Abstract
There is a growing body of evidence implicating aberrant circadian clock expression in the development of cancer. Based on our initial experiments identifying a putative interaction between BRCA1 and the clock proteins Per1 and Per2, as well as the reported involvement of the circadian clock in the development of cancer, we have performed an expression analysis of the circadian clock genes Per1 and Per2 in both sporadic and familial primary breast tumors and normal breast tissues using real-time polymerase chain reaction. Significantly decreased levels of Per1 were observed between sporadic tumors and normal samples (P < .00001), as well as a further significant decrease between familial and sporadic breast tumors for both Per1 (P < .00001) and Per2 (P < .00001). Decreased Per1 was also associated with estrogen receptor negativity (53% vs 15%, P = .04). These results suggest a role for both Per1 and Per2 in normal breast function and show for the first time that deregulation of the circadian clock may be an important factor in the development of familial breast cancer. Aberrant expression of circadian clock genes could have important consequences on the transactivation of downstream targets that control the cell cycle and on the ability of cells to undergo apoptosis, potentially promoting carcinogenesis.
Keywords: Circadian clock, gene expression, Per1, Per2, BRCA1
Introduction
Deregulation of the circadian clock has been implicated in many types of cancers, and large studies indicate that increased breast cancer risk is correlated with increased years of night shifts [1–3]. The circadian clock proteins Per1 and Per2 function in a series of positive and negative feedback loops that coordinate the circadian clock in both the brain and peripheral tissues [4]. Molecularly, several links between the circadian clock and DNA damage response have been drawn. Mice that are deficient in the mPer2 gene are not only deficient in their circadian clock rhythm but also cancer-prone and sensitive to γ-irradiation [5]. Additionally, thymocytes from mPer2 mutant mice either are less sensitive or have a slower apoptotic response to γ-irradiation [6]. Conversely, overexpression of mPer2 induces apoptosis and alters the expression levels of apoptosis-related genes in mouse tumor cells [7].
Moreover, overexpression of Per1 sensitizes HCT116 colon cancer cells to infrared-induced DNA apoptosis, and suppression of Per1 blunts apoptotic response [8]. Per1 physically interacts with ATM and Chk2, which are known to function in a DNA damage response complex along with other components such as BRCA1. Furthermore, Per1 expression is decreased in small lung cell carcinoma tumors, as well as in a small cohort of breast tumors [8].
Based on the reported involvement of the circadian clock in the development of several cancers, including breast cancer, as well as on experiments that have identified a putative interaction between BRCA1 and Per1 and Per2 in a yeast two-hybrid system, we used real-time polymerase chain reaction (PCR) to examine Per1 and Per2 in both familial and sporadic breast tumors and normal breast tissues to identify any changes in gene expression. We observed significant alterations in Per1 and Per2 expression suggesting that their deregulation may contribute to the development of sporadic or familial breast cancer, or both. Additionally, we analyzed the associations of Per1 or Per2 with breast tumor characteristics and found a significant association of Per1 expression with estrogen receptor (ER) status. Per1 and Per2 influence the transcription of genes and induce apoptosis; therefore, lack or misregulation of their expression could contribute to an improper apoptotic response and the development of breast cancer.
Materials and Methods
Tumor Samples
mRNA from 34 sporadic breast tumors were obtained as part of a prospective study of molecular alterations in auxiliary node-negative disease [9]. Eleven tumors were from familial breast cancer cases from the Ontario site of the Breast Cancer Family Registry [10].
DNA and RNA have previously been extracted by conventional techniques [11]. mRNA from normal breast tissue samples were obtained from 13 additional subjects with “sporadic” breast cancer. The normal breast tissue samples were selected by a pathologist from regions adjacent to tumors. All relevant institutional review boards approved the study protocol, and written informed consent was obtained from all participants.
Real-Time PCR Reactions
cDNA was reverse-transcribed, and real-time PCR was carried out as described [12]. HPRT-1, which has little variation in expression between tumors, was chosen for normalization. Primer/probe pairs used for the experiments were purchased from ABI (Foster City, CA) [BRCA1, cat no. HS 00173233-m1; Per1, cat no. HS 00242988-m1; Per2, cat no. HS 002561440m1; HPRT-1, cat no. 4326321E (endogenous control)].
