Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression.

The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression.

Germ line variants of human N-methylpurine DNA glycosylase show impaired DNA repair activity and facilitate 1,N6-ethenoadenine-induced mutations.

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.

Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered Kras(G12D) and Kras(G12D):Trp53(R172H) mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS-mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

ROS-mediated regulation of CXCR4 in cancer.

Oxidative stress and the accumulation of reactive oxygen species (ROS) play a role in cancer cells developing an advanced, phenotypic signature that associates with metastasis and progression. Increased ROS concentrations are involved in promoting cancer development and metastasis by inducing expression of oncogenes, suppressing activity of anti-survival molecules and by activating various cell survival and proliferation signaling pathways. Oxidative stress is higher in the epithelium of cancer patients than patients without the disease, and antioxidant trials are currently being explored as a therapeutic option. However, studies have shown that ROS increases expression of CXCR4 in cancer and immune cells. CXCR4 expression in tumors strongly correlates to metastasis and poor prognosis. Herein, we discuss an emerging relationship between ROS and CXCR4 in cancer cells.

Cysteine (C)-X-C Receptor 4 Regulates NADPH Oxidase-2 During Oxidative Stress in Prostate Cancer Cells.

Reactive oxygen species (ROS) are implicated in many human diseases, including cancer. We have previously demonstrated that ROS increased the expression and activity of the chemokine receptor, CXCR4, which enhanced metastatic functions in prostate cancer cells. Studies have also revealed that CXCR4 and its ligand, SDF-1α, promoted ROS accumulation; however the source of ROS was not investigated. Recent evidence suggested that ROS accumulation in prostate cancer cell lines was contributed by the NADPH oxidase (NOX) family of enzymes. Herein, we sought to determine whether the CXCR4/SDF-1α signaling axis mediates ROS production through NOX in prostate cancer. We observed an increase in intracellular ROS generation in prostate cancer cells upon SDF-1α stimulation compared to untreated samples. Conversely, lower levels of ROS were detected in cells treated with AMD3100 (CXCR4 antagonist) or the ROS scavenger, N-acetyl-cysteine (NAC). Markedly reduced levels of ROS were observed in cells treated with apocynin (NOX inhibitor) compared to rotenone (mitochondrial complex I inhibitor)-treated cells. Specifically, we determined that NOX2 responded to, and was regulated by, the SDF-1α/CXCR4 signaling axis. Moreover, chemical inhibition of the ERK1/2 and PI3K pathways revealed that PI3K/AKT signaling participated in CXCR4-mediated NOX activity, and that these collective signaling events resulted in enhanced cell movement towards a chemoattractant. Finally, NOX2 may be a potential therapeutic target, as Oncomine microarray database analysis of normal prostate, benign prostatic hyperplasia (BPH) and prostatic intraepithelial neoplasia (PIN) tissue samples determined a correlation between NOX2 expression and prostate cancer. Taken together, these results suggest that CXCR4/SDF-1α-mediated ROS production through NOX2 enzymes may be an emerging concept by which chemokine signaling progresses tumorigenesis.

Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinß1 and Transportinß1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

ROS-mediated activation of AKT induces apoptosis via pVHL in prostate cancer cells.

Reactive oxygen species (ROS) play a central role in oxidative stress, which leads to the onset of diseases, such as cancer. Furthermore, ROS contributes to the delicate balance between tumor cell survival and death. However, the mechanisms by which tumor cells decide to elicit survival or death signals during oxidative stress are not completely understood. We have previously reported that ROS enhanced tumorigenic functions in prostate cancer cells, such as transendothelial migration and invasion, which depended on CXCR4 and AKT signaling. Here, we report a novel mechanism by which ROS facilitated cell death through activation of AKT. We initially observed that ROS enhanced the expression of phosphorylated AKT (p-AKT) in 22Rv1 human prostate cancer cells. The tumor suppressor PTEN, a negative regulator of AKT signaling, was rendered catalytically inactive through oxidation by ROS, although the expression levels remained consistent. Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased, and contains a target site for p-AKT phosphorylation. pVHL and p-AKT associated in vitro, and knockdown of pVHL rescued HIF1α expression and the cells from apoptosis. Collectively, our study suggests that in the context of oxidative stress, p-AKT facilitated apoptosis by inducing pVHL function.

The phytoalexin camalexin mediates cytotoxicity towards aggressive prostate cancer cells via reactive oxygen species.

