Ninjurin1 suppresses metastatic property of lung cancer cells through inhibition of interleukin 6 signaling pathway.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell surface molecule that can mediate homophilic adhesion and promote neurite outgrowth from cultured dorsal root ganglion (DRG) neurons. Interestingly, Ninj1 overexpressed in human cancer; however, its role in metastasis is not clear. This study showed that inhibition of Ninj1 promotes lung cancer metastasis through interleukin 6 (IL-6)/STAT3 signaling. Ninj1 levels were relatively low in highly motile lung cancer cells. While inhibition of Ninj1 enhanced cell migration in lung cancer cells, overexpression of Ninj1 significantly suppressed it. We found that inhibition of Ninj1 significantly increased expression and secretion of IL-6 in A549 cells. We also found that inhibition of IL-6 decreased intercellular adhesion molecule 1 (ICAM-1) expression. In addition, inhibition of Ninj1 significantly increased cell motility and invasiveness of lung cancer cells. In an in vivo model, we found that Ninj1 suppression did not affect tumor growth but induced significant increase in incidence of lung metastasis, and sizes and number of tumor nodules. Taken together, our data clearly demonstrate that Ninj1 suppresses migration, invasion and metastasis of lung cancer via inhibition of the IL-6 signaling pathway in vitro and in vivo.

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway.

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer.

Fermented barley and soybean (BS) mixture enhances intestinal barrier function in dextran sulfate sodium (DSS)-induced colitis mouse model.

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic or relapsing immune system activation and inflammation within the gastrointestinal tract. The lack of safety and efficacy of standard therapies, the use of food supplements for managing IBD is increasing, and many studies have reported that various food supplements provide many beneficial effects for the IBD. METHODS: This study aimed to evaluate the anti-colitis effects of dietary supplementation with a fermented barley and soybean mixture (BS) on intestinal inflammation using a murine model of IBD. Female C57BL/6 mice were administered with either BS (100 and 200 mg/kg/day) or vehicle (PBS) control through oral gavages for 3 days and received 5% dextran sulfate sodium (DSS) drinking water to induce colitis. Mice body weight was measured every two days and disease activity index (DAI) score was determined on Day 15; mice were sacrificed and colons were analyzed by H & E staining and RT-PCR. We also measured intestinal barrier function in vitro using DSS-treated Caco-2 cells by assessing ZO-1 immunofluorescence staining and Western blotting and in vivo by measuring serum level of FITC-Dextran and by performing bacteria culture from mesenteric lymph nodes (MLN) extract. The gut microbiota was examined by real time PCR using fecal DNA. RESULTS: We found that BS alleviated the severity of colitis in a DSS-induced colitis mouse model, and suppressed levels of pro-inflammatory cytokines in colonic tissue. Moreover, BS prevented epithelial barrier dysfunction, inducing an increase of tight junction protein levels in colonic tissues, BS also inhibited FITC-dextran permeability, and suppressed bacterial translocation to MLNs. In addition, BS increased the levels of Lactobacilli and Bacteroides, which have anti-inflammatory properties. CONCLUSION: Our study suggests that BS has protective roles against inflammatory bowel disease through changes in inflammatory activity, tight junction protein expression, and gut microbiota composition in DSS-induced colitis.

The novel IGF-IR/Akt-dependent anticancer activities of glucosamine.

BACKGROUND: Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway. METHODS: We used various in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism. RESULTS: We found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress. CONCLUSIONS: Taken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer.

Novel NF-κB reporter mouse for the non-invasive monitoring of inflammatory diseases.

