George Sharbeen

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a dismal 5-year survival rate at 12%. The fibrotic PDAC desmoplasia is a major contributor to chemoresistance and metastasis that drive this poor prognosis. Cancer-associated fibroblasts (CAFs) generate PDAC tumour fibrosis and have been identified as therapeutic targets to remodel the stroma to a more drug-permissive microenvironment. We assessed the therapeutic potential of inhibiting the collagen chaperone, heat shock protein 47 (HSP47) in PDAC cells and CAFs. Collagen is a key component of PDAC fibrosis and requires the activity of HSP47 to ensure correct maturation and secretion. Herein, we show that HSP47 knockdown inhibits both PDAC cells and CAF proliferation in vitro. In vivo, therapeutic HSP47 knockdown in orthotopic PDAC tumours significantly reduced intratumoural fibrosis and opened intratumoural blood vessels, while stable HSP47 knockdown specifically in CAFs additionally reduced PDAC tumour growth. We observed that HSP47 is highly expressed in the stroma of >80% of patients in a PDAC cohort (Australian Pancreatic Cancer Genome Initiative), but that it was only prognostic of poorer overall survival in the tumour compartment. Functional relevance in the tumour compartment was further validated in 3D human PDAC explants. Our work demonstrates that HSP47 is a potential therapeutic target in both PDAC cells and CAFs and represents a robust target to interfere with tumour collagen deposition.

The microtubule protein βIII-tubulin is a prognostic, pro-survival, and chemoresistance factor in multiple malignancies, including pancreatic ductal adenocarcinoma (PDAC). However, the precise survival mechanisms controlled by βIII-tubulin in cancer remain unknown. Here, we discovered a link between βIII-tubulin and the activation of caspase 8-mediated extrinsic apoptosis. Silencing βIII-tubulin in PDAC cells activated caspase 8, leading to decreased cell viability and growth both in vitro and in vivo. βIII-tubulin knockdown also increased the sensitivity of PDAC cells to extrinsic cell death signals, including TNF-related apoptosis-inducing ligand (TRAIL), TNFα, and FasL. Furthermore, we demonstrated that βIII-tubulin knockdown in PDAC cells, in the absence or presence of TRAIL, increased diffusion and clustering of the TRAIL death receptor DR5 at the cell membrane, inducing extrinsic apoptosis. Nanoparticle delivery of βIII-tubulin siRNA to mouse PDAC tumours reduced tumour growth and increased responsiveness to TRAIL therapy. In patient-derived human PDAC explants, βIII-tubulin silencing reduced tumour cell frequency and improved sensitivity to TRAIL. Finally, we showed that high βIII-tubulin expression in the human PDAC stroma was independently prognostic for poor overall survival. Taken together, silencing βIII-tubulin represents an innovative strategy to activate a suicide signal in PDAC cells and render them more sensitive to microenvironment- and chemotherapy-derived death signals.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that urgently needs more effective therapies. Cancer-associated fibroblasts (CAFs) contribute to the aggressive and chemo-resistant nature of the disease by creating a drug-impeding fibrotic microenvironment. We developed novel compounds, the racemate CRO-05 and its active enantiomer CRO-67, which target both pancreatic tumor and CAF cells with robust anti-cancer activity. These compounds were designed using rational medicinal chemistry based on chromans, a class of anti-cancer drugs. Their therapeutic potential and efficacy were assessed in a clinically relevant patient-derived PDAC tumor explant model, which mimics the disease's 3-dimensional complexity. CRO-67 treatment in these explants significantly reduced tumor cell and αSMA⁺ CAF frequency, decreased cell proliferation and increased cell death. CRO-67 also significantly decreased cell proliferation and enhanced apoptosis by inhibiting cell cycle progression through G2/M phase in PDAC cells and patient-derived CAFs in vitro. CRO-67 treatment of orthotopic PDAC tumors in mice significantly reduced tumor growth in tumors with active growth (> 150% growth at endpoint), and remodeled tumor stroma (reduced αSMA⁺ CAF frequency, loosened tumor fibrosis and normalized tumor vasculature). Finally, CRO-67 sensitized PDAC cells to multiple standard-of-care chemotherapeutics in vitro, paving the way for future combination therapy development and validation.

Supplementary Figures S1-S4 from A Novel Role of Dipeptidyl Peptidase 9 in Epidermal Growth Factor Signaling

Validation of SLC7A11 antibodies and SLC7A11 knockdown in CAFs and PDAC cells in vitro.

Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.

Anti-proliferative effect of SLC7A11 knockdown in CAFs and the effect of SLC7A11 inhibition in MiaPaCa-2 PDAC cells and normal human pancreatic ductal epithelial (HPDE) cells.

SLC7A11 expression in iCAFs, myCAFs and quiescent PSCs.