Angiopoietin-2 mediates blood-brain barrier impairment and colonization of triple-negative breast cancer cells in brain.

Although the incidence of breast cancer metastasis (BCM) in brain has increased significantly in triple-negative breast cancer (TNBC), the mechanisms remain elusive. Using in vivo mouse models for BCM in brain, we observed that TNBC cells crossed the blood-brain barrier (BBB), lodged in the brain microvasculature and remained adjacent to brain microvascular endothelial cells (BMECs). Breaching of the BBB in vivo by TNBCs resulted in increased BBB permeability and changes in ZO-1 and claudin-5 tight junction (TJ) protein structures. Angiopoietin-2 expression was elevated in BMECs and was correlated with BBB disruption. Secreted Ang-2 impaired TJ structures and increased BBB permeability. Treatment of mice with the neutralizing Ang-2 peptibody trebananib prevented changes in the BBB integrity and BMEC destabilization, resulting in inhibition of TNBC colonization in brain. Thus, Ang-2 is involved in initial steps of brain metastasis cascade, and inhibitors for Ang-2 may serve as potential therapeutics for brain metastasis.

The cannabinoid CB2 receptor agonist AM1241 enhances neurogenesis in GFAP/Gp120 transgenic mice displaying deficits in neurogenesis.

BACKGROUND AND PURPOSE: HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo. HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems, we examined the protective effects of the CB2 receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH: We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS: AM1241 inhibited in vitro Gp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis, as indicated by reduction in BrdU⁺ cells and doublecortin⁺ (DCX⁺) cells, and a decrease in cells with proliferating cell nuclear antigen (PCNA), administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts, neuronal cells, BrdU⁺ cells and PCNA⁺ cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241, compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS: The CB2 receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus, CB2 receptor agonists may act as neuroprotective agents, restoring impaired neurogenesis in patients with HAD.

The proinflammatory peptide substance P promotes blood-brain barrier breaching by breast cancer cells through changes in microvascular endothelial cell tight junctions.

Neuropeptide substance P (SP) has been implicated in inflammation, pain, depression and breast cancer cell (BCC) growth. Here, we examined the role of SP in trafficking of BCCs (human MDA-MB-231 and MDA-MB-231BrM2 cells) across the blood-brain barrier (BBB) and brain microvascular endothelial cells (BMECs) using in vitro and in vivo models. SP was secreted from BCCs and mediated adhesion and transmigration of BCCs across human BMECs (HBMECs) in vitro. SP induced activation of HBMECs, leading to secretion of Tumor Necrosis Factor alpha (TNF-α) and angiopoietin-2 (Ang-2) from HBMECs, resulting in changes in localization and distribution of tight junction (TJ) ZO-1 (tight junction protein zonula occludins-1) and claudin-5 structures as well as increased permeability of HBMECs. Using spontaneous breast cancer metastasis mouse model (syngeneic) of GFP-4T1-BrM5 mammary tumor cells administered into mammary fat pads of Balb/c mice, SP inhibitor spantide III inhibited in vivo changes in permeability of the BBB and BMEC-TJs ZO-1 and claudin-5 structures as well as decreased tumor cell colonization in brain. Thus, SP secreted from BCCs induces transmigration of BCCs across the BBB, leading to activation of BMECs and secretion of TNF-α and Ang-2, resulting in BBB impairment and colonization of tumor cells in brain. Therefore, therapies based on SP inhibition in combination with other therapies may prevent breaching of the BBB by BCCs and their colonization in brain.

Regulation of hematopoietic stem cell trafficking and mobilization by the endocannabinoid system.