Bioluminescence imaging is useful for non-invasively monitoring inflammatory reactions associated with disease progression, and since NF-κB is a pivotal transcription factor that alters expressions of inflammatory genes, we generated novel NF-κB luciferase reporter (NF-κB-Luc) mice to understand the dynamics of inflammatory responses in whole body, and also in various type of cells by crossing NF-κB-Luc mice with cell-type specific Cre expressing mice (NF-κB-Luc:[Cre]). Bioluminescence intensity was significantly increased in NF-κB-Luc (NKL) mice exposed to inflammatory stimuli (PMA or LPS). Crossing NF-κB-Luc mice with Alb-cre mice or Lyz-cre mice generated NF-κB-Luc:Alb (NKLA) and NF-κB-Luc:Lyz2 (NKLL) mice, respectively. NKLA and NKLL mice showed enhanced bioluminescence in liver and macrophages, respectively. To confirm that our reporter mice could be utilized for the non-invasive monitoring of inflammation in preclinical models, we conducted a DSS-induced colitis model and a CDAHFD-induced NASH model in our reporter mice. In both models, our reporter mice reflected the development of these diseases over time. In conclusion, we believe that our novel reporter mouse can be utilized as a non-invasive monitoring platform for inflammatory diseases.

Dual-Armed Oncolytic Myxoma Virus Encoding IFN-γ and CD47 Promotes Lymphocyte Infiltration and Tumor Suppression of Syngeneic Murine Melanoma.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.

Insulin-like growth factor binding protein-3 suppresses vascular endothelial growth factor expression and tumor angiogenesis in head and neck squamous cell carcinoma.

Angiogenesis, the process by which new blood vessels are recruited to existing ones, is essential for tumor development. Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), which modulates bioavailability of IGF, has been studied for its potential role in angiogenesis during tissue regeneration and cancer development. In this study, we assessed the role of IGFBP-3 in tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC) and human umbilical vein endothelial cells (HUVECs) using adenoviral (Ad-BP3) and recombinant (rBP3) IGFBP-3. Using an in vivo orthotopic tongue tumor model, we confirmed that both Ad-BP3 and rBP3 suppress the growth of UMSCC38 HNSCC cells in vivo. Ad-BP3 inhibited vascularization in tongue tumors and chorio-allantoic membrane, and suppressed angiogenesis-stimulating activities in UMSCC38 cells. In HUVECs, Ad-BP3 decreased migration, invasion, and tube formation. rBP3 also suppressed production of vascular endothelial growth factor (VEGF) in HUVECs and UMSCC38 cells. IGFBP-3-GGG, a mutant IGFBP-3 with loss of IGF binding capacity, suppressed VEGF production. In addition, we found that IGFBP-3 suppressed VEGF expression, even in mouse embryonic fibroblasts from an IGF-1R-null mouse. Finally, we demonstrated that IGFBP-3-GGG inhibits tumor angiogenesis and growth to the same degree as wild-type IGFBP-3. Taken together, these results support the hypothesis that IGFBP-3 has anti-angiogenic activity in HNSCC, at least in part due to IGF-independent suppression of VEGF production from vascular endothelial cells and cancer cells.

Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NFκB signaling in HER2-overexpressing breast cancer cells.

Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are still unknown. This study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of sub G(0)/G(1) apoptotic fractions. Overexpression of HER2 did not confer resistance to apigenin in MCF-7 cells. Apigenin-induced extrinsic apoptosis pathway up-regulating the levels of cleaved caspase-8, and inducing the cleavage of poly (ADP-ribose) polymerase, whereas apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential maintaining red fluorescence and did not affect the levels of B-cell lymphoma 2 (BCL2) and Bcl-2-associated X protein. Moreover, apigenin reduced the tyrosine phosphorylation of HER2 (phospho-HER2 level) in MCF-7 HER2 cells, and up-regulated the levels of p53, phospho-p53 and p21 in MCF-7 vec and MCF-7 HER2 cells. This suggests that apigenin induces apoptosis through p53-dependent pathway. Apigenin also reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in MCF-7 vec and MCF-7 HER2 cells. Apigenin decreased the phosphorylation level of IκBα in the cytosol, and abrogated the nuclear translocation of p65 within the nucleus suggesting that it blocks the activation of NFκB signaling pathway in MCF-7 vec and MCF-7 HER2 cells. Our study indicates that apigenin could be a potential useful compound to prevent or treat HER2-overexpressing breast cancer.

