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Circadian Gene Expression and Clinicopathologic Correlates in Pancreatic Cancer

  • 2012 SSAT Plenary Presentation
  • Published:
Journal of Gastrointestinal Surgery Aims and scope

Abstract

Introduction

The circadian rhythm is responsible for physiologic homeostasis, behavior, and components of multiple metabolic processes. Disruption of the circadian rhythm is associated with cancer development, and several circadian clock genes have been implicated in loss of cell cycle control, impaired DNA damage repair, and subsequent tumor formation. Here, we investigated the expression profiles of several circadian clock genes in pancreatic ductal adenocarcinoma (PDA).

Methods

Quantitative real-time polymerase chain reaction was used to examine the circadian clock genes (brain-muscle-like (Bmal)-ARNTL, circadian locomotor output cycles kaput (Clock), cryptochrome 1 (Cry1), cryptochrome 2 (Cry2), casein kinase 1ε (CK1ε), period 1 (Per1), period 2 (Per2), period 3 (Per3), timeless (Tim), and timeless-interacting protein (Tipin)) in PDA, as well as matching adjacent and benign tissue. Logistic regression models with robust variance were used to analyze the gene expression levels, and Kaplan–Meier survival curves were generated based on gene expression.

Results

In the tumor tissue of PDA patients, compared to their matched adjacent tissue, expression levels of all circadian genes were lower, with statistical significance for Per1, Per2, Per3, Cry1, Cry2, Tipin, Tim, CK1ε, Bmal-ARNTL, and Clock (p < 0.025). PDA tumors also expressed significantly lower levels of the circadian genes when compared to benign lesions for Per1, Per2, Per3, Cry2, Tipin, and CK1ε. A significant association between low levels of expression in the tumors and reduced survival was found with Per1, Per2, Per3, Cry2, Tipin, CK1ε, Clock, and Bmal-ARNTL.

Conclusions

Our results reveal for the first time a dysregulated transcription of several circadian genes in PDA. Elevation of the gene levels in the benign and matched adjacent tissues may be indicative of their role during the process of tumorigenesis. The potential of using circadian genes as predictive markers of the outcomes and survival and distinguishing PDA from benign pancreas must be studied in larger populations to validate and demonstrate their eventual clinical utility.

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Author information

Authors and Affiliations

  1. Department of Surgery, Thomas Jefferson University, 1015 Walnut Street, Suite 618 Curtis, Philadelphia, PA, 19107, USA

    Daniel Relles, Galina Chipitsyna, Charles J. Yeo & Hwyda A. Arafat

  2. Department of Biostatistics, Thomas Jefferson University and the Jefferson Pancreatic, Biliary and Related Cancer Center, Philadelphia, PA, USA

    Jocelyn Sendecki & Terry Hyslop

Authors
  1. Daniel Relles
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  2. Jocelyn Sendecki
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  3. Galina Chipitsyna
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  5. Charles J. Yeo
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  6. Hwyda A. Arafat
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Corresponding author

Correspondence to Hwyda A. Arafat.

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Discussant

Circadian Genes in Pancreatic Ductal Adenocarcinoma: Alterations and Clinical Correlations

JOGS ID# TBD

Dr. John Allendorf (New York, NY): The authors have demonstrated a significant correlation between the level of circadian gene expression and patient survival. They have further demonstrated a significant reduction in expression of these genes within pancreatic ductal adenocarcinoma tissue compared to normal adjacent pancreas and benign tumors, suggesting that they may play a role in the mechanism of tumorigenesis or tumor progression and that expression levels of circadian genes may be a biomarker that can distinguish between benign and malignant lesions.

The authors should be commended for putting together a terrific paper and I would like to congratulate Dr. Relles on winning the resident competition this year here at the SSAT. I have several questions for the authors:

(1) Gene expression correlated with survival but did not correlate with several known pathologic predictors of survival, e.g., nodal status, perineural invasion, T stage. How do you explain that?

(2) Can you give us some sense about the anticipated normal variation in circadian gene expression in a normal pancreas over the course of a 24-h period? That is, would the differences you observed in this study be eclipsed by normal variation if the specimens were not all collected in the late morning, or was the magnitude of the differences well beyond the anticipated normal variation?

(3) As a potential biomarker, how much material do you need to perform your analysis and what is the cost? Can this be done on an FNA specimen? Did you have any trouble extracting high-quality RNA from an organ that makes RNase?

(4) Based on your work so far, have you identified any promising potential therapeutic targets for the treatment of pancreatic adenocarcinoma?

Closing Discussant

Dr. Daniel Relles: Thank you, Dr. Allendorf, for your insightful, thought-provoking questions, and for your kind words. While we’ve discussed some of the issues you raise, some of your ideas offer new potential directions for our work.

With respect to the discrepancy in the correlation with survival but not with pathologic predictors, we were surprised by this. This may be based on how we performed our statistical analysis, as we created a dichotomized variable for our survival analysis (above- and below-median gene expression) but did not do this for the pathology correlates. Perhaps, if we treated the other parameters in a similar matter, we might get statistical significance.

As for normal variation in human pancreas, this is an excellent question. There have not been studies evaluating changes in the circadian genes in normal human pancreas. A study on rat pancreas demonstrated significant changes in expression levels at various time points in the day. For our work, we assumed that within the 2–3-h time point when these tumors are removed and sent to pathology, the circadian gene expression could be considered a constant. However, physiological changes in the normal pancreatic circadian genes have not been studied. This warrants investigation of this point.

We had success in obtaining high-quality RNA. We either processed the samples for RNA isolation immediately after obtaining them from pathology, or we stored them in RNAlater, and that helped reducing the tissue degradation to a great extent. We need a very small amount of the tissue for this analysis. In our lab, we have several parallel projects looking at markers in FNA samples, and this is an excellent idea as a next step of our work. We will perform these studies shortly after we return from this conference.

In response to your final question regarding potential therapeutic targets, we are currently evaluating the functions of these genes. Preliminary results using siRNA studies implicate some of the circadian genes in tumor inflammation. We are currently working hard to delineate their function and regulation during the early stages of tumorigenesis.

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Relles, D., Sendecki, J., Chipitsyna, G. et al. Circadian Gene Expression and Clinicopathologic Correlates in Pancreatic Cancer. J Gastrointest Surg 17, 443–450 (2013). https://doi.org/10.1007/s11605-012-2112-2

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  • DOI: https://doi.org/10.1007/s11605-012-2112-2

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