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Ca2+/nuclear factor of activated T cells signaling is enriched in early-onset rectal tumors devoid of canonical Wnt activation

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Abstract

Our previous extensive analysis revealed a significant proportion of early-onset colorectal tumors from India to be localized to the rectum in younger individuals and devoid of deregulated Wnt/β-catenin signaling. In the current study, we performed a comprehensive genome-wide analysis of clinically well-annotated microsatellite stable early-onset sporadic rectal cancer (EOSRC) samples. Results revealed extensive DNA copy number alterations in rectal tumors in the absence of deregulated Wnt/β-catenin signaling. More importantly, transcriptome profiling revealed a (non-Wnt/β-catenin, non-MSI) genetic signature that could efficiently and specifically identify Wnt− rectal cancer. The genetic signature included a significant representation of genes belonging to Ca2+/NFAT signaling pathways that were validated in additional samples. The validated NFAT target genes exhibited significantly higher expression levels than canonical Wnt/β-catenin targets in Wnt− samples, an observation confirmed in other CRC expression data sets as well. We confirmed the validated genes to be transcriptionally regulated by NFATc1 by (a) evaluating their respective transcript levels and (b) performing promoter-luciferase and chromatin immunoprecipitation assays following ectopic expression as well as knockdown of NFATc1 in CRC cells. NFATc1 and its targets RUNX2 and GSN could drive increased migration in CRC cells. Finally, the validated genes were associated with poor survival in the cancer genome atlas CRC expression data set. This study is the first comprehensive molecular characterization of EOSRC that appears to be driven by noncanonical tumorigenesis pathways.

Key messages

  • Early-onset sporadic rectal cancer exhibits DNA gain and loss without Wnt activation.

  • Ca2+/NFAT signaling appears to be activated in the absence of Wnt activation.

  • An eight-gene genetic signature distinguishes Wnt+ and Wnt− rectal tumors.

  • NFAT and its target genes regulate tumorigenic properties in CRC cells.

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Acknowledgements

We are grateful to the patients and normal subjects for kindly agreeing to be a part of this study. We thank Dr. AS Raju, Research Associate, Laboratory of Molecular Oncology, CDFD for helping in microarray data analysis and for critical reading of the manuscript. The authors gratefully acknowledge the support from surgeons Dr. Mohana Vamsy, Dr. T Subramanyeshwar Rao, Dr. Sujit Patnaik, and Dr. KVVN Raju, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India; Prof Mukta Srinivasulu, MNJ Institute of Oncology and Regional Cancer Centre, Hyderabad, India; and Dr. N Bheerappa, Nizam’s Institute of Medical Sciences, Hyderabad, India; and from pathologists Dr. I Satish Rao, Krishna Institute of Medical Sciences, Hyderabad, India; Dr. Sudha Murthy, Basavatarakam Indo-American Cancer Hospital and Research Institute, Hyderabad, India; and Dr. K T Vijaya, Care Hospitals, Hyderabad, India, for evaluating FFPE and IHC sections. We thank Ms. Leena Bashyam and Prof Aparna Dutta Gupta, Genomics Facility, School of Life Sciences, University of Hyderabad, Hyderabad, India, for providing access to the Agilent scanner.

Funding

This work was supported by an NIH-FIRCA grant (TW007963) to JP and MDB and two grants from the Department of Biotechnology, Government of India (BT/PR8609/MED/12/626/2013 and the National Bioscience Award project) to MDB. RK and RR, registered PhD students of Manipal University, are grateful to the Council of Scientific and Industrial Research, Government of India, for junior and senior research fellowships.

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Author notes

  1. Saumyadipta Pyne

    Present address: Public Health Dynamics Laboratory and Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

Authors and Affiliations

  1. Laboratory of Molecular Oncology, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, 500001, India

    Raju Kumar, Ratheesh Raman, Viswakalyan Kotapalli & Murali D. Bashyam

  2. Graduate Studies, Manipal University, Manipal, Karnataka, India

    Raju Kumar & Ratheesh Raman

  3. Histopathology, Apollo Hospitals, Hyderabad, India

    Swarnalata Gowrishankar

  4. Public Health Foundation of India, Indian Institute of Public Health, Hyderabad, India

    Saumyadipta Pyne

  5. Stanford University School of Medicine, Stanford, CA, USA

    Jonathan R. Pollack

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Correspondence to Murali D. Bashyam.

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The study was performed as per the revised Helsinki declaration following approval of ethics committee of hospitals from where samples were collected as well as of the CDFD bioethics committee. Informed consent was obtained from patients.

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The authors declare that they have no conflict of interest.

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Kumar, R., Raman, R., Kotapalli, V. et al. Ca2+/nuclear factor of activated T cells signaling is enriched in early-onset rectal tumors devoid of canonical Wnt activation. J Mol Med 96, 135–146 (2018). https://doi.org/10.1007/s00109-017-1607-4

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