The exocytosis, also known as secretory trafficking involves the movement of molecules to intracellular destinations and plasma membrane for secretion. Proteins destined for secretion are synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus, where cargos are packaged into secretory vesicles that are transported to the plasma membrane through post-Golgi traffic.

This machinery is tightly orchestrated to organize cell microenvironment that regulates focal adhesion (FA) dynamics and immunity. The aberrant secretory trafficking leads to diverse diseases, including cancers. Secretory vesicle biogenesis and trafficking are coordinated by exocytotic Rabs, including Rab6, which promotes fission of exocytotic vesicles from Golgi membranes and activates vesicle trafficking toward the cell surface.

Rabs are small GTPases that cycle between inactive (GDP-bound) and active (GTP-bound) states. Cycling is dependent on coordinated interactions of Rabs with guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Most GEFs and GAPs were identified in yeast two-hybrid screens, only a few confirmatory studies have been carried out on mammalian cells. Despite the profound effects of Rab6 on secretory trafficking, only a few Rab6 GEFs (Ric1 and Rgp1), GAP (GAPCenA (RABGAP1)) and effectors (BICD2, p150glued, PIST, Rabkinesin-6 (KIF20A)) have been identified, and their mechanistic functions remain elusive.

• Do RGP1/RIC1 and RABGAP1 regulate Rab6A activity?

• Are there other Rab6A regulators?

• Developing Rab6A inhibitors?

Thus, we aim to determine the functions of Rab6 regulators in Rab6 activity and secretory trafficking regulation.

Epithelial-to-mesenchymal transition (EMT) underlies enhanced cell motility and immunosuppression thereby driving metastasis in epithelial malignancies. However, the way in which EMT orchestrates disparate biological processes remains unclear.

Recently, our group identified an EMT-activated membrane trafficking network that coordinates pro-migratory FA dynamics with an immunosuppressive secretory program in lung adenocarcinoma (LUAD).

The EMT-activating transcription factor ZEB1 drives secretory trafficking by relieving Rab6A, Rab8A, and GFEs from miR-148a-dependent silencing to enhance surface exposure of MMP14 that triggers extracellular matrix  (ECM) degradation, accelerates FA turnover, and promotes cancer cell migration.

Moreover, the ZEB1-driven changes in secretory trafficking increase the secretion of cytokines and pro-tumorigenic factors (e.g. autotaxin (ATX)) to generate an immunosuppressive tumor microenvironment (TME) and to promote metastasis. The factors involved in the ZEB1-driven secretory program, Rab6A and Rab8A, correlate with poor prognosis in LUAD patients.

The findings provide a rationale for targeting the pro-metastatic secretory program. We aim to identify potential factors that could serve as prognostic markers or therapeutic targets to suppress cancer cell motility and improve the efficacy of immunotherapy for cancer patients.

Our current working model demonstrates that cancer cells ‘adapt’ their membrane trafficking pathways to establish an immunosuppressive TME that drives cancer progression. 

To comprehensively dissect the adaptive membrane trafficking machinery in immunosuppression, we initiated CRISPRi in vivo screen to assess more than 2000 trafficking-related genes. In the preliminary results, we identified a panel of membrane trafficking regulators that specifically dropout in immunocompetent mice, suggesting their essential roles in the immunosuppressive TME.

We aim to further investigate the molecular functions of the identified factors in membrane trafficking dynamics, immunosuppression and cancer metastasis. The development of this project for the purpose of reversing resistance to immune checkpoint inhibition would have immediate clinical relevance.