Role of the tumor microenvironment in cancer hallmarks and targeted therapy (Review).

Genetic alterations drive tumor onset and progression. However, the cross­talk between tumor cells and the benign components of the surrounding stroma can also promote the initiation, progression and metastasis of solid tumors. These cellular and non­cellular stromal components form the tumor microenvironment (TME), which co­evolves with tumor cells. Their dynamic and mutualistic interactions are currently considered to be among the distinctive hallmarks of cancer. Biochemical and physical cues from the TME serve an essential role in regulating tumor onset and progression. They are also associated with resistance to treatment and poor prognosis in patients with cancer. Therefore, a deep understanding of the TME is vital for developing potent anticancer therapeutics and improving patient outcomes. The present review aims to review the biology of both cellular and non­cellular constituents of the TME and novel findings regarding their contribution to core as well as emerging cancer hallmarks. The present review also describes key TME markers that are either targeted in interventional clinical trials or serve as promising potential anticancer therapies. Understanding TME components and their intercellular interactions is key toward identifying the mechanisms of progression and treatment resistance. Such understanding is of utmost significance for personalized and effective cancer therapy strategies.

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42.

Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.

Human Recombinant Arginase I [HuArgI(Co)-PEG5000]-Induced Arginine Depletion Inhibits Pancreatic Cancer Cell Migration and Invasion Through Autophagy.

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.

StarD13 differentially regulates migration and invasion in prostate cancer cells.

Prostate cancer is the second most commonly diagnosed cancer in men and one of the main leading causes of cancer deaths among men worldwide. Rapid uncontrolled growth and the ability to metastasize to other sites are key hallmarks in cancer development and progression. The Rho family of GTPases and its activators the GTPase-activating proteins (GAPs) are required for regulating cancer cell proliferation and migration. StarD13 is a GAP for Rho GTPases, specifically for RhoA and Cdc42. We have previously shown that StarD13 acts as a tumor suppressor in astrocytoma as well as breast and colorectal cancer. In this study, we performed a functional comparative analysis of StarD13 targets/and or interacting molecules to understand the general role that StarD13 plays in cancers. Our data highlight the importance of StarD13 in modulating several hallmarks of cancer. Findings from database mining and immunohistochemistry revealed that StarD13 is underexpressed in prostate cancers, in addition knocking down Stard13 increased cancer cell proliferation, consistent with its role as a tumor suppressor. Stard13 depletion, however, led to an increase in cell adhesion, which inhibited 2D cell migration. Most interestingly, StarD13 depletion increases invasion and matrix degradation, at least in part, through its regulation of Cdc42. Altogether, the data presented suggest that StarD13 acts as a tumor suppressor inhibiting prostate cancer cell invasion.

Human recombinant arginase I [HuArgI (Co)-PEG5000]-induced arginine depletion inhibits ovarian cancer cell adhesion and migration through autophagy-mediated inhibition of RhoA.

Ovarian carcinoma is the second most common malignancy of the female reproductive system and the leading cause of death from female reproductive system malignancies. Cancer cells have increased proliferation rate and thus require high amounts of amino acids, including arginine. L-arginine is a non-essential amino acid synthesized from L-citrulline by the Arginosuccinate synthetase (ASS1) enzyme. We have previously shown that the ovarian cancer cells, SKOV3, are auxotrophic to arginine, and that arginine deprivation by treatment with the genetically engineered human arginase I (HuArgI (Co)-PEG5000) triggers the death of SKOV3 cells by autophagy. In this study we examine the effect of HuArgI (Co)-PEG5000 on ovarian cancer cell migration and we dissect the mechanism involved. Wound healing assays, 2D random cell migration assays and cell adhesion analysis indicate that arginine deprivation decreases SKOV3 cell migration and adhesion. This effect was mimicked when autophagy was induced through rapamycin and reversed with the autophagy inhibitor chloroquine when autophagy was inhibited. This proved that arginine deprivation leads to the inhibition of cancer cell migration through autophagy, in addition to cell death. In addition, we were able to establish through pull-down assays and reversal experiments, that arginine deprivation-mediated autophagy inhibits cell migration through a direct inhibition of RhoA, member of the Rho family of GTPases. In conclusion, here we identify, for the first time, an autophagy-mediated inhibition of RhoA that plays an important role in regulating ovarian cancer cells motility and adhesion in response to arginine depletion.

