Rac1 Inhibitor Research Articles

Does actin polymerization contribute to closure of the avian ductus arteriosus (Gallus gallus)?

The ductus arteriosus (DA) is a cardiovascular embryonic shunt present in all developing land vertebrates. This oxygen-sensitive blood vessel plays the important role of connecting the pulmonary artery to the aorta during development and directs blood flow toward the fetal respiratory organ (placenta in mammals or chorioallantois in birds and reptiles). Permanent vessel closure upon birth or hatch is necessary to establish proper circulation in the neonate. Increased arterial PO2 at birth or hatch stimulates DA closure via smooth muscle contraction. Smooth muscle contraction, such as in the DA, is governed by myosin light chain kinase (MLCK) stimulating increased cross-bridge cycling, and through increases in actin polymerization. The Ras homologous protein family (Rho) of GTPases are involved in calcium sensitization and contraction of vascular smooth muscle but have mostly been studied for their role in cell migration and signaling. Rho GTPases activate the Rho activated kinase (ROCK) pathway, which works in conjunction with MLCK to generate smooth muscle contraction as well as activating LIM kinase which inhibits the actin depolymerizer cofilin. Rac and Cdc42 GTPase pathways increase contractile strength in smooth muscle cells by activating the nucleator Arp2/3, leading to increased actin polymerization. The role of these GTPase pathways during closure of the DA is unknown. We examined the role of the Rho, Rac, and Cdc42 GTPase pathways leading to actin polymerization for their role in the closure of the avian DA in late-term chicken ( Gallus gallus) embryos. We hypothesized that inhibiting the pathways that lead to actin polymerization will inhibit tension development in the DA. Using a Danish Myo Technology wire myograph, DA physiology was examined when exposed to 25% oxygen followed by activators and inhibitors of the Rho, Rac, and Cdc42 GTPase pathways. Activation of Rho A pathways with CN01 (0.2 units/ml) produced contraction of the DA. Blocking ROCK pathways with Y-27632 (10 mM) removed the O2-sensitivity of the vessel, while the vessel was still able to respond to phenylephrine (100 mM). This suggests the Rho pathway is involved in maintaining baseline tension and contractile response to O2. Exposure to inhibitors of Rac (10 uM EHT 1864) and Cdc42 (via ML141) GTPases blocked the ability of vessels to contract in response to 25% oxygen, even after 3 hours of constant O2. Similarly, inhibition of the Arp2/3 complex (100 uM CK666) reduced the ability of the vessel to respond to oxygen. These findings suggest that Rho A, Rac, and Cdc42 pathways play a role in active tension of the chicken DA during development by potentially increasing actin polymerization. This research was funded by Grant R15HL14887 from NIH-NHLBI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cycloheptylprodigiosin from marine bacterium Spartinivicinus ruber MCCC 1K03745T induces a novel form of cell death characterized by Golgi disruption and enhanced secretion of cathepsin D in non-small cell lung cancer cell lines

Prodiginines have been studied extensively for their anticancer activity, however, the majority of the research has focused on prodigiosin. In this study, cycloheptylprodigiosin (S-1) is extracted from marine bacterium Spartinivicinus ruber MCCC 1K03745T, and its anticancer property was investigated. It exhibits remarkable cytotoxicity against a panel of human lung cancer cell lines, with the IC50 values ranging from 84.89 nM to 661.2 nM. After 6 h of treatment, S-1 gradually accumulates on mitochondria and lysosomes. While lower doses of S-1 induce cell cycle arrest, treatment with higher doses results in cell death in apoptotic independent manner in both NCI–H1299 and NCI–H460 cell lines. Interestingly, treatment with S-1 leads to the accumulation of LC3B-II via pathways that vary among different cell lines. In addition to its role as an autophagy inhibitor, S-1 also promotes autophagy initiation as demonstrated by the increment of EGFP fragment in the EGFP-LC3 degradation assay, however, inhibition of autophagy does not rescue cells from death induced by S-1. Mechanistically, S-1 impairs autophagic flux through disrupting acidic lysosomal pH and blocking the maturation of cathepsin D. Moreover, treatment with S-1 enhanced secretion of both pro- and mature forms of cathepsin D, coincident with disintegration of trans-Golgi network. Interestingly, S-1 does not induce ferroptosis, pyroptosis or necroptosis in NCI–H1299 cells. However, treatment of NCI–H460 cells with S-1 induces methuosis, which can be suppressed by Rac1 inhibitor EHT 1864. Our data demonstrate that S-1 is an effective anticancer agent with potential therapeutic application.

Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Abstract 7628: Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy

Abstract Metastatic disease drastically reduces clinical prognosis, and remains the primary cause of mortality from solid cancers, such as breast and pancreatic cancer. Since targeted therapeutic options are limited, we developed small molecule compounds to block activation (GTP loading) of the Rho GTPases Rac and Cdc42. Rac and Cdc42 are ideal molecular targets due to their regulation of cell migration, proliferation, invasion, and signaling in cancer and immunosuppressive cells in the tumor microenvironment. Our lead compound MBQ-167 is a dual Rac and Cdc42 inhibitor with IC50 of ~100 nM for inhibition of Rac/Cdc42 activation and the phosphorylation of their common downstream effector p21-activated kinase (PAK). MBQ-167 is highly effective in mouse models of breast cancer metastasis and is currently undergoing a Phase I clinical trial in advanced breast cancer patients. The purpose of this study is to investigate potential target molecules that can be used as biomarkers predictive of Rac/Cdc42 inhibitor efficacy. We recently published that in addition to inhibiting the growth and migration of breast and pancreatic cancer cells, MBQ-167 inhibits macrophage cell migration, immunosuppressive myeloid cell activation and release of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cancer. Transcriptomic data of tumors from mice treated with vehicle or MBQ-167 identified a number of immunomodulatory pathways downregulated by MBQ-167, including IL-6 and S100A family signaling, and Chitinase 3-like (CHI3L1/CHI4L1) genes coding for the human YKL-40 protein. We tested for the activation status of PAK (phospho (p)-PAK) as a diagnostic marker for Rac/Cdc42 inhibitor efficacy using breast cancer patient tissue in ex vivo culture treated with vehicle or MBQ-167 and found decreased p-PAK staining following immunohistochemistry of MBQ-167-treated tissue. From flow cytometry, using Alexa-488-tagged anti p-PAK antibody, we found that p-PAK was reduced from peripheral blood mononuclear cells (PBMCs) following MBQ-167. ELISA assays for YKL-40/CHI3L1 proteins after treatment of pancreatic cancer cell lines and macrophage-like cells with Rac/Cdc42 inhibitors show that MBQ-167 and the derivative MBQ-168 reduced CHI3L1 levels from conditioned media. These results demonstrate the potential of p-PAK and YKL-40/CHI3L1 from blood as biomarkers for Rac/Cdc42 inhibitor efficacy. This study impacts pharmacodynamic biomarker design for the planned Phase 2 clinical trials for MBQ-167 in breast and pancreatic cancer. Citation Format: Anamaris Torres-Sanchez, Ailed M. Cruz-Collazo, Michael Rivera-Robles, Olga Katsara, Stephanie Dorta-Estremera, Viviana Negron, Victor P. Carlo, Robert J. Schneider, Suranganie Dharmawardhane. Pharmacodynamic biomarkers for Rac and Cdc42 inhibitor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7628.

Abstract 677: Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1

Abstract The Rac1 signaling pathway is known to be hyperactivated in numerous solid tumors. We developed a Rac1 AlphaLISA assay that is amenable to high-throughput screening (HTS) to screen large libraries of small molecules. Based on molecular modeling of a putative allosteric binding site for several of the hits, we optimized a series of compounds that lead to RP-102124. RP-102124 engages with Rac1 in both biochemical and cellular target engagement assays. RP-102124 has greater potency than previously reported Rac1 inhibitors in biochemical assays, with potent inhibition of phenotypic characteristics of Rac signaling such as cell migration and cytoskeletal rearrangements. RP-102124 is a Racmulti inhibitor with activity against oncogenic Rac1b. RP-102124 is orally bioavailable and shows dose dependent inhibition of tumor growth in xenograft models at doses that are well-tolerated. RP-102124 is a first-in-class allosteric Racmulti inhibitor that shows promising therapeutic potential. Citation Format: Robert Silverman, Alan Cooper, Ronodip Kar, Mayuri V. Raman, Ramu Ravirala, Mubarak Hasan Shah, Sandeep Mahankali, Erik T. Goka. Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 677.