The cannabinoid receptors CB(1) and CB(2) are seven-transmembrane Gαi protein-coupled receptors and are expressed in certain mature hematopoietic cells. We recently showed that these receptors are expressed in murine and human hematopoietic stem cells (HSCs) and that CB(2) agonists induced chemotaxis, enhanced colony formation of marrow cells, as well as caused in vivo mobilization of murine HSCs with short- and long-term repopulating abilities. Based on these observations, we have further explored the role of CB(2) and its agonist AM1241 on hematopoietic recovery following sublethal irradiation in mice. Cannabinoid receptor 2 knockout mice (Cnr2(-/-) deficient mice) exhibited impaired recovery following sublethal irradiation as compared with irradiated wild-type (WT) mice, as determined by low colony-forming units and low peripheral blood counts. WT mice treated with CB(2) agonist AM1241 following sublethal irradiation demonstrated accelerated marrow recovery and increased total marrow cells (approximately twofold) and total lineage- c-kit(+) cells (approximately sevenfold) as well as enhanced HSC survival as compared with vehicle control-treated mice. When the CB(2) agonist AM1241 was administered to WT mice 12 days before their sublethal irradiation, analysis of hematopoiesis in these mice showed decreased apoptosis of HSCs, enhanced survival of HSCs, as well as increase in total marrow cells and c-kit+ cells in the marrow. Thus, CB(2) agonist AM1241 promoted recovery after sublethal irradiation by inhibiting apoptosis of HSCs and promoting survival, as well as enhancing the number of HSCs entering the cell cycle.

Cannabinoid receptor 2 and its agonists mediate hematopoiesis and hematopoietic stem and progenitor cell mobilization.

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB1 and CB2) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB2 receptors are expressed in human and murine HSPCs. On ligand stimulation with CB2 agonists, CB2 receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB2 agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB2 antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB2/CB2 agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB2 agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation.

Endocannabinoids are expressed in bone marrow stromal niches and play a role in interactions of hematopoietic stem and progenitor cells with the bone marrow microenvironment.

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.

Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

BACKGROUND: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. CONCLUSIONS/SIGNIFICANCE: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

Csk homologous kinase inhibits CXCL12-CXCR4 signaling in neuroblastoma.

Neuroblastoma is the second most common pediatric malignancy. The clinical course of this disease ranges from spontaneous regression and good survival to highly malignant therapy-resistant tumors. There is a continuous need for genetic and biologic markers for the diverse clinical phenotypes observed in neuroblastoma patients. One of the known markers in neuroblastoma is expression of the CXCR4 chemokine receptor. CXCR4 expression correlates with high-stage disease, and the autocrine stimulation of CXCR4 by its ligand (CXCL12) was shown to be necessary for the survival of some neuroblastoma cells in vitro. However, the mechanisms responsible for activation of the CXCL12-CXCR4 autocrine pathway in neuroblastoma remain uncertain. Our previous findings suggest that Csk homologous kinase (CHK) is a physiological inhibitor of CXCR4 expression. Since CHK is highly expressed in neurons, we evaluated changes in CHK expression in human neuroblastoma. CHK protein expression was below detectable levels based on Western blot analyses in 13 out of 16 human neuroblastoma cell lines and in 6 out of 16 primary neuroblastoma tissues. When CHK expression was restored in IMR32 neuroblastoma cells by retrovirus-mediated cDNA transfer, diminished CXCR4 mRNA and protein levels were observed, as assessed by RT-PCR and flow cytometry analyses, respectively. Furthermore, exogenous expression of CHK markedly suppressed the mRNA levels and secretion of the CXCL12 chemokine from IMR32 cells as well as inhibited the growth rate of these cells. Taken together, our data strongly suggest that CHK is capable of inhibiting the CXCL12-CXCR4 pathway in neuroblastoma.

Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells.

BACKGROUND: Characterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES) cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation. METHODS AND FINDINGS: The cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are members of the G-protein coupled receptor (GPCR) family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively) induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Delta(9)-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists. CONCLUSIONS: This work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell survival and hematopoietic differentiation.

Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1.