Statistical Analysis
A descriptive analysis comparing the frequency distributions of tumor characteristics between the Per1 and Per2 groups (high versus low) was performed by a pathologist using contingency tables. The association of each characteristic with Per1 or Per2 expression was investigated by Fisher's exact test [13]. The high-versus-low levels of Per1 or Per2 expression were established by dividing the tumor sets around the mean level of expression of each gene.
All statistical analyses were performed using SAS Statistical Software, version 8.2 (SAS, Inc., Cary, NC). R statistical software, version 1.9.1, was used to generate box plots. P < .05 was considered to be statistically significant for all analyses.
Results
Expression of Per1 and Per2 in Breast Tumors
Our preliminary results identifying the interaction of BRCA1 with Per1 and Per2 in the yeast two-hybrid system (data not shown) and the increasing amount of evidence that circadian clock perturbations may result in breast cancer led us to investigate the expression levels of BRCA1, as well as Per1 and Per2, in both sporadic and familial primary human breast tumors and normal breast tissues. Not surprisingly, low levels of BRCA1 mRNA were identified in sporadic tumors compared to both normal breast tissues and familial cancers, consistent with decreased expression, rather than mutation, being the means for BRCA1 inactivation in these tumors (Table 1) [14].
Table 1.
Summary of Gene Expression in Normal Tissues Versus Sporadic or Familial Breast Tumors for Per1 and Per2.
| Name of Gene | Tissue Type | Mean Expression* | Significance (P) |
| BRCA1 | Normal | 0.9 | |
| Sporadic | 0.6 | .01 | |
| Familial | 1.0 | .77 | |
| Per1 | Normal | 4.0 | |
| Sporadic | 1.8 | < .00001 | |
| Familial | 0.9 | < .00001 | |
| Per2 | Normal | 4.9 | |
| Sporadic | 3.6 | .005 | |
| Familial | 2.6 | < .00001 |
Test gene/HPRT-1 expression ratios as measured by quantitative real-time PCR.
Importantly, both Per1 and Per2 exhibit significantly decreased levels of expression in sporadic and familial tumors compared to normal breast tissues (Table 1). Furthermore, Per1 expression is also significantly decreased in familial tumors compared to sporadic tumors (Table 1), suggesting that deregulation of the circadian clock may contribute to the familial aspect of these tumors.
Association of Per1 and Per2 with Sporadic Tumor Characteristics
The use of primary human breast tumors allowed us to examine correlations between tumor or disease characteristics with Per1 and Per2 expression levels, and the results for Per1 are summarized in Table 2. The group of breast tumors with a “low” level of Per1 or Per2 mRNA was compared to those with “high” Per1 or Per2 expression levels, and we detected a weak association between low Per1 expression and ER status (P = .04), which needs to be investigated further in a larger sample set. Tumors with low Per1 levels were more likely to be negative for ER receptor than those with high Per1 levels (53% vs 15%). This association was not observed for Per2 (P = .30), and no other significant associations were identified for Per2 (not shown).
Table 2.
Tumor Characteristics and Association with Per1 Expression Levels (N = 33).
