Oral Presentation NSW State Cancer Conference 2023

βIII-Tubulin knockdown sensitises pancreatic tumours to the tumour-specific death ligand TRAIL. (#37)

George Sharbeen 1 , John Kokkinos 1 , Janet Youkhana 1 , Oliver Arkell 1 , Meagan Davis 1 , Keilah G Netto 1 , Cyrille Boyer 2 3 4 , David Goldstein 1 5 , Koroush S Haghighi 5 , Joshua A McCarroll 3 4 6 , Phoebe A Phillips 1 3
  1. Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, UNSW Sydney, Sydney, NSW, Australia
  2. Cluster for Advanced Macromolecular Design, School of Chemical Engineering, UNSW Sydney, Sydney, NSW, Australia
  3. Australian Centre for NanoMedicine, UNSW Sydney, Sydney, NSW, Australia
  4. UNSW RNAi Institute, Sydney, NSW, Australia
  5. Prince of Wales Hospital, School of Clinical Medicine, UNSW Sydney, Sydney, NSW, Australia
  6. Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, NSW, Australia

Background: Pancreatic Ductal Adenocarcinoma (PDAC) needs more effective therapies, with a 5-year survival rate <11%. We previously demonstrated βIII-tubulin knockdown in PDAC cells reduced tumour growth and metastasis in vivo, increased chemosensitivity, and induced apoptosis, making it a potent therapeutic target [McCarroll et al, Oncotarget, 6, 2235 (2015)]. Recently, we demonstrated that βIII-tubulin regulates extrinsic (caspase-8-dependent) apoptosis in PDAC cells and sensitises them to extrinsic apoptosis inducers in vitro, including the cancer-specific death ligand TRAIL. TRAIL induces extrinsic cell death by binding to the TRAIL-receptor DR5, inducing clustering and intracellular activation of caspase 8. We also showed βIII-tubulin knockdown in PDAC cells increased DR5 clustering. Building on these findings, we assessed whether these effects: (i) extended to PDAC cancer-associated fibroblasts (CAFs); (ii) are specifically linked to TRAIL receptor DR5; (iii) are reproducible in vivo. 

Aims: (1) Evaluate effect of βIII-tubulin knockdown ± TRAIL on CAF proliferation and apoptosis in vitro; (2) Determine effect of DR5 knockout in PDAC cells on βIII-tubulin knockdown-mediated sensitivity to TRAIL; (3) Assess effect of silencing βIII-tubulin combined with TRAIL in a PDAC xenograft mouse model.

Methods: (1) PDAC patient-derived CAFs were transfected with control-siRNA or βIII-tubulin-siRNA ± TRAIL and apoptosis measured (flow cytometry). (2) To confirm a functional link between βIII-tubulin and DR5, DR5 was knocked out in MiaPaCa2 PDAC cells using CRISPR, then experiments repeated as in aim 1. (3) PDAC sub-cutaneous tumours in mice were treated twice weekly with nanoparticles to deliver control-siRNA or βIII-tubulin-siRNA (intravenous) ± TRAIL (intratumoural; once weekly) for 4 weeks.

Results: (1) βIII-tubulin knockdown significantly reduced CAF proliferation and viable cell count (49.5±17.1% decrease relative to controls, p<0.05) but did not induce apoptosis in the presence or absence of TRAIL. (2) DR5 knockout in PDAC cells blocked the ability of βIII-tubulin knockdown to enhance TRAIL-mediated apoptosis. (3) βIII-tubulin knockdown combined with TRAIL treatment in PDAC tumours in vivo significantly increased the frequency of responders (tumour growth rate<average growth rate for controls; 88% responders), relative to either treatment alone (TRAIL, 63% responders; βIII-tubulin knockdown, 67% responders).

Conclusions: (1) Silencing βIII-tubulin represents a dual cell therapeutic target that inhibits both PDAC cell and CAF proliferation, and enhances PDAC cell sensitivity to the tumour death ligand TRAIL. (2) βIII-tubulin knockdown-mediated sensitisation of PDAC cells to TRAIL is DR5-dependent; (3) βIII-tubulin knockdown represents an innovative therapeutic strategy to unleash a suicide signal in PDAC cells and render them sensitive to a tumour-specific therapeutic.