Rac and Cdc42 inhibition in the breast and pancreatic cancer microenvironment

Abstract

Pancreatic cancer has a 13% survival rate when all the stages are combined, making it one of the deadliest cancers to date. It is expected that by 2030, it will be the second cause of cancer-related deaths. The only known curative treatment for this disease is surgical resection, however most patients don't qualify due to late diagnosis. Even though they demonstrate a strong initial response, chemotherapeutics FOLFIRINOX and Gemcitabine are hampered with toxicity and drug resistance. Which leaves researchers targeting the most prominent and driver mutation of pancreatic cancer, KRAS. Although there has been success in preclinical experiments, most of the putative therapeutics fail at clinical trials, mostly due to poor response and adverse events. One of the reasons for the failure stems from an immunosuppressive tumor microenvironment (TME), where inflammation of the pancreas is central to its disease etiology. Pancreatic ductal adenocarcinoma (PDAC) is comprised by dense extracellular matrix and immunosuppressive cells, such as tumor-associated macrophages (TAMs). These contribute to deposition of collagen, immune evasion, and migration of cancer cells. TAMs have also been correlated with poor prognosis and are more present in tumor than in healthy tissues. This leads to a need to develop novel therapeutic options for PDAC that are effective against the invasive PDAC cells and the accompanying inflammation that TAMs promote for disease progression.<br /> <br /> Rac and Cdc42 are ideal targets for PDAC due to their involvement of oncogenic signaling that contribute to transformation and invasion, leading to metastasis. In addition, Rac/Cdc42 signaling in tumor-associated immunosuppressive myeloid cells, such as TAMs, are essential for inflammation and related tumor progression. There have been attempts to develop inhibitors targeting Rac, Cdc42 and their common downstream effector PAK. However, they share the same downfall as PDAC's KRAS inhibitors where they are effective at high concentrations and show toxicity and poor availability. To fill the therapeutic need for anti-metastatic diseases, such as PDAC, our laboratory has developed several Rac and Cdc42 inhibitors. The first Rac inhibitor characterized by our laboratory was EHop-016, which inhibits activation of Rac by the GEF Vav. This was followed by MBQ-167, a more potent dual Rac/Cdc42 inhibitor that reduces both spontaneous and experimental metastasis in breast cancer mouse models, as well as pro-inflammatory molecules such as interleukin-6 (IL-6), Chitinase-3-like protein 1 (CH3L1), and S100A8/A9. The next generation inhibitors EHop-097 and MBQ-168 have demonstrated promising results against breast cancer cells and demonstrated to be not toxic to normal epithelial cells, like their parent compounds.<br /> <br /> We hypothesize that Rac/Cdcc42 inhibitors will simultaneously target the migration and activity of PDAC cells and macrophage-like cells in the TME. Two specific aims were established as goals for this Dissertation, which were: Aim 1. elucidate if Rac/Cdc42 inhibitors impacted PDAC and macrophage-like cells in-vitro (Chapter 3); Aim 2. identify a pharmacodynamic marker to measure Rac and Cdc42 inhibitor efficacy (Chapter 4).<br /> <br /> Mouse and human PDAC cell lines and macrophages were used to assess the effect of Rac/Cdc42 inhibitors on cell viability, immunofluorescence for the actin cytoskeleton and migration assays. Results show that MBQ-167 and MBQ-168 reduce active Rac/Cdc42, while EHop-097 only reduce Rac activation. These compounds also reduced pancreatic cell viability without affecting macrophage viability. Similarly, they also decrease pancreatic and macrophage-like cell migration. Macrophages are known for being phagocytes, and our inhibitors did not reduce this function below 50%. Out of the new Rac/Cdc42 inhibitors tested, MBQ-168 was the most effective at inhibiting pancreatic cancer cell migration in the presence of macrophages and reducing IL-6 levels in co cultures.<br /> <br /> To identify pharmacodynamic markers for Rac/Cdc42 therapeutic efficacy, we assessed the common downstream effector PAK, CHI3L1 and S100A8/A9, since these have been identified by us and are reduced MBQ-167. For this, we employed immunohistochemistry and flow cytometry to assess p-PAK levels in both human and murine samples. Moreover, we used ELISA's to assess CHI3L1 and S100A8/A9 levels on conditioned media and mice serum. Our results demonstrate that p-PAK is correlated with advanced PDAC staging, highlighting the benefit of our inhibitors for PDAC treatment. Moreover, we provide evidence that MBQ-167 reduces p-PAK levels in ex-vivo cultures of triple negative breast cancer (TNBC) patient tissue and peripheral blood mononuclear cells (PBMCs) from blood of mice that received Rac/Cdc42 inhibitors. Additionally, we provide evidence that by reducing Rac and Cdc42 activity, CHI3L1 is reduced significantly in breast and pancreatic cancer cells, as well as in stimulated macrophages. On the other hand, it was MBQ-168 that significantly decreased S100A8/A9 in pancreatic cancer cells and stimulated macrophages.<br /> <br /> Taken together, this Dissertation has provided an alternative strategy to treat PDAC and developed a methodology for assessment of pharmacodynamic markers for Rac/Cdc42 inhibitor efficacy. Our results show that similar to MBQ-167 in metastatic breast cancer, the derivative MBQ-168 has potential as an anti-cancer compound that can also reduce inflammatory signaling in pancreatic cancer. Additionally, we offer p-PAK as a potential predictive and pharmacodynamic marker for Rac/Cdc42 therapeutic efficacy. These results will impact the planned clinical trials for MBQ-167 in metastatic patients as well as future clinical trials for MBQ-168 in PDAC patients.