Epigenetics and pancreatic cancer
Pancreatic cancer is a devastating disease, usually first diagnosed at a late stage when the opportunity for the most effective therapy has been missed, and survival rates have not improved for decades. Early detection of the disease is crucial for giving patients the chance of therapy, but, so far, the search for serum protein biomarkers has not yielded satisfaction. Research has, however, uncovered a complex landscape of aberrant epigenetic regulation of gene expression. The use of panels of mutational and biomarker testing is beginning to see improving sensitivity and specificity results.
Epigenetics
The idea of ‘epigenesis’ (meaning ‘above genes’) was first mooted back in 1942, and has now become what we know as epigenetics. Epigenetics refers to mechanisms that regulate gene expression that occur without changes in the actual DNA sequence. Essentially, epigenetics concerns how gene expression is promoted or repressed through the accessibility, or lack thereof, of genes and their promoter regions to the proteins involved in gene expression, such as transcription factors and RNA polymerase. The epigenetic mechanisms that promote and repress gene expression are essen-tial in biology, and of course when they go wrong disease results. Once the epigenetic mechanisms of disease development are understood, we are then presented with targets for therapy. The impact of epigenetics on human disease has been reviewed recently in more detail than space allows here in Farsetti et al. [1]. The proteins involved in epigenetic regulation of gene expression have now been defined as ‘writers’ (add the chemical modification), ‘readers’ (recognize and interpret those modifications), and ‘erasers’ (remove the modification) [2].
DNA methylation
The presence of 5-methylcytosine was first found in 1948. It was much later, though, in the 1980s, that the high frequency of CpG dinucleotides (CpG islands) in the promoter regions of genes was observed, and that the methylation of these CpG islands resulted in the repression of gene expression (gene silencing) by preventing the binding of transcription factors and RNA polymerase [3]. Cytosine methylation and demethylation occurs via DNA methyl transferases (DNMTs; writers) and ten-eleven translocation (TET; erasers) dioxygenases, respectively. Methyl-binding domain (MBD)-containing proteins (readers) bind to CpG methylation sites on the DNA and coordinate with transcription activators and repressors to regulate gene expression.
Histone protein modification
Chromosomes exist in two forms: condensed, transcriptionally inactive heterochromatin; and less condensed euchromatin, which can be transcribed. Chromatin structure is altered by the different combinations of the many histone post-translational modifications, which involve acetylation, methylation, phosphorylation and ubiquitylation. Histone acetylation creates a more open chromatin structure, promoting gene expression, whereas histone methylation can result in either activation or repression of gene expression. Histone acetyl transferases (HATs; writers) and histone deacetylases (HDACs; erasers) are responsible for histone acetylation and deacetylation, respectively. The reader proteins include bromodomaincontaining proteins that recognize acetylated histones. Likewise, histone methyl transferases (HMTs) and histone demethylases are responsible for histone methylation and demethylation, respectively [1].
Noncoding RNAs
Additionally, epigenetic regulation of gene expression can occur through noncoding RNAs, in particular microRNAs (miRNAs), which are 17–25 nucleotides long, as well as long noncoding RNAs. miRNAs result in the downregulation of gene expression by binding to the target mRNA, which is then degraded. The expression of miRNAs themselves is subject to epigenetic regulation, as the promoter regions of many miRNA genes are associated with CpG islands [4].
Epigenetics in biology and disease
DNA methylation is crucial in embryo development, genomic imprinting (where only one gene allele is expressed) and in X-chromosome inactivation in females. Incorrect methylation patterns in embryonic development result in diseases such as Prader–Willi and Angelman syndromes. As epigenetics is key for proper gene expression, aberrant epigenetic modifications result in disease, which has so far been linked to cancer (particularly metastasis; reviewed in detail in Janin et al. [5]), type 2 diabetes, neurological disorders (Alzheimer’s and Parkinson’s diseases), cardiovascular disease and also COVID-19 syndrome. Naturally, understanding the involvement of epigenetic mechanisms in disease processes has led to the development of therapeutics that target different aspects of the pathways. For example, AraC and AzaC are cytosine analogues that inhibit DMTs. AraC is used in treating acute myeloid leukemia and other hematological malignancies. AzaC is used for treating myelodysplastic syndromes and certain leukemias. HDAC inhibitors have also been approved for treating hematological malignancies as well as being in clinical trials for use in Duchenne muscular dystrophy and Becker’s patients (See Farsetti and references therein for further discussion [1]).
Pancreatic cancer
The pancreas
The pancreas is a small organ, approximately 12–15cm long, located in the abdomen stretching from behind the stomach towards the left upper abdomen near the spleen. The pancreas is often described as feather-like in shape or similar to a flat pear, and it has both endocrine and exocrine functions. Its endocrine functions are involved with blood sugar regulation, secreting the hormones insulin, glucagon, somatostatin and pancreatic polypeptide, and hence it is a key organ in diabetes. However, the majority of its function is as an exocrine gland, releasing pancreatic juice into the duodenum. This pancreatic juice contains bicarbonate that neutralizes the acid entering the duodenum from the stomach as well as enzymes to break down carbohydrates, proteins and fats. Inflammation of the pancreas (pancreatitis) can be caused by chronic alcohol consumption and gallstones. Pancreatic cancer (PC) can develop following chronic pancreatitis or for other reasons.