Single-cell analysis of gastric pre-cancerous and cancer lesions reveals cell lineage diversity and intratumoral heterogeneity.

Single-cell transcriptomic profiles analysis has proposed new insights for understanding the behavior of human gastric cancer (GC). GC offers a unique model of intratumoral heterogeneity. However, the specific classes of cells involved in carcinogenetic passage, and the tumor microenvironment of stromal cells was poorly understood. We characterized the heterogeneous cell population of precancerous lesions and gastric cancer at the single-cell resolution by RNA sequencing. We identified 10 gastric cell subtypes and showed the intestinal and diffuse-type cancer were characterized by different cell population. We found that the intestinal and diffuse-type cancer cells have the differential metaplastic cell lineages: intestinal-type cancer cells differentiated along the intestinal metaplasia lineage while diffuse-type cancer cells resemble de novo pathway. We observed an enriched CCND1 mutation in premalignant disease state and discovered cancer-associated fibroblast cells harboring pro-stemness properties. In particular, tumor cells could be categorized into previously proposed molecular subtypes and harbored specific subtype of malignant cell with high expression level of epithelial-myofibroblast transition which was correlated with poor clinical prognosis. In addition to intratumoral heterogeneity, the analysis revealed different cellular lineages were responsible for potential carcinogenetic pathways. Single-cell transcriptomes analysis of gastric pre-cancerous lesions and cancer may provide insights for understanding GC cell behavior, suggesting potential targets for the diagnosis and treatment of GC.

The C-terminal region of Bfl-1 sensitizes non-small cell lung cancer to gemcitabine-induced apoptosis by suppressing NF-κB activity and down-regulating Bfl-1.

Gemcitabine is used to treat several cancers including lung cancer. However, tumor cells often escape gemcitabine-induced cell death via various mechanisms, which include modulating bcl-2 family members and NF-κB activation. We previously reported that the C-terminal region of Bfl-1 fused with GFP (BC) is sufficient to induce apoptosis in 293T cells. In the present study, we investigated the anti-tumor effect of combined BC gene therapy and gemcitabine chemotherapy in vitro and in vivo using non-small cell lung cancer cell lines and a xenograft model. Cell lines were resistant to low dose gemcitabine (4-40 ng/ml), which induced NF-κB activation and concomitant up-regulation of Bfl-1 (an NF-κB-regulated anti-apoptotic protein). BC induced the apoptosis of A549 and H157 cells with caspase-3 activation. Furthermore, co-treatment with BC and low dose gemcitabine synergistically and efficiently induced mitochondria-mediated apoptosis in these cells. When administered alone or with low dose gemcitabine, BC suppressed NF-κB activity, inhibited the nuclear translocation of p65/relA, and down-regulated Bfl-1 expression. Furthermore, direct suppression of Bfl-1 by RNA interference sensitized cells to gemcitabine-induced cell death, suggesting that Bfl-1 importantly regulates lung cancer cell sensitivity to gemcitabine. BC and gemcitabine co-treatment also showed a strong anti-tumor effect in a nude mouse/A549 xenograft model. These results suggest that lung cancer cells become resistant to gemcitabine via NF-κB activation and the subsequent overexpression of Bfl-1, and that BC, which has both pro-apoptotic and NF-κB inhibitory effects, could be harnessed as a gene therapy to complement gemcitabine chemotherapy in non-small cell lung cancer.

Ginsenoside Rp1 from Panax ginseng exhibits anti-cancer activity by down-regulation of the IGF-1R/Akt pathway in breast cancer cells.

Cancer prevention is effective and reduces health care costs because cancer is often a preventable disease that can be affected by lifestyle factors. Therefore, researchers are interested in discovering natural compounds that have anticancer activities, such as delaying the development of cancer and preventing its progression. One such natural agent is ginseng (Panax ginseng), which is traditionally used in some parts of the world as a popular remedy for various diseases including cancer. We hypothesized that the ginsenoside Rp1, a component of ginseng, reduces cancer cell proliferation through inhibition of the insulin-like growth factor 1 receptor (IGF-1R)/Akt pathway. We first tested the efficacy of Rp1 against human breast cancer cell lines. Treatment with Rp1 inhibited breast cancer cell proliferation and inhibited both anchorage-dependent and -independent breast cancer cell colony formation. In addition, treatment with 20 µM Rp1 induced cycle arrest and apoptosis-mediated cell growth suppression. Our findings further indicated that Rp1 decreased the stability of the IGF-1R protein in breast cancer cells. Therefore, we suggest that Rp1 has potential as an anticancer drug and that IGF-1R is an important target for treatment and prevention of breast cancer.

Radiation Potentiates Monocyte Infiltration into Tumors by Ninjurin1 Expression in Endothelial Cells.

Radiation is a widely used treatment for cancer patients, with over half the cancer patients receiving radiation therapy during their course of treatment. Considerable evidence from both preclinical and clinical studies show that tumor recurrence gets restored following radiotherapy, due to the influx of circulating cells consisting primarily of monocytes. The attachment of monocyte to endothelial cell is the first step of the extravasation process. However, the exact molecules that direct the transmigration of monocyte from the blood vessels to the tumors remain largely unknown. The nerve injury-induced protein 1 (Ninjurin1 or Ninj1) gene, which encodes a homophilic adhesion molecule and cell surface protein, was found to be upregulated in inflammatory lesions, particularly in macrophages/monocytes, neutrophils, and endothelial cells. More recently Ninj1 was reported to be regulated following p53 activation. Considering p53 has been known to be activated by radiation, we wondered whether Ninj1 could be increased in the endothelial cells by radiation and it might contribute to the recruiting of monocytes in the tumor. Here we demonstrate that radiation-mediated up-regulation of Ninj1 in endothelial cell lines such as human umbilical vein endothelial cells (HUVECs), EA.hy926, and immortalized HUVECs. Consistent with this, we found over-expressed Ninj1 in irradiated xenograft tumors, and increased monocyte infiltration into tumors. Radiation-induced Ninj1 was transcriptionally regulated by p53, as confirmed by transfection of p53 siRNA. In addition, Ninj1 over-expression in endothelial cells accelerated monocyte adhesion. Irradiation-induced endothelial cells and monocyte interaction was inhibited by knock-down of Ninj1. Furthermore, over-expressed Ninj1 stimulated MMP-2 and MMP-9 expression in monocyte cell lines, whereas the MMP-2 and MMP-9 expression were attenuated by Ninj1 knock-down in monocytes. Taken together, we provide evidence that Ninj1 is a key molecule that generates an interaction between endothelial cells and monocytes. This result suggests that radiation-mediated Ninj1 expression in endothelial cells could be involved in the post-radiotherapy recurrence mechanism.

Quercetin Is a Flavonoid Breast Cancer Resistance Protein Inhibitor with an Impact on the Oral Pharmacokinetics of Sulfasalazine in Rats.

The potential inhibitory effect of quercetin, a major plant flavonol, on breast cancer resistance protein (BCRP) activity was investigated in this study. The presence of quercetin significantly increased the cellular accumulation and associated cytotoxicity of the BCRP substrate mitoxantrone in human cervical cancer cells (HeLa cells) in a concentration-dependent manner. The transcellular efflux of prazosin, a stereotypical BCRP substrate, was also significantly reduced in the presence of quercetin in a bidirectional transport assay using human BCRP-overexpressing cells; further kinetic analysis revealed IC50 and Ki values of 4.22 and 3.91 µM, respectively. Moreover, pretreatment with 10 mg/kg quercetin in rats led to a 1.8-fold and 1.5-fold increase in the AUC8h (i.e., 44.5 ± 11.8 min∙µg/mL vs. 25.7 ± 9.98 min∙µg/mL, p < 0.05) and Cmax (i.e., 179 ± 23.0 ng/mL vs. 122 ± 23.2 ng/mL, p < 0.05) of orally administered sulfasalazine, respectively. Collectively, these results provide evidence that quercetin acts as an in vivo as well as in vitro inhibitor of BCRP. Considering the high dietary intake of quercetin as well as its consumption as a dietary supplement, issuing a caution regarding its food-drug interactions should be considered.