Camalexin is a phytoalexin that accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects in breast cancer and leukemia. We studied the cytotoxic effects of camalexin treatment on prostate cancer cell lines and whether this was mediated by reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an epithelial to mesenchymal transition (EMT) model and displays increased cell migration and tumorigenicity. We confirmed previous studies showing that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. Camalexin increased ROS, decreased cell proliferation, and increased apoptosis more significantly in C4-2 and ARCaP-Snail cells as compared to LNCaP and ARCaP-Neo cells, respectively, while normal prostate epithelial cells (PrEC) were unaffected. Increased caspase-3/7 activity and increased cleaved PARP protein shown by Western blot analysis was suggestive of increased apoptosis. The ROS scavenger N-acetyl cysteine (NAC) antagonized the effects of camalexin, whereas the addition of exogenous hydrogen peroxide potentiated the effects of camalexin, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin is more potent in aggressive prostate cancer cells that express high ROS levels, and this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer.

PTEN regulation of ERK1/2 signaling in cancer.

Since its discovery, the tumor suppressor phosphatase and tensin homolog (PTEN) has become a molecule with a wide spectrum of functions, which is typically meditated through its lipid phosphatase activity; however, PTEN also functions in a phosphatase-independent manner. It is well established that PTEN regulates several signaling pathways, such as phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), janus kinase (JAK)/signal transducers and activators of transcription (STAT), focal adhesion kinase (FAK), and more recent, extracellular signal-regulated kinase (ERK)1/2, where activation of these pathways typically leads to cancer development and progression. In regard to most of these pathways, the underlining molecular mechanism of PTEN-mediated regulation is well established, but not so much for the ERK1/2 pathway. Indeed, accumulating evidence has shown an inverse correlation between PTEN expression and ERK1/2 in several malignancies. However, the detailed mechanism by which PTEN regulates ERK1/2 is poorly understood. In this review, we discuss the role of PTEN in regulating ERK1/2 by directly targeting shc/Raf/MEK and PI3K/AKT cascades, and a putative cross-talk between the two.

ROS enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells.

Inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is heavily implicated in the tumorigenesis of prostate cancer. Conversely, the upregulation of the chemokine (CXC) receptor 4 (CXCR4) is associated with prostate cancer progression and metastasis. Studies have shown that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells. Loss of PTEN function is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Herein, we show that ROS accumulation permitted CXCR4-mediated functions through PTEN catalytic inactivation. ROS increased p-AKT and CXCR4 expression, which were abrogated by a ROS scavenger in prostate cancer cells. ROS mediated PTEN inactivation but did not affect expression, yet enhanced cell migration and invasion in a CXCR4-dependent manner. Collectively, our studies add to the body of knowledge on the regulatory role of PTEN in CXCR4-mediated cancer progression, and hopefully, will contribute to the development of therapies that target the tumor microenvironment, which have great potential for the better management of a metastatic disease.

Loss of PTEN permits CXCR4-mediated tumorigenesis through ERK1/2 in prostate cancer cells.

Loss of PTEN is frequently observed in androgen-independent prostate cancer, resulting in the deregulation of metastatic events. SDF1α activation of CXCR4 induces signaling pathways that have been implicated in prostate metastasis and progression to an advanced disease. The pathways of CXCR4 and PTEN converge, leading to the promotion and regulation of tumorigenesis, respectively. However, loss of PTEN may permit CXCR4 to progress prostate cancer to an advanced disease. In the present study, we investigated the involvement of PTEN in CXCR4-mediated tumorigenesis. When screening advanced metastatic prostate cancer cell lines for PTEN, we observed a loss of expression in PC3 and LNCaP cells whereas Du145 expressed wild-type PTEN. All three cell lines were positive for surface expression of CXCR4. Reconsitution of PTEN induced a mesenchymal to epithelial like morphologic change and inhibited CXCR4-mediated migration and proliferation in PC3 cells. Downregulation of PTEN by siRNA enhanced the CXCR4-mediated migratory behavior of Du145 cells. By Western blot analysis, we observed that PTEN inhibited basal AKT phosphorylation but not ERK1/2 phosphorylation in PTEN-expressing cells. Upon CXCR4 stimulation, PTEN inhibited ERK1/2 phosphorylation but not phosphorylation of AKT. The CXCR4-mediated migration of PC3 cells was through the ERK1/2 pathway, as confirmed by chemical inhibitors. On the basis of these studies, we suggest that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells through the ERK1/2 pathway. Antagonizing CXCR4 and downstream signaling cascades may provide an efficient approach for treating patients with advanced prostate cancer when hormone therapy fails to the stop the growth and containment of tumors.