In the present study we have used a quantitative high-throughput methylation analysis to show that the promoter of the SPG20 gene is hypermethylated in the vast majority of colorectal carcinomas and adenomas. The methylation was highly tumor-specific, underscored by lack of SPG20 methylation in normal colorectal mucosa samples. The findings have been validated in clinically independent sample series, altogether counting 505 tissue samples. Using ROC SPG20 methylation reached a very high area under the curve (AUC) value for carcinomas and for adenomas versus normal mucosa.
SPG20 methylation was also detected in a pilot series of stool samples from patients with colorectal carcinoma, underscoring its potential as a biomarker for early detection of colorectal cancer. Our finding that SPG20 promoter hypermethylation shuts down the expression of Spartin, resulting in cytokinesis arrest, suggests a potential role for SPG20 hypermethylation in genomic instability.
In contrast to genetic tests, which in most cases will require several parallel analyses in order to establish the mutation status of a single target gene (such as TP53
), DNA promoter methylation occurs at a defined region of the gene and is easily analyzed in a single cost-effective reaction, thereby demanding less biological starting material. Indeed, results from several studies of stool and serum DNA underscore the potential of using a non-invasive DNA methylation tests for early detection and/or screening for colorectal cancer (reviewed by Zitt et al. (2007)
and Kim et al. (2010)
). However, in spite of a few promising results (Müller et al., 2004
; Chen et al., 2005
; Zhang et al., 2007
; Devos et al., 2009
), specific and sensitive biomarkers to be included in an effective non-invasive test are still lacking. SPG20
promoter hypermethylation has previously been shown in ovarian cancers and proposed as a methylated target in prostate cancer based on genome-wide analyses (Goh et al., 2007
; Kron et al., 2009
). To the best of our knowledge, this is the first report identifying and validating the methylation status of SPG20
in a large cancer patient series, here shown for colorectal cancer. The resulting high sensitivity and specificity identifies SPG20
as one of the most promising single biomarkers for early detection of colorectal cancer.
By using real-time quantitative gene expression analyses we showed that promoter hypermethylation of SPG20
was associated with loss of gene expression. This was confirmed when the gene expression of SPG20
was restored in originally hypermethylated and non-expressing colon cancer cell lines by removal of DNA methylation using AZA. As expected, no Spartin protein expression could be seen in the hypermethylated colon cancer cell lines either by western blot analyses or by immunofluorescence confocal microscopy. Spartin expression was, however, seen in dividing control cells and in the unmethylated HeLa and hTERT RPE-1 cell lines where it, in accordance with previous findings, was located to the centrosome and midbody structures (Robay et al., 2006
Cytokinesis is the final part of the M-phase of the cell cycle, where the cytoplasm is divided between two daughter cells. It is initiated by the assembly of a contractile ring, which constricts the cell membrane into generating a cleavage furrow. This ingresses until a structure of densely packed microtubules, the midbody, is formed. Subsequent cleavage of the midbody completes the cytokinesis (reviewed by Sagona and Stenmark (2010)
). Microtubule severing during the abscission stage of cytokinesis is mediated by the AAA ATPase Spastin, which is found at the midbody during cytokinesis (Errico et al., 2004
; Connell et al., 2009
). Interestingly, Spastin contains an MIT domain that is closely related to that of Spartin (Ciccarelli et al., 2003
). Spastin is encoded by the SPG4
gene, which is mutated in the most frequent form of autosomal dominant spastic paraplegia (Hazan et al., 1999
). Thus, Spastin not only has structural similarity to Spartin, but also shares its association with spastic paraplegia. This opens the possibility that these two proteins could function in the same pathway.
Based on the midbody localization of Spartin as well as its common features with Spastin, we hypothesized that Spartin as well could have a role in cytokinesis. To provide insight into the underlying biology of SPG20
hypermethylation in cancer cells, we investigated cell proliferation in Spartin-deficient colon cancer cell lines and discovered that the ‘slower' proliferating cell lines showed significantly more cytokinesis profiles and convoluted midbody structures compared with the highly proliferative cell lines. HeLa and hTERT RPE-1 cells in which Spartin expression was knocked down by siRNA confirmed the phenotype seen among the ‘slower' growing colon cancer cell lines by showing cytokinesis arrest as well as convoluted midbodies. Interestingly, a strikingly similar midbody morphology has been reported in cells with knockdown of Spastin (Connell et al., 2009
). When the Spartin protein was re-expressed in initially non-expressing HT29 colon cancer cells, the number of cells arrested in late cytokinesis was significantly reduced. Taken together, these observations show that the multifunctional Spartin protein also has a role in cytokinesis. This novel role of the Spartin protein was recently also reported by Renvoise et al. (2010)
, who showed that Spartin colocalized with Ist1 (an endosomal sorting complex required for transport; ESCRT protein) at the midbody of HeLa cells and that depletion of Spartin with siRNA markedly impaired cytokinesis.
