Purpose: Colorectal cancer (CRC) is the third most common type of cancer worldwide, and it represents over half of all gastrointestinal cancer deaths. Knowing that cancer cells have a high proliferation rate, they require high amounts of amino acids, including arginine. In addition, several tumor types have been shown to downregulate ASS-1 expression, becoming auxotrophic for arginine. Therefore, Arginine deprivation is one of the promising therapeutic approaches to target cancer cells. This can be achieved through the use of a recombinant human arginase, HuArgI(Co)-PEG5000, an arginine degrading enzyme. Methods: In this present study, the cytotoxic effect of HuArgI(Co)-PEG5000 on CRC cell lines (HT-29, Caco-2, Sw837) is examined though cytotoxicity assays. Wound healing assays, invasion assays, and adhesion assays were also performed to detect the effect on metastasis. Results: Wound healing and invasion assays revealed a decrease in cell migration and invasion after treatment with arginase. Cells that were treated with arginase also showed a decrease in adhesion, which coincided with a decrease in RhoA activation, demonstrated though the use of a FRET biosensor to detect RhoA activation in a single cell assay, and a decrease in MMP-9 expression. Treating cells with both arginase and L-citrulline, which significantly restores intracellular arginine levels, reversed the effect of HuArgI(Co)-PEG5000 on cell viability, migration, and invasion. Conclusion: We can, therefore, conclude that colorectal cancer is partially auxotrophic to arginine and that arginine depletion is a potential selective inhibitory approach for motility and invasion in colon cancer cells.

Glioblastoma multiforme (GBM) is one of the most common and deadly cancers of the central nervous system (CNS). It is characterized by the presence of hypoxic regions, especially in the core, leading to an increase in vascularity. This increased vascularization is driven by the expression of the major angiogenic inducer VEGF and the indirect angiogenic inducer Epidermal growth factor (EGF), which stimulates VEGF expression. In this study, we examine the regulation of VEGF by both hypoxia and the EGF signaling pathway. We also examine the involvement of pathways downstream from EGF signaling, including the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway and the Phosphatidylinositol-3-kinase/RhoA/C (PI3K/RhoA/C) pathway in this regulation. Our results show that VEGF expression and secretion levels increase following either hypoxia or EGF stimulation, with the two stimuli signaling in parallel. We also observed an increase in ERK and protein kinase B (Akt) phosphorylation, in response to EGF stimulation, with kinetics that correlated with the kinetics of the effect on VEGF. Using pharmacological inhibitors against ERK and PI3K and small interfering RNAs (siRNAs) against RhoA and RhoC, we found that both the ERK and the PI3K/RhoA/C pathways have to cooperate in order to lead to an increase in VEGF expression, downstream from EGF. In response to hypoxia, however, only ERK was involved in the regulation of VEGF. Hypoxia also led to a surprising decrease in the activation of PI3K and RhoA/C. Finally, the decrease in the activation of these Rho-GTPases was found to be mediated through a hypoxia-driven overexpression of the Rho-GTPase GTPase activating protein (GAP), StarD13. Therefore, while under normoxic conditions, EGF stimulates the activation of both the PI3K and the MAPK pathways and the induction of VEGF, in glioblastoma cells, hypoxic conditions lead to the suppression of the PI3K/RhoA/C pathway and an exclusive switch to the MAPK pathway.

Background Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. Methods We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. Results We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. Conclusion In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases.

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Objectives: Pancreatic cancer is one of the most aggressive solid cancers and the fourth leading cause of cancer death in men and women. We previously showed that arginine depletion, using arginase I [HuArgI(Co)-PEG5000], selectively triggers cell death by autophagy in PANC-1 pancreatic cancer cells. The mechanism of action of [HuArgI(Co)-PEG5000], however, has remained poorly understood. In this study, we investigated the effects of arginine depletion on PANC-1 cell migration, adhesion, and invasion and determined the main molecular targets, which mediate PANC-1 cell response to treatment with HuArgI(Co)-PEG5000.Methods: This was done through examining 2-dimensional (2D) cell motility assays (wound healing and time lapse), cell adhesion, and cell invasion assays, as well as immunostaining for focal adhesions and invadopodia in cells without or with the treatment with arginase. Results:We demonstrate that arginine depletion decreases PANC-1 2D cell migration, adhesion, and 3D invasion. Moreover, our data suggest that these effects are mediated by autophagy and subsequent decrease in the activation of members of Ras homolog gene family (Rho) GTPase family.Conclusions: Altogether, these findings uncover the mechanism of action of [HuArgI(Co)-PEG5000] and highlight the promising and selective anticancer potential for arginine depletion in the treatment of pancreatic cancer cells.