoring of treatment response is essential in order to improve outcome. In this respect, the use of non-invasive tools for screening and monitoring has gained increasing interest and the clinical applicability of reliable, tumor-related substances that can be detected as tumor markers in easily accessible body fluids is subject of intense investigation. Some of these indicators, such as high LDH levels in serum as a reflection of the disease, have been in use for a long time as a general tumor marker. To allow for improved monitoring of the efficacy of new therapeutic modalities and for accurate subtyping, there is a strong need for specific and sensitive markers that are more directly related to the biology and behavior of small cell lung cancer. In this review the current status of these potential markers, like CEA, NSE, ProGRP, CK-BB, SCC, CgA, NCAM and several cytokeratins will be critically analyzed with respect to their performance in blood based assays. Based on known cleavage sites for cytoplasmic and extracellular proteases, a prediction of stable fragments can be obtained and used for optimal test design. Furthermore, insight into the synthesis of specific splice variants and neo-epitopes resulting from protein modification and cleavage, offers further opportunities for improvement of tumor assays.
Finally, we discuss the possibility that detection of SCLC related autoantibodies in paraneoplastic disease can be used as a very early indicator of SCLC.
Potential diagnostic utility of CD56 and claudin-1 in papillary thyroid carcinoma and solitary follicular thyroid nodules
2012, Journal of the Egyptian National Cancer Institute
The pathological diagnosis of papillary thyroid carcinoma (PTC) is usually easily achieved. However distinguishing the follicular variant of papillary carcinoma (FVPC) from other follicular thyroid lesions is an area of controversy. In this study we investigated the role of CD56 and claudin-1 in discriminating the FVPCs from other solitary follicular patterned nodules. We also evaluated the application of these two markers in reclassifying the controversial cases of the well differentiated tumors of unknown malignant potential (WDTs-UMP).
The immunohistochemical expression of CD56 and claudin-1 was evaluated in 86 samples of thyroid lesions together with 10 samples of normal thyroid tissue. Thyroid lesions included: 29 PTCs [classic papillary carcinoma (n = 13) and FVPC (n = 16)], 47 solitary follicular patterned nodules [follicular adenomas (n = 12), hyperplastic nodules (n = 32) and follicular tumor of unknown malignant potential (n = 3)] and 10 WDTs-UMP.
The statistical analysis showed significantly different expressions of each of CD56 and claudin-1 in the FVPCs versus other solitary follicular patterned nodules. Claudin-1 sensitivity (100%) was higher than CD56 sensitivity (81.3%). However claudin-1 specificity (80.9%) was < CD56 specificity (89.4%). The combined use of CD56 and claudin-1(claudin-1+/CD56−) showed specificity (100%), positive predictive value (100%) and sensitivity (81.3%) in the differentiation between the FVPCs and other follicular nodules. In the light of this statistical outcome, 5/10 cases of WDTs-UMP expressing the (claudin-1+/CD56−) panel could be rediagnosed as PTC.
Combined utility of CD56 and claudin-1 is helpful in diagnosing the FVPC and its differentiation from other follicular patterned nodules. Application of these two markers may greatly aid in the reevaluation of the WDTs-UMP and interpretation of their expected behavior.

View all citing articles on Scopus

^*: Reprint requests: Edward H. Livingston, MD, Chief, Department of Surgery (10H2), VA Greater Los Angeles Health Care System, 11301 Wilshire Blvd, Los Angeles, CA 90073.

View full text

Original CommunicationsAlternative mRNA splicing in colon cancer causes loss of expression of neural cell adhesion molecule*

Abstract

Section snippets

Colon cancer cell lines

Cell-line screening

Discussion

Curr Opin Cell Biol

Cell

Semin Cell Biol

J Biol Chem

Am J Surg

Curr Opin Cell Biol

Am J Pathol

Cell

Cell

Genomics

Neuron

Neuron

General mechanisms of metastasis

Cancer

Tumor cell surface determinants in host immunity against metastases

Semin Cancer Biol

Cell motility in angiogenesis and tumor metastasis

Cancer Invest

Cellular basis of site-specific tumor metastasis

N Engl J Med

The distribution of the deleted in colon cancer (DCC) protein in human tissues

Cancer Res

Expression and alternative splicing of the deleted in colorectal cancer (DCC) gene in normal and malignant tissues

Cancer Res

Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM 1986

J Cell Biol

Original Communications
Alternative mRNA splicing in colon cancer causes loss of expression of neural cell adhesion molecule*