Ductal 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.

Enhanced invasiveness, a critical determinant of metastasis and poor prognosis, has been observed in cancer cells that survive cancer therapy, including radiotherapy. Here, we show that invasiveness in radiation-surviving cancer cells is associated with alterations in lysosomal exocytosis caused by the enhanced activation of Arl8b, a small GTPase that regulates lysosomal trafficking. The binding of Arl8b with its effector, SKIP, is increased after radiation through regulation of BORC-subunits. Knockdown of Arl8b or BORC-subunits decreases lysosomal exocytosis and the invasiveness of radiation-surviving cells. Notably, high expression of ARL8B and BORC-subunit genes is significantly correlated with poor prognosis in breast cancer patients. Sp1, an ATM-regulated transcription factor, is found to increase BORC-subunit genes expression after radiation. In vivo experiments show that ablation of Arl8b decreases IR-induced invasive tumor growth and distant metastasis. These findings suggest that BORC-Arl8b-mediated lysosomal trafficking is a target for improving radiotherapy by inhibiting invasive tumor growth and metastasis.

CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

Radiotherapy is used extensively in cancer treatment, but radioresistance and the metastatic potential of cancer cells that survive radiation remain critical issues. There is a need for novel treatments to improve radiotherapy. Here, we evaluated the therapeutic benefit of λ-carrageenan (CGN) to enhance the efficacy of radiation treatment and investigated the underlying molecular mechanism. CGN treatment decreased viability in irradiated cancer cells and enhanced reactive oxygen species accumulation, apoptosis, and polyploid formation. Additionally, CGN suppressed radiation-induced chemoinvasion and invasive growth in 3D lrECM culture. We also screened target molecules using a gene expression microarray analysis and focused on Rac GTPase-activating protein 1 (RacGAP1). Protein expression of RacGAP1 was upregulated in several cancer cell lines after radiation, which was significantly suppressed by CGN treatment. Knockdown of RacGAP1 decreased cell viability and invasiveness after radiation. Overexpression of RacGAP1 partially rescued CGN cytotoxicity. In a mouse xenograft model, local irradiation followed by CGN treatment significantly decreased tumor growth and lung metastasis compared to either treatment alone. Taken together, these results suggest that CGN may enhance the effectiveness of radiation in cancer therapy by decreasing cancer cell viability and suppressing both radiation-induced invasive activity and distal metastasis through downregulating RacGAP1 expression.

Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial-mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.

This study investigated variations in the relative biological effectiveness (RBE) values among various sarcoma and normal-tissue-derived cell lines (normal cell line) in proton beam and carbon-ion irradiations. We used a consistent protocol that specified the timing of irradiation after plating cells and detailed the colony formation assay. We examined the cell type dependence of RBE for proton beam and carbon-ion irradiations using four human sarcoma cell lines (MG63 osteosarcoma, HT1080 fibrosarcoma, SW872 liposarcoma, and SW1353 chondrosarcoma) and three normal cell lines (HDF human dermal fibroblast, hTERT-HME1 mammary gland, and NuLi-1 bronchus epithelium). The cells were irradiated with gamma rays, proton beams at the center of the spread-out Bragg peak, or carbon-ion beams at 54.4 keV/μm linear energy transfer. In all sarcoma and normal cell lines, the average RBE values in proton beam and carbon-ion irradiations were 1.08 ± 0.11 and 2.08 ± 0.36, which were consistent with the values of 1.1 and 2.13 used in current treatment planning systems, respectively. Up to 34% difference in the RBE of the proton beam was observed between MG63 and HT1080. Similarly, a 32% difference in the RBE of the carbon-ion beam was observed between SW872 and the other sarcoma cell lines. In proton beam irradiation, normal cell lines had less variation in RBE values (within 10%), whereas in carbon-ion irradiation, RBE values differed by up to 48% between hTERT-HME1 and NuLi-1. Our results suggest that specific dose evaluations for tumor and normal tissues are necessary for treatment planning in both proton and carbon-ion therapies.

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.

The cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated.

In lung cancer, immune checkpoint inhibitors (ICIs) are often inadequate for tumor growth inhibition. Angiogenic inhibitors (AIs) are required to normalize tumor vasculature for improved immune cell infiltration. However, in clinical practice, ICIs and cytotoxic antineoplastic agents are simultaneously administered with an AI when tumor vessels are abnormal. Therefore, we examined the effects of pre-administering an AI for lung cancer immunotherapy in a mouse lung cancer model. Using DC101, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody, a murine subcutaneous Lewis lung cancer (LLC) model was used to determine the timing of vascular normalization. Microvessel density (MVD), pericyte coverage, tissue hypoxia, and CD8-positive cell infiltration were analyzed. The effects of an ICI and paclitaxel after DC101 pre-administration were investigated. On Day 3, increased pericyte coverage and alleviated tumor hypoxia represented the highest vascular normalization. CD8+ T-cell infiltration was also highest on Day 3. When combined with an ICI, DC101 pre-administration significantly reduced PD-L1 expression. When combined with an ICI and paclitaxel, only DC101 pre-administration significantly inhibited tumor growth, but simultaneous administration did not. AI pre-administration, and not simultaneous administration, may increase the therapeutic effects of ICIs due to improved immune cell infiltration.

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.

The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology.

Recent studies have demonstrated a relationship between oral bacteria and systemic inflammation. Endothelial cells (ECs), which line blood vessels, control the opening and closing of the vascular barrier and contribute to hematogenous metastasis; however, the role of oral bacteria-induced vascular inflammation in tumor metastasis remains unclear. In this study, we examined the phenotypic changes in vascular ECs following Streptococcus mutans (S. mutans) stimulation in vitro and in vivo. The expression of molecules associated with vascular inflammation and barrier-associated adhesion was analyzed. Tumor metastasis was evaluated after intravenous injection of S. mutans in murine breast cancer hematogenous metastasis model. The results indicated that S. mutans invaded the ECs accompanied by inflammation and NF-κB activation. S. mutans exposure potentially disrupts endothelial integrity by decreasing vascular endothelial (VE)-cadherin expression. The migration and adhesion of tumor cells were enhanced in S. mutans-stimulated ECs. Furthermore, S. mutans-induced lung vascular inflammation promoted breast cancer cell metastasis to the lungs in vivo. The results indicate that oral bacteria promote tumor metastasis through vascular inflammation and the disruption of vascular barrier function. Improving oral hygiene in patients with cancer is of great significance in preventing postoperative pneumonia and tumor metastasis.

Neutrophils rapidly migrate to infection sites after the recognition of invaders. During chemotaxis, neutrophils require energy supplied by mitochondria oxidative phosphorylation (OXPHOS), whereas neutrophils rely heavily on glycolysis under normal conditions. Mitochondrial OXPHOS correlates with mitochondrial morphology. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. Furthermore, MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. These results suggest that MFN2 is involved in chemotaxis of differentiated HL-60 cells depending on mitochondria.

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.

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 small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex.

TP53 mutation (i.e., loss of normal-p53) may evoke epithelial-mesenchymal transition (EMT), which was previously attributed to loss of certain miRNAs. However, not all epithelial cells undergo EMT upon TP53 mutation, and the p53-miRNA axis may not fully explain p53 function in epithelial integrity. We here show two modes of epithelial integrity: one involves p53-binding to a nucleotide region and the other does not. In the former, p53 binds to the CDH1 (encoding E-cadherin) locus to antagonize EZH2-mediated H3K27 trimethylation (H3K27me3) to maintain high levels of acetylation of H3K27 (H3K27ac). In the latter, the same locus is not highly acetylated at H3K27, and does not allow p53-binding, nor needs to antagonize EZH2. We moreover demonstrated that although the CDH1 locus in the p53-independent cells, but not in fibroblasts, becomes high-H3K27ac by butyrate and allows p53-biniding, their CDH1 expression does not become dependent on p53. Our results identified novel modes of the epithelial integrity, in which the same epithelial-specific gene locus exhibits different requirement for p53 with different histone modifications among different epithelial cells to warrant its expression.

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.

Mitochondria dynamically alter their subcellular localization during cell movement, although the underlying mechanisms remain largely elusive. The small GTPase Arf6 and its signaling pathway involving AMAP1 promote cell invasion via integrin recycling. Here we show that the Arf6-AMAP1 pathway promote the anterograde trafficking of mitochondria. Blocking the Arf6-based pathway causes mitochondrial aggregation near the microtubule-organizing center, and subsequently induces detrimental reactive oxygen species (ROS) production, likely via a mitochondrial ROS-induced ROS release-like mechanism. The Arf6-based pathway promotes the localization of ILK to focal adhesions to block RhoT1-TRAK2 association, which controls mitochondrial retrograde trafficking. Blockade of the RhoT1-TRAK1 machinery, rather than RhoT1-TRAK2, impairs cell invasion, but not two-dimensional random cell migration. Weakly or non-invasive cells do not notably express TRAK proteins, whereas they clearly express their mRNAs. Our results identified a novel association between cell movement and mitochondrial dynamics, which is specific to invasion and is necessary for avoiding detrimental ROS production.