Jin-Min Nam

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.

EphA2 engages Git1 to suppress Arf6 activity modulating epithelial cell-cell contacts.

ADP-ribosylation factor (Arf) 6 activity is crucially involved in the regulation of E-cadherin-based cell-cell adhesions. Erythropoietin-producing hepatocellular carcinoma (Eph)-family receptors recognize ligands, namely, ephrins, anchored to the membrane of apposing cells, and they mediate cell-cell contact-dependent events. Here, we found that Arf6 activity is down-regulated in Madin-Darby canine kidney cells, which is dependent on cell density and calcium ion concentration, and we provide evidence of a novel signaling pathway by which ligand-activated EphA2 suppresses Arf6 activity. This EphA2-mediated suppression of Arf6 activity was linked to the induction of cell compaction and polarization, but it was independent of the down-regulation of extracellular signal-regulated kinase 1/2 kinase activity. We show that G protein-coupled receptor kinase-interacting protein (Git) 1 and noncatalytic region of tyrosine kinase (Nck) 1 are involved in this pathway, in which ligand-activated EphA2, via its phosphorylated Tyr594, binds to the Src homology 2 domain of Nck1, and then via its Src homology 3 domain binds to the synaptic localizing domain of Git1 to suppress Arf6 activity. We propose a positive feedback loop in which E-cadherin-based cell-cell contacts enhance EphA-ephrinA signaling, which in turn down-regulates Arf6 activity to enhance E-cadherin-based cell-cell contacts as well as the apical-basal polarization of epithelial cells.

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.

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.

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.

The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial–mesenchymal transition

Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial–mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-β-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-α or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT.

Evaluation of the relative biological effectiveness of spot-scanning proton irradiation in vitro

Variations in relative biological effectiveness (RBE) from a fixed value of 1.1 are critical in proton beam therapy. To date, studies estimating RBE at multiple positions relative to the spread-out Bragg peak (SOBP) have been predominantly performed using passive scattering methods, and limited data are available for spot-scanning beams. Thus, to investigate the RBE of spot-scanning beams, Chinese hamster fibroblast V79 cells were irradiated using the beam line at the Hokkaido University Hospital Proton Therapy Center. Cells were placed at six different depths, including the entrance of the proton beam and the proximal and distal part of the SOBP. Surviving cell fractions were analyzed using colony formation assay, and cell survival curves were obtained by the curve fitted using a linear–quadratic model. RBE10 and RBE37 were 1.15 and 1.21 at the center of the SOBP, respectively. In contrast, the distal region showed higher RBE values (1.50 for RBE10 and 1.85 for RBE37). These results are in line with those of previous studies conducted using passive scattering proton beams. Taken together, these data strongly suggest that variations in RBE should be considered during treatment planning for spot-scanning beams as well as for passive scattering proton beams.

High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue

BackgroundSquamous cell carcinoma of the tongue (tongue SCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer under current therapeutics. ARF6 and its effector AMAP1 are often overexpressed in different types of cancers, such as breast cancer and renal cancer, and in these cancers, AMAP1 binds to EPB41L5 to promote invasion, metastasis, and drug resistance. EPB41L5 is a mesenchymal-specific protein, normally induced during epithelial-mesenchymal transition (EMT) to promote focal adhesion dynamics. Similarly to breast cancer and renal cancer, the acquisition of mesenchymal phenotypes is the key process that drives the malignancy of HNSCC. We previously showed that the overexpression of AMAP1 in tongue SCC is statistically correlated with the poor outcome of patients. In this study, we examined whether tongue SCC also expresses EPB41L5 at high levels.ResultsImmunohistochemical staining of clinical specimens of tongue SCC demonstrated that high expression levels of EPB41L5 statistically correlate with poor disease-free survival and poor overall survival rates of patients. The tongue SCC cell line SCC-9, which overexpress Arf6 and AMAP1, also expressed EPB41L5 at high levels to promote invasiveness, whereas the weakly invasive SCC-25 cells did not express EPB41L5 at notable levels. Among the different EMT-associated transcriptional factors, ZEB1 was previously found to be most crucial in inducing EPB41L5 in breast cancer and renal cancer. In contrast, expression levels of ZEB1 did not correlate with the expression levels of EPB41L5 in tongue SCC, whereas KLF8 and FOXO3 levels showed positive correlations with EPB41L5 levels. Moreover, silencing of EPB41L5 only marginally improved the drug resistance of SCC-9 cells, even when coupled with ionizing radiation.ConclusionOur results indicate that activation of the cancer mesenchymal program in tongue SCC, which leads to EPB41L5 expression, closely correlates with the poor prognosis of patients. However, ZEB1 was not the major inducer of EPB41L5 in tongue SCC, unlike in breast cancer and renal cancer. Thus, processes that trigger the mesenchymal program of tongue SCC, which drives their malignancies, seem to be substantially different from those of other cancers.