GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy.Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.     

The EGFR-GEP100-Arf6 pathway in breast cancer

Arf6 and its effector AMAP1 are overexpressed in malignant breast cancer cells, and are involved in their invasion and metastasis. We recently revealed that GEP100, a guanine nucleotide exchanging factor, is responsible for the activation of Arf6 which induces invasion and metastasis. GEP100 associated directly with ligand-activated epidermal growth factor receptor (EGFR) to be activated. Disruption of E-cadherin-mediated cell-cell adhesion is one of the major steps involved in acquisition of invasive phenotypes of most carcinomas. The EGFR-GEP100-Arf6 pathway not only activated matrix invasion activity but also perturbed E-cadherin function. GEP100 was found to be expressed in more than 80% of invasive ductal carcinomas. However, 60% of ductal carcinomas in situ were also positive for GEP100, in which GEP100 was preferentially coexpressed with EGFR in their malignant cases. Microenvionments have been highly implicated in the development of tumor malignancy. Our results reveal an aspect of the precise molecular mechanism of cancer invasion and metastasis, in which full invasiveness is not acquired just by alterations of cancer cells themselves, but their microenvironments may also play pivotal roles.     

Rab5c promotes AMAP1-PRKD2 complex formation to enhance β1 integrin recycling in EGF-induced cancer invasion

Epidermal growth factor receptor (EGFR) signaling is one of the crucial factors in breast cancer malignancy. Breast cancer cells often overexpress Arf6 and its effector, AMAP1/ASAP1/DDEF1; in these cells, EGFR signaling may activate the Arf6 pathway to induce invasion and metastasis. Active recycling of some integrins is crucial for invasion and metastasis. Here, we show that the Arf6-AMAP1 pathway links to the machinery that recycles β1 integrins, such as α3β1, to promote cell invasion upon EGFR stimulation. We found that AMAP1 had the ability to bind directly to PRKD2 and hence to make a complex with the cytoplasmic tail of the β1 subunit. Moreover, GTP-Rab5c also bound to AMAP1, and activation of Rab5c by EGFR signaling was necessary to promote the intracellular association of AMAP1 and PRKD2. Our results suggest a novel mechanism by which EGFR signaling promotes the invasiveness of some breast cancer cells via integrin recycling.     

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.     

Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.     

CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery

Expression of AMAP1 correlates well with the invasive phenotypes and malignancy of human primary breast carcinomas. AMAP1 recruits its binding proteins, such as cortactin and paxillin, to sites of Arf6 activation to form invadopodia. A mouse ortholog of AMAP1, ASAP1, is known to bind to CIN85, a binding partner of an E3 ligase, Cbl. Here, we found that CIN85 colocalizes with AMAP1 at invadopodia, and binding of AMAP1 with CIN85 is important for the invasive activities of breast cancer cells, including MDA-MB-231. siRNA-mediated silencing of CIN85, as well as Cbl, also inhibited the invasion. We moreover found that AMAP1 is monoubiquitinated, rather than polyubiquitinated, by virtue of Cbl and provide evidence that the ability of AMAP1 to be monoubiquitinated is important for its involvement in invasion. Our results indicate that CIN85, as well as Cbl, which is a well-known suppressor of growth factor receptor signaling, can be positively involved in tumor invasion, and suggest that a complex epigenetic process is involved in AMAP1 function in breast cancer cell invasion.     

A novel mode of action of an ArfGAP, AMAP2/PAG3/Papa lpha, in Arf6 function

Previously we reported that AMAP2/PAG3/Papalpha/KIAA0400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, whereas it was shown to exhibit efficient GAP activities for other Arf isoforms in vitro. Here, we found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6 but not to GDP-Arf6 or other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function; it exhibits efficient catalytic GAP activity for the class I and II Arfs and yet is involved in the cellular function of the class III Arf without immediate GAP activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.     

