Post-transcriptional modification in ceramide synthase 2 enzyme leads to tumor progression in breast cancer

Trishna Pani, Kajal Rajput and Animesh Kar joint first author interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Here, authors talk about their first author research article titled “Alternative splicing of ceramide synthase 2 alters levels of specific ceramides and modulates cancer cell proliferation and migration in Luminal B breast cancer subtype” published in Cell Death Dis (2021).

Trishna Pani did her post-graduation in biotechnology from Vellore Institute of Technology. Presently she is pursuing her Ph.D. from Amity University, Manesar, under the supervision of Dr. Ujjaini Dasgupta and co-supervision of Dr. Avinash Bajaj (RCB, Faridabad). Her area of interest is to look for the post transcriptional modification of sphingolipid genes in different breast cancer subtypes and use these genes as potential markers for subtypes detection and better therapeutic approach.

Kajal Rajput has completed her graduation and post-graduation in biotechnology from Maharishi Dayanand University (MDU), Rohtak, India. She is doing her Ph.D. from Amity University, Gurgaon under the supervision of Dr. Ujjaini Dasgupta. She’s passionately focussed in the area of cancer biology with the goal of identification of sphingolipid biomarker in various subtypes of breast cancer, and pharmacologically and genetically manipulating them for designing efficient therapeutic strategies.

Animesh Kar did his under graduation in Biomedical Science from University of Delhi, India. After that, he did his Master’s in Biophysics from All India Institute of Medical Sciences, Delhi, India. Currently, he is pursuing his Ph.D. under the supervision of Dr. Avinash Bajaj from Regional Centre for Biotechnology, Faridabad, India. His research focuses on understanding the identification and modulation of various molecular signatures in response to engineered nanotherapeutics in different cancer models.

How would you explain your paper’s key results to the non-scientific community?

Cell signalling allows cells to co-ordinate their cellular activities. These signals activate receptors present on the cell surface, which in turn activates the signalling pathways. Any deregulation during this process hinders the normal cellular functions leading to diseases like cancer. Among different types of cancers, breast cancer is the most common cancer in females. Heterogeneity in breast cancer makes the therapeutic approaches more challenging. Broadly, breast cancer categorised into four subtypes depending on the presence of different receptors such as Luminal A, Luminal B, Triple Negative, and HER2+. Each cancer subtypes exhibit a distinct gene expression pattern and show a distinct clinical outcome. So, we were curious to study the potential molecular mechanism that make each subtype unique. Sphingolipids are a family of lipids with diverse structural and signal transduction roles. Emerging data suggested that altered sphingolipid metabolism contributes to cancer initiation, progression, and metastasis. Our group previous data showed that each breast cancer subtype has a unique sphingolipid profile. In this study we extensively analysed RNA sequencing data from The Cancer Genome Atlas (TGCA). The BRCA cohort exhibited subtype-specific alternative splicing (AS) events in breast cancer patients. The AS has two events 1) Cassette exon (functional amino acid coding gene segment/ exon retained in the final transcript). 2) Intron retention (non-functional amino acid coding segment/ intron retained in the final transcript). We found an enzyme Ceramide synthase 2 (CERS2) undergoing a cassette exon event (exon 8), specifically in the Luminal B subtype. CERS2 is a key enzyme, which catalyzes very-long-chain ceramides synthesis. These very-long-chain ceramides prevent metastasis and proliferation in tumor cells. Exon 8 in CERS2 corresponds to the Lag1P catalytic domain, which determines its substrate specificity. Exon 8 loss in the protein alters ceramide chain length and decreases functional very-long-chain ceramide pool. This ceramide pool imbalance unable to reduce cancer cell proliferation and migration. We validated altered ceramide levels in Indian Luminal B tumor samples and their derived cancer cell lines.

Figure 1. Unique post-transcriptional regulation in luminal B subtype-specific cells affect the very-long-chain ceramide synthesis leading to tumor progression through increased cell proliferation and migration.

What are the possible consequences of these findings for your research area?

Our research focuses on sphingolipids role in tumor microenvironment for potential cancer therapy. Since we have interesting results related to CERS2, it is very important to see its role in a large patient cohort. Promising data from a larger cohort will give the confidence to develop this post-transcriptional event into a diagnostic marker and eventually develop it into cancer therapy.

“[…] we have interesting results related to CERS2, it is very important to see its role in a large patient cohort.”

What was the exciting moment (eureka moment) during your research?

Trishna Pani: The most exciting moment for me was when we could show the presence of both isoforms of CERS2 in the protein lysate of Indian patient tumor samples through western blotting and especially, the high abundance of the alternatively spliced CERS2 protein in the tumor as compared to its matched control.

Kajal Rajput: The eureka moment for me in this study was when we found the phenotypic effect of the overexpression of the spliced isoform in cancer cell line matched our original hypothesis.

Animesh Kar: For me the most exciting moment was to prove the existence of the spliced transcript of CERS2. When we ran the qRT-PCR amplified products of the CERS2 transcripts, not only we found the two bands of full length and spliced isoforms, but the size difference between the isoforms was exactly as we predicted.

What do you hope to do next?

A more in-depth study on the mechanism behind this post-transcriptional modification in CERS2 is our next aim. Further, we would like to do in vivo studies for the phenotypic effects of the alternatively spliced isoform of CERS2 in mice models.

Where do you seek scientific inspiration?

Trishna Pani: I was keen on science from my early school days. I was blessed to place under a professor in my post-graduation studies who inspired me to take up research as a career. My supervisor Dr. Ujjaini Dasgupta has been a perfect role model for me who has always guided me and taught me how a never give up attitude can lead to success. Dr. Avinash Bajaj inspires me a lot with his immense enthusiasm and passion for science. With all these inspiring people around me, here I am doing my research to giving back to society.

Kajal Rajput: Despite advance in medical research, cancer disease continues. This is why I am strongly attracted to cancer research and would like to solve existed complex problems. My PhD supervisors (Dr. Ujjaini Dasgupta and Dr. Avinash Bajaj) are my inspirational source, they guided me to think critically and do useful research

Animesh Kar: It gives me immense pleasure and joy to find new things, solve unanswered questions or develop possible therapeutic agents for humans. Renowned planetary scientist and science communicator, Neil DeGrasse Tyson always makes me fall in love with science. Also, I draw inspiration from both my PhD supervisors Dr. Avinash Bajaj and Dr. Ujjaini Dasgupta.

How do you intend to help Indian science improve?

Though the Indian science sector is rapidly growing with technologies and innovations, still India battel with cancer. It indicates India needs safer and targeted cancer therapies. As a researcher, I will contribute my acquired knowledge to Indian science.

Reference

Pani, T., Rajput, K., Kar, A. et al. Alternative splicing of ceramide synthase 2 alters levels of specific ceramides and modulates cancer cell proliferation and migration in Luminal B breast cancer subtype. Cell Death Dis 12, 171 (2021). https://doi.org/10.1038/s41419-021-03436-x

Email: trishna.pani@rcb.res.in; rkajal157@gmail.com; animesh@rcb.res.in

Dr. Avinash Bajaj lab: https://www.rcb.res.in/index.php?param=empdetails/133

Dr. Ujjaini Dasgupta lab: https://www.amity.edu/gurugram/aib/faculty_details.aspx?mpgid=634&pgidtrail=634&facultyid=1639

Edited by: Govinda Raju Yedida (Volunteer, Bio Patrika)

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