Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.     

Dvl2 facilitates the coordination of NF‐κB and Wnt signaling to promote colitis‐associated colorectal progression

Colitis‐associated colorectal cancer (CAC) arises due to prolonged inflammation and has distinct molecular events compared with sporadic colorectal cancer (CRC). Although inflammatory NF‐κB signaling was activated by pro‐inflammatory cytokines (such as TNFα) in early stages of CAC, Wnt/β‐catenin signaling later appears to function as a key regulator of CAC progression. However, the exact mechanism responsible for the cross‐regulation between these 2 pathways remains unclear. Here, we found reciprocal inhibition between NF‐κB and Wnt/β‐catenin signaling in CAC samples, and the Dvl2, an adaptor protein of Wnt/β‐catenin signaling, is responsible for NF‐κB inhibition. Mechanistically, Dvl2 interacts with the C‐terminus of tumor necrosis factor receptor 1 (TNFRI) and mediates TNFRI endocytosis, leading to NF‐κB signal inhibition. In addition, increased infiltration of the pro‐inflammatory cytokine interleukin‐13 (IL‐13) is responsible for upregulating Dvl2 expression through STAT6. Targeting STAT6 effectively decreases Dvl2 levels and restrains colony formation of cancer cells. These findings demonstrate a unique role for Dvl2 in TNFRI endocytosis, which facilitates the coordination of NF‐κB and Wnt to promote CAC progression.     

IKK phosphorylation of NF-κB at serine 536 contributes to acquired cisplatin resistance in head and neck squamous cell cancer

Current treatment methods for advanced head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy and chemotherapy. For recurrent and metastatic HNSCC, cisplatin is the most common treatment option, but most of patients will eventually develop cisplatin resistance. Therefore, it is imperative to define the mechanisms involved in cisplatin resistance and find novel therapeutic strategies to overcome this deadly disease. In order to determine the role of nuclear factor-kappa B (NF-κB) in contributing to acquired cisplatin resistance in HNSCC, the expression and activity of NF-κB and its upstream kinases, IKKα and IKKβ, were evaluated and compared in three pairs of cisplatin sensitive and resistant HNSCC cell lines, including a pair of patient derived HNSCC cell line. The experiments revealed that NF-κB p65 activity was elevated in cisplatin resistant HNSCC cells compared to that in their parent cells. Importantly, the phosphorylation of NF-κB p65 at serine 536 and the phosphorylation of IKKα and IKKβ at their activation loops were dramatically elevated in the resistant cell lines. Furthermore, knockdown of NF-κB or overexpression of p65-S536 alanine (p65-S536A) mutant sensitizes resistant cells to cisplatin. Additionally, the novel IKKβ inhibitor CmpdA has been shown to consistently block the phosphorylation of NF-κB at serine 536 while also dramatically improving the efficacy of cisplatin in inhibition of cell proliferation and induction of apoptosis in the cisplatin resistant cancer cells. These results indicated that IKK/NF-κB plays a pivotal role in controlling acquired cisplatin resistance and that targeting the IKK/NF-κB signaling pathway may provide a possible therapeutic method to overcome the acquired resistance to cisplatin in HNSCC.     

Dual role of ERK2/NF-κB signaling in TRAIL sensitivity

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.     

CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine–glycine–aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3^low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbβ3 activation appears to be a prerequisite for proper α2β1 activation. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulates α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.     

IL-32θ gene expression in acute myeloid leukemia suppresses TNF-α production

The proinflammatory cytokine TNF-α is highly expressed in patients with acute myeloid leukemia (AML) and has been demonstrated to induce rapid proliferation of leukemic blasts. Thus suppressing the production of TNF-α is important because TNF-α can auto-regulate own expression through activation of NF-κB and p38 mitogen-activated protein kinase (MAPK). In this study, we focused on the inhibitory effect of IL-32θ on TNF-α production in acute myeloid leukemia. Approximately 38% of patients with AML express endogenous IL-32θ, which is not expressed in healthy individuals. Furthermore, plasma samples were classified into groups with or without IL-32θ; then, we measured proinflammatory cytokine TNF-α, IL-1β, and IL-6 levels. TNF-α production was not increased in patients with IL-32θ expression than that in the no-IL-32θ group. Using an IL-32θ stable expression system in leukemia cell lines, we found that IL-32θ attenuated phorbol 12-myristate 13-acetate (PMA)-induced TNF-α production. IL-32θ inhibited phosphorylation of p38 MAPK, inhibitor of κB (IκB), and nuclear factor κB (NF-κB), which are key positive regulators of TNF-α expression, and inhibited nuclear translocation of NF-κB. Moreover, the presence of IL-32θ attenuated TNF-α promoter activity and the binding of NF-κB with the TNF-α promoter. In addition, IL-32γ-induced TNF-α production has no correlation with inhibition of TNF-α via IL-32θ expression. Thus, IL-32θ may serve as a potent inhibitor of TNF-α in patients with AML.     

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer

Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.     

TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases

NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.