Heterodimerization of insulin-like growth factor receptor/epidermal growth factor receptor and induction of survivin expression counteract the antitumor action of erlotinib.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been used to treat non-small cell lung cancer (NSCLC). However, the overall response rate to EGFR TKIs is limited, and the mechanisms mediating resistance to the drugs are poorly understood. Here, we report that insulin-like growth factor-I receptor (IGF-IR) activation interferes with the antitumor activity of erlotinib, an EGFR TKI. Treatment with erlotinib increased the levels of EGFR/IGF-IR heterodimer localized on cell membrane, activated IGF-IR and its downstream signaling mediators, and stimulated mammalian target of rapamycin (mTOR)-mediated de novo protein synthesis of EGFR and survivin in NSCLC cells. Inhibition of IGF-IR activation, suppression of mTOR-mediated protein synthesis, or knockdown of survivin expression abolished resistance to erlotinib and induced apoptosis in NSCLC cells in vitro and in vivo. Our data suggest that enhanced synthesis of survivin protein mediated by the IGFR/EGFR heterodimer counteracts the antitumor action of erlotinib, indicating the needs of integration of IGF-IR-targeted agents to the treatment regimens with EGFR TKI for patients with lung cancer.

Ninjurin1 Plays a Crucial Role in Pulmonary Fibrosis by Promoting Interaction between Macrophages and Alveolar Epithelial Cells.

The transmembrane nerve injury-induced protein 1 (Ninjurin1 or Ninj1) is involved in progressing inflammatory diseases. In this study, we aimed to investigate a novel function of Ninj1 in pulmonary fibrosis. We found that the expression of Ninj1 in a patient cohort was upregulated in the lung specimens of idiopathic pulmonary fibrosis patients as well as mice with bleomycin-induced pulmonary fibrosis. In addition, the BLM-injected Ninj1 KO mice exhibited a mild fibrotic phenotype, as compared to WT mice. Therefore, we hypothesized that Ninj1 would play an important role in the development of pulmonary fibrosis. We discovered that Ninj1 expression increased in BLM-treated macrophages and alveolar epithelial cells (AECs). Interestingly, macrophages bound to BLM-treated AECs were activated. However, when Ninj1 expression was suppressed in either of AECs or macrophages, contact-dependent activation of macrophages with AECs was diminished. In addition, introduction of recombinant mouse Ninj11-50 to macrophages triggered an inflammatory response, but did not stimulate Ninj1-deficient macrophages. In conclusion, we propose that Ninj1 may contribute to activation of macrophages by enhancing interaction with AECs having elevated Ninj1 expression due to injury-inducing stimuli. Consequently, Ninj1 may be involved in the development of pulmonary fibrosis by enhancing inflammatory response of macrophages.

Essential role of Ahnak in adipocyte differentiation leading to the transcriptional regulation of Bmpr1α expression.