Activation of autophagy following [HuArgI (Co)-PEG5000]-induced arginine deprivation mediates cell death in colon cancer cells.

Deregulating cellular energetics by reprogramming metabolic pathways, including arginine metabolism, is critical for cancer cell onset and survival. Drugs that target the specific metabolic requirements of cancer cells have emerged as promising targeted cancer therapeutics. In this study, we investigate the therapeutic potential of targeting colon cancer cells using arginine deprivation induced by a pegylated cobalt-substituted recombinant human Arginase I [HuArgI (Co)-PEG5000]. Four colon cancer cell lines were tested for their sensitivity to [HuArgI (Co)-PEG5000] as well as for their mechanism of cell death following arginine deprivation. All four cell lines were sensitive to arginine deprivation induced by [HuArgI (Co)-PEG5000]. All cells expressed ASS1 and were rescued from arginine deprivation-induced cytotoxicity by the addition of excess L-citrulline, indicating they are partially auxotrophic for arginine. Mechanistically, cells treated with [HuArgI (Co)-PEG5000] were negative for AnnexinV and lacked caspase activation. Further investigation revealed that arginine deprivation leads to a marked and prolonged activation of autophagy in both Caco-2 and T84 cell lines. Finally, we show that [HuArgI (Co)-PEG5000] causes cell death by sustained activation of autophagy as evidenced by the decrease in cell cytotoxicity upon treatment with chloroquine, an autophagy inhibitor. Altogether, these data demonstrate that colon cancer cells are partially auxotrophic for arginine and sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation. They also show that the activation of autophagy does not play protective roles but rather, induces cytotoxicity and leads to cell death.

The Role of Rho GTPases in VEGF Signaling in Cancer Cells.

Vascular endothelial growth factors (VEGFs) consist of five molecules (VEGFA through D as well as placental growth factor) which are crucial for regulating key cellular and tissue functions. The role of VEGF and its intracellular signaling and downstream molecular pathways have been thoroughly studied. Activation of VEGF signal transduction can be initiated by the molecules' binding to two classes of transmembrane receptors: (1) the VEGF tyrosine kinase receptors (VEGF receptors 1 through 3) and (2) the neuropilins (NRP1 and 2). The involvement of Rho GTPases in modulating VEGFA signaling in both cancer cells and endothelial cells has also been well established. Additionally, different isoforms of Rho GTPases, namely, RhoA, RhoC, and RhoG, have been shown to regulate VEGF expression as well as blood vessel formation. This review article will explore how Rho GTPases modulate VEGF signaling and the consequences of such interaction on cancer progression.

Hypoxia and EGF Stimulation Regulate VEGF Expression in Human Glioblastoma Multiforme (GBM) Cells by Differential Regulation of the PI3K/Rho-GTPase and MAPK Pathways.

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.

Thymoquinone enhances the anticancer activity of doxorubicin against adult T-cell leukemia in vitro and in vivo through ROS-dependent mechanisms.

