The Adenosine diphosphate-Ribosylation Factor (ARF) family belongs to the RAS superfamily of small GTPases and is involved in a wide variety of physiological processes, such as cell proliferation, motility and differentiation by regulating membrane traffic and associating with the cytoskeleton. Like other members of the RAS superfamily, ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. Several studies have reported that cancer cells are able to subvert membrane traffic regulators to enhance migration and invasion. Indeed, members of the ARF family, including ARF-Like (ARL) proteins have been implicated in tumorigenesis and progression of several types of cancer. Here, we review the role of ARF family members, their GEFs/GAPs and effectors in tumorigenesis and cancer progression, highlighting the ones that can have a pro-oncogenic behavior or function as tumor suppressors. Moreover, we propose possible mechanisms and approaches to target these proteins, toward the development of novel therapeutic strategies to impair tumor progression.

Colorectal cancer is the second leading cause of cancer-related mortality. Many current therapies rely on chemotherapeutic agents with poor specificity for tumor cells. The clinical success of cisplatin has prompted the research and design of a huge number of metal-based complexes as potential chemotherapeutic agents. In this study, two zinc(II) complexes, [ZnL₂] and [ZnL(AcO)], where AcO is acetate and L is an organic compound combining 8-hydroxyquinoline and a benzothiazole moiety, were developed and characterized. Analytical and spectroscopic studies, namely, NMR, FTIR, and UV-Vis allowed us to establish the complexes’ structures, demonstrating the ligand-binding versatility: tetradentate in [ZnL(AcO)] and bidentate in [ZnL₂]. Complexes were screened in vitro using murine and human colon cancer cells cultured in 2D and 3D settings. In 2D cells, the IC₅₀ values were <22 µM, while in 3D settings, much higher concentrations were required. [ZnL(AcO)] displayed more suitable antiproliferative properties than [ZnL₂] and was chosen for further studies. Moreover, based on the weak selectivity of the zinc-based complex towards cancer cell lines in comparison to the non-tumorigenic cell line, its incorporation in long-blood-circulating liposomes was performed, aiming to improve its targetability. The resultant optimized liposomal nanoformulation presented an I.E. of 76% with a mean size under 130 nm and a neutral surface charge and released the metal complex in a pH-dependent manner. The antiproliferative properties of [ZnL(AcO)] were maintained after liposomal incorporation. Preliminary safety assays were carried out through hemolytic activity that never surpassed 2% for the free and liposomal forms of [ZnL(AcO)]. Finally, in a syngeneic murine colon cancer mouse model, while free [ZnL(AcO)] was not able to impair tumor progression, the respective liposomal nanoformulation was able to reduce the relative tumor volume in the same manner as the positive control 5-fluorouracil but, most importantly, using a dosage that was 3-fold lower. Overall, our results show that liposomes were able to solve the solubility issues of the new metal-based complex and target it to tumor sites.

Breast cancer (BC) is the most common type of cancer in women and has a high rate of relapse and death. Notch signaling is crucial for normal breast development and homeostasis. Dysregulation of Notch receptors and ligands has been detected in different BC subtypes and shown to be implicated in tumor development, progression, drug resistance, and recurrence. However, the effects of Notch ligands in various types of BC remain poorly understood. In this study, we investigated the effects of the Notch ligand DLL1 in three different human BC cell lines: MCF-7, BT474, and MDA-MB-231. We showed that DLL1 expression is higher in MCF-7 and BT474 than in MDA-MB-231 cells, and that these cells respond differently to DLL1 downregulation. Functional assays in MCF-7 cells demonstrated that siRNA-mediated DLL1 downregulation reduced colony formation efficiency, migration, proliferation, caused cell cycle arrest at the G1 phase, and induced apoptosis. Gene expression studies revealed that these effects in MCF-7 cells were associated with increased expression of the cell cycle arrest p21 gene and decreased expression of genes that promote cell cycle progression (CDK2, SKP2), and survival (BCL2, BIRC5), unravelling possible mechanisms whereby DLL1 downregulation exerts some of its effects. Moreover, our results demonstrate that treatment with recombinant DLL1 increased MCF-7 cell proliferation and migration, confirming that DLL1 contributes to these processes in this BC cell line. DLL1 downregulation reduced the colony formation efficiency of BT474 cells and decreased the migration and invasion abilities of MDA-MB-231 cells but showed no effects in the proliferation and survival of these cells. These findings provide further evidence that DLL1 exerts carcinogenic effects in BC cells. The dissimilar effects of DLL1 downregulation observed amongst MCF-7, BT474, and MDA-MB-231 cells is likely due to their distinctive genetic and biologic characteristics, suggesting that DLL1 contributes to BC through various mechanisms.

Breast cancer is the first cause of cancer-related mortality among women worldwide, according to the most recent estimates. This mortality is mainly caused by the tumors’ ability to form metastases. Cancer cell migration and invasion are essential for metastasis and rely on the interplay between actin cytoskeleton remodeling and cell adhesion. Therefore, understanding the mechanisms by which cancer cell invasion is controlled may provide new strategies to impair cancer progression. We investigated the role of the ADP-ribosylation factor (Arf)-like (Arl) protein Arl13b in breast cancer cell migration and invasion in vitro, using breast cancer cell lines and in vivo, using mouse orthotopic models. We show that Arl13b silencing inhibits breast cancer cell migration and invasion in vitro, as well as cancer progression in vivo. We also observed that Arl13b is upregulated in breast cancer cell lines and patient tissue samples. Moreover, we found that Arl13b localizes to focal adhesions (FAs) and interacts with β3-integrin. Upon Arl13b silencing, β3-integrin cell surface levels and FA size are increased and integrin-mediated signaling is inhibited. Therefore, we uncover a role for Arl13b in breast cancer cell migration and invasion and provide a new mechanism for how ARL13B can function as an oncogene, through the modulation of integrin-mediated signaling.