BACKGROUND: While vascular endothelial growth factor (VEGF) expression in breast tumors has been correlated with a poor outcome in the pathogenesis of breast cancer, the expression, localization, and function of VEGF receptors VEGFR1 (also known as FLT1) and VEGFR2 (also known as KDR or FLK1), as well as neuropilin 1 (NRP1), in breast cancer are controversial. METHODS AND FINDINGS: We investigated the expression and function of VEGF and VEGF receptors in breast cancer cells. We observed that VEGFR1 expression was abundant, VEGFR2 expression was low, and NRP1 expression was variable. MDA-MB-231 and MCF-7 breast cancer cells, transfected with antisense VEGF cDNA or with siVEGF (VEGF-targeted small interfering RNA), showed a significant reduction in VEGF expression and increased apoptosis as compared to the control cells. Additionally, specifically targeted knockdown of VEGFR1 expression by siRNA (siVEGFR1) significantly decreased the survival of breast cancer cells through down-regulation of protein kinase B (AKT) phosphorylation, while targeted knockdown of VEGFR2 or NRP1 expression had no effect on the survival of these cancer cells. Since a VEGFR1-specific ligand, placenta growth factor (PGF), did not, as expected, inhibit the breast cancer cell apoptosis induced by siVEGF, and since VEGFR1 antibody also had no effects on the survival of these cells, we examined VEGFR1 localization. VEGFR1 was predominantly expressed internally in MDA-MB-231 and MCF-7 breast cancer cells. Specifically, VEGFR1 was found to be colocalized with lamin A/C and was expressed mainly in the nuclear envelope in breast cancer cell lines and primary breast cancer tumors. Breast cancer cells treated with siVEGFR1 showed significantly decreased VEGFR1 expression levels and a lack of VEGFR1 expression in the nuclear envelope. CONCLUSIONS: This study provides, to our knowledge for the first time, evidence of a unique survival system in breast cancer cells by which VEGF can act as an internal autocrine (intracrine) survival factor through its binding to VEGFR1. These results may lead to an improved strategy for tumor therapy based on the inhibition of angiogenesis.

Process elongation of oligodendrocytes is promoted by the Kelch-related protein MRP2/KLHL1.

Oligodendrocytes (OLGs) are generated by progenitor cells that are committed to differentiating into myelin-forming cells of the central nervous system. Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by OLGs. Here, we have characterized a new member of the Kelch-related protein family termed MRP2 (for Mayven-related protein 2) that is specifically expressed in brain. MRP2/KLHL1 is expressed in oligodendrocyte precursors and mature OLGs, and its expression is up-regulated during OLG differentiation. MRP2/KLHL1 expression was abundant during the specific stages of oligodendrocyte development, as identified by A2B5-, O4-, and O1-specific oligodendrocyte markers. MRP2/KLHL1 was localized in the cytoplasm and along the cell processes. Moreover, a direct endogenous association of MRP2/KLHL1 with actin was observed, which was significantly increased in differentiated OLGs compared with undifferentiated OLGs. Overexpression of MRP2/KLHL1 resulted in a significant increase in the process extension of rat OLGs, whereas MRP2/KLHL1 antisense reduced the process length of primary rat OLGs. Furthermore, murine OLGs isolated from MRP2/KLHL1 transgenic mice showed a significant increase in the process extension of OLGs compared with control wild-type murine OLGs. These studies provide insights into the role of MRP2/KLHL1, through its interaction with actin, in the process elongation of OLGs.

CHK negatively regulates Lyn kinase and suppresses pancreatic cancer cell invasion.

Among the most important signaling pathways operating in pancreatic cancer cells are those resulting from mutations in the Ras oncogene or from overexpression of ErbB-2 and associated Src-family kinases. In this study, we aimed to characterize CHK expression and function in pancreatic cancer. Our data demonstrated CHK expression in human pancreatic cancer tissues, and also showed that CHK associated with ErbB-2 via its SH2 domain in human PANC-1 pancreatic cancer cells. PANC-1 cells were found to express both Src kinase and Lyn kinase, although the expression of Lyn kinase was more abundant. Furthermore, CHK downregulated Lyn kinase activity and significantly inhibited the in vitro growth and invasion of PANC-1 cells upon EGF stimulation. These results indicate that CHK is a negative regulator of ErbB-2 and Lyn kinase signaling in pancreatic cancer cells.