Abstract 1881: Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds

Abstract Metastasis is responsible for >90% of cancer-related deaths and is a key cause of failure of cancer therapy. The development of effective targeted therapy to prevent and treat metastasis has been challenging. Homologous Rho GTPases Rac and Cdc42 are viable metastasis targets because they have a crucial role in the signaling pathways that regulate cell survival, proliferation, and migration, so dysregulation of these proteins leads to cancer progression and metastasis of diverse cancer types. Therefore, targeting these regulatory proteins results in anticancer and antimetastatic activity, as observed with our dual Rac/Cdc42 inhibitor, MBQ-167, in triple-negative breast cancer. MBQ-167 has an IC50 of ~100nM for Rac and Cdc42 activation inhibition and a GI50 of 130nM. MBQ-167 inhibits the binding of guanine nucleotides to Rac and Cdc42 with similar efficacy. The purpose of this study was to identify MBQ-167 derivatives with similar or greater efficacy and specificity, with a range of inhibitory mechanisms, that may be specific to the different guanine nucleotide exchange factors expressed in distinct cancer cell types. Currently, over 30 different compounds with potential Rac1 and Cdc42 inhibitory activity are being evaluated. So far, some of the evaluated compounds have shown comparable GI50 values to MBQ-167 in metastatic breast cancer cell lines. From this screening, CPV-337 was selected as a compound with a GI50 of ~55nM and ~70% Rac activation inhibition at 50nM, therefore being 2X more effective than our leading compound MBQ-167. However, evaluation of CPV-337 in the viability of the MCF-10 non-tumorigenic epithelial cell line demonstrated a GI50 of ~63nM, which is similar to that of breast cancer cells. Therefore, CPV-337 may have cytotoxic effects on breast cancer. We are continuing to screen the library of MBQ-167 derivatives in breast, pancreatic, lung, and prostate cancer cell lines. Citation Format: Jessica Colón González, María C. Santa María Fuentes, Cornelis Vlaar, Suranganie Dharmawardhane. Screening of Rac1 and Cdc42 inhibitors as anti-metastatic compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1881.

Abstract 1678: Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells

Abstract Prostate cancer (PCa) is a major health burden that affects the quality of life of men worldwide. In the United States, PCa is the second leading cause of mortality among cancers that affect men. Treatment of advanced PCa aims to suppress androgen signaling and proliferation in cancer cells using anti-androgens and chemotherapy. However, the development of drug resistance is a major limitation to curing advanced prostate cancer and strategies to block mechanisms of therapy resistance would enhance treatment efficacy and potentially improve patient prognosis. Cdc42 and Rac1, members of the family of Rho GTPases, have been associated with the progression of different types of hormone-associated cancer, such as breast, ovary, and prostate, among others. The current study was designed to evaluate the role of Cdc42 and Rac1 in chemoresistant prostate cancer cells, including under castration-resistant and androgen-independent conditions. We did not observe significant differences in active (Rac-GTP) or total Rac1 levels in chemoresistant compared to parental PC3 or DU145 cells; similar findings were observed for Ccd42. Treatment of drug-naïve cells with a sublethal dose of docetaxel (10uM) induced gradual increases in Rac1-GTP up to 30 minutes in 22rv1, 60 minutes in LNCaP and DU145, and 24 hours in PC3 cells. Cdc42-GTP levels also increased with subacute docetaxel treatment up to 24 hours in 22rv1 and DU145. Chemoresistant 22rv1, PC3, and DU145 exhibited similar sensitivity to Rac1 and Ccd42 inhibitors including MB1-167, MBQ-168, EHop-097, with an IC50 of about 1uM for each compound and line. Ongoing studies are evaluating the role of these Rho-GTPases in 3-dimensional growth and survival. Citation Format: Andrea Paola López González, Jessica Colón González, María C. Santa María Fuentes, Surangani Dharmawardhane, Carlos J. Diaz Osterman. Docetaxel induces Rac1 and Cdc42 Rho GTPase activation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1678.

