ADP-Ribosylation Factor 6 Pathway Acts as a Key Executor of Mesenchymal Tumor Plasticity.

Despite the "big data" on cancer from recent breakthroughs in high-throughput technology and the development of new therapeutic modalities, it remains unclear as to how intra-tumor heterogeneity and phenotypic plasticity created by various somatic abnormalities and epigenetic and metabolic adaptations orchestrate therapy resistance, immune evasiveness, and metastatic ability. Tumors are formed by various cells, including immune cells, cancer-associated fibroblasts, and endothelial cells, and their tumor microenvironment (TME) plays a crucial role in malignant tumor progression and responses to therapy. ADP-ribosylation factor 6 (ARF6) and AMAP1 are often overexpressed in cancers, which statistically correlates with poor outcomes. The ARF6-AMAP1 pathway promotes the intracellular dynamics and cell-surface expression of various proteins. This pathway is also a major target for KRAS/TP53 mutations to cooperatively promote malignancy in pancreatic ductal adenocarcinoma (PDAC), and is closely associated with immune evasion. Additionally, this pathway is important in angiogenesis, acidosis, and fibrosis associated with tumor malignancy in the TME, and its inhibition in PDAC cells results in therapeutic synergy with an anti-PD-1 antibody in vivo. Thus, the ARF6-based pathway affects the TME and the intrinsic function of tumors, leading to malignancy. Here, we discuss the potential mechanisms of this ARF6-based pathway in tumorigenesis, and novel therapeutic strategies.

KRAS, MYC, and ARF6: inseparable relationships cooperatively promote cancer malignancy and immune evasion.

Mutations in the KRAS gene and overexpression of protein products of the MYC and ARF6 genes occur frequently in cancer. Here, the inseparable relationships and cooperation of the protein products of these three genes in cancer malignancy and immune evasion are discussed. mRNAs encoded by these genes share the common feature of a G-quadruplex structure, which directs them to be robustly expressed when cellular energy production is increased. These three proteins are also functionally inseparable from each other, as follows.　1) KRAS induces MYC gene expression, and may also promote eIF4A-dependent MYC and ARF6 mRNA translation, 2) MYC induces the expression of genes involved in mitochondrial biogenesis and oxidative phosphorylation, and 3) ARF6 protects mitochondria from oxidative injury. ARF6 may moreover promote cancer invasion and metastasis, and also acidosis and immune checkpoint. Therefore, the inseparable relationships and cooperation of KRAS, MYC, and ARF6 appear to result in the activation of mitochondria and the driving of ARF6-based malignancy and immune evasion. Such adverse associations are frequent in pancreatic cancer, and appear to be further enhanced by TP53 mutations. Video Abstract.

Oncological Effects and Prognostic Value of AMAP1 in Gastric Cancer.

PURPOSE: We examined the diagnostic significance, prognostic value, and potential function of AMAP1 in gastric cancer (GC). METHODS: Comprehensive bioinformatic analysis was conducted to investigate differential expression of AMAP1 mRNA and protein in GC. Meta-analyses were utilized to determine the overall prognostic correlation of AMAP1 mRNA in patients with GC. A panel of vitro assays was applied to assess target microRNA and AMAP1 protein in GC cell lines and tissues, respectively. RESULTS: AMAP1 mRNA and protein levels were upregulated in GC specimens, compared to matched normal tissues. AMAP1 mRNA exhibited promising results regarding differential diagnosis of GC and normal tissue. Meta-analysis based on the TCGA and GEO databases revealed that high AMAP1 mRNA abundance was associated with poor overall survival (HR = 1.42; 95% CI: 1.06-1.89) and was correlated with reduced progression-free survival (HR = 1.89; 95% CI: 1.51-2.36) in GC patients. Moreover, AMAP1 was negatively correlated with miR-192-3p (r = -0.3843; P < 0.0001). A dual-luciferase assay revealed that miR-192-3p targeted AMAP1. Levels of miR-192-3p were significantly higher in GC tissues and GC cells than in normal tissues and cells. Moreover, AMAP1 silencing resulted in reduced GC proliferation, migration, and invasion. CONCLUSION: AMAP1 is a novel oncogene in GC and is negatively correlated with by miR-192-3p. AMAP1 may act as a diagnostic and prognostic marker of GC.

Inhibition of mutant KRAS-driven overexpression of ARF6 and MYC by an eIF4A inhibitor drug improves the effects of anti-PD-1 immunotherapy for pancreatic cancer.

Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating ß1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations. Video abstract.

High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer.

BACKGROUND: Not merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, ß1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis. METHODS: Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed. RESULTS: Elevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients. CONCLUSIONS: Together with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK. Video abstract.

Epithelial-specific histone modification of the miR-96/182 locus targeting AMAP1 mRNA predisposes p53 to suppress cell invasion in epithelial cells.

BACKGROUND: TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1. METHODS: Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells. RESULTS: We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3'-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells. CONCLUSION: Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.

Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model.

BACKGROUND: The small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion. METHODS: Expression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated. RESULTS: We found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases. CONCLUSIONS: PyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.

CCL18-dependent translocation of AMAP1 is critical for epithelial to mesenchymal transition in breast cancer.

AMAP1 was a GTPase-activating protein that regulates cytoskeletal structures in focal adhesions, circular dorsal ruffles, and promote cell differentiation in tumor cells. But the activation and function of AMAP1 in breast cancer remain largely unexplored. Here we show that AMAP1 was phosphorylated and translocated to plasma membrane and formed a stable complex with Pyk2 in response to CCL18. Moreover, CCL18-dependent AMAP1 translocation interfered the AMAP1-IKK-ß interaction, resulting in nuclear factor-kappaB (NF-κB) activation. Depletion of AMAP1 expression by RNAi efficiently reversed the CCL18-induced epithelial to mesenchymal transition (EMT) of breast cancer cells and as well as CCL18-induced adhesion, migration and invasion. Strikingly, AMAP1 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognosis in breast cancers. Given that AMAP1 mediated CCL18-induce activation of NF-κB and promoted breast cancer metastasis, AMAP1 may represent a therapeutic target for the eradication of breast cancer metastasis.

Inhibition of epithelial-mesenchymal transition by cetuximab via the EGFR-GEP100-Arf6-AMAP1 pathway in head and neck cancer.

BACKGROUND: Despite improved survival by the addition of a monoclonal antibody against epidermal growth factor receptor (EGFR), cetuximab, to chemotherapy or radiotherapy for squamous cell carcinoma of the head and neck (SCCHN), cetuximab by itself is not a potent antiproliferative agent against SCCHN. We aimed to elucidate working mechanism of cetuximab in SCCHN. METHODS: The effect of cetuximab on the proliferation, migration, invasion, epithelial-mesenchymal transition, and signaling events downstream of the EGFR were investigated in 4 SCCHN cell lines. The in vivo efficacy of cetuximab was evaluated in a xenotransplant model. RESULTS: Cetuximab inhibited migration, invasion, epithelial-mesenchymal transition, and lymph node metastasis by suppressing EGFR-GEP100-Arf6-AMAP1 pathway, but it did not inhibit cancer cell proliferation. CONCLUSION: The improved survival by the addition of cetuximab is likely to be attributable to the antiepithelial-mesenchymal transition action of cetuximab via inhibiting EGFR-GEP100-Arf6-AMAP1 pathway. © 2016 Wiley Periodicals, Inc. Head Neck 39: 476-485, 2017.