The Functions of Ras—part two

Morphogenesis and cell polarity
Changes in the morphology and polarity of cells are clearly essential for tumor progression and metastasis. In two-dimensional (2D) culture, it has been observed that oncogenic K-Ras expression disruptsapical polarity and causes multilayering of Madin-Darby canine kidney (MDCK) cells that normally grow as a monolayer. It is not clear, however, whether this is due to a loss of contact inhibition of growth, or to a direct effect of Ras signaling to polarity machinery. In the mouse embryo, increased ERK1/2 signaling disrupted proper mitotic spindle orientation, and it is possible that a defect in spindle orientation could underlie the multilayering phenotype.
Tight junctions are known to play an important role in maintaining cell polarity. It has been shown that oncogenic Ras disrupts localization of the tight junction components ZO-1 and occludin as well as inhibits E-cadherin expression and that this can be rescued by MEK inhibition). These results are consistent with another study indicating that oncogenic Raf downregulates occludin to disrupt tight junctions. Whether Ras expression affects the barrier function of tight junctions is not clear and may depend on the cell type. Induction of epithelial-to-mesenchymal transition (EMT) is an important hallmark of cancers and is associated with a complete loss of epithelial characteristics including cell-cell adhesion. A more mesenchymal morphology is clearly seen in MDCK cells expressing active Ras or Raf and is sometimes associated with decreased E-cadherin. Either alone, or in cooperation with the TGFβ pathway, oncogenic Ras represses transcription of E-cadherin via the SNAIL and SLUG transcription factors. Ras also promotes methylation of the E-cadherin promoter as well as increases E-cadherin degradation. Other aspects of EMT which contribute to invasion and metastasis will be discussed fiirther in Section 1B.4.7.

Evasion of immune response
Oncogenic Ras reduces the ability of the immune system to recognize tumor cells. First, Ras reduces the expression of major immunohistocompatibility complexes (MHCs) that are normally present on the surface of all cells, reducing the immunogenicity of tumor cells. This does not occur by changes in transcriptional regulation of MHCs, but rather by compromised peptide processing machinery. Second, T cells that are specific to mutant Ras are often anergic, perhaps because of the recruitment of regulatory T cells that are immunosuppressive.
Remodeling of microenvironment
Tumor cells remodel their microenvironment by promoting growth factors required for angiogenesis, which allow tumor cells to receive the oxygen and nutrients they require, as well as degrading the extracellular matrix (ECM) to permit vessel formation and aid tumor cell invasion. Oncogenic Ras has been shown to regulate both these processes. Downstream of Raf, for instance, active ERK phosphorylates HIFla, increasing HIF transcriptional activity to upregulate vascular endothelial growth factor (VEGF) expression, while PI3K activation has a similar effect. In addition, Ras increases cyclooxygenase-2 (COX-2) levels, which produces prostaglandins and leads to increased expression of VEGF.

Oncogenic Ras plays a role in the loss of cell adhesion to matrix that is characteristic of EMT. For instance, oncogenic Ras leads to downregulation of integrin (31 chain maturation in HD6-4 colon epithelial cells. In addition, oncogenic Ras reduces cell surface expression of aipi integrin receptor that binds collagen and laminin as well as the a5pi integrin receptor that binds fibronectin in SW 480 colon adenocarcinoma cells.

In order for tumor cells to invade and metastasize, many changes are necessary in the actin and microtubule cytoskeletons among other pathways to promote frontrear polarity and cell migration. As Rho GTPases are master cytoskeletal regulators, it is likely through these proteins that oncogenic Ras stimulates cytoskeletal changes and
establishes asymmetry within cells. It is already known, for instance, that PI3K activates multiple Rac GEFs that result in the activation of Rac, a Rho GTPase family member. Ras also directly regulates the Rac GEF Tiaml. Furthermore, RalA and RalB, which are activated downstream of the Ras effector RalGDS, regulate the Rho GAP domain containing protein RalBPl. Finally, Ras promotes Rho activation downstream of MEK, by translocating pi 90 Rho-GAP out of the cytoplasm and into an inaccessible cytoskeletal compartment. Another important requirement for tumor cells to successful invade through the basement membrane and metastasize is to avoid matrix deprivation-induced apoptosis, also known as anoikis. Multiple studies report that oncogenic Ras prevents anoikis by downregulating proapoptotic Bak and preventing the downregulation of the antiapoptotic BC1-XL. The effects of Ras on Bak are downstream of PI3K, but oftenthe effects are surprisingly independent of both PI3K and Raf.