Role of SRC kinases in Neu-induced tumorigenesis: challenging the paradigm using Csk homologous kinase transgenic mice.

Amplification of the HER-2/neu (ErbB2) gene is observed in approximately 30% of human breast cancers, correlating with a poor clinical prognosis. Src kinases are also involved in the etiology of breast cancer, and their activation was suggested to be necessary for Neu-induced oncogenesis. To address whether Src activity is essential for Neu-mediated tumorigenesis, we used a physiologic inhibitor of Src kinase activity, the Csk homologous kinase (CHK), expressed as a mammary tissue-specific transgene. Our data, using a physiologic inhibitor of Src activity (CHK), showed that blocking of Neu-induced Src activity without altering Src expression levels had no significant effects on Neu-mediated mammary tumorigenesis in vivo. This contradicts the current paradigm that activation of Src kinases is essential for Neu-induced oncogenesis. This study is the first to distinguish between the kinase-dependent and kinase-independent actions of Src and shows that its kinase-dependent properties are not requisite for Neu-induced tumorigenesis.

Csk homologous kinase (CHK), unlike Csk, enhances MAPK activation via Ras-mediated signaling in a Src-independent manner.

Substantial evidence exists supporting the notion that Csk and CHK, two negative regulatory kinases of the Src tyrosine kinase family, play distinct roles during development of the nervous system. One of the differences relies on the effects of both kinases on the MAPK transduction pathway. Specifically, CHK was shown to enhance MAPK signaling, while the role of Csk was unclear. In this work, we compared the effect of CHK versus Csk on MAPK signaling and elucidated the signaling pathway mediated by CHK leading to the activation of Erk1/2. Exogenous expression of wild-type CHK, but not Csk or a dead-kinase mutant of CHK, resulted in enhanced Erk1/2 phosphorylation in PC12 cells. CHK inhibited Src activity following stimulation of the cells with NGF. However, stimulation of Erk1/2 activation by CHK was independent of the NGF stimulation or the inhibition of Src kinase by CHK. CHK induced a complex formation between SHP-2 and Grb2, subsequently leading to the increased activity of Ras as well as Erk1/2 activation via the Raf/MEK1/2 pathway. Down-regulation of the expression of endogenous CHK by RNAi in PC12 cells led to a significant decrease in MAPK activation following NGF stimulation. Stimulation of CHK-overexpressing PC12 cells with EGF induced neurite outgrowth in the majority of cells. Taken together, this study describes for the first time the Src-independent actions of CHK and provides novel insights into CHK function in neural cells.

Mayven induces c-Jun expression and cyclin D1 activation in breast cancer cells.

Mayven is a member of the kelch-related superfamily of proteins, characterized by a series of 'kelch' repeats at their carboxyl terminus and a BTB/POZ domain at their NH2-terminus. Little is known about the role of Mayven in cancer. Here, we report that Mayven expression was abundant and diffuse in primary human epithelial breast tumor cells as compared to normal breast epithelial cells, where Mayven was detected in the normal breast layer of the mammary ducts. Overexpression of Mayven resulted in an induction of c-Jun protein levels, as well as increased AP-1 (activating protein 1) transcriptional activity in MCF-7 and T47D breast cancer cells through its BTB/POZ domain. Furthermore, Mayven activated c-Jun N-terminal kinase in breast cancer cells. Mayven, through its BTB/POZ domain, induced cyclin D1 expression and cyclin D1 promoter activity and promoted cell cycle progression from the G1 to S phase. MCF-7 cells transduced with the recombinant retroviral sense Mayven (pMIG-W-Mayven) showed significant induction of c-Jun and cyclin D1 mRNA expression and activities as compared to the retroviral vector alone, while MCF-7 cells transduced by the recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) demonstrated a significant decrease in c-Jun and cyclin D1 expression and activities. Given the crucial functions of cyclin D1 and AP-1 signaling in oncogenesis, our results strongly suggest that overexpression of Mayven may promote tumor growth through c-Jun and cyclin D1.

