Deregulated expression of cryptochrome genes in human colorectal cancer

Abstract

Background: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far.

Methods: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines.

Results: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes.

Conclusion: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.

Fig. 1

Expression of CRY1 and CRY2 mRNA and protein levels in colorectal cancer tissue and survival rates. a CRY1 and CRY2 mRNA levels analysed by qRT-PCR in colorectal cancer tissue and compared with matched normal tissue, with GAPDH expression used as the calibrator. A box and whisker plot is shown representing the interquartile range (IQR) with median, 25th and 75th percentile, minimum and maximum values, as well as outliers indicated by dots. b x-y plot showing regression lines with 95 % confidence limits between CRY1 and CRY2 mRNA expression levels in tumour tissues of CRC patients (n = 50, r = 0.607, p < 0.0001) c) CRY1 and CRY2 protein level evaluated by western blotting in a panel of matched specimens of tumour tissue and non-tumorous tissue. A box and whisker plot is shown representing the interquartile range (IQR) with median, 25th and 75th percentile, minimum and maximum values; d) Cumulative survival of CRC patients according to CRY1 (left) and CRY2 (right) expression levels. A significant difference was found in cumulative survival rates of CRC patients splitted at the median value according to CRY1 expression (p = 0.042) and CRY2 expression (p = 0.043). Patients with high expression levels showed significantly poorer survival rates

Fig. 2

Time related patterns of cryptochrome gene expression in colon cancer cell lines. Relative expression of CRY1 mRNA level in CaCo2, HCT116, HT29 and SW480 cells with and withour synchronization with serum shock (SS). Two biological replicates were each assayed in triplicate and results were expressed as mean ± standard deviation (SD)

Fig. 3

Time related patterns of cryptochrome gene expression in colon cancer cell lines. Relative expression of CRY2 mRNA level in CaCo2, HCT116, HT29 and SW480 cells with and withour synchronization with serum shock (SS). Two biological replicates were each assayed in triplicate and results were expressed as mean ± standard deviation (SD)

Fig. 4

Evaluation of CRY1 and CRY2 protein levels in colon cancer cell lines. a Immunoblot detection of CRY1 and CRY2 protein in CaCo2, HCT116, HT29 and SW480 cells harvested 22 h after synchronization with serum shock; b-c) Immunoblot detection of CRY1 and CRY2 protein in CaCo2, HCT116, HT29 and SW480 cells with and without cryptochrome gene ectopic expression. Western blot analysis was performed to detect the protein expression levels of CRY1 and CRY2 upon transfection using anti-CRY1 and anti-CRY2 antibody as well as anti-FLAG antibody. β-Actin antibody was used as control. d basal p53 levels in CaCo2, HCT116, HT29 and SW480 cells; e) p53 levels upon CRY1 and CRY2 ectopic expression in the four colon cancer cell lines; bars, standard deviation (SD); *P < 0.05; **P < 0.01. Each western blot analysis was performed five times

Fig. 5

FISH experiments performed on colon cancer cell lines. a-b UCSC maps of the BAC clones used for CRY1 (a) and CRY2 (b) copy number alterations. Genes are reported in blue at the bottom of the figures. c-f FISH results for CRY1 and CRY2 probes (in green and red, respectively) obtained in HCT116 (c), SW480 (d); HT29 (e), and CACO-2 (f) cells. See text for details

Fig. 6

Evaluation of apoptosis changes upon CRY1 and CRY2 ectopic transfection in colon cancer cell lines. a-b Apoptosis was evaluated by AnnexinV-FITC and Propidium Iodide stained cells using Apoptosis Detection Kit (BD Biosciences), according to the manufacturer’s protocols. All flow cytometry results were analyzed with FACSuite Software v.1.0.5.3841 (BD Biosciences); bars, standard deviation (SD); *P < 0.05; **P < 0.01; three biological replicates were prepared and each assayed in triplicate and the results expressed as mean ± standard deviation (SD)

Fig. 7

Evaluation of proliferation and cell cycle changes upon CRY1 and CRY2 ectopic expression in colon cancer cell lines. a MTT assays performed on CaCo2, HCT116, HT29 and SW480 cells transfected with CRY1 or CRY2 constructs. Wells containing transfected cells with reagents alone (Mock) were used as negative control; b) cell counts performed on CaCo2, HCT116, HT29 and SW480 cells transfected with CRY1 or CRY2 constructs. Wells containing transfected cells with reagents alone (Mock) were used as negative control; three biological replicates were prepared and each assayed in triplicate; the results were reported as mean ± standard deviation (SD); c) cell cycle analysis was performed three days after transfection on both attached and floating cells using the Cell-Cycle Test (BD Biosciences). Propidium Iodide stained cells (>20.000 events) were analyzed on FACSVerse flow cytometer (BD Biosciences); bars, standard deviation (SD); *P < 0.05; **P < 0.01; four biological replicates were each assayed in triplicate and results were expressed as mean ± SD

Fig. 8

Patterns of response to chemotherapeutic agents in colon cancer cell lines. a apoptotic and pre-apoptotic response to treatment with 5-fluorouracil (5-FU) or oxaliplatin (OXA) in CaCo2, HCT116, HT29 and SW480 cells; b) Effect of CRY1 or CRY2 ectopic expression on response to treatment with 5FU or OXA in colon cancer cell lines. The cell lines were treated with 1 μM, 5 μM and 10 μM of 5FU or OXA for 24 h at 37 °C in 5 % CO₂ atmosphere 48 h after transfection with CRY1 or CRY2 constructs. Wells containing transfected cells with CRY1 or CRY2 constructs were used as negative control (Mock). Cell viability was determined by MTT assay. bars, standard deviation (SD); *P < 0.05; **P < 0.01; three biological replicates were each assayed in triplicate and results were expressed as mean ± SD