Failure of cytokinesis and subsequent aneuploidy has been shown to be associated with carcinogenesis (reviewed by Sagona and Stenmark, 2010
). In cells experiencing cytokinesis arrest, a regression of the cytokinesis furrow can result in binuclearity (Fujiwara et al., 2005
; Ganem et al., 2007
; Steigemann et al., 2009
). Although a significant increase in the number of multinucleated Spartin-depleted HeLa cells was reported by Renvoise et al. (2010)
, binuclear Spartin-deficient cells were not observed in the present study either in HeLa cells or colon cancer cells. It is also worth noting that Spastin-deficient cells, similarly to Spartin-deficient cells, show cytokinesis arrest without a binuclear phenotype (Connell et al., 2009
), which further emphasizes the possible functional relationship between these two proteins. The lack of binuclear cells might suggest that the cells become arrested at a very late stage of cytokinesis where abortion of cytokinesis is no longer possible. The functional consequences of the cytokinesis arrest induced by Spartin downregulation therefore remain somewhat enigmatic. Cytokinesis failure in the absence of midbody regression has three alternative outcomes—an eventual (albeit delayed) midbody abscission, activation of apoptotic cell death (Bu et al., 2008
; Connell et al., 2009
) or proliferation of cells that remain interconnected by thin intercellular bridges. Delayed abscission is associated with chromosomal instability (Fujiwara et al., 2005
; Ganem et al., 2007
), whereas interconnected cells tend to show synchronized responses to proliferation and differentiation cues (Ventelä et al., 2003
; Vidulescu et al., 2004
; Pointis et al., 2010
). Both these mechanisms might thus contribute to carcinogenesis. Interestingly, both the ‘slower' proliferating colon cancer cell lines included in the present study (HT29 and SW480) have a chromosomal unstable phenotype (Lengauer et al., 1997
In the present study, we observed that, even though colon cancer cell lines with convoluted midbodies have a lower proliferation rate than cancer lines without cells arrested in late cytokinesis, they still proliferate significantly faster than control cells. This might be explained by the presence of multiple additional aberrations accumulated during tumorigenesis, which provides the cancer cells with a selective advantage. The nature of such additional aberrations might also explain why we observed a delay in cytokinesis and the presence of several cytokinesis bridges only in half of the colon cancer cell lines tested, although all of them lacked expression of Spartin. The MSS and MSI developmental pathways are characterized by accumulation of distinct aberrations. The latter group, which accounts for approximately 15% of sporadic colorectal cancers, has a defect in the DNA mismatch repair system leading to frequent aberrations in microsatellites, but the tumors are typically near diploid. On the other hand, the majority of sporadic colorectal carcinomas (~85%) are microsatellite stable (MSS), but typically contain multiple chromosomal aberrations (that is, aneuploid). The molecular basis of the MSS phenotype in Spartin-deficient colon cancer cells is not clarified, but a possible mechanism is that aberrant cytokinesis may lead to chromosome-containing intercellular bridges that eventually lead to reduced fidelity in chromosome distribution (Steigemann et al., 2009
A number of additional roles have been described for the Spartin protein, including in endocytosis and degradation of epidermal growth factor receptor (Bakowska et al., 2007
), and regulation of the size and number of lipid droplets (Eastman et al., 2009
; Hooper et al., 2010
). Cells lacking expression of Spartin will therefore suffer aberrations in several cellular mechanisms, possibly creating a cellular ‘lability', which makes them more prone to accumulate additional aberrations. In the present study, we showed that hypermethylation of SPG20
and subsequent loss of gene and protein expression is an early and frequent event in colorectal tumorigenesis. SPG20
hypermethylation was present in the majority of early precursor lesions, adenomas. Only a small fraction of these benign tumors, (the ones that accumulate aberrations necessary for malignant transformation) will progress into malignant carcinomas.
In summary, detection and quantification of SPG20 promoter methylation is a highly specific and sensitive biomarker for colorectal tumors. Analyses of a pilot of stool samples from colon cancer patients indicate that SPG20 promoter methylation can be detected in such samples, which is very promising for further development of SPG20 as a biomarker in non-invasive tests. This should be explored further using stool or plasma samples from large test groups. We further show that promoter hypermethylation of SPG20 is associated with loss of gene expression and subsequent lack of Spartin protein. The presence of aberrant cytokinesis profiles as well as convoluted midbodies in Spartin-depleted HeLa and hTERT RPE-1 cells, and Spartin-deficient ‘slower' growing colon cancer cell lines reveals a function for the Spartin protein also in cytokinesis. Our data not only identify a promising biomarker for colorectal cancer, but also reveal a cellular mechanism that could be involved in the early carcinogenic process.