Differential expression of Csk homologous kinase (CHK) in normal brain and brain tumors.

BACKGROUND: Substantial evidence supports the suggestion that carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK), two negative regulatory kinases of the Src tyrosine kinase family, play distinct roles during development of the central nervous system (CNS). METHODS: To further examine the individual roles of CHK and Csk in the CNS, the authors compared the expression patterns of Csk and CHK during differentiation of primary hippocampal neurons. RESULTS: Opposite patterns of expression were observed for CHK and Csk. The authors observed an increase in CHK expression and a decline in Csk expression during differentiation of primary hippocampal neurons, but no difference in Src expression levels. CHK was also expressed in astrocytes and oligodendrocytes in a differentiation-dependent manner. However, CHK expression was not detected in brain endothelial cells, transformed neuroblastoma or astrocytoma cell lines, nor in primary human neuroblastoma and glioblastoma tumors (10 cases), indicating that loss of CHK expression is associated with human brain tumors. Treatment with antisense CHK oligodeoxynucleotides blocked the neuronal process formation of primary hippocampal neurons and neuronal differentiation of PC12 cells. CHK overexpression in primary hippocampal neurons using recombinant adenovirus infection resulted both in increased CHK kinase activity and changes in neuronal morphology. In addition, CHK overexpression in neuroblastoma and astrocytoma cells inhibited their growth and proliferation. CONCLUSIONS: These findings strongly suggested that CHK may play a role in tumorigenesis and in the terminal differentiation of neurons within the CNS.

Role of the aging vasculature and Erb B-2 signaling in epidermal growth factor-dependent intravasion of breast carcinoma cells.

BACKGROUND: The risks for developing breast carcinoma and dying from the disease increase with age. Mortality from breast carcinoma usually is due to metastatic disease. Metastatic cells are able to invade into the vascular tissue in a growth factor-dependent manner. Because breast carcinoma mortality increases with age, examination of breast carcinoma interactions with young and aged endothelial cells is essential. METHODS: We studied a series of breast epithelial cells (HMT-3522 cells) that exhibited either noninvasive characteristics (S-1 cells) or epidermal growth factor (EGF)-dependent invasive characteristics (T4-2 cells). RESULTS: Increased invasion of HMT-3522 cells was observed across an aged rat brain microvascular endothelial cell (BMEC) monolayer that was isolated from aged rats (24 months) compared with young rats (age 1 month). This increased invasion was inhibited by the specific EGF receptor inhibitor, AG1478, and by the Erb B-2-specific inhibitor, AG825. To analyze further the contribution of Erb B-2 to the EGF-dependent invasion of HMT-3522 cells, T4-2 cells were treated with the Erb B-2-specific therapeutic antibody trastuzumab and with the specific inhibitor AG825 and were then assayed for invasion. Both inhibitors led to a significant decrease in EGF-dependent invasion. Erb B-2 expression was found to be elevated in T4-2 cells ( approximately 5-fold higher) compared with S-1 cells. However, treatment of T4-2 cells with the specific Erb B-2 inhibitor, AG825, failed to inhibit EGF-mediated signaling to phosphatidylinositol 3-kinase or extracellular-regulated kinases 1 and 2. CONCLUSIONS: The current study findings indicate that aging of endothelium may contribute to the invasive phenotype of breast carcinoma cells and that "cross-talk" between Erb B-2 and EGF receptor is required for the intravasion of these cells into the surrounding vasculature.

Coupling of RAFTK/Pyk2 kinase with c-Abl and their role in the migration of breast cancer cells.

