Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells

// Remya Raja 1, * , Nandini A. Sahasrabuddhe 1, * , Aneesha Radhakrishnan 1, 2, * , Nazia Syed 1, 2 , Hitendra S. Solanki 1, 3 , Vinuth N. Puttamallesh 1, 4 , Sai A. Balaji 5 , Vishalakshi Nanjappa 1, 4 , Keshava K. Datta 1, 3 , Niraj Babu 1 , Santosh Renuse 1, 4 , Arun H. Patil 1, 3 , Evgeny Izumchenko 6 , T.S. Keshava Prasad 1, 4, 11, 12 , Xiaofei Chang 6 , Annapoorni Rangarajan 5 , David Sidransky 6 , Akhilesh Pandey 7, 8, 9, 10 , Harsha Gowda 1, 11 , Aditi Chatterjee 1, 11 1 Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India 2 Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, 605014, India 3 School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India 4 Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India 5 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India 6 Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21231, USA 7 McKusick-Nathans Institute of Genetic Medicine, Baltimore, Maryland, 21205, USA 8 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 9 Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 10 Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA 11 YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, 575018, India 12 NIMHANS-IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neurosciences, Bangalore, 560029, India * These authors contributed equally to this work Correspondence to: Aditi Chatterjee, email: aditi@ibioinformatics.org Keywords: mass spectrometry, NSCLC, p21 (RAC1)-activated kinase 6, smoking Received: January 27, 2016 Accepted: August 08, 2016 Published: August 16, 2016 ABSTRACT Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.