RalB Loss in E-Cadherin-Deficient Tumor Cells Promotes 3D Tissue Hyperproliferation

Background: Events triggering the development of oral squamous cell carcinoma from premalignant lesions are not well understood. In classical, monolayer culture models of cancer cell transformation, Ras-like RalB has been demonstrated to be necessary for tumor cell migration. While we have previously shown that loss of cell-cell adhesion can trigger the conversion from precancer to cancer in 3D tissues, it is still not clear whether RalB is also required to activate a switch to a more aggressive malignant phenotype.

Objectives: To study the influence of alterations in RalB in the Ras signaling pathway on the behavior of E-cadherin-deficient tumor cells in 2D cultures and 3D engineered human tissues that mimic their in vivo counterpart

Methods: Lentiviral shRNA-mediated gene silencing was used to selectively knock down the expression of RalB in E-cadherin-deficient or E-cadherin-competent tumor cells. After confirming protein knockdown by western blot, the sh-RalB cell lines were grown both in 2D monolayer cultures and in 3D tissues that mimic precancer. Tissue morphology as well as tumor cell fate were analyzed using hemotoxylin and eosin and immunofluorescent stainings.

Results: Tissues comprised of E-cadherin-deficient tumor cells that were knocked down in RalB did not demonstrate inhibition of tumor cell migration into the underlying matrix. However, these tissues demonstrated increased thickness that was found to be a result of enhanced cell proliferation as seen by Ki67 and BrdU immunostainings. Furthermore, the enhanced proliferation led to increased cell death as detected by TUNEL and cleaved Caspase-3 immunostaining.

Conclusion: These results suggest that RalB plays a protective role by blocking Ras-mediated hyperproliferation and apoptosis of transformed cells. Further studies are needed to define the mechanistic basis of RalB's antagonistic role in Ras-mediated transformation of E-cadherin-deficient tumor cells and carcinoma progression.