The role of Ahnak in obesity has been reported previously. Loss of Ahnak leads to decreased Bmp4/Smad1 signaling, resulting in the downregulation of adipocyte differentiation. However, the biological significance of Ahnak remains largely unknown. In this study, we demonstrate that Ahnak-mediated impaired adipogenesis results in decreased Bmpr1α transcriptional expression. To confirm this, Ahnak siRNA was used to knock-down Ahnak in C3H10T1/2 and primary stromal vascular fraction cells. Ahnak siRNA transfected cells showed suppression of Bmpr1α expression and decreased BMP4/ Bmpr1α signaling. The differential adipogenesis was further confirmed by knock-down of Bmpr1α in C3H10T1/2 cells, which resulted in reduced adipogenesis. Moreover, stable Ahnak knock-out C3H10T1/2 cells stably transfected with Ahnak CRISPR/Cas9 plasmid suppressed expression of Bmpr1α and prevented differentiation into adipocytes. Furthermore, we developed immortalized pre-adipocytes from wild-type or Ahnak Knock-out mice's stromal vascular fraction (SVF) to confirm the function of Ahnak in pre-adipocyte transition. Immortalized Ahnak knock-out SVF cells showed lower level of Bmpr1α expression, evidence by their impaired BMP4/Bmpr1α signaling. Upon adipogenic induction, immortalized Ahnak knock-out SVF cells exhibited a marked decrease in adipocyte differentiation compared with immortalized wild-type pre-adipocytes. Furthermore, over-expression of Bmpr1α restored the adipogenic activity of Ahnak knock-out C3H10T1/2 cells and immortalized Ahnak knock-out SVF cells. Our data reveal the missing link in Ahnak-mediated adipose tissue remodeling and suggest that precise regulation of Ahnak in adipose tissue might have a therapeutic advantage for metabolic disease treatment.

AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia and Lung Tumor Development.

AHNAK is known to be a tumor suppressor in breast cancer due to its ability to activate the TGFß signaling pathway. However, the role of AHNAK in lung tumor development and progression remains unknown. Here, the Ahnak gene was disrupted to determine its effect on lung tumorigenesis and the mechanism by which it triggers lung tumor development was investigated. First, AHNAK protein expression was determined to be decreased in human lung adenocarcinomas compared with matched nonneoplastic lung tissues. Then, Ahnak -/- mice were used to investigate the role of AHNAK in pulmonary tumorigenesis. Ahnak -/- mice showed increased lung volume and thicker alveolar walls with type II pneumocyte hyperplasia. Most importantly, approximately 20% of aged Ahnak -/- mice developed lung tumors, and Ahnak -/- mice were more susceptible to urethane-induced pulmonary carcinogenesis than wild-type mice. Mechanistically, Ahnak deficiency promotes the cell growth of lung epithelial cells by suppressing the TGFß signaling pathway. In addition, increased numbers of M2-like alveolar macrophages (AM) were observed in Ahnak -/- lungs, and the depletion of AMs in Ahnak -/- lungs alleviated lung hyperplastic lesions, suggesting that M2-like AMs promoted the progression of lung hyperplastic lesions in Ahnak-null mice. Collectively, AHNAK suppresses type II pneumocyte proliferation and inhibits tumor-promoting M2 alternative activation of macrophages in mouse lung tissue. These results suggest that AHNAK functions as a novel tumor suppressor in lung cancer.Implications: The tumor suppressor function of AHNAK, in murine lungs, occurs by suppressing alveolar epithelial cell proliferation and modulating lung microenvironment. Mol Cancer Res; 16(8); 1287-98. ©2018 AACR.

SH003 reverses drug resistance by blocking signal transducer and activator of transcription 3 (STAT3) signaling in breast cancer cells.

Overcoming drug resistance is an important task for investigators and clinician to achieve successful chemotherapy in cancer patients. Drug resistance is caused by various factors, including the overexpression of P-glycoprotein (P-gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. SH003 is extracted from the mixture of three different herbs, and its anticancer effect has been revealed in different cancer cell types. In the present study, we investigated whether SH003 is able to reverse drug resistance using paclitaxel-resistant breast cancer cells (MCF-7/PAC). In our experiments, SH003 significantly decreased cell growth and colony formation in MCF-7/PAC cells and parental MCF-7 cells. This growth inhibition was related to the accumulation of cells in the sub-G0/G1 apoptotic population and an increase in the number of apoptotic cells. SH003 reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated proteins (MRPs) in MCF-7/PAC cells. SH003 also down-regulated the expression of P-gp. SH003 reversed drug efflux from MCF-7/PAC cells, resulting in rhodamine123 (Rho123) accumulation. Inhibition of drug resistance by SH003 is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. SH003 decreased STAT3 activation (p-STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP-2, which are STAT3 target genes. An STAT3 inhibitor, JAK inhibitor I and an HIF-1α inhibitor decreased cell growth in MCF-7 and MCF-7/PAC cells. Taken together, these results demonstrate that SH003 can overcome drug resistance, and SH003 might be helpful for chemotherapy in cancer patients.