Abstract 1671: Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in the United States. KRAS is an oncogene mutated in 90% of PDAC patients. Oncogenic KRAS leads to aberrant cell proliferation and survival. KRAS is considered hard to target because resistance mechanisms to KRAS inhibitors are common highlighting the need to understand alternative strategies. PDAC tumor microenvironment evolves with cancer cell progression leading to vascular remodeling and blood vessel collapse. This makes the tumors nutrient deplete. To circumvent this, cells employ KRAS-dependent macropinocytosis- a nutrient scavenging pathway. Macropinocytosis is the process by which cells take up extracellular material by membrane ruffling to form vesicles which then fuse with lysosomes to release nutrients. Targeting KRAS-driven macropinocytosis can be crucial to limiting nutrients to the cell to prevent tumor growth. Protein Phosphatase 2A (PP2A), a heterotrimeric Serine/Threonine phosphatase, is a downstream regulator of KRAS. Its activity is repressed by mutated KRAS. We have seen that the activation of PP2A with small molecular activator, DT061, prevents macropinosomes from fusing with lysosomes which leads to cell death. But how DT061 regulated PP2A activity affects the macropinocytosis pathway is still unknown. The purpose of this study is to understand the mechanism of cell death brough upon by the activation of PP2A with DT061. Membrane ruffling is critical for the initiation of macropinocytosis. Rac is a GTPase usually involved with membrane ruffling. To show that Rac is needed for DT061 mediated macropinocytosis, we used EHT 1864, a Rac family inhibitor. Cells were treated with EHT 1864 before being treated with DT061 and stained with crystal violet. The inhibition of Rac prevented DT061 driven cell death, indicating that Rac is crucial to DT061 driven cell death. High molecular weight TMR-Dextran can only be taken up by the cells through macropinocytosis. Cells were treated with TMR-Dextran and EHT 1864. Cells with Rac inhibition showed lower uptake of TMR-Dextran than cells that were treated with DT061. Together these findings indicate to us that Rac plays a crucial role in the initiation of macropinocytosis and the regulation of cell death caused by the activation of PP2A. Understanding the pathway of DT061 mediated cell death can be vital to developing this concept as a therapeutic method of treatment for PDAC patients who currently have the lowest 5-year survival rate (12%) of all cancers. Citation Format: Indiraa Doraivel, Garima Baral, Claire M. Pfeffer, Brittany L. Allen-Petersen. Exploring the mechanism of PP2A regulated cell death via macropinocytosis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1671.

Inhibition of RAC attenuates Adriamycin-induced podocyte injury

Minimal Change Disease (MCD), which is associated with podocyte injury, is the leading cause of nephrotic syndrome in children. A considerable number of patients experience relapses and require prolonged use of prednisone and immunosuppressants. Multi-drug resistance and frequent relapses can lead to disease progression to focal and segmental glomerulosclerosis (FSGS).To identify potential targets for therapy of podocyte injury, we examined microarray data of mRNAs in glomerular samples from both MCD patients and healthy donors, obtained from the GEO database. Differentially expressed genes (DEGs) were used to construct the protein-protein interactions (PPI) network through the application of the search tool for the retrieval of interacting genes (STRING) tool. The most connected genes in the network were ranked using cytoHubba. 16 hub genes were selected and validated by qRT-PCR. RAC2 was identified as a potential therapeutic target for further investigation. By downregulating RAC2, Adriamycin (ADR)-induced human podocytes (HPCs) injury was attenuated. EHT-1864, a small molecule inhibitor that targets the RAC (RAC1, RAC2, RAC3) family, proved to be more effective than RAC2 silencing in reducing HPCs injury. In conclusion, our research suggests that EHT-1864 may be a promising new molecular drug candidate for patients with MCD and FSGS.

Hyperglycemic stress induces oxidative damage of enteric glial cells by triggering redoxosomes/p66SHC activation.

