Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1ß (IL-1ß) production and mtDNA release in mice.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Oncogenic Y-box binding protein-1 as an effective therapeutic target in drug-resistant cancer.

Y-box binding protein-1 (YBX1), a multifunctional oncoprotein containing an evolutionarily conserved cold shock domain, dysregulates a wide range of genes involved in cell proliferation and survival, drug resistance, and chromatin destabilization by cancer. Expression of a multidrug resistance-associated ATP binding cassette transporter gene, ABCB1, as well as growth factor receptor genes, EGFR and HER2/ErbB2, was initially discovered to be transcriptionally activated by YBX1 in cancer cells. Expression of other drug resistance-related genes, MVP/LRP, TOP2A, CD44, CD49f, BCL2, MYC, and androgen receptor (AR), is also transcriptionally activated by YBX1, consistently indicating that YBX1 is involved in tumor drug resistance. Furthermore, there is strong evidence to support that nuclear localization and/or overexpression of YBX1 can predict poor outcomes in patients with more than 20 different tumor types. YBX1 is phosphorylated by kinases, including AKT, p70S6K, and p90RSK, and translocated into the nucleus to promote the transcription of resistance- and malignancy-related genes. Phosphorylated YBX1, therefore, plays a crucial role as a potent transcription factor in cancer. Herein, a novel anticancer therapeutic strategy is presented by targeting activated YBX1 to overcome drug resistance and malignant progression.

N-myc downstream-regulated gene 1 promotes tumor inflammatory angiogenesis through JNK activation and autocrine loop of interleukin-1α by human gastric cancer cells.

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.

Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.

The antitumor effect of a novel angiogenesis inhibitor (an octahydronaphthalene derivative) targeting both VEGF receptor and NF-κB pathway.

Development of a novel type of angiogenesis inhibitor will be essential for further improvement of therapeutics against cancer patients. We examined whether an octahydronaphthalene derivative, AMF-26, which was screened as an inhibitor of intercellular adhesion molecule-1 (ICAM-1) production stimulated by inflammatory stimuli in vascular endothelial cells, could block angiogenesis in response to vascular endothelial growth factor (VEGF) and/or inflammatory cytokines. Low dose AMF-26 effectively inhibited the tumor necrosis factor-α (TNF-α)- or the interleukin-1ß (IL-1ß)-induced production of ICAM-1 in human umbilical vascular endothelial cells (HUVECs). We found that the TNF-α-induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and nuclear translocation of p65 were impaired by AMF-26 in both endothelial cells and cancer cells. AMF-26 was found to inhibit the phosphorylation of VEGF receptor 1 (VEGFR1), VEGFR2 and the downstream signaling molecules Akt, extracellular signal-regulated kinase (ERK)1/2 stimulated by VEGF in HUVECs. Therefore, the VEGF-induced proliferation, migration and tube formation of vascular endothelial cells was highly susceptible to inhibition by AMF-26. Oral administration of AMF-26 significantly blocked VEGF- or IL-1ß-induced angiogenesis in the mouse cornea, and also tumor angiogenesis and growth. Together, our results indicate that AMF-26 inhibits angiogenesis through suppression of both VEGFR1/2 and nuclear factor-κB (NF-κB) signaling pathways when stimulated by VEGF or inflammatory cytokines. AMF-26 could be a promising novel candidate drug for cancer treatments.

Macrophage infiltration predicts a poor prognosis for human ewing sarcoma.

Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.

AXL/CDCP1/SRC axis confers acquired resistance to osimertinib in lung cancer.

Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

Identification of sites subjected to serine/threonine phosphorylation by SGK1 affecting N-myc downstream-regulated gene 1 (NDRG1)/Cap43-dependent suppression of angiogenic CXC chemokine expression in human pancreatic cancer cells.

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Four new cembrane diterpenes isolated from an Okinawan soft coral Lobophytum crassum with inhibitory effects on nitric oxide production.

