Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well-established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared to the normal pancreas and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette-smoke-extract (CSE) and nicotine, which is the major component of CS, significantly up-regulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. Additionally, we demonstrated that nicotine-mediated MUC4 up-regulation promotes the PC cell migration through the activation of the downstream effectors such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in-vivo studies demonstrated a dramatic increase in the mean pancreatic tumor weight [low-dose (100 mg/m3 TSP), p=0.014; high-dose (247 mg/m3 TSP), p=0.02] and significant tumor metastasis to various distant organs in the CS-exposed-mice, orthotopically implanted with luciferase-transfected PC cells, as compared to the sham-controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine [low-dose, 155.88±35.96 ng/ml; high-dose, 216.25±29.95 ng/ml] and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS up-regulates the MUC4 mucin in PC via α7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of pancreatic cancer.
MUC4; Mucin; Cigarette-smoke; Nicotine; Metastasis and Pancreatic Cancer
Smoking is a significant risk factor for pancreatic cancer, but the molecular mechanisms by which tobacco smoke components promote the growth and progression of these cancers are not fully understood. While nicotine, the addictive component of tobacco smoke, is not a carcinogen, it has been shown to promote the growth of non-small cell lung and pancreatic cancers in a receptor-dependent fashion. Here, we show that stimulation of pancreatic cancer cells with nicotine concentrations that are within the range of human exposure results in activation of Src kinase, which facilitated the induction of the inhibitor of differentiation-1 (Id1) transcription factor. Depletion of Id1 prevented nicotine-mediated induction of proliferation and invasion of pancreatic cancer cells, indicating that it is a major mediator of nicotine function. Nicotine could promote the growth and metastasis of pancreatic cancers orthotopically implanted into SCID mice; in addition, cells stably expressing a short hairpin RNA for Id1 did not grow or metastasize in response to nicotine. Nicotine could also confer resistance to apoptosis induced by gemcitabine in pancreatic cancer cells in vitro and depletion of Src or Id1 rendered the cells sensitive to gemcitabine. Further, nicotine could effectively inhibit the chemotherapeutic effects of gemcitabine on pancreatic tumors xenografted into mice. Clinical analyses of resected pancreatic cancer specimens demonstrated a statistically significant correlation between Id1 expression and phospho-Src, tumor grade/differentiation, and worsening overall patient survival. These results demonstrate that exposure to tobacco smoke components might promote pancreatic cancer progression, metastasis, and chemoresistance and highlight the role of Id1 in these processes.
Atherosclerosis involves a specialized inflammatory process regulated by an intricate network of cytokine and chemokine signaling. Atherosclerotic lesions lead to the release of cytokines that can have multiple affects on various vascular cell functions either promoting lesion expansion or alternatively retard progression. TNF-α is one such cytokine that can activate both cell survival and cell death mechanisms simultaneously. Here we show that TNFα induces apoptosis in human aortic endothelial cells, while it promotes the proliferation of vascular smooth muscle cells. Both events involved the activation of the Rb-E2F1 transcriptional regulatory pathway. Stimulation of HAECs with TNF-α led to an increased expression of p73 protein and a reduction in the levels of p53. This involved ASK1 mediated inactivation of Rb and its dissociation from the p73 promoter. In contrast, TNF-α stimulation of vascular smooth muscle cells enhanced the association of E2F1 with proliferative promoters like thymidylate synthase and cdc25A, while Rb was dissociated. ASK1 kinase plays a critical role in the apoptotic process, since its depletion or dissociation from Rb reduced TNF-α induced apoptosis. These results show that the cytokine TNF-α can elicit diametrically opposite responses in vascular endothelial cells and vascular smooth muscle cells, utilizing the Rb-E2F pathway.
TNF-α; endothelial cells; apoptosis; E2F1; p73; ASK1
The membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3.
Chromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer.
Our studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells.
Mucin 4; Pancreatic cancer; Cell proliferation and invasion invasion; Src Kinase; Akt pathway.
