Pablo Mendoza

Rab5 activation promotes focal adhesion disassembly, migration and invasiveness in tumor cells

Summary Migration and invasion are essential steps associated with tumor cell metastasis and increasing evidence points towards endosome trafficking being essential in this process. Indeed, the small GTPase Rab5, a crucial regulator of early endosome dynamics, promotes cell migration in vitro and in vivo. Precisely how Rab5 participates in these events remains to be determined. Considering that focal adhesions represent structures crucial to cell migration, we specifically asked whether Rab5 activation promoted focal adhesion disassembly and thereby facilitated migration and invasion of metastatic cancer cells. Pulldown and biosensor assays revealed that Rab5-GTP loading increased at the leading edge of migrating tumor cells. Additionally, targeting of Rab5 by different shRNA sequences, but not control shRNA, decreased Rab5-GTP levels, leading to reduced cell spreading, migration and invasiveness. Re-expression in knockdown cells of wild-type Rab5, but not the S34N mutant (GDP-bound), restored these properties. Importantly, Rab5 association with the focal adhesion proteins vinculin and paxillin increased during migration, and expression of wild-type, but not GDP-bound Rab5, accelerated focal adhesion disassembly, as well as FAK dephosphorylation on tyrosine 397. Finally, Rab5-driven invasiveness required focal adhesion disassembly, as treatment with the FAK inhibitor number 14 prevented Matrigel invasion and matrix metalloproteinase release. Taken together, these observations show that Rab5 activation is required to enhance cancer cell migration and invasion by promoting focal adhesion disassembly.

Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion

ABSTRACT Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5–GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85&agr; (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85&agr;, precluding p85&agr;-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1–Rab5–Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

Hypoxia promotes Rab5 activation, leading to tumor cell migration, invasion and metastasis.

Hypoxia, a common condition of the tumor microenvironment, is associated with poor patient prognosis, tumor cell migration, invasion and metastasis. Recent evidence suggests that hypoxia alters endosome dynamics in tumor cells, leading to augmented cell proliferation and migration and this is particularly relevant, because endosomal components have been shown to be deregulated in cancer. The early endosome protein Rab5 is a small GTPase that promotes integrin trafficking, focal adhesion turnover, Rac1 activation, tumor cell migration and invasion. However, the role of Rab5 and downstream events in hypoxia remain unknown. Here, we identify Rab5 as a critical player in hypoxia-driven tumor cell migration, invasion and metastasis. Exposure of A549 human lung carcinoma, ZR-75, MDA-MB-231 and MCF-7 human breast cancer and B16-F10 mouse melanoma cells to hypoxia increased Rab5 activation, followed by its re-localization to the leading edge and association with focal adhesions. Importantly, Rab5 was required for hypoxia-driven cell migration, FAK phosphorylation and Rac1 activation, as shown by shRNA-targeting and transfection assays with Rab5 mutants. Intriguingly, the effect of hypoxia on both Rab5 activity and migration was substantially higher in metastatic B16-F10 cells than in poorly invasive B16-F0 cells. Furthermore, exogenous expression of Rab5 in B16-F0 cells predisposed to hypoxia-induced migration, whereas expression of the inactive mutant Rab5/S34N prevented the migration of B16-F10 cells induced by hypoxia. Finally, using an in vivo syngenic C57BL/6 mouse model, Rab5 expression was shown to be required for hypoxia-induced metastasis. In summary, these findings identify Rab5 as a key mediator of hypoxia-induced tumor cell migration, invasion and metastasis.

Rab5 activation as a tumor cell migration switch

Increased cell migration is an acquired feature of metastatic cancer cells and relies on derailed signal transduction pathways. Intracellular vesicular trafficking plays a key role in cell migration due to its intricate involvement in cargo transport and membrane composition. In the last decade, endocytosis has been implicated in cell migration and found to be responsible for the internalization of membrane receptors at the plasma membrane, where integrin trafficking and fine-tuning of receptor tyrosine kinase signaling by internalization are major mechanisms. Accumulating evidence has suggested a link between endosome dynamics, cell migration, and invasion, in which small GTPases of the Rab family have central roles. We have recently determined that Rab5 activation is a crucial event in promoting focal adhesion disassembly, which is concomitant with the migration and invasion of metastatic cancer cells. The mechanisms underlying this novel role for Rab5 are currently unclear, and their elucidation will provide insight into the role of Rab5 function in cancer cell metastasis.

The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis

Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin‐1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts in vitro. However, the participation of histatin‐1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin‐1 promotes angiogenesis, as shown in vivo, using the chick chorioallantoic membrane model, and by an in vitro tube formation assay, using both human primary cultured endothelial cells (HUVECs) and the EA.hy926 cell line. Specifically, histatin‐1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin‐1 increased the recruitment of RIN2, a Rab5–guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin‐1‐dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin‐1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin‐1 is a novel proangiogenic factor that may contribute to oral wound healing.—Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina‐Berrios, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin‐1 promotes endothelial cell adhesion, migration, and angiogenesis. FASEB J. 31, 4946–4958 (2017). www.fasebj.org

ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis

Neisseria gonorrhoeae (Ngo) has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM). Here, we investigate the role of adenosine triphosphate (ATP) in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P < 0.01). However, we did not observe any changes in inflammasome transcriptional activation of speck-like protein containing a caspase recruitment domain (CARD) (ASC, P > 0.05) and caspase-1 (CASP1, P > 0.05). In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P > 0.01). Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation.

A novel caveolin-1/p85α/Rab5/Tiam1/Rac1 signaling axis in tumor cell migration and invasion

The small GTPase Rab5 has been frequently studied in the context of intracellular trafficking, but evidence obtained more recently has implicated Rab5 as a critical regulator of cell adhesion, migration and invasion in both normal and tumor cells. These recent findings showing that Rab5 promotes Rac1 activation and focal adhesion dynamics have highlighted the question as to what the upstream regulators of Rab5 activity might be and how these are connected to cell migration. The efforts to shed light on this issue identified in metastatic cancer cells a novel Caveolin‑1/p85α/Rab5/Tiam1/Rac1 signaling axis relevant to cancer cell migration and invasion. In this addendum, we highlight aspects concerning Rab5 regulation in this context.

Calpain2 mediates Rab5-driven focal adhesion disassembly and cell migration

ABSTRACT The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitates with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions. In addition, Rab5 silencing decreased calpain2 activity, as shown by cleavage of the fluorogenic substrate tBOC-LM-CMAC and the endogenous substrate talin. Accordingly, Rab5 promoted focal adhesion disassembly in a calpain2-dependent manner, as expression of GFP-Rab5 accelerated focal adhesion disassembly in nocodazole-synchronized cells, whereas pharmacological inhibition of calpain2 with N-acetyl-Leu-Leu-Met prevented both focal adhesion disassembly and cell migration induced by Rab5. In summary, these data uncover Rab5 as a novel regulator of calpain2 activity and focal adhesion proteolysis leading to cell migration.