Incidence and survival rates
Recent audits and research show increasing rates of PC over the last two decades [6–8], possibly due to increased longevity and increased diabetes incidence. According to Cancer Research UK, PC is the 10th most common cancer in the UK, with around 10 500 people being diagnosed each year. However, PC is the 5th most common cause of cancer death in the UK, with 10-year survival rates at only 5%, a number that has barely improved in the last 50 years [6].
Symptoms and risk factors
The low survival rates are the result of late diagnosis (stage 4; where metastasis has already occurred), as the main symptoms of abdominal pain, weight loss, jaundice, nausea/vomiting and decreased appetite often only appear at that late stage [9].
There are few specific risk factors for PC, with a family history of the disease being a factor in only 10% of patients. Generally, being older, overweight, a smoker, a heavy drinker, having diabetes, gallstones and chronic pancreatitis can all increase risk of PC [6].
Usual diagnosis and treatment
Currently, diagnosis of PC is made through a series of observations via blood tests, and more definitively by CT or MRI scan, although detection rates drop for early stage disease. The blood tests check liver/kidney function (as well as rule out other causes of the symptoms). The best treatment option is surgical resection followed by chemotherapy, but again only if the disease is detected early enough.
Biomarkers for PC
Blood tests can also assess cancer antigen (CA)19-9 levels, which is the most extensively validated biomarker for PC with a sensitivity and specificity of 79–81% and 82–90% respectively in late stage disease, but is not useful as an early screening marker because of a low positive predictive value (0.5–0.9%) [10]. However, CA19-9 levels can also be raised by a number of other malignant, benign and gastrointestinal diseases [11]. The combination of CA19-9, carcinoembryonic antigen (CEA), CA125, and CA242 showed high sensitivity and specificity for PC diagnosis, with up to 90.4% and 93.8%, respectively, significantly higher than the accuracy of a single serum marker [12]. In recent years, CP4, CP4B, PFAA, MUC5AC, and OPNT+TIMP-1 have shown promise for earlier detection, staging and disease differentiation, but further work is needed [13].
Role of epigenetics in PC
Whole-genome and whole-exome sequencing studies have revealed that a core of driver mutations in KRAS, TP53, CDKN2A and SMAD4 are important in early progression of PC. However, there is extensive genetic heterogeneity in PC patients, and there is now much evidence that chromatin remodelling and epigenetic mechanisms are involved in the progression to metastasis. This has recently been discussed in detail in Pandey et al. (See Pandey et al. and references therein [14]). The number of genes involved and their knock-on effects is too large to discuss individually here, but the following examples provide an idea of how fundamental epigenetic regulation is to the development of PC. Figure 2 in Pandey et al. provides an overview of how epigenetic mechanisms contribute to PC (https://www.futuremedicine.com/doi/10.2217/epi-2022-0177) [14].
It is well known that CpG methylation patterns are abnormal in PC. Increased levels of expression of DNA methylation writers (DNMT1, DNMT3a and DNMT3b) and decreased expression of DNA methylation erasers (TET1 and TET2) are associated with poor prognosis in PC patients. The methylation reader, MeCP2 has also been found to be upregulated in PC tissues.
The involvement of histone acetylation in PC is complex. Increased acetylation at the promoters of genes in the LOCK regions are associated with an increase in expression of genes associated with metastasis has been seen in PC. Also, expression of HDAC1 and 2 has been associated with PC metastasis, and a small clinical trial noted improved results when HDAC inhibitors were provided with conventional chemotherapy.
Abnormal expression of noncoding RNAs has been seen to be prominent at all stages of PC from initiation through to metastasis and chemoresistance. One study to identify a miRNA expression signature in PC, identified statistically significant upregulation of miR-21, miR-27a, miRNA-146a, miRNA-196a and miRNA200a, and downregulation of miR-20a, miR-96 and miR-217. Additionally, increased expression of long noncoding RNAs MALAT1 and HOTAIR, has been seen in PC.
Role of epigenetic markers in PC diagnosis
For a disease where most diagnoses take place at an advanced stage, too late for the patient to benefit from the only real therapy option of surgical resection, it is clear that currently there is no single biomarker that can identify disease early enough to improve survival rates. However, some success is being achieved by combining mutational and biomarker testing, such as evidenced by Cohen et al. in 2017 and 2018 [15,16], where the Johns Hopkins CancerSEEK panel gave specificity of >99% and sensitivity of 69% for PC patients. Adding analysis of cfDNA methylation may yet improve results enough to allow detection of early stage PC. Additionally, concentrations of plasma miR-221 have been shown to correlate in a statistically significant manner with PC disease progression, potentially providing another avenue to diagnosis, therapy decision-making and monitoring [14,17].
Conclusion
PC is a devastating disease, usually detected too late for meaningful therapy, associated with very poor survival rates and is predicted to become the second deadliest malignancy in the USA by 2030 [18]. However, much work has been done to understand the drivers of PC and the epigenetic landscape associated with it, and the use of panels of markers is improving the specificity and sensitivity of testing for early diagnosis. However, perhaps the real challenge will be in working out how to roll out a screen for a disease that has few obvious risk factors and is virtually asymptomatic in the early stages.
The author
Alison Sleigh PhD
Clinical Laboratory International
References
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