Small changes in gene expression influence urothelial cancer patient survival

Siddharth Shukla, 3rd year PhD candidate in Biochemistry

Cancer is a deadly disease that strikes with vengeance, and unless preventative action is taken at the earliest, a diagnosis often means a short survival span for the patient. In order to be able to predict cancer susceptibility, the knowledge of presumptive risk factors is necessary. While environmental factors play a role in the onset of cancer, often, risk factors manifest themselves in the form of small changes in the human genome, which are called mutations. A mutation can disrupt the normal function of a benign cell, turning it cancerous. This makes screening for mutations highly informative in the context of a disease like cancer.

You might have heard of Angelina Jolie’s decision to undergo a preventative double mastectomy in 2013, as she is a carrier for a potential breast cancer-causing mutation in the gene BRCA1. If other such cancer markers were identified, a person with a family history of cancer could undergo genetic testing to understand whether she or he were at risk, which could save much heartache later on for the both the patient and the doctor.

But where do you even start looking for such mutations? Doctors usually rely on family history for such investigations, but an unscreened family member could diminish the chances of identifying cancer-causing mutations with confidence. However, if a large number of unrelated patients all test positive for the same mutation, this gives the researchers something to probe deeper in order to establish causality. The concept of being able to identify the role of recurring mutations in unrelated patients intrigued Dr. Tom Cech’s lab in the Department of Chemistry & Biochemistry. In particular, they became interested in looking at the role of mutations in the promoter region of the TERT gene in cancer.

The DNA is double stranded, and with each round 
of cell replication, one strand called the lagging strand 
keeps getting shorter due to peculiarities of the replication 
machinery. The telomerase complex can extend the template 
strand so that the lagging strand can now be extended, and
 keeps the cell in an active proliferation state. 
Source: oregonstate.edu

The TERT gene codes for the TERT protein, which is the enzymatic component of a protein complex called telomerase. The telomerase enzyme complex’s activity is akin to a top-up when your cell phone’s balance is about to run out. Except that here, the cell phone’s balance is your DNA, packed into linear chromosomes, which is constantly getting shorter with each round of cell division (explained nicely in our previous post). When the cell divides, the ends of the chromosomes, called the telomeres, get shorter, and eventually pass a threshold that leads to cell death. This phenomenon is also the reason behind ageing, one of the central dilemmas of life. The telomerase complex can ‘top-up’ the telomeres and increase the ‘balance’ of your cell, but its activity is tightly regulated in the body and is limited to cells that are required to rejuvenate rapidly, such as stem cells.

However, many cancer cells have figured out a way to maintain their telomeres. The majority of them do so by activating the telomerase complex, thereby doing continuous top-ups of their DNA and allowing them to remain immortal. The telomerase complex is therefore a central component of a cancerous cell’s survival machinery.

Point mutations are single nucleotide
changes that occur in the genome for 
a variety of reasons. Such changes can 
affect the sequence of the protein made, 
as well as turn off protein production 
completely as well. In case of the TERT 
promoter mutations, these changes 
affect the level of the messenger 
RNA made from the DNA.
Source: academic.brooklyn.cuny.edu

Because of the important role of the telomerase complex in cancer, the Cech lab started by looking at the effect of TERT mutations on the telomerase complex. In an expansive and collaborative effort led by a post-doc, Dr. Sumit Borah, and a graduate student, Linghe Xi, the group screened 23 bladder cancer cell lines and identified two recurrent point (single nucleotide) mutations in the the TERT gene.

Interestingly, they found that these mutations were actually not in the coding sequence, or the sequence that carries information passed on to the protein, but rather were upstream in a sequence known as the ‘promoter’ of a gene. The promoter promotes the transcription of the DNA information in the gene to its mRNA counterpart, which in turn is translated into protein. The group found that these mutations increased the levels of the TERT mRNA 18-fold over that of a normal control.

Around the same time that the group started working on this project, graduate student Linghe was working on a method to quantitate the amount of telomerase complex components in the cell. According to Linghe, her previous work segued nicely into the urothelial cancer project. Using the method she had previously developed, she was able to quantify the levels of the telomerase protein and its activity in the cancer cell lines, which were found to be two-fold higher in the cancer cell lines bearing the promoter mutations compared to the controls. In other words, cells lines containing the TERT promoter mutations had more of the TERT mRNA and protein, which in turn led to more activity of the telomerase complex in these cells. The group hypothesized that an increase in TERT mRNA levels and telomerase activity could be a potential marker for urothelial cancer. Indeed, of the cell lines screened, those that contained the highest levels of TERT mRNA and protein showed longer telomeres, suggesting enhanced telomerase reactivation.

However, one thing didn’t make sense. The group was confounded by the apparent lack of direct association between TERT mRNA levels and patient survival in the Cancer Genome Atlas cohort (TCGA), one of the largest projects underway in the United States to establish causality of cancer based on genetic mutations. With the help of collaborators, the group realized that TCGA fails to collate disease-specific survival of patients; that is, the cohort does not separate patients who died of the reported disease from those who died in an accident or due to some other cause. The group therefore switched their focus to only those patients who died of cancer. Further, patients who underwent chemotherapy were also removed from the analysis to exclude secondary effects on survival, and only those patients who had the tumor surgically removed were included.

Once these criteria were used, the group found that high levels of TERT mRNA inversely correlated with disease-specific survival in urothelial cancer patients in two separate cohorts; that is, patients who had high levels of TERT mRNA in their tumors were more likely to die from the disease. According to Sumit, establishing this causality was very important since it points to the significance of the biochemical data generated by the group in a clinical setting. Therefore, it is now theoretically possible to screen for TERT mRNA levels and telomerase activity in cancer patients to get an idea of the aggressiveness of the cancer, since these parameters are likely to correlate with each other.

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The group is greatly indebted to its collaborators who provided them with the cell lines along with the background information, and also those who performed the statistical analysis of the patient cohorts. It must also be mentioned that a senior member of the lab, Art Zaug, made an invaluable contribution to the project through the analysis of the 23 different cell lines, which was no small task in itself. The full text of their article is available at: TERT promoter mutations and Telomerase reactivation in urothelial cancer, http://www.sciencemag.org/content/347/6225/1006.long.