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Regulation of lysophosphatidate-induced mechanisms of chemo-resistance

  • Author / Creator
    Venkatraman, Ganesh
  • Systemic chemotherapy in combination with local intervention through surgery and radiotherapy are effective treatments for breast cancers. Chemotherapy is often used in patients with early signs of disease to effectively shrink the tumor and prevent metastasis before surgical excision of the tumor. However, relapse occurs in some of these patients due to the presence of remnant cancer cells that are resistant to chemotherapy. These cancer cells may acquire additional resistance mechanisms resulting in multi-drug resistance and treatment failure. Aggressive tumors show inherently poor sensitivity to chemotherapeutics. Studies primarily based on cell culture models have identified mechanisms of chemo-resistance. These mechanisms include alterations in drug accumulation, increased drug metabolism, altered DNA damage response, evasion of cell-death and decreased ceramide accumulation. In animal models of cancer, additional complexity arises from signaling cross-talk among the cancer cells, stroma, extracellular matrix and the vasculature in the tumor microenvironment that contribute to the development of multi-drug resistance. Cytokines, chemokines and growth factors secreted into the tumor microenvironment represent a hurdle to successful chemotherapy by making the tumors inherently resistant and contributing to development of additional resistance. We examined the mechanism by which extracellular lysophosphatidate (LPA), which is produced by the secreted enzyme, autotaxin (ATX), contributes to multi-drug resistance using breast, thyroid, liver and lung cancer cells. LPA acts through its G-protein coupled receptor, LPA1-6, to promote survival and proliferation in cancers. We discovered that LPA increased the stability and nuclear localization of the transcription factor Nuclear Factor, Erythroid 2-Like 2 or Nrf2. Nrf2, a master regulator of the antioxidant response, promotes resistance to chemotherapeutics through increased metabolism, conjugation and export of drugs from the cell. We showed that LPA, through the activation of LPA1 receptors and phosphatidylinositol 3-kinase (PI3K), increased Nrf2 stabilization and the expression of multi-drug resistance transporters (MDRT) and antioxidant genes. LPA increased the efflux of substrates of the MDRT, which includes chemotherapeutics such as doxorubicin. Consequentially, LPA protected cancer cells from doxorubicin- and etoposide-induced apoptosis. We tested these results in vivo using a syngeneic 4T1 breast cancer model. Blocking LPA production with ONO-8430506, a competitive ATX inhibitor, decreased the expression of Nrf2 and Nrf2-regulated genes in breast tumors. Combining 4 mg/kg doxorubicin every third day with 10 mg/kg ONO-8430506 every day decreased tumor growth and metastasis to lungs and liver by >70%, whereas doxorubicin alone had no significant effect on tumor growth. Additionally, we show increased expression of Nrf2 in the primary tumors of breast cancer patients, who have a recurrence following surgery and chemotherapy. We also demonstrate a novel concept of chemotherapy-induced increases in inflammation and ATX production as a mediator of resistance to oxidative damage in the 4T1 tumors. Increased expression of Nrf2 and its targets were also observed in tamoxifen-treated breast cancer cells and tumors. Inhibition of ATX overcomes this vicious cycle of inflammation, LPA production and resistance to oxidative damage. Finally, we examined another aspect of LPA signaling that contributes to increased resistance to chemotherapeutics. This involves increased activation and expression of sphingosine kinase 1 (SK1), which results in formation of sphingosine 1-phosphate (S1P) in the cells. LPA-induced translocation of SK1 to membranes, which constitutes an activation step, is higher in doxorubicin-resistant cancer cells when compared to their isogenic controls. Additionally, the doxorubicin-resistant cancer cells have increased expression of the MDRT and S1P receptors. We propose that extracellular LPA coordinates S1P signaling in cancer cells. This is through activation of SK1, secretion of S1P through the MDRT and increased signaling of secreted S1P through the S1P receptors. Overall, our studies have demonstrated a potentially important role for LPA signaling in increasing resistance to chemotherapies and development of multi-drug resistance. This is through the increased expression of Nrf2 and transcription of antioxidant and MDRT genes. Our study also provides a practical strategy for targeting LPA signaling in cancers by blocking LPA production with ATX inhibitors. There are no ATX inhibitors in the clinic. Inhibition of ATX could be a useful strategy in improving the efficacy of existing cancer therapies and to prevent the development of chemo-resistance in patients.

  • Subjects / Keywords
  • Graduation date
    2015-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3BN9X79K
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Brindley, David (Biochemistry)
  • Examining committee members and their departments
    • Natarajan, Vishwanathan (Pharmacology)
    • Murray, David (Oncology)
    • Brindley, David (Biochemistry)
    • Berthiaume, Luc (Cell Biology)
    • Schang, Luis (Biochemistry)