Lectin, Galactoside-Binding Soluble 3 Binding Protein Promotes 17-N-Allylamino-17-demethoxygeldanamycin Resistance through PI3K/Akt Pathway in Lung Cancer Cell Line.

Heat shock protein 90 (HSP90) stabilizing oncoproteins has been an attractive target in cancer therapy. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, was tested in phase II/III clinical trials, but due to lack of efficacy, clinical evaluation of 17-AAG has achieved limited success, which led to resistance to 17-AAG. However, the mechanism of 17-AAG resistance has not clearly been identified. Here, we identified LGALS3BP (Lectin, galactoside-binding soluble 3 binding protein), a secretory glycoprotein, as a 17-AAG resistance factor. In the clinical reports, it was suggested that LGALS3BP was associated with low survival rate, development of cancer progression, and enhancement of metastasis in human cancers. As we confirmed that the LGALS3BP level was increased in 17-AAG-resistant cells (H1299_17R) compared with that of the parental cell line (H1299_17P), knockdown of LGALS3BP expression increased sensitivity to 17-AAG in H1299_17R cells. Overexpression of LGALS3BP also augmented PI3K/Akt and ERK signaling pathways. Furthermore, we determined that the PI3K/Akt signaling pathway was involved in LGALS3BP-mediated 17-AAG resistance in vitro and in vivo, demonstrating that LGALS3BP mediates the resistance against 17-AAG through PI3K/Akt activation rather than ERK activation. These findings suggest that LGALS3BP would be a target to overcome resistance to 17-AAG in lung cancer. For example, the combination of 17-AAG and PI3K/Akt inhibitor would effectively suppress acquired resistance to 17-AAG. In conclusion, targeting of LGALS3BP-mediated-specific survival signaling pathway in resistant cells may provide a novel therapeutic model for the cancer therapy. Mol Cancer Ther; 16(7); 1355-65. ©2017 AACR.

Daurinol blocks breast and lung cancer metastasis and development by inhibition of focal adhesion kinase (FAK).

FAK overexpression has been reported in diverse primary and metastatic tumor tissues, supporting its pro-tumorigenic and pro-metastatic roles. Therefore, we have developed a neo-treatment strategy using daurinol to effectively treat cancer metastasis. Daurinol blocked cancer cell migration and invasion in vitro and exhibited anti-metastatic activity in an experimental metastasis model of breast and lung cancer. Daurinol selectively inhibited phosphorylation of FAK at Tyr925, Tyr576/577, and Tyr397 sites in a dose- and time-dependent manner. Daurinol effectively suppressed migration and invasion of MDA-MB-231 and A549 cancer cells. These data were associated with inhibition of expression and secretion of invasion factors, including matrix metalloproteinase (MMP) 2, MMP9, and urokinase plasminogen activator (uPA). Consistent with these in vitro results, daurinol (10 mg/kg; Oral gavage) effectively inhibited breast and lung cancer metastasis in a mouse model. In addition, daurinol showed strong suppressive activity of cell survival as revealed by colony formation assays. Analysis of cellular phenotypes revealed that inhibition of FAK phosphorylation in cancer cells limited colony formation, cell migration, and invasion, thereby reducing the cell proliferation rate. Furthermore, daurinol significantly reduced tumor development in 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)/benzo(a)pyrene (BaP)-treated A/J mice. Our results suggest that daurinol suppresses lung metastasis through inhibition of migration and survival via blockade of FAK activity.