First person – Atanu Ghorai

Atanu Ghorai

How would you explain the main findings of your paper in lay terms?

One of the major treatment modalities in cancer is radiation therapy. Radiation induces irreparable DNA damage followed by cell death to kill cancer cells. However, there are several radiation-induced cellular and molecular events that are still not clearly understood, but which can greatly impact the outcome of radiotherapy. One such event, therapy-induced senescence (TIS, a state of growth cessation), has been associated with ‘tumour dormancy’ and is one of the mechanisms of survival of residual resistant cancer cells during cancer therapy. The underlying mechanisms of TIS upon radiation treatment are poorly understood. In this study, we uncovered that cells from the most aggressive brain tumours (glioblastoma) exhibited a ‘bi-phasic’ mode of DNA damage upon radiation treatment. The second phase of DNA damage was highly associated with TIS. To break this effect down further, we explored whether prolonged replication stress (disturbance in the process of duplicating genetic material) might be the source of the second phase of DNA damage. Indeed, these cancer cells showed signs of massive replication stress with non-canonical stress management. Moving ahead, we pinpointed the involvement of ATR (a major surveillance protein involved in the replication process) by using genetic ablation and chemical inhibitors. In the end, we showed that cells with compromised ATR levels and activity were highly susceptible to Olaparib (a PARP-1 inhibitor), which is used clinically in treatments for other cancers. Thus, our study unveiled the mechanisms of residual resistant cell survival via TIS and highlighted a newer and more effective strategy to combat such deadly residual glioblastoma cells.

Were there any specific challenges associated with this project? If so, how did you overcome them?

In our earlier studies, we established that glioblastoma cells enter a senescence phase (therapy-induced senescence, or TIS) upon radiation treatment if they grow for a longer time (e.g. several days). This non-proliferative senescence phase lasts from day 6 or 7 to day 12 or 14 post-irradiation, depending upon cell type. In this study, we showed that replication stress with persistent phosphorylated ATR (pATR) somehow facilitated the senescence state. However, it was a great challenge to perform EdU and BrdU assays or DNA fiber assays in these cells, as the uptake of nucleotide analogues was very low. This resulted in only a small fraction of cells suitable for analysis, which was not sufficient to establish our hypothesis. To overcome this, we irradiated multiple 10 cm culture plates, increasing the amount of starting material, so that we could get more cells positive for nucleotide analogue labelling. This strategy allowed us to push this project forward and ultimately confirmed that replication stress management was the important mediator of therapy-induced senescence.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

Early in the project in 2017, I observed that if U87MG glioblastoma cells were kept in culture for 14 days or more after irradiation, they showed a ‘bi-phasic’ γH2AX response, a marker of DNA damage. Although the first peak of DNA damage (an immediate effect of radiation) was expected, the second peak was not clearly understood, but was associated with radiation-induced senescence, a survival mechanism of radio-resistant glioblastoma cells. We then postulated that the second peak of γH2AX might be due to the accumulation of replication stress. The ‘eureka’ moment came when we indeed observed faulty replication events as detected by a DNA fibre assay. Our study reached a crucial moment when my co-author Bhawna elegantly showed that the second peak of γH2AX was primarily associated with S-phase cells (cells positive for EdU, labelled via click chemistry). We also found that pATR, a key player in replication stress management, was consistently high during the second phase of DNA damage. Inhibition of ATR reduced the senescence phenotype. By that time, I was confident that this would be an interesting story to investigate in detail.

Why did you choose Journal of Cell Science for your paper?

JCS is one of the top-rated and prestigious journals in the field of cell biology, acknowledged by many renowned researchers. It is always enjoyable to read articles in JCS because of its high quality, lucidness and attractive website design. Our lab has published a few papers in JCS in recent years and we have had a great publishing experience. The current study talks about the fundamental cellular and molecular understanding of therapy-induced senescence in glioblastoma, which perfectly aligns with JCS's focus areas. In addition, the ‘First Person’ section is also beneficial for early career independent researchers like me. Overall, I have a high regard for JCS, and I would love to publish here again.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

I am privileged to have had many successful and brilliant researchers as mentors throughout my scientific career. During my doctoral training, Dr Utpal Ghosh (University of Kalyani, West Bengal), Dr Asitikantha Sarma (Inter-University Accelerator Centre, New Delhi) and Prof. Nitai P Bhattacharyya (Saha Institute of Nuclear Physics, Kolkata) played instrumental roles in launching me into the scientific world and providing solid career building blocks. My first postdoc advisors, Prof. B J Rao and Prof. Ullas S Kolthur (Tata Institute of Fundamental Research, Mumbai), were the most versatile, intense, dedicated and hard-working researchers I have ever met. Their impact on my research career trajectory is immeasurable! My last postdoc advisors, Prof. Shilpee Dutt and Prof. Amit Dutt, actually introduced me to cancer research at the Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, and this current study was carried out under the supervision of Prof. Shilpee Dutt. They both provided valuable advice beyond research and helped me to find independent positions after my postdoc tenure.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

The liberty involved in doing any sort of research is the main ‘unique selling point’ for pursuing a career in science. Along with that freedom, the regular scientific challenges keep motivating me to be more productive and innovative. Furthermore, I have been inspired by observing my mentors' enthusiasm, dedication and passion for science. My first postdoc at the Tata Institute of Fundamental Research, Mumbai, shaped my research career to where I am now. My tenure there in Prof. B J Rao's lab taught me to nurture scientific ideas and implement them in a focused way. During this period, I benefitted from relentless scientific talks and discussions and became profoundly motivated to make research my professional career.

Who are your role models in science? Why?

All my teachers (from school to university) and mentors (during my PhD and postdocs) are my role models in science. Their knowledge, unique teaching styles and dedication to creating next-generation scientists have unequivocally crowned them as role models. Additionally, I must mention my childhood role model. I was mesmerized by Dr Mani Lal Bhaumik, a renowned physicist known for his seminal work in excimer laser technology used in Lasik eye surgery and other applications, who used to visit our school. I grew up reading his biography and seeing how he became a famous scientist from a small village (that is also my birthplace!). That definitely planted the nascent desire inside me to be a scientist in the future!

What's next for you?

This work was a major part of my postdoc work done in Shilpee Dutt's lab. As an independent researcher, one of my goals is to further explore the fascinating cellular and molecular roles of ATR beyond its well-known functions associated with replication stress. My next plan is to investigate its clinical significance in the context of cancer and how it can be targeted for better therapeutics.

Tell us something interesting about yourself that wouldn't be on your CV

I love cooking Indian cuisine with more perfection than I apply to my research work! Whenever my cooking is ‘peer-reviewed’, it receives applause without any ‘revisions’! Also, it is my dream to learn to play at least one musical instrument.