Diabetic gastrointestinal dysfunction (DGD) is a serious complication of diabetic mellitus (DM), affecting the enteric nervous system (ENS), particular enteric glial cells (EGCs). This study aimed to elucidate the effects and underlying molecular mechanisms of hyperglycemic stress on EGCs in in vitro and in vivo models of DM. In in vitro studies, enteric glial cell line CRL-2690 was exposed to hyperglycemia stress, and cell viability, cell apoptosis and oxidative damage were assessed. In in vivo studies, STZ-induced diabetic mice were constructed, and cell apoptosis and oxidative damage of EGCs in the duodenum of DM mice were assessed. The results showed that hyperglycemic stress markedly induced oxidative damage of EGCs in in vitro and in vivo models of DM. This damage was found to be dependent on the activation of redoxosomes, which involved the phosphorylation of SRC and Vav2, the up-regulation of active RAC1-GTP, and the activation of NADPH oxidase (NOX). Moreover, inhibitors of redoxosomes, such as the RAC1 inhibitor NSC23766 and the NOX inhibitor VAS2870, effectively mitigated the hyperglycemic stress-induced oxidative damage of EGCs. Additionally, inhibition of p66SHC, a downstream target of redoxosomes, attenuated oxidative damage of EGCs under hyperglycemic stress. Our findings suggest that the redoxosomes/p66SHC signaling is involved in the oxidative damage of EGCs during the pathological process of DGD. This signaling cascade may represent a potential therapeutic target for the treatment of DGD.

FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes invitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Quercetin suppresses ROS production and migration by specifically targeting Rac1 activation in gliomas.

P66Shc and Rac1 proteins are responsible for tumor-associated inflammation, particularly in brain tumors characterized by elevated oxidative stress and increased reactive oxygen species (ROS) production. Quercetin, a natural polyphenolic flavonoid, is a well-known redox modulator with anticancer properties. It has the capacity to cross the blood-brain barrier and, thus, could be a possible drug against brain tumors. In this study, we explored the effect of quercetin on Rac1/p66Shc-mediated tumor cell inflammation, which is the principal pathway for the generation of ROS in brain cells. Glioma cells transfected with Rac1, p66Shc, or both were treated with varying concentrations of quercetin for different time points. Quercetin significantly reduced the viability and migration of cells in an ROS-dependent manner with the concomitant inhibition of Rac1/p66Shc expression and ROS production in naïve and Rac1/p66Shc-transfected cell lines, suggestive of preventing Rac1 activation. Through molecular docking simulations, we observed that quercetin showed the best binding compared to other known Rac1 inhibitors and specifically blocked the GTP-binding site in the A-loop of Rac1 to prevent GTP binding and, thus, Rac1 activation. We conclude that quercetin exerts its anticancer effects via the modulation of Rac1-p66Shc signaling by specifically inhibiting Rac1 activation, thus restraining the production of ROS and tumor growth.

∆Np63α inhibits Rac1 activation and cancer cell invasion through suppression of PREX1

ΔNp63α, a member of the p53 family of transcription factors, plays a critical role in maintaining the proliferative potential of stem cells in the stratified epithelium. Although ΔNp63α is considered an oncogene and is frequently overexpressed in squamous cell carcinoma, loss of ΔNp63α expression is associated with increased tumor cell invasion and metastasis. We recently identified a ΔNp63α/miR-320a/PKCγ signaling axis that regulates cancer cell invasion by inhibiting phosphorylation of the small GTPase Rac1, a master switch of cell motility that positively regulates cell invasion in multiple human cancers. In this study, we identified a novel mechanism by which ΔNp63α negatively regulates Rac1 activity, by inhibiting the expression of the Rac-specific Guanine Exchange Factor PREX1. ΔNp63α knockdown in multiple squamous cell carcinoma cell lines leads to increased Rac1 activation, which is abrogated by treatment with the Rac1 inhibitor NSC23766. Furthermore, ΔNp63α negatively regulates PREX1 transcript and protein levels. Using a Rac-GEF activation assay, we also showed that ΔNp63α reduces the levels of active PREX1. The inhibition of the PREX1-Rac1 signaling axis by ΔNp63α leads to impaired cell invasion, thus establishing the functional relevance of this link. Our results elucidated a novel molecular mechanism by which ΔNp63α negatively affects cancer cell invasion and identifies the ΔNp63α/Rac1 axis as a potential target for metastasis.

