Investigating cell death resistance in tumor cells

NGP. Please tell us about the research currently being carried out in your laboratory.

HF. My main research interest is in understanding how cell survival and death are controlled in normal cells and in cancer cells. Specifically, we are looking at the cell survival and death machinery – the cross-talk machinery – that is controlled by the family of proteins called the 14-3-3 proteins. We’d then like to translate that into clinically relevant applications. In addition, also based on this research and its clinical significance, we’d like to identify small molecule modulators of those pathways, using our Chemical Biology Discovery Center.

NGP. You’ve discovered that by cutting off a key gene, lung cancer cells are left homeless and incapable of surviving on their own. Could you talk us through the behavioral characteristics of cancer cells in general?

HF. Cancer cells are derived from normal cells through the acquisition of a series of genetic or epigenetic changes. Those changes allow cancer cells to gain the capability for uncontrolled cell proliferation, the ability to invade other tissues, and the ability to spread to other tissue sites. We and others have found that 14-3-3 proteins are up-regulated in lung cancer cells and also several other types of cancer cells. But does this mean they play a role in the tumorigenesis process – in invasion and spreading?

One of the molecular features of tumor cell invasion and spreading is the ability to resist anoikis, the Greek word for homelessness. Normal cells, when detached from the extracellular matrix, will trigger a suicidal program called anoikis, and then die. That’s a safeguard mechanism, because it eliminates abnormal cells. Tumor cells, due to disregulated pathways, can resist this cell suicide program upon cell detachment and therefore they survive in the absence of adhesion to the matrix and spread to other parts of the body.

So we are interested in molecular events that allow cancer cells to do that, and we have found that 14-3-3ζ plays an important role in this process. We are trying to understand the molecular mechanism that controls this property of the cancer cell.

NGP. Could you give us more specific information about the gene in question and how it is involved in the development of lung cancer?

HF. 14-3-3ζ is a member of the 14-3-3 protein family, which play a critical role in diverse signaling pathways that control important physiological processes such as cell growth. A previous study showed that the 14-3-3ζ gene seems up-regulated in lung cancer. We carried out an extensive study in our group, along with my physician scientist collaborator, Dr. Fadlo Khuri, to demonstrate that this gene is also up-regulated in lung cancer patient tissues. That made us think this gene might play an important role in this process. So we examined multiple aspects of tumorigenesis and discovered that actually 14-3-3ζ plays a critical role in supporting anoikis resistance in lung cancer cells.

We did that by specifically knocking down a single isoform of 14-3-3ζ in lung cancer cells and then examining the impact of removal of the ζ isoform on tumorigenic activity of these lung cancer cells.

NGP. What was challenging in your quest to progress this study?

HF. To find the phenotype of 14-3-3ζ knockdown cells. Only when we challenged the cells by withdrawing serum and removing cells from our matrix, did we see a dramatic difference between parental and knockdown cells. This is a discovery process, and the key issue is the molecular mechanism that underlines its phenotype. That’s how we discovered the involvement of the BH3-only proteins in this important process.

NGP. You managed to silence the 14-3-3ζ gene using RNA interference. Can you please explain what this involves?

HF. We wanted to see the effect of decreasing 14-3-3ζ on tumorigenesis. We used a very popular technique called RNA interference, or RNAi. We targeted the three regions of the 14-3-3ζ messenger RNA, using a viral vector to introduce into cells small hairpin RNA that can bind to the specific 14-3-3 ζ messenger RNA, leading to its degradation and the subsequently decreased expression of the 14-3-3ζ protein.

NGP. You’ve already touched on how all this affected cancer cell growth – could you please summarize that?

HF. The major phenotype of 14-3-3ζ knockdown is decreased – what we call anchorage-independent growth of lung cancer cells. The parental cells – the lung cancer cells, A549 – can readily form colonies in the absence of matrix support. Those cells with decreased 14-3-3ζ either form only small colonies or fail to form colonies.

NGP. You mentioned that you would now be looking to take this into a clinically relevant direction. How will you now partner up with the pharmaceutical industry so they can benefit from your research findings?

HF. One benefit is that our findings offer a new class of targets for pharmaceutical interventions. 14-3-3 appears to control multiple pathways. Cancer therapy often uses a combination of agents to target multiple pathways or uses an agent that impacts multiple targets, such as Sorafnib or Hsp90 inhibitors. Here you have one target that can impact multiple significant pathways – I believe, this is a new class of target with tremendous potential. We are in the process of developing small molecule inhibitors, which hopefully will lead to future collaborations with interested partners.

NGP. Do your findings have any implications beyond lung cancer; and if so, which other cancers could be affected?

HF. We are extending our work to other cancer types like head and neck cancer and breast cancer. It has been reported that 14-3-3ζ is upregulated in several other cancers such as oral cancer and stomach cancers. So our discovery may have relevance to those cancers as well.

NGP. Moving on to what is still ahead of you, what are the next steps in your research and what are the challenges you will face?

HF. We want to move into a more clinically relevant direction for translational research. We also want to identify – which we are doing now – effective 14-3-3 inhibitors for potential therapeutic interventions. For the translational research aspect, there’s a recent report in Cancer Research by another group that strongly supports our direction. The authors showed that up-regulated 14-3-3ζ is associated with poor survival outcome of lung cancer patients. Our next step is to determine the prognostic value of 14-3-3ζ in lung cancer, studying two large lung cancer patient populations through our trans-continental collaborations with Dr. Fadlo Khuri at Emory and Dr. Rafael Rosell in Spain. Through these studies, we will establish a correlation between 14-3-3ζ and the responsiveness of lung cancer patients to different therapeutic options.

Certainly, we will continue researching the role of 14-3-3ζ in cell survival and death, in order to provide the mechanistic basis for small molecule inhibitor discovery and for the translational research.

And we will continue working with our academic team, including physician scientists and chemists to move this project forward. I’m sure that down the road, we’ll form new teams of academics and experts from the pharmaceutical industry working together to move meaningful discoveries into therapies that will benefit patients.

About Dr. Haian Fu

Dr. Haian Fu is Professor of Pharmacology, Hematology and Oncology at Emory University School of Medicine. He also serves as Director of the Emory Chemical Biology Discovery Center and co-principal investigator, along with Dr. Fadlo Khuri, in the National Cancer Institute-funded program project ‘Targeting Cell Signaling in Lung Cancer to Enhance Therapeutic Efficacy’.