Stat3, a member of the signal transducer and activator of transcription family, has the potential to mediate cell survival, growth and differentiation. Stat3 is constitutively activated in numerous cancers, including more than 50% of breast cancers. Previous studies demonstrated that constitutively activated Stat3 plays an important role in breast cancer development and progression by promoting cell proliferation and inhibiting apoptosis. The present study was designed to investigate the potential use of RNA interference (RNAi) to block Stat3 expression and activation, as well as the subsequent effect on human breast cancer cell growth. Our studies show that knockdown of STAT3 expression by siRNA reduced expression of Bcl-xL and survivin in MDA-MB-231 cells, and also led to Fas mediated intrinsic apoptotic pathway by activating Caspases -8, -9, -3 and PARP 1 cleavage. In nude mice, pRNAi-Stat3 significantly suppressed tumor growth compared with controls. It also suppressed Stat3 expression, and downregulated BcL-xL and upregulated Fas, Fas-L and cleaved Caspase 3 expression within the tumor, which significantly induced apoptosis and led to tumor suppression. Thus, targeting Stat3 signaling using siRNA may serve as a novel therapeutic strategy for the treatment of breast cancers expressing constitutively activated Stat3.
Stat3; apoptosis; Fas/Fas-L
Matrix metalloproteinase-2 (MMP-2) expression is often upregulated in advanced cancers and known to play important role in tumor angiogenesis. We previously showed that adenoviral-mediated delivery of siRNA for MMP-2 (Ad-MMP-2-Si) inhibited lung cancer growth, angiogenesis and metastasis. In this study, we investigated the signaling mechanisms involved in Ad-MMP-2-Si-mediated inhibition of angiogenesis. Ad-MMP-2-Si treatment inhibited neo-vascularization in vivo as determined by mouse dorsal air sac model, and conditioned medium from Ad-MMP-2-Si-infected A549 lung cancer cells (Ad-MMP-2-Si-CM) inhibited endothelial tube formation in vitro. Ad-MMP-2-Si-CM decreased proliferation as determined by Ki-67 immunofluorescence and induced apoptosis in endothelial cells as determined by TUNEL assay. Furthermore, Ad-MMP-2-Si-CM inhibited AKT phosphorylation and induced phosphorylation of ERK-MAPKs in endothelial cells. Overexpression of constitutively active-AKT reversed the Ad-MMP-2-Si-CM-mediated inhibition of tube formation and induction of ERK phosphorylation. Conversely, Ad-MMP-2-Si-CM induced TIMP-3 expression, and the interaction of VEGFR2 and TIMP-3 was determined by co-immunoprecipitation experiments. TIMP-3 induction was mediated by ERK activation. In addition, electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrate that Sp1 transcription factor mediated Ad-MMP-2-Si-CM-stimulated increase of TIMP-3. Vasculature destruction was confirmed with co-localization studies with TUNEL and an endothelial marker, CD31, in tumor sections of Ad-MMP-2-Si-treated mice. Our data collectively suggest MMP-2 inhibition induces endothelial apoptosis in vivo and inhibits endothelial tube formation. These experiments provide the first evidence that inhibition of p-AKT and induction of p-ERK1/2 are crucial events in the induction of TIMP-3-mediated endothelial apoptosis in MMP-2 inhibited lung tumors.
ERK; TIMP-3; MMP-2; siRNA; angiogenesis; apoptosis; lung cancer
Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (Ad-MMP-9), affects breast cancer sensitivity to radiation.
In the present study, we used antisense MMP-9 adenoviral construct (Ad-MMP-9) to downregulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro prior to irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were performed with orthotopic breast tumors and the radiosensitivity evaluated both in vitro and in vivo.
Ad-MMP-9 infection resulted in downregulation of radiation-induced levels of hypoxia-inducible factor 1 alpha (HIF1α) and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9 in combination with radiation decreased levels of the transcription factors NF-κB and AP 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas-L apoptotic cascade, which resulted in the cleavage of PARP-1 and caspases 10, 3 and 7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model.
In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.
Ad-MMP-9; Radiation; Orthotopic breast tumor; Hypoxia; Tumor growth
We previously showed that MMP-9 inhibition using an adenoviral-mediated delivery of MMP-9 siRNA (Ad-MMP-9), caused senescence in medulloblastoma cells. Regardless of whether or not, Ad-MMP-9 would induce apoptosis, the possible signaling mechanism is still obscure. In this report, we demonstrate that Ad-MMP-9 induced apoptosis in DAOY cells as determined by propidium iodide and TUNEL staining. Ad-MMP-9 infection induced the release of cytochrome-c, activation of caspases-9-3, and cleavage of PARP. Ad-MMP-9 infection stimulated ERK, and EMSA indicated an increase in NF-κB activation. ERK inhibition, using kinase dead mutant for ERK, ameliorated NF-κB activation and caspase-mediated apoptosis in Ad-MMP-9 infected cells. β1-integrin expression in Ad-MMP-9 infected cells also increased, and this increase was reversed by the reintroduction of MMP-9. We found that, addition of β1 blocking antibodies inhibited Ad-MMP-9-induced ERK activation. Taken together, our results indicate that MMP-9 inhibition induces apoptosis due to altered β1 integrin expression in medulloblastoma. In addition, ERK activation plays an active role in this process and functions upstream of NF-κB activation to initiate the apoptotic signal.
apoptosis; cytochrome C; ERK; Integrin β1; MMP-9; NFκB
The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA (siRNA) to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.
RNAi; uPAR; MMP-9; Invasion; Angiogenesis; Tumor growth
Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas where it promotes invasion and delays tumor growth, both in vitro and in vivo. SPARC, which interacts at the cell surface, has an impact on intracellular signaling and downstream gene expression changes, which might account for some of its effects on invasion and growth. Additional in vitro studies demonstrated that SPARC delays growth, increases attachment, and modulates migration of tumor cells in an extracellular matrix-specific and concentration-dependent manner. Because the signaling aspect of this migration is neither well understood nor characterized, we overexpressed SPARC in both the minimally-invasive U87 cell line and in the most aggressive invasive cell line, SNB19. We first performed RT-PCR analysis and observed an upregulation of uPA and its receptor, uPAR. We also observed increased expression levels of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9). Western blot analysis confirmed these results, and the enzymatic activity of the metalloproteinases and uPA was further supported by zymography.
Downstream of the uPA-uPAR interaction, upregulation of PI3-K occurred in cells overexpressing SPARC. Using GST-TRBD, we showed the upregulation of active GTP-bound RhoA, but neither Rac1 nor Cdc42 were activated. The inhibition of uPA and uPAR downregulated PI3-K activity and cell migration, as shown by matrigel invasion assay. A dorsal skin-fold chamber model revealed the high angiogenic activity of SPARC, though the proliferation of SPARC overexpressing cells was unaffected. Our results show that the small GTPase RhoA was a critical mediator of invasion or migration in the uPA-uPAR/PI3-K signaling pathway.
Glioblastoma; SPARC; uPA-uPAR signaling; small GTPase RhoA; Migration; SPARC Secreted protein acidic and rich in cysteine, MMP-2 and MMP-9 matrix metalloproteinases-2 and -9, RNAi, RNA interference; siRNA, short interfering RNA; uPA, urokinase-type plasminogen activator (receptor); uPAR, urokinase-type plasminogen activator receptor; CMV, cytomegalovirus; PBS, phosphate-buffered saline; FITC, fluorescein 5-isothiocyanate; EV, empty vector; H&E, hematoxylin & eosin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; pU2, plasmid siRNA vector for uPA and uPAR; pGFP, plasmid siRNA for GFP; puPAR,plasmid siRNA vector for uPAR; puPA,plasmid siRNA vector for uPA; PI3-K/Akt, Phosphoinositide 3-kinase/Serine and threonine kinase; FAK, focal adhesion kinase; RT-PCR, reverse transcriptase polymerase chain reaction; ECM, extra-cellular matrix; VEGF, vascular endothelial growth factor; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GPCR, G-protein coupled receptors; BFGF, Basic fibroblast growth factor; TNF-α, tumor necrosis factor; ANG-1 Angiopoietin-1; HBEGF, heparin-binding EGF-like growth factor; IGF-1 insulin-like growth factor 1