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. 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. 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. 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.

Since the time of Rudolf Virchow in the 19th century, it has been well-known that cancer-associated inflammation contributes to tumor initiation and progression. However, it remains unclear whether a collapse of the balance between the antitumor immune response via the immunological surveillance system and protumor immunity due to cancer-related inflammation is responsible for cancer malignancy. The majority of inflammatory signals affect tumorigenesis by activating signal transducer and activation of transcription 3 (STAT3) and nuclear factor-κB. Persistent STAT3 activation in malignant cancer cells mediates extremely widespread functions, including cell growth, survival, angiogenesis, and invasion and contributes to an increase in inflammation-associated tumorigenesis. In addition, intracellular STAT3 activation in immune cells causes suppressive effects on antitumor immunity and leads to the differentiation and mobilization of immature myeloid-derived cells and tumor-associated macrophages. In many cancer types, STAT3 does not directly rely on its activation by oncogenic mutations but has important oncogenic and malignant transformation-associated functions in both cancer and stromal cells in the tumor microenvironment (TME). We have reported a series of studies aiming towards understanding the molecular mechanisms underlying the proliferation of various types of tumors involving signal-transducing adaptor protein-2 as an adaptor molecule that modulates STAT3 activity, and we recently found that AT-rich interactive domain-containing protein 5a functions as an mRNA stabilizer that orchestrates an immunosuppressive TME in malignant mesenchymal tumors. In this review, we summarize recent advances in our understanding of the functional role of STAT3 in tumor progression and introduce novel molecular mechanisms of cancer development and malignant transformation involving STAT3 activation that we have identified to date. Finally, we discuss potential therapeutic strategies for cancer that target the signaling pathway to augment STAT3 activity.

Despite recent advances in cancer therapeutics in general, the survival of patients with head and neck squamous cell carcinomas (HNSCCs) has not improved substantially over the past few decades. HNSCC cells often exhibit invasive and metastatic phenotypes, and expression of epidermal growth factor receptor (EGFR) and cortactin has been highly implicated in the development of malignancy in HNSCCs. We have shown previously that an Arf6 pathway, in which Arf6 is activated by GEP100 and employs AMAP1 (also called DDEF1 or ASAP1) as its downstream effector, is pivotal for the invasion and metastasis of different breast cancer cells. This pathway is activated by receptor tyrosine kinases, including EGFR; and moreover, AMAP1 physically associates with cortactin, in which inhibition of this binding effectively blocks invasion and metastasis. We here investigated whether the expression of Arf6 pathway components correlates with the poor prognosis of HNSCC patients. We have shown previously that AMAP1 protein levels are not correlated with its mRNA levels, and hence we here employed immunohistochemical staining of HNSCC clinical specimens to investigate AMAP1 protein levels. We found that high levels of AMAP1 protein expression on its own, as well as its co-overexpression with EGFR statistically correlates with poor disease-free survival and poor overall survival, while high levels of cortactin expression or its co-expression with EGFR did not. Our identification of predictive biomarkers, together with our previous findings on the coherent signaling pathway that these biomarkers ultimately generate should be powerful information for the further development of HNSCC therapeutics.

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, β1-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.

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 β1-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.

Squamous 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. Immunohistochemical 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. Our 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.