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Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivo and are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146 dependent manner.

Background Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

The malignant brain cancer, glioblastoma multiforme (GBM), is heterogeneous, infiltrative, and associated with chemo- and radioresistance. Despite pharmacological advances, prognosis is poor. Delivery into the brain is hampered by the blood-brain barrier (BBB), which limits the efficacy of both conventional and novel therapies at the target site. Current treatments for GBM remain palliative rather than curative; therefore, innovative delivery strategies are required and nanoparticles (NPs) are at the forefront of future solutions. Since the FDA approval of Doxil® (1995) and Abraxane (2005), the first generation of nanomedicines, development of nano-based therapies as anti-cancer treatments has escalated. A new generation of NPs has been investigated to efficiently deliver therapeutic agents to the brain, overcoming the restrictive properties of the BBB. This review discusses obstacles encountered with systemic administration along with integration of NPs incorporated with conventional and emerging treatments. Barriers to brain drug delivery, NP transport mechanisms across the BBB, effect of opsonisation on NPs administered systemically, and peptides as NP systems are addressed.

RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.

Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional highresolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.

Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.

BackgroundRalA is a member of the Ras superfamily of small GTPases. The Anti-RalA autoantibodies (s-RalA-Abs) act as tumor markers in various types of cancer and are negatively associated with the p53 autoantibodies (s-p53-Abs). This study aimed to evaluate the relationship between s-RalA-Abs and s-p53-Abs in various types of cancer.MethodsA total of 1833 cancer patients (esophageal cancer, 172; hepatocellular carcinoma, 91; lung cancer, 269; gastric cancer, 317; colon cancer, 262; breast cancer, 364; and prostate cancer, 358) and 73 healthy subjects were enrolled in the study. The levels of s-RalA-Abs and s-p53-Abs were analyzed using enzyme-linked immunosorbent assay, and the positivity rates and relations between the two autoantibodies were evaluated. The cutoff values for s-RalA abs and s-p53 abs were set as mean + 2 standard deviation and the values higher than the cutoff values were defined as positive.ResultsThe titers in all cancer types were significantly higher than those in the controls (P < 0.01). The positivity rates for s-RalA-Abs ranged between 11.7 and 21.5%, and those for s-p53-Abs ranged between 12 and 28.5%. A combined assay of the two antibodies revealed positivity rates of 20.9 and 44.2%. In Stage 0/I/II tumors, the positivity rates of the combination of the two antibodies ranged between 21.5 and 42.3%. The two autoantibodies were complementary to each other in the prostate and breast cancers, but independent in other carcinomas.ConclusionThe combined use of s-RalA-Abs and s-p53-Abs tended to increase the positivity rate in all cancers, including Stage 0/I/II cancers.

Purpose: In our group's previous study, we identified the first somatic variant of RALA c.G482A that encodes a small GTPase of the Ras superfamily in the postoperative brain tissue from an epilepsy patient with focal cortical dysplasia type II (FCD II) using deep whole-exome sequencing and targeted amplicon sequencing. To date, the role of RALA in brain development is not yet known. Here, we report that the RALA somatic variant led to FCD type II through activation of the mTOR pathways. Methods: HEK293T cells were transfected in vitro to analyze the expression of the RalA protein, as well as phosphorylated S6 (P-S6), one of the major markers of mTOR pathway activation, RalA GTPase activity, and the interaction between RalA and its downstream binding effectors. In vivo, wild-type, and mutant RALA plasmids were transfected into the local cortex of mice using in utero electroporation to evaluate the effect of RALA c.G482A on neuronal migration. Results: The RALA c.G482A mutation increased RalA protein expression, the abnormal activation of the mTOR pathways, RalA GTPase activity, and binding to downstream effectors. RALA c.G482A local transfection in the embryonic brain in utero induced abnormal cortical neuron migration in mice. Conclusions: This study demonstrated for the first time that the somatic gain-of-function variant of RALA activates the mTOR pathway and leads to neuronal migration disorders in the brain, facilitating the development of FCD II. Therefore, RALA brain somatic mutation may be one of the pathogenic mechanisms leading to FCD II that is always related to drug-resistant epilepsy in children. However, more somatic variations of this gene are required to be confirmed in more FCD II patient brain samples.