Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

// Pawan Upadhyay 1, * , Sudhir Nair 2, * , Ekjot Kaur 3 , Jyotirmoi Aich 1 , Prachi Dani 1 , Vidyalakshmi Sethunath 1 , Nilesh Gardi 1 , Pratik Chandrani 1 , Mukul Godbole 1 , Kavita Sonawane 2 , Ratnam Prasad 1 , Sadhana Kannan 4 , Beamon Agarwal 5 , Shubhada Kane 6 , Sudeep Gupta 7 , Shilpee Dutt 3 , Amit Dutt 1 1 Integrated Genomics Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 2 Division of Head and Neck Oncology, Department of Surgical Oncology, Tata Memorial Hospital, Tata Memorial Centre,Mumbai- 4100012, India 3 Shilpee Laboratory, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 4 Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 5 Department of Pathology, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Navi Mumbai- 410210, India 6 Department of Pathology, Tata Memorial Hospital, Tata Memorial Centre, Mumbai- 400012, India 7 Department of Medical Oncology, Advanced Centre for Treatment, Research and Education In Cancer, Tata Memorial Centre, Mumbai- 400012, India * These authors have contributed equally to this work Correspondence to: Amit Dutt, email: adutt@actrec.gov.in Keywords: early stage tongue cancer, exome and transcriptome sequencing, IHC based expression analysis, cancer stem cell-like feature, Notch pathway inhibitors Received: January 25, 2016 Accepted: June 07, 2016 Published: July 06, 2016 ABSTRACT Background: Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC). Patients and Methods: We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays. Results: We show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1 , when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P =0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P =0.029). Conclusion: We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer.