Hypoxia‐inducible factor‐1α and nuclear factor‐κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non‐small‐cell lung cancer cells

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

Repression of NF‐κB and activation of AP‐1 enhance apoptosis in prostate cancer cells

TNFα and TRAIL, 2 members of the tumor necrosis factor family, share many common signaling pathways to induce apoptosis. Although many cancer cells are sensitive to these proapoptotic agents, some develop resistance. Recently, we have demonstrated that upregulation of c‐Fos/AP‐1 is necessary, but insufficient for cancer cells to undergo TRAIL‐induced apoptosis. Here we present a prostate cancer model with differential sensitivity to TNFα and TRAIL. We show that inhibition of NF‐κB or activation of AP‐1 can only partially sensitize resistant prostate cancer cells to proapoptotic effects of TNFα or TRAIL. Inhibition of NF‐κB by silencing TRAF2, by silencing RIP or by ectopic expression of IκB partially sensitized resistant prostate cancer. Similarly, activation of c‐Fos/AP‐1 only partially sensitized resistant cancer cells to proapoptotic effects of TNFα or TRAIL. However, concomitant repression of NF‐κB and activation of c‐Fos/AP‐1 significantly enhanced the proapoptotic effects of TNFα and TRAIL in resistant prostate cancer cells. Therefore, multiple molecular pathways may need to be modified, to overcome cancers that are resistant to proapoptotic therapies. © 2008 Wiley‐Liss, Inc.     

Endoplasmic reticulum stress pathway PERK‐eIF2α confers radioresistance in oropharyngeal carcinoma by activating NF‐κB

Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis and development of malignant tumors, as well as in the regulation of radiochemoresistance and chemoresistance in many malignancies. ERS signaling pathway protein kinase RNA‐like endoplasmic reticulum kinase (PERK)‐eukaryotic initiation factor‐2 (eIF2α) may induce aberrant activation of nuclear factor‐κB (NF‐κB). Our previous study showed that NF‐κB conferred radioresistance in lymphoma cells. However, whether PERK‐eIF2α regulates radioresistance in oropharyngeal carcinoma through NF‐κB activation is unknown. Herein, we showed that PERK overexpression correlated with a poor prognosis for patients with oropharyngeal carcinoma (P < 0.01). Meanwhile, the percentage of the high expression level of PERK in oropharyngeal carcinoma patients resistant to radiation was higher than in patients sensitive to radiation (77.7 and 33.3%, respectively; P < 0.05). Silencing PERK and eIF2α increased the radiosensitivity in oropharyngeal carcinoma cells and increased radiation‐induced apoptosis and G2/M phase arrest. PERK‐eIF2α silencing also inhibited radiation‐induced NF‐κB phosphorylation and increased the DNA double strand break‐related proteins ATM phosphorylation. NF‐κB activator TNF‐α and the ATM inhibitor Ku55933 offset the regulatory effect of eIF2α on the expression of radiation‐induced cell apoptosis‐related proteins and the G2/M phase arrest‐related proteins. These data indicate that PERK regulates radioresistance in oropharyngeal carcinoma through NF‐kB activation‐mediated phosphorylation of eIF2α, enhancing X‐ray‐induced activation of DNA DSB repair, cell apoptosis inhibition and G2/M cell cycle arrest.     

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)

NADPH oxidase/dual‐oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa‐B (NFκB)‐mediated inflammation and inflammation‐associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). In the PhIP‐induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB‐p50 and NFκB‐p65 were all highly overexpressed compared with their levels in adjacent normal‐looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco‐2 cells there was a strong apoptotic response, with increased levels of cleaved caspase‐3, ‐6, ‐7 and poly(ADP‐ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide‐induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP‐induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.     

Targeting nuclear factor‐κB suppresses the negative effect of Toll‐like receptor 4 signaling on antimetastasis therapy based on targeting αvβ3

The targeting of αvβ3 is a promising therapeutic strategy for suppressing tumor metastasis. However, it is unclear whether the therapeutic efficacy could be influenced by metastasis‐promoting factor(s) in vivo. Here we report that Toll‐like receptor 4 (TLR4) ligand released from damaged tumor cells or bacteria had a negative effect on the therapeutic effect of a recombinant CBD‐HepII polypeptide of fibronectin (CH50) that suppresses tumor metastasis by targeting αvβ3. The TLR4 ligand could antagonize the inhibitory effect of CH50 on tumor cell adhesion and invasion by promoting the expression and activity of αvβ3 in tumor cells. The TLR4 ligand also reduced the antimetastasis effect of CH50 by promoting tumor cell survival in circulation. Moreover, TLR4 ligands released by tumor cells in circulation could increase the survival and proliferation capacity of tumor cells after extravasation, resulting in the formation of more metastatic nodules. The effect of TLR4 signaling was mainly mediated by nuclear factor‐κB (NF‐κB). Inhibiting NF‐κB could abrogate the negative effect of TLR4 ligand, and augment the inhibitory effect of CH50 on tumor metastasis. Consistently, the combination of NF‐κB inhibitor and CH50 significantly inhibited metastasis of tumor cells in vivo and prolonged the survival of mice. The findings in this study suggest that the combination of NF‐κB inhibitor and αvβ3 antagonist would be a novel therapeutic option for the prevention of tumor metastasis. (Cancer Sci 2012; 103: 1319–1326)     

Oncogenic activity of BIRC2 and BIRC3 mutants independent of nuclear factor‐κB‐activating potential

BIRC2 and BIRC3 are closely related members of the inhibitor of apoptosis (IAP) family of proteins and play pivotal roles in regulation of nuclear factor‐κB (NF‐κB) signaling and apoptosis. Copy number loss for and somatic mutation of BIRC2 and BIRC3 have been frequently detected in lymphoid malignancies, with such genetic alterations being thought to contribute to carcinogenesis through activation of the noncanonical NF‐κB signaling pathway. Here we show that BIRC2 and BIRC3 mutations are also present in a wide range of epithelial tumors and that most such nonsense or frameshift mutations confer direct transforming potential. This oncogenic function of BIRC2/3 mutants is largely independent of their ability to activate NF‐κB signaling. Rather, all of the transforming mutants lack an intact RING finger domain, with loss of ubiquitin ligase activity being essential for transformation irrespective of NF‐κB regulation. The serine‐threonine kinase NIK was found to be an important, but not exclusive, mediator of BIRC2/3‐driven carcinogenesis, although this function was independent of NF‐κB activation. Our data thus suggest that, in addition to the BIRC2/3–NIK–NF‐κB signaling pathway, BIRC2/3–NIK signaling targets effectors other than NF‐κB and thereby contributes directly to carcinogenesis. Identification of these effectors may provide a basis for the development of targeted agents for the treatment of lymphoid malignancies and other cancers with BIRC2/3 alterations.     

Retinoid X receptor α enhances human cholangiocarcinoma growth through simultaneous activation of Wnt/β‐catenin and nuclear factor‐κB pathways

Retinoid X receptor α (RXRα) plays important roles in the malignancy of several cancers such as human prostate tumor, breast cancer, and thyroid tumor. However, its exact functions and molecular mechanisms in cholangiocarcinoma (CCA), a chemoresistant carcinoma with poor prognosis, remain unclear. In this study we found that RXRα was frequently overexpressed in human CCA tissues and CCA cell lines. Downregulation of RXRα led to decreased expression of mitosis‐promoting factors including cyclin D1and cyclin E, and the proliferating cell nuclear antigen, as well as increased expression of cell cycle inhibitor p21, resulting in inhibition of CCA cell proliferation. Furthermore, RXRα knockdown attenuated the expression of cyclin D1 through suppression of Wnt/β‐catenin signaling. Retinoid X receptor α upregulated proliferating cell nuclear antigen expression through nuclear factor‐κB (NF‐κB) pathways, paralleled with downregulation of p21. Thus, the Wnt/β‐catenin and NF‐κB pathways account for the inhibition of CCA cell growth induced by RXRα downregulation. Retinoid X receptor α plays an important role in proliferation of CCA through simultaneous activation of Wnt/β‐catenin and NF‐κB pathways, indicating that RXRα might serve as a potential molecular target for CCA treatment.     

High expression levels of IKKα and IKKβ are necessary for the malignant properties of liver cancer

IKK‐NF‐κB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF‐κB signaling including IKKα, IKKβ, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c‐FLIP, Bcl‐xl, Stat3, Cip1 and Cip2 by real‐time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKα, IKKβ, RANK, Maspin, c‐FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKα and IKKβ were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC‐97L and MHCC‐97H in vitro, the numbers of BrdU positive cells were decreased in both IKKα and IKKβ knockdown cells. Levels of apoptosis were also investigated in IKKα and IKKβ knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKα and IKKβ in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl‐XL and c‐FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKα and IKKβ downregulation depends on extent of NF‐κB inhibition and the malignant nature of the HCC. We anticipate that IKK‐targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.     

Expression of platelet‐derived growth factor (PDGF)‐B and PDGF‐receptor β is associated with lymphatic metastasis in human gastric carcinoma

Recent study of murine fibrosarcoma has revealed that platelet‐derived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF‐R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF‐B and PDGF‐Rβ expression in four human gastric carcinoma cell lines (TMK‐1, MKN‐1, MKN‐45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF‐B and PDGF‐Rβ expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK‐1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF‐R tyrosine kinase inhibitor imatinib. PDGF‐B and PDGF‐Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse‐type carcinoma than in intestinal‐type carcinoma. In surgical specimens, tumor cells expressed PDGF‐B, but PDGF‐Rβ was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF‐B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK‐1 tumors, cancer cells expressed PDGF‐B but not PDGF‐Rβ. PDGF‐Rβ was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF‐B by gastric carcinoma cells and expression of PDGF‐Rβ by tumor‐associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF‐R signaling pathways may inhibit lymph node metastasis of gastric carcinoma. (Cancer Sci 2010)     

Up‐regulation of pro‐inflammatory genes as adaptation to hypoxia in MCF‐7 cells and in human mammary invasive carcinoma microenvironment

The role of tumor cells in synthesizing pro‐inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF‐7 human mammary adenocarcinoma cell line induced activation of hypoxia‐inducible factor 1α (HIF‐1α) and nuclear factor‐kappa B (NF‐κB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up‐regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute‐phase protein pentraxin‐3 (PTX3). Hypoxia also stimulated chemokine (C‐X‐C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal‐derived factor‐1α (SDF‐1α) increased invasion and migration of hypoxic MCF‐7 cells. Inhibition of HIF‐1α by chetomin and NF‐κB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF‐7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser‐capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF‐1α and NF‐κB and up‐regulation of IR, CXCR4, estrogen receptor α (ERα), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF‐7 cells acquire a pro‐inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF‐1α, NF‐κB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells. (Cancer Sci 2010; 101: 1014–1023)