Mitogen-induced changes in the actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several proteins in focal adhesions. In this study, we have investigated the role of RAFTK (also termed Pyk2/CAK-beta), a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Maximal phosphorylation of the proteins participating in this complex occurred within 2 h of HRG stimulation. Analyses of the members of the HRG-stimulated complex revealed that RAFTK associated with p190 RhoGAP (p190), RasGAP, c-Abl as well as with the focal adhesion molecules p130cas and paxillin. c-Abl was found to be associated with RAFTK through the region of RAFTK containing amino acids 419-1009. Site-directed mutagenesis of Y881 aa within the RAFTK sequence abolished the binding of RAFTK to c-Abl, indicating that the tyrosine residue 881 of RAFTK is the c-Abl binding site within the RAFTK molecule. Overexpression of wild-type RAFTK significantly enhanced breast cancer cell invasion, while overexpression of the mutants Tyr402 or Tyr881 of RAFTK inhibited this migration. Therefore, RAFTK serves as a mediator and an integration point between focal adhesion molecules in HRG-mediated signaling in T47D breast cancer cells.

The invasive phenotype in HMT-3522 cells requires increased EGF receptor signaling through both PI 3-kinase and ERK 1,2 pathways.

We studied the invasion of HMT-3522 breast epithelial cells in response to epidermal growth factor (EGF), and the associated signaling pathways. HMT-3522 T4-2 cells were shown to invade Matrigel-coated Transwell membranes in response to EGF while HMT-3522 S-1 cells failed to invade when treated with EGF. Studies utilizing specific molecular inhibitors showed the importance of beta1 integrin, phosphatidylinositol 3 kinase (PI 3-kinase), p38, extracellular regulated kinase 1, 2 (Erk 1,2) MAP kinases, and metalloproteinases in invasion and motility. T4-2 cell invasion was shown to be time-dependent and also gene transcription-dependent as shown by inhibition with Actinomycin D. T4-2 cells exhibited an increased activation of MAP kinases Erk 1,2 (2-fold), EGF receptor (3-fold), and PI 3-kinase (3- to 4-fold) when compared to the S-1 cells. In response to EGF, T4-2 cells showed a 5-fold greater secretion of matrix metalloproteinase-9 (MMP-9) as compared to S-1 cells, and this increase was largely dependent on the activity of PI 3-kinase. These findings indicate that expression of the invasive phenotype in these breast epithelial cells requires increased EGF receptor signaling, involving both PI 3-kinase and Erk 1,2 activities, which leads to multiple downstream effects, including enhanced secretion of MMP-9 and transcription of invasion-related genes.

Overexpression of the Csk homologous kinase facilitates phosphorylation of Akt/PKB in MCF-7 cells.

The serine/threonine kinase Akt has recently been the focus of intense research. Akt activation requires the phosphorylation of both Thr-308 and Ser-473. Src kinase was shown to induce activation of Akt, while Lyn kinase seems to inhibit this activation. In the present study, we investigated the effect of overexpressing the Csk homologous kinase (CHK), an inhibitor of Src-family kinases, on the phosphorylation of Akt induced by two different factors: heregulin or cisplatin. We used MCF-7 cells stably overexpressing the wild-type CHK [CHK(wt)] or dead-kinase CHK [CHK(dk)]. We observed that in MCF-7 CHK(wt) cells Lyn kinase activity was more profoundly inhibited than Src kinase activity. When the cells were stimulated with heregulin or cisplatin, Akt phosphorylation occurred more rapidly in MCF-7 CHK(wt) cells in comparison to the other clones used. Interestingly, MCF-7 CHK(wt) cells in vitro were markedly more resistant to cisplatin than the other clones used in the experiments, and surprisingly chemical inhibition of Akt phosphorylation did not influence this resistance. In summary, our results show facilitation of Akt phosphorylation by the overexpression of CHK, and provide new insight into the putative role of CHK in human cancer.