Cyclin D1 is frequently overexpressed in microsatellite unstable colorectal cancer, independent of CpG island methylator phenotype

Aims: Cyclin D1 and cyclin‐dependent kinases are commonly activated in colorectal cancer. Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. The aim was to clarify the relationship between cyclin D1, MSI and CIMP. Methods and results: Among 865 colorectal cancers with MSI and CIMP data, 246 tumours (28.4%) showed cyclin D1 overexpression by immunohistochemistry. DNA methylation in p14 and eight CIMP‐specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) was quantified by real‐time polymerase chain reaction (MethyLight). Both MSI‐high and CIMP‐high were associated with cyclin D1 overexpression (P < 0.0001). After tumours were stratified by MSI and CIMP status, the relationship between MSI‐high and cyclin D1 persisted (P ≤ 0.02), whereas the relationship between CIMP‐high and cyclin D1 did not. Cyclin D1 overexpression was correlated with BRAF mutation (P = 0.0001), p27 loss (P = 0.0007) and p16 loss (P = 0.02), and inversely with p53 expression (P = 0.0002) and p21 loss (P < 0.0001). After stratification by MSI status, the inverse relationship between cyclin D1 and p21 loss still persisted (P < 0.008). Conclusions: Cyclin D1 activation is associated with MSI and inversely with p21 loss in colorectal cancers. Cyclin D1 may play an important role in the development of MSI‐high tumours, independent of CIMP status.     

Cytoplasmic localization of p27 (cyclin-dependent kinase inhibitor 1B/KIP1) in colorectal cancer: inverse correlations with nuclear p27 loss, microsatellite instability, and CpG island methylator phenotype

Cytoplasmic mislocalization of p27 (CDKN1B/KIP1) is caused by activated AKT1 and has been associated with poor prognosis in various cancers. CIMP in colorectal cancer is characterized by extensive promoter methylation and is associated with MSI-MSI-H and BRAF mutations. We have recently shown a positive correlation between MSI/CIMP and loss of nuclear p27. However, no study has examined cytoplasmic p27 mislocalization in relation to CIMP and MSI in colorectal cancer. Using MethyLight assays, we quantified DNA methylation in 8 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) in 853 colorectal cancer samples obtained from 2 large prospective cohorts. We assessed expressions of nuclear and cytoplasmic p27 and nuclear p53 by immunohistochemistry. Cytoplasmic p27 expression was inversely associated with loss of nuclear p27 (P < .0001), CIMP-high (P < .0001), MSI-H (P < .0001), and BRAF mutations (P < .0001). The inverse association of cytoplasmic p27 with CIMP-high (or MSI-H) was independent of MSI (or CIMP) status. In addition, the inverse association of cytoplasmic p27 with CIMP-high was independent of KRAS/BRAF status. BRAF and CDKN2A (p16) methylation were not correlated with cytoplasmic p27 after stratification by CIMP status. The inverse associations of cytoplasmic p27 with MSI-H and CIMP-high were much more pronounced in p53-negative than p53-positive tumors. In conclusion, cytoplasmic p27 expression is inversely associated with MSI-H and CIMP-high, particularly in p53-negative tumors, suggesting interplay of functional losses of p27 and p53 in the development of various molecular subtypes of colorectal cancer.     

Fatty acid synthase overexpression in colorectal cancer is associated with microsatellite instability, independent of CpG island methylator phenotype

Overexpression of fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is observed in many cancers including colorectal cancer and is associated with poor clinical outcomes. Cellular FASN expression is physiologically upregulated in a state of energy excess. Obesity and excess energy balance have been known to be risk factors for colorectal cancer. High degree of microsatellite instability (MSI-H) is a distinct phenotype in colorectal cancer, associated with CpG island methylator phenotype (CIMP). Previous data suggest that obesity or altered energy balance may potentially modify risks for MSI-H cancers and microsatellite stable (MSS) cancers differently. However, the relationship between MSI and FASN overexpression has not been investigated. Using 976 cases of population-based colorectal cancer samples from 2 large prospective cohort studies, we correlated FASN expression (by immunohistochemistry) with MSI, KRAS and BRAF mutations, p53 expression (by immunohistochemistry), and CIMP status [determined by MethyLight for 8 CIMP-specific gene promoters including CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1]. Marked (2+) FASN overexpression was observed in 110 (11%) of the 976 tumors and was significantly more common in MSI-H tumors (21% [28/135]) than MSI-low (5.6% [4/72], P = .004) and MSS tumors (11% [72/678], P = .001). The association between FASN overexpression and MSI-H persisted even after stratification by CIMP status. In contrast, FASN overexpression was not correlated with CIMP after stratification by MSI status. Fatty acid synthase overexpression was not significantly correlated with sex, tumor location, p53, or KRAS/BRAF status. In conclusion, FASN overexpression in colorectal cancer is associated with MSI-H, independent of CIMP status.