Engagement of overexpressed Her2 with GEP100 induces autonomous invasive activities and provides a biomarker for metastases of lung adenocarcinoma

Overexpression of Her2/ErbB2/Neu in cancer is often correlated with recurrent distant metastasis, although the mechanism still remains largely elusive. We have previously shown that EGFR, when tyrosine-phosphorylated, binds to GEP100/BRAG2 to activate Arf6, which induces cancer invasion and metastasis. We now show that overexpressed Her2 in lung adenocarcinoma cells also employs GEP100. Like EGFR-GEP100 binding, this association is primarily mediated by the pleckstrin homology (PH) domain of GEP100 and Tyr1139/Tyr1196 of Her2. Tyr1139/Tyr1196 are autonomously phosphorylated, when Her2 is overexpressed. Accordingly, invasive activities mediated by the Her2-GEP100 pathway are not dependent on external factors. Blocking Her2-GEP100 binding, as well as its signaling pathway all inhibit cancer invasive activities. Moreover, our clinical study indicates that co-overexpression of Her2 with GEP100 in primary lung adenocarcinomas of patients is correlated with the presence of their node-metastasis with a statistical significance. Since the GEP100 PH domain interacts with both Her2 and EGFR, targeting this domain may provide novel cancer therapeutics.     

Co-Overexpression of GEP100 and AMAP1 Proteins Correlates with Rapid Local Recurrence after Breast Conservative Therapy

A major problem of current cancer research and therapy is prediction of tumor recurrence after initial treatment, rather than the simple biological characterization of the malignancy and proliferative properties of tumors. Breast conservation therapy (BCT) is a well-approved, standard treatment for patients with early stages of breast cancer, which consists of lumpectomy and whole-breast irradiation. In spite of extensive studies, only ''age'' and ''Ki-67 positivity'' have been identified to be well correlated with local recurrence after BCT. An Arf6 pathway, activated by GEP100 under receptor tyrosine kinases (RTKs) and employs AMAP1 as its effector, is crucial for invasion and metastasis of some breast cancer cells. This pathway activates β1 integrins and perturbs E-cadherin-based adhesions, hence appears to be integral for epithelial-mesenchymal transdifferentiation (EMT). We here show that expression of the Arf6 pathway components statistically correlates with rapid local recurrence after BCT. We retrospectively analyzed four hundred seventy-nine patients who received BCT in Hokkaido University Hospital, and found 20 patients had local recurrence. We then analyzed pathological samples of patients who experienced local recurrence by use of Kaplan-Meier analysis, Stepwise regression analysis and the t-test, coupled with immunostaining, and found that co-overexpression of GEP100 and AMAP1 correlates with rapidity of the local recurrence. Their margin-status, node-positivity, and estrogen receptor (ER)- or progesterone receptor (PgR)-positivity did not correlated with the rapidity. This study is the first to show that expression of a certain set of proteins correlates with the rapidity of local recurrence. Our results are useful not only for prediction, but highlight the possibility of developing novel strategies to block local recurrence. We also discuss why mRNAs encoding these proteins have not been identified to correlate with local recurrence by previous conventional gene expression profiling analyses.     

Ectodermal‐neural cortex 1 as a novel biomarker predicts poor prognosis and induces metastasis in breast cancer by promoting Wnt/β‐catenin pathway

Breast cancer, as the most common malignancy, is the second leading cause of cancer‐related death in women. One of the kelch family member ENC1 is involved in various pathophysiologic processes. But the role of ENC1 in breast cancer has not been investigated. The present study value the feature, clinical significance and the molecular mechanisms of ENC1 in breast cancer. The expression and prognosis value of ENC1 expression among breast cancer and normal breast tissue were investigated in The Cancer Genome Atlas database and human samples. ENC1 was knockdown to explore its function in various breast cancer cell lines. Western blot was performed to explore the potential molecular mechanisms. We observed that ENC1 was overexpressed in breast cancer tissues. ENC1 overexpression was associated with high metastasis and predicted a poor prognosis in patients with breast cancer. ENC1 Knockdown inhibits the growth, clone formation, migration and invasion of breast cancer cells. Mechanism analysis revealed ENC1 was strong associated with the metastasis by modulating β‐catenin pathway. Our study emphasizes that ENC1 is a potential prognostic and metastasis‐related marker of breast cancer, and may function as a possible therapeutic target against breast cancer.     

Akt1 inhibition promotes breast cancer metastasis through EGFR-mediated β-catenin nuclear accumulation

Background

Knockdown of Akt1 promotes Epithelial-to-Mesenchymal Transition in breast cancer cells. However, the mechanisms are not completely understood.

Methods

Western blotting, immunofluorescence, luciferase assay, real time PCR, ELISA and Matrigel invasion assay were used to investigate how Akt1 inhibition promotes breast cancer cell invasion in vitro. Mouse model of lung metastasis was used to measure in vivo efficacy of Akt inhibitor MK2206 and its combination with Gefitinib.

Results

Knockdown of Akt1 stimulated β-catenin nuclear accumulation, resulting in breast cancer cell invasion. β-catenin nuclear accumulation induced by Akt1 inhibition depended on the prolonged activation of EGFR signaling pathway in breast cancer cells. Mechanistic experiments documented that knockdown of Akt1 inactivates PIKfyve via dephosphorylating of PIKfyve at Ser³¹⁸ site, resulting in a decreased degradation of EGFR signaling pathway. Inhibition of Akt1 using MK2206 could induce an increase in the expression of EGFR and β-catenin in breast cancer cells. In addition, MK2206 at a low dosage enhance breast cancer metastasis in a mouse model of lung metastasis, while an inhibitor of EGFR tyrosine kinase Gefitinib could potentially suppress breast cancer metastasis induced by Akt1 inhibition.

Conclusion

EGFR-mediated β-catenin nuclear accumulation is critical for Akt1 inhibition-induced breast cancer metastasis.

     

Advances in targeting epidermal growth factor receptor signaling pathway in mammary cancer

Breast cancer is the most common malignancy among women worldwide. The role of epidermal growth factor receptor (EGFR) in many epithelial malignancies has been established, since it is dysregulated, overexpressed or mutated. Its overexpression has been associated with increased aggressiveness and metastatic potential in breast cancer. The well-established interplay between EGFR signaling pathway and estrogen receptors (ERs) as well as major extracellular matrix (ECM) mediators is crucial for regulating basic functional properties of breast cancer cells, including migration, proliferation, adhesion and invasion. EGFR activation leads to endocytosis of the receptor with implications in the regulation of downstream signaling effectors, the modulation of autophagy and cell survival. Therefore, EGFR is considered as a promising therapeutic target in breast cancer. Several anti-EGFR therapies (i.e. monoclonal antibodies and tyrosine kinase inhibitors) have been evaluated both in vitro and in vivo, making their way to clinical trials. However, the response rates of anti-EGFR therapies in the clinical trials is low mainly due to chemoresistance. Novel drug design, phytochemicals and microRNAs (miRNAs) are assessed as new therapeutic approaches against EGFR. The main goal of this review is to highlight the importance of targeting EGFR signaling pathway in terms of its crosstalk with ERs, the involvement of ECM effectors and epigenetics. Moreover, recent insights into the design of specialized delivery systems contributing in the development of novel diagnostic and therapeutic approaches in breast cancer are addressed.     

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.     

Osteopontin overexpression in breast cancer: knowledge gained and possible implications for clinical management

Osteopontin (OPN) is a secreted protein that is overexpressed in a number of human cancers, and has been associated with increased metastatic burden and poor prognosis in breast cancer patients. The OPN protein contains several conserved structural elements including heparin- and calcium-binding domains, a thrombin-cleavage site, a CD44 binding site, and two integrin-binding sites. Experimental studies have shown that the ability of OPN to interact with a diverse range of factors, including cell surface receptors (integrins, CD44), secreted proteases (matrix metalloproteinases, urokinase plasminogen activator), and growth factor/receptor pathways (TGFalpha/EGFR, HGF/Met) is central to its role in malignancy. These complex signaling interactions can result in changes in gene expression, which ultimately lead to alterations in cell properties involved in malignancy such as adhesion, migration, invasion, enhanced tumor cell survival, tumor angiogenesis, and metastasis. Therefore, OPN is not merely associated with cancer, but rather it plays a multi-faceted functional role via complex molecular cross-talk with other factors. This review will focus on the role of OPN in breast cancer, in particular on the malignancy-promoting aspects of OPN that may reveal opportunities for new approaches to the clinical management of breast cancer.