The Rac1-inhibitor EHop-016 attenuates AML cell migration and enhances the efficacy of daunorubicin in MOLM-13 transplanted zebrafish larvae

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a GTPase implicated in cell migration and homing of hematopoietic cells to the hematopoietic niche, and is commonly overexpressed in acute myeloid leukemia (AML). This can lead to quiescence of leukemic blasts in the niche and reduced response to therapy. We investigated the Rac1 inhibitor EHop-016 on AML by assessing its effects on MOLM-13 cells in vitro and in zebrafish larvae, regarding cell motility and therapeutic potential in combination with daunorubicin (DNR). In vitro assessment of proliferation and viability was by measurement of 3H-thymidine incorporation and detection of Annexin V/PI positive cells. Cell motility was evaluated by measurement of migration in a transwell system. Fluorescently stained MOLM-13 cells were injected into zebrafish larvae, and individual cells followed by confocal microscopy. Cell accumulation in the caudal hematopoietic tissue (CHT) was studied using a 12-hour timelapse, while in vivo efficacy of DNR, EHop-016 or a combination was investigated over 24 h.The in vitro results showed that EHop-016 acted synergistically in combination with DNR in reducing the viability of MOLM-13 cells (Bliss synergy score above 10 %). Non-toxic concentrations of EHop-016 reduced cell migration. These findings were reproduced in zebrafish larvae: larvae receiving both DNR and EHop-016 had significantly reduced tumor burden compared to the untreated control or single treatments. The accumulation of MOLM-13 cells in the CHT was reduced in larvae receiving EHop-016 treatment.Our findings demonstrate that targeting Rac1 in AML holds promise as a complementary treatment to established chemotherapy and should be further investigated.

Inhibition of esophageal cancer progression through HACE1-TRIP12 interaction and associated RAC1 ubiquitination and degradation.

Objective: This study investigated the significance of HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) in esophageal cancer (ESCA) and its underlying mechanism in ESCA regulation through the induction of RAC1 ubiquitination and degradation. Methods: Characterization studies of HACE1 in ESCA clinical tissues and cell lines were performed. Next, the effects of HACE1 on the biological behavior of ESCA cells were examined by silencing and overexpressing HACE1. Protein-protein interactions (PPIs) involving HACE1 were analyzed using data from the String website. The function of HACE1 in RAC1 protein ubiquitination was validated using the proteasome inhibitor MG132. The effects of HACE1 on ESCA cells through RAC1 were elucidated by applying the RAC1 inhibitor EHop-016 in a tumor-bearing nude mouse model. To establish the relationship between HACE1 and TRIP12, rescue experiments were conducted, mainly to evaluate the effect of TRIP12 silencing on HACE1-mediated RAC1 regulation in vitro and in vivo. The PPI between HACE1 and TRIP12 and their subcellular localization were further characterized through co-immunoprecipitation and immunofluorescence staining assays, respectively. Results: HACE1 protein expression was notably diminished in ESCA cells but upregulated in normal tissues. HACE1 overexpression inhibited the malignant biological behavior of ESCA cells, leading to restrained tumor growth in mice. This effect was coupled with the promotion of RAC1 protein ubiquitination and subsequent degradation. Conversely, silencing HACE1 exhibited contrasting results. PPI existed between HACE1 and TRIP12, compounded by their similar subcellular localization. Intriguingly, TRIP12 inhibition blocked HACE1-driven RAC1 ubiquitination and mitigated the inhibitory effects of HACE1 on ESCA cells, alleviating tumor growth in the tumor-bearing nude mouse model. Conclusion: HACE1 expression was downregulated in ESCA cells, suggesting that it curbs ESCA progression by inducing RAC1 protein degradation through TRIP12-mediated ubiquitination.

Roles of the mechanosensitive ion channel Piezo1 in the renal podocyte injury of experimental hypertensive nephropathy.

Glomerular podocyte injury plays an essential role in proteinuria pathogenesis, a hallmark of chronic kidney disease, including hypertensive nephropathy. Although podocytes are susceptible to mechanical stimuli, their mechanotransduction pathways remain elusive. Piezo proteins, including Piezo1 and 2, are mechanosensing ion channels that mediate various biological phenomena. Although renal Piezo2 expression and its alteration in rodent dehydration and hypertension models have been reported, the role of Piezo1 in hypertensive nephropathy and podocyte injury is unclear. In this study, we examined Piezo1 expression and localization in the kidneys of control mice and in those of mice with hypertensive nephrosclerosis. Uninephrectomized, aldosterone-infused, salt-loaded mice developed hypertension, albuminuria, podocyte injury, and glomerulosclerosis. RNAscope in situ hybridization revealed that Piezo1 expression was enhanced in the podocytes, mesangial cells, and distal tubular cells of these mice compared to those of the uninephrectomized, vehicle-infused control group. Piezo1 upregulation in the glomeruli was accompanied by the induction of podocyte injury-related markers, plasminogen activator inhibitor-1 and serum/glucocorticoid regulated kinase 1. These changes were reversed by antihypertensive drug. Exposure of Piezo1-expressing cultured podocytes to mechanical stretch activated Rac1 and upregulated the above-mentioned markers, which was antagonized by the Piezo1 blocker grammostola mechanotoxin #4 (GsMTx4). Administration of Piezo1-specific agonist Yoda1 mimicked the effects of mechanical stretch, which was minimized by the Yoda1-specific inhibitor Dooku1 and Rac inhibitor. Rac1 was also activated in the above-mentioned hypertensive mice, and Rac inhibitor downregulated gene expression of podocyte injury-related markers in vivo. Our results suggest that Piezo1 plays a role in mechanical stress-induced podocyte injury.

Efferocyte-Derived MCTRs Metabolically Prime Macrophages for Continual Efferocytosis via Rac1-Mediated Activation of Glycolysis.

Clearance of multiple rounds of apoptotic cells (ACs) through continual efferocytosis is critical in the maintenance of organ function, the resolution of acute inflammation, and tissue repair. To date, little is known about the nature of mechanisms and factors that govern this fundamental process. Herein, the authors reported that breakdown of ACs leads to upregulation of 12-lipoxygenase in macrophages. This enzyme converts docosahexaenoic acid to maresin conjugates in tissue regeneration (MCTRs). The levels of these autacoids are elevated at sites of high apoptotic burden in vivo and in efferocytosing macrophages in vitro. Abrogation of MCTR production using genetic approaches limits the ability of macrophages to perform continual efferocytosis both in vivo and in vitro, an effect that is rescued by add-back of MCTRs. Mechanistically, MCTR-mediated priming of macrophages for continual efferocytosis is dependent on alterations in Rac1 signalling and glycolytic metabolism. Inhibition of Rac1 abolishes the ability of MCTRs to increase glucose uptake and efferocytosis in vitro, whereas inhibition of glycolysis limits the MCTR-mediated increases in efferocytosis and tissue repair. Together, these findings demonstrate that upregulation of MCTRs by efferocytosing macrophages plays a central role in the regulation of continual efferocytosis via the autocrine and paracrine modulation of metabolic pathways.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Abstract B103: Statins decrease mesothelial clearance, an early step in ovarian cancer metastasis