| Characteristic | Low Per1 Levels (≤ 1.60) | High Per1 Levels (> 1.60) | P* (Fisher's Exact Test) |
| Number of patients [n (%)] | 20 (60.6) | 13 (39.4) | |
| Age (years) {mean (SD) [minimum, maximum]} | 55.2 (11.5) [35.1, 75.1] | 55.2 (11.7) [37.5, 69.7] | |
| Menopausal status [n (%)] | |||
| Premenopausal | 6 (30.0) | 4 (30.8) | 1.00 |
| Perimenopausal | 2 (10.0) | 1 (7.7) | |
| Postmenopausal | 11 (55.0) | 8 (61.5) | |
| Missing | 1 (5.0) | 0 (0.0) | |
| Maximum size of tumor (cm) [n (%)] | |||
| 0.5 to < 1.0 | 0 (0.0) | 0 (0.0) | .06 |
| 1.0 to < 2.0 | 2 (10.0) | 4 (30.8) | |
| 2.0 to < 5.0 | 17 (85.0) | 7 (53.8) | |
| ≥ 5.0 | 0 (0.0) | 2 (15.4) | |
| Missing | 1 (5.0) | 0 (0.0) | |
| ER status | |||
| Positive | 9 (45.0) | 9 (69.2) | .04 |
| Negative | 10 (50.0) | 2 (15.4) | |
| Equivocal | 0 (0.0) | 2 (15.4) | |
| Unknown/not determined | 0 (0.0) | 0 (0.0) | |
| Missing | 1 (5.0) | 0 (0.0) | |
| Progesterone receptor status | |||
| Positive | 5 (25.0) | 5 (38.5) | .86 |
| Negative | 12 (60.0) | 7 (53.8) | |
| Equivocal | 2 (10.0) | 1 (7.7) | |
| Unknown/not determined | 0 (0.0) | 0 (0.0) | |
| Missing | 1 (5.0) | 0 (0.0) | |
| Histologic grade | |||
| 1 | 1 (5.0) | 1 (7.7) | .90 |
| 2 | 6 (30.0) | 5 (38.5) | |
| 3 | 9 (45.0) | 4 (30.8) | |
| Subtype | 1 (5.0) | 0 (0.0) | |
| Unknown/not determined | 2 (10.0) | 3 (23.0) | |
| Missing | 1 (5.0) | 0 (0.0) | |
| Lymphatic invasion | |||
| Yes | 5 (25.0) | 5 (38.5) | .70 |
| No | 14 (70.0) | 8 (61.5) | |
| Missing | 1 (5.0) | 0 (0.0) |
Without tumors in the missing category.
Discussion
Deregulation of the circadian clock may lead to the development of several types of cancer, including breast cancer. Consequently, we performed a genetic analysis of BRCA1 and Per1 and Per2 in both sporadic and familial primary breast tumors and normal breast tissues to identify aberrations in their expression levels and to analyze whether changes in Per1 and Per2 expression are associated with breast tumor characteristics. Interestingly, both Per1 and Per2 exhibit significantly decreased levels of expression in both sporadic and familial tumors compared to normal breast tissues. Additionally, the expression of Per1 was significantly decreased in familial breast tumors compared to its expression in sporadic specimens, suggesting that a possible deregulation of the circadian clock could contribute to the familial aspect of these tumors. Our results identifying decreased Per1 and Per2 expression in tumors is supported by previous work showing that Per gene deregulation is caused by methylation of the Per1 or Per2 promoter in approximately 50% of analyzed breasts cancers in Taiwanese women [15]. Per1 and Per2 regulate transcription and promote apoptosis; therefore, decreased levels of Per1 and Per2 in breast tumors could have important ramifications on the expression of both circadian and other downstream genes and could potentially limit the apoptotic response of the cell.
The use of primary human breast tumors allowed us to examine correlations between tumor or disease characteristics and Per1 and Per2 expression levels. Interestingly, we observed a statistically significant association between low levels of Per1 expression and negative ER status, and a weak association between low levels of Per1 expression and tumor size using our small sample, which need to be investigated further in a larger sample set. ER levels are maintained by a number of mechanisms, including activation or repression of the ER promoter at the transcriptional level. Interestingly, tumors with BRCA1 mutations also tend to be negative for ER expression [16,17], and it is possible that Per1 acts with proteins such as BRCA1 to regulate ER transcription.
We have analyzed the expression levels of Per1 and Per2 in both sporadic and familial breast tumors, as well as in normal breast tissues, and have determined that there are significant decreases in Per1 and Per2 expression levels in these tumors compared to normal breast tissues. Additionally, we identified a further decrease in Per1 expression when comparing tumors from women with familial breast cancer to tumors from women with sporadic breast cancer, suggesting that circadian clock disruption may contribute to the inherited form of the disease. Furthermore, we have detected a significant association of decreased Per1 levels with ER-negative breast tumors. These results suggest a role for both Per1 and Per2 in normal breast function and the potential deregulation of Per1 and Per2 expression in breast cancer development.
Abbreviations
- Per
period
- ER
estrogen receptor
- BRCA1
breast cancer susceptibility gene
Footnotes
We gratefully acknowledge the support of the National Cancer Institute of Canada with funds from the Terry Fox Run (I.L.A.) and a predoctoral fellowship from the United States Army Medical Research and Materiel Command (DAMD017-02-1-0496; to S.L.W.).
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