Yasuhito Onodera

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.

Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin α5β1 and Fibronectin

Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D lrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D lrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha5beta1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha5beta1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha5beta1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha5beta1-integrin as targets for breast cancer therapy.

The EGFR‐GEP100‐Arf6‐AMAP1 Signaling Pathway Specific to Breast Cancer Invasion and Metastasis†

Tumors are tissue‐specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin β4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand‐activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR‐GEP100‐Arf6‐AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed.

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

EGF signaling activates Rab5c and promotes the intracellular association of AMAP1 and PRKD2 to enhance β1 integrin recycling and promote the invasiveness of breast cancer cells.

Lysophosphatidic acid activates Arf6 to promote the mesenchymal malignancy of renal cancer

Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial–mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-Gα12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients.

P53- and mevalonate pathway–driven malignancies require Arf6 for metastasis and drug resistance

The mevalonate pathway (MVP) is a metabolic pathway associated with tumor invasiveness and is known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. Hashimoto et al. show that MVP-driven cancers require activation of the GTPase Arf6 for invasion and that the MVP substrate Rab11 is required for Arf6 activation.

Intracellular trafficking of integrins in cancer cells.

Integrins are heterodimeric cell surface receptors, which principally mediate the interaction between cells and their extracellular microenvironments. Because of their pivotal roles in cancer proliferation, survival, invasion and metastasis, integrins have been recognized as promising targets for cancer treatment. As is the case with other receptors, the localization of integrins on the cell surface has provided opportunities to block their functions by various inhibitory monoclonal antibodies. A number of small molecule agents blocking integrin-ligand binding have also been established, and some such agents are currently on the market or in clinical trials for some diseases including cancer. This review exclusively focuses on another strategy for cancer therapy, which comes from the obligate localization of integrins on the cell surface; targeting the intracellular trafficking of integrins. A number of studies have shown the essential roles of integrin trafficking in hallmarks of cancer, such as activation of oncogenic signaling pathways as well as acquisition of invasiveness. Recent findings have shown that increased integrin recycling activity is associated with some types of gain-of-function mutations of p53, a common feature of diverse types of cancers, which also indicates that targeting integrin recycling could be widely applicable and effective against many cancers. We also discuss possible therapeutic contexts where integrin trafficking can be effectively targeted, and what molecular interfaces may hopefully be druggable.

β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

IntroductionDuctal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells.MethodsWe measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence.ResultsP-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, α5β1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-κB) to the β1-integrin promoter region, associated with up-regulation of α5β1-integrins. Inhibition of NF-κB or β1-integrin signaling abrogated emergence of the invasive activity.ConclusionsP-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by β1-integrin and NF-κB signaling.

ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance

Onset of the cancer mesenchymal program is closely associated with cancer malignancy and drug resistance. Among the different epithelial–mesenchymal transition (EMT)-associated transcriptional factors, ZEB1 has a key role in inducing the mesenchymal phenotypes and stem cell-like properties of different breast cancer cells. ARF6 and its effector AMAP1 are frequently overexpressed in breast cancer cells, and promote invasion, metastasis and drug resistance. EPB41L5 is induced during EMT, and mediates the disruption of E-cadherin-based cell–cell adhesion and the promotion of focal adhesion dynamics. Here we show that EPB41L5 is an integral component of the ARF6-based pathway, which is induced by ZEB1. We found that EPB41L5 is expressed at high levels in malignant breast cancer cells and binds to AMAP1. ZEB1 induced EPB41L5 both in cancer cells and normal cells. This relationship was recaptured with The Cancer Genome Atlas RNASeq data set, and correlated with the poor outcome of the patients. In contrast, diversified events, such as tumor growth factor β1 stimulation, expression of SNAI1 and TP53 mutation, can each cause the induction of ZEB1 and EPB41L5, depending on the cellular context. Our results demonstrated that the ZEB1-EPB41L5 axis is at the core of the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance of significant populations of primary breast cancers, and is tightly correlated with the poor outcomes of patients.

Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

Gold nanoparticles (AuNPs) have recently attracted attention as clinical agents for enhancing the effect of radiotherapy in various cancers. Although radiotherapy is a standard treatment for cancers, invasive recurrence and metastasis are significant clinical problems. Several studies have suggested that radiation promotes the invasion of cancer cells by activating molecular mechanisms involving integrin and fibronectin (FN). In this study, polyethylene-glycolylated AuNPs (P-AuNPs) were conjugated with Arg–Gly–Asp (RGD) peptides (RGD/P-AuNPs) to target cancer cells expressing RGD-binding integrins such as α5- and αv-integrins. RGD/P-AuNPs were internalized more efficiently and colocalized with integrins in the late endosomes and lysosomes of MDA-MB-231 cells. A combination of RGD/P-AuNPs and radiation reduced cancer cell viability and increased DNA damage compared to radiation alone in MDA-MB-231 cells. Moreover, the invasive activity of breast cancer cell lines after radiation treatment was significantly inhibited in the presence of RGD/P-AuNPs. Microarray analyses revealed that the expression of FN in irradiated cells was suppressed by combined use of RGD/P-AuNPs. Reduction of FN and downstream signaling may be involved in suppressing radiation-induced invasive activity by RGD/P-AuNPs. Our study suggests that RGD/P-AuNPs can target integrin-overexpressing cancer cells to improve radiation therapy by suppressing invasive activity in addition to sensitization. Thus, these findings provide a possible clinical strategy for using AuNPs to treat invasive breast cancer following radiotherapy.