Abstract Ovarian cancer has a low 5-year survival rate often associated with detection of late-stage cancer due to the difficulty of early detection. Ovarian cancer metastasis can occur via a distinct mechanism compared to other solid cancers. Ovarian cancer spheroids slough off from the primary tumor. In the ascites, these spheroids attach to and clear the protective layer of mesothelial cells that line the peritoneum. Once this mesothelial clearance occurs, the ovarian spheroids can invade the connective tissue and metastasize to further parts of the body or grow implanted in the peritoneum. Targeting mesothelial clearance may be a new mechanism to treat ovarian cancer. Previously, it was shown that the use of statins, drugs commonly used to control cholesterol, is associated with decreased ovarian cancer occurrence. In addition, statins have the potential to interfere with several signaling pathways that are known to play key roles in cancer including metastasis. The potential for statins to target metastasis could be beneficial in treating cancers like ovarian cancer that tend to be found in metastatic stages. Therefore, we hypothesized that statins would reduce mesothelial clearance by ovarian cancer spheroids. Using an in vitro mesothelial clearance assay, we have found that statins, such as simvastatin and lovastatin, decrease the mesothelial clearing abilities of ovarian cancer spheroids. To better understand the mechanism of mesothelial clearance inhibition, we analyzed several known downstream pathways. Targeting HMGCR by siRNA, the known molecular target of statins, reduced mesothelial clearance. In addition, targeting Rac1 and CDC42 using the inhibitor AZA1 has a similar inhibitory effect as simvastatin on mesothelial clearance. This suggests that inhibition of Rac1 by statins may be the mechanism that statins inhibit mesothelial clearance. To support this, siRNA knockdown of RAC1 suggests the importance of Rac1 for mesothelial clearance. Previously, we found that treatment of ovarian spheroids with geranylgeranyl-pyrophosphate partially rescued the inhibition of viability by statins. Therefore, our current working hypothesis is that statins disrupt the function of Rac1 by reducing the cellular supply of geranylgeranyl-pyrophosphate through inhibiting HMGCR and the mevalonate pathway. While we are currently further analyzing the mechanisms behind this pathway, we believe this evidence suggests an exciting new therapeutic route for treating ovarian cancer. Citation Format: Brendan M Reilly, Olivia R Martin, Allison F Mackey, Sarah R Walker. Statins decrease mesothelial clearance, an early step in ovarian cancer metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B103.

Abstract C069: Targeting the Bai1-driven signaling pathway as a therapeutic approach for breast cancer treatment

Abstract Breast cancer is the most prevalent cancer among women globally, and it remains the second leading cause of cancer-related mortality in this group. Notably, breast cancer exhibits a survival disparity between black and white women, with black women experiencing higher mortality rates. While the reasons for this disparity are multifaceted, differences in molecular mechanisms and signaling pathways driving disease progression may contribute to the observed survival gap. In this study, we investigate the role of cancer cell fusion-driven tumor heterogeneity and metastasis as a potential explanation for the survival disparity in breast cancer. We hypothesize that the mechanism of breast cancer fusion involves Bai1 activation-driven induction of the Elmo1/Dock 180/Rac 1 signaling pathway. Elmo1 and Dock 180 are overexpressed in breast cancer cell lines and Elmo1/Dock 180 activation is associated with breast cancer metastasis. Targeting this pathway could prevent cancer cell fusion-driven metastasis and death. To test our hypothesis, we used non-metastatic and metastatic breast cancer cells isolated from black (HCC 1806, MDA-MB-157) and white (T47D, MDA-MB-231) women, as well as mesenchymal stem cells (MSCs) (hMSC4099, hMSC39334) as fusion partners. We used Elmo1 and Dock 180 shRNAs and a Rac1 inhibitor to analyze the role of Elmo1/Dock 180/Rac 1 signaling in breast cancer cell fusion. Fusion products were scored using the Cre/loxP system. We observed that breast cancer cells isolated from black women are more prone to fuse with MSCs than cells isolated from white women (P<0.05). knocking down Bai1 significantly reduced the frequency of fusion across all cell lines (P<0.05) and more significantly in the non-metastatic breast cancer cell line isolated from black women (P<0.001). A notable reduction in the frequency of fusion between breast cancer cells and MSCs was observed when Elmo1, Dock180, or Rac1 was inhibited (P<0.05). These results suggest that the Bai1/Elmo1/Dock180/Rac1 signaling plays an important role in cancer cell fusion. Targeting this mechanism could lead to new drug development tactics for cancer treatment. Citation Format: Oluwatoyin V. Odubanjo, Paul B. Tchounwou, Brenda M. Ogle, Felicite K. Noubissi. Targeting the Bai1-driven signaling pathway as a therapeutic approach for breast cancer treatment [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C069.