Four new cembrane diterpenes (1-4) and fifteen known cembranoids (5-19) were isolated from an Okinawan soft coral Lobophytum crassum. The structures of these four new cembranoids were determined on the basis of spectroscopic evidence. In particular, the absolute stereochemistry of 1, 2, 5 and 6 were elucidated by the application of the modified Mosher's method and circular dichroism (CD) spectral data. The inhibitory effects of some isolates were evaluated on nitric oxide (NO) production against a murine macrophage-like cell line (Raw 264.7). Cembranoids consisting of alpha-methylene-gamma-lactone, exhibited the significant effect on NO production.

Selective Covalent Targeting of Mutated EGFR(T790M) with Chlorofluoroacetamide-Pyrimidines.

Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.

NDRG1 activates VEGF-A-induced angiogenesis through PLCγ1/ERK signaling in mouse vascular endothelial cells.

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1-/- mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.

Bidirectional Regulation between NDRG1 and GSK3ß Controls Tumor Growth and Is Targeted by Differentiation Inducing Factor-1 in Glioblastoma.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3ß levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3ß. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3ß levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3ß phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3ß expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3ß signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. SIGNIFICANCE: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

Targeting Phosphorylation of Y-Box-Binding Protein YBX1 by TAS0612 and Everolimus in Overcoming Antiestrogen Resistance.

Nuclear expression of Y-box-binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.

The augmented expression of the cytidine deaminase gene by 5-azacytidine predicts therapeutic efficacy in myelodysplastic syndromes.

5-Azacytidine (5AC), a hypomethylating agent, is clinically used for the treatment of patients with myelodysplastic syndromes (MDS). Cytidine deaminase (CDA) is a key enzyme in the detoxification of 5AC. We investigated whether the CDA expression could predict response to 5AC in MDS. Among leukemia-derived cell lines, MDS-L, an MDS-derived cell line with a relatively low CDA expression level, was found to be the most sensitive to 5AC. Combination with tetrahydrouridine, an inhibitor of CDA, synergistically potentiated the cytotoxic effect of 5AC. Treatment with 5AC markedly enhanced the expression level of CDA mRNA and showed demethylation at CpG sites in the 5'-flanking region of the CDA gene. We further compared the protein expression levels of CDA in matched clinical samples before and after treatment with 5AC in bone marrow cells from 8 MDS patients by an immunohistochemical analysis. The CDA expression level showed an approximately 2- to 3-fold increase after 5AC treatment in 3 of these cases, and these three patients with relatively higher CDA expression levels after 5AC treatment all showed better clinical responses to 5AC. In contrast, the 5 remaining patients, whose CDA expression showed no augmentation, observed no clinical benefit. Taken together, the optimized determination of the CDA expression levels before and after 5AC treatment, and the methylation status at CpG sites of 5'-flanking region of the CDA gene, may contribute to the development of precise 5AC therapy for MDS.

Role of macrophages in inflammatory lymphangiogenesis: Enhanced production of vascular endothelial growth factor C and D through NF-kappaB activation.

The close association of inflammation, angiogenesis and cancer progression is now highlighted, and in this study we especially focused on a close association of inflammation and lymphangiogenesis. We found that proinflammatory cytokine, interleukin-1beta (IL-1beta), could induce lymphangiogenesis in mouse cornea through enhanced production of potent lymphangiogenic factors, VEGF-A, VEGF-C and VEGF-D. IL-1beta-induced lymphangiogenesis, but not angiogenesis, was inhibited by administration of a selective anti-VEGF receptor-3 (VEGFR-3) neutralizing antibody. And in mouse cornea we observed recruitment of monocyte/macrophages and neutrophils by IL-1beta implanted cornea. Depletion of macrophages by a bisphosphonate encapsulated in liposomes inhibited this IL-1beta-induced lymphangiogenesis and also up-regulation of VEGF-A, VEGF-C, and VEGF-D. Furthermore, IL-1beta-induced lymphangiogenesis and angiogenesis were suppressed by NF-kappaB inhibition with marked suppression of VEGF-A, VEGF-C, and VEGF-D expression.

Y-box binding protein YBX1 and its correlated genes as biomarkers for poor outcomes in patients with breast cancer.

