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K. Hill



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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.01 - Protein Kinase C iota is required for maintenance of a tumor initiating cell phenotype in lung squamous cell carcinoma (ID 2644)

      10:30 - 10:35  |  Author(s): K. Hill

      • Abstract
      • Presentation
      • Slides

      Background
      We discovered that PKCι is an oncogene in non-small cell lung cancer (NSCLC), elucidated a major oncogenic PKCι signaling mechanism, and identified therapeutic agents that target oncogenic PKCι signaling. We have shown that PKCι signaling is genetically activated in approximately 70% of lung squamous cell carcinomas (LSCCs) through tumor-specific amplification of the PKCι gene, PRKCI. More recently, we have investigated the role of PKCι in bronchio-alveolar stem cells (BASCs), which are putative lung tumor-initiating cells (TICs). We demonstrated that PKCι is required for Kras-mediated transformation of BASCs in a mouse model of Kras-mediated lung adenocarcinoma. We hypothesize that PKCι plays a critical role in the development and maintenance of the TIC phenotype in LSCC by activating cell autonomous proliferative signaling mechanisms.

      Methods
      We isolated “oncospheres” from four human LSCC cell lines (H1703, H1299, Calu-1, and ChagoK1) grown in non-adherent culture in defined stem cell medium using established protocols. Lentiviral shRNA techniques were used to genetically knock down expression of PKCι to assess the effect of PKCι depletion on the TIC phenotype. Non-target (NT) and PKCι RNAi TICs were assessed for the ability to grow as non-adherent oncospheres, to clonally expand, express stem marker genes, form colonies in soft agar, and initiate tumors in immune deficient mice. The effect of the selective and potent PKCι signaling inhibitor auranofin on TIC behavior and PKCι signaling activity was assessed as was the mTOR inhibitor, rapamycin.

      Results
      LSCC oncospheres exhibited characteristics of cancer stem or tumor-initiating cells including the ability to redifferentiate into bulk tumor cells when returned to adherent culture. Oncosphere cells express elevated levels of stem genes, clonally expand, exhibit enhanced transformed growth, and efficiently initiate and maintain lung orthotopic tumors and metastases. Biochemical studies indicate that the oncogenic PKCι-Rac1-Ect2-MMP10 signaling axis is activated in LSCC TICs. To assess the role of PKCι in TIC growth, we knocked down PKCι in TIC cultures derived from the four LSCC cell lines described above. Whereas TICs expressing NT RNAi grew efficiently as anchorage-independent colonies in soft agar and clonally expanded, PKCι RNAi TICs were severely impaired in soft agar growth, clonal expansion, and tumorigenicity in vivo. Treatment of TICs with the potent and selective PKCι inhibitor auranofin (ANF) likewise led to inhibition of PKCι signaling, TIC growth, clonal expansion, and tumorigenicity. Combined inhibition of PKCι and mTOR with ANF plus rapamycin was synergistic against TIC proliferation in vitro.

      Conclusion
      Our data demonstrate that PKCι signaling is activated in LSCC TICs and that PKCι signaling is important for maintaining the TIC phenotype. We showed that the selective PKCι inhibitor ANF potently inhibits LSCC TIC behavior. Taken together, our data support the targeting of LSCC TICs through selective inhibition of PKCι for treatment of patients with LSCC. Based on these and earlier results showing synergistic tumor inhibition with combined PKCι and mTOR inhibition, a phase I clinical trial of auranofin with the mTOR inhibitor sirolimus has been instituted as maintenance therapy for LSCC patients who have completed initial chemotherapy.

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