Rac and Cdc42 inhibitors reduce macrophage function in breast cancer preclinical models

Background: Metastatic disease lacks effective treatments and remains the primary cause of mortality from epithelial cancers, especially breast cancer. The metastatic cascade involves cancer cell migration and invasion and modulation of the tumor microenvironment (TME). A viable anti-metastasis strategy is to simultaneously target the migration of cancer cells and the tumor-infiltrating immunosuppressive inflammatory cells such as activated macrophages, neutrophils, and myeloid-derived suppressor cells (MDSC). The Rho GTPases Rac and Cdc42 are ideal molecular targets that regulate both cancer cell and immune cell migration, as well as their crosstalk signaling at the TME. Therefore, we tested the hypothesis that Rac and Cdc42 inhibitors target immunosuppressive immune cells, in addition to cancer cells. Our published data demonstrate that the Vav/Rac inhibitor EHop-016 and the Rac/Cdc42 guanine nucleotide association inhibitor MBQ-167 reduce mammary tumor growth and prevent breast cancer metastasis from pre-clinical mouse models without toxic effects.

Methods: The potential of Rac/Cdc42 inhibitors EHop-016 and MBQ-167 to target macrophages was tested in human and mouse macrophage cell lines via activity assays, MTT assays, wound healing, ELISA assays, and phagocytosis assays. Immunofluorescence, immunohistochemistry, and flow cytometry were used to identify myeloid cell subsets from tumors and spleens of mice following EHop-016 or MBQ-167 treatment.

Results: EHop-016 and MBQ-167 inhibited Rac and Cdc42 activation, actin cytoskeletal extensions, migration, and phagocytosis without affecting macrophage cell viability. Rac/Cdc42 inhibitors also reduced tumor- infiltrating macrophages and neutrophils in tumors of mice treated with EHop-016, and macrophages and MDSCs from spleens and tumors of mice with breast cancer, including activated macrophages and monocytes, following MBQ-167 treatment. Mice with breast tumors treated with EHop-016 significantly decreased the proinflammatory cytokine Interleukin-6 (IL-6) from plasma and the TME. This was confirmed from splenocytes treated with lipopolysaccharide (LPS) where EHop-016 or MBQ-167 reduced IL-6 secretion in response to LPS.

Conclusion: Rac/Cdc42 inhibition induces an antitumor environment via inhibition of both metastatic cancer cells and immunosuppressive myeloid cells in the TME.

 

Comments:

The study described provides valuable insights into the potential of Rac and Cdc42 inhibitors, specifically EHop-016 and MBQ-167, as effective anti-metastasis strategies in breast cancer. The research focuses on targeting both cancer cells and immunosuppressive inflammatory cells within the tumor microenvironment (TME). Here's a summary of the findings and their implications:

**1. Targeting Rac and Cdc42:** Rac and Cdc42 are molecular targets that play crucial roles in regulating cancer cell and immune cell migration, as well as their interactions within the TME. Inhibiting these proteins can hinder cancer cell migration and invasion, as well as modulate the behavior of immunosuppressive immune cells.

**2. Inhibition of Macrophages:** EHop-016 and MBQ-167 were found to inhibit Rac and Cdc42 activation in macrophages without affecting cell viability. These inhibitors also suppressed actin cytoskeletal extensions, migration, and phagocytosis of macrophages, indicating their potential in modulating macrophage behavior within the TME.

**3. Reduction of Tumor-Infiltrating Immune Cells:** Treatment with Rac/Cdc42 inhibitors resulted in a reduction of tumor-infiltrating macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs). These cells are known to promote immunosuppression within the TME, and their reduction signifies a shift towards an anti-tumor immune environment.

**4. Cytokine Modulation:** Mice treated with EHop-016 showed decreased levels of the proinflammatory cytokine Interleukin-6 (IL-6) in both plasma and the TME. Additionally, Rac/Cdc42 inhibitors reduced IL-6 secretion in response to lipopolysaccharide (LPS) stimulation, indicating their role in modulating the inflammatory response.

**5. Implications:** The findings suggest that Rac/Cdc42 inhibition not only targets cancer cells but also remodels the TME by inhibiting immunosuppressive myeloid cells. This dual action creates an anti-tumor environment, making these inhibitors promising candidates for further development as anti-metastatic therapies, especially in breast cancer.

In summary, this research provides strong evidence supporting the use of Rac and Cdc42 inhibitors, such as EHop-016 and MBQ-167, as potential therapeutic agents for metastatic breast cancer. Their ability to simultaneously target cancer cells and immunosuppressive immune cells offers a novel approach in the fight against metastatic diseases, addressing both components of the metastatic cascade effectively.