AIMS: Adult T-cell leukemia (ATL) is a mature T-cell neoplasm associated with human T-cell lymphotropic virus (HTLV-1) infection. Major limitations in Doxorubicin (Dox) chemotherapy are tumor resistance and severe drug complications. Here, we combined Thymoquinone (TQ) with low concentrations of Dox and determined anticancer effects against ATL in cell culture and animal model. MAIN METHODS: HTLV-1 positive (HuT-102) and HTLV-1 negative (Jurkat) CD4+ malignant T-cell lines were treated with TQ, Dox and combinations. Viability and cell cycle effects were determined by MTT assay and flow cytometry analysis, respectively. Combination effects on mitochondrial membrane potential and generation of reactive oxygen species (ROS) were assessed. Expression levels of key cell death proteins were investigated by western blotting. A mouse xenograft model of ATL in NOD/SCID was used for testing drug effects and tumor tissues were stained for Ki67 and TUNEL. KEY FINDINGS: TQ and Dox caused greater inhibition of cell viability and increased sub-G1 cells in both cell lines compared to Dox or TQ alone. The combination induced apoptosis by increasing ROS and causing disruption of mitochondrial membrane potential. Pretreatment with N-acetyl cysteine (NAC) or pan caspase inhibitor significantly inhibited the apoptotic response suggesting that cell death is ROS- and caspase-dependent. TQ and Dox combination reduced tumor volume in NOD/SCID mice more significantly than single treatments through enhanced apoptosis without affecting the survival of mice. SIGNIFICANCE: Our combination model offers the possibility to use up to twofold lower doses of Dox against ATL while exhibiting the same cancer inhibitory effects.

Metformin Treatment Inhibits Motility and Invasion of Glioblastoma Cancer Cells.

Glioblastoma multiforme (GBM) is one of the most common and deadliest cancers of the central nervous system (CNS). GBMs high ability to infiltrate healthy brain tissues makes it difficult to remove surgically and account for its fatal outcomes. To improve the chances of survival, it is critical to screen for GBM-targeted anticancer agents with anti-invasive and antimigratory potential. Metformin, a commonly used drug for the treatment of diabetes, has recently emerged as a promising anticancer molecule. This prompted us, to investigate the anticancer potential of metformin against GBMs, specifically its effects on cell motility and invasion. The results show a significant decrease in the survival of SF268 cancer cells in response to treatment with metformin. Furthermore, metformin's efficiency in inhibiting 2D cell motility and cell invasion in addition to increasing cellular adhesion was also demonstrated in SF268 and U87 cells. Finally, AKT inactivation by downregulation of the phosphorylation level upon metformin treatment was also evidenced. In conclusion, this study provides insights into the anti-invasive antimetastatic potential of metformin as well as its underlying mechanism of action.

Thymoquinone: fifty years of success in the battle against cancer models.

Thymoquinone (TQ), the main active constituent of black seed essential oil, exhibits promising effects against inflammatory diseases and cancer. TQ, modulates signaling pathways that are key to cancer progression, and enhances the anticancer potential of clinical drugs while reducing their toxic side effects. Considering that TQ was isolated 50 years ago, this review focuses on TQ's chemical and pharmacological properties and the latest advances in TQ analog design and nanoformulation. We discuss our current state of knowledge of TQ's adjuvant potential and in vivo antitumor activity and highlight its ability to modulate the hallmarks of cancer.

Synergistic anticancer activities of the plant-derived sesquiterpene lactones salograviolide A and iso-seco-tanapartholide.

We have previously shown that the two sesquiterpene lactones, salograviolide A (Sal A) and iso-seco-tanapartholide (TNP), isolated from the Middle Eastern indigenous plants Centaurea ainetensis and Achillea falcata, respectively, possess selective antitumor properties. Here, we aimed to assess the anticancer effects of the separate compounds and their combination, study their potential to generate reactive oxygen species (ROS), and investigate their underlying antitumor mechanisms in human colon cancer cell lines. Cells were treated with Sal A and TNP alone or in combination, and cell viability, cell cycle profile, apoptosis, ROS generation and changes in protein expression were monitored. Sal A and TNP in combination caused 80% decrease in HCT-116 and DLD-1 cell viability versus only 25% reduction when the drugs were used separately. The antitumor mechanism involved triggering ROS-dependent apoptosis as well as disruption of the mitochondrial membrane potential. Further studies showed that apoptosis by the Sal A and TNP combination was caspase-independent and that ERK, JNK and p38 of the serine/threonine MAPKs signaling pathway were involved in the cell death mechanism. Taken together, our data suggest that the combination of Sal A and TNP may be of therapeutic interest against colon cancer.