The enhanced expression of the Y-box binding protein YBX1 is consistently correlated with poor outcomes or reduced survival of breast cancer patients. However, the mechanism underlying the association between increased YBX1 expression and poor outcomes has yet to be revealed. We searched a database for the top 500 genes that are positively or negatively correlated with YBX1 and with ESR1 in breast cancer patients. We further examined the association between YBX1-correlated genes and breast cancer outcomes in patients at Kyushu University Hospital. More than 60% of genes that are positively correlated with YBX1 are also negatively correlated with ESR1. The enhanced expression levels of the top 20 positively correlated genes mostly predict negative outcomes, while the enhanced expression levels of the top 20 negatively correlated genes mostly predict positive outcomes. Furthermore, in breast cancer patients at Kyushu University Hospital, the expression levels of YBX1 and YBX1-positively correlated genes were significantly higher and the expression levels of genes negatively correlated with YBX1 were significantly lower in patients who relapsed after their primary surgery than in those who did not relapse. The expression of YBX1 together with the expression of its positively or negatively correlated genes may help to predict outcomes as well as resistance to endocrine therapies in breast cancer patients. Determining the expression of YBX1 and its closely correlated genes will contribute to the development of precision therapeutics for breast cancer.

Inflammatory stimuli from macrophages and cancer cells synergistically promote tumor growth and angiogenesis.

The focus of the present study was whether and how infiltrating macrophages play a role in angiogenesis and the growth of cancer cells in response to the inflammatory cytokine interleukin (IL)-1beta. Lewis lung carcinoma cells overexpressing IL-1beta grew faster and induced greater neovascularization than a low IL-1beta-expressing counterpart in vivo. When macrophages were depleted using clodronate liposomes, both neovascularization and tumor growth were reduced in the IL-1beta-expressing tumors. Co-cultivation of IL-1beta-expressing cancer cells with macrophages synergistically augmented neovascularization and the migration of vascular endothelial cells. In these co-cultures, production of the angiogenic factors vascular endothelial growth factor-A and IL-8, monocyte chemoattractant protein-1, and matrix metalloproteinase-9 were increased markedly. The production of these factors, induced by IL-1beta-stimulated lung cancer cells, was blocked by a nuclear factor (NF)-kappaB inhibitor, and also by the knockdown of p65 (NF-kappaB) and c-Jun using small interference RNA, suggesting involvement of the transcription factors NF-kappaB and AP-1. These results demonstrated that macrophages recruited into tumors by monocyte chemoattractant protein-1 and other chemokines could play a critical role in promoting tumor growth and angiogenesis, through interactions with cancer cells mediated by inflammatory stimuli.

The activation of SRC family kinases and focal adhesion kinase with the loss of the amplified, mutated EGFR gene contributes to the resistance to afatinib, erlotinib and osimertinib in human lung cancer cells.

Second- and third-generation inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity (EGFR-TKIs) are improving the treatment of patients with non-small cell lung cancer. Here we established two sublines (BR1-8 and BR2-3) resistant to a second-generation inhibitor, afatinib, from the human lung cancer cell line HCC827 that harbors a mutation that activates the tyrosine kinase activity of EGFR. These afatinib-resistant sublines were resistant to first-generation EGFR-TKIs, gefitinib and erlotinib, and a third-generation EGFR-TKI, osimertinib. These resistant sublines showed markedly reduced levels of multiple EGFR family proteins, including the activated mutant EGFR, and complete loss of EGFR amplification as compared with their parental HCC827 cells harboring amplification of EGFR gene. Treatment with the multikinase inhibitor dasatinib or transfection with a SRC small interfering RNA inhibited cell survival and AKT phosphorylation in drug-resistant sublines to a greater extent compared with HCC827 cells. Further, the migration of drug-resistant cells was greater compared with that of HCC827 cells and was inhibited by dasatinib or an FAK inhibitor. These findings indicate that compensatory activation of SRC family kinases (SFKs) and FAK supports the survival and migration of afatinib-resistant cells when the expression of multiple EGFR family proteins was mostly abrogated. Combinations of potent drugs that target SFKs and FAK may overcome the resistance of lung cancer cells to second-generation TKIs.

Breast Cancer Resistance to Antiestrogens Is Enhanced by Increased ER Degradation and ERBB2 Expression.

Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545-56. ©2016 AACR.